**Meet the editors**

Professor Steffen Koschmieder is Professor of Translational Hematology and Oncology, Head of the Clinical Hematology Laboratory, and Attending Physician in the Department of Medicine (Oncology, Hematology, and Stem Cell Transplantation) at the University of Aachen, Germany. He is board-certified for Internal Medicine, Hematology and Medical Oncology, and Hemostaseol-

ogy. He has received his medical training at the Universities of Bochum, Mainz, Dijon, Houston, Salt Lake City, Frankfurt, and Münster as well as the Harvard Institutes of Medicine in Boston where he was a postdoctoral fellow in Prof. Daniel Tenen´s group. His group has made significant contributions to the field of Myeloid Leukemias through basic and clinical research, including the elucidation of critical mechanisms of leukemia pathogenesis and progression, generation of tetracycline-inducible transgenic models of disease, characterization of leukemic stem cell populations as well as the development of novel treatment approaches.

Dr. Utz Krug is Lecturer, Head of the Clinical Hematology Laboratory, Attending Physician, and Group Leader in the Department of Medicine (Hematology, Oncology, Hemostaseology, and Pulmonology) at the University of Münster, Germany. He is board-certified specialist in Internal Medicine, Hematology and Medical Oncology, Palliative Care, and Emergency Medicine. He attended

Medical School at the University of Bochum and at Hannover Medical School, both in Germany. He completed his medical training at Hannover Medical School and at the University of Münster, Germany and was a postdoctoral fellow in Professor H. Phillipp Koeffler's Lab at the University of California, Los Angeles/USA. His group has identified and studied important novel targets in acute myeloid leukemia (AML) and has developed a new clinically relevant risk score for elderly patients with AML.

Contents

**Preface IX** 

Chapter 1 **BCR-ABL Hits at Mitosis; Implications** 

**and Therapeutic Strategy 1**  Katarzyna Piwocka, Kamila Wolanin,

Chapter 2 **BCR/ABL1 Extra Fusions in Patients with** 

Chapter 3 **Causative Factors Involved in Development** 

Chapter 4 *De Novo* **Acquisition of BCR-ABL Mutations for CML Acquired Resistance 69** 

> **of Human Malignancies 85**  Adrian Woolfson and Xiaoyan Jiang

Chapter 6 **The Proteasome as a Therapeutic Target in Chronic Myeloid Leukemia 111**  Ignacio Pérez-Roger and María Pilar Albero

Chapter 7 **Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 123** 

**and Novel Strategies Designed to Override This Resistance 41**  Ellen Weisberg and James D. Griffin

Maria Teresa Vargas

**for Chromosomal Instability, Aneuploidy** 

**Chronic Myeloid Leukaemia (CML) 27** 

**of Resistance to Tyrosine Kinase Inhibition** 

WenYong Chen, Hongfeng Yuan and Zhiqiang Wang

Amanda M. Smith, Kathryn G. Roberts and Nicole M. Verrills

Chapter 5 **Targeting the Chronic Myeloid Leukemia Stem Cell: A Paradigm for the Curative Treatment** 

Monika Kusio-Kobialka and Paulina Podszywalow-Bartnicka

### Contents

#### **Preface XIII**


Amanda M. Smith, Kathryn G. Roberts and Nicole M. Verrills

X Contents


Contents VII

Chapter 19 **The Association of the DNA Repair Genes with** 

Chapter 20 **Apoptosis and Apoptosis Modulators in Myeloid Leukemia 409** 

Maha Abdullah and Zainina Seman

Maha Abdullah and Zainina Seman

Chapter 23 **Myeloid Leukemia: A Molecular Focus on Etiology** 

Muntaser E. Ibrahim and Emad-Aldin I. Osman

**and Risk Within Africa 465** 

Chapter 22 **Epigenetic Changes Associated with** 

**Acute Myeloid Leukemia: The Susceptibility and the Outcome After Therapy 385**

Claudia Bănescu, Carmen Duicu and Minodora Dobreanu

Chapter 21 **Role of Signaling Pathways in Acute Myeloid Leukemia 429**

**Chromosomal Translocation in Leukemia 449**  Soraya Gutierrez, Amjad Javed, Janet Stein, Gary Stein, Sandra Nicovani, Valentina Fernandez, Ricardo Alarcon, Marcela Stuardo, Milka Martinez, Marcela Hinojosa and Boris Rebolledo-Jaramillo


VI Contents

Chapter 8 **Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs):** 

Hsin-An Hou, Wen-Chien Chou and Hwei-Fang Tien

Theresa M. McDevitt and Matthew V. Lorenzi

Chapter 9 **Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia 163**

Chapter 10 **Bone Marrow Microenvironment in** 

Olga Blau

Xiao Tang Hu

**the Pathogenesis of AML 185** 

Chapter 12 **Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 215** Kimberly D. Klarmann, Ming Ji, Huajie Li, Ande Satyanarayana, Wonil Kim, Emily Bowers, Bjorg Gudmundsdottir and Jonathan R. Keller

Chapter 13 **PU.1, a Versatile Transcription Factor and** 

Shinichiro Takahashi

Chapter 15 **p15INK4b, a Tumor Suppressor in**

and Pedro Martin Cabrera

**Acute Myeloid Leukemia 289** Joanna Fares, Linda Wolff and Juraj Bies

Chapter 17 **Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 339**  Julia Schanda, Reinhard Henschler, Manuel Grez and Christian Wichmann

Emma Lång and Stig Ove Bøe

Chapter 18 **Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 363**

**a Suppressor of Myeloid Leukemia 239** 

Chapter 14 **Vav1: A Key Player in Agonist-Induced Differentiation of** 

Chapter 16 **New Molecular Markers in Acute Myeloid Leukemia 313**  Silvia de la Iglesia Iñigo, María Teresa Gómez Casares,

Carmen Elsa López Jorge, Jezabel López Brito

**Promyelocytes from Acute Myeloid Leukemia (APL) 263** Valeria Bertagnolo, Federica Brugnoli and Silvano Capitani

Chapter 11 **Distinct Inhibitory Effect of TGFβ on the Growth of Human Myeloid Leukemia Cells 197** 

**Rationale for Targeting the JAK-STAT Pathway in Other Hematological Malignancies and Solid Tumors 149**


Preface

selected for publication.

Novel BCR-ABL1 fusions

Promyelocytic Leukemia

proteins, PU.1, Vav1, and p15INK4b

overcome these

AML

Cells

cover the following:

Myeloid leukemias have been studied for decades, and considerable progress has been made in the elucidation of critical pathogenetic factors including transcription factor networks and signaling pathways and in the diagnosis and treatment of these leukemias. However, while the prognosis of a fraction of patients (particularly those with chronic myeloid leukemia in chronic phase) has improved dramatically with the advent of novel rationally designed therapies, the prognosis of many other patients (i.e. with most subtypes of acute myeloid leukemia) has not improved to the same degree and have been compounded by the fact that molecular targeted therapies are

The intention of this book is to provide a global scope on these issues. Following an open call, authors were invited to propose topics and send in an abstract of the chapter they wanted to contribute. After selection of appropriate abstracts, full chapters were provided and reviewed. Revised chapters were again reviewed and final chapters

The topics of the present book focus on basic mechanisms of leukemogenesis and

Mechanisms of resistance to tyrosine kinase inhibitors and strategies how to

Novel targets at the cellular and molecular level, including CML stem cells,

Genetic Alterations and new molecular markers in AML, including Acute

The role of the bone marrow microenvironment and TGFß in the pathogenesis of

Hematopoietic transcription factors and tumor suppressors, including the Id

Functional Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor

expensive and are not readily available in all parts of the world.

Chromosomal instability and DNA repair in CML and AML

proteasome inhibitors, and activators of phosphatases

Apoptosis and Apoptosis Modulators in Myeloid Leukemia

### Preface

Myeloid leukemias have been studied for decades, and considerable progress has been made in the elucidation of critical pathogenetic factors including transcription factor networks and signaling pathways and in the diagnosis and treatment of these leukemias. However, while the prognosis of a fraction of patients (particularly those with chronic myeloid leukemia in chronic phase) has improved dramatically with the advent of novel rationally designed therapies, the prognosis of many other patients (i.e. with most subtypes of acute myeloid leukemia) has not improved to the same degree and have been compounded by the fact that molecular targeted therapies are expensive and are not readily available in all parts of the world.

The intention of this book is to provide a global scope on these issues. Following an open call, authors were invited to propose topics and send in an abstract of the chapter they wanted to contribute. After selection of appropriate abstracts, full chapters were provided and reviewed. Revised chapters were again reviewed and final chapters selected for publication.

The topics of the present book focus on basic mechanisms of leukemogenesis and cover the following:

	- Role of Signaling Pathways in Myeloid Leukemia, including BCR-ABL1 and mutant JAK2
	- Epigenetic Changes Associated with Chromosomal Translocation in Leukemia
	- Etiology and Risk factors of AML in Africa

Each chapter is a sole-standing publication that reflects each author´s interpretation of the data. However, the unifying theme is myeloid leukemia. Thus, the book displays a multi-facetted picture of our current understanding of myeloid leukemia pathogenesis. In addition, the open access structure of the book will guarantee widespread access even in cases where resources required for subscription to more expensive scientific journals or books are limited. We encourage the readers to send their comments. This is an exciting new way of discussing science and to support the effort of increasing the alertness and education of patients and physicians all around the globe.

#### **Prof. Dr. Steffen Koschmieder**

Department of Medicine (Oncology, Hematology, and Stem Cell Transplantation) at the University of Aachen, Germany

**Dr. Utz Krug**  Department of Medicine (Hematology, Oncology, Hemostaseology, and Pulmonology) at the University of Münster, Germany

X Preface

the globe.

mutant JAK2

Etiology and Risk factors of AML in Africa

Role of Signaling Pathways in Myeloid Leukemia, including BCR-ABL1 and

Each chapter is a sole-standing publication that reflects each author´s interpretation of the data. However, the unifying theme is myeloid leukemia. Thus, the book displays a multi-facetted picture of our current understanding of myeloid leukemia pathogenesis. In addition, the open access structure of the book will guarantee widespread access even in cases where resources required for subscription to more expensive scientific journals or books are limited. We encourage the readers to send their comments. This is an exciting new way of discussing science and to support the effort of increasing the alertness and education of patients and physicians all around

**Prof. Dr. Steffen Koschmieder** 

Germany

Germany

**Dr. Utz Krug** 

Department of Medicine (Oncology, Hematology,

and Pulmonology) at the University of Münster,

and Stem Cell Transplantation) at the University of Aachen,

Department of Medicine (Hematology, Oncology, Hemostaseology,

Epigenetic Changes Associated with Chromosomal Translocation in Leukemia

**1** 

*Poland* 

**BCR-ABL Hits at Mitosis; Implications for** 

**Chromosomal Instability, Aneuploidy** 

Monika Kusio-Kobialka and Paulina Podszywalow-Bartnicka *Nencki Institute of Experimental Biology, Polish Academy of Sciences* 

An unstable genome is a common hallmark of nearly all solid tumors and most of leukemias in contrast to normal, healthy cells which are able to maintein genome integrity (Negrini et al., 2010). Genomic instability could result from changes in chromosome structure and number as well as changes on the DNA level. Chromosomal instability (CIN) arises from unproper chromosome segregation as well as division defects and leads to aneuploidy (Foijer, 2010), whereas accumulation of mutations and DNA alterations usually is an effect of the defective repair systems and DNA damage response in cancer cells (Economopoulou

Chronic myeloid leukemia (CML) cells expressing the BCR-ABL tyrosine kinase have been found to accumulate mutations as well as chromosomal abnormalities. One of the first indications that CML correlates with additional chromosome changes has been presented in 1987 (Alimena et al., 1987). Moreover, authors showed that the rate of chromosomal anomalies increased during the blastic transformation. In the next years this has been also confirmed by other authors (Hagemeijer, 1987; Johansson et al., 2002; Su et al., 1999; Suzukawa et al., 1997). Later, random aneuploidy rate between chromosomes 9 and 18 has been reported in CML patients – both, untreated as well as upon imatinib therapy (Amiel et al., 2006). In broader analysis of CML patients it was found that chromosomal instability caused by centrosomal aberrations significantly correlated with the disease progression (Giehl et al., 2005). In the chronic phase only one sample out of 18 showed additional karyotypic alterations, in contrast to blast crisis where 73% patients (11/16) displayed additional karyotype alterations. The observation that CML patients have karyotype aberrations was confirmed in other studies where complex chromosomal rearrangements (CCR) were investigated (Babicka et al., 2006). By using cytogenetics, the FISH, and multicolor FISH (mFISH) methods, a very high level of the genomic instability at the chromosomal level, in cells obtained from chronic myeloid leukemia patients was observed. Altogether, it was shown that the aberrations associated with the progression of BCR-ABLpositive CML chronic phase to the aggressive blast crisis include additional chromosomes (Ph1, +8, +19), isochromosome 17q (associated with the loss of p53), reciprocal

**1. Introduction** 

et al., 2011).

**1.1 Genomic and chromosomal instability in CML** 

**and Therapeutic Strategy** 

Katarzyna Piwocka, Kamila Wolanin,

### **BCR-ABL Hits at Mitosis; Implications for Chromosomal Instability, Aneuploidy and Therapeutic Strategy**

Katarzyna Piwocka, Kamila Wolanin, Monika Kusio-Kobialka and Paulina Podszywalow-Bartnicka *Nencki Institute of Experimental Biology, Polish Academy of Sciences Poland* 

#### **1. Introduction**

#### **1.1 Genomic and chromosomal instability in CML**

An unstable genome is a common hallmark of nearly all solid tumors and most of leukemias in contrast to normal, healthy cells which are able to maintein genome integrity (Negrini et al., 2010). Genomic instability could result from changes in chromosome structure and number as well as changes on the DNA level. Chromosomal instability (CIN) arises from unproper chromosome segregation as well as division defects and leads to aneuploidy (Foijer, 2010), whereas accumulation of mutations and DNA alterations usually is an effect of the defective repair systems and DNA damage response in cancer cells (Economopoulou et al., 2011).

Chronic myeloid leukemia (CML) cells expressing the BCR-ABL tyrosine kinase have been found to accumulate mutations as well as chromosomal abnormalities. One of the first indications that CML correlates with additional chromosome changes has been presented in 1987 (Alimena et al., 1987). Moreover, authors showed that the rate of chromosomal anomalies increased during the blastic transformation. In the next years this has been also confirmed by other authors (Hagemeijer, 1987; Johansson et al., 2002; Su et al., 1999; Suzukawa et al., 1997). Later, random aneuploidy rate between chromosomes 9 and 18 has been reported in CML patients – both, untreated as well as upon imatinib therapy (Amiel et al., 2006). In broader analysis of CML patients it was found that chromosomal instability caused by centrosomal aberrations significantly correlated with the disease progression (Giehl et al., 2005). In the chronic phase only one sample out of 18 showed additional karyotypic alterations, in contrast to blast crisis where 73% patients (11/16) displayed additional karyotype alterations. The observation that CML patients have karyotype aberrations was confirmed in other studies where complex chromosomal rearrangements (CCR) were investigated (Babicka et al., 2006). By using cytogenetics, the FISH, and multicolor FISH (mFISH) methods, a very high level of the genomic instability at the chromosomal level, in cells obtained from chronic myeloid leukemia patients was observed. Altogether, it was shown that the aberrations associated with the progression of BCR-ABLpositive CML chronic phase to the aggressive blast crisis include additional chromosomes (Ph1, +8, +19), isochromosome 17q (associated with the loss of p53), reciprocal

BCR-ABL Hits at Mitosis; Implications for Chromosomal

relapse and malignant progression.

Instability, Aneuploidy and Therapeutic Strategy 3

Altogether, there is no doubt, that defects in DNA repair mechanisms and genomic surveillance in CML cells are an effect of the expression of BCR-ABL itself. However, there was still an open question, whether occurrence of the genomic instability participates in the development of the blast crisis phase (Penserga & Skorski, 2007; Shet et al., 2002). This has been strongly indicated to play a significant role in the malignant progression of the disease by many authors (Burke & Carroll, 2010; Salleset al., 2011; Skorski, 2008; Skorski, 2011). Till now, convincing data was presented and it seems clear, that genetic instability, accumulation of mutations and additional chromosomal alterations are the major factors involved in the CML progression and resistance to cell death. This leads to an accumulation of additional genetic aberrations and changes in gene expression, which result in the expansion of differentiation-arrested and increasingly malignant cell clones. Importantly, genetic instability of tyrosine kinase refractory cells, including leukemia stem cells (LSCs) has also recently been proposed as a reason for their fast transformation leading to the generation of additional resistant clones and transformation to a blast phase (Skorski, 2011). This mechanism could be responsible for clonal evolution and expansion causing finally

The current model of blastic transformation proposed recently by Perotti (Perrotti et al., 2010), indicates that acquiring of additional genetic and epigenetic changes by LSCs or their progeny causes leukemia transformation from the chronic phase to the advanced phases. This can explain the complexity of the disease progression and blast crisis as well as the inability to find common features of cells in blast crisis and specific secondary genetic aberrations. Most likely different mutations and aberrations are cumulated to obtain the critical point allowing the disease to progress. Thus, it will be very difficult to plan the therapeutic strategy against genetically unstable LSCs, resistant to tyrosine kinase inhibitors,

It has been known for more than a century that neoplastic cells could exhibit disturbances of the cell division process (Boveri, 1902, 1914). Boveri observed that sea urchin embryos manipulated to undergo mitosis in the presence of multipolar spindles produced aneuploid progeny and proposed that tumors arise from normal cells becoming aneuploid as a result of aberrant mitoses. Boveri's theory that division errors and aneuploidy could lead to cancer development has been revisited during the last decade (Duesberg et al., 2006; Holland &

Today, it is commonly accepted that aberrant mitoses result in chromosomal instability (CIN), leading to the gain or loss of whole or large fragments of chromosomes, which are the main form of genomic instability in cancers. As it was mentioned in the previous chapter, it is fully convincing that expression of BCR-ABL leads to significant chromosomal aberrations. Moreover, these abnormalities increase along with the disease progression, participating in the blastic transformation. Below, we present current data concerning the role of BCR-ABL-mediated defects in the mechanisms controlling cell division as well as the

Centrosomes are small organelles with a crucial role in the formation of bipolar mitotic spindle, which is necessary for the accurate segregation of chromosomes (Fukasawa, 2007;

with the already used agents and probably novel therapies need to be developed.

**2. The role of aberrant divisions in CML cells** 

role of BRCA1 in the development of aneuploidy in CML.

Cleveland, 2009; Weaver & Cleveland, 2006).

**2.1 Centrosomal multiplication** 

translocations, loss-of-heterozygosity at 14q32, homozygous mutations/deletions of pRb and p16/ARF, and mutations in p53 and RAS (Calabretta & Perrotti, 2004). The possible mechanisms participating in the BCR-ABL-mediated aneuploidy will be broadly described and discussed in the next paragraphs.

BCR-ABL has been also indicated as a promoter of secondary DNA mutations in CML (Burke & Carroll, 2010). This is the effect of the defective DNA damage response and DNA repair mechanisms found in CML cells. DNA damage can occur as single-nucleotide alterations, single-strand breaks (SSB), or double-strand breaks (DSB). Double-strand breaks are proposed to be the most mutagenic, as neither strand remains intact to serve as a template for repair. Single-nucleotide alterations are repaired by mismatch repair (MMR) or nucleotide excision repair (NER) mechanisms. Single or double-strand breaks are repaired by either high-fidelity homologous recombination repair (HRR) or non-homologous endjoining (NHEJ), when a sister chromatid is not available as a template. The last mechanism is error-prone and can lead to short deletions in the repaired strands.

Data from different laboratories collectively indicate that BCR-ABL promotes dysfunctions of nearly all mechanisms participating in the DNA repair. It is known that BCR-ABL cells treated with genotoxic agents present higher levels of DNA damage and aberrant repair systems, leading to the accumulation of DNA errors (Brady, 2003; Laurent et al., 2003; Slupianek et al., 2002). Studies from Skorski's group clearly showed that expression of BCR-ABL affects different mechanisms participating in the DNA repair. They found that BCR-ABL modifies the repair of DNA double-strand breaks (Koptyra et al., 2008; Nowicki et al., 2004; Slupianek et al., 2006). Briefly, CML cells produced increased rate of DSBs in S and G2/M phases of the cell cycle, as a result of oxidative DNA damage caused by BCR-ABL. These breaks were repaired, however with a high mutation rate and large deletions, as a result of defective HRR and NHEJ repair systems, respectively. Moreover, they found that BCR-ABL is able to inhibit both, mismatch repair (MMR) and inhibit apoptosis as well as to induce point mutations (Stoklosa et al., 2008). Upon this, CML cells were able to survive treatment leading to generation of the O(6)-methylguanine and O(4)-methylthymine recognized by the MMR system, however they displayed 15-fold higher mutation frequency than parental counterparts.

Deutsch et al indicated that DNA-PKcs, a protein involved in the NHEJ repair system, may be downregulated by BCR-ABL (Deutsch et al., 2001). This decrease was proteasome- and tyrosine kinase-dependent, as it was reversed by proteasome as well as tyrosime kinase inhibitors. Alternatively, the role of DNA-PKcs has been recently indicated to switch on the backup-NHEJ system, which is more error-prone (Poplawski & Blasiak, 2010). It was also shown that BCR-ABL upregulates the error-prone DSB repair pathways, particularly singlestrand annealing and non-homologous end-joining due to an increased level of DNA-endprocessing factor CtIP (Salles et al., 2011). Additionally, BCR-ABL also promotes the DNA DSB repair by using the highly mutagenic single-strand annealing (SSA) pathway which involves single repeats (Fernandes et al., 2009). This required the active Ras and PI3K pathways, acting downstream of the Y177 site of BCR-ABL, which is a major regulatory site for ROS induction and is necessary for the optimal activation of the PI3K and Ras pathways. Moreover, using stromal cell lines authors also showed that the stromal cell-conditioned media increased the SSA frequency, measured in K562 cells in the presence or absence of imatinib. This supported the hypothesis that microenvironment additionaly promotes mutagenesis in CML cells.

translocations, loss-of-heterozygosity at 14q32, homozygous mutations/deletions of pRb and p16/ARF, and mutations in p53 and RAS (Calabretta & Perrotti, 2004). The possible mechanisms participating in the BCR-ABL-mediated aneuploidy will be broadly described

BCR-ABL has been also indicated as a promoter of secondary DNA mutations in CML (Burke & Carroll, 2010). This is the effect of the defective DNA damage response and DNA repair mechanisms found in CML cells. DNA damage can occur as single-nucleotide alterations, single-strand breaks (SSB), or double-strand breaks (DSB). Double-strand breaks are proposed to be the most mutagenic, as neither strand remains intact to serve as a template for repair. Single-nucleotide alterations are repaired by mismatch repair (MMR) or nucleotide excision repair (NER) mechanisms. Single or double-strand breaks are repaired by either high-fidelity homologous recombination repair (HRR) or non-homologous endjoining (NHEJ), when a sister chromatid is not available as a template. The last mechanism is

Data from different laboratories collectively indicate that BCR-ABL promotes dysfunctions of nearly all mechanisms participating in the DNA repair. It is known that BCR-ABL cells treated with genotoxic agents present higher levels of DNA damage and aberrant repair systems, leading to the accumulation of DNA errors (Brady, 2003; Laurent et al., 2003; Slupianek et al., 2002). Studies from Skorski's group clearly showed that expression of BCR-ABL affects different mechanisms participating in the DNA repair. They found that BCR-ABL modifies the repair of DNA double-strand breaks (Koptyra et al., 2008; Nowicki et al., 2004; Slupianek et al., 2006). Briefly, CML cells produced increased rate of DSBs in S and G2/M phases of the cell cycle, as a result of oxidative DNA damage caused by BCR-ABL. These breaks were repaired, however with a high mutation rate and large deletions, as a result of defective HRR and NHEJ repair systems, respectively. Moreover, they found that BCR-ABL is able to inhibit both, mismatch repair (MMR) and inhibit apoptosis as well as to induce point mutations (Stoklosa et al., 2008). Upon this, CML cells were able to survive treatment leading to generation of the O(6)-methylguanine and O(4)-methylthymine recognized by the MMR system, however they displayed 15-fold higher mutation frequency

Deutsch et al indicated that DNA-PKcs, a protein involved in the NHEJ repair system, may be downregulated by BCR-ABL (Deutsch et al., 2001). This decrease was proteasome- and tyrosine kinase-dependent, as it was reversed by proteasome as well as tyrosime kinase inhibitors. Alternatively, the role of DNA-PKcs has been recently indicated to switch on the backup-NHEJ system, which is more error-prone (Poplawski & Blasiak, 2010). It was also shown that BCR-ABL upregulates the error-prone DSB repair pathways, particularly singlestrand annealing and non-homologous end-joining due to an increased level of DNA-endprocessing factor CtIP (Salles et al., 2011). Additionally, BCR-ABL also promotes the DNA DSB repair by using the highly mutagenic single-strand annealing (SSA) pathway which involves single repeats (Fernandes et al., 2009). This required the active Ras and PI3K pathways, acting downstream of the Y177 site of BCR-ABL, which is a major regulatory site for ROS induction and is necessary for the optimal activation of the PI3K and Ras pathways. Moreover, using stromal cell lines authors also showed that the stromal cell-conditioned media increased the SSA frequency, measured in K562 cells in the presence or absence of imatinib. This supported the hypothesis that microenvironment additionaly promotes

error-prone and can lead to short deletions in the repaired strands.

and discussed in the next paragraphs.

than parental counterparts.

mutagenesis in CML cells.

Altogether, there is no doubt, that defects in DNA repair mechanisms and genomic surveillance in CML cells are an effect of the expression of BCR-ABL itself. However, there was still an open question, whether occurrence of the genomic instability participates in the development of the blast crisis phase (Penserga & Skorski, 2007; Shet et al., 2002). This has been strongly indicated to play a significant role in the malignant progression of the disease by many authors (Burke & Carroll, 2010; Salleset al., 2011; Skorski, 2008; Skorski, 2011).

Till now, convincing data was presented and it seems clear, that genetic instability, accumulation of mutations and additional chromosomal alterations are the major factors involved in the CML progression and resistance to cell death. This leads to an accumulation of additional genetic aberrations and changes in gene expression, which result in the expansion of differentiation-arrested and increasingly malignant cell clones. Importantly, genetic instability of tyrosine kinase refractory cells, including leukemia stem cells (LSCs) has also recently been proposed as a reason for their fast transformation leading to the generation of additional resistant clones and transformation to a blast phase (Skorski, 2011). This mechanism could be responsible for clonal evolution and expansion causing finally relapse and malignant progression.

The current model of blastic transformation proposed recently by Perotti (Perrotti et al., 2010), indicates that acquiring of additional genetic and epigenetic changes by LSCs or their progeny causes leukemia transformation from the chronic phase to the advanced phases. This can explain the complexity of the disease progression and blast crisis as well as the inability to find common features of cells in blast crisis and specific secondary genetic aberrations. Most likely different mutations and aberrations are cumulated to obtain the critical point allowing the disease to progress. Thus, it will be very difficult to plan the therapeutic strategy against genetically unstable LSCs, resistant to tyrosine kinase inhibitors, with the already used agents and probably novel therapies need to be developed.

#### **2. The role of aberrant divisions in CML cells**

It has been known for more than a century that neoplastic cells could exhibit disturbances of the cell division process (Boveri, 1902, 1914). Boveri observed that sea urchin embryos manipulated to undergo mitosis in the presence of multipolar spindles produced aneuploid progeny and proposed that tumors arise from normal cells becoming aneuploid as a result of aberrant mitoses. Boveri's theory that division errors and aneuploidy could lead to cancer development has been revisited during the last decade (Duesberg et al., 2006; Holland & Cleveland, 2009; Weaver & Cleveland, 2006).

Today, it is commonly accepted that aberrant mitoses result in chromosomal instability (CIN), leading to the gain or loss of whole or large fragments of chromosomes, which are the main form of genomic instability in cancers. As it was mentioned in the previous chapter, it is fully convincing that expression of BCR-ABL leads to significant chromosomal aberrations. Moreover, these abnormalities increase along with the disease progression, participating in the blastic transformation. Below, we present current data concerning the role of BCR-ABL-mediated defects in the mechanisms controlling cell division as well as the role of BRCA1 in the development of aneuploidy in CML.

#### **2.1 Centrosomal multiplication**

Centrosomes are small organelles with a crucial role in the formation of bipolar mitotic spindle, which is necessary for the accurate segregation of chromosomes (Fukasawa, 2007;

BCR-ABL Hits at Mitosis; Implications for Chromosomal

Instability, Aneuploidy and Therapeutic Strategy 5

feature of CML, they have been proposed as a cause of karyotype instability and aneuploidy in CML progenitor cells as well as a valuable prognostic factor. In the long-term *in vitro* studies, using a cellular model of the chronic phase of CML, authors confirmed, that expression of BCR-ABL leads to significant centrosomal hypertrophy visible already after 4 weeks of BCR-ABL expression (Giehl et al., 2007). This increased upon the next 10 weeks of

We also found, using a mouse cellular model of CML, that the stable expression of low or high level of BCR-ABL in mouse progenitor 32D cells leads to the generation of cells with supernumerary centrosomes (Wolanin et al., 2010). This was accompanied by increased percentage of cells with aberrant mitoses, particularly multipolar spindles, lagging chromosomes and multinuclei. The presence of aberrant cells correlated with the level of BCR-ABL expression, indicating that the BCR-ABL itself is responsible for these abnormalities. Interestingly, Patel and colleagues presented that CML cells have defects in the centrosome-centriole cycle (Patel & Gordon, 2009). They showed that p210 (BCR-ABL1) and p145 (ABL1) are both, centrosome-associated proteins and form a complex with the pericentriolar protein, pericentrin. Numerical and structural centrosomal abnormalities were found in CML cell lines and in primary CD34+ cells from CML patients as a result of an increased level of separase participating in the abnormalities in the centrosome-centriole cycle. They also confirmed the previous data that abnormal centrosome distribution,

Although the tyrosine kinase inhibitors are very potent, selective and successful therapeutic agents for treatment of leukemia as well as some solid tumors it can not be neglected that some reports indicated that they can lead to centrosome aberrations in cancer as well as normal cells (Fabarius et al., 2005; Fabarius et al., 2008; Giehl et al., 2010). This was caused by blocking cells in the G1/S transition and the inhibition of cell growth which was followed by centrosomal aberrations. This should be taken into consideration with regards to the potential side-effects as well as a possible reason of dangerous clonal chromosomal abnormalities observed in BCR-ABL-negative progenitor cells under imatinib therapy.

The spindle assembly checkpoint (SAC) plays a major role in the division control and segregation of sister chromatids, preventing occurence of aneuploidy (Chin & Yeong, 2010; Kops, 2008; Logarinho & Bousbaa, 2008; Nezi & Musacchio, 2009). SAC proteins, including Mad1 (mitotic arrest-deficient protein 1), Mad2, Bub1 (budding uninhibited by benzimidazoles 1), BubR1 (Bub1-related kinase 1) and Bub3 are recruited to unattached or tensionless kinetochores, forming mitotic checkpoint complex, which inhibits the anaphase promoting complex (APC). This protects cells from preearly anaphase entry and unproper segregation of chromatids. In physiological conditions the mitotic checkpoint is temporarily activated until the mitotic spindle is properly formed, whereas in anticancer therapy it is activated upon treatment with a group of microtubule damaging agents, such as taxanes and vinca alcaloids. Both interfere with tubulin organization and spindle formation, leading

It is known that the complete loss of the mitotic checkpoint function results in embryonic lethality, what was shown in *Caenorhabditis elegans* (Kitagawa & Rose, 1999) as well as in mammalian cells (Michel et al., 2001; Schliekelman et al., 2009). Alternatively, partial loss of its function leads to chromosomes missegregation and chromosomal instability (Bharadwaj

propagation and correlated with the clonal expantion of aneuploid cells.

amplification and loss are more evident in the advanced stages of CML.

to the cell cycle arrest in mitosis and eventually cell death.

**2.2 Mitotic checkpoint failure** 

Rusan & Rogers, 2009; Tanenbaum & Medema, 2010). Briefly, they are formed by paired centrioles surrounded by a protein matrix of pericentriolar material, including pericentrin. Their function is to nucleate and anchor microtubules to form an interphase cytoplasmicmicrotubule network and mitotic spindle. During the cell division, each daughter cell receives one centrosome, thus the centrosome has to duplicate before the next mitosis. This takes place during the S phase and is driven at least partially by the Cdk2-cyclin E complex. Coordination of the DNA and centrosome replication is crucial to avoid their overduplication. Two mature centrosomes are generated at the late G2 phase and they become the spindle poles. It was shown that the DNA damage checkpoint proteins, such as ATM, ATR, Chk1 and Chk2 and others also localize at the centrosomes (Zhang et al., 2007). It seems that these proteins interact with gamma-tubulin and are involved in the controlling of microtubule kinetics during the DNA damage response. It was reported that DNA damage leads to centrosome amplification in the G2 phase as a result of cell cycle arrest (Inanc et al., 2010). Studies performed by Dodson and colleagues showed the involvement of ATM in the centrosome amplification in response to DNA damage, however gene targeting of *ATM* reduced, but did not abrogate completely centrosome amplification (Dodson et al., 2004). Alternatively, data from lymphoid gamma-irradiated cells showed that neither ATM nor ATR kinases are involved in this process, however Chk1-dependent signaling seems to be crucial (Bourke et al., 2007). This issue still needs to be clarified.

It is commonly accepted that the appearance of supernumerary centrosomes is associated with aberrant mitoses and chromosomal instability. Multipolar mitoses, lagging chromosomes or multinuclei are observed in cells with overduplicated centrosomes. Cells with three centrosomes usually undergo cytokinesis and some of the generated cells are viable, however aneuploid. Cells with multipolar (>3) spindles fail to undergo cytokinesis and can become polyploid if they are p53-deficient and are able to continue the cell cycle (Godinho et al., 2009).

Centrosome abnormalities are commonly observed in cancers and participate in the chromosomal instability and tumorigenesis (Carroll et al., 1999; Duensing & Duensing, 2010; Pihan et al., 2001). As mentioned before, mutlipolar mitosis as a result of centrosome overduplication can lead to gross chromosome missegregation and cell death. Thus cancer cells with supernumerary centrosomes possess the ability to suppress multipolar mitoses due to the inactivation, clustering or asymmetric segregation of extra centrosomes (Brinkley, 2001; Godinhoet al., 2009). This results in the formation of a bipolar, functional, however not symmetric mitotic spindle and so called mitotic stability of aneuploid cancer cells.

Abnormalities in the number of centrosomes were also found in leukemias. It was reported that defects in the number of centrosomes caused by the p53 mutation and cyclin E overexpression, detected in bladder cancers, led to centrosome amplification and chromosomal instability (Kawamura et al., 2004). Moreover, the centrosome aberrations were proposed as one of the main factors responsible for aneuploidy in acute myeloid leukemia (Kramer et al., 2003; Neben et al., 2003). Studies of CD34+ Ph+ cells isolated from chronic myeloid leukemia patients showed that centrosome aberrations correlate with the stage of the disease and aneuploidy (Giehl et al., 2005). In these studies freshly isolated cells from CML patients, in the chronic phase or blast crisis, were stained for pericentrin and gamma-tubulin to analyse the number as well as the structure of centrosomes. Moreover, they were studied for additional karyotypic abnormalities. Importantly, a strong correlation between the increase of centrosome aberrations, CML progression and blastic transformation was found. As centrosome defects were indicated as en early detectable

Rusan & Rogers, 2009; Tanenbaum & Medema, 2010). Briefly, they are formed by paired centrioles surrounded by a protein matrix of pericentriolar material, including pericentrin. Their function is to nucleate and anchor microtubules to form an interphase cytoplasmicmicrotubule network and mitotic spindle. During the cell division, each daughter cell receives one centrosome, thus the centrosome has to duplicate before the next mitosis. This takes place during the S phase and is driven at least partially by the Cdk2-cyclin E complex. Coordination of the DNA and centrosome replication is crucial to avoid their overduplication. Two mature centrosomes are generated at the late G2 phase and they become the spindle poles. It was shown that the DNA damage checkpoint proteins, such as ATM, ATR, Chk1 and Chk2 and others also localize at the centrosomes (Zhang et al., 2007). It seems that these proteins interact with gamma-tubulin and are involved in the controlling of microtubule kinetics during the DNA damage response. It was reported that DNA damage leads to centrosome amplification in the G2 phase as a result of cell cycle arrest (Inanc et al., 2010). Studies performed by Dodson and colleagues showed the involvement of ATM in the centrosome amplification in response to DNA damage, however gene targeting of *ATM* reduced, but did not abrogate completely centrosome amplification (Dodson et al., 2004). Alternatively, data from lymphoid gamma-irradiated cells showed that neither ATM nor ATR kinases are involved in this process, however Chk1-dependent signaling seems to

It is commonly accepted that the appearance of supernumerary centrosomes is associated with aberrant mitoses and chromosomal instability. Multipolar mitoses, lagging chromosomes or multinuclei are observed in cells with overduplicated centrosomes. Cells with three centrosomes usually undergo cytokinesis and some of the generated cells are viable, however aneuploid. Cells with multipolar (>3) spindles fail to undergo cytokinesis and can become polyploid if they are p53-deficient and are able to continue the cell cycle

Centrosome abnormalities are commonly observed in cancers and participate in the chromosomal instability and tumorigenesis (Carroll et al., 1999; Duensing & Duensing, 2010; Pihan et al., 2001). As mentioned before, mutlipolar mitosis as a result of centrosome overduplication can lead to gross chromosome missegregation and cell death. Thus cancer cells with supernumerary centrosomes possess the ability to suppress multipolar mitoses due to the inactivation, clustering or asymmetric segregation of extra centrosomes (Brinkley, 2001; Godinhoet al., 2009). This results in the formation of a bipolar, functional, however not

Abnormalities in the number of centrosomes were also found in leukemias. It was reported that defects in the number of centrosomes caused by the p53 mutation and cyclin E overexpression, detected in bladder cancers, led to centrosome amplification and chromosomal instability (Kawamura et al., 2004). Moreover, the centrosome aberrations were proposed as one of the main factors responsible for aneuploidy in acute myeloid leukemia (Kramer et al., 2003; Neben et al., 2003). Studies of CD34+ Ph+ cells isolated from chronic myeloid leukemia patients showed that centrosome aberrations correlate with the stage of the disease and aneuploidy (Giehl et al., 2005). In these studies freshly isolated cells from CML patients, in the chronic phase or blast crisis, were stained for pericentrin and gamma-tubulin to analyse the number as well as the structure of centrosomes. Moreover, they were studied for additional karyotypic abnormalities. Importantly, a strong correlation between the increase of centrosome aberrations, CML progression and blastic transformation was found. As centrosome defects were indicated as en early detectable

symmetric mitotic spindle and so called mitotic stability of aneuploid cancer cells.

be crucial (Bourke et al., 2007). This issue still needs to be clarified.

(Godinho et al., 2009).

feature of CML, they have been proposed as a cause of karyotype instability and aneuploidy in CML progenitor cells as well as a valuable prognostic factor. In the long-term *in vitro* studies, using a cellular model of the chronic phase of CML, authors confirmed, that expression of BCR-ABL leads to significant centrosomal hypertrophy visible already after 4 weeks of BCR-ABL expression (Giehl et al., 2007). This increased upon the next 10 weeks of propagation and correlated with the clonal expantion of aneuploid cells.

We also found, using a mouse cellular model of CML, that the stable expression of low or high level of BCR-ABL in mouse progenitor 32D cells leads to the generation of cells with supernumerary centrosomes (Wolanin et al., 2010). This was accompanied by increased percentage of cells with aberrant mitoses, particularly multipolar spindles, lagging chromosomes and multinuclei. The presence of aberrant cells correlated with the level of BCR-ABL expression, indicating that the BCR-ABL itself is responsible for these abnormalities. Interestingly, Patel and colleagues presented that CML cells have defects in the centrosome-centriole cycle (Patel & Gordon, 2009). They showed that p210 (BCR-ABL1) and p145 (ABL1) are both, centrosome-associated proteins and form a complex with the pericentriolar protein, pericentrin. Numerical and structural centrosomal abnormalities were found in CML cell lines and in primary CD34+ cells from CML patients as a result of an increased level of separase participating in the abnormalities in the centrosome-centriole cycle. They also confirmed the previous data that abnormal centrosome distribution, amplification and loss are more evident in the advanced stages of CML.

Although the tyrosine kinase inhibitors are very potent, selective and successful therapeutic agents for treatment of leukemia as well as some solid tumors it can not be neglected that some reports indicated that they can lead to centrosome aberrations in cancer as well as normal cells (Fabarius et al., 2005; Fabarius et al., 2008; Giehl et al., 2010). This was caused by blocking cells in the G1/S transition and the inhibition of cell growth which was followed by centrosomal aberrations. This should be taken into consideration with regards to the potential side-effects as well as a possible reason of dangerous clonal chromosomal abnormalities observed in BCR-ABL-negative progenitor cells under imatinib therapy.

#### **2.2 Mitotic checkpoint failure**

The spindle assembly checkpoint (SAC) plays a major role in the division control and segregation of sister chromatids, preventing occurence of aneuploidy (Chin & Yeong, 2010; Kops, 2008; Logarinho & Bousbaa, 2008; Nezi & Musacchio, 2009). SAC proteins, including Mad1 (mitotic arrest-deficient protein 1), Mad2, Bub1 (budding uninhibited by benzimidazoles 1), BubR1 (Bub1-related kinase 1) and Bub3 are recruited to unattached or tensionless kinetochores, forming mitotic checkpoint complex, which inhibits the anaphase promoting complex (APC). This protects cells from preearly anaphase entry and unproper segregation of chromatids. In physiological conditions the mitotic checkpoint is temporarily activated until the mitotic spindle is properly formed, whereas in anticancer therapy it is activated upon treatment with a group of microtubule damaging agents, such as taxanes and vinca alcaloids. Both interfere with tubulin organization and spindle formation, leading to the cell cycle arrest in mitosis and eventually cell death.

It is known that the complete loss of the mitotic checkpoint function results in embryonic lethality, what was shown in *Caenorhabditis elegans* (Kitagawa & Rose, 1999) as well as in mammalian cells (Michel et al., 2001; Schliekelman et al., 2009). Alternatively, partial loss of its function leads to chromosomes missegregation and chromosomal instability (Bharadwaj

BCR-ABL Hits at Mitosis; Implications for Chromosomal

cytokinesis and mitotic exit.

Instability, Aneuploidy and Therapeutic Strategy 7

found at the centrosomes of interphase cells, at the poles of the bipolar spindle and in the midbody of the mitotic apparatus. All three Aurora kinases members are overexpressed in many human cancers. This correlated with chromosomal instability and clinically aggressive forms of disease (Fu et al., 2007; Meraldi et al., 2004). Aurora A is localized in centrosomes and is important for maturation, spindle assembly and metaphase I spindle orientation. It has two independent functions in centrosome maturation and asymmetric protein localization during mitosis. Ectopic overexpression of Aurora A was shown to induce oncogenic transformation (Katayama et al., 2003). Moreover, overexpression of Aurora A and aneuploidy have been proposed as predictors of poor outcome in serous ovarian carcinoma (Lassus et al., 2011). Also a high level of Aurora B has been reported to promote

tetraploidy and tumorigenesis in the mouse Xenograft model (Nguyen et al., 2009).

CML treatment (Gontarewicz et al., 2008), what will be discussed by us later.

High expression of Aurora A in leukemia cell lines and freshly isolated leukemia CML cells has been presented by Ochi T et al (Ochi et al., 2009). We also showed that the expression of BCR-ABL leads to the mislocalization of Aurora A in the chromosomal passenger complex (Wolanin et al., 2006). The importance of Aurora A-dependent signaling in CML has been shown in studies indicating that Aurora inhibitors seem to be very effective therapeutics for

Another family of tubulin-associated serine/threonine kinases, Polo-like, has also received significant attention regarding its participation in tumorigenesis. As far, in mammalian cells four members of this family have been identified (PLK1-4), and each one of them has a distinct function. PLK1 is essentially involved in the control of mitotic steps, PLK2 and PLK3 have been described as potential regulators of the G1 and early S phases of the cell cycle, PLK4 as a major centrosome duplication regulator. Polo-like kinase 1 (PLK1) is a key regulator of mitosis and participates in regulating this process from its entry to cytokinesis (Yuan et al., 2011). Transcription and translation of PLK1 is highly coordinated with cell cycle progression. *Plk1* mRNA and protein levels begin to accumulate in the S-phase and reach a peak at the G2/M transition and then decline upon mitotic exit (Lee et al., 1995). At the G2/M phase, PLK1 regulates the Cdk1/Cyclin B1 complex promoting mitotic entry and regulating mitotic progression due to regulation of phosphorylation of Cyclin B1, Cdk1, Myt1 and Cdc25C. PLK1 also plays a role in centrosome maturation by promoting increased recruitment of microtubules to the spindle pole bodies. It also regulates the localization of Aurora A to the centrosomes for proper maturation. It is known today that all mitotic kinases interplay with each other and form an extensive functional network, thus targeting any of them has tremendous consequences for cell physiology (Lens et al., 2010). Additionally, it was shown that PLK1 catalysis survivin priming phosphorylation at Ser20, what is necessary for survivin-mediated Aurora B docking to the centromere and activation (Chu et al., 2010). Expression of the non-phosphorylable survivin mutant prevented Aurora B activation and corrected spindle microtubule attachment. We also observed that silencing of survivin in CML cells significantly affected CPC function and mitosis as well as proper completion of cytokinesis leading to the formation of giant polyploid cells (Wolanin et al., 2006). PLK1 also regulates the spindle assembly checkpoint (Nezi & Musacchio, 2009) probably by phosphorylation of BubR1 and finally, regulates chromosome segregation,

PLK1, similarly to other mitotic kinases has been shown to be upregulated in cancers, including lymphomas. Studies of a big group of non-Hodgkin's lymphoma (NHLs) patients presented that the level of PLK1 expression was significantly lower in low-grade NHLs than

& Yu, 2004; Ito & Matsumoto, 2010). This was due to the inability to activate the mitotic checkpoint and to arrest in mitosis in response to some disturbances. Instead - further progression of mitosis eventually leads to aberrant divisions and unproper chromosomes segregation.

Dysfunctions of the mitotic checkpoint were reported in different types of cancers (Baker et al., 2005; Bannon & Mc Gee, 2009; Tanaka & Hirota, 2009). They correlated with aneuploidy, disease progression and the increase of aggressiveness. Interestingly, similar effects were observed in case of the upregulation or decreased expression of mitotic checkpoint members. For example, the Mad2 protein has been recently proposed as a critical factor leading to aneuploidy in cancers with defects in the Rb and p53 pathways (Schvartzman et al., 2011). Authors found that Mad2 expression is repressed by p53 *via* the Rb pathway, thus the cancer cells lacking the Rb protein require Mad2 upregulation leading to chromosomal instability and tumor progression *in vivo*. On the other hand, also Mad2 haplo-insufficiency caused chromosomal instability in human cancer cells and murine primary embryonic fibroblasts (Michelet al., 2001).

BubR1 dysfunctions has also been found as a cause of cancer-susceptible disorder mosaic variegated aneuploidy (MVA) (Suijkerbuijk et al., 2010). Similarly to Mad2, BubR1 can be also overexpressed in cancer, what was shown in hepatocellular carcinoma (HCC) (Liu et al., 2009). Authors suggest that BubR1 overexpression, which was found in 45% of patients correlated with later stages and was associated with worse prognosis, thus it can be used as a potential prognostic factor for HCC.

 There were indications that CML cells could have a dysfunctional mitotic checkpoint, as their resistance to spindle poisons was reported previously. In the K562 and Lama-84 CML cell lines, microtubule disruption caused either by paclitaxel, nocodazole or novel microtubule-targeting agent PBOX-6 led to polyploidization without the presence of significant apoptosis (Greene et al., 2007). Imatinib treatment minimized the formation of polyploid cells and enhanced the apoptotic index upon treatment of CML cells with spindle poisons. Resistance to paclitaxel was also shown in K562 cells (Blagosklonny, 2001), but mitotic checkpoint competence was not investigated. All these data suggested that BCR-ABL could somehow affect the response to microtubule disruption; however this issue was not discussed by the authors.

We have shown for the first time that the expression of BCR-ABL in mouse 32D cells decreases the expression of SAC proteins, such as Mad2, Bub1, Bub3 and BubR1, as well as their mRNA levels, what was estimated by real time RT-PCR (Wolanin et al., 2010). Decreased levels of the mitotic checkpoint proteins were associated with dysfunctions in the mitotic checkpoint competence observed upon nocodazole and paclitaxel treatment as well as resistance to cell death induced by these agents. We found that the inhibition of the BCR-ABL kinase activity by imatinib reversed the observed phenotype confirming the crucial role of BCR-ABL.

#### **2.3 Aberrant expression of mitotic kinases**

Mitotic kinases have also been implicated in the regulation of the centrosome cycle, spindle checkpoint and microtubule-kinetochore attachment, as well as spindle assembly and chromosome condensation. The family of Aurora kinases consists of the following proteins: Aurora A, B and C. The whole family has serine/threonine kinase activity which modifies microtubules during chromosome movement and segregation. Aurora kinases have been

& Yu, 2004; Ito & Matsumoto, 2010). This was due to the inability to activate the mitotic checkpoint and to arrest in mitosis in response to some disturbances. Instead - further progression of mitosis eventually leads to aberrant divisions and unproper chromosomes

Dysfunctions of the mitotic checkpoint were reported in different types of cancers (Baker et al., 2005; Bannon & Mc Gee, 2009; Tanaka & Hirota, 2009). They correlated with aneuploidy, disease progression and the increase of aggressiveness. Interestingly, similar effects were observed in case of the upregulation or decreased expression of mitotic checkpoint members. For example, the Mad2 protein has been recently proposed as a critical factor leading to aneuploidy in cancers with defects in the Rb and p53 pathways (Schvartzman et al., 2011). Authors found that Mad2 expression is repressed by p53 *via* the Rb pathway, thus the cancer cells lacking the Rb protein require Mad2 upregulation leading to chromosomal instability and tumor progression *in vivo*. On the other hand, also Mad2 haplo-insufficiency caused chromosomal instability in human cancer cells and murine primary embryonic

BubR1 dysfunctions has also been found as a cause of cancer-susceptible disorder mosaic variegated aneuploidy (MVA) (Suijkerbuijk et al., 2010). Similarly to Mad2, BubR1 can be also overexpressed in cancer, what was shown in hepatocellular carcinoma (HCC) (Liu et al., 2009). Authors suggest that BubR1 overexpression, which was found in 45% of patients correlated with later stages and was associated with worse prognosis, thus it can be used as

 There were indications that CML cells could have a dysfunctional mitotic checkpoint, as their resistance to spindle poisons was reported previously. In the K562 and Lama-84 CML cell lines, microtubule disruption caused either by paclitaxel, nocodazole or novel microtubule-targeting agent PBOX-6 led to polyploidization without the presence of significant apoptosis (Greene et al., 2007). Imatinib treatment minimized the formation of polyploid cells and enhanced the apoptotic index upon treatment of CML cells with spindle poisons. Resistance to paclitaxel was also shown in K562 cells (Blagosklonny, 2001), but mitotic checkpoint competence was not investigated. All these data suggested that BCR-ABL could somehow affect the response to microtubule disruption; however this issue was

We have shown for the first time that the expression of BCR-ABL in mouse 32D cells decreases the expression of SAC proteins, such as Mad2, Bub1, Bub3 and BubR1, as well as their mRNA levels, what was estimated by real time RT-PCR (Wolanin et al., 2010). Decreased levels of the mitotic checkpoint proteins were associated with dysfunctions in the mitotic checkpoint competence observed upon nocodazole and paclitaxel treatment as well as resistance to cell death induced by these agents. We found that the inhibition of the BCR-ABL kinase activity by imatinib reversed the observed phenotype confirming the crucial role

Mitotic kinases have also been implicated in the regulation of the centrosome cycle, spindle checkpoint and microtubule-kinetochore attachment, as well as spindle assembly and chromosome condensation. The family of Aurora kinases consists of the following proteins: Aurora A, B and C. The whole family has serine/threonine kinase activity which modifies microtubules during chromosome movement and segregation. Aurora kinases have been

segregation.

fibroblasts (Michelet al., 2001).

not discussed by the authors.

**2.3 Aberrant expression of mitotic kinases** 

of BCR-ABL.

a potential prognostic factor for HCC.

found at the centrosomes of interphase cells, at the poles of the bipolar spindle and in the midbody of the mitotic apparatus. All three Aurora kinases members are overexpressed in many human cancers. This correlated with chromosomal instability and clinically aggressive forms of disease (Fu et al., 2007; Meraldi et al., 2004). Aurora A is localized in centrosomes and is important for maturation, spindle assembly and metaphase I spindle orientation. It has two independent functions in centrosome maturation and asymmetric protein localization during mitosis. Ectopic overexpression of Aurora A was shown to induce oncogenic transformation (Katayama et al., 2003). Moreover, overexpression of Aurora A and aneuploidy have been proposed as predictors of poor outcome in serous ovarian carcinoma (Lassus et al., 2011). Also a high level of Aurora B has been reported to promote tetraploidy and tumorigenesis in the mouse Xenograft model (Nguyen et al., 2009).

High expression of Aurora A in leukemia cell lines and freshly isolated leukemia CML cells has been presented by Ochi T et al (Ochi et al., 2009). We also showed that the expression of BCR-ABL leads to the mislocalization of Aurora A in the chromosomal passenger complex (Wolanin et al., 2006). The importance of Aurora A-dependent signaling in CML has been shown in studies indicating that Aurora inhibitors seem to be very effective therapeutics for CML treatment (Gontarewicz et al., 2008), what will be discussed by us later.

Another family of tubulin-associated serine/threonine kinases, Polo-like, has also received significant attention regarding its participation in tumorigenesis. As far, in mammalian cells four members of this family have been identified (PLK1-4), and each one of them has a distinct function. PLK1 is essentially involved in the control of mitotic steps, PLK2 and PLK3 have been described as potential regulators of the G1 and early S phases of the cell cycle, PLK4 as a major centrosome duplication regulator. Polo-like kinase 1 (PLK1) is a key regulator of mitosis and participates in regulating this process from its entry to cytokinesis (Yuan et al., 2011). Transcription and translation of PLK1 is highly coordinated with cell cycle progression. *Plk1* mRNA and protein levels begin to accumulate in the S-phase and reach a peak at the G2/M transition and then decline upon mitotic exit (Lee et al., 1995). At the G2/M phase, PLK1 regulates the Cdk1/Cyclin B1 complex promoting mitotic entry and regulating mitotic progression due to regulation of phosphorylation of Cyclin B1, Cdk1, Myt1 and Cdc25C. PLK1 also plays a role in centrosome maturation by promoting increased recruitment of microtubules to the spindle pole bodies. It also regulates the localization of Aurora A to the centrosomes for proper maturation. It is known today that all mitotic kinases interplay with each other and form an extensive functional network, thus targeting any of them has tremendous consequences for cell physiology (Lens et al., 2010). Additionally, it was shown that PLK1 catalysis survivin priming phosphorylation at Ser20, what is necessary for survivin-mediated Aurora B docking to the centromere and activation (Chu et al., 2010). Expression of the non-phosphorylable survivin mutant prevented Aurora B activation and corrected spindle microtubule attachment. We also observed that silencing of survivin in CML cells significantly affected CPC function and mitosis as well as proper completion of cytokinesis leading to the formation of giant polyploid cells (Wolanin et al., 2006). PLK1 also regulates the spindle assembly checkpoint (Nezi & Musacchio, 2009) probably by phosphorylation of BubR1 and finally, regulates chromosome segregation, cytokinesis and mitotic exit.

PLK1, similarly to other mitotic kinases has been shown to be upregulated in cancers, including lymphomas. Studies of a big group of non-Hodgkin's lymphoma (NHLs) patients presented that the level of PLK1 expression was significantly lower in low-grade NHLs than

BCR-ABL Hits at Mitosis; Implications for Chromosomal

downregulation.

**A**

**C**

Instability, Aneuploidy and Therapeutic Strategy 9

lack of a significant decrease of mRNA confirmed the previous observation that BCR-ABL affects the posttranscriptional stages of protein expression. Incubation with the proteasome inhibitor MG132 did not lead to an increase at the BRCA1 protein level (Fig. 1C), thus excluding the possibility that increased degradation is responsible for the protein

Recently, it was shown that BCR-ABL interferes with the Fanconi Anemia/BRCA1 pathway, thus increasing the predisposition to DNA repair errors and development of centrosomal and chromosomal aberrations (Valeri et al., 2010). The interference of BCR-ABL with the formation of BRCA1 and FANCD2 nuclear foci was observed in hematopoietic progenitors from CML patients. These authors also showed that the ectopic expression of BRCA1 reverted the generation of aberrant centrosomes induced by BCR-ABL. This suggests, however not directly studied, that overexpression of BRCA1 could antagonize also other effects of BCR-ABL expression, if they are mediated by BRCA1 downregulation, indeed.

**B**

C4 cells

PARP

BRCA1

Fig. 1. The influence of BCR-ABL expression on the level of the BRCA1 Protein.

A. Expression of BCR-ABL leads to downregulation of the BRCA1 protein. The level of BRCA1 was determined by Western Blot in mouse progenitor 32D cells, control or stably

B. Imatinib treatment leads to upregulation of the BRCA1 protein level in cells expressing BCR-ABL. 32D and C4 cells were treated with 0.5 μM imatinib for 1, 2.5 or 4 hours (upper panel) or with 0.5 or 5 μM imatinib for 4 and 6 hours (lower panel) followed by estimation

C. BRCA1 downregulation caused by BCR-ABL is not a result of increased proteasomal degradation. 32D, C2 and C4 cells were treated with 10 μM proteasome inhibitor MG132 for

Altogether, there are strong evidences indicating that the decrease of the BRCA1 protein and the BRCA1-dependent signaling is caused by BCR-ABL expression and is also specific for chronic myeloid leukemia, in addition to other types of tumors. There is a number of intracellular processes crucial for cell physiology controlled by BRCA1, including DNA damage response as well as activation of the cell cycle checkpoints, chromatin remodelling, apoptosis and mitosis. Aberrations in any of them, lead to the accumulation of mutations, genomic instability and finally an increased risk of cancerogenesis. Thus, we postulate that the decrease of BRCA1 caused by BCR-ABL could have tremendous consequences due to defective control of genomic stability. The role of BRCA1 in the regulation of the DNA

6 hours, followed by determination of the BRCA1 protein level by Western Blot.


PARP BRCA1

expressing BCR-ABL at low (C2 cells) or high (C4 cells) level.

BCR-ABL actin

32D C2 C4

of the BRCA1 protein level.

32D C2 C4

0 1 2.5 4 0 1 2.5 4 [h] IM 0,5 μM

0 4 6 4 6 [h] 0,5 μM 5 μM IM

PARP BRCA1

BRCA1

32D C4

in high-grade and intermediate-grade NHLs. Moreover, PLK1 has been proposed as a valuable marker of proliferating cells, even better than the commonly used Ki67 (Mito et al., 2005). It was also described that PLK1 is overexpressed in AML cell lines as well as in primary cells and its inhibition preferentially targeted lymphoid cells, indicating an important role of the PLK1-mediated signaling (Renner et al., 2009). Importantly, healthy hematopoietic progenitor CD34+ cells were much less sensitive to growth inhibition caused by PLK1 targeting, indicating a high potential of this therapeutic strategy. This observation was confirmed by studies performed by Ikezoe and colleagues, who also found PLK1 overexpressed in a number of human leukemia cell lines and freshly isolated leukemia cells from individuals with acute myelogenous leukemia as well as acute lymphoblastic leukemia, in comparison with normal bone marrow mononuclear cells (Ikezoe et al., 2009). As previously, they indicated PLK1 inhibition as a potent way to inhibit proliferation and induce cell death in leukemia cells. Moreover, the functional link between PLK1 and mTOR pathway has been shown in AML cells (Renner et al., 2010). Abnormal growth of cells overexpressing the active form of PLK1 was reversed by rapamycin, a specific inhibitor of the TORC1 complex. This showed a novel aspect of PLK1's role in leukemia and opened new therapeutic possibilities.

In chronic myeloid leukemia, PLK1was found to be expressed in the phosphorylated form in the CML cell line K562 as well as in primary CML cells from patients (Gleixner et al., 2010). Studies presenting the potential of the PLK1 inhibitors in therapy against CML were performed and indicate an important role of PLK1 in CML development and progression. They will be discussed in a detailed way in the chapter dedicated to anti-mitotic therapies against leukemia.

#### **3. BCR-ABL-mediated downregulation of BRCA1**

BRCA1, a tumor suppressor isolated in 1994 (Miki et al., 1994) has been implicated in a broad range of cellular processes, including DNA repair, cell cycle checkpoint control, cell division and gene transcription (Linger & Kruk, 2010; Thompson, 2010; Wu et al., 2010; Yang & Xia, 2010). It is a known familiar ovarian and breast cancer-specific tumor suppressor, however today it seems that it is involved in the development of other types of cancers as well. The protein contains two motifs: a RING domain at the N-terminus and two tandem copies of BRCT domain at the C-terminus (Baer, 2001). *In vivo* it exists in a heterodimeric complex with the BRCA1-associated RING domain (BARD1) protein, which resembles BRCA1 (Wu et al., 1996).

The first observation that BRCA1 protein is nearly undetectable in leukemia cells from chronic myeloid leukemia (CML) patients has been made by Deutsch et al (Deutsch et al., 2003). They found a significant downregulation of BRCA1 in primary CD34+ cells obtained from both, the chronic phase and the blast crisis patients as well as in cell lines expressing BCR-ABL. This was not accompanied by a decrease of the *BRCA1* mRNA, what was studied by real-time RT-PCR in one of the investigated cell lines.

Our group studied the direct influence of BCR-ABL on the BRCA1 expression, using the previously mentioned mouse progenitor 32D cell line stably expressing with BCR-ABL, particularly in clones, expressing low and high BCR-ABL levels (Fig.1A), (Wolanin et al., 2010). We found that BCR-ABL expression leads to a strong decrease of BRCA1 at the protein level. This was reversed by treatment with imatinib, a specific inhibitor of the BCR-ABL tyrosine kinase, confirming dependence on the tyrosine kinase activity (Fig. 1B). The

in high-grade and intermediate-grade NHLs. Moreover, PLK1 has been proposed as a valuable marker of proliferating cells, even better than the commonly used Ki67 (Mito et al., 2005). It was also described that PLK1 is overexpressed in AML cell lines as well as in primary cells and its inhibition preferentially targeted lymphoid cells, indicating an important role of the PLK1-mediated signaling (Renner et al., 2009). Importantly, healthy hematopoietic progenitor CD34+ cells were much less sensitive to growth inhibition caused by PLK1 targeting, indicating a high potential of this therapeutic strategy. This observation was confirmed by studies performed by Ikezoe and colleagues, who also found PLK1 overexpressed in a number of human leukemia cell lines and freshly isolated leukemia cells from individuals with acute myelogenous leukemia as well as acute lymphoblastic leukemia, in comparison with normal bone marrow mononuclear cells (Ikezoe et al., 2009). As previously, they indicated PLK1 inhibition as a potent way to inhibit proliferation and induce cell death in leukemia cells. Moreover, the functional link between PLK1 and mTOR pathway has been shown in AML cells (Renner et al., 2010). Abnormal growth of cells overexpressing the active form of PLK1 was reversed by rapamycin, a specific inhibitor of the TORC1 complex. This showed a novel aspect of PLK1's role in leukemia and opened

In chronic myeloid leukemia, PLK1was found to be expressed in the phosphorylated form in the CML cell line K562 as well as in primary CML cells from patients (Gleixner et al., 2010). Studies presenting the potential of the PLK1 inhibitors in therapy against CML were performed and indicate an important role of PLK1 in CML development and progression. They will be discussed in a detailed way in the chapter dedicated to anti-mitotic therapies

BRCA1, a tumor suppressor isolated in 1994 (Miki et al., 1994) has been implicated in a broad range of cellular processes, including DNA repair, cell cycle checkpoint control, cell division and gene transcription (Linger & Kruk, 2010; Thompson, 2010; Wu et al., 2010; Yang & Xia, 2010). It is a known familiar ovarian and breast cancer-specific tumor suppressor, however today it seems that it is involved in the development of other types of cancers as well. The protein contains two motifs: a RING domain at the N-terminus and two tandem copies of BRCT domain at the C-terminus (Baer, 2001). *In vivo* it exists in a heterodimeric complex with the BRCA1-associated RING domain (BARD1) protein, which resembles

The first observation that BRCA1 protein is nearly undetectable in leukemia cells from chronic myeloid leukemia (CML) patients has been made by Deutsch et al (Deutsch et al., 2003). They found a significant downregulation of BRCA1 in primary CD34+ cells obtained from both, the chronic phase and the blast crisis patients as well as in cell lines expressing BCR-ABL. This was not accompanied by a decrease of the *BRCA1* mRNA, what was studied

Our group studied the direct influence of BCR-ABL on the BRCA1 expression, using the previously mentioned mouse progenitor 32D cell line stably expressing with BCR-ABL, particularly in clones, expressing low and high BCR-ABL levels (Fig.1A), (Wolanin et al., 2010). We found that BCR-ABL expression leads to a strong decrease of BRCA1 at the protein level. This was reversed by treatment with imatinib, a specific inhibitor of the BCR-ABL tyrosine kinase, confirming dependence on the tyrosine kinase activity (Fig. 1B). The

new therapeutic possibilities.

**3. BCR-ABL-mediated downregulation of BRCA1** 

by real-time RT-PCR in one of the investigated cell lines.

against leukemia.

BRCA1 (Wu et al., 1996).

lack of a significant decrease of mRNA confirmed the previous observation that BCR-ABL affects the posttranscriptional stages of protein expression. Incubation with the proteasome inhibitor MG132 did not lead to an increase at the BRCA1 protein level (Fig. 1C), thus excluding the possibility that increased degradation is responsible for the protein downregulation.

Recently, it was shown that BCR-ABL interferes with the Fanconi Anemia/BRCA1 pathway, thus increasing the predisposition to DNA repair errors and development of centrosomal and chromosomal aberrations (Valeri et al., 2010). The interference of BCR-ABL with the formation of BRCA1 and FANCD2 nuclear foci was observed in hematopoietic progenitors from CML patients. These authors also showed that the ectopic expression of BRCA1 reverted the generation of aberrant centrosomes induced by BCR-ABL. This suggests, however not directly studied, that overexpression of BRCA1 could antagonize also other effects of BCR-ABL expression, if they are mediated by BRCA1 downregulation, indeed.

Fig. 1. The influence of BCR-ABL expression on the level of the BRCA1 Protein. A. Expression of BCR-ABL leads to downregulation of the BRCA1 protein. The level of BRCA1 was determined by Western Blot in mouse progenitor 32D cells, control or stably expressing BCR-ABL at low (C2 cells) or high (C4 cells) level.

B. Imatinib treatment leads to upregulation of the BRCA1 protein level in cells expressing BCR-ABL. 32D and C4 cells were treated with 0.5 μM imatinib for 1, 2.5 or 4 hours (upper panel) or with 0.5 or 5 μM imatinib for 4 and 6 hours (lower panel) followed by estimation of the BRCA1 protein level.

C. BRCA1 downregulation caused by BCR-ABL is not a result of increased proteasomal degradation. 32D, C2 and C4 cells were treated with 10 μM proteasome inhibitor MG132 for 6 hours, followed by determination of the BRCA1 protein level by Western Blot.

Altogether, there are strong evidences indicating that the decrease of the BRCA1 protein and the BRCA1-dependent signaling is caused by BCR-ABL expression and is also specific for chronic myeloid leukemia, in addition to other types of tumors. There is a number of intracellular processes crucial for cell physiology controlled by BRCA1, including DNA damage response as well as activation of the cell cycle checkpoints, chromatin remodelling, apoptosis and mitosis. Aberrations in any of them, lead to the accumulation of mutations, genomic instability and finally an increased risk of cancerogenesis. Thus, we postulate that the decrease of BRCA1 caused by BCR-ABL could have tremendous consequences due to defective control of genomic stability. The role of BRCA1 in the regulation of the DNA

BCR-ABL Hits at Mitosis; Implications for Chromosomal

**4.2 BRCA1 in the regulation of the mitotic checkpoint** 

mediated tumor suppression.

Cdc20 were downregulated.

in CML cells.

Instability, Aneuploidy and Therapeutic Strategy 11

TFIIH transcription factors and blocking the initiation of mRNA synthesis. Amphiregulin (AREG) and early growth response-1 (EGR-1) are examples of genes repressed by BRCA1 in breast cancers. This phenomen could be broader and may contribute to the BRCA1-

The role of BRCA1 in the regulation of the mitotic checkpoint has been indicated. BRCA1 was identified as a mitotic target of the Chk2 kinase in the absence of DNA damage (Stolz et al., 2010). Accordingly, loss of BRCA1 or its Chk2-mediated phosphorylation led to defects in the spindle formation and chromosomal instability (CIN) due to generation of lagging chromosomes and chromosome missegregation. It was shown that MCF-7 cells transfected with BRCA1 siRNA display a reduced mitotic index followed by premature cyclin B1 degradation upon paclitaxel treatment. This suggested that BRCA1 depletion results in the inactivation of the spindle checkpoint (Chabalier et al., 2006). They presented that BRCA1 up-regulates the expression of the protein kinase BubR1, an essential component of the functional spindle checkpoint. This indicated that BRCA1 directly influences the expression of the mitotic checkpoint components. It was also shown that BRCA1, due to an interaction with the transcription factor OCT-1, mediates the transactivation of Mad2 (mitotic arrest deficient protein 2) (Wang et al., 2004). The studies of BRCA1 knock-down in human prostate and breast cancer cell lines, by using the microarray technique, showed that BRCA1 depletion caused downregulation of many genes involved in mitosis progression (Bae et al., 2005). Specifically, mitotic checkpoint components (Bub1, STK6), proteins involved in the chromosome segregation and centrosome function as well as cytokinesis (including PLK) and finally proteins regulating mitosis entry and progression, such as cyclin B1, Cdc2 and

The influence of BRCA1 on the expression of components of the mitotic checkpoint was also confirmed in our studies (Wolanin et al., 2010). We showed that the downregulation of BRCA1, caused either by BCR-ABL expression or by gene silencing using siRNA, resulted in the downregulation of Mad2 as well as BubR1 and Bub3 gene expression, which all belong to the mitotic checkpoint complex and undergo common regulation. Decreased levels of these proteins finally led to dysfunctions of the mitotic checkpoint and increased occurence of aberrant mitoses and chromosomal instability. Moreover, we observed the increased rate of supernumerary centrosomes as well as aberrant divisions in cells expressing BCR-ABL. We propose that decrease of the BRCA1 protein caused by BCR-ABL could be an important factor participating in the development of genomic instability due to the generation of chromosomally unstable cells. We added the regulation of mitotic checkpoint to the repertoir of BRCA1-mediated mechanisms participating in the development of aneuploidy

Due to its function in the regulation of mitotic checkpoint competence, BRCA1 has been shown to correlate with the sensitivity to spindle poisons (Byrski et al., 2008; Quinn et al., 2007). As mentioned before, cells ability to activate the mitotic checkpoint is necessary for the sensitivity to spindle poisons. BRCA1 downregulation resulted in resistance to microtubule damage due to the inability to efficiently activate the mitotic checkpoint, block cells in mitosis and induce apoptosis. In our studies, cells expressing BCR-ABL with a significantly decreased BRCA1 level were resistant to cell death activated by nocodazole or paclitaxel (Wolanin et al., 2010). This was reversed by imatinib treatment, resulting in

damage response and cell cycle checkpoint control has been already well explained (Huen et al., 2010; Kim & Chen, 2008; Wuet al., 2010; Yang & Xia, 2010; Zhang & Powell, 2005). The detailed role of BRCA1 in the regulation of mechanisms participating in the occurence of genomic instability as a result of mitosis dysfunctions, referred as a CIN (chromosomal instability) will be discussed in the next paragraphs.

#### **4. The role of BRCA1 in mitosis**

#### **4.1 BRCA1 in the transcriptional regulation**

Currently, there is a lot of evidence suggesting that BRCA1 is involved in the transcriptional regulation. This opens a new list of possible interactions with intracellular processes (Murray et al., 2007). It has been shown that BRCA1 is a component of the RNA polymerase II (pol II) holoenzyme (Scully et al., 1997). Authors developed a purification strategy for the mammalian pol II holoenzyme to search for specific transcription factors and they found that the wild-type BRCA1 protein was copurified. Moreover, immunopurification of BRCA1 complexes also contained TFIIF, TFIIE and TFIIH transcription factors, which were previously reported to form a complex with the pol II holoenzyme (Maldonado et al., 1996). This strongly suggested that one of the BRCA1 functions is to regulate genes expression.

Unlike many enhancer-specific activators, BRCA1 does not appear to require the specific DNA binding domain to stimulate gene transcription, what was shown by investigation of the p53-responsive promoter *MDM2* (Nadeau et al., 2000). BRCA1 interacts rather with multiple transcription factors. Among them we can name ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family. BRCA1 stimulates its transcription from a natural promoter as well as reporter systems (Houvras et al., 2000). Moreover, BRCA1 significantly enhanced the transcription of NFkappaB target genes due to the binding to p65/RelA, one of the two subunits of the transcription factor NF-kappaB (Benezra et al., 2003). Authors suggested that BRCA1 acts as a coactivator and proposed a model in which BRCA1 interacts physically with p65/RelA, CBP as well as with RNA polymerase II and enhances transcriptional activation of the NFkappaB target genes. Additionally, MacLachlan reported that p53 can be stabilized by BRCA1 in response to DNA damage and by this selectively transactivated towards genes involved in the growth arrest and DNA repair (MacLachlan et al., 2002). The role of BRCA1 in the regulation of p53-dependent gene expression has been also shown by other groups (Ouchi et al., 1998; Zhang et al., 1998).

BRCA1 is also able to interact with components of the histone deacetylase complex, particularly with HDAC1 and HDAC2 (Yarden & Brody, 1999). It was shown to interact *in vitro* and *in vivo* with the Rb protein as well as with the RB-binding proteins, RBAp46 and RBAp48, which are components of the histone deacetylase complexes and are involved in chromatin remodelling. Involvement of BRCA1 in chromatin remodelling suggests its important role in the regulation of transcription, replication, recombination and others. BRCA1-mediated activation of specific genes may result from sequestration of histone deacetylases from DNA promoters. It was also reported that BRCA1 interacts with the hGCN5/TRAP histone acetyltransferase complex (Oishi et al., 2006), which co-activates the transactivation function of BRCA1.

More recently, BRCA1 has also been shown to play a role in the transcriptional repression by ubiquitin-dependent mechanism (Horwitz et al., 2007). It leads to ubiquitination of the transcriptional preinitiation complex, thus preventing the stable association of TFIIE and

damage response and cell cycle checkpoint control has been already well explained (Huen et al., 2010; Kim & Chen, 2008; Wuet al., 2010; Yang & Xia, 2010; Zhang & Powell, 2005). The detailed role of BRCA1 in the regulation of mechanisms participating in the occurence of genomic instability as a result of mitosis dysfunctions, referred as a CIN (chromosomal

Currently, there is a lot of evidence suggesting that BRCA1 is involved in the transcriptional regulation. This opens a new list of possible interactions with intracellular processes (Murray et al., 2007). It has been shown that BRCA1 is a component of the RNA polymerase II (pol II) holoenzyme (Scully et al., 1997). Authors developed a purification strategy for the mammalian pol II holoenzyme to search for specific transcription factors and they found that the wild-type BRCA1 protein was copurified. Moreover, immunopurification of BRCA1 complexes also contained TFIIF, TFIIE and TFIIH transcription factors, which were previously reported to form a complex with the pol II holoenzyme (Maldonado et al., 1996). This strongly suggested that one of the BRCA1 functions is to regulate genes expression. Unlike many enhancer-specific activators, BRCA1 does not appear to require the specific DNA binding domain to stimulate gene transcription, what was shown by investigation of the p53-responsive promoter *MDM2* (Nadeau et al., 2000). BRCA1 interacts rather with multiple transcription factors. Among them we can name ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family. BRCA1 stimulates its transcription from a natural promoter as well as reporter systems (Houvras et al., 2000). Moreover, BRCA1 significantly enhanced the transcription of NFkappaB target genes due to the binding to p65/RelA, one of the two subunits of the transcription factor NF-kappaB (Benezra et al., 2003). Authors suggested that BRCA1 acts as a coactivator and proposed a model in which BRCA1 interacts physically with p65/RelA, CBP as well as with RNA polymerase II and enhances transcriptional activation of the NFkappaB target genes. Additionally, MacLachlan reported that p53 can be stabilized by BRCA1 in response to DNA damage and by this selectively transactivated towards genes involved in the growth arrest and DNA repair (MacLachlan et al., 2002). The role of BRCA1 in the regulation of p53-dependent gene expression has been also shown by other groups

BRCA1 is also able to interact with components of the histone deacetylase complex, particularly with HDAC1 and HDAC2 (Yarden & Brody, 1999). It was shown to interact *in vitro* and *in vivo* with the Rb protein as well as with the RB-binding proteins, RBAp46 and RBAp48, which are components of the histone deacetylase complexes and are involved in chromatin remodelling. Involvement of BRCA1 in chromatin remodelling suggests its important role in the regulation of transcription, replication, recombination and others. BRCA1-mediated activation of specific genes may result from sequestration of histone deacetylases from DNA promoters. It was also reported that BRCA1 interacts with the hGCN5/TRAP histone acetyltransferase complex (Oishi et al., 2006), which co-activates the

More recently, BRCA1 has also been shown to play a role in the transcriptional repression by ubiquitin-dependent mechanism (Horwitz et al., 2007). It leads to ubiquitination of the transcriptional preinitiation complex, thus preventing the stable association of TFIIE and

instability) will be discussed in the next paragraphs.

**4.1 BRCA1 in the transcriptional regulation** 

**4. The role of BRCA1 in mitosis** 

(Ouchi et al., 1998; Zhang et al., 1998).

transactivation function of BRCA1.

TFIIH transcription factors and blocking the initiation of mRNA synthesis. Amphiregulin (AREG) and early growth response-1 (EGR-1) are examples of genes repressed by BRCA1 in breast cancers. This phenomen could be broader and may contribute to the BRCA1 mediated tumor suppression.

#### **4.2 BRCA1 in the regulation of the mitotic checkpoint**

The role of BRCA1 in the regulation of the mitotic checkpoint has been indicated. BRCA1 was identified as a mitotic target of the Chk2 kinase in the absence of DNA damage (Stolz et al., 2010). Accordingly, loss of BRCA1 or its Chk2-mediated phosphorylation led to defects in the spindle formation and chromosomal instability (CIN) due to generation of lagging chromosomes and chromosome missegregation. It was shown that MCF-7 cells transfected with BRCA1 siRNA display a reduced mitotic index followed by premature cyclin B1 degradation upon paclitaxel treatment. This suggested that BRCA1 depletion results in the inactivation of the spindle checkpoint (Chabalier et al., 2006). They presented that BRCA1 up-regulates the expression of the protein kinase BubR1, an essential component of the functional spindle checkpoint. This indicated that BRCA1 directly influences the expression of the mitotic checkpoint components. It was also shown that BRCA1, due to an interaction with the transcription factor OCT-1, mediates the transactivation of Mad2 (mitotic arrest deficient protein 2) (Wang et al., 2004). The studies of BRCA1 knock-down in human prostate and breast cancer cell lines, by using the microarray technique, showed that BRCA1 depletion caused downregulation of many genes involved in mitosis progression (Bae et al., 2005). Specifically, mitotic checkpoint components (Bub1, STK6), proteins involved in the chromosome segregation and centrosome function as well as cytokinesis (including PLK) and finally proteins regulating mitosis entry and progression, such as cyclin B1, Cdc2 and Cdc20 were downregulated.

The influence of BRCA1 on the expression of components of the mitotic checkpoint was also confirmed in our studies (Wolanin et al., 2010). We showed that the downregulation of BRCA1, caused either by BCR-ABL expression or by gene silencing using siRNA, resulted in the downregulation of Mad2 as well as BubR1 and Bub3 gene expression, which all belong to the mitotic checkpoint complex and undergo common regulation. Decreased levels of these proteins finally led to dysfunctions of the mitotic checkpoint and increased occurence of aberrant mitoses and chromosomal instability. Moreover, we observed the increased rate of supernumerary centrosomes as well as aberrant divisions in cells expressing BCR-ABL. We propose that decrease of the BRCA1 protein caused by BCR-ABL could be an important factor participating in the development of genomic instability due to the generation of chromosomally unstable cells. We added the regulation of mitotic checkpoint to the repertoir of BRCA1-mediated mechanisms participating in the development of aneuploidy in CML cells.

Due to its function in the regulation of mitotic checkpoint competence, BRCA1 has been shown to correlate with the sensitivity to spindle poisons (Byrski et al., 2008; Quinn et al., 2007). As mentioned before, cells ability to activate the mitotic checkpoint is necessary for the sensitivity to spindle poisons. BRCA1 downregulation resulted in resistance to microtubule damage due to the inability to efficiently activate the mitotic checkpoint, block cells in mitosis and induce apoptosis. In our studies, cells expressing BCR-ABL with a significantly decreased BRCA1 level were resistant to cell death activated by nocodazole or paclitaxel (Wolanin et al., 2010). This was reversed by imatinib treatment, resulting in

BCR-ABL Hits at Mitosis; Implications for Chromosomal

nucleation.

Instability, Aneuploidy and Therapeutic Strategy 13

expressing BCR-ABL oncogene (Wolanin et al., 2010). This was in contrast with the hypothesis that BRCA1 defects lead to centrosome amplification in breast cells but not in other types of cells (Starita et al., 2004). This idea has been based on the data obtained using the transient expression of the BRCA1-inhibiting BIF peptide in nine different cell lines, where four nonbreast cell lines - prostate (PC3), cervix (HeLa), colon (DLD-1) and osteosarcoma (U2OS), did not accumulate extra centrosomes. However, lymphoid cells were not included in these studies. To date, there were other indications, apart from ours, that the loss or mutation of BRCA1 could affect the centrosome number also in other types of cells. Recently, it was shown that BCR-ABL intereferes with the Fanconi Anemia (FA)/BRCA pathway and the ectopic expression of BRCA1 in CD34+ progenitor cells reversed the appearance of aberrant

The direct mechanism of BRCA1-mediated control of centrosome number is still not fully clear, although the BRCA1-dependent ubiquitination of gamma-tubulin is proposed to be involved in the regulation of centrosome function (Staritaet al., 2004). Gamma-tubulin is an important protein involved in the initiation of microtubule nucleation by centrosomes. Gamma-tubulin's lysines 48 and 344 have been indicated as crucial in the regulation of centrosome duplication and microtubule nucleation function, respectively (Sankaran et al., 2005). Cells with mutated lysines on gamma-tubulin, unable to be ubiquitinated, were characterized by centrosome amplification. On the other hand, the same phenotype was observed after inhibition of the enzymatic activity of BRCA1 by transfection of the BRCA1 (I26A) ligase-defective mutant (Sankaran et al., 2006). Additionally, *in vitro* experiments using *Xenopus* extracts, purified centrosomes and BRCA1 together with ubiquitination factors confirmed that BRCA1 is involved in the microtubule nucleation. It seems that BRCA1 controls the centrosome number by preventing reduplication due to ubiquitination of lysines of gamma-tubulin, which needs to be phosphorylated to prevent reduplication (Ko et al., 2006). Loss of BRCA1 did not affect centrosome duplication in the early S phase but rather caused a second round of duplication just prior to mitosis. The model has been proposed, in which BRCA1 marks centrosomes as already duplicated *via* the BRCA1 mediated ubiqutination of gamma-tubulin (Wong & Stearns, 2003). This issue is still not fully clarified, however there is no doubt about the significant role of the BRCA1-mediated ubiquitination of gamma-tubulin in this process (Kais & Parvin, 2008). Altogether, this led to the conclusion that the E3 ubiquitin ligase activity of BRCA1 is crucial for the effects on the biology of centrosomes, and controls centrosome duplication as well as microtubules

Recently, it was demonstrated that BRCA1 interacts with centrosomal protein Nlp (nineinlike protein) (Jin et al., 2009), which is a fast turnover protein and plays a role in the centrosome maturation and spindle formation (Casenghi et al., 2005). Authors found that Nlp is a BRCA1-associated protein and colocalizes with BRCA1 in different types of cancer cells, including HeLa and U2OS cells. Moreover, Nlp expression and stability depends on normal cellular BRCA1 function. A variety of different types of cells expressing the mutated BRCA1 or silenced for BRCA1 exhibited disrupted Nlp colocalization to centrosomes as well as enhanced Nlp degradation. This data was consistent with our observations concerning the role of BRCA1 in different types of cancers. The lack of Nlp protein led to centrosome amplification, aberrant chromosome segregation, cytokinesis failure and appearance of miltinuclei, thus resembling the phenotype upon BRCA1 disruption. Recent studies showed that Nlp is recruited by the Aurora B protein and localizes at the midbody during

centrosomes, thus confirming our previous observations (Valeri et al., 2010).

BRCA1 upregulation. In ovarian cancer it was suggested that BRCA1 can act as a predictive marker of response to chemotherapy (Quinn et al., 2009) and dysfunctional BRCA1 resulted in resistance to taxanes and other chemotherapeutics. On the other hand, reconstitution of BRCA1 into ovarian cancer cells, carrying BRCA1 mutation, reversed the resistance and sensitized cells to paclitaxel (Zhou et al., 2003). BRCA1 was also proposed as a predicive marker of drug sensitivity in breast cancer treatment (Mullan et al., 2006). As resistance to spindle poisons has been reported for CML cells, this supports the previously proposed idea, that the overexpression of BRCA1 diminishes some effects of BCR-ABL expression. In our opinion, BRCA1 level could serve as a prognostic marker of sensitivity to different therapies also those used in leukemias.

#### **4.3 BRCA1 in the regulation of centrosome number and function**

The first observation that BRCA1 localizes to centrosomes has been made by Hsu et al (Hsu & White, 1998), who showed that BRCA1 is associated with centrosomes during mitosis in a cell cycle-dependent manner. Moreover, they found that BRCA1 forms a complex with gamma-tubulin, which is preferentially associated with the hypophosphorylated form of BRCA1. Gamma-tubulin is a crucial component of centrosomes and is responsible for nucleation of microtubules. Therefore, this confirmed the idea that BRCA1 could play a role in the regulation of centrosome amplification and function and led to the later findings that a BF3 domain of BRCA1 (BRCA1 fragment no. 3, amino acids 504-803) is responsible for the gamma-tubulin binding (Hsu et al., 2001). Overexpression of the BF3 domain in COS-7 cells resulted in the accumulation of mitotic cells with supernumerrary centrosomes and abnormal spindles, what is known to lead to aneuploidization.

The role of BRCA1 in the regulation of centrosome number has been indicated by experiments using the mutated forms of BRCA1. Centrosomal amplification was shown in mouse embryonic fibroblasts carrying a targeted deletion of exon 11 of BRCA1 (Xu et al., 1999) and in a BRCA1-mutant breast cancer cell line HCC1937 (Schlegel et al., 2003). What is important, Waever et al showed that mouse embryonic fibroblasts carrying different BRCA1 defects show supernumerary centrosomes and other features similar to human breast cancer cells, indicating that the mechanisms are conserved between mice and humans (Weaver et al., 2002).

Moreover, immunohistochemical analysis of 50 samples from breast cancer patients showed that numerical centrosome aberrations were signicantly associated with the negative BRCA1 expression as well as with the BRCA1 germline mutation, whereas there was no significant correlation with the centrosome aberrations in size (Shimomura et al., 2009). This suggests that BRCA1 plays a role rather in the regulation of centrosome duplication and defects in its expression or function result in numerical aberrations. Very recently, direct studies of 14 different missense mutations in the RING domain of BRCA1 and their influence on the control of centrosome number were performed (Kais et al., 2011). Authors showed that only 2 out of the 14 BRCA1 variant proteins were neutral in the centrosome duplication assay. The others were either very effective and resulted in mutated BRCA1 proteins that caused centrosome amplification (C24R, C27A, C39Y, H41F, C44F, C47G, M18T and I42V) or had an intermediate, however still significant effect on centrosome duplication (I21V, I31M, L52F and D67Y).

Interestingly, we also observed a correlation between the loss of BRCA1 expression and increased percentage of cells with supernumerary centrosomes in murine lymphoid cells

BRCA1 upregulation. In ovarian cancer it was suggested that BRCA1 can act as a predictive marker of response to chemotherapy (Quinn et al., 2009) and dysfunctional BRCA1 resulted in resistance to taxanes and other chemotherapeutics. On the other hand, reconstitution of BRCA1 into ovarian cancer cells, carrying BRCA1 mutation, reversed the resistance and sensitized cells to paclitaxel (Zhou et al., 2003). BRCA1 was also proposed as a predicive marker of drug sensitivity in breast cancer treatment (Mullan et al., 2006). As resistance to spindle poisons has been reported for CML cells, this supports the previously proposed idea, that the overexpression of BRCA1 diminishes some effects of BCR-ABL expression. In our opinion, BRCA1 level could serve as a prognostic marker of sensitivity to different

The first observation that BRCA1 localizes to centrosomes has been made by Hsu et al (Hsu & White, 1998), who showed that BRCA1 is associated with centrosomes during mitosis in a cell cycle-dependent manner. Moreover, they found that BRCA1 forms a complex with gamma-tubulin, which is preferentially associated with the hypophosphorylated form of BRCA1. Gamma-tubulin is a crucial component of centrosomes and is responsible for nucleation of microtubules. Therefore, this confirmed the idea that BRCA1 could play a role in the regulation of centrosome amplification and function and led to the later findings that a BF3 domain of BRCA1 (BRCA1 fragment no. 3, amino acids 504-803) is responsible for the gamma-tubulin binding (Hsu et al., 2001). Overexpression of the BF3 domain in COS-7 cells resulted in the accumulation of mitotic cells with supernumerrary centrosomes and

The role of BRCA1 in the regulation of centrosome number has been indicated by experiments using the mutated forms of BRCA1. Centrosomal amplification was shown in mouse embryonic fibroblasts carrying a targeted deletion of exon 11 of BRCA1 (Xu et al., 1999) and in a BRCA1-mutant breast cancer cell line HCC1937 (Schlegel et al., 2003). What is important, Waever et al showed that mouse embryonic fibroblasts carrying different BRCA1 defects show supernumerary centrosomes and other features similar to human breast cancer cells, indicating that the mechanisms are conserved between mice and humans (Weaver et

Moreover, immunohistochemical analysis of 50 samples from breast cancer patients showed that numerical centrosome aberrations were signicantly associated with the negative BRCA1 expression as well as with the BRCA1 germline mutation, whereas there was no significant correlation with the centrosome aberrations in size (Shimomura et al., 2009). This suggests that BRCA1 plays a role rather in the regulation of centrosome duplication and defects in its expression or function result in numerical aberrations. Very recently, direct studies of 14 different missense mutations in the RING domain of BRCA1 and their influence on the control of centrosome number were performed (Kais et al., 2011). Authors showed that only 2 out of the 14 BRCA1 variant proteins were neutral in the centrosome duplication assay. The others were either very effective and resulted in mutated BRCA1 proteins that caused centrosome amplification (C24R, C27A, C39Y, H41F, C44F, C47G, M18T and I42V) or had an intermediate, however still significant effect on centrosome duplication (I21V, I31M, L52F

Interestingly, we also observed a correlation between the loss of BRCA1 expression and increased percentage of cells with supernumerary centrosomes in murine lymphoid cells

therapies also those used in leukemias.

al., 2002).

and D67Y).

**4.3 BRCA1 in the regulation of centrosome number and function** 

abnormal spindles, what is known to lead to aneuploidization.

expressing BCR-ABL oncogene (Wolanin et al., 2010). This was in contrast with the hypothesis that BRCA1 defects lead to centrosome amplification in breast cells but not in other types of cells (Starita et al., 2004). This idea has been based on the data obtained using the transient expression of the BRCA1-inhibiting BIF peptide in nine different cell lines, where four nonbreast cell lines - prostate (PC3), cervix (HeLa), colon (DLD-1) and osteosarcoma (U2OS), did not accumulate extra centrosomes. However, lymphoid cells were not included in these studies. To date, there were other indications, apart from ours, that the loss or mutation of BRCA1 could affect the centrosome number also in other types of cells. Recently, it was shown that BCR-ABL intereferes with the Fanconi Anemia (FA)/BRCA pathway and the ectopic expression of BRCA1 in CD34+ progenitor cells reversed the appearance of aberrant centrosomes, thus confirming our previous observations (Valeri et al., 2010).

The direct mechanism of BRCA1-mediated control of centrosome number is still not fully clear, although the BRCA1-dependent ubiquitination of gamma-tubulin is proposed to be involved in the regulation of centrosome function (Staritaet al., 2004). Gamma-tubulin is an important protein involved in the initiation of microtubule nucleation by centrosomes. Gamma-tubulin's lysines 48 and 344 have been indicated as crucial in the regulation of centrosome duplication and microtubule nucleation function, respectively (Sankaran et al., 2005). Cells with mutated lysines on gamma-tubulin, unable to be ubiquitinated, were characterized by centrosome amplification. On the other hand, the same phenotype was observed after inhibition of the enzymatic activity of BRCA1 by transfection of the BRCA1 (I26A) ligase-defective mutant (Sankaran et al., 2006). Additionally, *in vitro* experiments using *Xenopus* extracts, purified centrosomes and BRCA1 together with ubiquitination factors confirmed that BRCA1 is involved in the microtubule nucleation. It seems that BRCA1 controls the centrosome number by preventing reduplication due to ubiquitination of lysines of gamma-tubulin, which needs to be phosphorylated to prevent reduplication (Ko et al., 2006). Loss of BRCA1 did not affect centrosome duplication in the early S phase but rather caused a second round of duplication just prior to mitosis. The model has been proposed, in which BRCA1 marks centrosomes as already duplicated *via* the BRCA1 mediated ubiqutination of gamma-tubulin (Wong & Stearns, 2003). This issue is still not fully clarified, however there is no doubt about the significant role of the BRCA1-mediated ubiquitination of gamma-tubulin in this process (Kais & Parvin, 2008). Altogether, this led to the conclusion that the E3 ubiquitin ligase activity of BRCA1 is crucial for the effects on the biology of centrosomes, and controls centrosome duplication as well as microtubules nucleation.

Recently, it was demonstrated that BRCA1 interacts with centrosomal protein Nlp (nineinlike protein) (Jin et al., 2009), which is a fast turnover protein and plays a role in the centrosome maturation and spindle formation (Casenghi et al., 2005). Authors found that Nlp is a BRCA1-associated protein and colocalizes with BRCA1 in different types of cancer cells, including HeLa and U2OS cells. Moreover, Nlp expression and stability depends on normal cellular BRCA1 function. A variety of different types of cells expressing the mutated BRCA1 or silenced for BRCA1 exhibited disrupted Nlp colocalization to centrosomes as well as enhanced Nlp degradation. This data was consistent with our observations concerning the role of BRCA1 in different types of cancers. The lack of Nlp protein led to centrosome amplification, aberrant chromosome segregation, cytokinesis failure and appearance of miltinuclei, thus resembling the phenotype upon BRCA1 disruption. Recent studies showed that Nlp is recruited by the Aurora B protein and localizes at the midbody during

BCR-ABL Hits at Mitosis; Implications for Chromosomal

tyrosine kinase inhibitors, such as imatinib or dasatinib.

Petronczki, 2010).

Instability, Aneuploidy and Therapeutic Strategy 15

the CPC proteins and have a typical localization, however they are not members of the core complex. The complex undergoes a characteristic scenario of translocations during mitosis – they localize at the inner centromeres in prometaphase and metaphase then, at anaphase onset, they leave the chromosomes and transfer to the kinetochores at the central spindle to finally move to the midbody at cytokinesis. It plays a crucial role in the regulation of chromatin condensation, kinetochore function, mitotic checkpoint competence as well as cytokinesis (Terada, 2001; Vaderet al., 2006). Very recently it was presented that the chromosomal passenger complex is essential for correcting the non-bipolar chromosome attachments and for cytokinesis (Becker et al., 2010). To do this, Aurora B and INCENP have to be localized to centromeres. This is a very important finding as it indicates the supportive role of the CPC complex in case of mitotic checkpoint failure. Moreover, the translocation of Aurora B and other CPC proteins from centromeres to the spindle midzone in anaphase is necessary to prevent mitotic checkpoint engagement at anaphase (Vazquez-Novelle &

Members of the CPC complex have been proposed as very potent therapeutic targets. Treatment of imatinib-resistant CML cells carrying the T315I mutation with small molecule inhibitor, PHA-739358, which selectively targets BCR-ABL and Aurora kinases led to strong antiproliferative and apoptotic effects (Gontarewicz et al., 2008). Moreover, this has also been observed in CD34+ cells derived from untreated CML patients and from imatinibresistant patients in the chronic phase or blast crisis, including those harbouring the T315I mutation. Similar effects were obtained by combined treatment of imatinib resistant CML cells with vorinostat together with Aurora kinase inhibitor MK-0457 (Dai et al., 2008). Effectivity of this combined treatment has been shown against primary CD34+ CML cells, murine Ba/F3 cells with various BCR-ABL mutations (T315I, E255K, and M351T), as well as in imatinib-resistant K562 cells with BCR-ABL-independent, Lyn-dependent resistance. The same combination of therapy was presented also in other studies by Fiskus et al (Fiskus et al., 2008). Authors studied different CML cell lines, murine cells expressing BCR-ABL as well as primary CML and AML cells and came to the same conclusions. Novel inhibitors of Aurora kinases are still investigated in the anti-leukemia therapy of imatinib-resistant cells (Fei et al., 2010; Kelly et al., 2010). Usually they are proposed to be used together with

Survivin has been also proposed as a universal target for anticancer therapy (Andersen et al., 2007). Several trials are currently undergoing, using different methodologies, from small molecule antagonists to immunotherapy (Kanwar et al., 2010). However, the development of survivin inhibitors is not as advanced as other therapeutic small inhibitors. It is important to point that anti-survivin therapy should be probably combined with other treatments, as it is known that survivin depletion uncovers the function of the mitotic as well as post-mitotic p53-p21-dependent checkpoints, which protect from polyploidization upon mitosis disturbances (Beltrami et al., 2004). In case of cancers with the defective p53 function, survivin silencing led to reduced mitotic arrest and enhanced polyploidy, what is a very unwanted and dangerous side-effect. Also in our studies, specific depletion of survivin by siRNA approach in CML cells with checkpoints defects, resulted in strong polyploidization and chromosomal instability (Wolanin et al., 2006). However, when we used a natural compound – curcumin, which has been shown as a broadly acting, very potent anticancer agent, we found that it affects the CPC proteins and induces mitotic catastrophe, however without polyploidization. Curcumin decreased the level of survivin and caused unproper localization of Aurora B, leading to perturbances in mitosis and defective cytokinesis.

cytokinesis, thus its depletion or increased degradation triggers aborted division and subsequently leads to multinucleated phenotypes (Yan et al., 2010).

Altogether, this data strongly supported the idea that BRCA1 is one of the key elements controlling mitosis and the loss of BRCA1 could result in very severe dysfunctions of cell division. We propose that this can significantly participate in the generation of aneuploidy, CML progression and blastic transformation. A proposed model showing the influence of BCR-ABL-mediated downregulation of BRCA1 on the occurrence of genomic instability and aneuploidy in CML cells is presented in Figure 2.

Fig. 2. Proposed scheme of the mechanisms influenced by BCR-ABL-mediated BRCA1 downregulation; role in the genomic instability and generation of aneuploid cells.

#### **5. Therapeutic targeting of mitosis in CML cells**

The effects of the improper control of mitosis in the development and progression of leukemias, including chronic myeloid leukemia has been already described above. The importance of these processes and their potential as targets for therapy is already obvious. In general, looking for new treatment options or combined therapies is still necessary to overcome the insensitivity or resistance to tyrosine kinase inhibitors, often developed in CML patients. In our opinion, targeting the chromosomal passenger complex and mitotic kinases is a very promising trend in the development of novel anti-leukemia therapeutic strategies. We will discuss the current data and implications for the future.

#### **5.1 Chromosomal passenger complex and Aurora kinases**

The chromosomal passenger complex (CPC) is a group of proteins, which are involved in the regulation of nearly all stages of mitosis (Vader et al., 2006; Vagnarelli & Earnshaw, 2004; Yanet al., 2010). In most organisms, the chromosomal passenger complex is formed by four main proteins: Aurora B kinase, INCENP, Survivin and Borealin/Dasra-B (Ruchaud et al., 2007). Other proteins, like telophase disk 60 kDa (TD-60) have been shown to interact with

cytokinesis, thus its depletion or increased degradation triggers aborted division and

Altogether, this data strongly supported the idea that BRCA1 is one of the key elements controlling mitosis and the loss of BRCA1 could result in very severe dysfunctions of cell division. We propose that this can significantly participate in the generation of aneuploidy, CML progression and blastic transformation. A proposed model showing the influence of BCR-ABL-mediated downregulation of BRCA1 on the occurrence of genomic instability and

Bcr -Abl

BRCA1 expression

Centrosomes amplification

Dysfunctions of DNA damage response

Defective cell cycle checkpoints function Error-prone DNA repair

Mitotic spindle abnormalities Unproper chromosomes segregation

**High rate of genomic/chromosomal instability Increased susceptibility to generate aneuploid clones**

The effects of the improper control of mitosis in the development and progression of leukemias, including chronic myeloid leukemia has been already described above. The importance of these processes and their potential as targets for therapy is already obvious. In general, looking for new treatment options or combined therapies is still necessary to overcome the insensitivity or resistance to tyrosine kinase inhibitors, often developed in CML patients. In our opinion, targeting the chromosomal passenger complex and mitotic kinases is a very promising trend in the development of novel anti-leukemia therapeutic

The chromosomal passenger complex (CPC) is a group of proteins, which are involved in the regulation of nearly all stages of mitosis (Vader et al., 2006; Vagnarelli & Earnshaw, 2004; Yanet al., 2010). In most organisms, the chromosomal passenger complex is formed by four main proteins: Aurora B kinase, INCENP, Survivin and Borealin/Dasra-B (Ruchaud et al., 2007). Other proteins, like telophase disk 60 kDa (TD-60) have been shown to interact with

Fig. 2. Proposed scheme of the mechanisms influenced by BCR-ABL-mediated BRCA1 downregulation; role in the genomic instability and generation of aneuploid cells.

strategies. We will discuss the current data and implications for the future.

**5.1 Chromosomal passenger complex and Aurora kinases** 

**5. Therapeutic targeting of mitosis in CML cells** 

subsequently leads to multinucleated phenotypes (Yan et al., 2010).

aneuploidy in CML cells is presented in Figure 2.

Decreased expression of BRCA1 transcriptional targets (BubR1, Mad2, Bub1, Bub3, cyclin B1)

Lower mitotic checkpoint competence

the CPC proteins and have a typical localization, however they are not members of the core complex. The complex undergoes a characteristic scenario of translocations during mitosis – they localize at the inner centromeres in prometaphase and metaphase then, at anaphase onset, they leave the chromosomes and transfer to the kinetochores at the central spindle to finally move to the midbody at cytokinesis. It plays a crucial role in the regulation of chromatin condensation, kinetochore function, mitotic checkpoint competence as well as cytokinesis (Terada, 2001; Vaderet al., 2006). Very recently it was presented that the chromosomal passenger complex is essential for correcting the non-bipolar chromosome attachments and for cytokinesis (Becker et al., 2010). To do this, Aurora B and INCENP have to be localized to centromeres. This is a very important finding as it indicates the supportive role of the CPC complex in case of mitotic checkpoint failure. Moreover, the translocation of Aurora B and other CPC proteins from centromeres to the spindle midzone in anaphase is necessary to prevent mitotic checkpoint engagement at anaphase (Vazquez-Novelle & Petronczki, 2010).

Members of the CPC complex have been proposed as very potent therapeutic targets. Treatment of imatinib-resistant CML cells carrying the T315I mutation with small molecule inhibitor, PHA-739358, which selectively targets BCR-ABL and Aurora kinases led to strong antiproliferative and apoptotic effects (Gontarewicz et al., 2008). Moreover, this has also been observed in CD34+ cells derived from untreated CML patients and from imatinibresistant patients in the chronic phase or blast crisis, including those harbouring the T315I mutation. Similar effects were obtained by combined treatment of imatinib resistant CML cells with vorinostat together with Aurora kinase inhibitor MK-0457 (Dai et al., 2008). Effectivity of this combined treatment has been shown against primary CD34+ CML cells, murine Ba/F3 cells with various BCR-ABL mutations (T315I, E255K, and M351T), as well as in imatinib-resistant K562 cells with BCR-ABL-independent, Lyn-dependent resistance. The same combination of therapy was presented also in other studies by Fiskus et al (Fiskus et al., 2008). Authors studied different CML cell lines, murine cells expressing BCR-ABL as well as primary CML and AML cells and came to the same conclusions. Novel inhibitors of Aurora kinases are still investigated in the anti-leukemia therapy of imatinib-resistant cells (Fei et al., 2010; Kelly et al., 2010). Usually they are proposed to be used together with tyrosine kinase inhibitors, such as imatinib or dasatinib.

Survivin has been also proposed as a universal target for anticancer therapy (Andersen et al., 2007). Several trials are currently undergoing, using different methodologies, from small molecule antagonists to immunotherapy (Kanwar et al., 2010). However, the development of survivin inhibitors is not as advanced as other therapeutic small inhibitors. It is important to point that anti-survivin therapy should be probably combined with other treatments, as it is known that survivin depletion uncovers the function of the mitotic as well as post-mitotic p53-p21-dependent checkpoints, which protect from polyploidization upon mitosis disturbances (Beltrami et al., 2004). In case of cancers with the defective p53 function, survivin silencing led to reduced mitotic arrest and enhanced polyploidy, what is a very unwanted and dangerous side-effect. Also in our studies, specific depletion of survivin by siRNA approach in CML cells with checkpoints defects, resulted in strong polyploidization and chromosomal instability (Wolanin et al., 2006). However, when we used a natural compound – curcumin, which has been shown as a broadly acting, very potent anticancer agent, we found that it affects the CPC proteins and induces mitotic catastrophe, however without polyploidization. Curcumin decreased the level of survivin and caused unproper localization of Aurora B, leading to perturbances in mitosis and defective cytokinesis.

BCR-ABL Hits at Mitosis; Implications for Chromosomal

**7. Acknowledgment** 

**8. References** 

168(1): 22-9.

*Biol* 8(10): 822-4.

72.

generation tyrosine kinase inhibitors, such as dasatinib or nilotinib.

Centre in Ireland for providing the 32D, C2 and C4 cell lines.

blastic crisis. *Cancer Genet Cytogenet* 26(1): 39-50.

*Cancer Genet Cytogenet* 168(2): 120-3.

progression. *Cell Cycle* 4(11): 1641-66.

Instability, Aneuploidy and Therapeutic Strategy 17

that the decrease of BRCA1 caused by BCR-ABL in CML cells could be a critical factor determining the generation of supernumerary centrosomes, aberrant mitotic spindles, mitosis and cytokinesis failures, finally leading to aneuploidization. All data presented in this review convincingly show that activity of the BCR-ABL kinase is directly responsible for the promotion of chromosomally unstable phenotype. As chromosomal instability seems to play a crucial role in the disease progression, mitosis is a prospective target for treatment in CML. This opens new possibilities for therapeutical intervention based on the targeting processes involved in the control of mitosis. It can be an alternative strategy for alone or combined treatment of leukemia cells, which developed resistance to imatinib or second-

This work has been partially superted by grants from the Ministry of Science and Higher Education: 2P04A 05729 and N N301 425938 (for K.P.) and Iuventus Plus IP2010 032870 (for P.P-B.) Authors would like to thank Dr. Sharon McKenna from Cork Cancer Research

Alimena, G., De Cuia, M. R., Diverio, D., Gastaldi, R.&Nanni, M. (1987). The karyotype of

Amiel, A., Yukla, M., Gaber, E., Leopold, L., Josef, G., Fejgin, M.&Lishner, M. (2006).

Andersen, M. H., Svane, I. M., Becker, J. C.&Straten, P. T. (2007). The universal character of the tumor-associated antigen survivin. *Clin Cancer Res* 13(20): 5991-4. Babicka, L., Zemanova, Z., Pavlistova, L., Brezinova, J., Ransdorfova, S., Houskova, L.,

Bae, I., Rih, J. K., Kim, H. J., Kang, H. J., Haddad, B., Kirilyuk, A., Fan, S., Avantaggiati, M.

Baer, R. (2001). With the ends in sight: images from the BRCA1 tumor suppressor. *Nat Struct* 

Baker, D. J., Chen, J.&van Deursen, J. M. (2005). The mitotic checkpoint in cancer and aging:

Bannon, J. H.&Mc Gee, M. M. (2009). Understanding the role of aneuploidy in

Becker, M., Stolz, A., Ertych, N.&Bastians, H. (2010). Centromere localization of INCENP-

Beltrami, E., Plescia, J., Wilkinson, J. C., Duckett, C. S.&Altieri, D. C. (2004). Acute ablation

Aurora B is sufficient to support spindle checkpoint function. *Cell Cycle* 9(7): 1360-

of survivin uncovers p53-dependent mitotic checkpoint functions and control of

what have mice taught us? *Curr Opin Cell Biol* 17(6): 583-9.

tumorigenesis. *Biochem Soc Trans* 37(Pt 4): 910-3.

mitochondrial apoptosis. *J Biol Chem* 279(3): 2077-84.

Random aneuploidy in CML patients at diagnosis and under imatinib treatment.

Moravcova, J., Klamova, H.&Michalova, K. (2006). Complex chromosomal rearrangements in patients with chronic myeloid leukemia. *Cancer Genet Cytogenet*

L.&Rosen, E. M. (2005). BRCA1 regulates gene expression for orderly mitotic

Thus, the status of the mitotic checkpoint competence as well as p53 should be taken into consideration when anti-survivin therapy is proposed. Another natural compound, 16 hydroxycleroda-3,13-dien-15,16-olide (PL3), one of the clerodane diterpenoid compounds isolated from *Polyalthia longifolia*, induced degradation of Aurora B, mitotic checkpoint dysfunctions and finally led to cell death of CML cells, including the T315I-mutated BCR-ABL+ BA/F3 cells (Lin et al., 2011). Additionally, it reversed the sensitivity to imatinib of T315I-mutated CML cells in comparizon to treatment only with imatinib.

Recently, Aurora inhibitors were indicated as promising agents for acute myeloid and chronic myeloid leukemias (Moore et al., 2010). The most promising data was obtained for FLT3-mutated AML and imatinib-resistant Ph+ CML, particularly with the T315I mutation. Clinical trials investigating these agents have been already initiated (Cheung et al., 2011).

#### **5.2 Polo-like kinases**

Polo-like kinases are of strong interest according to potential anticancer therapy, as similarly to Aurora kinases, they can be targeted with selective small molecule inhibitors (Warner et al., 2008). Additionally, many natural compounds with the ability to prevent cancerogenesis, such as wortmanin, quercetin, thymoquinone, genistein, indirubin and others, have been shown to modulate PLK1 level or activity. It is proposed that naturally occurring PLK1 inhibitors with low or no toxicity should be considered as interesting agents in prevention as well as treatment of cancer (Schmit et al., 2010).

As described before, PLK1 has been upregulated in different cancers, including leukemias. Its inhibition or silencing resulted in cell cycle arrest, decrease of cell viability and induction of apoptosis in various cancer cells. Inhibition of PLK1 by different small molecule compounds in acute myeloid leukemia (AML) cells led to mitotic accumulation and apoptosis (Didier et al., 2008). Comprehensive studies of PLK1 silencing and inhibition using the novel selective inhibitor GW843682X in a broad range of different leukemia cell lines and primary cells led to the conclusion that PLK1targeting can be a promising strategy (Ikezoeet al., 2009). This observation was confirmed by other studies of leukemia primary cells (Renneret al., 2009). The potential of PLK1 inhibition to improve the chemotherapy or irradiation of resistant leukemia cells has been also investigated in primary patient cells as well as *in vivo,* in mouse Xenograft models of B-lineage ALL studies, with the same conclusions (Uckun et al., 2010). Currently, several PLK1 inhibitors are in different phases of clinical development for anticancer therapy (Chopra et al., 2010; Schoffski, 2009). Data from one of the first clinical trials indicated that the PLK1 inhibitor BI 2536 was well tolerated and showed antitumor activity in patients with advanced solid tumors and refractory or relapsed AML (Wasch et al., 2010). According to CML, this scheme of therapy was not very intensively studied till now, however it also seems to be very potent and prospective. PLK1 inhibitor BI 2536 in a low, nanomolar concentration was able to induce growth inhibition and mitotic arrest followed by apoptosis in CML cells, including cell lines and primary cells from patients (Gleixneret al., 2010). Importantly, this agent was very effective not only against imatinib-sensitive CML cells, but also imatinib-resistant cells carrying the T315I mutation. Treatment with BI2536 together with imatinib or nilotinib showed synergistic effect, indicating possibility of a combined therapeutic application.

#### **6. Final conclusions**

Taken together, it is already clear that BRCA1 due to its multifunctional nature, is one of the key molecules controlling mitosis on the different levels of organization. There is no doubt that the decrease of BRCA1 caused by BCR-ABL in CML cells could be a critical factor determining the generation of supernumerary centrosomes, aberrant mitotic spindles, mitosis and cytokinesis failures, finally leading to aneuploidization. All data presented in this review convincingly show that activity of the BCR-ABL kinase is directly responsible for the promotion of chromosomally unstable phenotype. As chromosomal instability seems to play a crucial role in the disease progression, mitosis is a prospective target for treatment in CML. This opens new possibilities for therapeutical intervention based on the targeting processes involved in the control of mitosis. It can be an alternative strategy for alone or combined treatment of leukemia cells, which developed resistance to imatinib or secondgeneration tyrosine kinase inhibitors, such as dasatinib or nilotinib.

#### **7. Acknowledgment**

16 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Thus, the status of the mitotic checkpoint competence as well as p53 should be taken into consideration when anti-survivin therapy is proposed. Another natural compound, 16 hydroxycleroda-3,13-dien-15,16-olide (PL3), one of the clerodane diterpenoid compounds isolated from *Polyalthia longifolia*, induced degradation of Aurora B, mitotic checkpoint dysfunctions and finally led to cell death of CML cells, including the T315I-mutated BCR-ABL+ BA/F3 cells (Lin et al., 2011). Additionally, it reversed the sensitivity to imatinib of

Recently, Aurora inhibitors were indicated as promising agents for acute myeloid and chronic myeloid leukemias (Moore et al., 2010). The most promising data was obtained for FLT3-mutated AML and imatinib-resistant Ph+ CML, particularly with the T315I mutation. Clinical trials investigating these agents have been already initiated (Cheung et al., 2011).

Polo-like kinases are of strong interest according to potential anticancer therapy, as similarly to Aurora kinases, they can be targeted with selective small molecule inhibitors (Warner et al., 2008). Additionally, many natural compounds with the ability to prevent cancerogenesis, such as wortmanin, quercetin, thymoquinone, genistein, indirubin and others, have been shown to modulate PLK1 level or activity. It is proposed that naturally occurring PLK1 inhibitors with low or no toxicity should be considered as interesting agents in prevention

As described before, PLK1 has been upregulated in different cancers, including leukemias. Its inhibition or silencing resulted in cell cycle arrest, decrease of cell viability and induction of apoptosis in various cancer cells. Inhibition of PLK1 by different small molecule compounds in acute myeloid leukemia (AML) cells led to mitotic accumulation and apoptosis (Didier et al., 2008). Comprehensive studies of PLK1 silencing and inhibition using the novel selective inhibitor GW843682X in a broad range of different leukemia cell lines and primary cells led to the conclusion that PLK1targeting can be a promising strategy (Ikezoeet al., 2009). This observation was confirmed by other studies of leukemia primary cells (Renneret al., 2009). The potential of PLK1 inhibition to improve the chemotherapy or irradiation of resistant leukemia cells has been also investigated in primary patient cells as well as *in vivo,* in mouse Xenograft models of B-lineage ALL studies, with the same conclusions (Uckun et al., 2010). Currently, several PLK1 inhibitors are in different phases of clinical development for anticancer therapy (Chopra et al., 2010; Schoffski, 2009). Data from one of the first clinical trials indicated that the PLK1 inhibitor BI 2536 was well tolerated and showed antitumor activity in patients with advanced solid tumors and refractory or relapsed AML (Wasch et al., 2010). According to CML, this scheme of therapy was not very intensively studied till now, however it also seems to be very potent and prospective. PLK1 inhibitor BI 2536 in a low, nanomolar concentration was able to induce growth inhibition and mitotic arrest followed by apoptosis in CML cells, including cell lines and primary cells from patients (Gleixneret al., 2010). Importantly, this agent was very effective not only against imatinib-sensitive CML cells, but also imatinib-resistant cells carrying the T315I mutation. Treatment with BI2536 together with imatinib or nilotinib showed synergistic

Taken together, it is already clear that BRCA1 due to its multifunctional nature, is one of the key molecules controlling mitosis on the different levels of organization. There is no doubt

T315I-mutated CML cells in comparizon to treatment only with imatinib.

**5.2 Polo-like kinases** 

**6. Final conclusions** 

as well as treatment of cancer (Schmit et al., 2010).

effect, indicating possibility of a combined therapeutic application.

This work has been partially superted by grants from the Ministry of Science and Higher Education: 2P04A 05729 and N N301 425938 (for K.P.) and Iuventus Plus IP2010 032870 (for P.P-B.) Authors would like to thank Dr. Sharon McKenna from Cork Cancer Research Centre in Ireland for providing the 32D, C2 and C4 cell lines.

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**2** 

*Spain* 

Maria Teresa Vargas

**BCR/ABL1 Extra Fusions in Patients with** 

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph), which is originated by a balanced translocation between chromosomes 9 and 22, that is, t(9;22)(q34;q11) results in production of a BCR-ABL1 fusion protein with constitutive Abl kinase activity. In experimental models, the expression of said activity causes expansion of pluripotent stem cells, preferentially favours myeloid lineage differentiation, and inhibits

The BCR/ABL1 fusion in 22q11 is observed in ~95% of patients with chronic myeloid leukemia (CML). The BCR/ABL1 fusion signal on the derivative chromosome 22 is present

 In 5-10% of cases, this signal is generated by variant rearrangements (variant Ph) that involve other genomic regions [3-7]. More than 600 CML cases with variant rearrangements have been reported. The breakpoint distribution clearly exhibits a non-random pattern, with marked clustering to some chromosome bands, such as 1p36, 3p21, 5q13, 6p21, 9q22, 11q13, 12p13, 17p13, 17q21, 17q25, 19q13, 21q22, 22q12, and 22q13, which suggests that these regions may be particularly prone to breakage. In addition, some specific variants are also known to be more frecuent, with the two translocations t(3;9;22)(p21;q34;q11) and

The occurrence of additional chromosomal aberrations (ACA) in Ph-positive CML is strongly associated with disease progression and has been interpreted as a sign of clonal evolution as well as chromosomal instability. Mitelman et al. [9] identified +Ph, +8, and i(17q) as the most common secondary changes that occur in nearly 90% of the cases with additional abnormalities. These three abnormalities were proposed to follow the 'major route' of clonal evolution, whereas other changes, which evolved more rarely, were suggested to follow the 'minor route' [9]; the terms 'major' and 'minor' refer to frequencies of the aberrations but not to the pathogenetic impact. Thus the three major route changes, followed by +19, were the most common ACA. Other additional chromosomal abnormalities take place in less than 10% of the cases, the most frequent being –Y, +21, +17, –7, and –17. On the other hand, no apparent differences in ACA between CML with standard Ph and CML cases with Ph variants are observed. The most common additional chromosomal changes are +8 (34%), +Ph (30%), i(17q) (20%), +19 (13%), –Y (8% of

t(17;22)(q25;q11) both having been reported in more than 10 cases [8].

**1. Introduction** 

erythroid differentiation [1,2].

in most CML patients.

**Chronic Myeloid Leukaemia (CML)** 

*Division of Genetics, Department of Pathology, Virgen Macarena University Hospital, Seville,* 

genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer. *Oncogene* 21(33): 5097-107.


### **BCR/ABL1 Extra Fusions in Patients with Chronic Myeloid Leukaemia (CML)**

Maria Teresa Vargas

*Division of Genetics, Department of Pathology, Virgen Macarena University Hospital, Seville, Spain* 

#### **1. Introduction**

26 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Wolanin, K., Magalska, A., Kusio-Kobialka, M., Podszywalow-Bartnicka, P., Vejda, S.,

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Yan, J., Jin, S., Li, J.&Zhan, Q. (2010). Aurora B interaction of centrosomal Nlp regulates

Yang, E. S.&Xia, F. (2010). BRCA1 16 years later: DNA damage-induced BRCA1 shuttling.

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Yuan, K., Huang, Y.&Yao, X. (2011). Illumination of mitotic orchestra during cell division: a

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Zhang, J.&Powell, S. N. (2005). The role of the BRCA1 tumor suppressor in DNA double-

Zhang, S., Hemmerich, P.&Grosse, F. (2007). Centrosomal localization of DNA damage

Zhou, C., Smith, J. L.&Liu, J. (2003). Role of BRCA1 in cellular resistance to paclitaxel and

W. S. (1998). BRCA1 physically associates with p53 and stimulates its

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expressing cells. *Mol Cancer Res* 4(7): 457-69.

cytokinesis. *J Biol Chem* 285(51): 40230-9.

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21(33): 5097-107.

38.

1(2): 117-23.

389-95.

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Polo view. *Cell Signal* 23(1): 1-5.

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genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer. *Oncogene*

McKenna, S. L., Mosieniak, G., Sikora, E.&Piwocka, K. (2010). Expression of oncogenic kinase Bcr-Abl impairs mitotic checkpoint and promotes aberrant divisions and resistance to microtubule-targeting agents. *Mol Cancer Ther* 9(5): 1328-

E.&Piwocka, K. (2006). Curcumin affects components of the chromosomal passenger complex and induces mitotic catastrophe in apoptosis-resistant Bcr-Abl-

Hwang, L. Y., Bowcock, A. M.&Baer, R. (1996). Identification of a RING protein that

C. X. (1999). Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. *Mol Cell* 3(3): Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph), which is originated by a balanced translocation between chromosomes 9 and 22, that is, t(9;22)(q34;q11) results in production of a BCR-ABL1 fusion protein with constitutive Abl kinase activity. In experimental models, the expression of said activity causes expansion of pluripotent stem cells, preferentially favours myeloid lineage differentiation, and inhibits erythroid differentiation [1,2].

The BCR/ABL1 fusion in 22q11 is observed in ~95% of patients with chronic myeloid leukemia (CML). The BCR/ABL1 fusion signal on the derivative chromosome 22 is present in most CML patients.

 In 5-10% of cases, this signal is generated by variant rearrangements (variant Ph) that involve other genomic regions [3-7]. More than 600 CML cases with variant rearrangements have been reported. The breakpoint distribution clearly exhibits a non-random pattern, with marked clustering to some chromosome bands, such as 1p36, 3p21, 5q13, 6p21, 9q22, 11q13, 12p13, 17p13, 17q21, 17q25, 19q13, 21q22, 22q12, and 22q13, which suggests that these regions may be particularly prone to breakage. In addition, some specific variants are also known to be more frecuent, with the two translocations t(3;9;22)(p21;q34;q11) and t(17;22)(q25;q11) both having been reported in more than 10 cases [8].

The occurrence of additional chromosomal aberrations (ACA) in Ph-positive CML is strongly associated with disease progression and has been interpreted as a sign of clonal evolution as well as chromosomal instability. Mitelman et al. [9] identified +Ph, +8, and i(17q) as the most common secondary changes that occur in nearly 90% of the cases with additional abnormalities. These three abnormalities were proposed to follow the 'major route' of clonal evolution, whereas other changes, which evolved more rarely, were suggested to follow the 'minor route' [9]; the terms 'major' and 'minor' refer to frequencies of the aberrations but not to the pathogenetic impact. Thus the three major route changes, followed by +19, were the most common ACA. Other additional chromosomal abnormalities take place in less than 10% of the cases, the most frequent being –Y, +21, +17, –7, and –17. On the other hand, no apparent differences in ACA between CML with standard Ph and CML cases with Ph variants are observed. The most common additional chromosomal changes are +8 (34%), +Ph (30%), i(17q) (20%), +19 (13%), –Y (8% of

BCR/ABL1 Extra Fusions in Patients with Chronic Myeloid Leukaemia (CML) 29

BC include: inv(3)(q21q26)/t(3;3)(q21;q26), t(3;21)(q26;q22), t(7;11)(p15;p15), t(8;21)(q22;q22),

Although secondary by definition, these ACA do not behave as ordinary secondary changes. Firstly, they are strongly related to quite specific phenotypic features as the i(17q) have been associated with marked basophilia and the presence of pseudo Pelger-Huët neutrophils [4], while other secondary aberrations are not. Secondly, they do not normally take place at the same time as other common additional changes, such as +8, i(17q), and +Ph, which clearly indicates that they follow a different evolutionary pathway. Rather than being secondary changes, they may be seen as second primary changes. It is worth noting that the fact that balanced aberrations characteristic of acute lymphoblastic leukemia (ALL), such as t(4;11)(q21;q23) and t(8;14)(q24;q32), have, to date, never been reported in CML blastic phase [12]. The idea that ALL-associated translocations cannot originate during the particular differentiation stage that characterizes the Ph-positive stem cell population is a possibility. On the other hand, and equally possible, ALLassociated translocations may not provide any selective advantage for cells expressing the

In 60–80% of the cases, ACA precede or accompany blastic phase [14, 15, 23-33], thus there is undoubtedly a strong association between secondary changes and transformation. Furthermore, early studies of splenic CML transformations give rise to strong support for the clinical importance of additional changes, showing that the secondary aberrations appeared in the spleen before bone marrow [34-36]. However, the prognostic impact of ACA has turned out to be difficult to assess. First, although most studies have reported that lack of ACA during blastic phase give rise to a better prognosis [32, 33, 37-41], some investigators have not found any prognostic differences between CML blastic phase cases with and without secondary changes [23, 30]. Second, it has been debated whether additional changes found during chronic phase constitute a dismal prognostic sign, announcing transformation. On one hand, as has been repeatedly highlighted, the emergence of new clones does not necessarily lead to disease progression and some clones appear only transiently [23, 42, 43]. On the other hand, the appearance of new ACA or increment of the clone with additional abnormalities has been shown to be generally associated with clinical deterioration [28, 37,

Moreover, Sokal et al. [45] reported that patients' survival with secondary changes already present at the time of diagnosis had a shorter survival rate, but with survival curves for those with and without additional changes not diverging until after the 2-year point. Third, the number and types of aberrations present during chronic phase and blastic phase may also play a role. Quite often the karyotypic changes observed in the chronic phase are single events, particularly the loss of the Y chromosome, +8, +Ph, and i(17q) [47], while they are multiple and complex in blastic phase [15,31,37,42,46]. The aberrations in i(17q), or other changes that result in loss of 17p seem particularly ominous [15,23,25,37,42.47-49]. Furthermore, trisomy 8 and +Ph that take place in the blastic phase have been associated with poor prognosis [15, 50-52] (table 1). Thus, the prognostic impact of secondary abnormalities in CML is complex, heterogeneous, and is probably related to several

Therefore, response to imatinib on the one hand, and resistance on the other, may also be conditioned by different types of ACAs. Enhanced response or resistance to treatment may be associated with the occurrence of different cytogenetic or molecular changes which are capable of activating the metabolic pathways with different outcomes [53,54]. Several

parameters such as time of appearance and specific abnormalities [52].

and inv(16) (p13q22) [12, 18-22].

BCR/ABL1 chimeric protein.

44].

anomalies), +21 (7%), +17 (5%), and –7 (5%), when these two CML groups are combined (Table 1).


Table 1. Secondary chromosome abnormality patterns in CML patients.

The chromosome bands frequently involved in secondary structural changes are similar in both groups. It is apparent that some chromosome segments are particularly 'breakprone': 1q, 3q21, 3q26, 7p, 9p, 11q23, 12p13, 13q11–14, 17p11, 17q10, 21q22, and 22q10 when breakpoint distribution is observed in CML with standard Ph.

The ACA are found in various combinations and it has been suggested that i(17q) followed by +8 are rather early changes, whereas trisomy 19 is a relatively late one [10,11]. Although this temporal order of ACA seems common, exceptions clearly exist. There are several examples of Ph-positive CML cases with +8 in all metaphases but i(17q) may also occur in only subclones, and Ph-positive CML cases with a trisomy 19 [12]. Moreover, some combinations are more frequent than others. While +8, +Ph, and i(17q) often occur together, the combination i(17q),+19 (without +8/+Ph) has only been reported in 2 cases; +8,i(17q),+19 (without +Ph) in 16 cases; and i(17q),+19,+Ph (without +8) in 2 cases[12]. Some combinations (+8, i(17q); +8,+19; +19,+Ph) are positively associated, while i(17q),+19 and i(17q),+Ph are negatively associated according to Hashimoto et al. [13].

Thus, the selective ACA appearance seems to vary, depending on other aberrations present in the combination. Furthermore, available data indicate that the most common secondary abnormalities occur in a step-wise, well-ordered manner, with a putative frequent pathway which starts with i(17q), then followed by +8 and +Ph, and lastly +19. Since three out of the four most frequent ACA involve gains, i.e. +8, +19, and +Ph, thus hyperdiploidy is the most common modal number in CML with ACA [14,15]; nearly 50% of the cases have 47–50 chromosomes. Pseudodiploidy (46 chromosomes with structural abnormalities) is observed in almost 40% of the cases, while other modal numbers are uncommon. It is worth bearing in mind that the distributions of ploidy levels are not different between CML cases with standard Ph and those with variant Ph translocations, which confirms a strong indication that there are not any major differences between these two groups when related to cytogenetic evolution patterns. Most of the ACA in CML are genomically unbalanced, such as trisomies, monosomies, and deletions. In fact, this is common in hematologic malignancies, therefore while the primary changes have been proposed as balanced, the secondary changes are said to be unbalanced [16]. However, there are some notable exceptions in CML, in 10 cases the presence of t(15;17)(q22;q12–21) has been reported [12]. Other balanced rearrangements which are characteristically found in acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) and occasionally also found in CML

anomalies), +21 (7%), +17 (5%), and –7 (5%), when these two CML groups are combined

+Ph classical +Ph classical +Ph variant + Ph variant + Ph, +8 +Ph, +8 +Ph, -Y +Ph, -Y +Ph, -21 +Ph, i(17q) +8 +19/ +17 /-7 Complex Karyotype Complex karyotype

The chromosome bands frequently involved in secondary structural changes are similar in both groups. It is apparent that some chromosome segments are particularly 'breakprone': 1q, 3q21, 3q26, 7p, 9p, 11q23, 12p13, 13q11–14, 17p11, 17q10, 21q22, and 22q10 when

The ACA are found in various combinations and it has been suggested that i(17q) followed by +8 are rather early changes, whereas trisomy 19 is a relatively late one [10,11]. Although this temporal order of ACA seems common, exceptions clearly exist. There are several examples of Ph-positive CML cases with +8 in all metaphases but i(17q) may also occur in only subclones, and Ph-positive CML cases with a trisomy 19 [12]. Moreover, some combinations are more frequent than others. While +8, +Ph, and i(17q) often occur together, the combination i(17q),+19 (without +8/+Ph) has only been reported in 2 cases; +8,i(17q),+19 (without +Ph) in 16 cases; and i(17q),+19,+Ph (without +8) in 2 cases[12]. Some combinations (+8, i(17q); +8,+19; +19,+Ph) are positively associated, while i(17q),+19 and

Thus, the selective ACA appearance seems to vary, depending on other aberrations present in the combination. Furthermore, available data indicate that the most common secondary abnormalities occur in a step-wise, well-ordered manner, with a putative frequent pathway which starts with i(17q), then followed by +8 and +Ph, and lastly +19. Since three out of the four most frequent ACA involve gains, i.e. +8, +19, and +Ph, thus hyperdiploidy is the most common modal number in CML with ACA [14,15]; nearly 50% of the cases have 47–50 chromosomes. Pseudodiploidy (46 chromosomes with structural abnormalities) is observed in almost 40% of the cases, while other modal numbers are uncommon. It is worth bearing in mind that the distributions of ploidy levels are not different between CML cases with standard Ph and those with variant Ph translocations, which confirms a strong indication that there are not any major differences between these two groups when related to cytogenetic evolution patterns. Most of the ACA in CML are genomically unbalanced, such as trisomies, monosomies, and deletions. In fact, this is common in hematologic malignancies, therefore while the primary changes have been proposed as balanced, the secondary changes are said to be unbalanced [16]. However, there are some notable exceptions in CML, in 10 cases the presence of t(15;17)(q22;q12–21) has been reported [12]. Other balanced rearrangements which are characteristically found in acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) and occasionally also found in CML

Table 1. Secondary chromosome abnormality patterns in CML patients.

i(17q),+Ph are negatively associated according to Hashimoto et al. [13].

breakpoint distribution is observed in CML with standard Ph.

**Accelerated and Blastic Phase**  (in order of appearance)

(Table 1).

**Chronic Phase**  (in order of appearance) BC include: inv(3)(q21q26)/t(3;3)(q21;q26), t(3;21)(q26;q22), t(7;11)(p15;p15), t(8;21)(q22;q22), and inv(16) (p13q22) [12, 18-22].

Although secondary by definition, these ACA do not behave as ordinary secondary changes. Firstly, they are strongly related to quite specific phenotypic features as the i(17q) have been associated with marked basophilia and the presence of pseudo Pelger-Huët neutrophils [4], while other secondary aberrations are not. Secondly, they do not normally take place at the same time as other common additional changes, such as +8, i(17q), and +Ph, which clearly indicates that they follow a different evolutionary pathway. Rather than being secondary changes, they may be seen as second primary changes. It is worth noting that the fact that balanced aberrations characteristic of acute lymphoblastic leukemia (ALL), such as t(4;11)(q21;q23) and t(8;14)(q24;q32), have, to date, never been reported in CML blastic phase [12]. The idea that ALL-associated translocations cannot originate during the particular differentiation stage that characterizes the Ph-positive stem cell population is a possibility. On the other hand, and equally possible, ALLassociated translocations may not provide any selective advantage for cells expressing the BCR/ABL1 chimeric protein.

In 60–80% of the cases, ACA precede or accompany blastic phase [14, 15, 23-33], thus there is undoubtedly a strong association between secondary changes and transformation. Furthermore, early studies of splenic CML transformations give rise to strong support for the clinical importance of additional changes, showing that the secondary aberrations appeared in the spleen before bone marrow [34-36]. However, the prognostic impact of ACA has turned out to be difficult to assess. First, although most studies have reported that lack of ACA during blastic phase give rise to a better prognosis [32, 33, 37-41], some investigators have not found any prognostic differences between CML blastic phase cases with and without secondary changes [23, 30]. Second, it has been debated whether additional changes found during chronic phase constitute a dismal prognostic sign, announcing transformation. On one hand, as has been repeatedly highlighted, the emergence of new clones does not necessarily lead to disease progression and some clones appear only transiently [23, 42, 43]. On the other hand, the appearance of new ACA or increment of the clone with additional abnormalities has been shown to be generally associated with clinical deterioration [28, 37, 44].

Moreover, Sokal et al. [45] reported that patients' survival with secondary changes already present at the time of diagnosis had a shorter survival rate, but with survival curves for those with and without additional changes not diverging until after the 2-year point. Third, the number and types of aberrations present during chronic phase and blastic phase may also play a role. Quite often the karyotypic changes observed in the chronic phase are single events, particularly the loss of the Y chromosome, +8, +Ph, and i(17q) [47], while they are multiple and complex in blastic phase [15,31,37,42,46]. The aberrations in i(17q), or other changes that result in loss of 17p seem particularly ominous [15,23,25,37,42.47-49]. Furthermore, trisomy 8 and +Ph that take place in the blastic phase have been associated with poor prognosis [15, 50-52] (table 1). Thus, the prognostic impact of secondary abnormalities in CML is complex, heterogeneous, and is probably related to several parameters such as time of appearance and specific abnormalities [52].

Therefore, response to imatinib on the one hand, and resistance on the other, may also be conditioned by different types of ACAs. Enhanced response or resistance to treatment may be associated with the occurrence of different cytogenetic or molecular changes which are capable of activating the metabolic pathways with different outcomes [53,54]. Several

BCR/ABL1 Extra Fusions in Patients with Chronic Myeloid Leukaemia (CML) 31

completely clear. Erythroblastic transformation of CML has been reported in 10% of patients in blastic crisis; nonetheless, this has not been clearly associated to specific chromosome

(a) (b)

Three cases have been described with additional BCR/ABL1 fusions onto 18p accompanied by other chromosome alterations [67, 68], two of which have been reported by the same authors [68]. In the case described by Tanaka et al. [67], the blast morphology was not specified. Khac et al. [68], however, described two patients with CML in blastic transformation with a duplication and transposition of BCR/ABL1 to 18p that presented loss of part of its short arm; both patients had erythroblastic transformations, which the authors believed to be associated with this particular cytological subtype of disease progression in CML. The present case supports the relationship suggested by Khac et al. [68] between a tendency for erythroblastic transformation and the presence of extra fused BCR/ABL1 onto 18p, as in the patient we described, with a duplication and transposition of BCR/ABL1 onto 18p in the majority line (95%) in blastic phase, with no other chromosome alterations, together with an increase of red cells, intense dyserythropoietic features and

A 31-year-old caucasian man, a smoker (10 cigarettes/day) and with a past appendectomy, presented with a history of abdominal pain with diarrhea and an abscess in the jawbone; he was attended at the Department of Hematology. Clinical examination revealed hepatosplenomegaly. Complementary tests revealed leukocytosis in a peripheral blood examination. His white blood cell count was 52x109/L, with a peripheral monocytosis of 18x109/L and a left shift. The number of platelets was 94x109/L; hemoglobin was normal; leukocyte alkaline phosphatase was reduced. In the biochemical

Fig. 1. (a) Metaphase cell FISH showing two fusions (a classic one in the Ph chromosome and another BCR/ABL fusion in 18p) (arrows), two orange signals (normal and residual ABL probe in 9q34), and one green signal (BCR in the normal 22 chromosome) (94% of cells). (b) Partial FISH metaphase with DAPI/ FITC showing the three copies of the BCR gene (in the Ph chromosome; in 18p, indicated by arrows; and in the normal chromosome 22) (94% of

aberrations [3-7, 61-66].

more than 100 cells).

**3. Case report nº 2** 

PAS in granules leading to a fatal outcome.

studies have assessed the impact of ACA on the clinical efficacy of imatinib by assuming that these abnormalities may confer BCR/ABL1-independent proliferation and decrease sensitivity to imatinib [50-55].

However, the presence of additional copies of the BCR/ABL1 fusion onto structurally abnormal chromosomes has rarely been reported in the chronic or blastic phase of CML patients.

We present two CML patients with additional copies of BCR/ABL1 fusions: the first patient with +Ph and BCR/ABL1 extra fusion in chromosome 18 in the blastic phase, and the second patient with four BCR/ABL1 fusion signals at diagnosis **.** 

#### **2. Case report nº 1**

A 34-year-old patient was diagnosed with CML. The hematologic features were: Hb, 96 g; WBC, 354x109/L with 2% blasts, 4% basophils; platelets, 716x109/L. Neutrophil alkaline phosphatase (NAF) activity was absent. The bone marrow aspirate was hypercellular with 1% blast cells.

A cytogenetic study was performed on bone marrow cells and revealed a 46,XY,t(9;22)(q34;q11) karyotype in all cells analyzed. FISH was performed on chromosome preparations using commercially available probe sets (LSI BCR/ABL-ES probe; Vysis, Downers Grove, IL) and based on a count of 100 cells (metaphases and interphases). The existence of the classic BCR/ABL1 fusion was proved. The patient was treated with hydroxyurea and *a*-interferon, with a satisfactory clinical, but unsatisfactory cytogenetic and FISH response , the maximum response reached, based on a count of 120 cells, was 27/120 cells with the BCR/ABL1 rearrangement during the 4 year follow-up . A bone marrow transplantation could not be performed because no compatible HLA-matched donor was available.

After the aforementioned, 4 years after the initial diagnosis, a decrease of Hb, hematocrit, and platelets took place in the peripheral blood: WBC was 4.8x109/L with 38% blasts. Bone marrow was hypercellular, with 30% of cells representing the erythroid lineage at all the maturation stages, and with intense dyserythropoietic changes: 21% in the myeloid series (myeloid/erythroid ratio 5:1.5); 9% in the lymphoplasmocytoid series; and 40% as large blasts with fine chromatin in the nucleus, with two nucleoli, and irregularly shaped nongranular cytoplasms. Blast cells gave positive histochemical reactions for peroxidise and naphthol AS-D chloracetate esterase (inhibited by fluoride) and were periodic acid Schiff (PAS) positive as granules. Analysis in bone marrow of more than 100 cells at this time revealed that 94% of the cells had two fused BCR/ABL signals: one on the Ph chromosome and the other on the short arm of a derivative chromosome 18, detected by metaphase FISH analysis with Vysis LSI-BCR/ABL-ES and Cytocell mBCR/ABL probes (Fig. 1). Karyotype in bone marrow samples showed one Ph chromosome and one normal chromosome 22, and one normal chromosome 18 and another with a deletion of part of its short arm. The remaining 6% of cells only had the usual BCR/ABL1 fusion, without any other abnormalities.

The patient was then treated with mitoxantrone and cytarabine without clinical response, and later with splenic radiotherapy, which led to an intense pancytopenia; the patient then died 4 years 8 months after the initial diagnosis.

The occurrence of additional chromosome changes is an ominous sign, indicating that illness progression is imminent; however, the etiology of blastic transformation is still not completely clear. Erythroblastic transformation of CML has been reported in 10% of patients in blastic crisis; nonetheless, this has not been clearly associated to specific chromosome aberrations [3-7, 61-66].

Fig. 1. (a) Metaphase cell FISH showing two fusions (a classic one in the Ph chromosome and another BCR/ABL fusion in 18p) (arrows), two orange signals (normal and residual ABL probe in 9q34), and one green signal (BCR in the normal 22 chromosome) (94% of cells). (b) Partial FISH metaphase with DAPI/ FITC showing the three copies of the BCR gene (in the Ph chromosome; in 18p, indicated by arrows; and in the normal chromosome 22) (94% of more than 100 cells).

Three cases have been described with additional BCR/ABL1 fusions onto 18p accompanied by other chromosome alterations [67, 68], two of which have been reported by the same authors [68]. In the case described by Tanaka et al. [67], the blast morphology was not specified. Khac et al. [68], however, described two patients with CML in blastic transformation with a duplication and transposition of BCR/ABL1 to 18p that presented loss of part of its short arm; both patients had erythroblastic transformations, which the authors believed to be associated with this particular cytological subtype of disease progression in CML. The present case supports the relationship suggested by Khac et al. [68] between a tendency for erythroblastic transformation and the presence of extra fused BCR/ABL1 onto 18p, as in the patient we described, with a duplication and transposition of BCR/ABL1 onto 18p in the majority line (95%) in blastic phase, with no other chromosome alterations, together with an increase of red cells, intense dyserythropoietic features and PAS in granules leading to a fatal outcome.

#### **3. Case report nº 2**

30 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

studies have assessed the impact of ACA on the clinical efficacy of imatinib by assuming that these abnormalities may confer BCR/ABL1-independent proliferation and decrease

However, the presence of additional copies of the BCR/ABL1 fusion onto structurally abnormal chromosomes has rarely been reported in the chronic or blastic phase of CML

We present two CML patients with additional copies of BCR/ABL1 fusions: the first patient with +Ph and BCR/ABL1 extra fusion in chromosome 18 in the blastic phase, and the

A 34-year-old patient was diagnosed with CML. The hematologic features were: Hb, 96 g; WBC, 354x109/L with 2% blasts, 4% basophils; platelets, 716x109/L. Neutrophil alkaline phosphatase (NAF) activity was absent. The bone marrow aspirate was hypercellular with

A cytogenetic study was performed on bone marrow cells and revealed a 46,XY,t(9;22)(q34;q11) karyotype in all cells analyzed. FISH was performed on chromosome preparations using commercially available probe sets (LSI BCR/ABL-ES probe; Vysis, Downers Grove, IL) and based on a count of 100 cells (metaphases and interphases). The existence of the classic BCR/ABL1 fusion was proved. The patient was treated with hydroxyurea and *a*-interferon, with a satisfactory clinical, but unsatisfactory cytogenetic and FISH response , the maximum response reached, based on a count of 120 cells, was 27/120 cells with the BCR/ABL1 rearrangement during the 4 year follow-up . A bone marrow transplantation could not be performed because no compatible HLA-matched donor was

After the aforementioned, 4 years after the initial diagnosis, a decrease of Hb, hematocrit, and platelets took place in the peripheral blood: WBC was 4.8x109/L with 38% blasts. Bone marrow was hypercellular, with 30% of cells representing the erythroid lineage at all the maturation stages, and with intense dyserythropoietic changes: 21% in the myeloid series (myeloid/erythroid ratio 5:1.5); 9% in the lymphoplasmocytoid series; and 40% as large blasts with fine chromatin in the nucleus, with two nucleoli, and irregularly shaped nongranular cytoplasms. Blast cells gave positive histochemical reactions for peroxidise and naphthol AS-D chloracetate esterase (inhibited by fluoride) and were periodic acid Schiff (PAS) positive as granules. Analysis in bone marrow of more than 100 cells at this time revealed that 94% of the cells had two fused BCR/ABL signals: one on the Ph chromosome and the other on the short arm of a derivative chromosome 18, detected by metaphase FISH analysis with Vysis LSI-BCR/ABL-ES and Cytocell mBCR/ABL probes (Fig. 1). Karyotype in bone marrow samples showed one Ph chromosome and one normal chromosome 22, and one normal chromosome 18 and another with a deletion of part of its short arm. The remaining 6% of cells only had the usual BCR/ABL1 fusion, without any other

The patient was then treated with mitoxantrone and cytarabine without clinical response, and later with splenic radiotherapy, which led to an intense pancytopenia; the patient then

The occurrence of additional chromosome changes is an ominous sign, indicating that illness progression is imminent; however, the etiology of blastic transformation is still not

second patient with four BCR/ABL1 fusion signals at diagnosis **.** 

sensitivity to imatinib [50-55].

**2. Case report nº 1** 

1% blast cells.

available.

abnormalities.

died 4 years 8 months after the initial diagnosis.

patients.

A 31-year-old caucasian man, a smoker (10 cigarettes/day) and with a past appendectomy, presented with a history of abdominal pain with diarrhea and an abscess in the jawbone; he was attended at the Department of Hematology. Clinical examination revealed hepatosplenomegaly. Complementary tests revealed leukocytosis in a peripheral blood examination. His white blood cell count was 52x109/L, with a peripheral monocytosis of 18x109/L and a left shift. The number of platelets was 94x109/L; hemoglobin was normal; leukocyte alkaline phosphatase was reduced. In the biochemical

BCR/ABL1 Extra Fusions in Patients with Chronic Myeloid Leukaemia (CML) 33

p210 BCR-ABL1 fusion transcript, identified using RT-PCR (Biomed protocol), decreased

Fig. 2. (A) Partial metaphase karyogram shows two derivative chromosomes 9 and two derivative chromosomes 22, along with one each of normal chromosomes 9 and 22 in case report nº 2 of chronic myeloid leukemia in chronic phase. (B) For the same patient, interphase FISH with LSI BCR/ABL-ES probes (Vysis; Abbott Molecular, Des Plaines, IL) shows four BCR/ABL1 fusion genes (arrows). Filter: 4', 6-diamidino-2-phenylindole-

A year after diagnosis, in a routine evaluation, the patient was still asymptomatic and in hematological remission. The RT-PCR study performed using FRET probes detected BCR/ABL1 transcripts. A quantification analysis (Biomed protocol) performed then was negative for p210 (b3a2), but p190 (e1a2) was found at 23.3%. Thus, the original sample was analyzed again, in which p190 was also positive. The original imatinib treatment was changed to dasatinib at 100 mg/day due to the disappearance of p210 and the presence of

At diagnosis, the patient in the present case had a karyotype with 48 chromosomes and four BCR/ABL1 fusion signals distributed between the two Ph chromosomes and two out of the three chromosomes 9 in 90% of all the cells analyzed by FISH. One possible explanation could be that the classical BCR/ABL1 fusion occurs initially, followed first by the duplication and translocation of the fusion to a derivative 9 chromosome and then by a duplication of both Ph chromosomes and 9q+ (the remaining chromosomes were unaltered). The possible poor prognostic effect of a BCR/ABL1 fusion located on chromosome 9 has been described [4, 5, 61, 70]. Nevertheless, other researchers have reported cases of patients with a chimerical BCR/ABL1 fusion gene on chromosome 9 in ~80% of CML samples [62] and a double fusion on both chromosomes 9 [71, 72], and the clinical course of the disease seemed unaffected by the positional regulation. In the present case, there was only hematologic remission after treatment. With regard to treatment with imatinib, additional copies of the Ph chromosome have been reported [73, 74]. The development of additional chromosomal aberrations (ACA) in Ph negative or Ph+ cells during treatment is a sign of clonal evolution and of treatment failure. Patients with ACA more frequently tended to have BCR/ABL1 mutations (53% *versus* 29%) and more resistant mutations (60% *versus* 38%) [75]. The acquisition of ACAs may confer resistance to imatinib mesylate a mechanism for imatinib resistance. Therefore, response to imatinib on one hand and resistance on the other may also be conditioned by different types of ACAs. Enhanced response or resistance to treatment may be associated with the occurrence of different cytogenetic or molecular

changes that are able to activate metabolic pathways with different outcomes [76-77].

to 1.66%.

rhodamine-fluoresceinisothiocyanate

p190 in 23.3%.

study (which included lipids and thyroid hormones) increased HDL, vitamin B12 and uric acid were observed; findings of the remaining studies were normal. The thoracoabdominal computed tomography scan revealed mild bilateral axillary adenopathy, right pleural effusion, hepatomegaly, and periaortic adenopathy; the gastroduodenal endoscopy was unremarkable. The patient's bone marrow was described as hypercellular, without visible fat spaces; megakaryocytes were present, normal in both number and morphology. The myeloid/erythroid ratio was 20:1. Myeloid precursors were high in number and left-shifted, with a relative increase in promyelocytes (7%) and eosinophils (6%). The blast percentage was 4%. Erythroid precursors were present in normal numbers.

Cytogenetic analysis with G-banding was performed on chromosomes obtained from 24 hour bone-marrow cell cultures following the standard procedures. Fluorescence in situ hybridization (FISH) was performed on chromosome preparations using a commercially available dual-color LSI BCR/ABL-ES probe (Abbott Molecular, Des Plaines, IL) labelled in SpectrumGreen and SpectrumOrange, respectively, to detect the BCR/ABL1 rearrangements in interphase and metaphase nuclei. To identify the chromosomes 9 correctly, a Vysis whole chromosome paint (WCP 9) was used, labeled in Spectrum- Orange. The hybridization was performed according to the manufacturer's protocols.

Molecular studies using real-time polymerase chain reaction (RT-PCR) from peripheral blood were performed. One microgram of the total RNA was used for cDNA synthesis, and later PCR was performed based on a Biomed protocol and using a LightCycler 2.0 [69] (Roche Diagnostics, Indianapolis, IN; Mannheim, Germany). A RT-PCR study with a fluorescence resonance energy transfer (FRET) probe quantification kit t(9;22) (Roche) that detected BCR/ABL1 fusion transcripts resulting from the major and minor breakpoint cluster regions (M-BCR and m-BCR) was also performed. The primers used in this kit were specifically designed to detect b3a2, b2a2, and e1a2 fusion transcripts, thereby covering >95% of the described t(9;22) translocations.

At diagnosis, cytogenetic studies indicated the following karyotype:

46,XY,der(22)t(9;22)(q34;q11.2)[3]/48,XY,+9,t(9;22)(q34;q11.2)x2,+22[40] (figure 2A).

FISH studies on 167 metaphase and interphase cells revealed the presence of a mosaicism formed by three distinct cellular clones at diagnosis. In the major clone, 150/167 cells (90%) presented four BCR/ABL1 fusion signals, with two signals on the two chromosomes der(9) and the other two signals on the two Ph chromosomes (Fig. 2B and 3). Three chromosomes 9 were observed with a WCP-9 probe. A sole BCR/ABL1 fusion signal, on chromosome 22, was observed in 5/167 cells (3%), and there were no rearrangements in the remaining cells analyzed (12/167, or 7%). The FISH technique was performed twice, confirming the results. The RT-PCR demonstrated a p210, b3a2 fusion transcript.

Once the patient was diagnosed as CML, treatment with imatinib was initiated at a dose of 400 mg/day. Two months later the patient was asymptomatic and in hematological remission. The b3a2 transcripts detected by RT-PCR were at 59%. The patient continued with the same imatinib treatment of 400 mg/day. Ten months after beginning therapy, a FISH follow-up study performed on a bone marrow sample revealed a cellular mosaicism, with partial remission. The initial majority line with four fusions was present in only 20/300 cells, only one BCR/ABL1 rearrangement was seen, on chromosome 22, in 25/300 cells; the remaining 255/300 cells were normal, without any fusion signals. The

study (which included lipids and thyroid hormones) increased HDL, vitamin B12 and uric acid were observed; findings of the remaining studies were normal. The thoracoabdominal computed tomography scan revealed mild bilateral axillary adenopathy, right pleural effusion, hepatomegaly, and periaortic adenopathy; the gastroduodenal endoscopy was unremarkable. The patient's bone marrow was described as hypercellular, without visible fat spaces; megakaryocytes were present, normal in both number and morphology. The myeloid/erythroid ratio was 20:1. Myeloid precursors were high in number and left-shifted, with a relative increase in promyelocytes (7%) and eosinophils (6%). The blast percentage was 4%. Erythroid precursors were present in

Cytogenetic analysis with G-banding was performed on chromosomes obtained from 24 hour bone-marrow cell cultures following the standard procedures. Fluorescence in situ hybridization (FISH) was performed on chromosome preparations using a commercially available dual-color LSI BCR/ABL-ES probe (Abbott Molecular, Des Plaines, IL) labelled in SpectrumGreen and SpectrumOrange, respectively, to detect the BCR/ABL1 rearrangements in interphase and metaphase nuclei. To identify the chromosomes 9 correctly, a Vysis whole chromosome paint (WCP 9) was used, labeled in Spectrum- Orange.

Molecular studies using real-time polymerase chain reaction (RT-PCR) from peripheral blood were performed. One microgram of the total RNA was used for cDNA synthesis, and later PCR was performed based on a Biomed protocol and using a LightCycler 2.0 [69] (Roche Diagnostics, Indianapolis, IN; Mannheim, Germany). A RT-PCR study with a fluorescence resonance energy transfer (FRET) probe quantification kit t(9;22) (Roche) that detected BCR/ABL1 fusion transcripts resulting from the major and minor breakpoint cluster regions (M-BCR and m-BCR) was also performed. The primers used in this kit were specifically designed to detect b3a2, b2a2, and e1a2 fusion transcripts, thereby covering

The hybridization was performed according to the manufacturer's protocols.

At diagnosis, cytogenetic studies indicated the following karyotype:

The RT-PCR demonstrated a p210, b3a2 fusion transcript.

46,XY,der(22)t(9;22)(q34;q11.2)[3]/48,XY,+9,t(9;22)(q34;q11.2)x2,+22[40] (figure 2A).

FISH studies on 167 metaphase and interphase cells revealed the presence of a mosaicism formed by three distinct cellular clones at diagnosis. In the major clone, 150/167 cells (90%) presented four BCR/ABL1 fusion signals, with two signals on the two chromosomes der(9) and the other two signals on the two Ph chromosomes (Fig. 2B and 3). Three chromosomes 9 were observed with a WCP-9 probe. A sole BCR/ABL1 fusion signal, on chromosome 22, was observed in 5/167 cells (3%), and there were no rearrangements in the remaining cells analyzed (12/167, or 7%). The FISH technique was performed twice, confirming the results.

Once the patient was diagnosed as CML, treatment with imatinib was initiated at a dose of 400 mg/day. Two months later the patient was asymptomatic and in hematological remission. The b3a2 transcripts detected by RT-PCR were at 59%. The patient continued with the same imatinib treatment of 400 mg/day. Ten months after beginning therapy, a FISH follow-up study performed on a bone marrow sample revealed a cellular mosaicism, with partial remission. The initial majority line with four fusions was present in only 20/300 cells, only one BCR/ABL1 rearrangement was seen, on chromosome 22, in 25/300 cells; the remaining 255/300 cells were normal, without any fusion signals. The

>95% of the described t(9;22) translocations.

normal numbers.

p210 BCR-ABL1 fusion transcript, identified using RT-PCR (Biomed protocol), decreased to 1.66%.

Fig. 2. (A) Partial metaphase karyogram shows two derivative chromosomes 9 and two derivative chromosomes 22, along with one each of normal chromosomes 9 and 22 in case report nº 2 of chronic myeloid leukemia in chronic phase. (B) For the same patient, interphase FISH with LSI BCR/ABL-ES probes (Vysis; Abbott Molecular, Des Plaines, IL) shows four BCR/ABL1 fusion genes (arrows). Filter: 4', 6-diamidino-2-phenylindolerhodamine-fluoresceinisothiocyanate

A year after diagnosis, in a routine evaluation, the patient was still asymptomatic and in hematological remission. The RT-PCR study performed using FRET probes detected BCR/ABL1 transcripts. A quantification analysis (Biomed protocol) performed then was negative for p210 (b3a2), but p190 (e1a2) was found at 23.3%. Thus, the original sample was analyzed again, in which p190 was also positive. The original imatinib treatment was changed to dasatinib at 100 mg/day due to the disappearance of p210 and the presence of p190 in 23.3%.

At diagnosis, the patient in the present case had a karyotype with 48 chromosomes and four BCR/ABL1 fusion signals distributed between the two Ph chromosomes and two out of the three chromosomes 9 in 90% of all the cells analyzed by FISH. One possible explanation could be that the classical BCR/ABL1 fusion occurs initially, followed first by the duplication and translocation of the fusion to a derivative 9 chromosome and then by a duplication of both Ph chromosomes and 9q+ (the remaining chromosomes were unaltered). The possible poor prognostic effect of a BCR/ABL1 fusion located on chromosome 9 has been described [4, 5, 61, 70]. Nevertheless, other researchers have reported cases of patients with a chimerical BCR/ABL1 fusion gene on chromosome 9 in ~80% of CML samples [62] and a double fusion on both chromosomes 9 [71, 72], and the clinical course of the disease seemed unaffected by the positional regulation. In the present case, there was only hematologic remission after treatment. With regard to treatment with imatinib, additional copies of the Ph chromosome have been reported [73, 74]. The development of additional chromosomal aberrations (ACA) in Ph negative or Ph+ cells during treatment is a sign of clonal evolution and of treatment failure. Patients with ACA more frequently tended to have BCR/ABL1 mutations (53% *versus* 29%) and more resistant mutations (60% *versus* 38%) [75]. The acquisition of ACAs may confer resistance to imatinib mesylate a mechanism for imatinib resistance. Therefore, response to imatinib on one hand and resistance on the other may also be conditioned by different types of ACAs. Enhanced response or resistance to treatment may be associated with the occurrence of different cytogenetic or molecular changes that are able to activate metabolic pathways with different outcomes [76-77].

BCR/ABL1 Extra Fusions in Patients with Chronic Myeloid Leukaemia (CML) 35

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Fig. 3. Metaphase FISH with Vysis LSI BCR/ABLeES probes shows four BCR/ABL fusion signals (yellow): two signals on the two der(9) chromosomes and two signals on the two der(22) chromosomes. The green and orange signals indicate, respectively, the BCR gene on the normal chromosome 22 and the ABL gene on the normal chromosome 9.

In the present case, all the cytogenetic aberrations began to decrease with imatinib treatment, and only a small percentage of cells maintained the initial karyotype.

At the molecular level, the patient showed a p210 and p190 fusion. A p210 decrease coincided with an important partial cytogenetical remission of the four BCR/ABL1 fusions. During evolution of the disease, the p210 protein disappeared with imatinib as first-line treatment, but p190 increased until treatment with dasatinib was started.

Five months after the start of treatment with dasatinib, RT-PCR showed that p210 remained negative and p190 had decreased to 1%. Two years after diagnosis, the patient was still clinically asymptomatic and hematologically in remission. New FISH studies, with 200 cells analyzed, also showed complete cytogenetic remission. Molecular studies remained negative for p210 and positive for p190 in only 0.09%.

Although interphase FISH is increasingly used for BCR/ABL1 gene rearrangement identification in CML, we believe, in agreement with Lim et al. [72] and Primo et al. [79] and supported by the present case, that the exact interpretation of any atypical interphase FISH pattern is dependent on FISH metaphase studies and molecular breakpoint definition of BCR/ABL1 fusion.

#### **4. References**


Fig. 3. Metaphase FISH with Vysis LSI BCR/ABLeES probes shows four BCR/ABL fusion signals (yellow): two signals on the two der(9) chromosomes and two signals on the two der(22) chromosomes. The green and orange signals indicate, respectively, the BCR gene on

In the present case, all the cytogenetic aberrations began to decrease with imatinib

At the molecular level, the patient showed a p210 and p190 fusion. A p210 decrease coincided with an important partial cytogenetical remission of the four BCR/ABL1 fusions. During evolution of the disease, the p210 protein disappeared with imatinib as first-line

Five months after the start of treatment with dasatinib, RT-PCR showed that p210 remained negative and p190 had decreased to 1%. Two years after diagnosis, the patient was still clinically asymptomatic and hematologically in remission. New FISH studies, with 200 cells analyzed, also showed complete cytogenetic remission. Molecular studies remained

Although interphase FISH is increasingly used for BCR/ABL1 gene rearrangement identification in CML, we believe, in agreement with Lim et al. [72] and Primo et al. [79] and supported by the present case, that the exact interpretation of any atypical interphase FISH pattern is dependent on FISH metaphase studies and molecular breakpoint definition of

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**3** 

*USA* 

**Causative Factors Involved in Development** 

**of Resistance to Tyrosine Kinase Inhibition** 

This article describes original work performed in our laboratory related to the characterization of kinase inhibitors developed to treat myeloid leukemia, as well as identified mechanisms that likely contribute to resistance to kinase inhibition. Specifically, we provide detailed overviews of the kinase inhibitors nilotinib, used in the treatment of chronic myeloid leukemia (CML) and midostaurin, which is currently under investigation in late stage clinical trials for the treatment of mutant FLT3-positive acute myeloid leukemia (AML). Presented as well is a description of studies investigating strategies that leukemic cells employ to escape killing through kinase inhibition, including alterations in the expression and composition of targeted proteins and chemoresistance conferred by the bone marrow microenvironment. We provide a general overview of kinase inhibitors in preclinical and clinical development for CML and AML, and also discuss the use of combination therapy as an approach to enhancing the efficacy of tyrosine kinase inhibition

**2. Development of BCR-ABL inhibitors and combination therapy studies** 

The development of resistance in leukemia patients to treatment with targeted tyrosine kinase inhibitors is a growing area of concern. For instance, the ABL inhibitor imatinib (STI571) (Druker et al., 1996; Buchdunger et al., 2001), which also inhibits KIT and plateletderived growth factor receptor (PDGFR), has proven to be a highly effective, front line therapy for CML, a hematopoietic malignancy caused by the product of a reciprocal t(9;22) chromosomal translocation, *BCR-ABL* (Deininger et al., 2000). This targeted therapy has changed how newly diagnosed CML patients are treated and has substantially improved their prognosis. However, patients in the more advance phases of CML (namely, accelerated or blast crisis phase CML patients) often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase

**geared toward overriding resistance to BCR-ABL inhibition** 

**1. Introduction** 

and reducing the incidence of residual disease.

*Imatinib resistance: Introduction* 

**and Novel Strategies Designed** 

**to Override This Resistance** 

Ellen Weisberg and James D. Griffin

*Dana Farber Cancer Institute* 

Philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications. Cancer Genet Cytogenet. 2010 Jun; 199 (2): 76-80.


### **Causative Factors Involved in Development of Resistance to Tyrosine Kinase Inhibition and Novel Strategies Designed to Override This Resistance**

Ellen Weisberg and James D. Griffin *Dana Farber Cancer Institute USA* 

#### **1. Introduction**

40 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

[78] Kim TD, Türkmen S, Schwarz M, Koca G, Nogai H, Bommer C, Dörken B, Daniel P, le

positive chronic myeloid leukemia. Haematologica. 2010; 95 (4): 582-8. [79] Primo D, Tabernero MD, Rasillo A, Sayague´s JM, Espinosa AB, Chillon MC, Garcia-

(2): 76-80.

Leukemia 2003; 17:1124-9.

Philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications. Cancer Genet Cytogenet. 2010 Jun; 199

Coutre P Impact of additional chromosomal aberrations and BCR-ABL kinase domain mutations on the response to nilotinib in Philadelphia chromosome-

Sanz R, Gutierrez N, Giralt M, Hagemeijer A, San Miguel JF, Orfao A. Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic abnormalities.

> This article describes original work performed in our laboratory related to the characterization of kinase inhibitors developed to treat myeloid leukemia, as well as identified mechanisms that likely contribute to resistance to kinase inhibition. Specifically, we provide detailed overviews of the kinase inhibitors nilotinib, used in the treatment of chronic myeloid leukemia (CML) and midostaurin, which is currently under investigation in late stage clinical trials for the treatment of mutant FLT3-positive acute myeloid leukemia (AML). Presented as well is a description of studies investigating strategies that leukemic cells employ to escape killing through kinase inhibition, including alterations in the expression and composition of targeted proteins and chemoresistance conferred by the bone marrow microenvironment. We provide a general overview of kinase inhibitors in preclinical and clinical development for CML and AML, and also discuss the use of combination therapy as an approach to enhancing the efficacy of tyrosine kinase inhibition and reducing the incidence of residual disease.

#### **2. Development of BCR-ABL inhibitors and combination therapy studies geared toward overriding resistance to BCR-ABL inhibition**

#### *Imatinib resistance: Introduction*

The development of resistance in leukemia patients to treatment with targeted tyrosine kinase inhibitors is a growing area of concern. For instance, the ABL inhibitor imatinib (STI571) (Druker et al., 1996; Buchdunger et al., 2001), which also inhibits KIT and plateletderived growth factor receptor (PDGFR), has proven to be a highly effective, front line therapy for CML, a hematopoietic malignancy caused by the product of a reciprocal t(9;22) chromosomal translocation, *BCR-ABL* (Deininger et al., 2000). This targeted therapy has changed how newly diagnosed CML patients are treated and has substantially improved their prognosis. However, patients in the more advance phases of CML (namely, accelerated or blast crisis phase CML patients) often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase

Causative Factors Involved in Development of Resistance to Tyrosine Kinase

3. Kinase inhibitor binds at sites distal from the ATP binding site.

imatinib therapy (Roche-Lestienne et al., 2002; Hofmann et al., 2003).

kinase activity (Liu and Gray et al., 2006):

terminus, of the activation loop) .

the "DFG-out" conformation.

indirect mechanism.

through an indirect mechanism.

Pluk et al., 2002).

Inhibition and Novel Strategies Designed to Override This Resistance 43

inhibitor binding modes that represent main mechanisms for pharmacological inhibition of

1. Kinase inhibitor directly competes with ATP in the ATP binding site. Inhibitors of this type are known as "Type I" and bind to the "Asp-Phe-Gly (DFG)-in" conformation (the "DFG" motif is highly conserved in protein kinases and sits near the beginning, or N-

2. Kinase inhibitor engages an adjacent allosteric binding site, typically accessible when the activation loop and/or alpha-c helix is folded away from the binding site, which leads to kinase inactivation. Inhibitors of this type are known as "Type II" and bind to

Imatinib, a "Type II" inhibitor, binds to the catalytically inactive conformation of the ABL kinase domain. BCR-ABL point mutations that destabilize the inactive conformations of the DFG motif diminish the binding capacity of imatinib, and therefore work through an

Over fifty different BCR-ABL point mutations have been identified (Rourmiantsev et al., 2002; Von Bubnoff et al., 2002; Shah et al., 2002; Hochhaus et al., 2002) and generally fall into different categorical regions that include the hinge region (which bridges the C- and Nterminal lobes of the kinase domain to develop the ATP-binding cleft), A-loop, and P-loop (or "nucleotide-binding loop"). The position of the A-loop, which includes amino acids 381- 402, regulates ABL kinase activity. Mutations in the A-loop typically confer moderate resistance. In contrast, P-loop mutations, which occur at the binding site for phosphates of ATP, include amino acids 244-255 of ABL, are highly imatinib-resistant, and confer a worse prognosis than other mutations (Soverini et al., 2005). Point mutations in the ABL kinase domain that destabilize the inactive conformations of the P-loop increase the free energy of the imatinib-ABL drug-protein complex, and in effect diminish imatinib binding. Gly250, Tyr 253, and Glu255 in the P-loop are examples of mutations that reduce imatinib binding

The vast majority of the BCR-ABL point mutations identified occur infrequently. Mutations such as those occurring at residues Thr315, Phe359, Met351, Gly250, Glu255, and Tyr253 comprise up to 70% of all existing mutations. It is possible that extremely rare levels of BCR-ABL point mutations pre-exist treatment with imatinib and increase in frequency during

In addition to detection of BCR-ABL point mutations in patients, BCR-ABL point mutations characterized as conferring imatinib resistance have been identified through *in vitro* random mutagenesis of BCR-ABL (Shah et al., 2002; Hochhaus et al., 2002). Some identified mutations were found to exist distal to the kinase domain, including the Src homology 2 (SH2) and SH3 domains and N-terminal cap region (Hochhaus et al., 2002; Azam et al., 2003;

The detection of a point mutation in the BCR-ABL kinase domain was first reported in imatinib-resistant CML patients: three out of nine patients were characterized by *BCR-ABL*  gene amplification, while six out of nine patients were characterized by an isoleucine substitution in the Thr315 residue located at the periphery of the nucleotide-binding site of ABL (also referred to as the "gatekeeper position") (Gorre et al., 2001). The Thr315 residue, located in the hinge region, participates through a hydroxymethylene side chain in a critical H-bond interaction between imatinib and its protein target (Schindler et al., 2000; Manley et al., 2002; Nagar et al., 2002). Mutation of threonine to isoleucine confers resistance to

domain that reduce sensitivity towards imatinib (Gorre et al., 2001) or amplification of the target gene (Weisberg and Griffin, 2000).

Imatinib resistance and disease relapse in some patients can also be due to BCR-ABLindependent mechanisms, as exemplified by the small residual pool of BCR-ABL-positive quiescent leukemic stem cells existing in CML patients that have achieved complete responses after imatinib treatment. This inability of imatinib to completely eradicate quiescent leukaemic stem cells occurs despite the high expression of BCR-ABL mRNA and protein in these cells (Michor et al., 2005; Roeder et al., 2006; Copland et al., 2006), which suggests that these leukemic stem cells are not dependent on BCR-ABL for viability. Therefore, a population of BCR-ABL-positive, quiescent stem cells continues to exist after imatinib therapy even in patients who have achieved complete responses, and the insensitivity of these cells to imatinib is believed to contribute to relapse observed in some patients following termination of imatinib treatment. The existence of this quiescent cell population is important to consider when attempting development of effective therapies for overriding intrinsic kinase inhibitor resistance and resistance due to stromal-mediated mechanisms. Such novel treatment approaches must be able to eradicate this cell population if maximum clinical benefit and complete disease-free survival is to be achieved.

Among strategies to override imatinib resistance are the direct targeting of the BCR-ABL protein and the targeting of mediators of signaling pathways downstream of BCR-ABL that are needed for transformation. Biorational drug design has led to the development of novel agents that overcome some forms of imatinib resistance, and impressive results obtained in clinical trials have led to rapid FDA approval of second generation compounds that are presently used to treat newly diagnosed CML patients.

#### *BCR-ABL point mutations as a mechanism of imatinib resistance*

Although early studies based on cell line models pointed toward *BCR-ABL* gene amplification and over-expression of BCR-ABL mRNA and protein as contributing to imatinib resistance (Weisberg and Griffin, 2000; Mahon et al., 2000; le Coutre et al., 2000), the most common mechanism of imatinib resistance was later discovered to be point mutations in the *BCR-ABL* gene that lead to changes in amino acids in the BCR-ABL protein catalytic domain. Of the mechanisms considered to be of major importance in the development of drug resistance, gene amplification accounts for approximately 10% of relapse in patients, whereas mutations in the kinase domain account for 40-90% of relapse in patients. Other important mechanisms include mutations or activation of other genes (i.e. Lyn) (Wu et al., 2008) and the contribution of organic cation transporter 1 or other related drug pumps (Engler et al., 2010). Mechanisms of minor importance or uncertain significance include the influence of multidrug resistance 1 gene (Gambacorti-Passerini et al., 2000) and alpha1 acidic glycoprotein (Mahon et al., 2000).

BCR-ABL point mutations, which diminish the binding of imatinib to its target, are often those that directly or indirectly impair the binding of imatinib to the BCR-ABL protein without compromising the function of the ABL tyrosine kinase domain. Point mutations that reduce imatinib binding through a direct mechanism are generally positioned around the imatinib binding site, and diminish imatinib binding through alterations in amino acid side chains or topographical changes that sterically hinder the binding of imatinib. Phe317 and Thr315 are examples of this type of BCR-ABL point mutation (Cowan-Jacob et al., 2004). Point mutations that reduce imatinib binding through an indirect mechanism utilize the distinct binding mode of a drug to BCR-ABL. Generally, there are three primary kinase

domain that reduce sensitivity towards imatinib (Gorre et al., 2001) or amplification of the

Imatinib resistance and disease relapse in some patients can also be due to BCR-ABLindependent mechanisms, as exemplified by the small residual pool of BCR-ABL-positive quiescent leukemic stem cells existing in CML patients that have achieved complete responses after imatinib treatment. This inability of imatinib to completely eradicate quiescent leukaemic stem cells occurs despite the high expression of BCR-ABL mRNA and protein in these cells (Michor et al., 2005; Roeder et al., 2006; Copland et al., 2006), which suggests that these leukemic stem cells are not dependent on BCR-ABL for viability. Therefore, a population of BCR-ABL-positive, quiescent stem cells continues to exist after imatinib therapy even in patients who have achieved complete responses, and the insensitivity of these cells to imatinib is believed to contribute to relapse observed in some patients following termination of imatinib treatment. The existence of this quiescent cell population is important to consider when attempting development of effective therapies for overriding intrinsic kinase inhibitor resistance and resistance due to stromal-mediated mechanisms. Such novel treatment approaches must be able to eradicate this cell population

if maximum clinical benefit and complete disease-free survival is to be achieved.

presently used to treat newly diagnosed CML patients. *BCR-ABL point mutations as a mechanism of imatinib resistance* 

acidic glycoprotein (Mahon et al., 2000).

Among strategies to override imatinib resistance are the direct targeting of the BCR-ABL protein and the targeting of mediators of signaling pathways downstream of BCR-ABL that are needed for transformation. Biorational drug design has led to the development of novel agents that overcome some forms of imatinib resistance, and impressive results obtained in clinical trials have led to rapid FDA approval of second generation compounds that are

Although early studies based on cell line models pointed toward *BCR-ABL* gene amplification and over-expression of BCR-ABL mRNA and protein as contributing to imatinib resistance (Weisberg and Griffin, 2000; Mahon et al., 2000; le Coutre et al., 2000), the most common mechanism of imatinib resistance was later discovered to be point mutations in the *BCR-ABL* gene that lead to changes in amino acids in the BCR-ABL protein catalytic domain. Of the mechanisms considered to be of major importance in the development of drug resistance, gene amplification accounts for approximately 10% of relapse in patients, whereas mutations in the kinase domain account for 40-90% of relapse in patients. Other important mechanisms include mutations or activation of other genes (i.e. Lyn) (Wu et al., 2008) and the contribution of organic cation transporter 1 or other related drug pumps (Engler et al., 2010). Mechanisms of minor importance or uncertain significance include the influence of multidrug resistance 1 gene (Gambacorti-Passerini et al., 2000) and alpha1

BCR-ABL point mutations, which diminish the binding of imatinib to its target, are often those that directly or indirectly impair the binding of imatinib to the BCR-ABL protein without compromising the function of the ABL tyrosine kinase domain. Point mutations that reduce imatinib binding through a direct mechanism are generally positioned around the imatinib binding site, and diminish imatinib binding through alterations in amino acid side chains or topographical changes that sterically hinder the binding of imatinib. Phe317 and Thr315 are examples of this type of BCR-ABL point mutation (Cowan-Jacob et al., 2004). Point mutations that reduce imatinib binding through an indirect mechanism utilize the distinct binding mode of a drug to BCR-ABL. Generally, there are three primary kinase

target gene (Weisberg and Griffin, 2000).

inhibitor binding modes that represent main mechanisms for pharmacological inhibition of kinase activity (Liu and Gray et al., 2006):


Imatinib, a "Type II" inhibitor, binds to the catalytically inactive conformation of the ABL kinase domain. BCR-ABL point mutations that destabilize the inactive conformations of the DFG motif diminish the binding capacity of imatinib, and therefore work through an indirect mechanism.

Over fifty different BCR-ABL point mutations have been identified (Rourmiantsev et al., 2002; Von Bubnoff et al., 2002; Shah et al., 2002; Hochhaus et al., 2002) and generally fall into different categorical regions that include the hinge region (which bridges the C- and Nterminal lobes of the kinase domain to develop the ATP-binding cleft), A-loop, and P-loop (or "nucleotide-binding loop"). The position of the A-loop, which includes amino acids 381- 402, regulates ABL kinase activity. Mutations in the A-loop typically confer moderate resistance. In contrast, P-loop mutations, which occur at the binding site for phosphates of ATP, include amino acids 244-255 of ABL, are highly imatinib-resistant, and confer a worse prognosis than other mutations (Soverini et al., 2005). Point mutations in the ABL kinase domain that destabilize the inactive conformations of the P-loop increase the free energy of the imatinib-ABL drug-protein complex, and in effect diminish imatinib binding. Gly250, Tyr 253, and Glu255 in the P-loop are examples of mutations that reduce imatinib binding through an indirect mechanism.

The vast majority of the BCR-ABL point mutations identified occur infrequently. Mutations such as those occurring at residues Thr315, Phe359, Met351, Gly250, Glu255, and Tyr253 comprise up to 70% of all existing mutations. It is possible that extremely rare levels of BCR-ABL point mutations pre-exist treatment with imatinib and increase in frequency during imatinib therapy (Roche-Lestienne et al., 2002; Hofmann et al., 2003).

In addition to detection of BCR-ABL point mutations in patients, BCR-ABL point mutations characterized as conferring imatinib resistance have been identified through *in vitro* random mutagenesis of BCR-ABL (Shah et al., 2002; Hochhaus et al., 2002). Some identified mutations were found to exist distal to the kinase domain, including the Src homology 2 (SH2) and SH3 domains and N-terminal cap region (Hochhaus et al., 2002; Azam et al., 2003; Pluk et al., 2002).

The detection of a point mutation in the BCR-ABL kinase domain was first reported in imatinib-resistant CML patients: three out of nine patients were characterized by *BCR-ABL*  gene amplification, while six out of nine patients were characterized by an isoleucine substitution in the Thr315 residue located at the periphery of the nucleotide-binding site of ABL (also referred to as the "gatekeeper position") (Gorre et al., 2001). The Thr315 residue, located in the hinge region, participates through a hydroxymethylene side chain in a critical H-bond interaction between imatinib and its protein target (Schindler et al., 2000; Manley et al., 2002; Nagar et al., 2002). Mutation of threonine to isoleucine confers resistance to

Causative Factors Involved in Development of Resistance to Tyrosine Kinase

O'Hare et al., 2005; Burgess et al., 2005).

*Other BCR-ABL inhibitors* 

mutation (Giles et al., 2007).

*vitro* and *in vivo* (Choi et al., 2010).

al., 2006).

Inhibition and Novel Strategies Designed to Override This Resistance 45

point mutants, with the notable exception of the T315I gatekeeper mutant (Shah et al., 2004;

Dasatinib received accelerated approval by the FDA in June 2006 and the European Medicines Agency (EMEA) in November 2006 for treatment of adults in all phase of CML with resistance or intolerance to imatinib treatment. This approval was based on positive results in early (Phase I and II) clinical trials (Talpaz et al., 2006; Cortes et al., 2007). Dasatinib was also approved for treatment of drug-resistant Ph+ acute lymphoblastic leukemia (ALL) patients, in which a 190 kDa BCR-ABL protein is expressed resulting from an alternative breakpoint in the *BCR* gene. As with nilotinib, dasatinib has been granted

Dual ABL and Src inhibitors, bosutinib (SKI-606) (Puttini et al., 2006; Golas et al., 2003), which is a Type I ATP competitive inhibitor that only occupies the region normally occupied by ATP, and INNO-406 (first developed as NS-187) (Kimura et al., 2005), are active against a panel of imatinib-resistant BCR-ABL point mutants with the exception of the T315I gatekeeper mutant. AZD0530 is another dual ABL and Src inhibitor and Type I ATP competitive inhibitor that only weakly inhibits BCR-ABL (Lockton et al., 2005; Hennequin et

Aurora kinase inhibitors, which work through inhibition of the Aurora family of serine/threonine kinases, members of which are important for mitotic progression, represent another class of second generation inhibitors of BCR-ABL. Examples include PHA-739358 (Nerviano Medical Sciences) (Fancelli et al., 2006), which has been tested in early clinical trials in patients that relapsed after treatment with imatinib. PHA-739358 is a Type I inhibitor that binds the ATP binding site directly. Another example of an Aurora kinase inhibitor is MK-0457 (first developed as VX-680), which inhibits the proliferation of cells expressing the T315I-BCR-ABL gatekeeper mutant at submicromolar concentrations (Carter et al., 2005). MK-0457 has shown activity in some patients harboring the T315I

Non-ATP-competitive BCR-ABL inhibitors represent another class of second generation BCR-ABL inhibitors that work either via a non-ATP competitive allosteric mechanism or by prevention of substrate binding. Examples include ON012380, which shows potent activity against BCR-ABL-positive cells (Gumireddy et al., 2005). Compounds of this class make a covalent bond to the kinase and act as irreversible inhibitors. One complication is the fact that these compounds bind the substrate binding sites of multiple kinases, and therefore are very cytotoxic. Consequently, no clinical compound has been derived from this series. Non-ATP competitive inhibitors are also exemplified by the GNF2 and GNF5 family of inhibitors, which bind to the myristate binding site of BCR-ABL and inhibit its activity by stabilizing a catalytically less competent conformation of the protein (Adrian et al., 2006; Zhang et al., 2010). In addition, a type II inhibitor, GNF-7, has been shown to inhibit the T315I mutant *in* 

As an approach to overriding imatinib resistance, we developed and characterized HG-7-85- 01, which is a Type II ATP competitive inhibitor that opens up a new chemical space in the field of kinase inhibitor development (Weisberg et al., 2010a). The structural inspiration for HG-7-85-01 is an amalgam of dasatinib and nilotinib, and is mostly utilization of the Type II nilotinib binding mode. Part of the structure of HG-7-85-01 binds to the ATP binding site, while the other part of its structure binds adjacent to the ATP binding site. In addition to

FDA approval for therapy of newly diagnosed CML patients (Kantarjian et al, 2010).

imatinib because of direct steric intrusion of the bulky isoleucine side chain, as well as loss of the hydrogen-bonding interaction in the ATP-cleft. An independent study of the crystal structure of an imatinib analog bound to ABL had shown Thr315 to be critical for the binding of imatinib to ABL, and the mutation of threonine to valine at this residue (T315V) was observed to confer imatinib resistance (Schindler et al., 2000; Corbin et al., 2002).

*Development of second generation BCR-ABL inhibitors designed to override imatinib resistance Nilotinib* 

As an approach to overriding imatinib resistance, we characterized and developed the phenylaminopyrimidine-based Type II ATP competitive inhibitor, nilotinib (NVP-AMN107-NX; 4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[[4- 3-pyridinyl)-2-pyrimidinyl]amino]benzamide), based on the crystal structures of inhibitors complexed with ABL (Cowan-Jacob et al., 2004). As with imatinib, another Type II ATP competitive inhibitor, part of nilotinib's structure binds the ATP binding site, while part of its structure binds adjacent to the ATP binding site. Nilotinib was designed to fit into the ATP-binding site of BCR-ABL with higher affinity than imatinib, and demonstrates significantly higher potency than imatinib in inducing apoptosis of BCR-ABL-expressing cells (IC50<10 nM) by directly inhibiting ABL tyrosine kinase (Weisberg et al., 2005). Nilotinib also shows activity against a panel of imatinib-resistant BCR-ABL mutants, with the exception of T315I, and is effective in preventing the onset of leukemia in a BCR-ABL+ leukemic mouse model, and in prolonging the survival of mice harboring imatinib-resistant BCR-ABL-expressing cells (Weisberg et al., 2005; O'Hare et al., 2005). Crystallographic analysis of nilotinib complexed with ABL shows that, like imatinib, nilotinib binds to the inactive conformation of ABL. This suggests that the superior topographical fit to the ABL protein, as well as differences in the binding nature of nilotinib to ABL, contribute to the higher potency of nilotinib versus imatinib.

Clinical trial results with nilotinib in imatinib-resistant patients were favorable (Kantarjian et al., 2006; le Coutre et al., 2006; Ottmann et al., 2006). A phase III randomized, open-label multicenter study was conducted as a direct comparison of imatinib and nilotinib in CML patients. Results showed nilotinib to be superior to imatinib in patients with newly diagnosed chronic phase CML (Saglio et al., 2010). On October 29, 2007, the FDA granted accelerated approval to nilotinib for use in the treatment of chronic phase and accelerated phase Philadelphia chromosome positive (Ph+) CML in adult patients resistant or intolerant to prior therapy that included imatinib. On June 17, 2010, the FDA granted accelerated approval to nilotinib for treatment of adult patients with newly diagnosed Ph+ CML in chronic phase.

#### *Dasatinib*

Dasatinib (BMS-354825) is a second generation Type I ATP competitive inhibitor of BCR-ABL that is both a potent ABL inhibitor and a potent Src family kinase inhibitor (Shah et al., 2004; O'Hare et al., 2005; Burgess et al., 2005; Das et al, 2006; Melnick et al., 2006). Overexpression and activation of Src family kinases, such as LYN and HCK, have been shown to be associated with imatinib resistance (Donato et al., 2003), which suggests that the simultaneous targeting of BCR-ABL and Src family kinases may be effective in overriding resistance to imatinib. Unlike imatinib and nilotinib, which bind to the inactive conformation of BCR-ABL, dasatinib binds to the active conformation (Tokarski et al., 2006). Similar to nilotinib, dasatinib is effective against a number of imatinib-resistant BCR-ABL point mutants, with the notable exception of the T315I gatekeeper mutant (Shah et al., 2004; O'Hare et al., 2005; Burgess et al., 2005).

Dasatinib received accelerated approval by the FDA in June 2006 and the European Medicines Agency (EMEA) in November 2006 for treatment of adults in all phase of CML with resistance or intolerance to imatinib treatment. This approval was based on positive results in early (Phase I and II) clinical trials (Talpaz et al., 2006; Cortes et al., 2007). Dasatinib was also approved for treatment of drug-resistant Ph+ acute lymphoblastic leukemia (ALL) patients, in which a 190 kDa BCR-ABL protein is expressed resulting from an alternative breakpoint in the *BCR* gene. As with nilotinib, dasatinib has been granted FDA approval for therapy of newly diagnosed CML patients (Kantarjian et al, 2010).

#### *Other BCR-ABL inhibitors*

44 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

imatinib because of direct steric intrusion of the bulky isoleucine side chain, as well as loss of the hydrogen-bonding interaction in the ATP-cleft. An independent study of the crystal structure of an imatinib analog bound to ABL had shown Thr315 to be critical for the binding of imatinib to ABL, and the mutation of threonine to valine at this residue (T315V)

As an approach to overriding imatinib resistance, we characterized and developed the phenylaminopyrimidine-based Type II ATP competitive inhibitor, nilotinib (NVP-AMN107-NX; 4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-[[4- 3-pyridinyl)-2-pyrimidinyl]amino]benzamide), based on the crystal structures of inhibitors complexed with ABL (Cowan-Jacob et al., 2004). As with imatinib, another Type II ATP competitive inhibitor, part of nilotinib's structure binds the ATP binding site, while part of its structure binds adjacent to the ATP binding site. Nilotinib was designed to fit into the ATP-binding site of BCR-ABL with higher affinity than imatinib, and demonstrates significantly higher potency than imatinib in inducing apoptosis of BCR-ABL-expressing cells (IC50<10 nM) by directly inhibiting ABL tyrosine kinase (Weisberg et al., 2005). Nilotinib also shows activity against a panel of imatinib-resistant BCR-ABL mutants, with the exception of T315I, and is effective in preventing the onset of leukemia in a BCR-ABL+ leukemic mouse model, and in prolonging the survival of mice harboring imatinib-resistant BCR-ABL-expressing cells (Weisberg et al., 2005; O'Hare et al., 2005). Crystallographic analysis of nilotinib complexed with ABL shows that, like imatinib, nilotinib binds to the inactive conformation of ABL. This suggests that the superior topographical fit to the ABL protein, as well as differences in the binding nature of nilotinib to ABL, contribute to the

Clinical trial results with nilotinib in imatinib-resistant patients were favorable (Kantarjian et al., 2006; le Coutre et al., 2006; Ottmann et al., 2006). A phase III randomized, open-label multicenter study was conducted as a direct comparison of imatinib and nilotinib in CML patients. Results showed nilotinib to be superior to imatinib in patients with newly diagnosed chronic phase CML (Saglio et al., 2010). On October 29, 2007, the FDA granted accelerated approval to nilotinib for use in the treatment of chronic phase and accelerated phase Philadelphia chromosome positive (Ph+) CML in adult patients resistant or intolerant to prior therapy that included imatinib. On June 17, 2010, the FDA granted accelerated approval to nilotinib for treatment of adult patients with newly diagnosed Ph+ CML in

Dasatinib (BMS-354825) is a second generation Type I ATP competitive inhibitor of BCR-ABL that is both a potent ABL inhibitor and a potent Src family kinase inhibitor (Shah et al., 2004; O'Hare et al., 2005; Burgess et al., 2005; Das et al, 2006; Melnick et al., 2006). Overexpression and activation of Src family kinases, such as LYN and HCK, have been shown to be associated with imatinib resistance (Donato et al., 2003), which suggests that the simultaneous targeting of BCR-ABL and Src family kinases may be effective in overriding resistance to imatinib. Unlike imatinib and nilotinib, which bind to the inactive conformation of BCR-ABL, dasatinib binds to the active conformation (Tokarski et al., 2006). Similar to nilotinib, dasatinib is effective against a number of imatinib-resistant BCR-ABL

was observed to confer imatinib resistance (Schindler et al., 2000; Corbin et al., 2002). *Development of second generation BCR-ABL inhibitors designed to override imatinib resistance* 

*Nilotinib* 

chronic phase. *Dasatinib* 

higher potency of nilotinib versus imatinib.

Dual ABL and Src inhibitors, bosutinib (SKI-606) (Puttini et al., 2006; Golas et al., 2003), which is a Type I ATP competitive inhibitor that only occupies the region normally occupied by ATP, and INNO-406 (first developed as NS-187) (Kimura et al., 2005), are active against a panel of imatinib-resistant BCR-ABL point mutants with the exception of the T315I gatekeeper mutant. AZD0530 is another dual ABL and Src inhibitor and Type I ATP competitive inhibitor that only weakly inhibits BCR-ABL (Lockton et al., 2005; Hennequin et al., 2006).

Aurora kinase inhibitors, which work through inhibition of the Aurora family of serine/threonine kinases, members of which are important for mitotic progression, represent another class of second generation inhibitors of BCR-ABL. Examples include PHA-739358 (Nerviano Medical Sciences) (Fancelli et al., 2006), which has been tested in early clinical trials in patients that relapsed after treatment with imatinib. PHA-739358 is a Type I inhibitor that binds the ATP binding site directly. Another example of an Aurora kinase inhibitor is MK-0457 (first developed as VX-680), which inhibits the proliferation of cells expressing the T315I-BCR-ABL gatekeeper mutant at submicromolar concentrations (Carter et al., 2005). MK-0457 has shown activity in some patients harboring the T315I mutation (Giles et al., 2007).

Non-ATP-competitive BCR-ABL inhibitors represent another class of second generation BCR-ABL inhibitors that work either via a non-ATP competitive allosteric mechanism or by prevention of substrate binding. Examples include ON012380, which shows potent activity against BCR-ABL-positive cells (Gumireddy et al., 2005). Compounds of this class make a covalent bond to the kinase and act as irreversible inhibitors. One complication is the fact that these compounds bind the substrate binding sites of multiple kinases, and therefore are very cytotoxic. Consequently, no clinical compound has been derived from this series. Non-ATP competitive inhibitors are also exemplified by the GNF2 and GNF5 family of inhibitors, which bind to the myristate binding site of BCR-ABL and inhibit its activity by stabilizing a catalytically less competent conformation of the protein (Adrian et al., 2006; Zhang et al., 2010). In addition, a type II inhibitor, GNF-7, has been shown to inhibit the T315I mutant *in vitro* and *in vivo* (Choi et al., 2010).

As an approach to overriding imatinib resistance, we developed and characterized HG-7-85- 01, which is a Type II ATP competitive inhibitor that opens up a new chemical space in the field of kinase inhibitor development (Weisberg et al., 2010a). The structural inspiration for HG-7-85-01 is an amalgam of dasatinib and nilotinib, and is mostly utilization of the Type II nilotinib binding mode. Part of the structure of HG-7-85-01 binds to the ATP binding site, while the other part of its structure binds adjacent to the ATP binding site. In addition to

Causative Factors Involved in Development of Resistance to Tyrosine Kinase

2010).

et al., 2007).

proposes.

Inhibition and Novel Strategies Designed to Override This Resistance 47

Since clonal resistance could potentially be overcome by combining two agents with different resistance profiles, we investigated the ability of HG-7-85-01 to positively combine with GNF-5, which is unable to potently inhibit T315I as a single agent (Zhang et al., 2010; Weisberg et al., 2010c). We demonstrated that combinations of HG-7-85-01 with GNF-5 have at least additive effects against both non-mutated BCR-ABL and BCR-ABL T315I *in vitro* and *in vivo* (Weisberg et al., 2010c). GNF-5 was also shown to enhance the activity of nilotinib against both non-mutated BCR-ABL and T315I-BCR-ABL *in vitro* and *in vivo* (Zhang et al.,

An important characteristic of leukemia that confers survival advantages is the common deregulation of one or more of the three signaling pathways: PI3K/PTEN/Akt/mTOR, RAS/Raf/MEK/ERK, and Jak/STAT, each frequently activated by mutations in upstream genes. The redundancy and simultaneous/cross-activation between the three pathways warrants consideration of the use of a multi-targeted therapeutic strategy, or the use of more than one type of signaling inhibitor. We have shown that the dual PI3K/PDK-1 inhibitor, BAG956, has the ability to enhance the inhibitory effects of imatinib and nilotinib, respectively, to effectively kill BCR-ABL-expressing cells both *in vitro* and *in vivo* (Weisberg et al., 2008a). Similarly, enhanced apoptosis of BCR-ABL-positive cells has been found to result from treatment with a STAT5 inhibitor and imatinib or nilotinib (Nelson et al., 2011). In addition, inhibitors of MAPK and PI3K/Akt signaling have been implicated in upregulation of proapoptotic BIM expression (Strasser et al., 2005). This is of potential importance with respect to imatinib resistance, as the proapoptotic BH3 domain-only protein, BIM, as well as proapoptotic BAD, mediate imatinib-induced apoptosis (Kuroda et al., 2006). Of relevance, the BH3 mimetic, ABT-737, overrides imatinib resistance due to loss of BIM and BAD (Kuroda et al., 2006). Inhibition of PI3K/Akt signaling also may be of potential benefit insofar as stromal-mediated chemoresistance is concerned, as Akt has been shown to be involved in leukemic cell survival in the bone marrow microenvironment (Tabe

**3. Development of inhibitors of FLT3, and combination therapy studies** 

The hematological malignancy, AML, is generally characterized by a block in cellular differentiation at different stages and aberrant proliferation of myeloid precursor cells. Early models have indicated that two types of mutations are responsible for the development of AML: One type of mutation is responsible for blocking terminal differentiation and the other type of mutation is responsible for promotion of growth and viability. However, full genome sequencing results suggest that up to 20 different mutations initiate AML, and disease development is thus likely more complicated than what the two mutation model

Approximately 30% of AML patients, as well as a portion of ALL patients, express a mutated form of the class III receptor tyrosine kinase, FLT3 (*F*ms-*L*ike *T*yrosine kinase-3; STK-1, human *S*tem Cell *T*yrosine *K*inase-1; FLK-2, *F*etal *L*iver *K*inase-2) (Stirewalt and Radich, 2003). The existence of a FLT3 mutation generally translates into a poorer prognosis in terms of overall survival and disease-free survival (Mattison et al., 2007). The fact that FLT3 mutations are only found in a portion of AML cells suggests that FLT3 mutations may

**geared toward overriding resistance to FLT3 inhibition** 

be involved in promotion of disease rather than initiation of disease.

*Mutant FLT3 as a therapeutic target for AML* 

inhibiting T315I-BCR-ABL in CML, HG-7-85-01 was observed to inhibit gatekeeper mutants of multiple kinases of therapeutic interest including T670I c-Kit and T674I-PDGFRalpha, which are clinically observed in gastrointestinal stromal tumor (GIST) and hypereosinophilic syndrome (HEL). Despite this broad profile with respect to 'gatekeeper' mutant kinases, HG-7-85-01 is not a promiscuous inhibitor, and no other inhibitor has been reported to possess this biological profile. Demonstration that this multi-gatekeeper mutant profile can be achieved is very important to the field because many pharmaceutical companies are reluctant to develop inhibitors for drug-resistant mutant kinases as they do not believe a sufficient number of patients are likely to benefit. The biological profile of HG-7-85-01 demonstrates that a single compound could be developed that is capable of simultaneously targeting drug-resistant target kinases relevant across a range of malignancies.

Progress is being made toward development of pan-BCR-ABL inhibitors that target all identified BCR-ABL point mutants, including T315I-BCR-ABL gatekeeper mutation (the socalled "third generation" of BCR-ABL inhibitors). However, as the potential for emergence of new drug-resistant BCR-ABL point mutations still exists, benefit is likely to be gained from further development of BCR-ABL inhibitors displaying higher potency and distinct mutagenicity profiles, as well as therapeutic approaches involving administration of more than one BCR-ABL inhibitor or combined use of signal transduction inhibitors and BCR-ABL inhibitors.

#### *Combination therapy as an approach to overriding resistance to BCR-ABL inhibition*

As it was anticipated that, similar to imatinib, resistance to nilotinib and dasatinib would likely also emerge due to arising BCR-ABL point mutations in CML and Ph+ ALL, mutagenesis screens were performed to identify nilotinib- and dasatinib-resistant BCR-ABL point mutations and showed overlapping but distinct mutagenicity profiles (Von Bubnoff et al., 2006; Bradeen et al., 2006; Ray et al., 2007). There has been substantial interest in testing the notion that simultaneous or sequential administration of more than one ABL inhibitor might delay the onset of emergence of drug-resistant BCR-ABL point mutations. For instance, we have shown beneficial paired combinations of imatinib and nilotinib against imatinib-resistant CML (Weisberg et al., 2007a). Specifically, we demonstrate the synergistic interaction between nilotinib and imatinib against both non-mutated BCR-ABL and imatinib-resistant BCR-ABL point mutants. It has been suggested that the differential uptake mechanisms for imatinib and nilotinib may contribute to the observed synergy between the two agents (White et al., 2006). Alternatively, synergy may be explained by the ability of imatinib to increase the intracellular uptake and retention of nilotinib (and therefore its overall concentration), possibly through imatinib inhibition of ABCB1-mediated efflux of nilotinib (White et al., 2007). The demonstrated synergy between imatinib and nilotinib complements other published reports showing synergistic interaction between imatinib and the dual Src/Abl inhibitors, dasatinib and AP23848 (O'Hare et al., 2005), as well as synergistic interactions between imatinib and a panel of standard chemotherapeutic agents (Liu et al., 2002). In addition, combinations of dasatinib and imatinib have proven to be effective in diminishing the frequency of occurrence of drug-resistant BCR-ABL mutants, with the exception of T315I (Shah et al., 2004; Burgess et al., 2005; Bradeen et al., 2006; Talpaz et al., 2006). As improvement of disease-free survival could potentially be achieved from combination therapy involving inhibitors from the same or diverse structural classes, these studies could serve as a guide for further clinical investigation.

inhibiting T315I-BCR-ABL in CML, HG-7-85-01 was observed to inhibit gatekeeper mutants of multiple kinases of therapeutic interest including T670I c-Kit and T674I-PDGFRalpha, which are clinically observed in gastrointestinal stromal tumor (GIST) and hypereosinophilic syndrome (HEL). Despite this broad profile with respect to 'gatekeeper' mutant kinases, HG-7-85-01 is not a promiscuous inhibitor, and no other inhibitor has been reported to possess this biological profile. Demonstration that this multi-gatekeeper mutant profile can be achieved is very important to the field because many pharmaceutical companies are reluctant to develop inhibitors for drug-resistant mutant kinases as they do not believe a sufficient number of patients are likely to benefit. The biological profile of HG-7-85-01 demonstrates that a single compound could be developed that is capable of simultaneously targeting drug-resistant target kinases relevant across a range of

Progress is being made toward development of pan-BCR-ABL inhibitors that target all identified BCR-ABL point mutants, including T315I-BCR-ABL gatekeeper mutation (the socalled "third generation" of BCR-ABL inhibitors). However, as the potential for emergence of new drug-resistant BCR-ABL point mutations still exists, benefit is likely to be gained from further development of BCR-ABL inhibitors displaying higher potency and distinct mutagenicity profiles, as well as therapeutic approaches involving administration of more than one BCR-ABL inhibitor or combined use of signal transduction inhibitors and BCR-

As it was anticipated that, similar to imatinib, resistance to nilotinib and dasatinib would likely also emerge due to arising BCR-ABL point mutations in CML and Ph+ ALL, mutagenesis screens were performed to identify nilotinib- and dasatinib-resistant BCR-ABL point mutations and showed overlapping but distinct mutagenicity profiles (Von Bubnoff et al., 2006; Bradeen et al., 2006; Ray et al., 2007). There has been substantial interest in testing the notion that simultaneous or sequential administration of more than one ABL inhibitor might delay the onset of emergence of drug-resistant BCR-ABL point mutations. For instance, we have shown beneficial paired combinations of imatinib and nilotinib against imatinib-resistant CML (Weisberg et al., 2007a). Specifically, we demonstrate the synergistic interaction between nilotinib and imatinib against both non-mutated BCR-ABL and imatinib-resistant BCR-ABL point mutants. It has been suggested that the differential uptake mechanisms for imatinib and nilotinib may contribute to the observed synergy between the two agents (White et al., 2006). Alternatively, synergy may be explained by the ability of imatinib to increase the intracellular uptake and retention of nilotinib (and therefore its overall concentration), possibly through imatinib inhibition of ABCB1-mediated efflux of nilotinib (White et al., 2007). The demonstrated synergy between imatinib and nilotinib complements other published reports showing synergistic interaction between imatinib and the dual Src/Abl inhibitors, dasatinib and AP23848 (O'Hare et al., 2005), as well as synergistic interactions between imatinib and a panel of standard chemotherapeutic agents (Liu et al., 2002). In addition, combinations of dasatinib and imatinib have proven to be effective in diminishing the frequency of occurrence of drug-resistant BCR-ABL mutants, with the exception of T315I (Shah et al., 2004; Burgess et al., 2005; Bradeen et al., 2006; Talpaz et al., 2006). As improvement of disease-free survival could potentially be achieved from combination therapy involving inhibitors from the same or diverse structural classes,

*Combination therapy as an approach to overriding resistance to BCR-ABL inhibition* 

these studies could serve as a guide for further clinical investigation.

malignancies.

ABL inhibitors.

Since clonal resistance could potentially be overcome by combining two agents with different resistance profiles, we investigated the ability of HG-7-85-01 to positively combine with GNF-5, which is unable to potently inhibit T315I as a single agent (Zhang et al., 2010; Weisberg et al., 2010c). We demonstrated that combinations of HG-7-85-01 with GNF-5 have at least additive effects against both non-mutated BCR-ABL and BCR-ABL T315I *in vitro* and *in vivo* (Weisberg et al., 2010c). GNF-5 was also shown to enhance the activity of nilotinib against both non-mutated BCR-ABL and T315I-BCR-ABL *in vitro* and *in vivo* (Zhang et al., 2010).

An important characteristic of leukemia that confers survival advantages is the common deregulation of one or more of the three signaling pathways: PI3K/PTEN/Akt/mTOR, RAS/Raf/MEK/ERK, and Jak/STAT, each frequently activated by mutations in upstream genes. The redundancy and simultaneous/cross-activation between the three pathways warrants consideration of the use of a multi-targeted therapeutic strategy, or the use of more than one type of signaling inhibitor. We have shown that the dual PI3K/PDK-1 inhibitor, BAG956, has the ability to enhance the inhibitory effects of imatinib and nilotinib, respectively, to effectively kill BCR-ABL-expressing cells both *in vitro* and *in vivo* (Weisberg et al., 2008a). Similarly, enhanced apoptosis of BCR-ABL-positive cells has been found to result from treatment with a STAT5 inhibitor and imatinib or nilotinib (Nelson et al., 2011). In addition, inhibitors of MAPK and PI3K/Akt signaling have been implicated in upregulation of proapoptotic BIM expression (Strasser et al., 2005). This is of potential importance with respect to imatinib resistance, as the proapoptotic BH3 domain-only protein, BIM, as well as proapoptotic BAD, mediate imatinib-induced apoptosis (Kuroda et al., 2006). Of relevance, the BH3 mimetic, ABT-737, overrides imatinib resistance due to loss of BIM and BAD (Kuroda et al., 2006). Inhibition of PI3K/Akt signaling also may be of potential benefit insofar as stromal-mediated chemoresistance is concerned, as Akt has been shown to be involved in leukemic cell survival in the bone marrow microenvironment (Tabe et al., 2007).

#### **3. Development of inhibitors of FLT3, and combination therapy studies geared toward overriding resistance to FLT3 inhibition**

#### *Mutant FLT3 as a therapeutic target for AML*

The hematological malignancy, AML, is generally characterized by a block in cellular differentiation at different stages and aberrant proliferation of myeloid precursor cells. Early models have indicated that two types of mutations are responsible for the development of AML: One type of mutation is responsible for blocking terminal differentiation and the other type of mutation is responsible for promotion of growth and viability. However, full genome sequencing results suggest that up to 20 different mutations initiate AML, and disease development is thus likely more complicated than what the two mutation model proposes.

Approximately 30% of AML patients, as well as a portion of ALL patients, express a mutated form of the class III receptor tyrosine kinase, FLT3 (*F*ms-*L*ike *T*yrosine kinase-3; STK-1, human *S*tem Cell *T*yrosine *K*inase-1; FLK-2, *F*etal *L*iver *K*inase-2) (Stirewalt and Radich, 2003). The existence of a FLT3 mutation generally translates into a poorer prognosis in terms of overall survival and disease-free survival (Mattison et al., 2007). The fact that FLT3 mutations are only found in a portion of AML cells suggests that FLT3 mutations may be involved in promotion of disease rather than initiation of disease.

Causative Factors Involved in Development of Resistance to Tyrosine Kinase

at diagnosis.

*Resistance to FLT3 inhibition* 

Inhibition and Novel Strategies Designed to Override This Resistance 49

generation inhibitor, CEP-701 (lestaurtinib) (Levis et al., 2002; Smith et al., 2004; Knapper et al., 2006), KW-2449 (Pratz and Levis, 2008; Pratz et al., 2009; Shiotsu et al., 2009), 3 substituted indolinones SU5416 (Semaxanib) and SU5614 (Yee et al., 2002; Fielder et al., 2003; Giles et al., 2003; O'Farrell et al., 2004), the indolinone derivative and first-generation inhibitor, SU11248 (SU011248, sunitinib) (Fielder et al., 2005; Kancha et al., 2007; O'Farrell et al., 2003a,b), and the piperazinyl quinazoline and first-generation inhibitor, MLN518 (tandutinib; CT53518) (Kelly et al., 2002b; Cheng and Paz, 2008). Among FLT3 inhibitors in early development are the *N-*(4-(3-amino-1H-indazol-4yl)phenyl-*N*1(2-fluoro-5 methylphenyl) urea ABT-869 (Albert et al., 2006; Shankar et al., 2007; Zhou et al., 2008), the benzimidalzole-quinoline CHIR-258 (TKI258) (Lopes de Menezes et al., 2005), the hydroxystyryl-acrylonitrile LS104 (Kasper et al., 2008) and AP24534 (Gozgit et al., 2011). AC220 (Chao et al., 2009; Zarrinkar et al., 2009) is a second-generation FLT3 inhibitor that exhibits significantly higher potency and selectivity than first-generation inhibitors, and is under clinical investigation for mutant FLT3-expressing AML and wild-type FLT3 harboring AML. A study by Pratz and colleagues suggested, however, that the potency and selectivity of agents targeting mutant FLT3 may not, in fact, be the best predictor of clinical efficacy (Pratz et al., 2010). In their study, AC220 was found to be the most potent inhibitor of several common FLT3 inhibitors tested (including lestaurtinib, midostaurin, sorafenib, and sunitinib) and had the highest index of selectivity. However, lestaurtinib- which had the lowest index of selectivity- was found to be the most effective when tested against FLT3- ITD-positive patient samples. Results shown in this study suggested that inhibition of FLT3 autophosphorylation in a FLT3-ITD specimen does not always induce death. For example, a side-by-side comparison of AC220 and lestaurtinib showed that while both compounds fully inhibited FLT3 autophosphorylation and suppressed downstream STAT5 activation in one patient sample, only lestaurtinib induced apoptosis. Relapsed AML samples were observed to be more sensitive to FLT3 inhibition than diagnostic AML, suggesting that some FLT3-ITD AML (i.e. diagnostic) may not be addicted to FLT3 signaling. This study suggested that more selective inhibitors, like AC220 may be less effective in the diagnostic setting while less selective inhibitors- like lestaurtinib or midostaurin- may be more effective

Thus far, none of the FLT3 inhibitors under clinical investigation has elicited a sustained clinical response when used as a single agent. As an example, a phase I clinical trial for KW-2449 showed that FLT3 inhibition in patients occurred only transiently to less than 20% of baseline levels (Pratz et al., 2009). It has been suggested that partial and transient FLT3 inhibition such as this applies to other FLT3 inhibitors in clinical development and may be

One putative resistance mechanism related to FLT3 inhibition is dysregulation of signaling molecules, such as those associated with the PI3K/Akt and Ras/MEK/MAPK pathways (Piloto et al., 2007). Also implicated in drug resistance are aberrant STAT signaling (Zhou et al., 2009), the aberrant expression of the STAT5 target gene, PIM (Kim et al., 2005; Kim et al., 2006), and high levels of phosphoprotein expression of the forkhead transcription factor, FOXO3A (Kornblau et al., 2010). Additional mechanisms include up-regulation of antiapoptotic proteins (Kohl et al., 2007; Brietenbuecher et al., 2009; Stolzel et al., 2010), upregulation of inhibitors of apoptosis proteins, and elevated FLT3 ligand levels (Zhou et al., 2009). The concentration of FLT3 ligand has been observed to be significantly higher in

responsible for their limited efficacy (Chu and Small, 2009).

The most prevalent form of constitutively activated FLT3, found in approximately 20-25% of AML patients (although in less than 5% of myelodysplastic syndrome patients), occurs as internal tandem duplications (ITDs) that are located within the juxtamembrane domain (Nakao et al., 1996; Horiike et al., 1997; Kiyoi et al., 1998; Kondo et al., 1999; Rombouts et al., 2000). This mutation has been shown to cause a rapidly lethal myeloproliferative disorder in mice in the absence of a block in differentiation, suggesting that its main role may be to cause hyper-proliferation of primitive myeloid cells (Kelly et al., 2002a). Mutant FLT3 is believed to interact with additional oncogenes that block differentiation, such as AML1/ETO (t(8;21)) PML/RARalpha (t(15;17).) to cause full leukemia development.

Occurring in approximately 7% of AML cases are gain-of-function FLT3 kinase domain point mutations, frequently at position 835 (Yamamoto et al., 2001). Also identified have been other kinase domain point mutations, including Y842C (Kindler et al., 2005) and N841I (Jiang et al., 2004). An activating point mutation with comparatively weaker transforming potential has also been discovered in a stretch of the FLT3 juxtamembrane domain (Reindl et al., 2006).

#### *Inhibitors of mutant FLT3*

There are several FLT3 inhibitors presently in clinical trials, and a number of novel agents under preclinical investigation. Unfortunately, the FLT3 inhibitors tested thus far clinically induce only partial and transient responses in patients- at best- when used as single agents.

We identified and developed a small molecule inhibitor, the *N*-indolo-carbazole midostaurin (PKC412; N-benzoylstaurosporin), (Weisberg et al., 2002), a broad-spectrum first generation inhibitor that targets constitutively activated mutant FLT3. Other targets of midostaurin include (PDGFRbeta), c-KIT and c-FMS (Weisberg et al., 2002). Midostaurin showed high potency against mutant FLT3 as a target *in vitro*, and progressive leukemia was prevented in mice transplanted with marrow transduced with a FLT3-ITD-expressing retrovirus (Weisberg et al., 2002).

In early clinical trials, midostaurin administered in sequential and simultaneous combinations with standard chemotherapeutic agents such as daunorubicin and cytarabine induction and high-dose cytarabine consolidation yielded clinical responses with transient and/or reversible side effects (Stone et al., 2004). Midostaurin is one of several FLT3 inhibitors presently undergoing clinical testing, and it is currently under investigation in late stage clinical trials for AML.

We have demonstrated the activity of midostaurin against imatinib-resistant GIST (Weisberg et al., 2006). Specifically, we demonstrated the potent activity of midostaurin against cells expressing two PDGFRalpha mutants associated GIST: the V561D juxtamembrane domain mutation and the less imatinib-sensitive D842V kinase domain mutation. We also demonstrated the ability of nilotinib to positively combine with either imatinib or midostaurin against cells expressing the D842V-PDGFRalpha mutant. Findings reported introduce midostaurin as a potential treatment option for mutant PDGFRalphapositive GIST and warrant its clinical testing for this disease target.

Additional multi-targeted inhibitors, such as dasatinib and the biaryl urea compound and first-generation inhibitor, sorafenib (BAY 43-9006), have been shown to have activity against mutant FLT3 and are under clinical investigation for AML (Auclair et al., 2007; Kancha et al., 2007; Lierman et al., 2007; Zhang et al., 2008; Metzelder et al., 2009; Guerrouahen et al., 2010). Other FLT3 inhibitors that have been clinically investigated and which elicited transient responses in early phase trials include the indolocarbazole alkaloid and firstgeneration inhibitor, CEP-701 (lestaurtinib) (Levis et al., 2002; Smith et al., 2004; Knapper et al., 2006), KW-2449 (Pratz and Levis, 2008; Pratz et al., 2009; Shiotsu et al., 2009), 3 substituted indolinones SU5416 (Semaxanib) and SU5614 (Yee et al., 2002; Fielder et al., 2003; Giles et al., 2003; O'Farrell et al., 2004), the indolinone derivative and first-generation inhibitor, SU11248 (SU011248, sunitinib) (Fielder et al., 2005; Kancha et al., 2007; O'Farrell et al., 2003a,b), and the piperazinyl quinazoline and first-generation inhibitor, MLN518 (tandutinib; CT53518) (Kelly et al., 2002b; Cheng and Paz, 2008). Among FLT3 inhibitors in early development are the *N-*(4-(3-amino-1H-indazol-4yl)phenyl-*N*1(2-fluoro-5 methylphenyl) urea ABT-869 (Albert et al., 2006; Shankar et al., 2007; Zhou et al., 2008), the benzimidalzole-quinoline CHIR-258 (TKI258) (Lopes de Menezes et al., 2005), the hydroxystyryl-acrylonitrile LS104 (Kasper et al., 2008) and AP24534 (Gozgit et al., 2011). AC220 (Chao et al., 2009; Zarrinkar et al., 2009) is a second-generation FLT3 inhibitor that exhibits significantly higher potency and selectivity than first-generation inhibitors, and is under clinical investigation for mutant FLT3-expressing AML and wild-type FLT3 harboring AML. A study by Pratz and colleagues suggested, however, that the potency and selectivity of agents targeting mutant FLT3 may not, in fact, be the best predictor of clinical efficacy (Pratz et al., 2010). In their study, AC220 was found to be the most potent inhibitor of several common FLT3 inhibitors tested (including lestaurtinib, midostaurin, sorafenib, and sunitinib) and had the highest index of selectivity. However, lestaurtinib- which had the lowest index of selectivity- was found to be the most effective when tested against FLT3- ITD-positive patient samples. Results shown in this study suggested that inhibition of FLT3 autophosphorylation in a FLT3-ITD specimen does not always induce death. For example, a side-by-side comparison of AC220 and lestaurtinib showed that while both compounds fully inhibited FLT3 autophosphorylation and suppressed downstream STAT5 activation in one patient sample, only lestaurtinib induced apoptosis. Relapsed AML samples were observed to be more sensitive to FLT3 inhibition than diagnostic AML, suggesting that some FLT3-ITD AML (i.e. diagnostic) may not be addicted to FLT3 signaling. This study suggested that more selective inhibitors, like AC220 may be less effective in the diagnostic setting while less selective inhibitors- like lestaurtinib or midostaurin- may be more effective at diagnosis.

#### *Resistance to FLT3 inhibition*

48 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

The most prevalent form of constitutively activated FLT3, found in approximately 20-25% of AML patients (although in less than 5% of myelodysplastic syndrome patients), occurs as internal tandem duplications (ITDs) that are located within the juxtamembrane domain (Nakao et al., 1996; Horiike et al., 1997; Kiyoi et al., 1998; Kondo et al., 1999; Rombouts et al., 2000). This mutation has been shown to cause a rapidly lethal myeloproliferative disorder in mice in the absence of a block in differentiation, suggesting that its main role may be to cause hyper-proliferation of primitive myeloid cells (Kelly et al., 2002a). Mutant FLT3 is believed to interact with additional oncogenes that block differentiation, such as

AML1/ETO (t(8;21)) PML/RARalpha (t(15;17).) to cause full leukemia development.

al., 2006).

*Inhibitors of mutant FLT3* 

retrovirus (Weisberg et al., 2002).

late stage clinical trials for AML.

Occurring in approximately 7% of AML cases are gain-of-function FLT3 kinase domain point mutations, frequently at position 835 (Yamamoto et al., 2001). Also identified have been other kinase domain point mutations, including Y842C (Kindler et al., 2005) and N841I (Jiang et al., 2004). An activating point mutation with comparatively weaker transforming potential has also been discovered in a stretch of the FLT3 juxtamembrane domain (Reindl et

There are several FLT3 inhibitors presently in clinical trials, and a number of novel agents under preclinical investigation. Unfortunately, the FLT3 inhibitors tested thus far clinically induce only partial and transient responses in patients- at best- when used as single agents. We identified and developed a small molecule inhibitor, the *N*-indolo-carbazole midostaurin (PKC412; N-benzoylstaurosporin), (Weisberg et al., 2002), a broad-spectrum first generation inhibitor that targets constitutively activated mutant FLT3. Other targets of midostaurin include (PDGFRbeta), c-KIT and c-FMS (Weisberg et al., 2002). Midostaurin showed high potency against mutant FLT3 as a target *in vitro*, and progressive leukemia was prevented in mice transplanted with marrow transduced with a FLT3-ITD-expressing

In early clinical trials, midostaurin administered in sequential and simultaneous combinations with standard chemotherapeutic agents such as daunorubicin and cytarabine induction and high-dose cytarabine consolidation yielded clinical responses with transient and/or reversible side effects (Stone et al., 2004). Midostaurin is one of several FLT3 inhibitors presently undergoing clinical testing, and it is currently under investigation in

We have demonstrated the activity of midostaurin against imatinib-resistant GIST (Weisberg et al., 2006). Specifically, we demonstrated the potent activity of midostaurin against cells expressing two PDGFRalpha mutants associated GIST: the V561D juxtamembrane domain mutation and the less imatinib-sensitive D842V kinase domain mutation. We also demonstrated the ability of nilotinib to positively combine with either imatinib or midostaurin against cells expressing the D842V-PDGFRalpha mutant. Findings reported introduce midostaurin as a potential treatment option for mutant PDGFRalpha-

Additional multi-targeted inhibitors, such as dasatinib and the biaryl urea compound and first-generation inhibitor, sorafenib (BAY 43-9006), have been shown to have activity against mutant FLT3 and are under clinical investigation for AML (Auclair et al., 2007; Kancha et al., 2007; Lierman et al., 2007; Zhang et al., 2008; Metzelder et al., 2009; Guerrouahen et al., 2010). Other FLT3 inhibitors that have been clinically investigated and which elicited transient responses in early phase trials include the indolocarbazole alkaloid and first-

positive GIST and warrant its clinical testing for this disease target.

Thus far, none of the FLT3 inhibitors under clinical investigation has elicited a sustained clinical response when used as a single agent. As an example, a phase I clinical trial for KW-2449 showed that FLT3 inhibition in patients occurred only transiently to less than 20% of baseline levels (Pratz et al., 2009). It has been suggested that partial and transient FLT3 inhibition such as this applies to other FLT3 inhibitors in clinical development and may be responsible for their limited efficacy (Chu and Small, 2009).

One putative resistance mechanism related to FLT3 inhibition is dysregulation of signaling molecules, such as those associated with the PI3K/Akt and Ras/MEK/MAPK pathways (Piloto et al., 2007). Also implicated in drug resistance are aberrant STAT signaling (Zhou et al., 2009), the aberrant expression of the STAT5 target gene, PIM (Kim et al., 2005; Kim et al., 2006), and high levels of phosphoprotein expression of the forkhead transcription factor, FOXO3A (Kornblau et al., 2010). Additional mechanisms include up-regulation of antiapoptotic proteins (Kohl et al., 2007; Brietenbuecher et al., 2009; Stolzel et al., 2010), upregulation of inhibitors of apoptosis proteins, and elevated FLT3 ligand levels (Zhou et al., 2009). The concentration of FLT3 ligand has been observed to be significantly higher in

Causative Factors Involved in Development of Resistance to Tyrosine Kinase

chemotherapy (Pratz and Levis, 2008).

chemotherapeutic agents.

transferase (Mollgard et al., 2008).

**chemoresistance to tyrosine kinase inhibition** 

Inhibition and Novel Strategies Designed to Override This Resistance 51

cyclophosphamide, etoposide, mitoxantrone, cytarabine, doxorubicin, and idarubicin (Mollgard et al., 2008; Furukawa et al., 2007). The sequence of administration of FLT3 inhibitors and standard chemotherapy appears to often, depending on the FLT3 inhibitor, be sequence-dependent, resulting in antagonism when the FLT3 inhibitor is administered prior to chemotherapy and resulting in synergy when the FLT3 inhibitor is administered after

One strategy geared toward overriding resistance to FLT3 inhibition includes the combined use of more than one FLT3 inhibitor, providing their interaction with the FLT3 protein target or FLT3 signaling pathway components is distinct enough for the two inhibitors to synergize. We demonstrated the potent and selective activity of the novel type II ATP competitive inhibitors, HG-7-85-01 and HG-7-86-01, against mutant FLT3, as well as their ability to synergize well with standard chemotherapeutic agents or midostaurin (Weisberg et al., 2010b). The ability of HG-7-85-01 and HG-7-86-01 to inhibit mutant FLT3, in addition to a number of gatekeeper mutants of multiple kinases of therapeutic interest, highlights their unique versatility and potential widespread clinical usefulness. Other potent FLT3 inhibitors, such as NVP-AST487, have the ability to effectively combine with midostaurin to kill drug-sensitive and insensitive mutant FLT3-expressing cells (Weisberg et al., 2008b). The ability of two targeted FLT3 inhibitors to synergize may translate into clinical benefit in terms of reducing toxicity, as clinical responses achieved with late-stage FLT3 inhibitors like midostaurin and CEP-701 depend on combined administration with standard

Alternatively, FLT3 inhibitors can be combined with small molecule inhibitors that interact with key mediators of major signaling pathways that play a significant role in AML. Crosstalk between the three main pathways activated by mutant FLT3 (RAS/Raf/MEK/ERK, Jak/STAT, and PI3K/PTEN/Akt/mTOR) warrants the development of multi-targeted approaches for treatment of mutant FLT3-positive AML (Brandts et al., 2005; Recher et al., 2005; Kornblau et al., 2006; Rocnik et al., 2006; Al Shaer et al., 2008). We have shown that signaling pathway inhibitors, such as the dual PI3K/PDK-1 inhibitor, BAG956, can positively combine with midostaurin against mutant FLT3-positive cells (Weisberg et al., 2008a). Of relevance, PKC412 and the mTOR inhibitor, rapamycin, synergize against PKC412-sensitive and –resistant mutant FLT3-expressing cells (Mohi et al., 2004). The rapamycin derivative, RAD001, enhances the anti-leukemic effects of sunitinib (Ikezoe et al., 2006), as does the MEK1/2 kinase inhibitor, AZD6244 (ARRY-142886) (Nishioka et al., 2008b). Other agents effective in killing mutant FLT3-expressing cells are heat shock protein 90 (Hsp90) inhibitors and histone deacetylase inhibitors, which disrupt the major signaling pathways (Al Shaer et al., 2008; Nishioka et al., 2008a), and drugs that inhibit farnesyl-

**4. Characterization of underlying mechanisms of stroma-mediated** 

In addition to identifying and developing potent kinase inhibitors representative of novel and unique structural classes with the ability to override drug resistance due to changes in the target protein, there is a push toward gaining a better understanding of the mechanisms underlying drug resistance in CML and AML as they relate to the leukemic cell microenvironment. Bone marrow is comprised of hematopoietic and stromal cells, in addition to other factors including extracellular matrix and blood vessels; growth factors

patients treated with chemotherapy or radiotherapy (Lyman et al., 1995; Wodnar-Filipowicz et al., 1996; Zwierzina et al., 1999; Bojko et al., 2002), and this is predicted to impede the action of FLT3 inhibitors administered after chemotherapy.

Another possible resistance mechanism is cytoprotection conferred by the bone marrow microenvironment. Indeed, FLT3 inhibition was found to actually enhance the survival of CD34+CD38-CD123+ leukemic stem cells and progenitor cells in a "niche-like" *in vitro*  microenvironment that was comprised of factors including IL-6, IL-3, stem cell factor (SCF), and immobilized fibronectin (Mony et al., 2008). Small molecule CXCR4 inhibitors may be effective in enhancing kinase inhibitor-induced apoptosis of stromal-protected leukemic cells, implicating a causal relationship between the chemokine receptor CXCR4 and stromaderived factor 1alpha (SDF-1a) interaction and drug-resistant leukemia (Zeng et al., 2006).

Pre-existing or acquired point mutations in the FLT3 kinase domain that interfere with drug binding may also contribute to drug resistance in patients (Cools et al., 2004; Heidel et al., 2006).

Point mutations in the FLT3 receptor are generally analogous to point mutations in BCR-ABL, the most prevalent mechanism of resistance to imatinib. However, there is little evidence for the existence of overlapping resistance profiles for FLT3 inhibitors, such as those demonstrated for imatinib, dasatinib, and nilotinib with respect to the T315I gatekeeper mutation (Von Bubnoff et al., 2006; Bradeen et al., 2006; Ray et al., 2007). Instead, non-overlapping mechanisms of resistance between PKC412, sorafenib, and SU5614 were found in a screening assay designed to study drug resistance profiles (Von Bubnoff et al., 2009). For example, SU5614 resistance was characterized predominantly as mutations in the D835 residue, while sorafenib resistance was characterized by unique mutations such as F691L and point mutations in the Y842 residue (Von Bubnoff et al., 2009). Another study confirmed the involvement of acquired mutations, D835N and Y842H, in resistance to SU5614 (Bagrintseva et al., 2004). In contrast, PKC412 resistance was characterized by mutations in the N676 residue, which was previously identified and characterized in a PKC412-resistant AML patient as the sole determinant of drug resistance (Heidel et al., 2006).

In addition to non-overlapping mutagenicity profiles between existing FLT3 inhibitors, each shows variable activity toward different mutations, for example activation loop mutants versus ITD mutations. As an example, sunitinib inhibits ITD and activation loop mutants with equal potency, whereas sorafenib is less efficacious toward activation loop mutants than ITD (Kancha et al., 2007). PKC412 shows comparable activity against different FLT3 activation loop mutants, whereas MLN518 shows variable activity (Clark et al., 2004; Barry et al., 2007).

#### *Combination therapy as an approach to treating mutant FLT3-positive disease*

As responses of patients to FLT3 inhibition is generally only partial and transient, there is a need for development of novel agents that can either be used effectively alone or combined with FLT3 inhibitors to suppress disease progression and prolong lifespan. FLT3 inhibitors have been tested in combination with standard chemotherapy agents as an approach to assessing the overall efficacy of combined therapies. For example, SU11248, combined with cytarabine or daunorubicin, exhibited additive-to-synergistic inhibitory effects on cells expressing mutant FLT3 (Yee et al., 2004). Similarly, CEP-701 showed synergy when combined with etoposide, mitoxantrone, daunorubicin, and cytarabine (Levis et al., 2004). PKC412 demonstrated synergy when combined with vincristine, 4-hydroperoxy-

patients treated with chemotherapy or radiotherapy (Lyman et al., 1995; Wodnar-Filipowicz et al., 1996; Zwierzina et al., 1999; Bojko et al., 2002), and this is predicted to impede the

Another possible resistance mechanism is cytoprotection conferred by the bone marrow microenvironment. Indeed, FLT3 inhibition was found to actually enhance the survival of CD34+CD38-CD123+ leukemic stem cells and progenitor cells in a "niche-like" *in vitro*  microenvironment that was comprised of factors including IL-6, IL-3, stem cell factor (SCF), and immobilized fibronectin (Mony et al., 2008). Small molecule CXCR4 inhibitors may be effective in enhancing kinase inhibitor-induced apoptosis of stromal-protected leukemic cells, implicating a causal relationship between the chemokine receptor CXCR4 and stromaderived factor 1alpha (SDF-1a) interaction and drug-resistant leukemia (Zeng et al., 2006). Pre-existing or acquired point mutations in the FLT3 kinase domain that interfere with drug binding may also contribute to drug resistance in patients (Cools et al., 2004; Heidel et al.,

Point mutations in the FLT3 receptor are generally analogous to point mutations in BCR-ABL, the most prevalent mechanism of resistance to imatinib. However, there is little evidence for the existence of overlapping resistance profiles for FLT3 inhibitors, such as those demonstrated for imatinib, dasatinib, and nilotinib with respect to the T315I gatekeeper mutation (Von Bubnoff et al., 2006; Bradeen et al., 2006; Ray et al., 2007). Instead, non-overlapping mechanisms of resistance between PKC412, sorafenib, and SU5614 were found in a screening assay designed to study drug resistance profiles (Von Bubnoff et al., 2009). For example, SU5614 resistance was characterized predominantly as mutations in the D835 residue, while sorafenib resistance was characterized by unique mutations such as F691L and point mutations in the Y842 residue (Von Bubnoff et al., 2009). Another study confirmed the involvement of acquired mutations, D835N and Y842H, in resistance to SU5614 (Bagrintseva et al., 2004). In contrast, PKC412 resistance was characterized by mutations in the N676 residue, which was previously identified and characterized in a PKC412-resistant AML patient as the sole determinant of drug resistance (Heidel et al.,

In addition to non-overlapping mutagenicity profiles between existing FLT3 inhibitors, each shows variable activity toward different mutations, for example activation loop mutants versus ITD mutations. As an example, sunitinib inhibits ITD and activation loop mutants with equal potency, whereas sorafenib is less efficacious toward activation loop mutants than ITD (Kancha et al., 2007). PKC412 shows comparable activity against different FLT3 activation loop mutants, whereas MLN518 shows variable activity (Clark et al., 2004; Barry

As responses of patients to FLT3 inhibition is generally only partial and transient, there is a need for development of novel agents that can either be used effectively alone or combined with FLT3 inhibitors to suppress disease progression and prolong lifespan. FLT3 inhibitors have been tested in combination with standard chemotherapy agents as an approach to assessing the overall efficacy of combined therapies. For example, SU11248, combined with cytarabine or daunorubicin, exhibited additive-to-synergistic inhibitory effects on cells expressing mutant FLT3 (Yee et al., 2004). Similarly, CEP-701 showed synergy when combined with etoposide, mitoxantrone, daunorubicin, and cytarabine (Levis et al., 2004). PKC412 demonstrated synergy when combined with vincristine, 4-hydroperoxy-

*Combination therapy as an approach to treating mutant FLT3-positive disease* 

action of FLT3 inhibitors administered after chemotherapy.

2006).

2006).

et al., 2007).

cyclophosphamide, etoposide, mitoxantrone, cytarabine, doxorubicin, and idarubicin (Mollgard et al., 2008; Furukawa et al., 2007). The sequence of administration of FLT3 inhibitors and standard chemotherapy appears to often, depending on the FLT3 inhibitor, be sequence-dependent, resulting in antagonism when the FLT3 inhibitor is administered prior to chemotherapy and resulting in synergy when the FLT3 inhibitor is administered after chemotherapy (Pratz and Levis, 2008).

One strategy geared toward overriding resistance to FLT3 inhibition includes the combined use of more than one FLT3 inhibitor, providing their interaction with the FLT3 protein target or FLT3 signaling pathway components is distinct enough for the two inhibitors to synergize. We demonstrated the potent and selective activity of the novel type II ATP competitive inhibitors, HG-7-85-01 and HG-7-86-01, against mutant FLT3, as well as their ability to synergize well with standard chemotherapeutic agents or midostaurin (Weisberg et al., 2010b). The ability of HG-7-85-01 and HG-7-86-01 to inhibit mutant FLT3, in addition to a number of gatekeeper mutants of multiple kinases of therapeutic interest, highlights their unique versatility and potential widespread clinical usefulness. Other potent FLT3 inhibitors, such as NVP-AST487, have the ability to effectively combine with midostaurin to kill drug-sensitive and insensitive mutant FLT3-expressing cells (Weisberg et al., 2008b). The ability of two targeted FLT3 inhibitors to synergize may translate into clinical benefit in terms of reducing toxicity, as clinical responses achieved with late-stage FLT3 inhibitors like midostaurin and CEP-701 depend on combined administration with standard chemotherapeutic agents.

Alternatively, FLT3 inhibitors can be combined with small molecule inhibitors that interact with key mediators of major signaling pathways that play a significant role in AML. Crosstalk between the three main pathways activated by mutant FLT3 (RAS/Raf/MEK/ERK, Jak/STAT, and PI3K/PTEN/Akt/mTOR) warrants the development of multi-targeted approaches for treatment of mutant FLT3-positive AML (Brandts et al., 2005; Recher et al., 2005; Kornblau et al., 2006; Rocnik et al., 2006; Al Shaer et al., 2008). We have shown that signaling pathway inhibitors, such as the dual PI3K/PDK-1 inhibitor, BAG956, can positively combine with midostaurin against mutant FLT3-positive cells (Weisberg et al., 2008a). Of relevance, PKC412 and the mTOR inhibitor, rapamycin, synergize against PKC412-sensitive and –resistant mutant FLT3-expressing cells (Mohi et al., 2004). The rapamycin derivative, RAD001, enhances the anti-leukemic effects of sunitinib (Ikezoe et al., 2006), as does the MEK1/2 kinase inhibitor, AZD6244 (ARRY-142886) (Nishioka et al., 2008b). Other agents effective in killing mutant FLT3-expressing cells are heat shock protein 90 (Hsp90) inhibitors and histone deacetylase inhibitors, which disrupt the major signaling pathways (Al Shaer et al., 2008; Nishioka et al., 2008a), and drugs that inhibit farnesyltransferase (Mollgard et al., 2008).

#### **4. Characterization of underlying mechanisms of stroma-mediated chemoresistance to tyrosine kinase inhibition**

In addition to identifying and developing potent kinase inhibitors representative of novel and unique structural classes with the ability to override drug resistance due to changes in the target protein, there is a push toward gaining a better understanding of the mechanisms underlying drug resistance in CML and AML as they relate to the leukemic cell microenvironment. Bone marrow is comprised of hematopoietic and stromal cells, in addition to other factors including extracellular matrix and blood vessels; growth factors

Causative Factors Involved in Development of Resistance to Tyrosine Kinase

viability signals may contribute to chemoresistance to FLT3 inhibitors

LCL161 is being considered for testing in clinical trials for leukemia.

overcome by inclusion of IAP inhibitor treatment (Weisberg et al., 2007b).

2007b).

**6. Conclusion** 

to override stromal-mediated chemoresistance *in vitro* (Weisberg et al., 2007b).

Inhibition and Novel Strategies Designed to Override This Resistance 53

synergize with midostaurin *in vivo* against progressive mutant FLT3-positive leukemia and

Clinical trial data with midostaurin show that while good responses are achievable in mutant FLT3-positive AML patient peripheral blood, patient bone marrow responses are more modest. This suggests that stromal cells provide viability signals to AML cells that protect the cells from the effects of the selective inhibitor being used. We investigated the responsiveness of Ba/F3-FLT3-ITD cells cultured in the presence and absence of WEHI (used as a source of IL-3) to the cytotoxic effects of midostaurin, and we found that the presence of IL-3 completely protected cells from midostaurin-inhibition of cellular proliferation (Weisberg et al., 2007b). In contrast to midostaurin, the IAP inhibitor, LBW242, inhibited Ba/F3-FLT3-ITD proliferation in both the absence and the presence of IL-3 (Weisberg et al., 2007b). Interestingly, LBW242 plus midostaurin inhibited the growth of Ba/F3-FLT3-ITD cells cultured in the presence of IL-3 to a greater extent than either agent alone; this inhibition was similar to that achieved with co-administration of both agents in the absence of IL-3, suggesting that an IAP inhibitor is able to enhance the effects of a FLT3 inhibitor and override chemoresistance due to provision of viability signals (Weisberg et al.,

In an attempt to more closely model putative stromal-AML cell interactions and stromalmediated viability signaling effects on the cytotoxic effects of midostaurin, we tested the human stroma cell line, HS-5, in combination with the mutant FLT3 AML line, MOLM-13-luc+ (luciferase was used to specifically quantify only the leukemia component in the assay using light emission). A confluent layer of HS-5 stromal cells enhanced the growth of MOLM13-luc+ cells and was partially protective against the inhibitory effects of midostaurin (Weisberg et al., 2007b). LBW242 enhanced the cytotoxic effects of midostaurin against MOLM13-luc+ cells in the presence of HS-5 human stromal cells, supporting the notion that stromal-mediated

(such as midostaurin) observed in marrow, as well as the idea that such resistance may be

We have also shown the ability of LCL161 to significantly delay disease recurrence in mice injected with BCR-ABL-expressing cells and treated for several weeks with a moderate dose of nilotinib (Weisberg et al., 2010d). Specifically, using *in vitro* models of intrinsic drug resistance and stromal-mediated chemoresistance, as well as functional mouse models of progressive and residual disease, we showed the ability of the novel IAP inhibitor, LCL161, to enhance the cytotoxic effects of tyrosine kinase inhibitors against leukemic disease. Importantly, we observed LCL161 to synergize *in vivo* with nilotinib to reduce leukemia burden significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib. Our results support the idea of using IAP inhibitors in conjunction with targeted tyrosine kinase inhibition to suppress or eradicate progressive and drug-resistant or residual disease. Phase I studies in advanced solid tumors are currently ongoing, and

Elucidation of mechanisms of resistance to tyrosine kinase inhibition is critical for the optimization of existing therapies and prolongation of patient survival via delaying or eradicating the recurrence of disease. For CML, the discovery and characterization of BCR-ABL point mutations in the kinase target of a number of inhibitors in clinical use has been a

and cell:cell interactions (Charbord et al. 1996). Bone marrow stroma and factors derived from stroma have been suggested to play a role in the long-term survival and proliferation of normal and leukemia cells (Ashley et al., 1994; Bradstock et al., 1996; Rafii et al., 1997; Lagneaux et al., 1998, Lagneaux et al., 1999; Konopleva et al., 2002; Litwin et al., 2002). Specifically, bone marrow stroma provides signals, such as granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and terminal differentiation of hematopoietic stem cells or mediate/support their proliferation (Verfaillie et al., 1993; Liesveld et al., 1996; Harrison et al., 1997; Breems et al., 1997; O'Prey et al., 1998; Leslie et al., 1998; Shih et al., 1999). SSpenic stroma, as well, has been implicated in enhanced survival of both normal and leukemic cells (Shaked et al., 2005; Despars and O'Neill, 2006). Clinical trial data with tyrosine kinase inhibitors show that while the peripheral blood of

patients responds well, bone marrow responds less well. It appears possible that small numbers of leukemic CD34+ cells can persist in the marrow microenvironment of leukemia patients after years of therapy with kinase inhibitors. Stromal cells have been implicated, as they provide viability signals to leukemic cells that protect them from inhibitor effects. Indeed, the quantity of leukemic stem cells that rely on stroma to survive is predictive of disease outcome (Kumagai et al., 1996).

We revealed highest tumor burden and residual disease to occur *in vivo* in stroma-associated tissues in imatinib/nilotinib-treated NCr nude mice, suggesting that significant reservoirs for tumor growth appear to be tissues that are able to support normal hematopoietic and malignant stem cell development (Weisberg et al., 2008c). These studies, which showed a pattern of leukemia distribution consistent with what is observed in imatinib- and nilotinibtreated chronic myeloid leukemia patients, were followed by a more in-depth analysis of stroma-leukemia cell interactions that lead to protection of leukemia cells from nilotinibinduced cytotoxicity. For the latter, we used the human BCR-ABL-positive cell line, KU812F, and the human bone marrow stroma cell line, HS-5, to more closely approximate the bone marrow-associated cytoprotection observed in drug-treated leukemia patients. Partial stromal-mediated protection of BCR-ABL-positive leukemic cells from nilotinib treatment involved the cooperative interaction of members of a select panel of stromal-secreted viability factors, including IL-6 and GM-CSF (Weisberg et al., 2008c). Similar results were observed with mutant FLT3-positive leukemia cells treated with midostaurin in the presence of HS-5-stromal-conditioned media versus the same panel of stromal-secreted viability factors (Weisberg et al., 2009).

#### **5. Potentiation of anti-leukemic effects of tyrosine kinase inhibitors by IAP inhibition: Implications for use of IAP inhibition to prevent or reduce residual disease following tyrosine kinase inhibition**

Deregulated signaling molecules associated with viability/apoptotic signaling represent attractive targets for therapeutic intervention, and several strategies have emerged that may be effective in preventing drug resistance due to this. One approach involves combining targeted inhibitors with small molecule inhibitors of key components of major signaling pathways affecting the viability/expansion of leukemic cells. Accordingly, we have developed effective inhibitors of the inhibitor of apoptosis (IAP) family of proteins (Liu et al., 2000; Wu et al., 2000), such as LBW242 (Weisberg et al., 2007b), and its structural analog, LCL161, which bind to and inhibit multiple IAPs (i.e. XIAP, c-IAP) to enhance the activity of different proapoptotic signaling pathways. We have demonstrated the ability of LBW242 to synergize with midostaurin *in vivo* against progressive mutant FLT3-positive leukemia and to override stromal-mediated chemoresistance *in vitro* (Weisberg et al., 2007b).

Clinical trial data with midostaurin show that while good responses are achievable in mutant FLT3-positive AML patient peripheral blood, patient bone marrow responses are more modest. This suggests that stromal cells provide viability signals to AML cells that protect the cells from the effects of the selective inhibitor being used. We investigated the responsiveness of Ba/F3-FLT3-ITD cells cultured in the presence and absence of WEHI (used as a source of IL-3) to the cytotoxic effects of midostaurin, and we found that the presence of IL-3 completely protected cells from midostaurin-inhibition of cellular proliferation (Weisberg et al., 2007b). In contrast to midostaurin, the IAP inhibitor, LBW242, inhibited Ba/F3-FLT3-ITD proliferation in both the absence and the presence of IL-3 (Weisberg et al., 2007b). Interestingly, LBW242 plus midostaurin inhibited the growth of Ba/F3-FLT3-ITD cells cultured in the presence of IL-3 to a greater extent than either agent alone; this inhibition was similar to that achieved with co-administration of both agents in the absence of IL-3, suggesting that an IAP inhibitor is able to enhance the effects of a FLT3 inhibitor and override chemoresistance due to provision of viability signals (Weisberg et al., 2007b).

In an attempt to more closely model putative stromal-AML cell interactions and stromalmediated viability signaling effects on the cytotoxic effects of midostaurin, we tested the human stroma cell line, HS-5, in combination with the mutant FLT3 AML line, MOLM-13-luc+ (luciferase was used to specifically quantify only the leukemia component in the assay using light emission). A confluent layer of HS-5 stromal cells enhanced the growth of MOLM13-luc+ cells and was partially protective against the inhibitory effects of midostaurin (Weisberg et al., 2007b). LBW242 enhanced the cytotoxic effects of midostaurin against MOLM13-luc+ cells in the presence of HS-5 human stromal cells, supporting the notion that stromal-mediated viability signals may contribute to chemoresistance to FLT3 inhibitors

(such as midostaurin) observed in marrow, as well as the idea that such resistance may be overcome by inclusion of IAP inhibitor treatment (Weisberg et al., 2007b).

We have also shown the ability of LCL161 to significantly delay disease recurrence in mice injected with BCR-ABL-expressing cells and treated for several weeks with a moderate dose of nilotinib (Weisberg et al., 2010d). Specifically, using *in vitro* models of intrinsic drug resistance and stromal-mediated chemoresistance, as well as functional mouse models of progressive and residual disease, we showed the ability of the novel IAP inhibitor, LCL161, to enhance the cytotoxic effects of tyrosine kinase inhibitors against leukemic disease. Importantly, we observed LCL161 to synergize *in vivo* with nilotinib to reduce leukemia burden significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib. Our results support the idea of using IAP inhibitors in conjunction with targeted tyrosine kinase inhibition to suppress or eradicate progressive and drug-resistant or residual disease. Phase I studies in advanced solid tumors are currently ongoing, and LCL161 is being considered for testing in clinical trials for leukemia.

#### **6. Conclusion**

52 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

and cell:cell interactions (Charbord et al. 1996). Bone marrow stroma and factors derived from stroma have been suggested to play a role in the long-term survival and proliferation of normal and leukemia cells (Ashley et al., 1994; Bradstock et al., 1996; Rafii et al., 1997; Lagneaux et al., 1998, Lagneaux et al., 1999; Konopleva et al., 2002; Litwin et al., 2002). Specifically, bone marrow stroma provides signals, such as granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and terminal differentiation of hematopoietic stem cells or mediate/support their proliferation (Verfaillie et al., 1993; Liesveld et al., 1996; Harrison et al., 1997; Breems et al., 1997; O'Prey et al., 1998; Leslie et al., 1998; Shih et al., 1999). SSpenic stroma, as well, has been implicated in enhanced survival of both normal and leukemic cells (Shaked et al., 2005; Despars and O'Neill, 2006). Clinical trial data with tyrosine kinase inhibitors show that while the peripheral blood of patients responds well, bone marrow responds less well. It appears possible that small numbers of leukemic CD34+ cells can persist in the marrow microenvironment of leukemia patients after years of therapy with kinase inhibitors. Stromal cells have been implicated, as they provide viability signals to leukemic cells that protect them from inhibitor effects. Indeed, the quantity of leukemic stem cells that rely on stroma to survive is predictive of

We revealed highest tumor burden and residual disease to occur *in vivo* in stroma-associated tissues in imatinib/nilotinib-treated NCr nude mice, suggesting that significant reservoirs for tumor growth appear to be tissues that are able to support normal hematopoietic and malignant stem cell development (Weisberg et al., 2008c). These studies, which showed a pattern of leukemia distribution consistent with what is observed in imatinib- and nilotinibtreated chronic myeloid leukemia patients, were followed by a more in-depth analysis of stroma-leukemia cell interactions that lead to protection of leukemia cells from nilotinibinduced cytotoxicity. For the latter, we used the human BCR-ABL-positive cell line, KU812F, and the human bone marrow stroma cell line, HS-5, to more closely approximate the bone marrow-associated cytoprotection observed in drug-treated leukemia patients. Partial stromal-mediated protection of BCR-ABL-positive leukemic cells from nilotinib treatment involved the cooperative interaction of members of a select panel of stromal-secreted viability factors, including IL-6 and GM-CSF (Weisberg et al., 2008c). Similar results were observed with mutant FLT3-positive leukemia cells treated with midostaurin in the presence of HS-5-stromal-conditioned media versus the same panel of stromal-secreted viability

**5. Potentiation of anti-leukemic effects of tyrosine kinase inhibitors by IAP inhibition: Implications for use of IAP inhibition to prevent or reduce residual** 

Deregulated signaling molecules associated with viability/apoptotic signaling represent attractive targets for therapeutic intervention, and several strategies have emerged that may be effective in preventing drug resistance due to this. One approach involves combining targeted inhibitors with small molecule inhibitors of key components of major signaling pathways affecting the viability/expansion of leukemic cells. Accordingly, we have developed effective inhibitors of the inhibitor of apoptosis (IAP) family of proteins (Liu et al., 2000; Wu et al., 2000), such as LBW242 (Weisberg et al., 2007b), and its structural analog, LCL161, which bind to and inhibit multiple IAPs (i.e. XIAP, c-IAP) to enhance the activity of different proapoptotic signaling pathways. We have demonstrated the ability of LBW242 to

disease outcome (Kumagai et al., 1996).

factors (Weisberg et al., 2009).

**disease following tyrosine kinase inhibition** 

Elucidation of mechanisms of resistance to tyrosine kinase inhibition is critical for the optimization of existing therapies and prolongation of patient survival via delaying or eradicating the recurrence of disease. For CML, the discovery and characterization of BCR-ABL point mutations in the kinase target of a number of inhibitors in clinical use has been a

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tremendous step forward in understanding an important underlying mechanism of drug resistance. While second generation BCR-ABL inhibitors, such as nilotinib and dasatinib, have proven to be highly effective in the clinic and override imatinib resistance to an extent, the existence of highly imatinib-resistant mutants, such as the T315I gatekeeper mutant, is a limiting factor. Even for third generation BCR-ABL inhibitors that are able to override T315I, the potential exists for the evolution of novel drug-resistant point mutations that impede the binding of drug to its target. Thus, there continues to be a need for development of BCR-ABL inhibitors with high potency and unique mutagenicity profiles, as well as a continued need for combination therapy approaches to overriding drug resistance.

For mutant FLT3-positive AML, due to the only transient and partial effects observed clinically with FLT3 inhibitors in late-stage development, there is an urgent need for the development of new treatment approaches that could potentially lead to improved clinical effectiveness. As with CML, the key to improved patient responsiveness may lie in continued development of compounds conferring a higher degree of potency, as well as novel combination therapy strategies.

For both CML and mutant FLT3-positive AML, elucidation of mechanisms of resistance that are associated with leukemic cell survival, such as stromal-mediated chemoresistance and up-regulation of viability signaling molecules, warrants the investigation of single agent activity of pro-apoptotic agents. It also warrants the development of treatment approaches that rely on administration of kinase inhibitors combined with other agents targeting components of leukemia:stromal cell interactions and stromal-derived viability factors.

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**4** 

*USA* 

*De Novo* **Acquisition of BCR-ABL Mutations** 

Chronic myelogenous leukemia (CML) is a rare and fatal neoplastic disease clinically presented as aberrant accumulation of myeloid cells in bone marrow, blood and spleen. CML occurs in 1 to 2 cases per 100,000 people, and is most common in older people with a median age at diagnosis of around 65. In the United States of America, there are 3500 to 5000 new cases per year (Jemal et al., 2010). If untreated, CML naturally progresses from initial chronic phase to accelerated phase and blast crisis that resembles acute myeloid leukemia. CML is one of the most extensively studied human cancers, and exemplifies that good

In early 1960s, a characteristic small chromosome of CML was identified by Peter Nowell and David Hungerford who worked in University of Pennsylvania and Fox Chase Cancer Center in Philadelphia, respectively, and was subsequently called the Philadelphia (Ph) chromosome (Nowell and Hungerford, 1960; Nowell and Hungerford, 1961). Ph chromosome is found in 95% CML patients. In 1973, Janet Rowley identified that Ph chromosome was a product of reciprocal translocation of chromosome 9 and 22, the t(9;22)(q34;q11) (Rowley, 1973). In early 1980s, the genes involved in translocation were identified as proto-oncogene ABL (human homolog of Abelson leukemia virus gene) on chromosome 9 that was fused to BCR (break cluster region) on chromosome 22 (Groffen et al., 1984), revealing molecular insight of CML. In early 1990s, aberrant tyrosine kinase activity of BCR-ABL and its essential roles in transformation of cells were established by several groups (Daley et al., 1990; Heisterkamp et al., 1990; Lugo et al., 1990). These molecular discoveries provide crucial foundation for drug development for CML treatment. In 1996, Brian Druker and colleagues reported the first specific ABL kinase inhibitor imatinib mesylate (STI571, CGP 57148, or Gleevec) that effectively inhibited growth of BCR-ABL positive cells (Druker et al., 1996). In 1998, a phase I clinical trial of imatinib was initiated in three centers in the United States (Druker et al., 2001). Imatinib shows magnificent effect on bringing CML patients, especially those in chronic phase, into remission and improving long-term survival and disease management (Druker et al., 2006),

However, in contrast to chronic CML, patients at advanced phases (accelerated phase and blast crisis) generally have only transient response to imatinib, and relapse quickly (Deininger and Druker, 2003). Even for chronic phase patients, imatinib does not eradicate the disease and it relapses rapidly if imatinib is discontinued (Michor et al., 2005). Multiple

scientific research can lead to successful treatment of a devastating disease.

and the drug is now the first line treatment for CML.

**1. Introduction** 

**for CML Acquired Resistance** 

WenYong Chen, Hongfeng Yuan and Zhiqiang Wang

*Department of Cancer Biology, Beckman Research Institute, City of Hope* 


### *De Novo* **Acquisition of BCR-ABL Mutations for CML Acquired Resistance**

WenYong Chen, Hongfeng Yuan and Zhiqiang Wang *Department of Cancer Biology, Beckman Research Institute, City of Hope USA* 

#### **1. Introduction**

68 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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Lyman, S.D. (1999). Endogenous FLT-3 ligand serum levels are associated with disease stage in patients with myelodysplastic syndromes. *Leukemia,* Apr;13(4), pp. Chronic myelogenous leukemia (CML) is a rare and fatal neoplastic disease clinically presented as aberrant accumulation of myeloid cells in bone marrow, blood and spleen. CML occurs in 1 to 2 cases per 100,000 people, and is most common in older people with a median age at diagnosis of around 65. In the United States of America, there are 3500 to 5000 new cases per year (Jemal et al., 2010). If untreated, CML naturally progresses from initial chronic phase to accelerated phase and blast crisis that resembles acute myeloid leukemia. CML is one of the most extensively studied human cancers, and exemplifies that good scientific research can lead to successful treatment of a devastating disease.

In early 1960s, a characteristic small chromosome of CML was identified by Peter Nowell and David Hungerford who worked in University of Pennsylvania and Fox Chase Cancer Center in Philadelphia, respectively, and was subsequently called the Philadelphia (Ph) chromosome (Nowell and Hungerford, 1960; Nowell and Hungerford, 1961). Ph chromosome is found in 95% CML patients. In 1973, Janet Rowley identified that Ph chromosome was a product of reciprocal translocation of chromosome 9 and 22, the t(9;22)(q34;q11) (Rowley, 1973). In early 1980s, the genes involved in translocation were identified as proto-oncogene ABL (human homolog of Abelson leukemia virus gene) on chromosome 9 that was fused to BCR (break cluster region) on chromosome 22 (Groffen et al., 1984), revealing molecular insight of CML. In early 1990s, aberrant tyrosine kinase activity of BCR-ABL and its essential roles in transformation of cells were established by several groups (Daley et al., 1990; Heisterkamp et al., 1990; Lugo et al., 1990). These molecular discoveries provide crucial foundation for drug development for CML treatment. In 1996, Brian Druker and colleagues reported the first specific ABL kinase inhibitor imatinib mesylate (STI571, CGP 57148, or Gleevec) that effectively inhibited growth of BCR-ABL positive cells (Druker et al., 1996). In 1998, a phase I clinical trial of imatinib was initiated in three centers in the United States (Druker et al., 2001). Imatinib shows magnificent effect on bringing CML patients, especially those in chronic phase, into remission and improving long-term survival and disease management (Druker et al., 2006), and the drug is now the first line treatment for CML.

However, in contrast to chronic CML, patients at advanced phases (accelerated phase and blast crisis) generally have only transient response to imatinib, and relapse quickly (Deininger and Druker, 2003). Even for chronic phase patients, imatinib does not eradicate the disease and it relapses rapidly if imatinib is discontinued (Michor et al., 2005). Multiple

*De Novo* Acquisition of BCR-ABL Mutations for CML Acquired Resistance 71

difference may suggest the increased functional selection for drug resistance in advanced

CML cells, mechanisms underlying such difference are unknown.

% of total mutations

adjacent to those regions.

M351T

M244V

E255K

Fig. 1. The top ten mutations of BCR-ABL in all or chronic CML patients

Y253H/F

F359V/I

Upper panel: Ten most frequent mutations in all CML. The profile was created by adding 344 mutation cases published between 2008 to 2010 to the previously published profile (Apperley, 2007). Lower panel: Ten most frequent mutations in chronic CML. The profile was created according to the previously compiled data containing 572 total mutation cases published from 2001 to 2009 (Klemm et al., 2009). Mutations in the regions of BCR-ABL kinase domain are color-coded: red for imatinib binding site, maroon for ATP binding site, green for catalytic domain, fuchsia for activation loop, and light blue for amino acids

Charles Sawyers and colleagues were also among the first to identify mutations of BCR-ABL kinase domain in CML patients before imatinib treatment (Roche-Lestienne et al., 2002; Shah et al., 2002), and proposed that BCR-ABL mutations in relapsed CML patients upon imatinib treatment are a result of selection of pre-existing mutant clones (Shah and Sawyers, 2003). This model has been validated by subsequent studies from multiple groups identifying such mutations (Ernst et al., 2008; Roche-Lestienne et al., 2003; Willis et al., 2005). Mathematical modeling further provides support for rapid expansion of pre-existing mutant clones for fast relapse in CML patients (Michor et al., 2005). However, the rate of BCR-ABL mutations found in CML patients prior to imatinib exposure is around 21%, even using high sensitivity

G250/A/E

T315I

H396R/P

E355G

F317L

% of total mutations

T315I

Y253H/F

E255D/K/R/V

G250A/E

M351T

F359C/L/V

H396P/R

F317C/L/V

E355/A/G

Chronic CML

 /K

all CML

M244V

mechanisms, both BCR-ABL dependent and independent, have been described or proposed for CML drug resistance, including amplification of BCR-ABL, mutations of BCR-ABL, drug pump, leukemia stem cell quiescence, as extensively reviewed before (Apperley, 2007). For the scope of this chapter, we will focus on CML acquired resistance, the resistance developed after initial remission of the disease, through BCR-ABL genetic mutations.

#### **2. Acquisition of genetic mutations of CML through drug selection of pre-existing mutants**

Charles Sawyers and colleagues first described that acquired resistance to imatinib is mediated primarily by BCR-ABL mutations, and to a less extent, by amplification of BCR-ABL gene and other mechanisms (Gorre et al., 2001; Shah et al., 2002). These mutations confer various degree of resistance of CML cells to imatinib. Compiled from published data from 2001 to 2007, over 50 unique mutations have been identified in all CML patients, and they are mainly located at the BCR-ABL kinase domain (Apperley, 2007). We have updated this mutation profile by adding data published from 2008 to 2010 (Chien et al., 2008; Jones et al., 2008; Kim et al., 2009; Lewandowski et al., 2009; Markose et al., 2009; Press, 2010). The ten most common mutations representing 74% of total mutations (Fig. 1) exhibit similar patterns as shown before (Apperley, 2007). T315I remains as the most frequent mutation followed by Y253 and E255 mutations. Some minor changes are noticed: G250A/E moves to the 4th place ahead of M351T whereas M244V moves to the 10th place.

The four critical regions of BCR-ABL kinase domain are the prime targets for these mutations: T315I mutation for imatinib binding site, mutations on Y253, E255 and G250 for ATP binding site or P-loop, mutations on M351, E355 and F359 for catalytic domain and mutations on H396 for activation loop (Nagar et al., 2002; Schindler et al., 2000). M244V mutation occurs in close proximity to the P-loop and F317 mutations close to imatinib binding site. Although it is not functionally clear about the significance of every mutation detected, the profile highlights the importance of those high frequency mutations. T315I mutant BCR-ABL is resistant to inhibition by the second generation of tyrosine kinase inhibitors nilotinib (von Bubnoff et al., 2006; Weisberg et al., 2005) and dasatinib (Shah et al., 2004), which inhibit most other BCR-ABL mutations. T315I and P-loop mutations are associated with reduced survival and poor prognosis in patients (Nicolini et al., 2006). Y253F and E255K mutations increase BCR-ABL transformation capability whereas T315I mutation affects oncogenicity by altering P-loop phosphorylation (Griswold et al., 2006; Skaggs et al., 2006).

Progression of chronic phase to advanced phases may require additional alterations of CML genome beyond BCR-ABL (Perrotti et al., 2010). It is perceived that mutation mechanisms may be different in chronic phase from that in blast crisis CML cells. We have plotted ten most frequent mutations in chronic phase CML (Fig. 1) collected from data published from 2001 to 2009 (according to Table S2 in Klemm et al., 2009). The ten most common mutations representing 78% of total mutations in chronic phase are the same amino acids at the BCR-ABL kinase domain as seen in the all CML profile. Noticeably, M351T, instead of T315I, mutation is the most frequent mutation in chronic CML. It is evident that there is substantial increase of T315I mutation and reduction of M244V mutation in all CML profile, suggesting T315I is more favored and M244V is less favored towards the advanced phases. In addition, the most frequent mutations are clustered more tightly according to the functional regions of the kinase domain in all CML profile than in chronic CML profile. Although the above

mechanisms, both BCR-ABL dependent and independent, have been described or proposed for CML drug resistance, including amplification of BCR-ABL, mutations of BCR-ABL, drug pump, leukemia stem cell quiescence, as extensively reviewed before (Apperley, 2007). For the scope of this chapter, we will focus on CML acquired resistance, the resistance developed after initial remission of the disease, through BCR-ABL genetic mutations.

Charles Sawyers and colleagues first described that acquired resistance to imatinib is mediated primarily by BCR-ABL mutations, and to a less extent, by amplification of BCR-ABL gene and other mechanisms (Gorre et al., 2001; Shah et al., 2002). These mutations confer various degree of resistance of CML cells to imatinib. Compiled from published data from 2001 to 2007, over 50 unique mutations have been identified in all CML patients, and they are mainly located at the BCR-ABL kinase domain (Apperley, 2007). We have updated this mutation profile by adding data published from 2008 to 2010 (Chien et al., 2008; Jones et al., 2008; Kim et al., 2009; Lewandowski et al., 2009; Markose et al., 2009; Press, 2010). The ten most common mutations representing 74% of total mutations (Fig. 1) exhibit similar patterns as shown before (Apperley, 2007). T315I remains as the most frequent mutation followed by Y253 and E255 mutations. Some minor changes are noticed: G250A/E moves to

The four critical regions of BCR-ABL kinase domain are the prime targets for these mutations: T315I mutation for imatinib binding site, mutations on Y253, E255 and G250 for ATP binding site or P-loop, mutations on M351, E355 and F359 for catalytic domain and mutations on H396 for activation loop (Nagar et al., 2002; Schindler et al., 2000). M244V mutation occurs in close proximity to the P-loop and F317 mutations close to imatinib binding site. Although it is not functionally clear about the significance of every mutation detected, the profile highlights the importance of those high frequency mutations. T315I mutant BCR-ABL is resistant to inhibition by the second generation of tyrosine kinase inhibitors nilotinib (von Bubnoff et al., 2006; Weisberg et al., 2005) and dasatinib (Shah et al., 2004), which inhibit most other BCR-ABL mutations. T315I and P-loop mutations are associated with reduced survival and poor prognosis in patients (Nicolini et al., 2006). Y253F and E255K mutations increase BCR-ABL transformation capability whereas T315I mutation affects oncogenicity by altering P-loop phosphorylation (Griswold et al., 2006; Skaggs et al.,

Progression of chronic phase to advanced phases may require additional alterations of CML genome beyond BCR-ABL (Perrotti et al., 2010). It is perceived that mutation mechanisms may be different in chronic phase from that in blast crisis CML cells. We have plotted ten most frequent mutations in chronic phase CML (Fig. 1) collected from data published from 2001 to 2009 (according to Table S2 in Klemm et al., 2009). The ten most common mutations representing 78% of total mutations in chronic phase are the same amino acids at the BCR-ABL kinase domain as seen in the all CML profile. Noticeably, M351T, instead of T315I, mutation is the most frequent mutation in chronic CML. It is evident that there is substantial increase of T315I mutation and reduction of M244V mutation in all CML profile, suggesting T315I is more favored and M244V is less favored towards the advanced phases. In addition, the most frequent mutations are clustered more tightly according to the functional regions of the kinase domain in all CML profile than in chronic CML profile. Although the above

**2. Acquisition of genetic mutations of CML through drug selection of** 

the 4th place ahead of M351T whereas M244V moves to the 10th place.

**pre-existing mutants** 

2006).

difference may suggest the increased functional selection for drug resistance in advanced CML cells, mechanisms underlying such difference are unknown.

Fig. 1. The top ten mutations of BCR-ABL in all or chronic CML patients Upper panel: Ten most frequent mutations in all CML. The profile was created by adding 344 mutation cases published between 2008 to 2010 to the previously published profile (Apperley, 2007). Lower panel: Ten most frequent mutations in chronic CML. The profile was created according to the previously compiled data containing 572 total mutation cases published from 2001 to 2009 (Klemm et al., 2009). Mutations in the regions of BCR-ABL kinase domain are color-coded: red for imatinib binding site, maroon for ATP binding site, green for catalytic domain, fuchsia for activation loop, and light blue for amino acids adjacent to those regions.

Charles Sawyers and colleagues were also among the first to identify mutations of BCR-ABL kinase domain in CML patients before imatinib treatment (Roche-Lestienne et al., 2002; Shah et al., 2002), and proposed that BCR-ABL mutations in relapsed CML patients upon imatinib treatment are a result of selection of pre-existing mutant clones (Shah and Sawyers, 2003). This model has been validated by subsequent studies from multiple groups identifying such mutations (Ernst et al., 2008; Roche-Lestienne et al., 2003; Willis et al., 2005). Mathematical modeling further provides support for rapid expansion of pre-existing mutant clones for fast relapse in CML patients (Michor et al., 2005). However, the rate of BCR-ABL mutations found in CML patients prior to imatinib exposure is around 21%, even using high sensitivity

*De Novo* Acquisition of BCR-ABL Mutations for CML Acquired Resistance 73

rate was found in other brands (Fig. 2).

Hyclone Brand B Brand C

Fig. 2. Tissue culture conditions affect BCR-ABL mutations

not absolutely required for acquisition of mutations.

ABL mutations that will be discussed further below.

maroon) were tested.

STI-resistant colony

number /well

cloning process. We have identified three clones that can acquire T315I, E255K and Y253H mutation, respectively. These three mutations have the highest frequency in human patients shown in Fig. 1. Third, the ability for acquisition of BCR-ABL mutations diminishes in parental KCL-22 cells after they are cultured continuously for more than 30 passages. Finally, we have found that the quality of fetal bovine serum can affect BCR-ABL mutation rate. We routinely use Hyclone characterized fetal bovine serum (Cat# SH30071.03) for mutation study, which typically produces consistent results among batches. However, when Hyclone serum was compared side by side with two other brands of fetal bovine serum, a noticeable reduction of BCR-ABL mutation

Colony number/well

Comparison of Hyclone characterized fetal bovine serum with two other brands of fetal bovine serum. KCL-22 cells were incubated with medium supplied with these sera, respectively, for two days, and then seeded onto soft agar for colony formation assay. Left panel, 1x106 cells seeded per well with 2.5 M imatinib. Right panel: plating efficiency with 500 cells seeded per well. Two independent batches of each serum (colored as blue and

4. Acquisition of BCR-ABL mutations depends on expression of BCR-ABL, since knockdown of BCR-ABL blocks KCL-22 cell relapse and mutations. By over-expression of kinase-inactive BCR-ABL, intriguingly, it was found that BCR-ABL kinase activity is

5. The endogenous BCR-ABL translocation locus significantly influences BCR-ABL mutations, as the mutation rate on the endogenous locus is nearly 10 times higher than that of the randomly integrated BCR-ABL cDNA as illustrated in Fig. 3. This finding reveals a previously unrecognized role of the translocation locus itself in driving BCR-ABL mutagenesis, suggesting possible involvement of the local epigenome for BCR-

Our extensive analyses of BCR-ABL mutations in KCL-22 cells lead to the conclusion that mutations can be acquired *de novo* after imatinib treatment, although precise molecular mechanisms are yet to be uncovered. Such *de novo* acquisition of mutations may provide an alternative mechanism for clinical resistance of CML to tyrosine kinase inhibitors (Fig. 4). This is particularly important given that the majority of CML patients at the time of diagnosis do not harbor detectable BCR-ABL mutations. *De novo* acquisition of BCR-ABL mutations does not necessarily have to operate exclusively from selection of pre-existing mutations. It is anticipated that these two modes of acquisition of BCR-ABL mutations may

0

Hyclone Brand B Brand C

50

100

150

200

methods for detection (Ernst et al., 2008; Roche-Lestienne et al., 2002; Willis et al., 2005), as compared to 40% to 90% in relapsed patients. This difference could be blamed to the insufficient sensitivity of detection methods. Alternatively, additional mechanisms for BCR-ABL mutations may exist and accentuate the mutations.

#### **3.** *De novo* **acquisition of genetic mutations for CML drug resistance**

To address mechanisms of CML acquired resistance, we have recently developed a novel tissue culture model using a naïve blast crisis CML cell line KCL-22 (Yuan et al., 2010). KCL-22 cells were derived from a female blast crisis CML patient, and were characterized as immature undifferentiated cells lacking lymphoid cell characteristics (Kubonishi and Miyoshi, 1983). KCL-22 cells are positive for myeloid cell markers including myeloid progenitor cell marker CD33. KCL-22 cells harbor two Ph chromosomes but do not have detectable BCR-ABL mutations (Yuan et al., 2010). In contrast to resistance of blast crisis CML in vivo, most blast crisis CML cell lines in culture are very sensitive to 1 M imatinib with exception of KCL-22 (Deininger et al., 1997). Imatinib also kills KCL-22 cells, but it requires higher concentrations and takes longer time (Yuan et al., 2010). This unique feature of KCL-22 cell line perhaps made us successful in establishing it as a new model for acquired resistance of CML in vitro. The key characteristics of this model are summarized as follows:


This model provides a novel tool for us to look into the process of acquisition of BCR-ABL mutations on the endogenous BCR-ABL in blast crisis CML cells, and has already provided valuable information in our initial study (Yuan et al., 2010) as summarized below.


methods for detection (Ernst et al., 2008; Roche-Lestienne et al., 2002; Willis et al., 2005), as compared to 40% to 90% in relapsed patients. This difference could be blamed to the insufficient sensitivity of detection methods. Alternatively, additional mechanisms for BCR-

To address mechanisms of CML acquired resistance, we have recently developed a novel tissue culture model using a naïve blast crisis CML cell line KCL-22 (Yuan et al., 2010). KCL-22 cells were derived from a female blast crisis CML patient, and were characterized as immature undifferentiated cells lacking lymphoid cell characteristics (Kubonishi and Miyoshi, 1983). KCL-22 cells are positive for myeloid cell markers including myeloid progenitor cell marker CD33. KCL-22 cells harbor two Ph chromosomes but do not have detectable BCR-ABL mutations (Yuan et al., 2010). In contrast to resistance of blast crisis CML in vivo, most blast crisis CML cell lines in culture are very sensitive to 1 M imatinib with exception of KCL-22 (Deininger et al., 1997). Imatinib also kills KCL-22 cells, but it requires higher concentrations and takes longer time (Yuan et al., 2010). This unique feature of KCL-22 cell line perhaps made us successful in establishing it as a new model for acquired resistance of CML in vitro. The key characteristics of this model are summarized as

1. High relevance: KCL-22 cells are incubated with in vivo therapeutic concentrations of imatinib, i.e. 2.5 to 10 M. Cells undergo gradually increasing apoptosis for about a week followed by re-growing (relapse) with homogenous acquisition of T315I mutation. 2. Easy to use: KCL-22 cell line is commercially available from German Collection of Cell Culture. Cells are easy to grow in standard RPMI 1640 medium supplied with 10% fetal bovine serum, and no additional growth factors are needed. One single dose of imatinib

3. Rapid turnaround: Relapse occurs in about two weeks with initial relapsed cells microscopically visible as small clusters of enlarged and sometimes irregularly shaped

4. High reproducibility: Multiple persons in the lab have used this model and it produces

This model provides a novel tool for us to look into the process of acquisition of BCR-ABL mutations on the endogenous BCR-ABL in blast crisis CML cells, and has already provided

1. Acquisition of BCR-ABL mutations does not require pre-existing mutant clones derived from the original patient, as clonal cells without an initial mutation can regenerate mutations and relapse on imatinib. The mutations do not derive from a fixed

2. Acquisition of BCR-ABL mutations does not provoke additional chromosomal rearrangement, is not a result of mutator phenotype of KCL-22 cells, and can not be

3. Acquisition of BCR-ABL mutations is a dynamic process influenced by culture and environmental conditions. This is supported by several lines of evidence. First, clonal KCL-22 cells exhibit great difference for BCR-ABL mutations whereas mutations on HPRT gene are relatively stable among clones. Second, clonal KCL-22 cells can acquire different single mutations for each clone, suggesting change of mutation hot spots after

valuable information in our initial study (Yuan et al., 2010) as summarized below.

**3.** *De novo* **acquisition of genetic mutations for CML drug resistance** 

ABL mutations may exist and accentuate the mutations.

follows:

treatment is sufficient.

cells after around eight days of drug treatment.

highly consistent results among them.

subpopulation of pre-mutant cells.

blocked by standard anti-oxidant treatment.

cloning process. We have identified three clones that can acquire T315I, E255K and Y253H mutation, respectively. These three mutations have the highest frequency in human patients shown in Fig. 1. Third, the ability for acquisition of BCR-ABL mutations diminishes in parental KCL-22 cells after they are cultured continuously for more than 30 passages. Finally, we have found that the quality of fetal bovine serum can affect BCR-ABL mutation rate. We routinely use Hyclone characterized fetal bovine serum (Cat# SH30071.03) for mutation study, which typically produces consistent results among batches. However, when Hyclone serum was compared side by side with two other brands of fetal bovine serum, a noticeable reduction of BCR-ABL mutation rate was found in other brands (Fig. 2).

Fig. 2. Tissue culture conditions affect BCR-ABL mutations Comparison of Hyclone characterized fetal bovine serum with two other brands of fetal bovine serum. KCL-22 cells were incubated with medium supplied with these sera, respectively, for two days, and then seeded onto soft agar for colony formation assay. Left panel, 1x106 cells seeded per well with 2.5 M imatinib. Right panel: plating efficiency with 500 cells seeded per well. Two independent batches of each serum (colored as blue and maroon) were tested.


Our extensive analyses of BCR-ABL mutations in KCL-22 cells lead to the conclusion that mutations can be acquired *de novo* after imatinib treatment, although precise molecular mechanisms are yet to be uncovered. Such *de novo* acquisition of mutations may provide an alternative mechanism for clinical resistance of CML to tyrosine kinase inhibitors (Fig. 4). This is particularly important given that the majority of CML patients at the time of diagnosis do not harbor detectable BCR-ABL mutations. *De novo* acquisition of BCR-ABL mutations does not necessarily have to operate exclusively from selection of pre-existing mutations. It is anticipated that these two modes of acquisition of BCR-ABL mutations may

*De Novo* Acquisition of BCR-ABL Mutations for CML Acquired Resistance 75

management caused by these two modes of mutation acquisition could be different. Whereas strong tyrosine kinase inhibitors may be needed to suppress expansion of preexisting mutant cells, prevention of *de novo* mutation acquisition by alternative strategies

So far, *de novo* acquisition of mutations has not yet been proved in patients. As described above, *de novo* mutation acquisition happens rapidly in the KCL-22 cell model in which early relapsed cells are microscopically visible after eight days of imatinib treatment. We have further found that mutations may likely form as early as two days after imatinib treatment (unpublished data). This rapid generation of mutations may prevent the appropriate use of mathematical models (Michor et al., 2005) to effectively distinguish between *de novo* mutation acquisition versus selection of pre-existing mutations. Separation of these two modes of mutation acquisition is further confounded by pre-existing mutations that may not be detected truly due to insufficient method sensitivity or by non-representative sample collection for analysis. It is currently unclear about the molecular signature of *de novo* mutation acquisition in CML patients or how to track this type of mutagenesis. However,

The mutation hot spots are different in three KCL-22 clones (clone L1 for E255K, clone L7 for Y253H and clone Ag 11 for T315I) (Yuan et al., 2010). When the codon changes are examined (TAC to CAC for Y253H, GAG to AAG for E255K, and ACT to ATT for T315I), a common molecular feature for these three mutations can be easily identified, namely, they are all transition mutations. It is important to note that both Y253 and E255 codons can acquire either transition or transversion mutation. Since these clonal cells are genetically identical, switching mutation hot spot from one transition mutation spot to another transition spot but not transversion, indicating that transition mutation is easier to be adapted on the BCR-ABL locus for kinase domain mutation. In line with this notion, frequencies for transition mutations for both Y253 and E255 codons are significantly higher than their transversion counterparts, Y253F and E255V in CML patients (Hochhaus et al., 2002; Press, 2010). Importantly, most of high frequency mutations shown in Fig. 1 are transition mutations. Further supporting this, a recent study shows that AID (activation-induced cytidine deaminase) promotes transition mutations in Ph+ B lymphoid blast crisis CML (Klemm et al., 2009). However, AID is activated in B lymphoid CML, but not in myeloid blast crisis CML including KCL-22 cells (Klemm et al., 2009), there may be additional mechanisms to enhance transitional mutations in myeloid blast crisis CML that makes up about 60% of all blast crisis CML (Calabretta and Perrotti, 2004). On the other hand, genetic mutations tend to have bias towards transition mutations (Wakeley, 1996). Therefore, additional studies including rigorous analysis of clinical samples are needed to explore if transition mutations

may indeed play a role in *de novo* acquisition of mutations in CML patients.

**selection of pre-existing mutations for drug resistance** 

**5. Mechanistic basis for integration of** *de novo* **mutation acquisition and** 

A key question about *de novo* acquisition of mutations is how it arises and integrates with selection of pre-existing mutations. The selection model for drug resistance is rooted in

**4. Search for molecular evidence of** *de novo* **acquisition of mutations in** 

may help block relapse of CML in this category as detailed later.

mutations in clonal KCL-22 cells may provide a clue.

**human CML patients** 

work in concert in certain patients leading to even faster relapse on the drug treatment. Therefore, we propose that *de novo* mutation acquisition and selection of pre-existing mutations are integrated processes for acquired resistance.

Fig. 3. Mutations on the endogenous BCR-ABL versus integrated BCR-ABL cDNA. Left, endogenous BCR-ABL (BA) was amplified by intron primers and integrated BCR-ABL was amplified by exon primers for mutation detection. Green for introns, blue for exons, asterisk for T315 mutation, and arrows for PCR primers. Right, relative mutation rate was the ratio of the number of clones bearing mutated endogenous BA or integrated BA cDNA divided by total number of clones, according to Yuan et al., 2010.

Fig. 4. Two modes of acquisition of BCR-ABL mutations for CML acquired resistance. Clinical relapse of CML on tyrosine kinase may be mediated by selection of pre-existing BCR-ABL mutant cells (green), *de novo* acquisition of BCR-ABL mutations (blue), or the combination of both. Non-mutant leukemia cells are in red. Two types of mutation acquisition could happen on the same amino acids.

Although the clinical readout for *de novo* acquisition and selection of pre-existing mutations are the same, i.e. relapse of the disease due to mutations, the implication for disease

work in concert in certain patients leading to even faster relapse on the drug treatment. Therefore, we propose that *de novo* mutation acquisition and selection of pre-existing

Relative mutation rate

Fig. 3. Mutations on the endogenous BCR-ABL versus integrated BCR-ABL cDNA.

divided by total number of clones, according to Yuan et al., 2010.

0

Left, endogenous BCR-ABL (BA) was amplified by intron primers and integrated BCR-ABL was amplified by exon primers for mutation detection. Green for introns, blue for exons, asterisk for T315 mutation, and arrows for PCR primers. Right, relative mutation rate was the ratio of the number of clones bearing mutated endogenous BA or integrated BA cDNA

> Selection of pre-existing mutation

de novo acquisition

*de novo*

Selection & de novo acquisition

*de novo*

Although the clinical readout for *de novo* acquisition and selection of pre-existing mutations are the same, i.e. relapse of the disease due to mutations, the implication for disease

Fig. 4. Two modes of acquisition of BCR-ABL mutations for CML acquired resistance. Clinical relapse of CML on tyrosine kinase may be mediated by selection of pre-existing BCR-ABL mutant cells (green), *de novo* acquisition of BCR-ABL mutations (blue), or the combination of both. Non-mutant leukemia cells are in red. Two types of mutation

acquisition could happen on the same amino acids.

0.1 0.2 0.3 0.4 0.5

> Endogenous BA translocation locus

Randomly integrated BA cDNA

mutations are integrated processes for acquired resistance.

**\***

Endogenous BA

Integrated BA cDNA

**\***

management caused by these two modes of mutation acquisition could be different. Whereas strong tyrosine kinase inhibitors may be needed to suppress expansion of preexisting mutant cells, prevention of *de novo* mutation acquisition by alternative strategies may help block relapse of CML in this category as detailed later.

#### **4. Search for molecular evidence of** *de novo* **acquisition of mutations in human CML patients**

So far, *de novo* acquisition of mutations has not yet been proved in patients. As described above, *de novo* mutation acquisition happens rapidly in the KCL-22 cell model in which early relapsed cells are microscopically visible after eight days of imatinib treatment. We have further found that mutations may likely form as early as two days after imatinib treatment (unpublished data). This rapid generation of mutations may prevent the appropriate use of mathematical models (Michor et al., 2005) to effectively distinguish between *de novo* mutation acquisition versus selection of pre-existing mutations. Separation of these two modes of mutation acquisition is further confounded by pre-existing mutations that may not be detected truly due to insufficient method sensitivity or by non-representative sample collection for analysis. It is currently unclear about the molecular signature of *de novo* mutation acquisition in CML patients or how to track this type of mutagenesis. However, mutations in clonal KCL-22 cells may provide a clue.

The mutation hot spots are different in three KCL-22 clones (clone L1 for E255K, clone L7 for Y253H and clone Ag 11 for T315I) (Yuan et al., 2010). When the codon changes are examined (TAC to CAC for Y253H, GAG to AAG for E255K, and ACT to ATT for T315I), a common molecular feature for these three mutations can be easily identified, namely, they are all transition mutations. It is important to note that both Y253 and E255 codons can acquire either transition or transversion mutation. Since these clonal cells are genetically identical, switching mutation hot spot from one transition mutation spot to another transition spot but not transversion, indicating that transition mutation is easier to be adapted on the BCR-ABL locus for kinase domain mutation. In line with this notion, frequencies for transition mutations for both Y253 and E255 codons are significantly higher than their transversion counterparts, Y253F and E255V in CML patients (Hochhaus et al., 2002; Press, 2010). Importantly, most of high frequency mutations shown in Fig. 1 are transition mutations. Further supporting this, a recent study shows that AID (activation-induced cytidine deaminase) promotes transition mutations in Ph+ B lymphoid blast crisis CML (Klemm et al., 2009). However, AID is activated in B lymphoid CML, but not in myeloid blast crisis CML including KCL-22 cells (Klemm et al., 2009), there may be additional mechanisms to enhance transitional mutations in myeloid blast crisis CML that makes up about 60% of all blast crisis CML (Calabretta and Perrotti, 2004). On the other hand, genetic mutations tend to have bias towards transition mutations (Wakeley, 1996). Therefore, additional studies including rigorous analysis of clinical samples are needed to explore if transition mutations may indeed play a role in *de novo* acquisition of mutations in CML patients.

#### **5. Mechanistic basis for integration of** *de novo* **mutation acquisition and selection of pre-existing mutations for drug resistance**

A key question about *de novo* acquisition of mutations is how it arises and integrates with selection of pre-existing mutations. The selection model for drug resistance is rooted in

*De Novo* Acquisition of BCR-ABL Mutations for CML Acquired Resistance 77

Epigenome

Acquisition of BCR-ABL mutations is a multi-step process that is regulated by BCR-ABL local epigenome. The epigenome is an important sensor of intrinsic oxidative stress signal (green) or extrinsic stress signal (red) including therapeutic or environmental stress, which in turn affects DNA damage, repair and cell survival, leading to maturation of genetic

**6. New opportunities for studying molecular mechanisms of acquired** 

In the coming years, it is anticipated that some steps of molecular regulation of mutation acquisition can be answered using the KCL-22 cell model. Here we discuss some steps that need obvious attention. Understanding these mutagenesis steps may help design new

1. **Regulation of DNA damage initiation.** The very origin of mutations, where DNA damage is from, remains to be clearly defined. Many chemotherapeutic agents deliberately induce DNA damage to kill cancer cells. In contrast, imatinib treatment reduces bulk ROS production and DNA damage response measured by H2AX (Yuan et al., 2010). It has been shown that anti-oxidants can inhibit BCR-ABL induced ROS production, DNA damage and mutations (Koptyra et al., 2006). However, anti-oxidants vitamin E and N-acetylcysteine, even in excessive amount, are unable to block acquisition of BCR-ABL mutations in KCL-22 cells (Yuan et al., 2010). There are several

Fig. 5. An epigenome model for acquisition of BCR-ABL mutations

Therapeutic stress

Spontaneous oxidative stress

Environmental stress and diet

mutations for acquired resistance of CML.

strategies to block BCR-ABL mutagenesis.

**resistance** 

DNA damage initiation

BCR-ABL locus

DNA damage repair

Mutant cell survival

CML relapse

Darwinian genetics, in which mutations can randomly form without providing survival advantage until environmental stress is imposed. It has been well recognized that mammalian genome is under constant genotoxic assault from intrinsic or extrinsic source, and DNA damage contributes significantly to genetic mutations (Friedberg, 2003; Wiseman and Halliwell, 1996). Transformation of BCR-ABL in hematopoietic progenitor cells increases production of reactive oxygen species (ROS) and DNA damage (Koptyra et al., 2006). Expression of BCR-ABL alters functions of cellular DNA damage repair machineries, especially for double-strand break (DSB) repair, which promotes CML genetic instability (Melo and Barnes, 2007). It is reasonable to believe that increased DNA damage and genetic instability would predispose CML cells to form mutations prior to drug treatment.

In the past two decades, significant progresses have been made in the understanding of epigenetics and epigenomics for their roles in shaping gene functions, particularly in cancer development (Jones and Baylin, 2007). Unlike genetic changes, epigenetic/ epigenomic changes are versatile and reversible, which bring dynamics to genetic codes and bridge genetic alterations to environmental changes. Epigenomics plays important roles in DNA damage repair, best studied for DSB repair. Several chromatin remodeling complexes are participated in modifying local chromatin structure to facilitate DNA damage repair (Rossetto et al., 2010). Epigenetic regulation of chemoresistance is a burgeoning field, and little is understood how it is involved in acquisition of genetic mutations for drug resistance.

We propose the following model as illustrated in Fig.5: Acquisition of BCR-ABL mutation is a multi-step process involving at least three core processes: DNA damage initiation, DNA damage repair and survival of newly mutated cells. These core processes are bound together through local epigenome that may serve as a sensor for stress signals and influence either exposure of local DNA to damage, repair process or cell survival. BCR-ABL locus may sustain constant DNA damage and repair, and the status of such DNA damage and repair determines whether the eventual relapse is mediated by selection of pre-existing mutations or *de novo* acquisition of mutations under stress.

Based on this model, we predict that under the steady state, the BCR-ABL locus has ongoing DNA damage and repair, and most of cells do not harbor mutations because most of DNA damage is repaired correctly. Inappropriate repair will lead to low incidence of mutations and allow selection of such pre-existing mutations for cell survival and relapse during chemotherapy. On the other hand, chemotherapy, including but not limited to imatinib, may provide an extrinsic stress signal through epigenome to the locus, which may either increase local DNA damage load or interfere functions of repair machineries, accentuating the mutagenesis process and resulting in *de novo* acquisition of mutations.

The definition of pre-existing mutations or *de novo* acquisition of mutations is based on the readout of DNA sequencing. However, DNA sequencing only detects completed mutations but not DNA damage. Therefore, a damaged base undetectable as a mutation by sequencing may emerge as a mutation, if it is not repaired on time or is repaired improperly. Alternatively, a mutation may emerge as a result of new DNA damage elicited by extrinsic stress. Consequently, *de novo* mutation acquisition would not require a fixed subpopulation as we have shown (Yuan et al., 2010), and it would be a dynamic process if environmental changes, such as cell growth conditions and nutrients, affect local epigenome and subsequently interfere the repair process.

Darwinian genetics, in which mutations can randomly form without providing survival advantage until environmental stress is imposed. It has been well recognized that mammalian genome is under constant genotoxic assault from intrinsic or extrinsic source, and DNA damage contributes significantly to genetic mutations (Friedberg, 2003; Wiseman and Halliwell, 1996). Transformation of BCR-ABL in hematopoietic progenitor cells increases production of reactive oxygen species (ROS) and DNA damage (Koptyra et al., 2006). Expression of BCR-ABL alters functions of cellular DNA damage repair machineries, especially for double-strand break (DSB) repair, which promotes CML genetic instability (Melo and Barnes, 2007). It is reasonable to believe that increased DNA damage and genetic

instability would predispose CML cells to form mutations prior to drug treatment.

mutations for drug resistance.

or *de novo* acquisition of mutations under stress.

subsequently interfere the repair process.

In the past two decades, significant progresses have been made in the understanding of epigenetics and epigenomics for their roles in shaping gene functions, particularly in cancer development (Jones and Baylin, 2007). Unlike genetic changes, epigenetic/ epigenomic changes are versatile and reversible, which bring dynamics to genetic codes and bridge genetic alterations to environmental changes. Epigenomics plays important roles in DNA damage repair, best studied for DSB repair. Several chromatin remodeling complexes are participated in modifying local chromatin structure to facilitate DNA damage repair (Rossetto et al., 2010). Epigenetic regulation of chemoresistance is a burgeoning field, and little is understood how it is involved in acquisition of genetic

We propose the following model as illustrated in Fig.5: Acquisition of BCR-ABL mutation is a multi-step process involving at least three core processes: DNA damage initiation, DNA damage repair and survival of newly mutated cells. These core processes are bound together through local epigenome that may serve as a sensor for stress signals and influence either exposure of local DNA to damage, repair process or cell survival. BCR-ABL locus may sustain constant DNA damage and repair, and the status of such DNA damage and repair determines whether the eventual relapse is mediated by selection of pre-existing mutations

Based on this model, we predict that under the steady state, the BCR-ABL locus has ongoing DNA damage and repair, and most of cells do not harbor mutations because most of DNA damage is repaired correctly. Inappropriate repair will lead to low incidence of mutations and allow selection of such pre-existing mutations for cell survival and relapse during chemotherapy. On the other hand, chemotherapy, including but not limited to imatinib, may provide an extrinsic stress signal through epigenome to the locus, which may either increase local DNA damage load or interfere functions of repair machineries, accentuating

The definition of pre-existing mutations or *de novo* acquisition of mutations is based on the readout of DNA sequencing. However, DNA sequencing only detects completed mutations but not DNA damage. Therefore, a damaged base undetectable as a mutation by sequencing may emerge as a mutation, if it is not repaired on time or is repaired improperly. Alternatively, a mutation may emerge as a result of new DNA damage elicited by extrinsic stress. Consequently, *de novo* mutation acquisition would not require a fixed subpopulation as we have shown (Yuan et al., 2010), and it would be a dynamic process if environmental changes, such as cell growth conditions and nutrients, affect local epigenome and

the mutagenesis process and resulting in *de novo* acquisition of mutations.

Fig. 5. An epigenome model for acquisition of BCR-ABL mutations Acquisition of BCR-ABL mutations is a multi-step process that is regulated by BCR-ABL local epigenome. The epigenome is an important sensor of intrinsic oxidative stress signal (green) or extrinsic stress signal (red) including therapeutic or environmental stress, which in turn affects DNA damage, repair and cell survival, leading to maturation of genetic mutations for acquired resistance of CML.

#### **6. New opportunities for studying molecular mechanisms of acquired resistance**

In the coming years, it is anticipated that some steps of molecular regulation of mutation acquisition can be answered using the KCL-22 cell model. Here we discuss some steps that need obvious attention. Understanding these mutagenesis steps may help design new strategies to block BCR-ABL mutagenesis.

1. **Regulation of DNA damage initiation.** The very origin of mutations, where DNA damage is from, remains to be clearly defined. Many chemotherapeutic agents deliberately induce DNA damage to kill cancer cells. In contrast, imatinib treatment reduces bulk ROS production and DNA damage response measured by H2AX (Yuan et al., 2010). It has been shown that anti-oxidants can inhibit BCR-ABL induced ROS production, DNA damage and mutations (Koptyra et al., 2006). However, anti-oxidants vitamin E and N-acetylcysteine, even in excessive amount, are unable to block acquisition of BCR-ABL mutations in KCL-22 cells (Yuan et al., 2010). There are several

*De Novo* Acquisition of BCR-ABL Mutations for CML Acquired Resistance 79

4. **Regulation of epigenome.** Epigenome is a central component in our model. Identifying epigenetic factors involved in acquired resistance will significantly help us understand the dynamic mutation acquisition process. DNA methylation plays an important role in genetic mutations (Jones and Baylin, 2002). Methylation of cytosine at CpG sites significantly increases C to T transition mutation because of the spontaneous hydrolytic deamination of methylated cytosine. Cytosine methylation in gene coding regions is associated with mutation hot spots of certain tumor suppressor genes, such as p53, in

Histone modifications are essential for DSB repair. Within minutes of DSB, histone H2A variant H2AX is phosphorylated and recruited to damage foci, which is followed by accumulation of DNA repair and chromatin remodeling factors (Rossetto et al., 2010). Several types of chromatin remodeling occur on and surround DSB sites. ATP-dependent chromatin remodeling complex INO80 is rapidly recruited by H2AX (Downs et al., 2004; Morrison et al., 2004; van Attikum et al., 2004). Recruitment of acetyltransferases and localized acetylation of histone H3 and H4 helps maintain open chromatin for repair (Tamburini and Tyler, 2005). Methylation of histone H4K20 also helps recruitment of repair factors through direct protein interaction (Botuyan et al., 2006; Sanders et al., 2004). When repair is completed, local deacetylation mediated by histone deacetylases restores chromatin structure. Importantly, the ability to modulate histone acetylation and deacetylation is essential for cell viability following HR repair, likely due to its signaling to prevent persistent activation of DNA damage checkpoint (Tamburini and Tyler, 2005). In addition, class III histone deacetylase SIRT1 is also recruited to DSB sites (O'Hagan et al., 2008) and regulates DNA damage response (Oberdoerffer et al., 2008; Yuan et al., 2007). It would be of great interest to define which and how chromatin modifiers response to therapeutic and

environmental stress signals to mediate DSB repair and BCR-ABL mutagenesis.

**7. Implication of** *de novo* **acquisition of genetic mutations in leukemia** 

Blocking outgrowth of mutant BCR-ABL clones has clear therapeutic significance, regardless how the mutations may be acquired. However, strategies used to overcome resistance may be different for pre-exiting mutations and *de novo* acquired mutations. The former demands more and more potent BCR-ABL inhibitors, which could lead to stronger resistance each time. This has been proved with sequential use of kinase inhibitors, resulting in selection of compound drug-resistant mutations (Shah et al., 2007). More potent third generation of tyrosine kinase inhibitors have been developed (Quintas-Cardama et al., 2010), including AP24534 that inhibits all tested BCR-ABL mutations and blocks outgrowth of mutant clones (O'Hare et al., 2009). Clinical resistance to the third generation of tyrosine kinase inhibitors

acquired resistance.

cancer.

**management** 

remains to be determined.

Marumoto et al., 2005). Aurora A over-expression overrides the mitotic spindle assembly checkpoint and promotes cancer cells resistance to chemotherapy (Anand et al., 2003). Interestingly, the pan-Aurora kinase inhibitor VX-680 not only inhibits Aurora kinases but also suppresses T315I BCR-ABL through distinct structural mechanisms (Carter et al., 2005; Young et al., 2006). Another mitotic kinase Polo-like kinase 1 (PLK1) is also known to mediate cancer chemoresistance (Luo et al., 2009). It remains to be determined if these and other mitotic regulators may play a role in CML

possibilities for this observation: these anti-oxidants do not have sufficient potency, the DNA damage is too severe to be blocked, the anti-oxidants are inappropriate for the type of stress and damage in these cells, and the BCR-ABL locus is not accessible for these agents in KCL-22 cells. It is formally possible that there might be other sources of DNA damage.

One interesting but not surprising observation is that after imatinib treatment, ROS level in early apoptotic cells surges by more than 100 folds (Yuan et al., 2010) (Fig. 6). This high level of ROS may initiate cell death pathway. It is provocative to speculate that mutant cells might be derived from rare early apoptotic cells as they somehow abandon apoptosis process but sustain substantial DNA damage, which could not be blocked by treatment with standard anti-oxidants, and would thus lead to genetic mutations.

Fig. 6. ROS levels in different apoptotic fractions of KCL-22 cells after imatinib treatment.


One interesting but not surprising observation is that after imatinib treatment, ROS level in early apoptotic cells surges by more than 100 folds (Yuan et al., 2010) (Fig. 6). This high level of ROS may initiate cell death pathway. It is provocative to speculate that mutant cells might be derived from rare early apoptotic cells as they somehow abandon apoptosis process but sustain substantial DNA damage, which could not be blocked by treatment with

100 101 102 103 104

Non-apoptotic Pre-apoptotic Late-apoptotic

Early apoptotic

Cou t

100 101 102 103 104

in acquisition of BCR-ABL mutations for acquired resistance to imatinib.

3. **Regulation of survival of emerging mutant cells.** The newly relapsed KCL-22 cells exhibit morphological enlargement and irregularity with significant increase of G2/M fraction (Yuan et al., 2010). This indicates that newly relapsed cells may likely suffer mitotic crisis as they slowly progress through cell cycle. This is perhaps the last and critical step for the completion of mutagenesis process to allow full outgrowth of mutant cells. Molecular machineries involved in mitotic regulation might play a role in survival of newly relapsed CML cells. Among them, Aurora serine/threonine kinases are critically involved in regulating mammalian cell division (Keen and Taylor, 2004;

Fig. 6. ROS levels in different apoptotic fractions of KCL-22 cells after imatinib treatment.

2. **Regulation of DNA damage repair**. BCR-ABL alters functions of both homologous recombination (HR) and non-homologous end joining (NHEJ) repair machineries for DSB repair (Melo and Barnes, 2007). NHEJ is the major repair pathway in higher eukaryotes (Khanna and Jackson, 2001). NHEJ repair consists of both classic and alternative pathways. Classic NHEJ has core components KU70/86 and DNA-PKcs. When classic NHEJ is inhibited, alternative NHEJ that is independent of KU70/86 and DNA-PKcs will be activated. BCR-ABL inhibits classic NHEJ by degrading the key NHEJ factor DNA-PKcs (Deutsch et al., 2001). Recently, it has been found that BCR-ABL activates alterative NHEJ through upregulation of Werner Syndrome protein (WRN) and DNA ligase IIIa in CML cells (Sallmyr et al., 2008). For HR repair, BCR-ABL increases expression of RAD51, a central HR factor, which abnormally increases DNA repair and enhances CML drug resistance (Slupianek et al., 2001). In addition, BCR-ABL expression compromises repair fidelity and increases DNA repair errors in CML cells (Nowicki et al., 2004). It will be interesting to determine whether the above mentioned repair mechanisms and factors, and perhaps other repair mechanisms, may be involved

standard anti-oxidants, and would thus lead to genetic mutations.

6.25%

ROS

100 101 102 103 104

Counts

DNA damage.

possibilities for this observation: these anti-oxidants do not have sufficient potency, the DNA damage is too severe to be blocked, the anti-oxidants are inappropriate for the type of stress and damage in these cells, and the BCR-ABL locus is not accessible for these agents in KCL-22 cells. It is formally possible that there might be other sources of Marumoto et al., 2005). Aurora A over-expression overrides the mitotic spindle assembly checkpoint and promotes cancer cells resistance to chemotherapy (Anand et al., 2003). Interestingly, the pan-Aurora kinase inhibitor VX-680 not only inhibits Aurora kinases but also suppresses T315I BCR-ABL through distinct structural mechanisms (Carter et al., 2005; Young et al., 2006). Another mitotic kinase Polo-like kinase 1 (PLK1) is also known to mediate cancer chemoresistance (Luo et al., 2009). It remains to be determined if these and other mitotic regulators may play a role in CML acquired resistance.

4. **Regulation of epigenome.** Epigenome is a central component in our model. Identifying epigenetic factors involved in acquired resistance will significantly help us understand the dynamic mutation acquisition process. DNA methylation plays an important role in genetic mutations (Jones and Baylin, 2002). Methylation of cytosine at CpG sites significantly increases C to T transition mutation because of the spontaneous hydrolytic deamination of methylated cytosine. Cytosine methylation in gene coding regions is associated with mutation hot spots of certain tumor suppressor genes, such as p53, in cancer.

Histone modifications are essential for DSB repair. Within minutes of DSB, histone H2A variant H2AX is phosphorylated and recruited to damage foci, which is followed by accumulation of DNA repair and chromatin remodeling factors (Rossetto et al., 2010). Several types of chromatin remodeling occur on and surround DSB sites. ATP-dependent chromatin remodeling complex INO80 is rapidly recruited by H2AX (Downs et al., 2004; Morrison et al., 2004; van Attikum et al., 2004). Recruitment of acetyltransferases and localized acetylation of histone H3 and H4 helps maintain open chromatin for repair (Tamburini and Tyler, 2005). Methylation of histone H4K20 also helps recruitment of repair factors through direct protein interaction (Botuyan et al., 2006; Sanders et al., 2004). When repair is completed, local deacetylation mediated by histone deacetylases restores chromatin structure. Importantly, the ability to modulate histone acetylation and deacetylation is essential for cell viability following HR repair, likely due to its signaling to prevent persistent activation of DNA damage checkpoint (Tamburini and Tyler, 2005). In addition, class III histone deacetylase SIRT1 is also recruited to DSB sites (O'Hagan et al., 2008) and regulates DNA damage response (Oberdoerffer et al., 2008; Yuan et al., 2007). It would be of great interest to define which and how chromatin modifiers response to therapeutic and environmental stress signals to mediate DSB repair and BCR-ABL mutagenesis.

#### **7. Implication of** *de novo* **acquisition of genetic mutations in leukemia management**

Blocking outgrowth of mutant BCR-ABL clones has clear therapeutic significance, regardless how the mutations may be acquired. However, strategies used to overcome resistance may be different for pre-exiting mutations and *de novo* acquired mutations. The former demands more and more potent BCR-ABL inhibitors, which could lead to stronger resistance each time. This has been proved with sequential use of kinase inhibitors, resulting in selection of compound drug-resistant mutations (Shah et al., 2007). More potent third generation of tyrosine kinase inhibitors have been developed (Quintas-Cardama et al., 2010), including AP24534 that inhibits all tested BCR-ABL mutations and blocks outgrowth of mutant clones (O'Hare et al., 2009). Clinical resistance to the third generation of tyrosine kinase inhibitors remains to be determined.

*De Novo* Acquisition of BCR-ABL Mutations for CML Acquired Resistance 81

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For mutations acquired *de novo*, however, a prevention strategy may be more appropriate and effective to block mutant cells from emergence at the beginning. With better understanding of mechanisms of mutations described above, it is expected that a new agent may be devised to modulate DNA damage initiation, repair, epigenome remodeling, or survival of newly emerged mutant cells, which will prevent CML acquired resistance in future. A combination therapy may be adapted with such a new agent and imatinib or other tyrosine kinase inhibitors. Alternatively, it is also reasonable to speculate that a multi-target agent can be developed to incorporate these features, for example, a drug that both inhibits BCR-ABL tyrosine kinase and modulates epigenome. We anticipate different ways to overcome resistance depending on which steps and factors the drug would target to block mutagenesis. We expect that such a strategy may also have broader implication for management of other types of leukemia to overcome drug resistance.

#### **8. Acknowledgements**

This study is supported by the following grants: a career development award from the STOPCANCER Foundation, W81XWH-06-1-0268 from US Department of Defense, a translational research grant from the V-Foundation, and NCI R01 CA143421 to W.Y.C. The core facilities used in this study were supported by NCI P30 CA033572. The contents are solely the responsibility of the authors and do not represent the official views of the National Cancer Institute or NIH.

#### **9. References**


For mutations acquired *de novo*, however, a prevention strategy may be more appropriate and effective to block mutant cells from emergence at the beginning. With better understanding of mechanisms of mutations described above, it is expected that a new agent may be devised to modulate DNA damage initiation, repair, epigenome remodeling, or survival of newly emerged mutant cells, which will prevent CML acquired resistance in future. A combination therapy may be adapted with such a new agent and imatinib or other tyrosine kinase inhibitors. Alternatively, it is also reasonable to speculate that a multi-target agent can be developed to incorporate these features, for example, a drug that both inhibits BCR-ABL tyrosine kinase and modulates epigenome. We anticipate different ways to overcome resistance depending on which steps and factors the drug would target to block mutagenesis. We expect that such a strategy may also have broader implication for

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Z., Hoffmann, T.K., Kim, Y.M., et al. (2009). The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia. Cancer Cell

M., Blasiak, J., and Skorski, T. (2006). BCR/ABL kinase induces self-mutagenesis via reactive oxygen species to encode imatinib resistance. Blood 108, 319-327. Kubonishi, I., and Miyoshi, I. (1983). Establishment of a Ph1 chromosome-positive cell line

from chronic myelogenous leukemia in blast crisis. International journal of cell

Sacha, T., Gniot, M., Majewski, M., Solarska, I., et al. (2009). Frequency of BCR-ABL gene mutations in Polish patients with chronic myeloid leukemia treated with imatinib: a final report of the MAPTEST study. Polskie Archiwum Medycyny

and Elledge, S.J. (2009). A genome-wide RNAi screen identifies multiple synthetic

Mathews, V., George, B., Viswabandya, A., Srivastava, A., and Chandy, M. (2009). Spectrum of BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia from India with suspected resistance to imatinib-mutations are rare and


**5** 

*1US 2Canada* 

**Targeting the Chronic Myeloid Leukemia** 

*2Terry Fox Laboratory, British Columbia Cancer Agency and Departments of Medical* 

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder of pluripotent hematopoietic stem/progenitor cells that has been paradigmatic to our understanding of the molecular and cellular basis of human malignancies. It has provided an excellent example of how a specific molecular abnormality can be targeted therapeutically to transform a lifethreatening malignancy into a chronic disease. The study of CML has been characterized by a number of 'firsts'. CML was the first malignancy to be: (i) associated with a specific chromosomal abnormality, (ii) associated with a specific molecular alteration (*BCR-ABL*) and (iii) successfully treated with a specifically designed targeted therapeutic agent. As such it seems natural that CML should be the first human malignancy in which a complete medical cure is achieved through the eradication of cancer stem cells. This should be realizable through combining the specific targeting of BCR-ABL and CML stem cells. Once this has been achieved, the challenge will be to successfully transfer the lessons learned from this relatively simple and well-characterized model system to the eradication of cancer stem

The treatment of CML has changed dramatically following the introduction into the clinic of the tyrosine kinase inhibitor (TKI) imatinib mesylate and second generation TKIs. These agents directly target the BCR-ABL oncoprotein product of the constitutively active BCR-ABL tyrosine kinase. The specific targeting of BCR-ABL induces durable clinical remission in a high proportion of chronic phase CML (CP-CML) subjects (5 year survival of 89%) (Druker et al, 2006). Although a major molecular response (defined as a 3-log reduction in *BCR-ABL* mutant allele burden) is obtained in many CP-CML patients, only a small number attain PCR negativity as determined by the absence of residual *BCR-ABL* transcripts (Hughes et al, 2003). This is because TKI therapy does not specifically target or eliminate leukemia stem cells (LSCs). Indeed TKI therapy alone is unlikely to ever be curative, as following treatment with TKIs LSCs persist in bone marrow (BM) stem cell niches where they harbor the potential for relapse. The emergence of resistance to TKI monotherapy

**1. Introduction** 

cells in more complex malignancies.

**Stem Cell: A Paradigm for the Curative** 

*Genetics and Medicine, University of British Columbia, Vancouver, BC,* 

**Treatment of Human Malignancies** 

*1Discovery Medicine and Clinical Pharmacology Oncology,* 

Adrian Woolfson1 and Xiaoyan Jiang2

*Bristol-Myers Squibb, Princeton,* 


### **Targeting the Chronic Myeloid Leukemia Stem Cell: A Paradigm for the Curative Treatment of Human Malignancies**

Adrian Woolfson1 and Xiaoyan Jiang2

*1Discovery Medicine and Clinical Pharmacology Oncology, Bristol-Myers Squibb, Princeton, 2Terry Fox Laboratory, British Columbia Cancer Agency and Departments of Medical Genetics and Medicine, University of British Columbia, Vancouver, BC, 1US 2Canada* 

#### **1. Introduction**

84 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Shah, N.P., and Sawyers, C.L. (2003). Mechanisms of resistance to STI571 in Philadelphia

Shah, N.P., Skaggs, B.J., Branford, S., Hughes, T.P., Nicoll, J.M., Paquette, R.L., and Sawyers,

Shah, N.P., Tran, C., Lee, F.Y., Chen, P., Norris, D., and Sawyers, C.L. (2004). Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305, 399-401. Skaggs, B.J., Gorre, M.E., Ryvkin, A., Burgess, M.R., Xie, Y., Han, Y., Komisopoulou, E.,

Slupianek, A., Schmutte, C., Tombline, G., Nieborowska-Skorska, M., Hoser, G., Nowicki,

van Attikum, H., Fritsch, O., Hohn, B., and Gasser, S.M. (2004). Recruitment of the INO80

von Bubnoff, N., Manley, P.W., Mestan, J., Sanger, J., Peschel, C., and Duyster, J. (2006). Bcr-

Wakeley, J. (1996). The excess of transitions among nucleotide substitutions: new methods of

Weisberg, E., Manley, P.W., Breitenstein, W., Bruggen, J., Cowan-Jacob, S.W., Ray, A., Huntly,

cytogenetic evolution but not response to therapy. Blood 106, 2128-2137. Wiseman, H., and Halliwell, B. (1996). Damage to DNA by reactive oxygen and nitrogen

Young, M.A., Shah, N.P., Chao, L.H., Seeliger, M., Milanov, Z.V., Biggs, W.H., 3rd, Treiber,

Yuan, H., Wang, Z., Gao, C., Chen, W., Huang, Q., Yee, J.K., Bhatia, R., and Chen, W.Y.

Yuan, Z., Zhang, X., Sengupta, N., Lane, W.S., and Seto, E. (2007). SIRT1 regulates the function of the Nijmegen breakage syndrome protein. Mol Cell 27, 149-162.

with DNA double-strand break repair. Cell 119, 777-788.

C.L. (2007). Sequential ABL kinase inhibitor therapy selects for compound drugresistant BCR-ABL mutations with altered oncogenic potency. J Clin Invest 117,

Brown, L.M., Loo, J.A., Landaw, E.M., et al. (2006). Phosphorylation of the ATPbinding loop directs oncogenicity of drug-resistant BCR-ABL mutants. Proc Natl

M.O., Pierce, A.J., Fishel, R., and Skorski, T. (2001). BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance. Mol Cell 8, 795-806. Tamburini, B.A., and Tyler, J.K. (2005). Localized histone acetylation and deacetylation

triggered by the homologous recombination pathway of double-strand DNA

complex by H2A phosphorylation links ATP-dependent chromatin remodeling

Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107).

estimating transition bias underscore its significance. Trends in ecology &

B., Fabbro, D., Fendrich, G., Hall-Meyers, E., et al. (2005). Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 7, 129-141. Willis, S.G., Lange, T., Demehri, S., Otto, S., Crossman, L., Niederwieser, D., Stoffregen, E.P.,

McWeeney, S., Kovacs, I., Park, B., et al. (2005). High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal

species: role in inflammatory disease and progression to cancer. Biochem J 313 ( Pt

D.K., Patel, H.K., Zarrinkar, P.P., Lockhart, D.J., et al. (2006). Structure of the kinase domain of an imatinib-resistant Abl mutant in complex with the Aurora kinase

(2010). BCR-ABL gene expression is required for its mutations in a novel KCL-22 cell culture model for acquired resistance of chronic myelogenous leukemia. J Biol

chromosome-associated leukemias. Oncogene 22, 7389-7395.

2562-2569.

Acad Sci U S A 103, 19466-19471.

repair. Mol Cell Biol 25, 4903-4913.

inhibitor VX-680. Cancer Res 66, 1007-1014.

Blood 108, 1328-1333.

evolution 11, 158-162.

Chem 285, 5085-5096.

1), 17-29.

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder of pluripotent hematopoietic stem/progenitor cells that has been paradigmatic to our understanding of the molecular and cellular basis of human malignancies. It has provided an excellent example of how a specific molecular abnormality can be targeted therapeutically to transform a lifethreatening malignancy into a chronic disease. The study of CML has been characterized by a number of 'firsts'. CML was the first malignancy to be: (i) associated with a specific chromosomal abnormality, (ii) associated with a specific molecular alteration (*BCR-ABL*) and (iii) successfully treated with a specifically designed targeted therapeutic agent. As such it seems natural that CML should be the first human malignancy in which a complete medical cure is achieved through the eradication of cancer stem cells. This should be realizable through combining the specific targeting of BCR-ABL and CML stem cells. Once this has been achieved, the challenge will be to successfully transfer the lessons learned from this relatively simple and well-characterized model system to the eradication of cancer stem cells in more complex malignancies.

The treatment of CML has changed dramatically following the introduction into the clinic of the tyrosine kinase inhibitor (TKI) imatinib mesylate and second generation TKIs. These agents directly target the BCR-ABL oncoprotein product of the constitutively active BCR-ABL tyrosine kinase. The specific targeting of BCR-ABL induces durable clinical remission in a high proportion of chronic phase CML (CP-CML) subjects (5 year survival of 89%) (Druker et al, 2006). Although a major molecular response (defined as a 3-log reduction in *BCR-ABL* mutant allele burden) is obtained in many CP-CML patients, only a small number attain PCR negativity as determined by the absence of residual *BCR-ABL* transcripts (Hughes et al, 2003). This is because TKI therapy does not specifically target or eliminate leukemia stem cells (LSCs). Indeed TKI therapy alone is unlikely to ever be curative, as following treatment with TKIs LSCs persist in bone marrow (BM) stem cell niches where they harbor the potential for relapse. The emergence of resistance to TKI monotherapy

Targeting the Chronic Myeloid Leukemia Stem Cell:

A Paradigm for the Curative Treatment of Human Malignancies 87

that has significantly enhanced and constitutive tyrosine kinase activity, which drives the pathogenic features of the disease (Druker et al., 1996; Lugo et al., 1990). It produces a range of biochemical changes that impact the growth-factor dependence, turnover, and genomic stability of primitive CD34+ leukemic cells, whilst at the same time having little impact on their ability to differentiate according to predefined molecular programs (Holyoake et al.,

The identification of an invariant molecular genetic alteration (*BCR-ABL*) in the vast majority of cases of CML has facilitated the development of rational targeted therapy focused on the selective inhibition of the dysregulated tyrosine kinase activity of the encoded BCR-ABL oncoprotein (Druker et al., 1996; Shah et al., 2004; Weisberg et al., 2005). Imatinib mesylate (IM, Novartis, Basel, Swizerland) was the first tyrosine inhibitor to be developed as a molecular targeted drug (Druker et al., 1996). It is a competitive inhibitor of the ATP-binding site of the ABL-kinase domain, and prevents a conformational change of the oncoprotein to its active form, resulting in the elimination of most *BCR-ABL*+ cells (Druker et al., 1996). This relatively selective agent (it also recognizes the ATP-binding site of the c-Kit and platelet-derived growth factor receptors) has been immensely effective in the treatment of subjects with CP-CML (Druker et al., 2006; Druker et al., 2001; Kantarjian et al., 2002; O'Brien et al., 2003). Nevertheless, early relapse and the emergence of IM resistance are observed in 10-20% of subjects in early CP-CML, and up to 40% of those with advanced phase disease including 1-3% of newly diagnosed CML patients that develop sudden blast crisis (Apperley, 2007; Deininger et al., 2005; Forrest et al., 2008; Kantarjian et al., 2003; O'Hare et al., 2006; Valent, 2008). The inability to successfully discontinue IM therapy following at least five years of therapy, the persistence of a reservoir of clonal leukemic stem cells following the attainment of a complete molecular response (CMR), and the uncertain safety profile of long-term TKI treatment, has led to differing views on the most appropriate choice of therapy in CP-CML (Mahon et al., 2010; Ross et al., 2010a; Ross et al., 2010b; Rousselot et al., 2007; Sobrinho-Simoes et al., 2010). Recently, second-line TKIs including Dasatinib (DA,Bristol-Myers Squibb, New York, NY, USA) and Nilotinib (NL, Novatis) have been licensed for use in this indication and represent alternative therapeutic options either first-line or for resistant or intolerant cases. Both drugs have increased potency against the *BCR-ABL* kinase domain mutants most commonly associated with IM resistance. This along with their differing spectrums of inhibitory activity across the human kinome, has translated into increased clinical efficacy in subjects with IM-resistant disease (Carter et al., 2005; Shah et al., 2004; Weisberg et al., 2005). The major cytogenetic response (MCyR) rate following therapy with DA or NL in subjects with IM-resistant CP-CML is approximately 60%, with a complete cytogenetic response (CCyR) rate of 50% (Hochhaus et al., 2008; Kantarjian et al., 2007). Two recent Phase 3 randomized trials in subjects with treatment naïve early stage CP-CML demonstrated that both drugs are more effective than IM at inducing MCyR and major molecular (MMR) responses (Kantarjian et al., 2010; Saglio et al., 2010). DA and NL were subsequently approved by the U.S. Food and Drug Administration (FDA) as first-line therapies in CP-CML. Clinical experience, however, has shown that some subjects experience inadequate responses to all existing TKI therapies, or have an initial response but then progress rapidly (Kantarjian et al., 2006; Talpaz et al., 2006). As is the case in 60% of subjects with IM-resistant disease, the recalcitrant T315I mutation also routinely dominates the observed resistance with DA and NL (Apperley, 2007; Goldman, 2007). As a result these agents have no benefit over IM in subjects whose resistance is thought to be mediated

2002; Jiang et al., 2007b; Penserga & Skorski, 2007; Sloma et al., 2010; Valent, 2008).

through the accumulation of somatic kinase domain mutations that interfere with the binding of TKIs to the BCR-ABL ATP-binding site accounts for around 60-90% of relapses (O'Hare et al, 2006). There is consequently a significant unmet medical need for more effective therapeutic strategies that following effective tumor debulking are able to: (i) inhibit the molecular mechanisms responsible for generating the LSC genomic instability phenotype, (ii) target the essential components of the stem cell niche and the BM microenvironment that generate, protect, and nurture LSCs and (iii) efficiently eradicate LSCs. The micro-evolution of TKI resistance in CML is driven by the intrinsic genomic instability of the LSC, which in the presence of the selective pressure of drug, results in the expansion of a relatively predictable and invariant quasispecies of somatic mutants, which have differing degrees of intrinsic and acquired TKI resistance. The frequency of each of these clones oscillates with time, with a unitary or oligoclonal set eventually dominating the structure of the population and the clinical response to TKI therapy. The spectrum of mutants in any individual may be characterized through mutational analysis and subsequently used to select the most appropriate TKI therapy. The persistence of LSCs, however, allows additional drug-resistant mutants to arise, creating a new repertoire of diversity from which the most resistant and fastest growing clones are selected. As a result the therapeutic effects of first- and second-line TKI therapy are eventually circumvented. Clearly the eradication of the LSCs must form both a necessary and essential component of any therapeutic strategies that aim to achieve a deep and sustained molecular and clinical response, and ultimately a cure. In what follows we outline a number of approaches to the characterization of the CML LSC. The profiling and characterization of the LSC phenotype is expected to contribute to the rational design of LSC-targeted therapy, and as such presents an opportunity to establish a general paradigm for the development of cancer stem celldirected cures for human malignancies.

#### **2. Clinical and therapeutic challenges in the management of CML**

CML is a clonal, multi-step and multi-lineage myeloproliferative disease that typically evolves through three phenotypically and clinically distinct stages (Goldman & Melo, 2003; Jiang X. 2007; Savona & Talpaz, 2008; Sloma et al., 2010). The first of these is an indolent chronic phase (CP) characterized at the time of diagnosis by the presence of a deregulated *BCR-ABL*+ clone. This expands inappropriately and comes to dominate the population of BM progenitor cells, while at the same time continuing to produce phenotypically normal mature blood cells. There is, as a result, an excessive output of myeloid precursors and mature granulocytes into the BM and peripheral blood (PB). The second stage is an accelerated phase (AP) characterized by an incremental increase in the disease burden as demonstrated by an increased frequency of leukemic myeloid progenitor/precursor cells. The third stage is a rapidly fatal acute blast crisis phase (BC) characterized by increased genomic instability, deregulated proliferation and loss of differentiation. BC-CML may be categorized as myeloid or lymphoid (pre-B) by the appearance of increased numbers of differentiation-arrested blast cells that reflect the growth of sub-clones of early myeloid or pre-B cells respectively that have acquired additional somatic mutations (Goldman & Melo, 2003; Jiang X. 2007; Savona & Talpaz, 2008; Sloma et al., 2010). The canonical feature of CML is the presence in hematopoietic stem cell (HSC) derived progeny of a Philadelphia chromosome (Ph+) containing a reciprocal t(9;22)(q34;q11) translocation, which generates a clone-specific *BCR-ABL* fusion oncogene. This encodes a chimeric BCR-ABL oncoprotein

through the accumulation of somatic kinase domain mutations that interfere with the binding of TKIs to the BCR-ABL ATP-binding site accounts for around 60-90% of relapses (O'Hare et al, 2006). There is consequently a significant unmet medical need for more effective therapeutic strategies that following effective tumor debulking are able to: (i) inhibit the molecular mechanisms responsible for generating the LSC genomic instability phenotype, (ii) target the essential components of the stem cell niche and the BM microenvironment that generate, protect, and nurture LSCs and (iii) efficiently eradicate LSCs. The micro-evolution of TKI resistance in CML is driven by the intrinsic genomic instability of the LSC, which in the presence of the selective pressure of drug, results in the expansion of a relatively predictable and invariant quasispecies of somatic mutants, which have differing degrees of intrinsic and acquired TKI resistance. The frequency of each of these clones oscillates with time, with a unitary or oligoclonal set eventually dominating the structure of the population and the clinical response to TKI therapy. The spectrum of mutants in any individual may be characterized through mutational analysis and subsequently used to select the most appropriate TKI therapy. The persistence of LSCs, however, allows additional drug-resistant mutants to arise, creating a new repertoire of diversity from which the most resistant and fastest growing clones are selected. As a result the therapeutic effects of first- and second-line TKI therapy are eventually circumvented. Clearly the eradication of the LSCs must form both a necessary and essential component of any therapeutic strategies that aim to achieve a deep and sustained molecular and clinical response, and ultimately a cure. In what follows we outline a number of approaches to the characterization of the CML LSC. The profiling and characterization of the LSC phenotype is expected to contribute to the rational design of LSC-targeted therapy, and as such presents an opportunity to establish a general paradigm for the development of cancer stem cell-

directed cures for human malignancies.

**2. Clinical and therapeutic challenges in the management of CML** 

CML is a clonal, multi-step and multi-lineage myeloproliferative disease that typically evolves through three phenotypically and clinically distinct stages (Goldman & Melo, 2003; Jiang X. 2007; Savona & Talpaz, 2008; Sloma et al., 2010). The first of these is an indolent chronic phase (CP) characterized at the time of diagnosis by the presence of a deregulated *BCR-ABL*+ clone. This expands inappropriately and comes to dominate the population of BM progenitor cells, while at the same time continuing to produce phenotypically normal mature blood cells. There is, as a result, an excessive output of myeloid precursors and mature granulocytes into the BM and peripheral blood (PB). The second stage is an accelerated phase (AP) characterized by an incremental increase in the disease burden as demonstrated by an increased frequency of leukemic myeloid progenitor/precursor cells. The third stage is a rapidly fatal acute blast crisis phase (BC) characterized by increased genomic instability, deregulated proliferation and loss of differentiation. BC-CML may be categorized as myeloid or lymphoid (pre-B) by the appearance of increased numbers of differentiation-arrested blast cells that reflect the growth of sub-clones of early myeloid or pre-B cells respectively that have acquired additional somatic mutations (Goldman & Melo, 2003; Jiang X. 2007; Savona & Talpaz, 2008; Sloma et al., 2010). The canonical feature of CML is the presence in hematopoietic stem cell (HSC) derived progeny of a Philadelphia chromosome (Ph+) containing a reciprocal t(9;22)(q34;q11) translocation, which generates a clone-specific *BCR-ABL* fusion oncogene. This encodes a chimeric BCR-ABL oncoprotein that has significantly enhanced and constitutive tyrosine kinase activity, which drives the pathogenic features of the disease (Druker et al., 1996; Lugo et al., 1990). It produces a range of biochemical changes that impact the growth-factor dependence, turnover, and genomic stability of primitive CD34+ leukemic cells, whilst at the same time having little impact on their ability to differentiate according to predefined molecular programs (Holyoake et al., 2002; Jiang et al., 2007b; Penserga & Skorski, 2007; Sloma et al., 2010; Valent, 2008). The identification of an invariant molecular genetic alteration (*BCR-ABL*) in the vast majority of cases of CML has facilitated the development of rational targeted therapy focused on the selective inhibition of the dysregulated tyrosine kinase activity of the encoded BCR-ABL oncoprotein (Druker et al., 1996; Shah et al., 2004; Weisberg et al., 2005). Imatinib mesylate (IM, Novartis, Basel, Swizerland) was the first tyrosine inhibitor to be developed as a molecular targeted drug (Druker et al., 1996). It is a competitive inhibitor of the ATP-binding site of the ABL-kinase domain, and prevents a conformational change of the oncoprotein to its active form, resulting in the elimination of most *BCR-ABL*+ cells (Druker et al., 1996). This relatively selective agent (it also recognizes the ATP-binding site of the c-Kit and platelet-derived growth factor receptors) has been immensely effective in the treatment of subjects with CP-CML (Druker et al., 2006; Druker et al., 2001; Kantarjian et al., 2002; O'Brien et al., 2003). Nevertheless, early relapse and the emergence of IM resistance are observed in 10-20% of subjects in early CP-CML, and up to 40% of those with advanced phase disease including 1-3% of newly diagnosed CML patients that develop sudden blast crisis (Apperley, 2007; Deininger et al., 2005; Forrest et al., 2008; Kantarjian et al., 2003; O'Hare et al., 2006; Valent, 2008). The inability to successfully discontinue IM therapy following at least five years of therapy, the persistence of a reservoir of clonal leukemic stem cells following the attainment of a complete molecular response (CMR), and the uncertain safety profile of long-term TKI treatment, has led to differing views on the most appropriate choice of therapy in CP-CML (Mahon et al., 2010; Ross et al., 2010a; Ross et al., 2010b; Rousselot et al., 2007; Sobrinho-Simoes et al., 2010). Recently, second-line TKIs including Dasatinib (DA,Bristol-Myers Squibb, New York, NY, USA) and Nilotinib (NL, Novatis) have been licensed for use in this indication and represent alternative therapeutic options either first-line or for resistant or intolerant cases. Both drugs have increased potency against the *BCR-ABL* kinase domain mutants most commonly associated with IM resistance. This along with their differing spectrums of inhibitory activity across the human kinome, has translated into increased clinical efficacy in subjects with IM-resistant disease (Carter et al., 2005; Shah et al., 2004; Weisberg et al., 2005). The major cytogenetic response (MCyR) rate following therapy with DA or NL in subjects with IM-resistant CP-CML is approximately 60%, with a complete cytogenetic response (CCyR) rate of 50% (Hochhaus et al., 2008; Kantarjian et al., 2007). Two recent Phase 3 randomized trials in subjects with treatment naïve early stage CP-CML demonstrated that both drugs are more effective than IM at inducing MCyR and major molecular (MMR) responses (Kantarjian et al., 2010; Saglio et al., 2010). DA and NL were subsequently approved by the U.S. Food and Drug Administration (FDA) as first-line therapies in CP-CML. Clinical experience, however, has shown that some subjects experience inadequate responses to all existing TKI therapies, or have an initial response but then progress rapidly (Kantarjian et al., 2006; Talpaz et al., 2006). As is the case in 60% of subjects with IM-resistant disease, the recalcitrant T315I mutation also routinely dominates the observed resistance with DA and NL (Apperley, 2007; Goldman, 2007). As a result these agents have no benefit over IM in subjects whose resistance is thought to be mediated

Targeting the Chronic Myeloid Leukemia Stem Cell:

A Paradigm for the Curative Treatment of Human Malignancies 89

al., 2002). In contrast, cells with replicative competency during the same interval were eliminated. A similar insensitivity of this subset of cells to DA and NL has also been demonstrated (Copland et al., 2006; Jorgensen et al., 2007). Subsequent studies using assays for long-term culture-initiating cells (LTC-ICs) and colony forming assays (CFCs) have

Fig. 1. Unique features of CML stem cells promoting their resistance to BCR-ABL-targeted

CD34+CD38+ CML cells and maintained for 3 weeks in the presence or absence of growth factors and variable concentrations of imatinib (IM). The differential IM sensitivity of lin-

growth factor-deprived conditions. (B) *BCR-ABL* transcript levels relative to *BCR* were measured in RNA isolated from different subsets of cells. Expression of *BCR-ABL* is highly

CD34+CD38+ CML cells to IM *in vitro* is markedly enhanced under

CD34+CD38+) and differentiated cells (lin+CD34-

*ABCB1* and *ABCG2* transcript levels relative to *GAPDH* were measured in different subsets of CP-CML and normal BM cells. A reduced level of *OCT1* and elevated levels of *ABCB1* and

more mature progenitor cells. The combination of very low expression of *OCT1* (low IM uptake), highly elevated expression of *ABCB1* and *ABCG2* (high efflux of IM and other drugs) and elevated expression of *BCR-ABL* in *CML* stem cells indicates that their general insensitivity to IM and other therapeutics is likely to be explained by multiple abnormal mechanisms.

stem-cell enriched population as compared to their more

stem-cell enriched population as compared to their

CD34+CD38-

). (C) *OCT1*,

and lin-

therapies. (A) Suspension cultures were initiated with FACS-purified lin-

CD34+CD38-

CD34+CD38-

deregulated in lin-

and lin-

mature progenitor cells (lin-

*ABCG2* were detected in lin-

CD34+CD38-

principally by T315I somatic mutants. In spite of this, each TKI has a unique spectrum of activity with respect to most of the other commonly encountered mutations that confer resistance to TKI therapy. Subjects with the V299L, T315A, and F317L/V/I/C mutations, for example, are less sensitive to DA (Muller et al., 2009; Soverini et al., 2011; Soverini et al., 2006), whereas the Y253H, E255K/V and F359V/C/I mutations are less sensitive to treatment with NL (Hughes et al., 2009; Soverini et al., 2011). Several third-generation tyrosine kinase inhibitors have been developed, including ponatinib (AP24534, ARIAD, Cambridge, Massachusetts, US) (O'Hare et al., 2009), which is an orally active multi-targeted kinase inhibitor that targets both the wild type and a broad spectrum of mutant forms of BCR-ABL. It was specifically designed to inhibit the autophosphorylation of wild-type and T315I mutant BCR-ABL and is active against most of the commonly encountered IMresistant mutations including G250E, Y253F and E255K (O'Hare et al., 2009). In a phase 1 study 38 patients with CP-CML, 66% achieved a MCyR and 53% a CCyR. Most significantly, a total of 89% (nine subjects) of the subjects harboring a T315I mutation attained a CCyR (Santos & Quintas-Cardama, 2011). The effectiveness of long-term therapy with ponatinib in IM-resistant patients, however, has yet to be determined. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only therapeutic option for CML that has curative potential. Its use, however, is restricted to subjects of less than 50 years that have a suitable donor, and even this highly selected group has a high risk of procedure-related morbidity and mortality (Forrest et al., 2008). The salvage rate for subjects with advanced phase disease, especially BC-CML, is poor even with allo-HSCT, with the vast majority dying as a result of their disease within a few years. There is consequently a significant unmet medical need for therapeutic options that prevent the emergence of resistant subclones and that can be administered with curative intent as a result of the selective targeting of LSCs.

#### **3. Properties of CML stem/progenitor cells that generate TKI resistance**

Primitive quiescent CML stem cells are relatively unresponsive to TKIs (Copland et al., 2006; Graham et al., 2002; Jorgensen et al., 2007) and possess unique features that predispose them to intrinsic and acquired resistance to BCR-ABL targeted therapeutics (Chu et al., 2005; Jiang et al., 2007a; Jiang et al., 2007b; Jiang et al., 2007c; Konig et al., 2008; Sorel et al., 2004). Evidence further suggests that LSCs are responsible for relapse following the discontinuation of IM therapy (Mahon et al., 2010; Ross et al., 2010a; Ross et al., 2010b; Rousselot et al., 2007; Sobrinho-Simoes et al., 2010). The elimination of the LSCs responsible for disease perpetuation and for the intrinsic and acquired TKI resistance observed in CML therefore represents the next logical step in the treatment of CML. An essential component of this enterprise, involves the molecular characterization of the CML stem cell phenotype, BM microenvironment and the stem niche that foster the origin, development, growth and survival of LSCs.

The relative insensitivity of primitive CML cells to treatment with IM was first reported in a quiescent subset of CD34+ CML cells using a carboxy-fluorescein diacetate succinimidyl diester (CFSE) staining cell division tracking assay (Graham et al., 2002; Holyoake et al., 1999). This demonstrated that 3 days of exposure of CML cells *in vitro* to concentrations of IM that were several-fold higher than those achieved in the plasma of subjects treated with 400 mg IM daily, failed to eliminate most of the primitive quiescent CML cells (Graham et

principally by T315I somatic mutants. In spite of this, each TKI has a unique spectrum of activity with respect to most of the other commonly encountered mutations that confer resistance to TKI therapy. Subjects with the V299L, T315A, and F317L/V/I/C mutations, for example, are less sensitive to DA (Muller et al., 2009; Soverini et al., 2011; Soverini et al., 2006), whereas the Y253H, E255K/V and F359V/C/I mutations are less sensitive to treatment with NL (Hughes et al., 2009; Soverini et al., 2011). Several third-generation tyrosine kinase inhibitors have been developed, including ponatinib (AP24534, ARIAD, Cambridge, Massachusetts, US) (O'Hare et al., 2009), which is an orally active multi-targeted kinase inhibitor that targets both the wild type and a broad spectrum of mutant forms of BCR-ABL. It was specifically designed to inhibit the autophosphorylation of wild-type and T315I mutant BCR-ABL and is active against most of the commonly encountered IMresistant mutations including G250E, Y253F and E255K (O'Hare et al., 2009). In a phase 1 study 38 patients with CP-CML, 66% achieved a MCyR and 53% a CCyR. Most significantly, a total of 89% (nine subjects) of the subjects harboring a T315I mutation attained a CCyR (Santos & Quintas-Cardama, 2011). The effectiveness of long-term therapy with ponatinib in IM-resistant patients, however, has yet to be determined. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only therapeutic option for CML that has curative potential. Its use, however, is restricted to subjects of less than 50 years that have a suitable donor, and even this highly selected group has a high risk of procedure-related morbidity and mortality (Forrest et al., 2008). The salvage rate for subjects with advanced phase disease, especially BC-CML, is poor even with allo-HSCT, with the vast majority dying as a result of their disease within a few years. There is consequently a significant unmet medical need for therapeutic options that prevent the emergence of resistant subclones and that can be administered with curative intent as a result of the selective targeting

**3. Properties of CML stem/progenitor cells that generate TKI resistance** 

Primitive quiescent CML stem cells are relatively unresponsive to TKIs (Copland et al., 2006; Graham et al., 2002; Jorgensen et al., 2007) and possess unique features that predispose them to intrinsic and acquired resistance to BCR-ABL targeted therapeutics (Chu et al., 2005; Jiang et al., 2007a; Jiang et al., 2007b; Jiang et al., 2007c; Konig et al., 2008; Sorel et al., 2004). Evidence further suggests that LSCs are responsible for relapse following the discontinuation of IM therapy (Mahon et al., 2010; Ross et al., 2010a; Ross et al., 2010b; Rousselot et al., 2007; Sobrinho-Simoes et al., 2010). The elimination of the LSCs responsible for disease perpetuation and for the intrinsic and acquired TKI resistance observed in CML therefore represents the next logical step in the treatment of CML. An essential component of this enterprise, involves the molecular characterization of the CML stem cell phenotype, BM microenvironment and the stem niche that foster the origin, development, growth and

The relative insensitivity of primitive CML cells to treatment with IM was first reported in a quiescent subset of CD34+ CML cells using a carboxy-fluorescein diacetate succinimidyl diester (CFSE) staining cell division tracking assay (Graham et al., 2002; Holyoake et al., 1999). This demonstrated that 3 days of exposure of CML cells *in vitro* to concentrations of IM that were several-fold higher than those achieved in the plasma of subjects treated with 400 mg IM daily, failed to eliminate most of the primitive quiescent CML cells (Graham et

of LSCs.

survival of LSCs.

al., 2002). In contrast, cells with replicative competency during the same interval were eliminated. A similar insensitivity of this subset of cells to DA and NL has also been demonstrated (Copland et al., 2006; Jorgensen et al., 2007). Subsequent studies using assays for long-term culture-initiating cells (LTC-ICs) and colony forming assays (CFCs) have

Fig. 1. Unique features of CML stem cells promoting their resistance to BCR-ABL-targeted therapies. (A) Suspension cultures were initiated with FACS-purified lin-CD34+CD38 and lin-CD34+CD38+ CML cells and maintained for 3 weeks in the presence or absence of growth factors and variable concentrations of imatinib (IM). The differential IM sensitivity of lin-CD34+CD38 and lin-CD34+CD38+ CML cells to IM *in vitro* is markedly enhanced under growth factor-deprived conditions. (B) *BCR-ABL* transcript levels relative to *BCR* were measured in RNA isolated from different subsets of cells. Expression of *BCR-ABL* is highly deregulated in lin-CD34+CD38 stem-cell enriched population as compared to their more mature progenitor cells (lin-CD34+CD38+) and differentiated cells (lin+CD34- ). (C) *OCT1*, *ABCB1* and *ABCG2* transcript levels relative to *GAPDH* were measured in different subsets of CP-CML and normal BM cells. A reduced level of *OCT1* and elevated levels of *ABCB1* and *ABCG2* were detected in lin-CD34+CD38 stem-cell enriched population as compared to their more mature progenitor cells. The combination of very low expression of *OCT1* (low IM uptake), highly elevated expression of *ABCB1* and *ABCG2* (high efflux of IM and other drugs) and elevated expression of *BCR-ABL* in *CML* stem cells indicates that their general insensitivity to IM and other therapeutics is likely to be explained by multiple abnormal mechanisms.

Targeting the Chronic Myeloid Leukemia Stem Cell:

acquisition in subjects with IM-treated CML.

the genome-wide instability of primitive CML cells.

**CML LSCs** 

A Paradigm for the Curative Treatment of Human Malignancies 91

treated with IM (White et al., 2010; White et al., 2006). This suggests that measurement of OCT1 expression might provide a useful predictor of the long-term risk of resistance

In order to determine whether the unique properties of CD34+ stem/progenitor cells derived from treatment-naïve CML patients correlate with the subsequent clinical response of the patients to IM therapy, we conducted a retrospective analysis of pre-treatment CD34+ PB cells obtained from 25 IM-treated CP-CML subjects with documented clinical outcomes (Jiang et al., 2010). Following the isolation of CD34+ cells from pre-treatment samples, we measured their *in vitro* CFC sensitivity to IM, expression levels of *BCR-ABL*, *OCT1*, *ABCB1/MDR*, and *ABCG2*, and the frequency of *BCR-ABL* tyrosine kinase domain somatic mutations. The data were segregated and analyzed according to whether they were from the 11 clinically defined IM-responders or the 14 IM non-responders. This confirmed the reported features of CD34+ CML cells, and identified two further features that differed significantly between the two groups. These were the responses of the pre-treatment CFCs to IM exposure *in vitro* (P<0.0001) and the frequency of mutant *BCR-ABL* transcripts in CD34+ cells (P=0.0025) suggesting that these parameters might form the basis of a

prospective test for the optimization of CP-CML management (Jiang et al., 2010).

**4. Genomic instability and a mutator phenotype as an invariant feature of** 

The genomic instability of primitive CML cells induced by the presence of the *BCR-ABL* fusion oncogene has long been thought to be a critical feature of CML. However, it is only in the last decade that data from CML cell lines and transgenic mice have provided definitive evidence that the *BCR-ABL* oncogene is necessary for the induction of genomic instability in hematopoietic cells (Brain et al., 2002; Brain et al., 2003; Canitrot et al., 1999). *BCR-ABL* is able, for example, to induce a mutator phenotype in *BCR-ABL* transfected murine cells. It also produces elevated levels of reactive oxygen species (ROS)-dependent DNA damage as compared with non-transfected controls. This damage to genomic DNA is likely to contribute to the accumulation of the somatic point mutations found in the ATP-binding site of the constitutively expressed BCR-ABL tyrosine kinase, and which are responsible for most cases of TKI resistance (Koptyra et al., 2006; Skorski, 2008). Interestingly, the ROSdependent mutations in *BCR-ABL* transfected cells were principally detected in a gene encoding the Na+K+ATPase *Atp1a1* (Koptyra et al., 2006). Mutant *ATP1A1* transcripts including those containing point mutations, insertions and deletions have been reported at high frequency (15-34%) in CD34+ CML cells rescued from IM nonresponders, a mutational rate similar to that observed in the *BCR-ABL* kinase domain of CML subjects. These mutations are not seen in CD34+ BM cells derived from healthy controls (Jiang et al., 2010). This and other data suggest that *BCR-ABL* induces a mutator phenotype, which results in

Investigations into the cellular and molecular causes of IM resistance have shown that the acquisition of somatic mutations in the *BCR-ABL* kinase-encoding domain that reduce the efficiency of IM binding to the ATP binding site represents the most common mechanism of resistance (accounting for 60-80% of relapses) (Deininger et al., 2005; O'Hare et al., 2006; Soverini et al., 2011; Valent, 2008). Copy number amplifications of *BCR-ABL* in contrast are rare (<10% of cases) (Gorre et al., 2001; Hochhaus et al., 2002; Tauchi & Ohyashiki, 2004).

indicated that although IM is able to inhibit the proliferation of primitive CML cells *in vitro*, it does not induce concurrent apoptosis (Holtz et al., 2005; Holtz et al., 2002). We have shown that the ability of IM to inhibit primitive CML cells depends on their differentiation status (Jiang et al., 2007c). In these experiments, stem cell and progenitor cell enriched CML cell fractions (lin-CD34+CD38- and lin-CD34+CD38+ cells, respectively) were isolated and cultured in the presence of varying concentrations of IM for a more prolonged period than in the earlier studies with primary quiescent CML cells (3 weeks vs. 3 to 12 days). The inhibitory effect of IM on the yield of viable cells following a 3 week exposure period was found to be much less (~10- to 20-fold) pronounced in cultures initiated with more primitive lin-CD34+CD38- CML cells as compared with the more differentiated lin-CD34+CD38+ cells (Figure 1A). Taken together, these findings suggest that the most primitive CML cells are much less sensitive to IM than the differentiated bulk population. The fact that Ph+CD34+ CFCs and LTC-ICs remain detectable in subjects with CML that have achieved hematological remission following treatment with IM, suggests that these *in vitro* findings are likely to translate into the clinic (Bhatia et al., 2003; Chu et al., 2005). They are further supported by a recent report revealing the presence of *BCR-ABL*+ cells in LTC-ICs of CML patients that have achieved prolonged clinical remission following treatment with either interferon-alpha, IM or DA (Chomel et al., 2011). However, whereas most CML cells are oncogene addicted and sensitive to TKI treatment, the growth and survival of CML LSCs do not appear to be BCR-ABL tyrosine kinase activity dependent (Corbin et al., 2011). This suggests that alternative pathways may be active in CML LSCs that drive their proliferation and self-renewal in a BCR-ABL independent manner. Combination therapies aimed at targeting critical components of these pathways are likely to be of key importance in the derivation of a logical CML LSC eradication strategy.

Other studies have shown that CML stem/progenitor cells have multiple unique features that would be expected to contribute to the observed intrinsic and acquired resistance to *BCR-ABL* directed therapeutics (Copland, 2009; Engler et al., 2010; Jiang et al., 2007a; Jiang et al., 2007b; Jiang et al., 2007c). These include: (i) elevated levels of *BCR-ABL* expression and kinase activity in CML stem cells as compared with their more mature progeny (Figure 1B) in a manner that is cell cycle status independent (Barnes et al., 2005; Copland et al., 2006; Jamieson et al., 2004; Jiang et al., 2007b; Jiang et al., 2008; Jiang et al., 2007c), (ii) a corresponding reduced (almost undetectable) level of the transporter gene *OCT1* that is the principal regulator of IM uptake (Figure 1C) (Thomas et al., 2004; White et al., 2006) and whose decreased levels would be expected to reduce the ability of cells to take up IM (Engler et al., 2010; Jiang et al., 2007c) and (iii) elevated levels of expression of the ABC transporter genes *ABCB1(MDR)* and *ABCG2* (Figure 1C) which enhance the cellular efflux of IM and other drugs (Jiang et al., 2007c; Jordanides et al., 2006; Lepper et al., 2005). The combination of an exceptionally low level of *OCT1* expression which impedes cellular IM uptake, a highly elevated expression of *ABCB1* and *ABCG2* which produces a high rate of cellular IM efflux, and elevated expression of the *BCR-ABL* oncogene in CML stem cells, indicates that their insensitivity to IM and other therapeutics is in part explained by a unique portfolio of protective mechanisms (Jiang et al., 2007b; Jiang et al., 2007c). Interestingly studies in a cohort of 30 CML patients have shown that IM-non-responders have lower *OCT1* transcript levels than IM-responders (Crossman et al., 2005), and that the functional activity of the encoded OCT1 protein, as measured by the OCT1-mediated influx of IM into primary CML cells, is predictive of the long-term outcome of CP-CML subjects

indicated that although IM is able to inhibit the proliferation of primitive CML cells *in vitro*, it does not induce concurrent apoptosis (Holtz et al., 2005; Holtz et al., 2002). We have shown that the ability of IM to inhibit primitive CML cells depends on their differentiation status (Jiang et al., 2007c). In these experiments, stem cell and progenitor cell enriched CML

cultured in the presence of varying concentrations of IM for a more prolonged period than in the earlier studies with primary quiescent CML cells (3 weeks vs. 3 to 12 days). The inhibitory effect of IM on the yield of viable cells following a 3 week exposure period was found to be much less (~10- to 20-fold) pronounced in cultures initiated with more primitive

(Figure 1A). Taken together, these findings suggest that the most primitive CML cells are much less sensitive to IM than the differentiated bulk population. The fact that Ph+CD34+ CFCs and LTC-ICs remain detectable in subjects with CML that have achieved hematological remission following treatment with IM, suggests that these *in vitro* findings are likely to translate into the clinic (Bhatia et al., 2003; Chu et al., 2005). They are further supported by a recent report revealing the presence of *BCR-ABL*+ cells in LTC-ICs of CML patients that have achieved prolonged clinical remission following treatment with either interferon-alpha, IM or DA (Chomel et al., 2011). However, whereas most CML cells are oncogene addicted and sensitive to TKI treatment, the growth and survival of CML LSCs do not appear to be BCR-ABL tyrosine kinase activity dependent (Corbin et al., 2011). This suggests that alternative pathways may be active in CML LSCs that drive their proliferation and self-renewal in a BCR-ABL independent manner. Combination therapies aimed at targeting critical components of these pathways are likely to be of key importance in the

Other studies have shown that CML stem/progenitor cells have multiple unique features that would be expected to contribute to the observed intrinsic and acquired resistance to *BCR-ABL* directed therapeutics (Copland, 2009; Engler et al., 2010; Jiang et al., 2007a; Jiang et al., 2007b; Jiang et al., 2007c). These include: (i) elevated levels of *BCR-ABL* expression and kinase activity in CML stem cells as compared with their more mature progeny (Figure 1B) in a manner that is cell cycle status independent (Barnes et al., 2005; Copland et al., 2006; Jamieson et al., 2004; Jiang et al., 2007b; Jiang et al., 2008; Jiang et al., 2007c), (ii) a corresponding reduced (almost undetectable) level of the transporter gene *OCT1* that is the principal regulator of IM uptake (Figure 1C) (Thomas et al., 2004; White et al., 2006) and whose decreased levels would be expected to reduce the ability of cells to take up IM (Engler et al., 2010; Jiang et al., 2007c) and (iii) elevated levels of expression of the ABC transporter genes *ABCB1(MDR)* and *ABCG2* (Figure 1C) which enhance the cellular efflux of IM and other drugs (Jiang et al., 2007c; Jordanides et al., 2006; Lepper et al., 2005). The combination of an exceptionally low level of *OCT1* expression which impedes cellular IM uptake, a highly elevated expression of *ABCB1* and *ABCG2* which produces a high rate of cellular IM efflux, and elevated expression of the *BCR-ABL* oncogene in CML stem cells, indicates that their insensitivity to IM and other therapeutics is in part explained by a unique portfolio of protective mechanisms (Jiang et al., 2007b; Jiang et al., 2007c). Interestingly studies in a cohort of 30 CML patients have shown that IM-non-responders have lower *OCT1* transcript levels than IM-responders (Crossman et al., 2005), and that the functional activity of the encoded OCT1 protein, as measured by the OCT1-mediated influx of IM into primary CML cells, is predictive of the long-term outcome of CP-CML subjects

CD34+CD38- CML cells as compared with the more differentiated lin-

CD34+CD38+ cells, respectively) were isolated and

CD34+CD38+ cells

cell fractions (lin-

lin-

CD34+CD38- and lin-

derivation of a logical CML LSC eradication strategy.

treated with IM (White et al., 2010; White et al., 2006). This suggests that measurement of OCT1 expression might provide a useful predictor of the long-term risk of resistance acquisition in subjects with IM-treated CML.

In order to determine whether the unique properties of CD34+ stem/progenitor cells derived from treatment-naïve CML patients correlate with the subsequent clinical response of the patients to IM therapy, we conducted a retrospective analysis of pre-treatment CD34+ PB cells obtained from 25 IM-treated CP-CML subjects with documented clinical outcomes (Jiang et al., 2010). Following the isolation of CD34+ cells from pre-treatment samples, we measured their *in vitro* CFC sensitivity to IM, expression levels of *BCR-ABL*, *OCT1*, *ABCB1/MDR*, and *ABCG2*, and the frequency of *BCR-ABL* tyrosine kinase domain somatic mutations. The data were segregated and analyzed according to whether they were from the 11 clinically defined IM-responders or the 14 IM non-responders. This confirmed the reported features of CD34+ CML cells, and identified two further features that differed significantly between the two groups. These were the responses of the pre-treatment CFCs to IM exposure *in vitro* (P<0.0001) and the frequency of mutant *BCR-ABL* transcripts in CD34+ cells (P=0.0025) suggesting that these parameters might form the basis of a prospective test for the optimization of CP-CML management (Jiang et al., 2010).

#### **4. Genomic instability and a mutator phenotype as an invariant feature of CML LSCs**

The genomic instability of primitive CML cells induced by the presence of the *BCR-ABL* fusion oncogene has long been thought to be a critical feature of CML. However, it is only in the last decade that data from CML cell lines and transgenic mice have provided definitive evidence that the *BCR-ABL* oncogene is necessary for the induction of genomic instability in hematopoietic cells (Brain et al., 2002; Brain et al., 2003; Canitrot et al., 1999). *BCR-ABL* is able, for example, to induce a mutator phenotype in *BCR-ABL* transfected murine cells. It also produces elevated levels of reactive oxygen species (ROS)-dependent DNA damage as compared with non-transfected controls. This damage to genomic DNA is likely to contribute to the accumulation of the somatic point mutations found in the ATP-binding site of the constitutively expressed BCR-ABL tyrosine kinase, and which are responsible for most cases of TKI resistance (Koptyra et al., 2006; Skorski, 2008). Interestingly, the ROSdependent mutations in *BCR-ABL* transfected cells were principally detected in a gene encoding the Na+K+ATPase *Atp1a1* (Koptyra et al., 2006). Mutant *ATP1A1* transcripts including those containing point mutations, insertions and deletions have been reported at high frequency (15-34%) in CD34+ CML cells rescued from IM nonresponders, a mutational rate similar to that observed in the *BCR-ABL* kinase domain of CML subjects. These mutations are not seen in CD34+ BM cells derived from healthy controls (Jiang et al., 2010). This and other data suggest that *BCR-ABL* induces a mutator phenotype, which results in the genome-wide instability of primitive CML cells.

Investigations into the cellular and molecular causes of IM resistance have shown that the acquisition of somatic mutations in the *BCR-ABL* kinase-encoding domain that reduce the efficiency of IM binding to the ATP binding site represents the most common mechanism of resistance (accounting for 60-80% of relapses) (Deininger et al., 2005; O'Hare et al., 2006; Soverini et al., 2011; Valent, 2008). Copy number amplifications of *BCR-ABL* in contrast are rare (<10% of cases) (Gorre et al., 2001; Hochhaus et al., 2002; Tauchi & Ohyashiki, 2004).

Targeting the Chronic Myeloid Leukemia Stem Cell:

FACS-purified lin-

in freshly isolated lin-

with IM.

CD34+CD38-

itself evolves as the disease progresses from CP to BC.

A Paradigm for the Curative Treatment of Human Malignancies 93

Fig. 2. CML stem cells are highly unstable and generate a high frequency of *BCR-ABL* kinase domain mutations both *in vitro* and *in vivo*. (A) Suspension cultures were initiated with

growth of primitive CML cells *in vitro*. (C) Chromosomal abnormalities, such as 17 p+, could be observed from single colonies generated from the cells present after 3 weeks in culture

in early CML and also the large deletions seen in later-stage CML. This is supported by evidence which suggests that key protein components of the major NHEJ pathway, WRN and DNA ligase IIIα that form a molecular complex which is recruited to DSBs, are upregulated in CML (Sallmyr et al., 2008). There is also evidence that ROS-induced DSBs are repaired, at least to some extent, by single-strand annealing (SSA). This is a rare and very unfaithful repair mechanism whose activity has been shown to be stimulated by *BCR-ABL*. Interestingly the activation of this repair mechanism is attenuated by IM therapy (Cramer et al., 2008). The presence of multiple alternative error-prone mechanisms for repairing DSBs, raises the possibility that the differential recruitment of these alternative repair pathways

One approach to identifying the generative mechanism that underlies the observed genomic instability in CML, is to generate a compendium of *BCR-ABL* kinase domain somatic mutations to determine if the mutator phenotype observed in LSCs is associated with a distinct mutational signature. In order to establish whether CML is characterized by a

the presence or absence of growth factors and IM. The cells harvested from the 3-week cultures were then assayed for CFCs. Mutational analysis was then performed for the detection of *BCR-ABL* kinase domain mutations in freshly isolated lin-CD34+CD38- cells and their CFC progenies. (B) Pre-existing *BCR-ABL* kinase domain mutations could be detected

CD34+CD38- stem cell-enriched population and maintained for 3 weeks in

stem cells and new mutations appeared during the

More than 90 different *BCR-ABL* kinase domain point mutations have been reported at varying frequencies in IM-resistant subjects (Apperley, 2007; Hughes et al., 2006; Shah et al., 2002; Soverini et al., 2011). Of these 15 specific amino acid substitutions account for more than 85% of the mutations at the protein sequence level. The mutations responsible for 66% of reported cases, furthermore, have been shown to occur at only six different positions (G250E, Y253F/H, E255K/V, T315I, F359V, H396R/P). The T315I mutation, which confers resistance to IM, DA and NL, is the most frequently detected mutation in IM-resistant patients and is the hardest mutant to treat (Apperley, 2007; Carter et al., 2005; Shah et al., 2004; Soverini et al., 2011; Weisberg et al., 2005). *BCR-ABL* kinase domain mutations are found in IM-naïve patients but not in the germline of healthy controls, indicating that at least some of the mutations are somatically generated prior to the presence of drug selection, and that the increased frequency of mutations observed following TKI therapy occurs as a result of drug-induced selection and associated clonal expansion (Roche-Lestienne et al., 2003; Roche-Lestienne et al., 2002; Willis et al., 2005). The somatic mutations themselves arise as a consequence of underlying genomic instability, which may reflect impaired processes of DNA repair in LSCs. Although some of the mutants are adaptive under the selective pressure of drug exposure, the majority diminish in frequency in the presence of TKI selection. We and others have demonstrated that the *BCR-ABL* fusion gene in CD34+ leukemic progenitor cells (Chu et al., 2005; Jiang et al., 2010) and CD34+CD38- stem cell-enriched cells is itself highly unstable (Jiang et al., 2007a) This is reflected in the unusually high frequency of *BCR-ABL* mutations that accumulate in the CD34+CD38 compartment in the presence or absence of IM selection. The rapid acquisition of somatic mutations in *BCR-ABL* is also observed in the progeny of CML cells stimulated to proliferate and differentiate *in vitro* (Figure 2) (Jiang et al., 2007a). The rapid and prolific generation of *BCR-ABL* somatic mutants in primary CML cells *in vitro* extends recent findings in *BCR-ABL*-transduced murine BaF3 cells (von Bubnoff et al., 2005), and adult BM cells (Flamant & Turhan, 2005) and indicates that primitive leukemic CML cells have an intrinsically high rate of mutation, and a tendency to fix new somatic point mutations irrespective of the presence or absence of drug. The nature and timing of these apparently stochastic events relative to the size of the primary LSC clone at the time diagnosis and the timing, nature and extent of TKI therapy, may to some extent explain the variable clinical responses observed in different subjects (Jiang et al., 2007a; Roche-Lestienne et al., 2003; Shah et al., 2002; Sorel et al., 2004). A recent study shows that CML subjects defined retrospectively as either IM responders or IM non-responders display significant differences in the frequency of mutant *BCR-ABL* transcripts present in their pre-treatment CD34+ cells (P=0.0025), with some of the highly resistant *BCR-ABL* kinase domain somatic mutants such as T315I being amplified from the CD34+ cells of IM non-responsive subjects (Jiang et al., 2010). Overall this suggests that primary CML stem/progenitor cells have a high degree of focal and possibly genome-wide instability, emphasizing the importance of taking the properties of these cells into account when considering new therapeutic approaches. The unique properties of leukemia stem/progenitor cells may, furthermore, help predict individual responses to TKI therapies and in so doing improve clinical management by facilitating personalized treatment decisions. One approach to targeting the LSC involves defining and inhibiting the generative mechanism causal to the observed genomic instability in LSCs. The ROS that are induced by *BCR-ABL* are known to cause many types of DNA damage including double-strand breaks (DSBs). It has been shown that the error-prone repair of DSBs by non-homologous endjoining (NHEJ) may be responsible for at least some of the somatic point mutations observed

More than 90 different *BCR-ABL* kinase domain point mutations have been reported at varying frequencies in IM-resistant subjects (Apperley, 2007; Hughes et al., 2006; Shah et al., 2002; Soverini et al., 2011). Of these 15 specific amino acid substitutions account for more than 85% of the mutations at the protein sequence level. The mutations responsible for 66% of reported cases, furthermore, have been shown to occur at only six different positions (G250E, Y253F/H, E255K/V, T315I, F359V, H396R/P). The T315I mutation, which confers resistance to IM, DA and NL, is the most frequently detected mutation in IM-resistant patients and is the hardest mutant to treat (Apperley, 2007; Carter et al., 2005; Shah et al., 2004; Soverini et al., 2011; Weisberg et al., 2005). *BCR-ABL* kinase domain mutations are found in IM-naïve patients but not in the germline of healthy controls, indicating that at least some of the mutations are somatically generated prior to the presence of drug selection, and that the increased frequency of mutations observed following TKI therapy occurs as a result of drug-induced selection and associated clonal expansion (Roche-Lestienne et al., 2003; Roche-Lestienne et al., 2002; Willis et al., 2005). The somatic mutations themselves arise as a consequence of underlying genomic instability, which may reflect impaired processes of DNA repair in LSCs. Although some of the mutants are adaptive under the selective pressure of drug exposure, the majority diminish in frequency in the presence of TKI selection. We and others have demonstrated that the *BCR-ABL* fusion gene in CD34+ leukemic progenitor cells (Chu et al., 2005; Jiang et al., 2010) and CD34+CD38- stem cell-enriched cells is itself highly unstable (Jiang et al., 2007a) This is reflected in the unusually high frequency of *BCR-ABL* mutations that accumulate in the CD34+CD38 compartment in the presence or absence of IM selection. The rapid acquisition of somatic mutations in *BCR-ABL* is also observed in the progeny of CML cells stimulated to proliferate and differentiate *in vitro* (Figure 2) (Jiang et al., 2007a). The rapid and prolific generation of *BCR-ABL* somatic mutants in primary CML cells *in vitro* extends recent findings in *BCR-ABL*-transduced murine BaF3 cells (von Bubnoff et al., 2005), and adult BM cells (Flamant & Turhan, 2005) and indicates that primitive leukemic CML cells have an intrinsically high rate of mutation, and a tendency to fix new somatic point mutations irrespective of the presence or absence of drug. The nature and timing of these apparently stochastic events relative to the size of the primary LSC clone at the time diagnosis and the timing, nature and extent of TKI therapy, may to some extent explain the variable clinical responses observed in different subjects (Jiang et al., 2007a; Roche-Lestienne et al., 2003; Shah et al., 2002; Sorel et al., 2004). A recent study shows that CML subjects defined retrospectively as either IM responders or IM non-responders display significant differences in the frequency of mutant *BCR-ABL* transcripts present in their pre-treatment CD34+ cells (P=0.0025), with some of the highly resistant *BCR-ABL* kinase domain somatic mutants such as T315I being amplified from the CD34+ cells of IM non-responsive subjects (Jiang et al., 2010). Overall this suggests that primary CML stem/progenitor cells have a high degree of focal and possibly genome-wide instability, emphasizing the importance of taking the properties of these cells into account when considering new therapeutic approaches. The unique properties of leukemia stem/progenitor cells may, furthermore, help predict individual responses to TKI therapies and in so doing improve clinical management by facilitating personalized treatment decisions. One approach to targeting the LSC involves defining and inhibiting the generative mechanism causal to the observed genomic instability in LSCs. The ROS that are induced by *BCR-ABL* are known to cause many types of DNA damage including double-strand breaks (DSBs). It has been shown that the error-prone repair of DSBs by non-homologous endjoining (NHEJ) may be responsible for at least some of the somatic point mutations observed

Fig. 2. CML stem cells are highly unstable and generate a high frequency of *BCR-ABL* kinase domain mutations both *in vitro* and *in vivo*. (A) Suspension cultures were initiated with FACS-purified lin-CD34+CD38- stem cell-enriched population and maintained for 3 weeks in the presence or absence of growth factors and IM. The cells harvested from the 3-week cultures were then assayed for CFCs. Mutational analysis was then performed for the detection of *BCR-ABL* kinase domain mutations in freshly isolated lin-CD34+CD38- cells and their CFC progenies. (B) Pre-existing *BCR-ABL* kinase domain mutations could be detected in freshly isolated lin-CD34+CD38 stem cells and new mutations appeared during the growth of primitive CML cells *in vitro*. (C) Chromosomal abnormalities, such as 17 p+, could be observed from single colonies generated from the cells present after 3 weeks in culture with IM.

in early CML and also the large deletions seen in later-stage CML. This is supported by evidence which suggests that key protein components of the major NHEJ pathway, WRN and DNA ligase IIIα that form a molecular complex which is recruited to DSBs, are upregulated in CML (Sallmyr et al., 2008). There is also evidence that ROS-induced DSBs are repaired, at least to some extent, by single-strand annealing (SSA). This is a rare and very unfaithful repair mechanism whose activity has been shown to be stimulated by *BCR-ABL*. Interestingly the activation of this repair mechanism is attenuated by IM therapy (Cramer et al., 2008). The presence of multiple alternative error-prone mechanisms for repairing DSBs, raises the possibility that the differential recruitment of these alternative repair pathways itself evolves as the disease progresses from CP to BC.

One approach to identifying the generative mechanism that underlies the observed genomic instability in CML, is to generate a compendium of *BCR-ABL* kinase domain somatic mutations to determine if the mutator phenotype observed in LSCs is associated with a distinct mutational signature. In order to establish whether CML is characterized by a

Targeting the Chronic Myeloid Leukemia Stem Cell:

resistant mutations, DCC-2036 alone is unlikely to target LSCs.

2010; Park et al., 2003; Rizo et al., 2010; Zhao et al., 2007).

CD34+ progenitor cells and CD34+CD38-

A Paradigm for the Curative Treatment of Human Malignancies 95

targeting of the mutant clonal population with a second generation TKI will be necessary if molecular cure is to be achieved. This process will be facilitated by the development of new and more effective classes of debulking agents. A switch pocket inhibitor DCC-2036, for example, has been developed which targets the hydrophobic pockets distant from the catalytic region that regulate the transition between the active and inactive state of BCR-ABL. This drug appears to successfully inhibit the majority of TKI-resistant mutants, including the critically important T315I gatekeeper mutation (Chan et al., 2011). DCC-2036 inhibits the BCR-ABL oncoprotein in both its active and inactive conformation by inducing and stabilizing the type II inactive conformation. It is highly effective in suppressing the growth of transduced wild-type *BCR-ABL* murine cells, including cells transduced with the T315I mutant both *in vitro* and *in vivo* (Chan et al., 2011). A phase I trial of DCC-2036 in IM resistant subjects carrying either the T315I mutation or two or more alternative TKI mutations is currently underway. However, although useful in the management of TKI-

Global gene expression analysis and transcriptome profiling, including the identification of deregulated micro-RNAs (miRs) and the genes they target using next-generation sequencing technologies, have been applied in order to facilitate the identification of new molecular targets and biomarkers able to predict TKI responsivity in primary CML cells, IM resistant cells and *BCR-ABL*-transduced cells. Several studies have compared the transcriptome of

counterparts. These studies have confirmed the functional relevance of the activation of the JAK/STAT, PI3K/AKT, RAS/MAPK and NFκB pathways in LSCs (Janssen et al., 2005; Jongen-Lavrencic et al., 2005; Kronenwett et al., 2005; Nowicki et al., 2003; Radich et al., 2006; Salesse & Verfaillie, 2003; Yong et al., 2006; Zhao et al., 2008). These studies have also identified differentially expressed genes that are involved in the regulation of DNA repair, cell cycle control, cell adhesion, and homing, as well as genes and transcription factors involved in drug metabolism (Diaz-Blanco et al., 2007; Kronenwett et al., 2005; Salesse & Verfaillie, 2003; Yong et al., 2006; Zhao et al., 2008). Several miRs and their target genes, including *miR-203, miR-328* and *miR-17-92* cluster, have been shown to regulate *BCR-ABL* and the expression of other genes known to be critical to the generation and maintenance of the CML phenotype (Bueno et al., 2008; Eiring et al., 2010; Venturini et al., 2007), indicating the potential utility of Micro-RNA profiling in the identification of novel targets for LSCdirected therapies. Of note, several new potential targets have been identified which regulate the maintenance of self-renewal, quiescence and expansion of CML stem and progenitor cells. These include promyelocytic leukemia protein (PML), β-Catenin, RNAbinding proteins (RBPs) and the *BMI1* and *FOXO* transcription factors, suggesting that the specific targeting of these proteins in conjunction with TKIs, may help eliminate residual CML LSCs (Copland, 2009; Eiring et al., 2008; Hu et al., 2009; Ito et al., 2008; Naka et al.,

The systematic applications of these and other new technologies have resulted in significant advances in our understanding of the molecular properties of CML stem and progenitor cells. They are also helping to define the nature of the BM microenvironment and stem cell niche that support the growth and differentiation of LSCs. The specific targeting of the BM microenvironment and stem cell niche represent alternative and indirect strategies for the elimination of LSCs (Figure 3). The chemokine receptor CXCR4, for example, which is central to stem cell localization and a known chemo-attractant for hematopoietic cells, is induced by IM and causes CML cell migration to the BM and promotes the survival of

stem cell-enriched leukemic cells with their healthy

distinct mutational imprint, we analyzed *BCR-ABL* tyrosine kinase domain sequence data from 15 IM-naïve and 316 IM-resistant CP-CML subjects (Grant et al., 2010). This revealed a distinct and non-random distribution of *BCR-ABL* kinase domain mutations with apparent hot spot regions located at codon positions 1 and 2, and several other distinct features that are commensurate with the activity of a distinct mutator. These include a propensity for transitions relative to unselected regions of the human genome across all codon positions, a T-to-C mutational hotspot at codon position 2, a near lack of mutations at codon position 3, and an overall under-representation of C-to-T mutations. These results provide evidence for the activity of a distinct mutator that is active in LSCs, and it is interesting that the frequently observed M244V and D276G mutations arise from T-to-C transitions, both of which are predicted consequences of putative CML LSC mutator activity. The clinically most problematic mutation, T315I, is interestingly generated by a C-to-T transition, indicating the imprint of intense drug-mediated selection. This characteristic mutational signature may provide insights into the mechanism that contributes to the observed genomic instability in LSCs, and which may act in concert with the error-prone repair of DSBs. Candidate mutators include the *MYC* oncogene, which is known to result in aberrant DNA synthesis and which has been shown to be over-expressed in CML subjects at the time of diagnosis. Higher expression levels have, furthermore, been shown to correlate with a poor clinical response to IM. Interestingly *MYC* levels do not directly correlate with *BCR-ABL* levels in subjects treated with IM (Albajar et al., 2011).

#### **5. Strategies for the eradication of leukemic stem cells**

The existence of both intrinsic and acquired resistance to TKIs in CML stem cells has prompted considerable interest in identifying multi-targeted therapeutic strategies able to combat the emergence of resistant clones by eliminating the LSCs that generate them. It has been hypothesized that combination strategies able to target both proliferating and primitive quiescent leukemic cells will significantly improve clinical outcomes in CML (Jiang X. 2007; Savona & Talpaz, 2008; Sloma et al., 2010). There is no doubt that the development of stem cell-directed therapies will be critical to the attainment of prolonged remission and ultimately TKI cessation and cure. As expected, the use of molecularly targeted agents such as IM results in the elimination of the majority of the more differentiated leukemic cells, but leaves primitive stem cells largely untouched. So although able to effect a significant initial reduction in leukemic cells of a more mature differentiation stage, LSCs eventually repopulate the malignant cell population following or during ongoing TKI therapy, generating new resistant mutants that leads to disease persistence and clinical recurrence. This situation recapitulates the elimination of rapidly cycling cells and sparing of quiescent leukemic stem cells seen in the deployment of conventional chemotherapeutics. Whereas monotherapy using agents that directly target LSCs may result in only a minimal observable initial response, in the absence of LSCs the leukemia is not expected to be maintained or expanded. Most importantly, the micro-evolutionary process that continuously and dynamically generates a spectrum of mutant clones will be disabled. As a result the disease burden would be expected to decline incrementally, providing significantly improved long-term outcomes. Combination with TKIs or other agents that target proliferating cells of a more mature phenotype will though continue to be essential for debulking. In the situation where resistant clones with *BCR-ABL* kinase domain mutations and/or other critical mutations are already present at high frequency, the simultaneous

distinct mutational imprint, we analyzed *BCR-ABL* tyrosine kinase domain sequence data from 15 IM-naïve and 316 IM-resistant CP-CML subjects (Grant et al., 2010). This revealed a distinct and non-random distribution of *BCR-ABL* kinase domain mutations with apparent hot spot regions located at codon positions 1 and 2, and several other distinct features that are commensurate with the activity of a distinct mutator. These include a propensity for transitions relative to unselected regions of the human genome across all codon positions, a T-to-C mutational hotspot at codon position 2, a near lack of mutations at codon position 3, and an overall under-representation of C-to-T mutations. These results provide evidence for the activity of a distinct mutator that is active in LSCs, and it is interesting that the frequently observed M244V and D276G mutations arise from T-to-C transitions, both of which are predicted consequences of putative CML LSC mutator activity. The clinically most problematic mutation, T315I, is interestingly generated by a C-to-T transition, indicating the imprint of intense drug-mediated selection. This characteristic mutational signature may provide insights into the mechanism that contributes to the observed genomic instability in LSCs, and which may act in concert with the error-prone repair of DSBs. Candidate mutators include the *MYC* oncogene, which is known to result in aberrant DNA synthesis and which has been shown to be over-expressed in CML subjects at the time of diagnosis. Higher expression levels have, furthermore, been shown to correlate with a poor clinical response to IM. Interestingly *MYC* levels do not directly correlate with *BCR-*

The existence of both intrinsic and acquired resistance to TKIs in CML stem cells has prompted considerable interest in identifying multi-targeted therapeutic strategies able to combat the emergence of resistant clones by eliminating the LSCs that generate them. It has been hypothesized that combination strategies able to target both proliferating and primitive quiescent leukemic cells will significantly improve clinical outcomes in CML (Jiang X. 2007; Savona & Talpaz, 2008; Sloma et al., 2010). There is no doubt that the development of stem cell-directed therapies will be critical to the attainment of prolonged remission and ultimately TKI cessation and cure. As expected, the use of molecularly targeted agents such as IM results in the elimination of the majority of the more differentiated leukemic cells, but leaves primitive stem cells largely untouched. So although able to effect a significant initial reduction in leukemic cells of a more mature differentiation stage, LSCs eventually repopulate the malignant cell population following or during ongoing TKI therapy, generating new resistant mutants that leads to disease persistence and clinical recurrence. This situation recapitulates the elimination of rapidly cycling cells and sparing of quiescent leukemic stem cells seen in the deployment of conventional chemotherapeutics. Whereas monotherapy using agents that directly target LSCs may result in only a minimal observable initial response, in the absence of LSCs the leukemia is not expected to be maintained or expanded. Most importantly, the micro-evolutionary process that continuously and dynamically generates a spectrum of mutant clones will be disabled. As a result the disease burden would be expected to decline incrementally, providing significantly improved long-term outcomes. Combination with TKIs or other agents that target proliferating cells of a more mature phenotype will though continue to be essential for debulking. In the situation where resistant clones with *BCR-ABL* kinase domain mutations and/or other critical mutations are already present at high frequency, the simultaneous

*ABL* levels in subjects treated with IM (Albajar et al., 2011).

**5. Strategies for the eradication of leukemic stem cells** 

targeting of the mutant clonal population with a second generation TKI will be necessary if molecular cure is to be achieved. This process will be facilitated by the development of new and more effective classes of debulking agents. A switch pocket inhibitor DCC-2036, for example, has been developed which targets the hydrophobic pockets distant from the catalytic region that regulate the transition between the active and inactive state of BCR-ABL. This drug appears to successfully inhibit the majority of TKI-resistant mutants, including the critically important T315I gatekeeper mutation (Chan et al., 2011). DCC-2036 inhibits the BCR-ABL oncoprotein in both its active and inactive conformation by inducing and stabilizing the type II inactive conformation. It is highly effective in suppressing the growth of transduced wild-type *BCR-ABL* murine cells, including cells transduced with the T315I mutant both *in vitro* and *in vivo* (Chan et al., 2011). A phase I trial of DCC-2036 in IM resistant subjects carrying either the T315I mutation or two or more alternative TKI mutations is currently underway. However, although useful in the management of TKIresistant mutations, DCC-2036 alone is unlikely to target LSCs.

Global gene expression analysis and transcriptome profiling, including the identification of deregulated micro-RNAs (miRs) and the genes they target using next-generation sequencing technologies, have been applied in order to facilitate the identification of new molecular targets and biomarkers able to predict TKI responsivity in primary CML cells, IM resistant cells and *BCR-ABL*-transduced cells. Several studies have compared the transcriptome of CD34+ progenitor cells and CD34+CD38 stem cell-enriched leukemic cells with their healthy counterparts. These studies have confirmed the functional relevance of the activation of the JAK/STAT, PI3K/AKT, RAS/MAPK and NFκB pathways in LSCs (Janssen et al., 2005; Jongen-Lavrencic et al., 2005; Kronenwett et al., 2005; Nowicki et al., 2003; Radich et al., 2006; Salesse & Verfaillie, 2003; Yong et al., 2006; Zhao et al., 2008). These studies have also identified differentially expressed genes that are involved in the regulation of DNA repair, cell cycle control, cell adhesion, and homing, as well as genes and transcription factors involved in drug metabolism (Diaz-Blanco et al., 2007; Kronenwett et al., 2005; Salesse & Verfaillie, 2003; Yong et al., 2006; Zhao et al., 2008). Several miRs and their target genes, including *miR-203, miR-328* and *miR-17-92* cluster, have been shown to regulate *BCR-ABL* and the expression of other genes known to be critical to the generation and maintenance of the CML phenotype (Bueno et al., 2008; Eiring et al., 2010; Venturini et al., 2007), indicating the potential utility of Micro-RNA profiling in the identification of novel targets for LSCdirected therapies. Of note, several new potential targets have been identified which regulate the maintenance of self-renewal, quiescence and expansion of CML stem and progenitor cells. These include promyelocytic leukemia protein (PML), β-Catenin, RNAbinding proteins (RBPs) and the *BMI1* and *FOXO* transcription factors, suggesting that the specific targeting of these proteins in conjunction with TKIs, may help eliminate residual CML LSCs (Copland, 2009; Eiring et al., 2008; Hu et al., 2009; Ito et al., 2008; Naka et al., 2010; Park et al., 2003; Rizo et al., 2010; Zhao et al., 2007).

The systematic applications of these and other new technologies have resulted in significant advances in our understanding of the molecular properties of CML stem and progenitor cells. They are also helping to define the nature of the BM microenvironment and stem cell niche that support the growth and differentiation of LSCs. The specific targeting of the BM microenvironment and stem cell niche represent alternative and indirect strategies for the elimination of LSCs (Figure 3). The chemokine receptor CXCR4, for example, which is central to stem cell localization and a known chemo-attractant for hematopoietic cells, is induced by IM and causes CML cell migration to the BM and promotes the survival of

Targeting the Chronic Myeloid Leukemia Stem Cell:

A Paradigm for the Curative Treatment of Human Malignancies 97

quiescent CML stem cells (Figure 3). This study also suggests that an IM/HDAC combination inhibits several genes that regulate hematopoietic stem cell maintenance and survival (Zhang et al., 2010). Another study demonstrates that the targeting of autophagy, a process that allows cells to adapt to environmental stresses, enhances the effects of TKIs in *BCR-ABL*+ cell lines and in primary CML stem and progenitor cells (Bellodi et al., 2009). IM induces autophagy in BC-CML cell lines and in primary CML cells, and is associated with endoplasmic reticulum (ER) stress that is mechanistically non-overlapping with IM-induced apoptosis. Combination treatment with TKIs and inhibitors of autophagy such as chloroquine, is more effective in eliminating CML stem and progenitor cells *in vitro* than TKIs or autophagy inhibitors alone (Bellodi et al., 2009). The effectiveness of this combination treatment in eliminating primary CML stem and progenitor cells *in vivo*, however, remains to be seen. Other promising therapeutic strategies include the induction of protein phosphatase-2A activation by FTY720, which inhibits the survival and selfrenewal of CML progenitor cells (Neviani et al., 2007; Neviani et al., 2005). A farnesyltransferase inhibitor (BMS-214662) was also found to target primitive quiescent

CML cells, indicating a possible role for this class of inhibitors (Copland et al., 2008).

We have been pursuing a strategy based on the targeting of JAK2 with a view to inhibiting the activity of a biologically important multi-molecular complex that we have identified comprising AHI-1 (a novel signalling molecule encoded by the Abelson helper integration site 1 gene), the BCR-ABL fusion oncoprotein and JAK2 kinase (Zhou et al., 2008). *AHI-1* is upregulated in highly enriched populations of CML stem cells in which the levels of *BCR-ABL* transcripts are also elevated (Jiang et al., 2004; Jiang et al., 2007c). Interestingly, overexpression of *AHI-1* confers a growth advantage *in vitro* and results in leukemia *in vivo*, synergizing with *BCR-ABL* to enhance these outcomes (Zhou et al., 2008). Conversely, the stable suppression of *AHI-1* in CD34+ CML cells using small interfering RNA, reduces their growth autonomy *in vitro.* Importantly, this newly defined AHI-1-BCR-ABL-JAK2 molecular interaction complex appears to mediate leukemic stem cell transformation and plays an important role in the TKI response/resistance of primary CML stem and progenitor cells. JAK2 itself is known to interact with the C-terminus of BCR-ABL, and is one of the most prominent targets of BCR-ABL in *BCR-ABL* transformed CML cells (Miyamoto et al., 2001; Samanta et al., 2006; Xie et al., 2001). BCR-ABL has also been found to interact with the IL-3/GM-CSF receptor, which subsequently contributes to the downstream activation of JAK2 (Wilson-Rawls et al., 1997). Furthermore, in primitive CML cells, *BCR-ABL* expression stimulates the production of IL-3, G-CSF and GM-CSF which, following binding to their cognate receptors, further contributes to the CML progenitor cell resistance to TKIs via the activation of the JAK2/STAT5 pathway (Jiang et al., 1999; Wang et al., 2007). High *STAT5* levels have also been shown to mediate acquired IM-resistance in CML cells and the STAT5 inhibitor pimozide was shown to reduce their survival (Nelson et al., 2011; Warsch et al., 2011). Therefore, targeting the activity of JAK2 could provide an excellent strategy to complement the inhibition of BCR-ABL kinase activity in primary CML stem cells (Figure 4). Indeed, recent studies have demonstrated that JAK2 inhibitors (TG101209, WP1193) and a dual kinase inhibitor of JAK2 and ABL kinases (ON044580) induced apoptosis in IMsensitive and IM-resistant CML cell lines (Samanta et al., 2011; Samanta et al., 2010) and that treatment with TKIs in combination with TG101209 results in greater inhibition of CML stem and progenitor cells as compared to when the same cells are treated with either TKIs or TG101209 alone or a combination of TKIs (DeGeer et al., 2010; DeGeer et al., 2009). Several

quiescent CML progenitors (Jin et al., 2008). This suggests a possible mechanism of IM resistance working through the cross-talk between CML stem/progenitor cells and their BM microenvironment niches, and suggests a rationale for the combination of CXCR4 antagonists with TKIs so as to more effectively eliminate IM-resistant LSCs.

Fig. 3. **Targeting the CML stem cell.** Schematic diagram of the stem cell compartment and stem cell niche, indicating the specific targets of conventional first line TKI drugs, second and third generation TKIs, and the opportunities for targeting the CML stem cells either directly, or through the targeting and manipulation of the stem cell niche or bone marrow microenvironment.

Components of the sonic hedgehog signalling pathway, including the smoothened transmembrane protein (SMO), have similarly been shown to play a critical role in normal and leukemic stem cell development, proliferation and self-renewal, including the regulation of the epithelial-mesenchymal transition (Varjosalo & Taipale, 2008). Knockdown or inhibition of *SMO* impairs HSC self-renewal and abrogates or delays the appearance of CML in several *in vitro* and *in vivo* models (Dierks et al., 2008; Zhao et al., 2009). Conversely, SMO over-activity has been demonstrated in CML cells, with their proliferation being more SMO-dependent than that of healthy HSCs. Clinical trials evaluating the effects of SMO pathway antagonists developed by a number of different pharmaceutical companies and the relevance of this pathway are currently under way in a range of different malignant indications. The combination of NL with the SMO inhibitor LDE225 and DA with PF-04449913 has been reported as having additive effects on the inhibition of primitive CML cells *in vitro* and *in vivo* (Mar et al., 2011). Another study indicates that a combination of histone deacetylase inhibitors (HDACis, e.g. LAQ824) with IM is effective in targeting

quiescent CML progenitors (Jin et al., 2008). This suggests a possible mechanism of IM resistance working through the cross-talk between CML stem/progenitor cells and their BM microenvironment niches, and suggests a rationale for the combination of CXCR4

Fig. 3. **Targeting the CML stem cell.** Schematic diagram of the stem cell compartment and stem cell niche, indicating the specific targets of conventional first line TKI drugs, second and third generation TKIs, and the opportunities for targeting the CML stem cells either directly, or through the targeting and manipulation of the stem cell niche or bone marrow

Components of the sonic hedgehog signalling pathway, including the smoothened transmembrane protein (SMO), have similarly been shown to play a critical role in normal and leukemic stem cell development, proliferation and self-renewal, including the regulation of the epithelial-mesenchymal transition (Varjosalo & Taipale, 2008). Knockdown or inhibition of *SMO* impairs HSC self-renewal and abrogates or delays the appearance of CML in several *in vitro* and *in vivo* models (Dierks et al., 2008; Zhao et al., 2009). Conversely, SMO over-activity has been demonstrated in CML cells, with their proliferation being more SMO-dependent than that of healthy HSCs. Clinical trials evaluating the effects of SMO pathway antagonists developed by a number of different pharmaceutical companies and the relevance of this pathway are currently under way in a range of different malignant indications. The combination of NL with the SMO inhibitor LDE225 and DA with PF-04449913 has been reported as having additive effects on the inhibition of primitive CML cells *in vitro* and *in vivo* (Mar et al., 2011). Another study indicates that a combination of histone deacetylase inhibitors (HDACis, e.g. LAQ824) with IM is effective in targeting

microenvironment.

antagonists with TKIs so as to more effectively eliminate IM-resistant LSCs.

quiescent CML stem cells (Figure 3). This study also suggests that an IM/HDAC combination inhibits several genes that regulate hematopoietic stem cell maintenance and survival (Zhang et al., 2010). Another study demonstrates that the targeting of autophagy, a process that allows cells to adapt to environmental stresses, enhances the effects of TKIs in *BCR-ABL*+ cell lines and in primary CML stem and progenitor cells (Bellodi et al., 2009). IM induces autophagy in BC-CML cell lines and in primary CML cells, and is associated with endoplasmic reticulum (ER) stress that is mechanistically non-overlapping with IM-induced apoptosis. Combination treatment with TKIs and inhibitors of autophagy such as chloroquine, is more effective in eliminating CML stem and progenitor cells *in vitro* than TKIs or autophagy inhibitors alone (Bellodi et al., 2009). The effectiveness of this combination treatment in eliminating primary CML stem and progenitor cells *in vivo*, however, remains to be seen. Other promising therapeutic strategies include the induction of protein phosphatase-2A activation by FTY720, which inhibits the survival and selfrenewal of CML progenitor cells (Neviani et al., 2007; Neviani et al., 2005). A farnesyltransferase inhibitor (BMS-214662) was also found to target primitive quiescent CML cells, indicating a possible role for this class of inhibitors (Copland et al., 2008).

We have been pursuing a strategy based on the targeting of JAK2 with a view to inhibiting the activity of a biologically important multi-molecular complex that we have identified comprising AHI-1 (a novel signalling molecule encoded by the Abelson helper integration site 1 gene), the BCR-ABL fusion oncoprotein and JAK2 kinase (Zhou et al., 2008). *AHI-1* is upregulated in highly enriched populations of CML stem cells in which the levels of *BCR-ABL* transcripts are also elevated (Jiang et al., 2004; Jiang et al., 2007c). Interestingly, overexpression of *AHI-1* confers a growth advantage *in vitro* and results in leukemia *in vivo*, synergizing with *BCR-ABL* to enhance these outcomes (Zhou et al., 2008). Conversely, the stable suppression of *AHI-1* in CD34+ CML cells using small interfering RNA, reduces their growth autonomy *in vitro.* Importantly, this newly defined AHI-1-BCR-ABL-JAK2 molecular interaction complex appears to mediate leukemic stem cell transformation and plays an important role in the TKI response/resistance of primary CML stem and progenitor cells. JAK2 itself is known to interact with the C-terminus of BCR-ABL, and is one of the most prominent targets of BCR-ABL in *BCR-ABL* transformed CML cells (Miyamoto et al., 2001; Samanta et al., 2006; Xie et al., 2001). BCR-ABL has also been found to interact with the IL-3/GM-CSF receptor, which subsequently contributes to the downstream activation of JAK2 (Wilson-Rawls et al., 1997). Furthermore, in primitive CML cells, *BCR-ABL* expression stimulates the production of IL-3, G-CSF and GM-CSF which, following binding to their cognate receptors, further contributes to the CML progenitor cell resistance to TKIs via the activation of the JAK2/STAT5 pathway (Jiang et al., 1999; Wang et al., 2007). High *STAT5* levels have also been shown to mediate acquired IM-resistance in CML cells and the STAT5 inhibitor pimozide was shown to reduce their survival (Nelson et al., 2011; Warsch et al., 2011). Therefore, targeting the activity of JAK2 could provide an excellent strategy to complement the inhibition of BCR-ABL kinase activity in primary CML stem cells (Figure 4). Indeed, recent studies have demonstrated that JAK2 inhibitors (TG101209, WP1193) and a dual kinase inhibitor of JAK2 and ABL kinases (ON044580) induced apoptosis in IMsensitive and IM-resistant CML cell lines (Samanta et al., 2011; Samanta et al., 2010) and that treatment with TKIs in combination with TG101209 results in greater inhibition of CML stem and progenitor cells as compared to when the same cells are treated with either TKIs or TG101209 alone or a combination of TKIs (DeGeer et al., 2010; DeGeer et al., 2009). Several

Targeting the Chronic Myeloid Leukemia Stem Cell:

across a broad range of different malignancies.

**8. Conflict-of-interest disclosure** 

**7. Acknowledgment** 

Research Scholar.

in this chapter.

**9. References** 

A Paradigm for the Curative Treatment of Human Malignancies 99

for cancer stem cell eradication and the consequent provision for the basis of medical cures

The preparation of this book chapter was enabled by support from research grants from the Canadian Cancer Society (Grant# 700289), the leukemia & Lymphoma Society of Canada and the Cancer Research Society (X. J). X. Jiang is Michael Smith Foundation for Health

A. Woolfson is an employee of Bristol-Myers Squibb, some of whose products are discussed

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JAK2 inhibitors with varying degrees of intra-JAK family and intra-kinome selectivity are currently in various stages of clinical development. However, the myelosuppressive effects of these inhibitors on normal hematopoietic stem/progenitor cells remain a concern. The development of highly selective and less toxic (fewer hits across the kinome) JAK2 inhibitors in combination with first or second generation TKIs provides an attractive option for the specific targeting of LSCs (Figure 3 and 4). The permanent eradication of the leukemia stem cell in conjunction with tumor debulking, is expected to result in a functional cure, and in so doing to provide the study of CML with its ultimate crowning achievement. Importantly it should also provide molecular medicine with a general paradigm for the medical cure of cancer through stem cell eradication that with appropriate modifications should be applicable to multiple tumor types.

Fig. 4. Model of the targeting of the AHI-1-BCR-ABL-JAK2 complex in CML stem cells by combination treatment of TKI and JAK2 inhibitors. Schematic diagram of the AHI-1-BCR-ABL-JAK2 interaction complex that regulates constitutive activation of BCR-ABL and JAK2/STAT5 and results in increased proliferation and a reduced TKI response in CML stem and progenitor cells. Targeting both BCR-ABL and JAK2 activities to destabilize this protein interaction complex may represent a potential therapeutic option for CML.

#### **6. Conclusion**

The discovery of tyrosine kinase inhibitors marked a major advance in CML therapy and other cancers. Although highly successful, selective tyrosine kinase inhibition has not resulted in a functional cure. As CML is driven by genetically unstable pluripotent leukemic stem cells, therapeutic approaches that target these cells will be required for definitive curative therapies. The systematic characterization of the unique biological properties of CML LSCs promises to deliver new insights into the process of malignant transformation and disease progression. As it comprises a relatively simple and well-understood model system, it is envisaged that the elimination of LSCs in CML will provide a general paradigm for cancer stem cell eradication and the consequent provision for the basis of medical cures across a broad range of different malignancies.

#### **7. Acknowledgment**

98 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

JAK2 inhibitors with varying degrees of intra-JAK family and intra-kinome selectivity are currently in various stages of clinical development. However, the myelosuppressive effects of these inhibitors on normal hematopoietic stem/progenitor cells remain a concern. The development of highly selective and less toxic (fewer hits across the kinome) JAK2 inhibitors in combination with first or second generation TKIs provides an attractive option for the specific targeting of LSCs (Figure 3 and 4). The permanent eradication of the leukemia stem cell in conjunction with tumor debulking, is expected to result in a functional cure, and in so doing to provide the study of CML with its ultimate crowning achievement. Importantly it should also provide molecular medicine with a general paradigm for the medical cure of cancer through stem cell eradication that with appropriate modifications should be

Fig. 4. Model of the targeting of the AHI-1-BCR-ABL-JAK2 complex in CML stem cells by combination treatment of TKI and JAK2 inhibitors. Schematic diagram of the AHI-1-BCR-ABL-JAK2 interaction complex that regulates constitutive activation of BCR-ABL and JAK2/STAT5 and results in increased proliferation and a reduced TKI response in CML stem and progenitor cells. Targeting both BCR-ABL and JAK2 activities to destabilize this protein interaction complex may represent a potential therapeutic option for CML.

The discovery of tyrosine kinase inhibitors marked a major advance in CML therapy and other cancers. Although highly successful, selective tyrosine kinase inhibition has not resulted in a functional cure. As CML is driven by genetically unstable pluripotent leukemic stem cells, therapeutic approaches that target these cells will be required for definitive curative therapies. The systematic characterization of the unique biological properties of CML LSCs promises to deliver new insights into the process of malignant transformation and disease progression. As it comprises a relatively simple and well-understood model system, it is envisaged that the elimination of LSCs in CML will provide a general paradigm

applicable to multiple tumor types.

**6. Conclusion** 

The preparation of this book chapter was enabled by support from research grants from the Canadian Cancer Society (Grant# 700289), the leukemia & Lymphoma Society of Canada and the Cancer Research Society (X. J). X. Jiang is Michael Smith Foundation for Health Research Scholar.

#### **8. Conflict-of-interest disclosure**

A. Woolfson is an employee of Bristol-Myers Squibb, some of whose products are discussed in this chapter.

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**6** 

*Spain* 

**The Proteasome as a Therapeutic Target** 

Chronic Myeloid Leukemia (CML) appears as a consequence of the reciprocal translocation between chromosomes 9 and 22 which results in the expression of the Bcr-Abl oncoprotein in a hematopoietic stem cell. The tyrosine kinase activity of Bcr-Abl is the direct cause of the disease. Therefore, inhibitors of this kinase activity are being used in the treatment of CML. Imatinib was the first of such inhibitors and is the first line treatment in CML. However, the appearance of Bcr-Abl mutants that are resistant to these inhibitors has emerged as a problem in the treatment of CML. Consequently, the search for new drugs or therapeutic

The proteasome is a multiprotein complex where proteins are degraded in a much regulated manner. Protein degradation at the proteasome is dependent upon ubiquitylation, which, in turn, is mediated by ubiquitin-ligase complexes. By degrading specific proteins, the proteasome is involved in cellular processes as important as cell cycle regulation, proliferation, differentiation and survival. As such, its activity is also related to the transformation of tumor cells. In fact, it has been shown that tumor cells show an increase in proteasome activity. Specifically, Bcr-Abl tyrosine kinase activity results in an increase of proteasome activity and degradation of some negative regulators of cell cycle progression as

p27kip1. Therefore, proteasome inhibitors could be an alternative in CML treatment.

**2. Bcr-Abl and its control of the cell cycle and apoptosis** 

In this chapter, we will discuss the available data suggesting that proteasome inhibition could be used in the treatment of CML, including those cases in which Bcr-Abl inhibition is

In the t(9;22)(q34;q11) translocation found in CML, the *bcr* (Breakpoint Cluster Region) and *abl* (Abelson Leukemia) genes get fused. As a result, the chimeric tyrosine kinase oncoprotein Bcr-Abl is expressed (Ben-Neriah et al., 1986). Unlike c-Abl that shuttles between the nucleus and the cytoplasm and whose kinase activity is finely tuned, Bcr-Abl is cytoplasmic and its kinase activity is increased and deregulated (Salesse & Verfaillie, 2002). Through phosphorylation of different substrates, Bcr-Abl activates different signal transduction pathways that participate in the control of cellular proliferation, differentiation, migration, adhesion and survival (Afar et al., 1994; Andreu et al., 2005; Bedi et al., 1994;

**1. Introduction** 

targets is underway.

not a possibility.

**in Chronic Myeloid Leukemia** 

Ignacio Pérez-Roger and María Pilar Albero

*Universidad CEU Cardenal Herrera* 

Zhou, L.L., Zhao, Y., Ringrose, A., DeGeer, D., Kennah, E., Lin, A.E., Sheng, G., Li, X.J., Turhan, A. & Jiang, X. (2008). AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells. *J. Exp. Med.,* Vol.205, 2657-2671

### **The Proteasome as a Therapeutic Target in Chronic Myeloid Leukemia**

Ignacio Pérez-Roger and María Pilar Albero *Universidad CEU Cardenal Herrera Spain* 

#### **1. Introduction**

110 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Zhou, L.L., Zhao, Y., Ringrose, A., DeGeer, D., Kennah, E., Lin, A.E., Sheng, G., Li, X.J.,

*Exp. Med.,* Vol.205, 2657-2671

Turhan, A. & Jiang, X. (2008). AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells. *J.* 

> Chronic Myeloid Leukemia (CML) appears as a consequence of the reciprocal translocation between chromosomes 9 and 22 which results in the expression of the Bcr-Abl oncoprotein in a hematopoietic stem cell. The tyrosine kinase activity of Bcr-Abl is the direct cause of the disease. Therefore, inhibitors of this kinase activity are being used in the treatment of CML. Imatinib was the first of such inhibitors and is the first line treatment in CML. However, the appearance of Bcr-Abl mutants that are resistant to these inhibitors has emerged as a problem in the treatment of CML. Consequently, the search for new drugs or therapeutic targets is underway.

> The proteasome is a multiprotein complex where proteins are degraded in a much regulated manner. Protein degradation at the proteasome is dependent upon ubiquitylation, which, in turn, is mediated by ubiquitin-ligase complexes. By degrading specific proteins, the proteasome is involved in cellular processes as important as cell cycle regulation, proliferation, differentiation and survival. As such, its activity is also related to the transformation of tumor cells. In fact, it has been shown that tumor cells show an increase in proteasome activity. Specifically, Bcr-Abl tyrosine kinase activity results in an increase of proteasome activity and degradation of some negative regulators of cell cycle progression as p27kip1. Therefore, proteasome inhibitors could be an alternative in CML treatment.

> In this chapter, we will discuss the available data suggesting that proteasome inhibition could be used in the treatment of CML, including those cases in which Bcr-Abl inhibition is not a possibility.

#### **2. Bcr-Abl and its control of the cell cycle and apoptosis**

In the t(9;22)(q34;q11) translocation found in CML, the *bcr* (Breakpoint Cluster Region) and *abl* (Abelson Leukemia) genes get fused. As a result, the chimeric tyrosine kinase oncoprotein Bcr-Abl is expressed (Ben-Neriah et al., 1986). Unlike c-Abl that shuttles between the nucleus and the cytoplasm and whose kinase activity is finely tuned, Bcr-Abl is cytoplasmic and its kinase activity is increased and deregulated (Salesse & Verfaillie, 2002). Through phosphorylation of different substrates, Bcr-Abl activates different signal transduction pathways that participate in the control of cellular proliferation, differentiation, migration, adhesion and survival (Afar et al., 1994; Andreu et al., 2005; Bedi et al., 1994;

The Proteasome as a Therapeutic Target in Chronic Myeloid Leukemia 113

division, differentiation and development, all of them related directly or indirectly to

For a protein to be degraded at the proteasome, it must be labelled with ubiquitin. Ubiquitin is a small polypeptide of 76 aminoacids which is highly conserved in all eukaryotes. Protein ubiquitylation not only can lead to degradation but is also a mechanism involved in intracellular transport. Ubiquitin can be added to proteins as single molecules to one (monoubiquitylation) or several (multiubiquitylation) Lysine residues or as polyubiquitin chains (polyubiquitylation) using one of several Lysine residues of the ubiquitin molecule to

Ubiquitylation requires the sequential activity of three enzymes. The C-terminal Glycine of ubiquitin is activated in an ATP dependent process by an E1 ubiquitin-activating enzyme. This reaction results in the formation of a thioester covalent linkage between ubiquitin and a Cysteine residue in E1. Activated ubiquitin is then transferred to the ubiquitin-conjugating enzyme E2 by a trans-esterification reaction. Finally, a ubiquitin-ligase E3 enzyme transfers the ubiquitin molecule to the specific target protein to be degraded, thus acting as the substrate recognizing enzyme. This conserved ubiquitylation cascade allows for different combinations of E1-E2-E3 molecules leading to the recognition, labeling and degradation of

Along the cell cycle, two different E3 complexes are involved in the degradation of regulatory proteins. The SCF complex contains four different subunits: Skp1, Cul1, Roc1/Rbx1 and one of several different F-box proteins. This E3 complex is necessary, for instance, for the degradation of G1 cyclins, leading to the G1/S transition. G2 cyclins, on the other hand, are ubiquitylated by the E3 APC/C (Anaphase Promoting Complex/Cyclosome). The APC/C complex is activated by phosphorylation and its activity

The F-box protein in the SCF complexes interacts with specific substrates targeting them for degradation. Interaction with Skp1 requires the conserved F-box region. The substrate that is to be degraded needs to be previously phosphorylated. SCF complexes containing the Fbox protein β-Trcp (SCFβ-Trcp) are involved in the degradation of proteins of the NF-κB pathway and of β-catenin. Another important F-box protein is Skp2 (S-phase kinaseinteracting protein 2), identified as a protein interacting with cyclin A/Cdk2 complexes (Zhang et al., 1995). Many key regulators of the cell cycle, including the cell cycle inhibitor p27kip1, are ubiquitylated by SCFSkp2 complexes (Carrano et al., 1999; Sutterluty et al., 1999;

The cell cycle inhibitor p27kip1 binds to Cdk2-containing complexes, inhibiting its kinase activity and therefore blocking cell cycle progression along G1 and into the S phase. It can also bind to cyclin D-containing complexes, where p27kip1 is "sequestered" and maintained away from active cyclin E/Cdk2 complexes (Bouchard et al., 1999; Perez-Roger et al., 1999). p27kip1 levels in tumors are useful as a prognostic marker. Degradation of p27kip1 at the proteasome requires its previous phosphorylation (at Threonine 187) by the same cyclin E/Cdk2 complexes it inhibits. Next, the SCFSkp2 complex recognizes the phosphorylated protein and labels it with a polyubiquitin chain leading to its degradation (Carrano et al., 1999; Montagnoli et al., 1999; Nguyen et al., 1999; Perez-Roger et al., 1999; Perez-Roger et al., 1997; Sheaff et al., 1997; Sutterluty et al., 1999; Tsvetkov et al., 1999; Vlach et al., 1997).

tumoral transformation (DeSalle & Pagano, 2001).

multiple target proteins (Hershko & Ciechanover, 1998).

**3.2 The proteasome and the cell cycle control** 

is required for mitosis (Murray, 2004).

Tsvetkov et al., 1999).

form the chain.

Gordon et al., 1987; Horita et al., 2000; Jiang et al., 2000; Puil et al., 1994; Salesse & Verfaillie, 2003; Sawyers, 1993).

Several different substrates and partners of Bcr-Abl have been identified so far. Current efforts are directed at linking these pathways to the specific pathologic defects that characterize CML. These defects include increased proliferation and/or decreased apoptosis of a hematopoietic stem cell or progenitor cell (which leads to a massive increase in myeloid cell numbers), premature release of immature myeloid cells into the circulation (probably due to a defect in adherence of myeloid progenitors to marrow stroma) and genetic instability which results in disease progression (Druker, 2008).

Pathways activated by Bcr-Abl include the Ras-Raf-MAPK, the PI3K/Akt and the JAK/STAT pathways. The Ras-Raf-MAPK pathway links Bcr-Abl to the increased proliferative rate. The PI3K/Akt pathway induces survival signals through different mechanisms such as the Ras-Raf pathway, the phosphorylation of Bad, the induction of BclxL expression and the inhibition of caspase activity. Besides, it controls the activity of several transcription factors involved in the expression of cell cycle regulatory proteins, such as FoxO, belonging to the Forkhead family of transcription factors, and NF-κB (Manning & Cantley, 2007; Medema et al., 2000). Akt also regulates cell growth through mTOR and the control of protein translation and synthesis (Manning & Cantley, 2007). The JAK/STAT pathway is also a target of Bcr-Abl kinase activity. Bcr-Abl induces the expression of the antiapoptotic protein Bcl-xL through the activation of STAT5, which binds to the promoter of the *bcl-xL* gene, thus increasing survival of Bcr-Abl expressing cells (Horita et al., 2000).

Bcr-Abl regulates the expression and activity of the cell cycle inhibitor p27kip1 through the PI3K/Akt pathway. The inhibition of Bcr-Abl and the inhibition of the PI3K/Akt pathway both result in an increase of p27kip1 levels. Bcr-Abl also induces the expression of cyclins D and E and the activity of the cyclin E associated kinase Cdk2, resulting in the phosphorylation and inactivation of the tumor suppressor Retinoblastoma protein (Rb). Consequently, Bcr-Abl expressing cells show a deregulation of one of the main mechanisms controlling cell cycle progression along G1 and into S phase (Andreu et al., 2005).

Therefore, by activating several different signal transduction pathways, Bcr-Abl induces cell proliferation and suppresses apoptosis in CML cells.

#### **3. The proteasome as a therapeutic target**

Protein synthesis and degradation are key mechanisms by which protein levels are regulated inside cells. In fact, specific protein degradation has emerged as a very active field of research. Two different machineries are involved in this process: the autophagosome (which will not be considered in this chapter) and the proteasome.

#### **3.1 Protein degradation at the proteasome**

The 26S proteasome is a multiprotein complex that can be found in the cytoplasm and in the nucleus of eukaryotic cells. It is composed of a catalytic 20S core and two 19S regulatory subunits. Proteasome assembly and protein degradation are both ATP dependent (Almond & Cohen, 2002; Baumeister et al., 1997; Baumeister et al., 1998). The catalytic core has three activities: chymotryptic, tryptic, and post-glutamyl peptide hydrolytic-like activities.The regulatory subunits are responsible for substrate recognition and unfolding. The proteasome is involved in a wide variety of cellular processes including apoptosis, cell cycle and

Gordon et al., 1987; Horita et al., 2000; Jiang et al., 2000; Puil et al., 1994; Salesse & Verfaillie,

Several different substrates and partners of Bcr-Abl have been identified so far. Current efforts are directed at linking these pathways to the specific pathologic defects that characterize CML. These defects include increased proliferation and/or decreased apoptosis of a hematopoietic stem cell or progenitor cell (which leads to a massive increase in myeloid cell numbers), premature release of immature myeloid cells into the circulation (probably due to a defect in adherence of myeloid progenitors to marrow stroma) and genetic

Pathways activated by Bcr-Abl include the Ras-Raf-MAPK, the PI3K/Akt and the JAK/STAT pathways. The Ras-Raf-MAPK pathway links Bcr-Abl to the increased proliferative rate. The PI3K/Akt pathway induces survival signals through different mechanisms such as the Ras-Raf pathway, the phosphorylation of Bad, the induction of BclxL expression and the inhibition of caspase activity. Besides, it controls the activity of several transcription factors involved in the expression of cell cycle regulatory proteins, such as FoxO, belonging to the Forkhead family of transcription factors, and NF-κB (Manning & Cantley, 2007; Medema et al., 2000). Akt also regulates cell growth through mTOR and the control of protein translation and synthesis (Manning & Cantley, 2007). The JAK/STAT pathway is also a target of Bcr-Abl kinase activity. Bcr-Abl induces the expression of the antiapoptotic protein Bcl-xL through the activation of STAT5, which binds to the promoter of the *bcl-xL* gene, thus increasing survival of Bcr-Abl expressing cells (Horita et al., 2000). Bcr-Abl regulates the expression and activity of the cell cycle inhibitor p27kip1 through the PI3K/Akt pathway. The inhibition of Bcr-Abl and the inhibition of the PI3K/Akt pathway both result in an increase of p27kip1 levels. Bcr-Abl also induces the expression of cyclins D and E and the activity of the cyclin E associated kinase Cdk2, resulting in the phosphorylation and inactivation of the tumor suppressor Retinoblastoma protein (Rb). Consequently, Bcr-Abl expressing cells show a deregulation of one of the main mechanisms

controlling cell cycle progression along G1 and into S phase (Andreu et al., 2005).

proliferation and suppresses apoptosis in CML cells.

**3. The proteasome as a therapeutic target** 

**3.1 Protein degradation at the proteasome** 

(which will not be considered in this chapter) and the proteasome.

Therefore, by activating several different signal transduction pathways, Bcr-Abl induces cell

Protein synthesis and degradation are key mechanisms by which protein levels are regulated inside cells. In fact, specific protein degradation has emerged as a very active field of research. Two different machineries are involved in this process: the autophagosome

The 26S proteasome is a multiprotein complex that can be found in the cytoplasm and in the nucleus of eukaryotic cells. It is composed of a catalytic 20S core and two 19S regulatory subunits. Proteasome assembly and protein degradation are both ATP dependent (Almond & Cohen, 2002; Baumeister et al., 1997; Baumeister et al., 1998). The catalytic core has three activities: chymotryptic, tryptic, and post-glutamyl peptide hydrolytic-like activities.The regulatory subunits are responsible for substrate recognition and unfolding. The proteasome is involved in a wide variety of cellular processes including apoptosis, cell cycle and

instability which results in disease progression (Druker, 2008).

2003; Sawyers, 1993).

division, differentiation and development, all of them related directly or indirectly to tumoral transformation (DeSalle & Pagano, 2001).

For a protein to be degraded at the proteasome, it must be labelled with ubiquitin. Ubiquitin is a small polypeptide of 76 aminoacids which is highly conserved in all eukaryotes. Protein ubiquitylation not only can lead to degradation but is also a mechanism involved in intracellular transport. Ubiquitin can be added to proteins as single molecules to one (monoubiquitylation) or several (multiubiquitylation) Lysine residues or as polyubiquitin chains (polyubiquitylation) using one of several Lysine residues of the ubiquitin molecule to form the chain.

Ubiquitylation requires the sequential activity of three enzymes. The C-terminal Glycine of ubiquitin is activated in an ATP dependent process by an E1 ubiquitin-activating enzyme. This reaction results in the formation of a thioester covalent linkage between ubiquitin and a Cysteine residue in E1. Activated ubiquitin is then transferred to the ubiquitin-conjugating enzyme E2 by a trans-esterification reaction. Finally, a ubiquitin-ligase E3 enzyme transfers the ubiquitin molecule to the specific target protein to be degraded, thus acting as the substrate recognizing enzyme. This conserved ubiquitylation cascade allows for different combinations of E1-E2-E3 molecules leading to the recognition, labeling and degradation of multiple target proteins (Hershko & Ciechanover, 1998).

#### **3.2 The proteasome and the cell cycle control**

Along the cell cycle, two different E3 complexes are involved in the degradation of regulatory proteins. The SCF complex contains four different subunits: Skp1, Cul1, Roc1/Rbx1 and one of several different F-box proteins. This E3 complex is necessary, for instance, for the degradation of G1 cyclins, leading to the G1/S transition. G2 cyclins, on the other hand, are ubiquitylated by the E3 APC/C (Anaphase Promoting Complex/Cyclosome). The APC/C complex is activated by phosphorylation and its activity is required for mitosis (Murray, 2004).

The F-box protein in the SCF complexes interacts with specific substrates targeting them for degradation. Interaction with Skp1 requires the conserved F-box region. The substrate that is to be degraded needs to be previously phosphorylated. SCF complexes containing the Fbox protein β-Trcp (SCFβ-Trcp) are involved in the degradation of proteins of the NF-κB pathway and of β-catenin. Another important F-box protein is Skp2 (S-phase kinaseinteracting protein 2), identified as a protein interacting with cyclin A/Cdk2 complexes (Zhang et al., 1995). Many key regulators of the cell cycle, including the cell cycle inhibitor p27kip1, are ubiquitylated by SCFSkp2 complexes (Carrano et al., 1999; Sutterluty et al., 1999; Tsvetkov et al., 1999).

The cell cycle inhibitor p27kip1 binds to Cdk2-containing complexes, inhibiting its kinase activity and therefore blocking cell cycle progression along G1 and into the S phase. It can also bind to cyclin D-containing complexes, where p27kip1 is "sequestered" and maintained away from active cyclin E/Cdk2 complexes (Bouchard et al., 1999; Perez-Roger et al., 1999). p27kip1 levels in tumors are useful as a prognostic marker. Degradation of p27kip1 at the proteasome requires its previous phosphorylation (at Threonine 187) by the same cyclin E/Cdk2 complexes it inhibits. Next, the SCFSkp2 complex recognizes the phosphorylated protein and labels it with a polyubiquitin chain leading to its degradation (Carrano et al., 1999; Montagnoli et al., 1999; Nguyen et al., 1999; Perez-Roger et al., 1999; Perez-Roger et al., 1997; Sheaff et al., 1997; Sutterluty et al., 1999; Tsvetkov et al., 1999; Vlach et al., 1997).

The Proteasome as a Therapeutic Target in Chronic Myeloid Leukemia 115

expression of Skp2 is silenced in Bcr-Abl expressing cells, the levels of p27kip1 increase, showing again the inverse relationship between these two proteins. The regulation of p27kip1 expression by Bcr-Abl depends mainly on the PI3K/Akt pathway. This can be demonstrated because when cells are treated with either the Bcr-Abl inhibitor Imatinib or the PI3K inhibitor LY294002 the effects are quite similar. These effects are threefold (Fig. 1): first, Akt phosphorylates and inactivates FoxO transcription factors, involved in the regulation of p27kip1 gene transcription; second, Bcr-Abl induces the expression of Skp2 through the Akt pathway, which results in the proteasomal degradation of p27kip1 (Andreu et al., 2005); third, Akt also phosphorylates p27kip1 directly, preventing its translocation to the nucleus where it

inhibits the cyclin/Cdk complexes controlling the cell cycle (Liang et al., 2002).

Fig. 1. Bcr-Abl regulation of the level and activity of p27kip1 includes transcriptional

Apart from the regulation of key proteins controlling cell cycle progression, there is a direct relationship between Bcr-Abl and the proteasome. Exogenous expression of Bcr-Abl in cells directly increases the activity of the proteasome and siRNA knock down of Bcr-Abl expression in K562 cells is associated with a decrease of all three proteasome activities

As Bcr-Abl regulates the activity of the proteasome and induces the proteasomal degradation of several proteins, Bortezomib was proposed as a therapeutic alternative to

Proteasome inhibition with Bortezomib in Bcr-Abl expressing cell models or primary CD34+ cells from CML patients results in a cell cycle inhibition and induction of apoptosis (Albero et al., 2010; Gatto et al., 2003; Jagani et al., 2009). Different key regulators are affected by Bortezomib leading to the detection of apoptosis markers, such as the activation (cleavage) of caspases (3, 8 and 9), or the down-regulation of Bcl-xL and induction of Bim. Among these

inhibition, misslocalization and proteasomal degradation.

Imatinib, especially in CML cases of Imatinib resistance.

**4.2 Molecular changes in CML cells treated with Bortezomib** 

(Crawford et al., 2009).

Skp2 expression changes along the cell cycle, being higher in S and G2 and lower in M and G1. Induction of Skp2 in the G1-S transition promotes the degradation of p27kip1, allowing cell cycle progression (Andreu et al., 2005; Carrano et al., 1999; Marti et al., 1999; Schneider et al., 2006; Wirbelauer et al., 2000; Zhang et al., 1995). Skp2 is degraded at the proteasome in mitosis, when the APC/C complex becomes active (Wirbelauer et al., 2000). Ectopic expression of Skp2 in quiescent cells is sufficient to induce cell cycle entry, making Skp2 a potent oncoprotein. In many tumors, the levels of Skp2 and of p27kip1 are inversely proportional: low Skp2 correlates with high p27kip1 and slow growing tumors, whereas high Skp2 correlates with low p27kip1 and fast growing tumors.

#### **3.3 Proteasome inhibition: Bortezomib**

The relationship between the ubiquitin-proteasome system (UPS) and the regulation of cellular proliferation, differentiation, survival and transformation led to considering the proteasome as a suitable target in cancer therapy (Adams, 2004). Proteasome inhibitors block protein degradation, causing the accumulation of damaged proteins and inducing the heat shock response and cell death (Concannon et al., 2007; Friedman & Xue, 2004).

Among the several proteasome inhibitors described, Bortezomib has been approved by the FDA for treatment of Multiple Myeloma and Mantle Cell Lymphoma (Ocio et al., 2008; Perez-Galan et al., 2006). Bortezomib is a dipeptdyl boronic acid that inhibits specifically and reversibly the chymotrypsine like activity of the proteasome, thought to be the rate limiting step of proteasome degradation (Eldridge & O'Brien, 2010; Jung et al., 2004; Richardson et al., 2006).

Surprisingly, Bortezomib appears to be quite selective against tumor cells. For some reason, normal cells seem to be more resistant to proteasome inhibition. One of the molecular effects of Bortezomib in several (but not all) cell types is the inhibition of the NF-κB pathway involved in suppressing apoptosis and increasing cell survival and resistance to cytotoxic agents (Baud & Karin, 2009). Some cases of Multiple Myeloma show an increase in NF-κB signaling making them especially sensitive to Bortezomib. Other probably important targets of Bortezomib are p53, p21, p27, Bax, Smac/Diablo and p44/42MAPK, all of them involved in the regulation of cell proliferation and/or survival (Voorhees et al., 2003).

#### **4. Inhibition of the proteasome in CML**

Proteasome activity is necessary for all cells to maintain the appropriate balance between protein synthesis and degradation. Therefore, proteasome inhibitors have a highly cytotoxic effect. However, cancer cells seem to be more sensitive to proteasome inhibition than control or normal cells.

#### **4.1 Bcr-Abl and the proteasome**

Bcr-Abl activity down-regulates the level of the cell cycle inhibitor p27kip1 in different cell models expressing Bcr-Abl endogenously (like K562) or exogenously (like Mo7e and BaF/3) and in primary CML CD34+ cells. Moreover, exogenous p27kip1 expression partially antagonizes the effect of Bcr-Abl on proliferation (Albero et al., 2010; Andreu et al., 2005; Bretones et al., 2011; Jonuleit et al., 2000). By inhibiting the tyrosine kinase activity of Bcr-Abl with Imatinib, the half-life of p27kip1 is increased, meaning that Bcr-Abl induces the degradation of p27kip1. At the same time, the expression of Skp2 decreases and when the

Skp2 expression changes along the cell cycle, being higher in S and G2 and lower in M and G1. Induction of Skp2 in the G1-S transition promotes the degradation of p27kip1, allowing cell cycle progression (Andreu et al., 2005; Carrano et al., 1999; Marti et al., 1999; Schneider et al., 2006; Wirbelauer et al., 2000; Zhang et al., 1995). Skp2 is degraded at the proteasome in mitosis, when the APC/C complex becomes active (Wirbelauer et al., 2000). Ectopic expression of Skp2 in quiescent cells is sufficient to induce cell cycle entry, making Skp2 a potent oncoprotein. In many tumors, the levels of Skp2 and of p27kip1 are inversely proportional: low Skp2 correlates with high p27kip1 and slow growing tumors, whereas high

The relationship between the ubiquitin-proteasome system (UPS) and the regulation of cellular proliferation, differentiation, survival and transformation led to considering the proteasome as a suitable target in cancer therapy (Adams, 2004). Proteasome inhibitors block protein degradation, causing the accumulation of damaged proteins and inducing the

Among the several proteasome inhibitors described, Bortezomib has been approved by the FDA for treatment of Multiple Myeloma and Mantle Cell Lymphoma (Ocio et al., 2008; Perez-Galan et al., 2006). Bortezomib is a dipeptdyl boronic acid that inhibits specifically and reversibly the chymotrypsine like activity of the proteasome, thought to be the rate limiting step of proteasome degradation (Eldridge & O'Brien, 2010; Jung et al., 2004;

Surprisingly, Bortezomib appears to be quite selective against tumor cells. For some reason, normal cells seem to be more resistant to proteasome inhibition. One of the molecular effects of Bortezomib in several (but not all) cell types is the inhibition of the NF-κB pathway involved in suppressing apoptosis and increasing cell survival and resistance to cytotoxic agents (Baud & Karin, 2009). Some cases of Multiple Myeloma show an increase in NF-κB signaling making them especially sensitive to Bortezomib. Other probably important targets of Bortezomib are p53, p21, p27, Bax, Smac/Diablo and p44/42MAPK, all of them involved

Proteasome activity is necessary for all cells to maintain the appropriate balance between protein synthesis and degradation. Therefore, proteasome inhibitors have a highly cytotoxic effect. However, cancer cells seem to be more sensitive to proteasome inhibition than control

Bcr-Abl activity down-regulates the level of the cell cycle inhibitor p27kip1 in different cell models expressing Bcr-Abl endogenously (like K562) or exogenously (like Mo7e and BaF/3) and in primary CML CD34+ cells. Moreover, exogenous p27kip1 expression partially antagonizes the effect of Bcr-Abl on proliferation (Albero et al., 2010; Andreu et al., 2005; Bretones et al., 2011; Jonuleit et al., 2000). By inhibiting the tyrosine kinase activity of Bcr-Abl with Imatinib, the half-life of p27kip1 is increased, meaning that Bcr-Abl induces the degradation of p27kip1. At the same time, the expression of Skp2 decreases and when the

heat shock response and cell death (Concannon et al., 2007; Friedman & Xue, 2004).

in the regulation of cell proliferation and/or survival (Voorhees et al., 2003).

Skp2 correlates with low p27kip1 and fast growing tumors.

**3.3 Proteasome inhibition: Bortezomib** 

**4. Inhibition of the proteasome in CML** 

**4.1 Bcr-Abl and the proteasome** 

Richardson et al., 2006).

or normal cells.

expression of Skp2 is silenced in Bcr-Abl expressing cells, the levels of p27kip1 increase, showing again the inverse relationship between these two proteins. The regulation of p27kip1 expression by Bcr-Abl depends mainly on the PI3K/Akt pathway. This can be demonstrated because when cells are treated with either the Bcr-Abl inhibitor Imatinib or the PI3K inhibitor LY294002 the effects are quite similar. These effects are threefold (Fig. 1): first, Akt phosphorylates and inactivates FoxO transcription factors, involved in the regulation of p27kip1 gene transcription; second, Bcr-Abl induces the expression of Skp2 through the Akt pathway, which results in the proteasomal degradation of p27kip1 (Andreu et al., 2005); third, Akt also phosphorylates p27kip1 directly, preventing its translocation to the nucleus where it inhibits the cyclin/Cdk complexes controlling the cell cycle (Liang et al., 2002).

Fig. 1. Bcr-Abl regulation of the level and activity of p27kip1 includes transcriptional inhibition, misslocalization and proteasomal degradation.

Apart from the regulation of key proteins controlling cell cycle progression, there is a direct relationship between Bcr-Abl and the proteasome. Exogenous expression of Bcr-Abl in cells directly increases the activity of the proteasome and siRNA knock down of Bcr-Abl expression in K562 cells is associated with a decrease of all three proteasome activities (Crawford et al., 2009).

As Bcr-Abl regulates the activity of the proteasome and induces the proteasomal degradation of several proteins, Bortezomib was proposed as a therapeutic alternative to Imatinib, especially in CML cases of Imatinib resistance.

#### **4.2 Molecular changes in CML cells treated with Bortezomib**

Proteasome inhibition with Bortezomib in Bcr-Abl expressing cell models or primary CD34+ cells from CML patients results in a cell cycle inhibition and induction of apoptosis (Albero et al., 2010; Gatto et al., 2003; Jagani et al., 2009). Different key regulators are affected by Bortezomib leading to the detection of apoptosis markers, such as the activation (cleavage) of caspases (3, 8 and 9), or the down-regulation of Bcl-xL and induction of Bim. Among these

The Proteasome as a Therapeutic Target in Chronic Myeloid Leukemia 117

As Bortezomib inhibition of the cell cycle and induction of apoptosis is independent of Bcr-Abl kinase activity it should be effective even in Imatinib resistant cases. Several different reports show the effect of Bortezomib on cells resistant to Imatinib, using cell lines exogenously expressing wild type and mutant forms of Bcr-Abl, primary CD34+ cells and cell lines derived from patients in blast crisis resistant to Imatinib, or CML cell lines selected for Imatinib resistance by chronic exposure to the inhibitor. In all these cases, Bortezomib induces apoptosis of Bcr-Abl expressing cells regardless of the mutational status of the protein, and at concentrations at which control cells are not sensitive (Albero et al., 2010; Colado et al., 2008; Crawford et al., 2009; Gatto et al., 2003; Heaney et al., 2010; Jagani et al., 2009). As these studies include the highly resistant gate keeper mutant T315I, it is clear that

Bortezomib could be an alternative to Imatinib when this treatment is ineffective.

Another source of Imatinib resistant cells is the population of primitive, quiescent hematopoietic CML stem cells (Graham et al., 2002). During Imatinib treatment, there is an initial phase in which Bcr-Abl transcripts decrease rapidly, followed by a second phase in which this decrease is slower. This is probably due to the different sensitivity of CML cell subpopulations to Imatinib. Differentiated cells are more sensitive to Imatinib, whereas primitive CML stem cells are less sensitive to Imatinib. This difference could be due to the almost quiescent state of CML stem cells. This subpopulation of cells (Lin-CD34+) represents approximately 0.5% of the CD34+ cells but are likely to be responsible for the minimal residual disease found in Imatinib treated patients and to relapse if the treatment is interrupted. It is estimated that up to 106 lekemia cells may remain in patients with no detectable Bcr-Abl transcripts and therefore it is recommended that Imatinib treatment is not interrupted (Bhatia et al., 2003; Graham et al., 2002; Holyoake et al., 1999; Holyoake et

Bortezomib is also effective against the quiescent CML stem cells in different assays (Heaney et al., 2010). Bortezomib treatment induces apoptosis of CD34+ cells from chronic phase CML patients at diagnosis, with the accumulation of ubiquitylated proteins but with no effect on the kinase activity of Bcr-Abl. The primitive CD34+CD38- population that includes the quiescent CML stem cells is also sensitive to Bortezomib, which reduces the long-term colony formation capability of the cells. Finally, Bortezomib treatment also leads to a significant reduction in the engraftment potential of human CD34+ CML cells, although in this experiment there were still detectable Bcr-Abl positive cells. However, the effect of Bortezomib is not specific for CML cells, as it induces apoptosis also in normal CD34+CD38-

Imatinib is the first and so far the best example of rational design of anticancer therapeutic drugs. However, it is not able to cure and in some cases its efficacy is reduced due to Bcr-Abl dependent and independent mechanisms of resistance. In this context, the relationship between Bcr-Abl and the proteasome is worth exploiting. In fact, the proteasome inhibitor Bortezomib has shown its potential *in vitro* and in animal models. Nevertheless, proteasome inhibition is cytotoxic to all cell types. Therefore, the future of Bortezomib and other "nonspecific" inhibitors or drugs may lie in the search for synergies with other "specific" drugs aimed to the molecular events characteristic of the tumor. In Chronic Myeloid Leukemia,

**4.3 Effect of Bortezomib on Imatinib resistant CML cells** 

al., 2001; Quintas-Cardama et al., 2009).

hematopoietic stem cells (Heaney et al., 2010).

**5. Conclusion** 

key events, Bortezomib treatment reverses the Bcr-Abl suppression of FoxO proteins and blocks the activation of NF-κB.

Although apoptosis induction is a commonplace of Bortezomib treatment, the inhibition of the cell cycle can be at the G1/S transition or at the G2/M boundary, depending on the cell models used (Albero et al., 2010; Gatto et al., 2003). The cell cycle inhibition at the G1/S transition can be explained by the accumulation of p27kip1 in Bortezomib treated cells (Fig. 2). This accumulation inhibits the kinase activity of cyclin/Cdk complexes, which results in the dephosphorylation (activation) of Rb, blocking the cell cycle at the restriction point by the sequestration and inactivation of E2F transcription factors. The dephosphorylation of Rb is followed by its caspase-dependent processing and degradation, something associated with the induction of apoptosis. As a consequence of the impact on Rb phosphorylation, Bortezomib treatment also results in the down-regulation of cyclin A expression (Albero et al., 2010).

In cell lines derived from CML patients in blast crisis, Bortezomib induces the accumulation of IκBα, correlating with a decrease in the NF-κB DNA binding activity in the nucleus at the same time (Gatto et al., 2003). However, this effect was only transient and perhaps does not explain the cellular effects of Bortezomib. In contrast, in Baf/3-derived cell lines exogenously expressing Bcr-Abl, there was no effect of Bortezomib on NF-κB activity (Albero et al., 2010). However, in this same study, the non-canonical NF-κB pathway was analyzed and the results show that Bortezomib prevents the activation of NF-κB2. This protein is activated by proteolysis from a 100 kDa precursor to yield the 52 kDa active form in a proteasome dependent manner (Barre & Perkins, 2007). As NF-κB2 is not activated, the expression of two of its regulated targets, Skp2 and Myc, is also inhibited (Albero et al., 2010; Barre & Perkins, 2007; Schneider et al., 2006). This effect, in turn, would reinforce the effect of Bortezomib on the accumulation of p27kip1 (Fig. 2) leading to the dephosphorylation of Rb and the cell cycle inhibition at the G1/S transition (Albero et al., 2010).

Fig. 2. Possible molecular mechanism leading to cell cycle arrest in G1 and apoptosis in CML cells treated with Bortezomib, as proposed (Albero et al., 2010).

#### **4.3 Effect of Bortezomib on Imatinib resistant CML cells**

As Bortezomib inhibition of the cell cycle and induction of apoptosis is independent of Bcr-Abl kinase activity it should be effective even in Imatinib resistant cases. Several different reports show the effect of Bortezomib on cells resistant to Imatinib, using cell lines exogenously expressing wild type and mutant forms of Bcr-Abl, primary CD34+ cells and cell lines derived from patients in blast crisis resistant to Imatinib, or CML cell lines selected for Imatinib resistance by chronic exposure to the inhibitor. In all these cases, Bortezomib induces apoptosis of Bcr-Abl expressing cells regardless of the mutational status of the protein, and at concentrations at which control cells are not sensitive (Albero et al., 2010; Colado et al., 2008; Crawford et al., 2009; Gatto et al., 2003; Heaney et al., 2010; Jagani et al., 2009). As these studies include the highly resistant gate keeper mutant T315I, it is clear that Bortezomib could be an alternative to Imatinib when this treatment is ineffective.

Another source of Imatinib resistant cells is the population of primitive, quiescent hematopoietic CML stem cells (Graham et al., 2002). During Imatinib treatment, there is an initial phase in which Bcr-Abl transcripts decrease rapidly, followed by a second phase in which this decrease is slower. This is probably due to the different sensitivity of CML cell subpopulations to Imatinib. Differentiated cells are more sensitive to Imatinib, whereas primitive CML stem cells are less sensitive to Imatinib. This difference could be due to the almost quiescent state of CML stem cells. This subpopulation of cells (Lin-CD34+) represents approximately 0.5% of the CD34+ cells but are likely to be responsible for the minimal residual disease found in Imatinib treated patients and to relapse if the treatment is interrupted. It is estimated that up to 106 lekemia cells may remain in patients with no detectable Bcr-Abl transcripts and therefore it is recommended that Imatinib treatment is not interrupted (Bhatia et al., 2003; Graham et al., 2002; Holyoake et al., 1999; Holyoake et al., 2001; Quintas-Cardama et al., 2009).

Bortezomib is also effective against the quiescent CML stem cells in different assays (Heaney et al., 2010). Bortezomib treatment induces apoptosis of CD34+ cells from chronic phase CML patients at diagnosis, with the accumulation of ubiquitylated proteins but with no effect on the kinase activity of Bcr-Abl. The primitive CD34+CD38- population that includes the quiescent CML stem cells is also sensitive to Bortezomib, which reduces the long-term colony formation capability of the cells. Finally, Bortezomib treatment also leads to a significant reduction in the engraftment potential of human CD34+ CML cells, although in this experiment there were still detectable Bcr-Abl positive cells. However, the effect of Bortezomib is not specific for CML cells, as it induces apoptosis also in normal CD34+CD38 hematopoietic stem cells (Heaney et al., 2010).

#### **5. Conclusion**

116 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

key events, Bortezomib treatment reverses the Bcr-Abl suppression of FoxO proteins and

Although apoptosis induction is a commonplace of Bortezomib treatment, the inhibition of the cell cycle can be at the G1/S transition or at the G2/M boundary, depending on the cell models used (Albero et al., 2010; Gatto et al., 2003). The cell cycle inhibition at the G1/S transition can be explained by the accumulation of p27kip1 in Bortezomib treated cells (Fig. 2). This accumulation inhibits the kinase activity of cyclin/Cdk complexes, which results in the dephosphorylation (activation) of Rb, blocking the cell cycle at the restriction point by the sequestration and inactivation of E2F transcription factors. The dephosphorylation of Rb is followed by its caspase-dependent processing and degradation, something associated with the induction of apoptosis. As a consequence of the impact on Rb phosphorylation, Bortezomib treatment also results in the down-regulation of cyclin A expression (Albero et al., 2010). In cell lines derived from CML patients in blast crisis, Bortezomib induces the accumulation of IκBα, correlating with a decrease in the NF-κB DNA binding activity in the nucleus at the same time (Gatto et al., 2003). However, this effect was only transient and perhaps does not explain the cellular effects of Bortezomib. In contrast, in Baf/3-derived cell lines exogenously expressing Bcr-Abl, there was no effect of Bortezomib on NF-κB activity (Albero et al., 2010). However, in this same study, the non-canonical NF-κB pathway was analyzed and the results show that Bortezomib prevents the activation of NF-κB2. This protein is activated by proteolysis from a 100 kDa precursor to yield the 52 kDa active form in a proteasome dependent manner (Barre & Perkins, 2007). As NF-κB2 is not activated, the expression of two of its regulated targets, Skp2 and Myc, is also inhibited (Albero et al., 2010; Barre & Perkins, 2007; Schneider et al., 2006). This effect, in turn, would reinforce the effect of Bortezomib on the accumulation of p27kip1 (Fig. 2) leading to the dephosphorylation

of Rb and the cell cycle inhibition at the G1/S transition (Albero et al., 2010).

Fig. 2. Possible molecular mechanism leading to cell cycle arrest in G1 and apoptosis in CML

cells treated with Bortezomib, as proposed (Albero et al., 2010).

blocks the activation of NF-κB.

Imatinib is the first and so far the best example of rational design of anticancer therapeutic drugs. However, it is not able to cure and in some cases its efficacy is reduced due to Bcr-Abl dependent and independent mechanisms of resistance. In this context, the relationship between Bcr-Abl and the proteasome is worth exploiting. In fact, the proteasome inhibitor Bortezomib has shown its potential *in vitro* and in animal models. Nevertheless, proteasome inhibition is cytotoxic to all cell types. Therefore, the future of Bortezomib and other "nonspecific" inhibitors or drugs may lie in the search for synergies with other "specific" drugs aimed to the molecular events characteristic of the tumor. In Chronic Myeloid Leukemia,

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inhibiting the proteasome with Bortezomib and inhibiting the kinase activity of Bcr-Abl with Imatinib or one of the new compounds may prove the right combination to tackle the problems of Bcr-Abl mutations and the Imatinib resistant leukemia stem cell. This could be a good starting point for this kind of combined therapies in other tumors where the search for a "specific" therapy is still ongoing.

#### **6. Acknowledgement**

Financial support: IPR receives grants from the Instituto de Salud Carlos III (PI10/00109) and from the Universidad CEU Cardenal Herrera (Programa Banco Santander Copernicus and PRUCH).

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**7** 

*Australia* 

**Ser/Thr Phosphatases: The New Frontier** 

Amanda M. Smith, Kathryn G. Roberts and Nicole M. Verrills

Myeloid leukemias are characterised by mutation and altered expression of a range of tyrosine kinases. Over 90% of chronic myeloid leukemias (CML) harbour the Philadelphia chromosome, resulting in expression of the BCR/ABL fusion protein, a constitutively active tyrosine kinase that is essential for survival of the CML cells. Acute myeloid leukemia (AML) is a heterogeneous disease characterised by mutations and dysregulation of a range of tyrosine kinases including the receptors Fms-like tyrosine kinase (Flt-3), c-KIT and platelet derived growth factor receptor (PDGFR). Tyrosine kinases represent powerful therapeutic targets, as the archetypical example of imatinib has shown for CML. However, many patients develop resistance to imatinib and other second generation inhibitors. Furthermore, trials of tyrosine kinase inhibitors for AML have thus far proven disappointing. Thus novel therapeutic targets are needed in order to improve the survival of

Oncogenic tyrosine kinases induce activation of a variety of signaling pathways required for the growth and survival of leukemia cells, such as the Ras/MAPK, PI3K/Akt, and JAK/STAT pathways. In addition to protein kinases, the rate and duration of protein phosphorylation is tightly regulated by the activity of protein phosphatases, and in normal cells the reversal of protein phosphorylation by phosphatases is essential for providing the fine-tuning of signaling pathways and maintaining a balance in cellular physiology. While much of the focus for targeted therapies in leukemia therapy has concentrated on the kinases responsible for phosphorylation events, relatively little attention has been given to the role that protein phosphatases play. However, research over the past decade has now begun to highlight the importance of protein phosphatases in leukemia and their potential as targets for novel therapies. In particular, the ser/thr phosphatase PP2A has emerged as an important tumor suppressor in myeloid leukemias and strategies aimed at reactivating this complex enzyme show great promise for a new generation of leukemia therapies.

Signal transduction via a network of cellular communication pathways enables modulation of essential cellular functions such as proliferation, differentiation, survival, adhesion, motility and death. Phosphorylation is the most common mechanism for the propagation of

**1. Introduction** 

myeloid leukemia patients.

**2. Protein phosphorylation in cellular signaling** 

**for Myeloid Leukemia Therapy?** 

*School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW,* 


### **Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy?**

Amanda M. Smith, Kathryn G. Roberts and Nicole M. Verrills *School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW, Australia* 

#### **1. Introduction**

122 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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182

pp. 915-925

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essential elements of the cyclin A-CDK2 S phase kinase. Cell, Vol.82, No.6, (Sep 22),

Myeloid leukemias are characterised by mutation and altered expression of a range of tyrosine kinases. Over 90% of chronic myeloid leukemias (CML) harbour the Philadelphia chromosome, resulting in expression of the BCR/ABL fusion protein, a constitutively active tyrosine kinase that is essential for survival of the CML cells. Acute myeloid leukemia (AML) is a heterogeneous disease characterised by mutations and dysregulation of a range of tyrosine kinases including the receptors Fms-like tyrosine kinase (Flt-3), c-KIT and platelet derived growth factor receptor (PDGFR). Tyrosine kinases represent powerful therapeutic targets, as the archetypical example of imatinib has shown for CML. However, many patients develop resistance to imatinib and other second generation inhibitors. Furthermore, trials of tyrosine kinase inhibitors for AML have thus far proven disappointing. Thus novel therapeutic targets are needed in order to improve the survival of myeloid leukemia patients.

Oncogenic tyrosine kinases induce activation of a variety of signaling pathways required for the growth and survival of leukemia cells, such as the Ras/MAPK, PI3K/Akt, and JAK/STAT pathways. In addition to protein kinases, the rate and duration of protein phosphorylation is tightly regulated by the activity of protein phosphatases, and in normal cells the reversal of protein phosphorylation by phosphatases is essential for providing the fine-tuning of signaling pathways and maintaining a balance in cellular physiology. While much of the focus for targeted therapies in leukemia therapy has concentrated on the kinases responsible for phosphorylation events, relatively little attention has been given to the role that protein phosphatases play. However, research over the past decade has now begun to highlight the importance of protein phosphatases in leukemia and their potential as targets for novel therapies. In particular, the ser/thr phosphatase PP2A has emerged as an important tumor suppressor in myeloid leukemias and strategies aimed at reactivating this complex enzyme show great promise for a new generation of leukemia therapies.

#### **2. Protein phosphorylation in cellular signaling**

Signal transduction via a network of cellular communication pathways enables modulation of essential cellular functions such as proliferation, differentiation, survival, adhesion, motility and death. Phosphorylation is the most common mechanism for the propagation of

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 125

(Bruecher-Encke *et al.* 2001; Lorenz *et al.* 1996; Zhang *et al.* 2000), and these interactions exert primarily inhibitory effects on the signaling pathways. Silencing of the SHP1 gene via promotor methylation has been identified as a common event in a range of leukemias and lymphomas, and has been suggested as a potential marker for disease progression (Chim *et al.* 2004; Johan *et al.* 2005; Oka *et al.* 2002; Zhang *et al.* 2000). Other PTPs implicated as tumor suppressors in myeloid leukemias include PTEN, PTPN2, DEP-1, and DUSP-16. In contrast, SHP2 displays oncogenic properties as it promotes growth and survival pathways due to its dephosphorylating activity toward negative regulators of the Ras/Erk and PI3K/Akt signaling pathways (Neel *et al.* 2003). Somatic mutations in the SHP2 gene occur in around 30% of sporadic juvenile myelomonocytic leukemia (JMML) cases (Tartaglia *et al.* 2003), 6% of childhood acute lymphoblastic leukemia (ALL) (Tartaglia *et al.* 2004) and 5% of AML (Bentires-Alj *et al.* 2004; Tartaglia and Gelb 2005; Tartaglia *et al.* 2004; Tartaglia *et al.* 2005). Other PTPs with potential oncogenic roles in leukemia include CD45, Cdc25, and DUSP-7.

In the early 1980s Ingebritsen and Cohen utilised a number of characteristics of phosphatases to pioneer nomenclature classes for the various enzymes (Ingebritsen and Cohen 1983a). Biochemical assays, sensitivity to endogenous inhibitors and the limited knowledge of substrate specificity at the time were originally used to classify the Ser/Thr phosphatases as either type 1 (PP1) or type 2 (PP2). PP1 specifically dephosphorylates the β subunit of phosphorylase kinase and is inhibited by nanomolar concentrations of the two small heat- and acid-stable proteins, termed inhibitor-1 (I-1) (Nimmo and Cohen 1978) and inhibitor-2 (I-2) (Foulkes and Cohen 1980). PP2 phosphatases preferentially dephosphorylate the α-subunit of phosphorylase kinase and are insensitive to I-1 and I-2 (Cohen 1989; Ingebritsen and Cohen 1983a; Ingebritsen and Cohen 1983b). PP2 phosphatases could in turn be subclassified into three distinct enzymes, PP2A, PP2B, and PP2C in a number of ways, but most simply by their metal-ion requirement. PP2A does not require a metal ion, however, PP2B and PP2C are Ca2+ and Mg2+ dependent respectively (Ingebritsen and Cohen 1983a; Ingebritsen and Cohen 1983b; Moorhead *et al.* 2007). More recently the Ser/Thr phosphatases were re-defined according to their structurally distinct gene family as part of the phosphoprotein phosphatase (PPP) family comprising PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7 catalytic subunits, or protein phosphatase Mg2+- or Mn2+ dependent (PPM) family primarily composed of the PP2C catalytic subunit. The majority of Ser/Thr phosphatase activity in vivo is accounted for by the PPP family members PP1, PP2A and PP2B, together with PP2C of the PPM family (Barford *et al.* 1998). In the majority of cases they operate in hetero-oligomeric complexes interacting with an increasing diversity

The past decade has seen a surge in research into the PPP family member, PP2A, most notably due to the discoveries indicating its important role as a tumor suppressor. In particular PP2A has been implicated as a major player in myeloid leukemias and as a potential target for novel therapeutic strategies. In order to study the role of PP2A in leukemogenesis, understanding the structure and function of this complex enzyme is

of targeting and regulatory subunits (Gallego and Virshup 2005).

**6. Protein phosphatase 2A (PP2A)** 

imperative, and as such is summarised below.

**5. Ser/Thr phosphatases** 

intracellular signals. The net phosphorylation state relies on a delicate balance between protein kinases, which catalyse phosphate addition, and protein phosphatases, catalysing phosphate removal. The role of protein kinases in the cellular signaling pathways controlling biological functions has been extensively studied and protein kinases are currently the pharmaceutical industry's second largest drug target (Cohen 2002). In contrast, the role of phosphatases in disease has only recently come to the forefront of research.

Proteins are primarily phosphorylated on Serine (Ser), Threonine (Thr) and Tyrosine (Tyr) residues, each accounting for approximately 86, 12 and 2% of the human phosphoproteome respectively (Olsen *et al.* 2006). Around 2% of the human genome encodes a protein kinase gene, totalling 518 genes, of which 428 are known or predicted to phosphorylate Ser or Thr residues, and 90 encode protein tyrosine kinases (Alonso *et al.* 2004; Manning *et al.* 2002). In contrast, the human genome only encodes 147 phosphatase catalytic subunit genes. Of these, 107 encode a protein tyrosine phosphatase (PTP), a number that is comparable with the opposing tyrosine kinase genes (Alonso *et al.* 2004). Interestingly, while 98% of phosphoprotein sites are Ser and Thr residues, only a handful of the total protein phosphatases are specific for these amino acids (Moorhead *et al.* 2007). The unique way in which Ser/Thr phosphatases are regulated explains the difference in the number of Ser/Thr phosphatase catalytic subunits compared to PTPs. The evolution of PTPs has developed through the addition of discrete modular domains onto a core catalytic domain that define the function of the enzyme. In contrast, Ser/Thr phosphatases consist of a relatively simple catalytic subunit that reversibly binds to additional regulatory or interacting partner proteins which target the complex to specific subcellular locations and substrates, and ultimately control their activity.

#### **3. Classification of protein phosphatases**

Protein phosphatases can be classified into three main classes based on characteristics such as sequence, structure and phosphoamino-acid specificity. According to substrate specificity the largest phosphatase class is the ser/thr specific phosphoprotein phosphatase (PPP) family including PP1, PP2A, PP2B and PP4-PP7. The metallo-protein phosphatase dependant (PPM) family, made up mainly of PP2C, also functions against serine and threonine residues. Protein tyrosine phosphatases (PTP) form the second group and the aspartate-based or dual specificity protein phosphatases (DUSPs) the third. Genetic sequencing and analysis maintained these rules of partition, however it has recently been shown that particular Ser/Thr phosphatases can also dephosphorylate Tyr residues and various enzymes that fall into the dual specificity category based on their genetic sequence can selectively function on Ser, Thr, Tyr, phosphoinositides or RNA (Alonso *et al.* 2004; Begley and Dixon 2005).

#### **4. PTPs in myeloid leukemias**

The role of PTPs in cancer has been recently reviewed (Jiang and Zhang 2008; Julien *et al.* 2011; Lopez-Ruiz *et al.* 2011; Ruela-de-Sousa *et al.* 2011) and will not be discussed in detail here. However it should be noted that PTPs can act as either tumor suppressors or oncogenes in both solid tumors and leukaemais. For example, SHP1, a non-receptor PTP, displays tumor suppressive properties. SHP1 associates with a number of signaling molecules including CD5, the IL-3 receptor, CD22, the B-cell receptor, c-KIT and BCR/ABL (Bruecher-Encke *et al.* 2001; Lorenz *et al.* 1996; Zhang *et al.* 2000), and these interactions exert primarily inhibitory effects on the signaling pathways. Silencing of the SHP1 gene via promotor methylation has been identified as a common event in a range of leukemias and lymphomas, and has been suggested as a potential marker for disease progression (Chim *et al.* 2004; Johan *et al.* 2005; Oka *et al.* 2002; Zhang *et al.* 2000). Other PTPs implicated as tumor suppressors in myeloid leukemias include PTEN, PTPN2, DEP-1, and DUSP-16. In contrast, SHP2 displays oncogenic properties as it promotes growth and survival pathways due to its dephosphorylating activity toward negative regulators of the Ras/Erk and PI3K/Akt signaling pathways (Neel *et al.* 2003). Somatic mutations in the SHP2 gene occur in around 30% of sporadic juvenile myelomonocytic leukemia (JMML) cases (Tartaglia *et al.* 2003), 6% of childhood acute lymphoblastic leukemia (ALL) (Tartaglia *et al.* 2004) and 5% of AML (Bentires-Alj *et al.* 2004; Tartaglia and Gelb 2005; Tartaglia *et al.* 2004; Tartaglia *et al.* 2005). Other PTPs with potential oncogenic roles in leukemia include CD45, Cdc25, and DUSP-7.

#### **5. Ser/Thr phosphatases**

124 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

intracellular signals. The net phosphorylation state relies on a delicate balance between protein kinases, which catalyse phosphate addition, and protein phosphatases, catalysing phosphate removal. The role of protein kinases in the cellular signaling pathways controlling biological functions has been extensively studied and protein kinases are currently the pharmaceutical industry's second largest drug target (Cohen 2002). In contrast, the role of phosphatases in disease has only recently come to the forefront of research. Proteins are primarily phosphorylated on Serine (Ser), Threonine (Thr) and Tyrosine (Tyr) residues, each accounting for approximately 86, 12 and 2% of the human phosphoproteome respectively (Olsen *et al.* 2006). Around 2% of the human genome encodes a protein kinase gene, totalling 518 genes, of which 428 are known or predicted to phosphorylate Ser or Thr residues, and 90 encode protein tyrosine kinases (Alonso *et al.* 2004; Manning *et al.* 2002). In contrast, the human genome only encodes 147 phosphatase catalytic subunit genes. Of these, 107 encode a protein tyrosine phosphatase (PTP), a number that is comparable with the opposing tyrosine kinase genes (Alonso *et al.* 2004). Interestingly, while 98% of phosphoprotein sites are Ser and Thr residues, only a handful of the total protein phosphatases are specific for these amino acids (Moorhead *et al.* 2007). The unique way in which Ser/Thr phosphatases are regulated explains the difference in the number of Ser/Thr phosphatase catalytic subunits compared to PTPs. The evolution of PTPs has developed through the addition of discrete modular domains onto a core catalytic domain that define the function of the enzyme. In contrast, Ser/Thr phosphatases consist of a relatively simple catalytic subunit that reversibly binds to additional regulatory or interacting partner proteins which target the complex to specific subcellular locations and substrates, and

Protein phosphatases can be classified into three main classes based on characteristics such as sequence, structure and phosphoamino-acid specificity. According to substrate specificity the largest phosphatase class is the ser/thr specific phosphoprotein phosphatase (PPP) family including PP1, PP2A, PP2B and PP4-PP7. The metallo-protein phosphatase dependant (PPM) family, made up mainly of PP2C, also functions against serine and threonine residues. Protein tyrosine phosphatases (PTP) form the second group and the aspartate-based or dual specificity protein phosphatases (DUSPs) the third. Genetic sequencing and analysis maintained these rules of partition, however it has recently been shown that particular Ser/Thr phosphatases can also dephosphorylate Tyr residues and various enzymes that fall into the dual specificity category based on their genetic sequence can selectively function on Ser, Thr, Tyr, phosphoinositides or RNA (Alonso *et al.* 2004;

The role of PTPs in cancer has been recently reviewed (Jiang and Zhang 2008; Julien *et al.* 2011; Lopez-Ruiz *et al.* 2011; Ruela-de-Sousa *et al.* 2011) and will not be discussed in detail here. However it should be noted that PTPs can act as either tumor suppressors or oncogenes in both solid tumors and leukaemais. For example, SHP1, a non-receptor PTP, displays tumor suppressive properties. SHP1 associates with a number of signaling molecules including CD5, the IL-3 receptor, CD22, the B-cell receptor, c-KIT and BCR/ABL

ultimately control their activity.

Begley and Dixon 2005).

**4. PTPs in myeloid leukemias** 

**3. Classification of protein phosphatases** 

In the early 1980s Ingebritsen and Cohen utilised a number of characteristics of phosphatases to pioneer nomenclature classes for the various enzymes (Ingebritsen and Cohen 1983a). Biochemical assays, sensitivity to endogenous inhibitors and the limited knowledge of substrate specificity at the time were originally used to classify the Ser/Thr phosphatases as either type 1 (PP1) or type 2 (PP2). PP1 specifically dephosphorylates the β subunit of phosphorylase kinase and is inhibited by nanomolar concentrations of the two small heat- and acid-stable proteins, termed inhibitor-1 (I-1) (Nimmo and Cohen 1978) and inhibitor-2 (I-2) (Foulkes and Cohen 1980). PP2 phosphatases preferentially dephosphorylate the α-subunit of phosphorylase kinase and are insensitive to I-1 and I-2 (Cohen 1989; Ingebritsen and Cohen 1983a; Ingebritsen and Cohen 1983b). PP2 phosphatases could in turn be subclassified into three distinct enzymes, PP2A, PP2B, and PP2C in a number of ways, but most simply by their metal-ion requirement. PP2A does not require a metal ion, however, PP2B and PP2C are Ca2+ and Mg2+ dependent respectively (Ingebritsen and Cohen 1983a; Ingebritsen and Cohen 1983b; Moorhead *et al.* 2007). More recently the Ser/Thr phosphatases were re-defined according to their structurally distinct gene family as part of the phosphoprotein phosphatase (PPP) family comprising PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7 catalytic subunits, or protein phosphatase Mg2+- or Mn2+ dependent (PPM) family primarily composed of the PP2C catalytic subunit. The majority of Ser/Thr phosphatase activity in vivo is accounted for by the PPP family members PP1, PP2A and PP2B, together with PP2C of the PPM family (Barford *et al.* 1998). In the majority of cases they operate in hetero-oligomeric complexes interacting with an increasing diversity of targeting and regulatory subunits (Gallego and Virshup 2005).

#### **6. Protein phosphatase 2A (PP2A)**

The past decade has seen a surge in research into the PPP family member, PP2A, most notably due to the discoveries indicating its important role as a tumor suppressor. In particular PP2A has been implicated as a major player in myeloid leukemias and as a potential target for novel therapeutic strategies. In order to study the role of PP2A in leukemogenesis, understanding the structure and function of this complex enzyme is imperative, and as such is summarised below.

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 127

absence of pMEK (Fig. 3A) (Letourneux *et al.* 2006). More recent evidence indicates that PP2A activates Ras-dependent MAPK signaling at the level of Raf-1 and its scaffolding protein Kinase Suppressor of Ras-1 (KSR1) (Abraham *et al.* 2000; Jaumot and Hancock 2001; Kubicek *et al.* 2002; Ory *et al.* 2003). Dephosphorylation of KSR1 (Ser329) and Raf-1 (Ser259) by B55α-containing PP2A complexes induces membrane translocation and increases the kinase activity of both proteins in several mammalian cell models, including NIH3T3, COS and HEK293 cells (Fig. 3B) (Abraham *et al.* 2000; Adams *et al.* 2005; Dougherty *et al.* 2005; Ory *et al.* 2003). Taken together, these studies provide biochemical mechanisms for how PP2A functions as a negative and positive regulator of MAPK signaling, depending on the

specific regulatory subunit and substrate involved.

interaction and induces transcriptional activation.

**6.2.2 PP2A and PI3K/Akt signaling** 

**6.2.3 PP2A and Wnt/β-catenin signaling** 

Fig. 2. Schematic overview of MAPK signaling regulation by PP2A

**A)** PP2A holoenzymes containing B56 family members negatively regulate MAPK signaling by inactivating MEK and ERK. **B)** In the inactive state, Raf and KSR1 are phosphorylated on Ser259 and Ser329, respectively, and are located within the cytoplasm. Upon stimulation, B55-containing PP2A complexes dephosphorylate these residues, which results in protein

The normal function of Akt is tightly modulated by phosphorylation events on Thr308 or Ser473 (Sarbassov *et al.* 2005; Vanhaesebroeck and Alessi 2000), and PP2A is the major phosphatase targeting these residues *in vitro* (Borgatti *et al.* 2003; Ivaska *et al.* 2002; Resjo *et al.* 2002). Over-expression of B55α-containing PP2A holoenzymes in the pro-lymphoid FL5.12 cell line substantially dephosphorylates Akt at Thr308 and results in subsequent growth suppression (Kuo *et al.* 2008). Conversely, Aα downregulation impairs Akt phosphorylation in neuronal cells, implicating PP2A as a positive regulator of the PI3K/Akt survival signaling cascade (Strack *et al.* 2004). An intriguing study by Andrabi *et al*. , demonstrated that Akt can act as a pro- or anti-apoptotic protein depending on environmental stimuli, and this is governed by PP2A (Andrabi *et al.* 2007). The specific PP2A regulatory subunits controlling this apparent switch remain undefined; however, this study reinforces the importance of

The function of PP2A in Wnt/β-catenin signaling is similar to its role in the MAPK pathway, with individual PP2A subunits exerting either positive or negative effects (Fig. 3). B56

activation and translocation to the plasma membrane. This facilitates the Ras/Raf

understanding PP2A regulated cell signaling in a context dependent manner.

#### **6.1 Structure and regulation of PP2A**

PP2A is not a single phosphatase, but essentially encompasses a group of oligomeric enzymes consisting of a well conserved catalytic and structural subunit, together with the addition of one of a variety of regulatory subunits (Fig. 1). It makes up 1% of total cellular proteins and along with PP1, accounts for over 90% of serine/threonine phosphatase activity in the cell (Eichhorn *et al.* 2009). The PP2A core enzyme consists of a structural subunit (PP2A-A/PR65) and a catalytic subunit (PP2Ac). In mammals, two distinct genes (α and β) encode closely related versions of both the A (Hemmings *et al.* 1990) and C subunits (Arino *et al.* 1988). A third regulatory subunit (PP2A B) binds to the AC heterodimer, and determines both the substrate specificity and cellular localisation of PP2A holoenzyme complexes. Three B subunit families have been identified to date: B/B55/PR55 (Mayer *et al.* 1991; Strack *et al.* 1999; Zolnierowicz *et al.* 1994), B'/B56/PR61 (McCright *et al.* 1996; McCright and Virshup 1995), B"/PR72/130/PR70/48 (Hendrix *et al.* 1993; Stevens *et al.* 2003; Yan *et al.* 2000). Regulation of PP2A activity is accomplished primarily by members of its regulatory subunits. An additional level of regulation is introduced by post-translational modification of the catalytic subunit, which can undergo methylation and phosphorylation (Janssens *et al.* 2008), as well as the interaction with a vast array of other cellular and viral proteins such as SET, CIP2A and the SV40 small T antigen (Janssens and Goris 2001; Sablina and Hahn 2008). The use of specific PP2A inhibitors, PP2A activators and molecular genetics tools both *in vitro* and *in vivo* have exposed a role for PP2A in cell morphology, cell cycle regulation, development and apoptosis.

Fig. 1. Schematic of PP2A holoenzymes

#### **6.2 PP2A in cellular signaling**

PP2A has been implicated in a wide range of cellular signaling pathways, many of which are involved in cellular proliferation, apoptosis and differentiation, and as such are important in tumorigenesis. Since PP2A is one of the most abundant cellular phosphatases, it is not surprising that PP2A can exert opposing roles on similar pathways by acting at different levels. Recent work indicates that this substrate specificity is mediated by distinct PP2A complexes.

#### **6.2.1 PP2A and MAPK signaling**

A major function of PP2A is regulation of the MAPK signaling pathway. Interestingly, PP2A can exert both inhibitory and activating effects in a context-dependent manner (Fig. 2). PP2A acts negatively via the dephosphorylation of MEK (Heriche *et al.* 1997; Sontag *et al.* 1993) and ERK both *in vitro* and in mammalian cells (Alessi *et al.* 1995; Wang *et al.* 2003; Zhou *et al.* 2002). Specific knockdown of B56β and B56γ, but not B55 family subunits in NIH3T3 mouse fibroblasts increases basal ERK activation and prolongs ERK signal during stimulation in the

PP2A is not a single phosphatase, but essentially encompasses a group of oligomeric enzymes consisting of a well conserved catalytic and structural subunit, together with the addition of one of a variety of regulatory subunits (Fig. 1). It makes up 1% of total cellular proteins and along with PP1, accounts for over 90% of serine/threonine phosphatase activity in the cell (Eichhorn *et al.* 2009). The PP2A core enzyme consists of a structural subunit (PP2A-A/PR65) and a catalytic subunit (PP2Ac). In mammals, two distinct genes (α and β) encode closely related versions of both the A (Hemmings *et al.* 1990) and C subunits (Arino *et al.* 1988). A third regulatory subunit (PP2A B) binds to the AC heterodimer, and determines both the substrate specificity and cellular localisation of PP2A holoenzyme complexes. Three B subunit families have been identified to date: B/B55/PR55 (Mayer *et al.* 1991; Strack *et al.* 1999; Zolnierowicz *et al.* 1994), B'/B56/PR61 (McCright *et al.* 1996; McCright and Virshup 1995), B"/PR72/130/PR70/48 (Hendrix *et al.* 1993; Stevens *et al.* 2003; Yan *et al.* 2000). Regulation of PP2A activity is accomplished primarily by members of its regulatory subunits. An additional level of regulation is introduced by post-translational modification of the catalytic subunit, which can undergo methylation and phosphorylation (Janssens *et al.* 2008), as well as the interaction with a vast array of other cellular and viral proteins such as SET, CIP2A and the SV40 small T antigen (Janssens and Goris 2001; Sablina and Hahn 2008). The use of specific PP2A inhibitors, PP2A activators and molecular genetics tools both *in vitro* and *in vivo* have exposed a role for PP2A in cell morphology, cell cycle

PP2A has been implicated in a wide range of cellular signaling pathways, many of which are involved in cellular proliferation, apoptosis and differentiation, and as such are important in tumorigenesis. Since PP2A is one of the most abundant cellular phosphatases, it is not surprising that PP2A can exert opposing roles on similar pathways by acting at different levels. Recent work indicates that this substrate specificity is mediated by distinct

A major function of PP2A is regulation of the MAPK signaling pathway. Interestingly, PP2A can exert both inhibitory and activating effects in a context-dependent manner (Fig. 2). PP2A acts negatively via the dephosphorylation of MEK (Heriche *et al.* 1997; Sontag *et al.* 1993) and ERK both *in vitro* and in mammalian cells (Alessi *et al.* 1995; Wang *et al.* 2003; Zhou *et al.* 2002). Specific knockdown of B56β and B56γ, but not B55 family subunits in NIH3T3 mouse fibroblasts increases basal ERK activation and prolongs ERK signal during stimulation in the

**6.1 Structure and regulation of PP2A** 

regulation, development and apoptosis.

Fig. 1. Schematic of PP2A holoenzymes

**6.2 PP2A in cellular signaling** 

**6.2.1 PP2A and MAPK signaling** 

PP2A complexes.

absence of pMEK (Fig. 3A) (Letourneux *et al.* 2006). More recent evidence indicates that PP2A activates Ras-dependent MAPK signaling at the level of Raf-1 and its scaffolding protein Kinase Suppressor of Ras-1 (KSR1) (Abraham *et al.* 2000; Jaumot and Hancock 2001; Kubicek *et al.* 2002; Ory *et al.* 2003). Dephosphorylation of KSR1 (Ser329) and Raf-1 (Ser259) by B55α-containing PP2A complexes induces membrane translocation and increases the kinase activity of both proteins in several mammalian cell models, including NIH3T3, COS and HEK293 cells (Fig. 3B) (Abraham *et al.* 2000; Adams *et al.* 2005; Dougherty *et al.* 2005; Ory *et al.* 2003). Taken together, these studies provide biochemical mechanisms for how PP2A functions as a negative and positive regulator of MAPK signaling, depending on the specific regulatory subunit and substrate involved.

Fig. 2. Schematic overview of MAPK signaling regulation by PP2A

**A)** PP2A holoenzymes containing B56 family members negatively regulate MAPK signaling by inactivating MEK and ERK. **B)** In the inactive state, Raf and KSR1 are phosphorylated on Ser259 and Ser329, respectively, and are located within the cytoplasm. Upon stimulation, B55-containing PP2A complexes dephosphorylate these residues, which results in protein activation and translocation to the plasma membrane. This facilitates the Ras/Raf interaction and induces transcriptional activation.

#### **6.2.2 PP2A and PI3K/Akt signaling**

The normal function of Akt is tightly modulated by phosphorylation events on Thr308 or Ser473 (Sarbassov *et al.* 2005; Vanhaesebroeck and Alessi 2000), and PP2A is the major phosphatase targeting these residues *in vitro* (Borgatti *et al.* 2003; Ivaska *et al.* 2002; Resjo *et al.* 2002). Over-expression of B55α-containing PP2A holoenzymes in the pro-lymphoid FL5.12 cell line substantially dephosphorylates Akt at Thr308 and results in subsequent growth suppression (Kuo *et al.* 2008). Conversely, Aα downregulation impairs Akt phosphorylation in neuronal cells, implicating PP2A as a positive regulator of the PI3K/Akt survival signaling cascade (Strack *et al.* 2004). An intriguing study by Andrabi *et al*. , demonstrated that Akt can act as a pro- or anti-apoptotic protein depending on environmental stimuli, and this is governed by PP2A (Andrabi *et al.* 2007). The specific PP2A regulatory subunits controlling this apparent switch remain undefined; however, this study reinforces the importance of understanding PP2A regulated cell signaling in a context dependent manner.

#### **6.2.3 PP2A and Wnt/β-catenin signaling**

The function of PP2A in Wnt/β-catenin signaling is similar to its role in the MAPK pathway, with individual PP2A subunits exerting either positive or negative effects (Fig. 3). B56

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 129

Sears 2006). In addition, a novel PP2A-interacting protein, designated cancerous inhibitor of PP2A (CIP2A), was found to selectively target the catalytic activity of PP2A-B56α associated with c-Myc, and protect c-Myc from Ser62 dephosphorylation. Accordingly, depletion of CIP2A results in significantly increased PP2A activity measured from c-Myc

Collectively, these results illustrate the dynamic interaction of the PP2A holoenzyme with signaling cascades involved in fundamental cellular processes such as proliferation, survival and development. The fact that PP2A is involved in both the negative and positive regulation of these pathways highlights the exquisite nature of PP2A modulation and underscores the importance of investigating specific complexes when determining PP2A function. Identification of critical subunits that are aberrantly regulated during transformation may ultimately lead to the development of novel treatments for cancer

The fundamental evidence that implicated PP2A as a tumor suppressor was the discovery that okadaic acid, a tumor promoter (Fujiki and Suganuma 2009; Suganuma *et al.* 1988), potently inhibits the phosphatase activity of PP2A (Bialojan and Takai 1988; Haystead *et al.* 1989). In addition, the oncogenic polyomavirus middle and small tumor (ST) antigens, along with the simian virus 40 (SV40) ST antigen, transforms mammalian cells by inhibiting PP2A (Pallas *et al.* 1990). Transformation of the normal human fibroblast kidney epithelial cell line, HEK293, required several key genetic elements; human telomerase catalytic subunit, an oncogenic allele of H-Ras, and the SV40 large T (LT) and ST antigens (HEK-TER) (Hahn *et al.* 1999). Whilst expression of LT enables the cells to bypass senescence, complete tumor formation requires the addition of ST and thus inhibition of PP2A (Yu *et al.* 2001). Accordingly, ST mutants lacking the PP2A binding domain fail to induce tumorigenic transformation of HEK-TER cells (Hahn *et al.* 2002). Structural insights reveal that ST interacts with the PP2A-A subunit which overlaps B56 subunit binding site and results in its displacement from the core enzyme. Functionally, expression of ST activates Akt signaling in human cells *in vitro* (Rodriguez-Viciana *et al.* 2006; Yuan *et al.* 2002; Zhao *et al.* 2003). Taken together, these observations indicate that complete transformation of human cells

A somewhat confusing aspect of PP2A function in cancer arises because PP2A plays important roles in promoting cell cycle progression and cell survival (Li *et al.* 2002; Lin *et al.* 1998; Mayer-Jaekel *et al.* 1993), which are functions usually associated with tumor initiation and progression rather than suppression. Therefore, cellular transformation will most likely occur through the disruption of PP2A holoenzymes that normally exert negative regulation

Whilst some contradiction exists as to the importance of PP2A scaffolding subunits in cancer development, several mutations have been identified in spontaneously arising human cancers. Notably, somatic alterations of the gene encoding Aβ (PPP2R1B) have been detected in up to 8-15% of colon, 15% of lung and 13% of breast cancers (Calin *et al.* 2000; Takagi *et al.* 2000; Tamaki *et al.* 2004; Wang *et al.* 1998). Mutations of the more abundant Aα subunit have been observed, albeit at a lower frequency (Calin *et al.* 2000) and cancerassociated A subunit mutants exhibit differential defects in binding to the B and C subunits, which correlates with impaired PP2A activity (Chen *et al.* 2005; Ruediger *et al.* 2001a;

immunoprecipitates and correlates with c-Myc destabilisation (Junttila *et al.* 2007).

patients.

**6.3 PP2A as a tumor suppressor** 

requires the perturbation of PP2A, for example by ST.

on oncogenic pathways.

family members associate with APC (Seeling *et al.* 1999; Yamamoto *et al.* 2001) and axin (Hsu *et al.* 1999; Li *et al.* 2001) to impair Wnt signaling; a function that is critical for normal dorsal/ventral axis formation in Xenopus development (Li *et al.* 2001). Further studies demonstrate that association of the PP2A PR72 subunit with Naked cuticle is critical for the inhibitory function of this protein on the Wnt pathway (Fig. 3A) (Creyghton *et al.* 2005).

PP2A is also an important positive regulator of Wnt signaling (Bajpai *et al.* 2004; Gotz *et al.* 2000; Ratcliffe *et al.* 2000; Willert *et al.* 1999). Loss of function analysis suggests that B56ε is required for Wnt-mediated development in Xenopus embryogenesis (Yang *et al.* 2003). Purified B55α-containing PP2A holoenzymes directly dephosphorylate β-catenin *in vitro*. Accordingly, specific knockdown of B55α in SW480 colon cancer cells significantly elevates β-catenin phosphorylation, which induces protein degradation and inhibits the Wnt pathway (Zhang *et al.* 2009). Surprisingly, PR130 opposes the action of PR72 and modulates Wnt signal transduction by restricting the ability of Naked to function as a Wnt inhibitor (Fig. 4B) (Creyghton *et al.* 2006). These studies illustrate an excellent example whereby specific PP2A regulatory subunits determine holoenzyme function and provide the fine control on important cellular processes.

#### **6.2.4 PP2A and p53 regulation**

The tumor suppressor, p53, plays a critical role in mediating cellular responses to various types of stress, such as DNA damage, by inducing growth arrest or programmed cell death. The stability and activity of p53 is regulated by phosphorylation which, under normal cellular growth conditions, targets the protein for proteasome-mediated degradation (Vogelstein *et al.* 2000). PP2A B56γ holoenzymes dephosphorylate p53 on Ser37 (Dohoney *et al.* 2004) and Thr55 (Li *et al.* 2007) following γ radiation; an event which stabilises p53 in response to DNA damage and contributes to apoptosis in mammalian cells. Furthermore, ataxia-telangiectasia mutated (ATM) directly phosphorylates and specifically regulates B56γ and B56δ. Phosphorylation of B56γ3 at Ser510 after DNA damage increases B56γ-PP2A complexes, and directs PP2A phosphatase activity toward p53, activating its tumorsuppressive functions (Shouse *et al.* 2010). Conversely, RNAi knockdown of B56γ reduces p53 stability and inhibits cell death (Li *et al.* 2007). B56ε also regulates the p53-dependent apoptotic pathway by controlling the stability of p53 protein (Jin *et al.* 2010).

Other findings implicate an important role for B56α-containing PP2A complexes in p53 degradation. One target of p53, cyclin G, recruits B56α into a quaternary complex with the E3 ubiquitin ligase, mouse double minute 2 (Mdm2) (Okamoto *et al.* 1996; Okamoto *et al.* 2002). The subsequent dephosphorylation and activation of Mdm2 leads to ubiquitination and degradation of p53 (Haupt *et al.* 1997), thus allowing the cell to proliferate. In this context, PP2A serves as a negative regulator.

#### **6.2.5 PP2A and c-Myc regulation**

PP2A also plays a prominent role in controlling the accumulation of the proto-oncoprotein, c-Myc (Yeh *et al.* 2004). Aberrant regulation of c-Myc has been linked to transformation in up to 70% of human tumors; therefore tight control of this protein is crucial for maintaining cellular homeostasis (Nesbit *et al.* 1999). c-Myc stability is regulated, in part, through phosphorylation at two residues, Ser62 and Thr58 (Sears *et al.* 2000). Whilst ERK-mediated phosphorylation at Ser62 stabilises c-Myc, specific B56-containing PP2A complexes reverse these effects, leading to destabilisation and ubiquitin-mediated degradation (Arnold and Sears 2006). In addition, a novel PP2A-interacting protein, designated cancerous inhibitor of PP2A (CIP2A), was found to selectively target the catalytic activity of PP2A-B56α associated with c-Myc, and protect c-Myc from Ser62 dephosphorylation. Accordingly, depletion of CIP2A results in significantly increased PP2A activity measured from c-Myc immunoprecipitates and correlates with c-Myc destabilisation (Junttila *et al.* 2007).

Collectively, these results illustrate the dynamic interaction of the PP2A holoenzyme with signaling cascades involved in fundamental cellular processes such as proliferation, survival and development. The fact that PP2A is involved in both the negative and positive regulation of these pathways highlights the exquisite nature of PP2A modulation and underscores the importance of investigating specific complexes when determining PP2A function. Identification of critical subunits that are aberrantly regulated during transformation may ultimately lead to the development of novel treatments for cancer patients.

#### **6.3 PP2A as a tumor suppressor**

128 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

family members associate with APC (Seeling *et al.* 1999; Yamamoto *et al.* 2001) and axin (Hsu *et al.* 1999; Li *et al.* 2001) to impair Wnt signaling; a function that is critical for normal dorsal/ventral axis formation in Xenopus development (Li *et al.* 2001). Further studies demonstrate that association of the PP2A PR72 subunit with Naked cuticle is critical for the inhibitory function of this protein on the Wnt pathway (Fig. 3A) (Creyghton *et al.* 2005). PP2A is also an important positive regulator of Wnt signaling (Bajpai *et al.* 2004; Gotz *et al.* 2000; Ratcliffe *et al.* 2000; Willert *et al.* 1999). Loss of function analysis suggests that B56ε is required for Wnt-mediated development in Xenopus embryogenesis (Yang *et al.* 2003). Purified B55α-containing PP2A holoenzymes directly dephosphorylate β-catenin *in vitro*. Accordingly, specific knockdown of B55α in SW480 colon cancer cells significantly elevates β-catenin phosphorylation, which induces protein degradation and inhibits the Wnt pathway (Zhang *et al.* 2009). Surprisingly, PR130 opposes the action of PR72 and modulates Wnt signal transduction by restricting the ability of Naked to function as a Wnt inhibitor (Fig. 4B) (Creyghton *et al.* 2006). These studies illustrate an excellent example whereby specific PP2A regulatory subunits determine holoenzyme function and provide the fine

The tumor suppressor, p53, plays a critical role in mediating cellular responses to various types of stress, such as DNA damage, by inducing growth arrest or programmed cell death. The stability and activity of p53 is regulated by phosphorylation which, under normal cellular growth conditions, targets the protein for proteasome-mediated degradation (Vogelstein *et al.* 2000). PP2A B56γ holoenzymes dephosphorylate p53 on Ser37 (Dohoney *et al.* 2004) and Thr55 (Li *et al.* 2007) following γ radiation; an event which stabilises p53 in response to DNA damage and contributes to apoptosis in mammalian cells. Furthermore, ataxia-telangiectasia mutated (ATM) directly phosphorylates and specifically regulates B56γ and B56δ. Phosphorylation of B56γ3 at Ser510 after DNA damage increases B56γ-PP2A complexes, and directs PP2A phosphatase activity toward p53, activating its tumorsuppressive functions (Shouse *et al.* 2010). Conversely, RNAi knockdown of B56γ reduces p53 stability and inhibits cell death (Li *et al.* 2007). B56ε also regulates the p53-dependent

Other findings implicate an important role for B56α-containing PP2A complexes in p53 degradation. One target of p53, cyclin G, recruits B56α into a quaternary complex with the E3 ubiquitin ligase, mouse double minute 2 (Mdm2) (Okamoto *et al.* 1996; Okamoto *et al.* 2002). The subsequent dephosphorylation and activation of Mdm2 leads to ubiquitination and degradation of p53 (Haupt *et al.* 1997), thus allowing the cell to proliferate. In this

PP2A also plays a prominent role in controlling the accumulation of the proto-oncoprotein, c-Myc (Yeh *et al.* 2004). Aberrant regulation of c-Myc has been linked to transformation in up to 70% of human tumors; therefore tight control of this protein is crucial for maintaining cellular homeostasis (Nesbit *et al.* 1999). c-Myc stability is regulated, in part, through phosphorylation at two residues, Ser62 and Thr58 (Sears *et al.* 2000). Whilst ERK-mediated phosphorylation at Ser62 stabilises c-Myc, specific B56-containing PP2A complexes reverse these effects, leading to destabilisation and ubiquitin-mediated degradation (Arnold and

apoptotic pathway by controlling the stability of p53 protein (Jin *et al.* 2010).

control on important cellular processes.

context, PP2A serves as a negative regulator.

**6.2.5 PP2A and c-Myc regulation** 

**6.2.4 PP2A and p53 regulation** 

The fundamental evidence that implicated PP2A as a tumor suppressor was the discovery that okadaic acid, a tumor promoter (Fujiki and Suganuma 2009; Suganuma *et al.* 1988), potently inhibits the phosphatase activity of PP2A (Bialojan and Takai 1988; Haystead *et al.* 1989). In addition, the oncogenic polyomavirus middle and small tumor (ST) antigens, along with the simian virus 40 (SV40) ST antigen, transforms mammalian cells by inhibiting PP2A (Pallas *et al.* 1990). Transformation of the normal human fibroblast kidney epithelial cell line, HEK293, required several key genetic elements; human telomerase catalytic subunit, an oncogenic allele of H-Ras, and the SV40 large T (LT) and ST antigens (HEK-TER) (Hahn *et al.* 1999). Whilst expression of LT enables the cells to bypass senescence, complete tumor formation requires the addition of ST and thus inhibition of PP2A (Yu *et al.* 2001). Accordingly, ST mutants lacking the PP2A binding domain fail to induce tumorigenic transformation of HEK-TER cells (Hahn *et al.* 2002). Structural insights reveal that ST interacts with the PP2A-A subunit which overlaps B56 subunit binding site and results in its displacement from the core enzyme. Functionally, expression of ST activates Akt signaling in human cells *in vitro* (Rodriguez-Viciana *et al.* 2006; Yuan *et al.* 2002; Zhao *et al.* 2003). Taken together, these observations indicate that complete transformation of human cells requires the perturbation of PP2A, for example by ST.

A somewhat confusing aspect of PP2A function in cancer arises because PP2A plays important roles in promoting cell cycle progression and cell survival (Li *et al.* 2002; Lin *et al.* 1998; Mayer-Jaekel *et al.* 1993), which are functions usually associated with tumor initiation and progression rather than suppression. Therefore, cellular transformation will most likely occur through the disruption of PP2A holoenzymes that normally exert negative regulation on oncogenic pathways.

Whilst some contradiction exists as to the importance of PP2A scaffolding subunits in cancer development, several mutations have been identified in spontaneously arising human cancers. Notably, somatic alterations of the gene encoding Aβ (PPP2R1B) have been detected in up to 8-15% of colon, 15% of lung and 13% of breast cancers (Calin *et al.* 2000; Takagi *et al.* 2000; Tamaki *et al.* 2004; Wang *et al.* 1998). Mutations of the more abundant Aα subunit have been observed, albeit at a lower frequency (Calin *et al.* 2000) and cancerassociated A subunit mutants exhibit differential defects in binding to the B and C subunits, which correlates with impaired PP2A activity (Chen *et al.* 2005; Ruediger *et al.* 2001a;

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 131

Fig. 3. Model of PP2A inhibition in myeloid leukemias. (A) PP2A activity is inhibited by BCR/ABL and oncogenic mutant c-KIT, enabling the activation of signaling pathways leading to leukemogenesis. (B) Pharmacological activation of PP2A induces de-activation (dephosphorylation) of BCR/ABL and c-KIT, and inhibition of downstream signaling

Functional inactivation of PP2A by BCR/ABL is essential for the development of CML-BC and Ph+ ALL, and is thought to result from upregulation of the endogenous PP2A inhibitor SET (I2PP2A or TAF-1β) protein (Neviani *et al.* 2007; Neviani *et al.* 2005). Although its physiological function remains incompletely understood, abnormal expression of SET mRNA has been documented in solid tumors and haematological disorders (Carlson *et al.* 1998; Fornerod *et al.* 1995; Li *et al.* 1996). Expression of BCR/ABL in mouse myeloid precursor cells stimulates SET expression and correlates with a loss of PP2A activity (Neviani *et al.* 2005). Conversely, inhibition of BCR/ABL with imatinib treatment dramatically reduces SET expression, which results in the restoration of PP2A activity back to untransfected levels and results in the dephosphorylation of several substrates which are shared by BCR/ABL and PP2A (Neviani *et al.* 2005). These include STAT5, ERK1/2, Akt, BAD and JAK2 (Calabretta and Perrotti 2004; Janssens and Goris 2001). Importantly, molecular or pharmacological reactivation of PP2A resulted in growth suppression, enhanced apoptosis, restored differentiation, and decreased *in vivo* leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cell lines and primary CML-BC cells. Furthermore, reactivation of PP2A, by overexpression of PP2AC or 1,9-dideoxyforskolin treatment (a cAMP-independent PP2A activator) (see section 6.5.1), reduced the activity and expression of BCR/ABL in both imatinib-sensitive and -resistant BCR/ABL cells, suggesting that BCR/ABL itself is a target for PP2A activity. Indeed, reactivation of PP2A results in PP2AC association with BCR/ABL via the SHP1 tyrosine phosphatase, BCR/ABL dephosphorylation, and proteasomal degradation (Neviani *et al.* 2005). These findings establish an important link between an oncogenic kinase and a tumor suppressing phosphatase, and indicate that pharmacologic enhancement of PP2A is a powerful therapeutic strategy for imatinib-resistant CML. Taken together, these results suggest that SET-dependent inhibition of PP2A is required for the transduction of aberrant mitogenic, survival and anti-differentiation signals that contribute to the development of CML from the

pathways, leading to inhibition of leukemogenesis.

chronic phase into blast crisis (Neviani *et al.* 2005).

**6.4.1 PP2A inhibition in CML** 

Ruediger *et al.* 2001b; Sablina *et al.* 2007). Even in the absence of mutations, reduced protein expression of Aα has been found in 25 out of 58 brain tumors (Colella *et al.* 2001). Decreased levels of Aβ have also been observed in 16 of 32 cancer cell lines derived from human lung, colon and breast cancer, as well as primary glioblastoma and B-CLL patient samples compared to normal tissue (Kalla *et al.* 2007; Suzuki and Takahashi 2003; Takagi *et al.* 2000; Zhou *et al.* 2003). Importantly, Ruediger *et al*., recently generated knock-in mice with cancer associated A mutations, and A knockouts, both of which exhibited increased incidence of lung cancer when treated with benzopyrene, thus supporting the role of PP2A as a tumor suppressor (Ruediger *et al.* 2011).

Pivotal studies using the HEK-TER transformation model demonstrated that suppression of B56γ, but not B55α, functionally mimicked the introduction of ST and resulted in partial tumorigenic transformation (Chen *et al.* 2004; Moreno *et al.* 2004). Moreover, depletion of B56γ containing complexes leads to activation of the anti–apoptotic Akt pathway (Chen *et al.* 2005). These observations were the first to demonstrate that PP2A complexes containing B56γ modulate the phosphorylation of substrates associated with transformation. Loss of B56γ has been demonstrated in some human cancers. Decreased expression was identified in primary human melanoma samples compared to melanocytic nevi (Deichmann *et al.* 2001), and in human lung cancer cell lines where subsequent overexpression of B56γ reversed the tumorigenic phenotype (Chen *et al.* 2004). Reduced transcript levels of B56γ have also been documented in patients with aggressive B-CLL compared to those with stable disease (Falt *et al.* 2005). A mutation in B56γ has also been observed in lung cancer, and this was shown to disrupt the interaction of B56γ with p53 (Shouse *et al.* 2010). In contrast, higher expression levels of B56γ1 mRNA were reported in human melanoma cell lines compared to normal melanocytes (Francia *et al.* 1999).

Recently the HEK293T system was further utilised to systematically examine all PP2A regulatory subunits (Sablina *et al.* 2010). In addition to B56γ, suppression of B56α, PR72/PR130, and PTPA (protein phosphatase 2A activator), replaced the expression of ST in transformation. Interestingly, the effects on signaling pathways differed depending on the regulatory subunit suppressed. Knockdown of B56γ and PTPA, but not B56α or PR72/PR130, led to enhanced Akt phosphorylation, while knockdown of B56α, PR72/PR130, and PTPA, but not B56γ, resulted in increased c-Myc expression. Moreover, suppression of B56γ and PTPA led to increased β-catenin activity, whereas PR72/PR130 suppression decreased β-catenin activity (Sablina *et al.* 2010). Overexpression of CIP2A in the HEK-TER model can also replace ST in inducing transformation (Junttila *et al.* 2007), and increased CIP2A has been detected in myeloid leukemia patient samples (Wang and Li 2011), gastric and colon cancer samples (Khanna *et al.* 2009; Li *et al.* 2008; Soo Hoo *et al.* 2002)

#### **6.4 PP2A in myeloid leukemias**

An emerging body of evidence implicates PP2A as an important tumor suppressor in myeloid leukemias (Table 1). In particular, recent work from our laboratory and others has revealed a common theme in myeloid leukemias: inactivation of PP2A by leukemia associated tyrosine kinases (Fig. 3). Importantly, PP2A inhibition is essential for leukemias driven by these oncogenic tyrosine kinases, as re-activation of PP2A results in dephosphorylation, and thus deactivation, of the kinase and subsequent inhibition of leukemogenesis. Hence, re-activation of PP2A is an attractive strategy for leukemia therapy.

Fig. 3. Model of PP2A inhibition in myeloid leukemias. (A) PP2A activity is inhibited by BCR/ABL and oncogenic mutant c-KIT, enabling the activation of signaling pathways leading to leukemogenesis. (B) Pharmacological activation of PP2A induces de-activation (dephosphorylation) of BCR/ABL and c-KIT, and inhibition of downstream signaling pathways, leading to inhibition of leukemogenesis.

#### **6.4.1 PP2A inhibition in CML**

130 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Ruediger *et al.* 2001b; Sablina *et al.* 2007). Even in the absence of mutations, reduced protein expression of Aα has been found in 25 out of 58 brain tumors (Colella *et al.* 2001). Decreased levels of Aβ have also been observed in 16 of 32 cancer cell lines derived from human lung, colon and breast cancer, as well as primary glioblastoma and B-CLL patient samples compared to normal tissue (Kalla *et al.* 2007; Suzuki and Takahashi 2003; Takagi *et al.* 2000; Zhou *et al.* 2003). Importantly, Ruediger *et al*., recently generated knock-in mice with cancer associated A mutations, and A knockouts, both of which exhibited increased incidence of lung cancer when treated with benzopyrene, thus supporting the role of PP2A as a tumor

Pivotal studies using the HEK-TER transformation model demonstrated that suppression of B56γ, but not B55α, functionally mimicked the introduction of ST and resulted in partial tumorigenic transformation (Chen *et al.* 2004; Moreno *et al.* 2004). Moreover, depletion of B56γ containing complexes leads to activation of the anti–apoptotic Akt pathway (Chen *et al.* 2005). These observations were the first to demonstrate that PP2A complexes containing B56γ modulate the phosphorylation of substrates associated with transformation. Loss of B56γ has been demonstrated in some human cancers. Decreased expression was identified in primary human melanoma samples compared to melanocytic nevi (Deichmann *et al.* 2001), and in human lung cancer cell lines where subsequent overexpression of B56γ reversed the tumorigenic phenotype (Chen *et al.* 2004). Reduced transcript levels of B56γ have also been documented in patients with aggressive B-CLL compared to those with stable disease (Falt *et al.* 2005). A mutation in B56γ has also been observed in lung cancer, and this was shown to disrupt the interaction of B56γ with p53 (Shouse *et al.* 2010). In contrast, higher expression levels of B56γ1 mRNA were reported in human melanoma cell

Recently the HEK293T system was further utilised to systematically examine all PP2A regulatory subunits (Sablina *et al.* 2010). In addition to B56γ, suppression of B56α, PR72/PR130, and PTPA (protein phosphatase 2A activator), replaced the expression of ST in transformation. Interestingly, the effects on signaling pathways differed depending on the regulatory subunit suppressed. Knockdown of B56γ and PTPA, but not B56α or PR72/PR130, led to enhanced Akt phosphorylation, while knockdown of B56α, PR72/PR130, and PTPA, but not B56γ, resulted in increased c-Myc expression. Moreover, suppression of B56γ and PTPA led to increased β-catenin activity, whereas PR72/PR130 suppression decreased β-catenin activity (Sablina *et al.* 2010). Overexpression of CIP2A in the HEK-TER model can also replace ST in inducing transformation (Junttila *et al.* 2007), and increased CIP2A has been detected in myeloid leukemia patient samples (Wang and Li 2011), gastric and colon cancer samples (Khanna *et al.* 2009; Li *et al.* 2008; Soo Hoo *et al.* 2002)

An emerging body of evidence implicates PP2A as an important tumor suppressor in myeloid leukemias (Table 1). In particular, recent work from our laboratory and others has revealed a common theme in myeloid leukemias: inactivation of PP2A by leukemia associated tyrosine kinases (Fig. 3). Importantly, PP2A inhibition is essential for leukemias driven by these oncogenic tyrosine kinases, as re-activation of PP2A results in dephosphorylation, and thus deactivation, of the kinase and subsequent inhibition of leukemogenesis. Hence, re-activation of PP2A is an attractive strategy for leukemia

suppressor (Ruediger *et al.* 2011).

**6.4 PP2A in myeloid leukemias** 

therapy.

lines compared to normal melanocytes (Francia *et al.* 1999).

Functional inactivation of PP2A by BCR/ABL is essential for the development of CML-BC and Ph+ ALL, and is thought to result from upregulation of the endogenous PP2A inhibitor SET (I2PP2A or TAF-1β) protein (Neviani *et al.* 2007; Neviani *et al.* 2005). Although its physiological function remains incompletely understood, abnormal expression of SET mRNA has been documented in solid tumors and haematological disorders (Carlson *et al.* 1998; Fornerod *et al.* 1995; Li *et al.* 1996). Expression of BCR/ABL in mouse myeloid precursor cells stimulates SET expression and correlates with a loss of PP2A activity (Neviani *et al.* 2005). Conversely, inhibition of BCR/ABL with imatinib treatment dramatically reduces SET expression, which results in the restoration of PP2A activity back to untransfected levels and results in the dephosphorylation of several substrates which are shared by BCR/ABL and PP2A (Neviani *et al.* 2005). These include STAT5, ERK1/2, Akt, BAD and JAK2 (Calabretta and Perrotti 2004; Janssens and Goris 2001). Importantly, molecular or pharmacological reactivation of PP2A resulted in growth suppression, enhanced apoptosis, restored differentiation, and decreased *in vivo* leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cell lines and primary CML-BC cells. Furthermore, reactivation of PP2A, by overexpression of PP2AC or 1,9-dideoxyforskolin treatment (a cAMP-independent PP2A activator) (see section 6.5.1), reduced the activity and expression of BCR/ABL in both imatinib-sensitive and -resistant BCR/ABL cells, suggesting that BCR/ABL itself is a target for PP2A activity. Indeed, reactivation of PP2A results in PP2AC association with BCR/ABL via the SHP1 tyrosine phosphatase, BCR/ABL dephosphorylation, and proteasomal degradation (Neviani *et al.* 2005). These findings establish an important link between an oncogenic kinase and a tumor suppressing phosphatase, and indicate that pharmacologic enhancement of PP2A is a powerful therapeutic strategy for imatinib-resistant CML. Taken together, these results suggest that SET-dependent inhibition of PP2A is required for the transduction of aberrant mitogenic, survival and anti-differentiation signals that contribute to the development of CML from the chronic phase into blast crisis (Neviani *et al.* 2005).

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 133

and reduced survival (Muller *et al.* 2008). While imatinib has shown remarkable success in treating c-KIT+ GIST patients (Demetri *et al.* 2002), the majority of c-KIT mutations expressed in AML patients are intrinsically resistant to imatinib, and as such, many CBF-AML and systemic mastocytosis patients are unresponsive to imatinib therapy (Cairoli *et al.* 2005; Pardanani *et al.* 2003). Our laboratory has recently shown that myeloid precursor cells expressing oncogenic mutant c-KIT receptors display significantly reduced PP2A activity compared to c-KIT-negative or WT-c-KIT expressing cells (Roberts *et al.* 2010). Inhibition of PP2A by mutant c-KIT is associated with reduced protein expression of PP2A subunits, together with altered expression of the endogenous PP2A inhibitory protein SET. Overexpression of PP2A-A caused growth inhibition and apoptosis, suggesting that PP2A inhibition is functionally important in c-KIT mediated leukemogenesis. Importantly, reactivation of PP2A resulted in dephosphorylation (and hence deactivation) of the mutant c-KIT receptor itself, as well as inactivation of downstream signaling proteins required for c-KIT induced leukemogenesis (e.g. Akt, ERK and STAT5) (Roberts *et al.* 2010). Furthermore, activation of PP2A by the pharmacological agent FTY720, inhibited leukemogenesis driven

A number of recent studies further support a tumor suppressive role for PP2A in AML. Reduced expression of the PP2A-B55α subunit and increased Akt phophorylation in AML patient blasts was associated with shorter complete remission (Ruvolo *et al.* 2011). Hyperphosphorylation of PP2Ac, known to inhibit PP2A activity, has been observed in 78% of AML patients. This was associated with enhanced expression of PP2A inhibitors SET, CIP2A, and/or SET binding protein 1 (SETBP1) (Cristobal *et al.* 2011). Reduced expression of PP2A-A and a number of PP2A regulatory subunits was also observed in a number of patients. Increased expression of SETBP1 was also shown to be induced by a novel translocation t(12;18)(p13;q12) involving ETV6 in a patient with AML (Cristobal *et al.* 2010). SETBP1 overexpression protected SET from protease cleavage, leading to the formation of a SETBP1-SET-PP2A complex that results in PP2A inhibition, promoting proliferation of the leukemic cells. SETBP1 was further shown to be overexpressed in over 27% of AML patients and correlated with significantly shorter overall survival, in particular in patients over 60 years (Cristobal *et al.* 2010). Thus SETBP1 confers a negative prognostic impact and may be a predictive factor in any future clinical trials with PP2A activators. Increased expression of another PP2A inhibitory protein, CIP2A, has also been observed in diagnosis and relapse AML patients compared to patients in remission or healthy controls (Wang and Li 2011).

Forskolin, a diterpene isolated from the roots of *Coleus forskohlii*, is primarily known to stimulate the adenylate cyclase system, which results in elevated levels of cyclic AMP (cAMP) and subsequent activation of protein kinase A (PKA) (Seamon and Daly 1981; Seamon *et al.* 1981). The anticancer properties of this compound were initially demonstrated through potent inhibition of growth and tumor colonisation of the highly metastatic BL6 melanoma cell line (Agarwal and Parks 1983). Further studies indicated its potential use against ALL (Gutzkow *et al.* 2002) and CML cell lines (Taetle and Li-en 1984). However, more recent findings demonstrate that forskolin also activates PP2A (Feschenko *et al.* 2002); a mechanism which contributes to induction of apoptosis in B-CLL, CML-BC and AML patient samples (Cristobal *et al.* 2011; Moon and Lerner 2003; Neviani *et al.* 2005). Neviani *et* 

by mutant c-KIT (See section 6.5.2).

**6.5 Targeting PP2A for anti-leukemia therapy** 

**6.5.1 Forskolin** 


Table 1. Ser/Thr Phosphatases in Leukaemia

#### **6.4.2 PP2A inhibition in AML**

Oncogenic c-KIT signaling also requires inhibition of PP2A for leukemogenesis (Roberts *et al.* 2010). c-KIT is a receptor tyrosine kinase that activates similar proliferation, differentiation and survival signaling pathways as BCR/ABL, such as the PI3K, ERK and JAK/STAT pathways (Linnekin 1999). Gain-of-function *c-KIT* mutations occur in a range of malignancies, including AML, systemic mastocytosis, testicular seminoma, gastrointestinal stromal tumors (GIST) and melanomas, making c-KIT an excellent target for anti-cancer therapies (Kitamura and Hirota 2004; Masson and Ronnstrand 2009). These mutants induce hyperphosphorylation of c-KIT and constitutive activation of downstream signaling pathways (Masson and Ronnstrand 2009). Activating c- KIT mutations occur in up to 48% of core binding factor-AML (CBF-AML) patients, and are associated with increased relapse and reduced survival (Muller *et al.* 2008). While imatinib has shown remarkable success in treating c-KIT+ GIST patients (Demetri *et al.* 2002), the majority of c-KIT mutations expressed in AML patients are intrinsically resistant to imatinib, and as such, many CBF-AML and systemic mastocytosis patients are unresponsive to imatinib therapy (Cairoli *et al.* 2005; Pardanani *et al.* 2003). Our laboratory has recently shown that myeloid precursor cells expressing oncogenic mutant c-KIT receptors display significantly reduced PP2A activity compared to c-KIT-negative or WT-c-KIT expressing cells (Roberts *et al.* 2010). Inhibition of PP2A by mutant c-KIT is associated with reduced protein expression of PP2A subunits, together with altered expression of the endogenous PP2A inhibitory protein SET. Overexpression of PP2A-A caused growth inhibition and apoptosis, suggesting that PP2A inhibition is functionally important in c-KIT mediated leukemogenesis. Importantly, reactivation of PP2A resulted in dephosphorylation (and hence deactivation) of the mutant c-KIT receptor itself, as well as inactivation of downstream signaling proteins required for c-KIT induced leukemogenesis (e.g. Akt, ERK and STAT5) (Roberts *et al.* 2010). Furthermore, activation of PP2A by the pharmacological agent FTY720, inhibited leukemogenesis driven by mutant c-KIT (See section 6.5.2).

A number of recent studies further support a tumor suppressive role for PP2A in AML. Reduced expression of the PP2A-B55α subunit and increased Akt phophorylation in AML patient blasts was associated with shorter complete remission (Ruvolo *et al.* 2011). Hyperphosphorylation of PP2Ac, known to inhibit PP2A activity, has been observed in 78% of AML patients. This was associated with enhanced expression of PP2A inhibitors SET, CIP2A, and/or SET binding protein 1 (SETBP1) (Cristobal *et al.* 2011). Reduced expression of PP2A-A and a number of PP2A regulatory subunits was also observed in a number of patients. Increased expression of SETBP1 was also shown to be induced by a novel translocation t(12;18)(p13;q12) involving ETV6 in a patient with AML (Cristobal *et al.* 2010). SETBP1 overexpression protected SET from protease cleavage, leading to the formation of a SETBP1-SET-PP2A complex that results in PP2A inhibition, promoting proliferation of the leukemic cells. SETBP1 was further shown to be overexpressed in over 27% of AML patients and correlated with significantly shorter overall survival, in particular in patients over 60 years (Cristobal *et al.* 2010). Thus SETBP1 confers a negative prognostic impact and may be a predictive factor in any future clinical trials with PP2A activators. Increased expression of another PP2A inhibitory protein, CIP2A, has also been observed in diagnosis and relapse AML patients compared to patients in remission or healthy controls (Wang and Li 2011).

#### **6.5 Targeting PP2A for anti-leukemia therapy**

#### **6.5.1 Forskolin**

132 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Oncogenic c-KIT signaling also requires inhibition of PP2A for leukemogenesis (Roberts *et al.* 2010). c-KIT is a receptor tyrosine kinase that activates similar proliferation, differentiation and survival signaling pathways as BCR/ABL, such as the PI3K, ERK and JAK/STAT pathways (Linnekin 1999). Gain-of-function *c-KIT* mutations occur in a range of malignancies, including AML, systemic mastocytosis, testicular seminoma, gastrointestinal stromal tumors (GIST) and melanomas, making c-KIT an excellent target for anti-cancer therapies (Kitamura and Hirota 2004; Masson and Ronnstrand 2009). These mutants induce hyperphosphorylation of c-KIT and constitutive activation of downstream signaling pathways (Masson and Ronnstrand 2009). Activating c- KIT mutations occur in up to 48% of core binding factor-AML (CBF-AML) patients, and are associated with increased relapse

Table 1. Ser/Thr Phosphatases in Leukaemia

**6.4.2 PP2A inhibition in AML** 

Forskolin, a diterpene isolated from the roots of *Coleus forskohlii*, is primarily known to stimulate the adenylate cyclase system, which results in elevated levels of cyclic AMP (cAMP) and subsequent activation of protein kinase A (PKA) (Seamon and Daly 1981; Seamon *et al.* 1981). The anticancer properties of this compound were initially demonstrated through potent inhibition of growth and tumor colonisation of the highly metastatic BL6 melanoma cell line (Agarwal and Parks 1983). Further studies indicated its potential use against ALL (Gutzkow *et al.* 2002) and CML cell lines (Taetle and Li-en 1984). However, more recent findings demonstrate that forskolin also activates PP2A (Feschenko *et al.* 2002); a mechanism which contributes to induction of apoptosis in B-CLL, CML-BC and AML patient samples (Cristobal *et al.* 2011; Moon and Lerner 2003; Neviani *et al.* 2005). Neviani *et* 

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 135

and showing no signs of leukemia. In contrast, all saline-treated mice were sacrificed 5 weeks post-tumor cell injection (Neviani *et al.* 2007). No toxic side effects were observed with administration of FTY720, highlighting the safety and therapeutic relevance of utilising

FTY720 also activates PP2A in mutant c-KIT myeloid cells, leading to growth inhibition and induction of apoptosis *in vitro* (Roberts *et al.* 2010). Activation of PP2A is required for the anti-leukaemic effects, as PP2A inhibition with okadaic acid inhibits these effects. FTY720 induced PP2A activity results in inhibition of c-KIT phosphorylation, and inactivation of downstream signaling proteins regulated by both c-KIT and PP2A. Importantly, FTY720 also inhibited the *in vivo* tumor growth of mutant c-KIT myeloid cells in a syngeneic mouse model (Roberts *et al.* 2010). Thus FTY720 may also be a useful therapeutic agent for CBF-

PP1 activity has been found to alter according to AML blast differentiation. Yamamoto *et al.,*  found that AML patients with well differentiated leukemia had higher PP1 activity than patients with poorly differentiated AML-M1 (Yamamoto *et al.* 1999). AML blast PP1 activity also correlated with patient prognosis where individuals with significantly low PP1 activity had lower overall survival than those with high PP1 activity, with a median survival for each group of 8 and 27 months respectively (Nishikawa *et al.* 1994). Thus low PP1 activity may be a prognostic indicator of poor prognosis. The functional consequence of low PP1 activity in AML is not known, however PP1 was recently found to dephosphorylate Akt at T405, and PP1 overexpression induced Akt dephosphorylation, promoted cell survival and inhibited differentiation (Xiao *et al.* 2010). PP1 also promotes survival by negative regulation of p53 (Li *et al.* 2006), and is implicated in regulating Wnt/β-catenin (Jiang *et al.* 2009), TGF-β (Shi *et al.* 2004), and NF-κB (Li *et al.* 2008) signaling pathways. Luo *et al.,* showed that arsenic sulfide, a therapy historically used for treating CML in China, inhibited proliferation and induced differentiation of a human APL HL-60 cell line down a monocytic pathway. This correlated with increasing PP1 and PP2A activity, and co-treatment with a concentration of OA that inhibits both phosphatases suppressed the arsenic sulfide induced differentiation (Luo *et al.* 2006). Furthermore, while the mechanism was unknown at the time, other studies had previously shown similar effect in an APL NB4 cell model (Bai and Huang 1998; Lu and Wang 2002) and in chronic myeloid leukemia K562 cells (Yin *et al.* 2004). These studies suggest that PP1 plays a role in the growth inhibition and differentiation of AML, and may

PP2B (calcineurin) is probably best known for its role in immunity where it is activated upon T cell receptor stimulation. Indeed, calcineurin is a well established target of prophylactic agents used in transplantation, such as Cyclosporin A and FK506, where calcineurin inhibition suppresses IL-2 production. Calcineurin was recently implicated as an important oncogene in lymphoid leukemias (Medyouf *et al.* 2007; Medyouf and Ghysdael 2008; Muller and Rao 2007). Sustained calcineurin activity was observed in human B- and Tcell lymphomas and in a range of mouse models of lymphoid malignancies. Moreover,

PP2A-activating drugs in leukemia patients.

**7.1 PP1** 

**7.2 PP2B** 

AML patients harbouring activating c-KIT mutations.

also provide a useful prognostic tool and/or therapy target.

**7. Other serine/threonine phosphatases in myeloid leukemia** 

*al*., was the first to highlight the therapeutic relevance of using PP2A-activators to specifically target leukemia cells (Neviani *et al.* 2005). Forskolin inhibited the *in vivo* leukemogenesis of imatinib sensitive and resistant BCR/ABL+ 32Dcl3 cells in mice, resulting in significantly prolonged survival. Furthermore, treatment with 1,9-dideoxyforskolin, which lacks adenylate cyclase activity, impaired the clonogenic potential of BCR/ABL+ 32Dcl3 cells to a similar degree as forskolin, suggesting that the anti-leukaemic effects of forskolin and its derivative depends on the induction of PP2A activity rather than cAMP . Restoration of PP2A activity with forskolin was also found to inhibit Akt and ERK activity, block proliferation and induce caspase-dependant apoptosis in AML cell lines (Cristobal *et al.* 2011; Neviani *et al.* 2005). Furthermore, forskolin had an additive effect with common AML induction therapy drugs Idarubicin and Ara-c (Cristobal *et al.* 2011).

#### **6.5.2 FTY720**

FTY720 was first synthesised by structural modifications of myriocin (ISP-1), a fungal metabolite isolated from *Isaria sinclairii* culture broth (Fujita *et al.* 1994), and is structurally similar to sphingosine (Albert *et al.* 2005; Kiuchi *et al.* 2005). It is effectively phosphorylated *in vivo* by SphK2 to yield the biologically active FTY720-phosphate (FTY720-P) (Brinkmann *et al.* 2002; Zemann *et al.* 2006). Interaction of FTY720-P with one of the five known S1P receptors induces receptor internalisation and degradation (Graler and Goetzl 2004; Matloubian *et al.* 2004). The S1P signal is required for the migration of lymphocytes from secondary lymphoid tissues back into the efferent lymphatics and systemic circulation (Cyster 2005; Matloubian *et al.* 2004) and prolonged S1PR downregulation by FTY720-P inhibits the immune response by sequestering functional lymphocytes within secondary lymphoid organs (Brinkmann *et al.* 2002; Mandala *et al.* 2002). The use of FTY720 as an immunomodulator is currently being evaluated in Phase III trials for multiple sclerosis (Cohen *et al.* 2009; Takabe *et al.* 2008).

A more recent mechanism of action identified for FTY720 is its activation of purified PP2A trimers *in vitro* (Matsuoka *et al.* 2003) and as loss of PP2A phosphatase activity contributes to the pathophysiology of BCR/ABL-driven leukemias (Neviani *et al.* 2007) a logical prediction would be that restoration of PP2A levels reverses the leukaemic phenotype. Indeed, an extensive study has shown that pharmacological reactivation of PP2A with FTY720 inhibits the proliferation, enhances apoptosis, restores differentiation and impairs colony formation of imatinib-sensitive and –resistant BCR-ABL+ cell lines and CML-BC patient blasts (Neviani *et al.* 2007). Notably, FTY720 promotes BCR/ABL tyrosine dephosphorylation and proteolytic degradation, together with reduced phosphorylation of the PP2A targets Akt, ERK1/2 and STAT5 (Neviani *et al.* 2007). Co-treatment with okadaic acid or transduction of SV40 ST reverses the enhancement of PP2A activity and restores substrate phosphorylation, strongly indicating that FTY720 functions through a PP2A-dependent mechanism.

Importantly, the *in vitro* efficacy of FTY720 translated into an *in vivo* model markedly suppressing both imatinib-sensitive (WT) and –resistant (T315I) BCR/ABL+ leukemogenesis (Neviani *et al.* 2007). After 4 weeks of treatment, saline-treated mice contained a large number of undifferentiated myeloid cells within the peripheral circulation, representing an overt AML phenotype with extensive blast infiltration of distal organs. In contrast, FTY720 treated mice displayed undetectable levels of BCR/ABL+ cells in the systemic circulation and secondary organs. Accordingly, these effects were sustained long term with 80% and 50% of WT and T315I BCR/ABL+ FTY720-treated mice, respectively, still alive at 27 weeks and showing no signs of leukemia. In contrast, all saline-treated mice were sacrificed 5 weeks post-tumor cell injection (Neviani *et al.* 2007). No toxic side effects were observed with administration of FTY720, highlighting the safety and therapeutic relevance of utilising PP2A-activating drugs in leukemia patients.

FTY720 also activates PP2A in mutant c-KIT myeloid cells, leading to growth inhibition and induction of apoptosis *in vitro* (Roberts *et al.* 2010). Activation of PP2A is required for the anti-leukaemic effects, as PP2A inhibition with okadaic acid inhibits these effects. FTY720 induced PP2A activity results in inhibition of c-KIT phosphorylation, and inactivation of downstream signaling proteins regulated by both c-KIT and PP2A. Importantly, FTY720 also inhibited the *in vivo* tumor growth of mutant c-KIT myeloid cells in a syngeneic mouse model (Roberts *et al.* 2010). Thus FTY720 may also be a useful therapeutic agent for CBF-AML patients harbouring activating c-KIT mutations.

#### **7. Other serine/threonine phosphatases in myeloid leukemia**

#### **7.1 PP1**

134 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

*al*., was the first to highlight the therapeutic relevance of using PP2A-activators to specifically target leukemia cells (Neviani *et al.* 2005). Forskolin inhibited the *in vivo* leukemogenesis of imatinib sensitive and resistant BCR/ABL+ 32Dcl3 cells in mice, resulting in significantly prolonged survival. Furthermore, treatment with 1,9-dideoxyforskolin, which lacks adenylate cyclase activity, impaired the clonogenic potential of BCR/ABL+ 32Dcl3 cells to a similar degree as forskolin, suggesting that the anti-leukaemic effects of forskolin and its derivative depends on the induction of PP2A activity rather than cAMP . Restoration of PP2A activity with forskolin was also found to inhibit Akt and ERK activity, block proliferation and induce caspase-dependant apoptosis in AML cell lines (Cristobal *et al.* 2011; Neviani *et al.* 2005). Furthermore, forskolin had an additive effect with

common AML induction therapy drugs Idarubicin and Ara-c (Cristobal *et al.* 2011).

FTY720 was first synthesised by structural modifications of myriocin (ISP-1), a fungal metabolite isolated from *Isaria sinclairii* culture broth (Fujita *et al.* 1994), and is structurally similar to sphingosine (Albert *et al.* 2005; Kiuchi *et al.* 2005). It is effectively phosphorylated *in vivo* by SphK2 to yield the biologically active FTY720-phosphate (FTY720-P) (Brinkmann *et al.* 2002; Zemann *et al.* 2006). Interaction of FTY720-P with one of the five known S1P receptors induces receptor internalisation and degradation (Graler and Goetzl 2004; Matloubian *et al.* 2004). The S1P signal is required for the migration of lymphocytes from secondary lymphoid tissues back into the efferent lymphatics and systemic circulation (Cyster 2005; Matloubian *et al.* 2004) and prolonged S1PR downregulation by FTY720-P inhibits the immune response by sequestering functional lymphocytes within secondary lymphoid organs (Brinkmann *et al.* 2002; Mandala *et al.* 2002). The use of FTY720 as an immunomodulator is currently being evaluated in Phase III trials for multiple sclerosis

A more recent mechanism of action identified for FTY720 is its activation of purified PP2A trimers *in vitro* (Matsuoka *et al.* 2003) and as loss of PP2A phosphatase activity contributes to the pathophysiology of BCR/ABL-driven leukemias (Neviani *et al.* 2007) a logical prediction would be that restoration of PP2A levels reverses the leukaemic phenotype. Indeed, an extensive study has shown that pharmacological reactivation of PP2A with FTY720 inhibits the proliferation, enhances apoptosis, restores differentiation and impairs colony formation of imatinib-sensitive and –resistant BCR-ABL+ cell lines and CML-BC patient blasts (Neviani *et al.* 2007). Notably, FTY720 promotes BCR/ABL tyrosine dephosphorylation and proteolytic degradation, together with reduced phosphorylation of the PP2A targets Akt, ERK1/2 and STAT5 (Neviani *et al.* 2007). Co-treatment with okadaic acid or transduction of SV40 ST reverses the enhancement of PP2A activity and restores substrate phosphorylation,

strongly indicating that FTY720 functions through a PP2A-dependent mechanism.

Importantly, the *in vitro* efficacy of FTY720 translated into an *in vivo* model markedly suppressing both imatinib-sensitive (WT) and –resistant (T315I) BCR/ABL+ leukemogenesis (Neviani *et al.* 2007). After 4 weeks of treatment, saline-treated mice contained a large number of undifferentiated myeloid cells within the peripheral circulation, representing an overt AML phenotype with extensive blast infiltration of distal organs. In contrast, FTY720 treated mice displayed undetectable levels of BCR/ABL+ cells in the systemic circulation and secondary organs. Accordingly, these effects were sustained long term with 80% and 50% of WT and T315I BCR/ABL+ FTY720-treated mice, respectively, still alive at 27 weeks

**6.5.2 FTY720** 

(Cohen *et al.* 2009; Takabe *et al.* 2008).

PP1 activity has been found to alter according to AML blast differentiation. Yamamoto *et al.,*  found that AML patients with well differentiated leukemia had higher PP1 activity than patients with poorly differentiated AML-M1 (Yamamoto *et al.* 1999). AML blast PP1 activity also correlated with patient prognosis where individuals with significantly low PP1 activity had lower overall survival than those with high PP1 activity, with a median survival for each group of 8 and 27 months respectively (Nishikawa *et al.* 1994). Thus low PP1 activity may be a prognostic indicator of poor prognosis. The functional consequence of low PP1 activity in AML is not known, however PP1 was recently found to dephosphorylate Akt at T405, and PP1 overexpression induced Akt dephosphorylation, promoted cell survival and inhibited differentiation (Xiao *et al.* 2010). PP1 also promotes survival by negative regulation of p53 (Li *et al.* 2006), and is implicated in regulating Wnt/β-catenin (Jiang *et al.* 2009), TGF-β (Shi *et al.* 2004), and NF-κB (Li *et al.* 2008) signaling pathways. Luo *et al.,* showed that arsenic sulfide, a therapy historically used for treating CML in China, inhibited proliferation and induced differentiation of a human APL HL-60 cell line down a monocytic pathway. This correlated with increasing PP1 and PP2A activity, and co-treatment with a concentration of OA that inhibits both phosphatases suppressed the arsenic sulfide induced differentiation (Luo *et al.* 2006). Furthermore, while the mechanism was unknown at the time, other studies had previously shown similar effect in an APL NB4 cell model (Bai and Huang 1998; Lu and Wang 2002) and in chronic myeloid leukemia K562 cells (Yin *et al.* 2004). These studies suggest that PP1 plays a role in the growth inhibition and differentiation of AML, and may also provide a useful prognostic tool and/or therapy target.

#### **7.2 PP2B**

PP2B (calcineurin) is probably best known for its role in immunity where it is activated upon T cell receptor stimulation. Indeed, calcineurin is a well established target of prophylactic agents used in transplantation, such as Cyclosporin A and FK506, where calcineurin inhibition suppresses IL-2 production. Calcineurin was recently implicated as an important oncogene in lymphoid leukemias (Medyouf *et al.* 2007; Medyouf and Ghysdael 2008; Muller and Rao 2007). Sustained calcineurin activity was observed in human B- and Tcell lymphomas and in a range of mouse models of lymphoid malignancies. Moreover,

Ser/Thr Phosphatases: The New Frontier for Myeloid Leukemia Therapy? 137

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Albert, R., K. Hinterding, et al. (2005). "Novel immunomodulator FTY720 is phosphorylated

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expression of constitutively active mutant calcineurin favored leukemia progression, while treatment with Cyclosporin A or FK506 induced apoptosis of leukemic cells and rapid tumor clearance, and significantly improved mouse survival (Medyouf *et al.* 2007). Thus inhibiting calcineurin is a potential therapeutic strategy for lymphoid leukemias. In contrast, a specific role for calcineurin in myeloid leukemias has not been reported, however one study investigating its activity in AML patients of different FAB subtype found that calcineurin activity is relatively low in leukemic blasts arresting at the stage of early pluripotent stem cells, and increases during the course of myelomonocytic commitment and maturation (Yamamoto *et al.* 1999).

#### **7.3 PP2C**

Like PP2B, little information exists regarding the role of PP2C in myeloid leukemia. One study revealed it had low expression in AML patient blood cells and its activity and expression were relatively constant in various leukemic cell types from AML, ALL, and CLL patients (Yamamoto *et al.* 1999), suggesting it may not play a remarkable role in leukemogenesis.

#### **8. Conclusions**

There is no doubt that PP2A acts as a tumor suppressor in myeloid leukemias, and targeting its re-activation, either directly or via inhibition of an endogenous inhibitory protein such as SET, is a promising therapeutic strategy. Whether the pre-clinical promise will translate to improved survival of myeloid leukemia patients is currently unknown, but clinical trials of PP2A activators are eagerly awaited. Emerging studies indicate that other phosphatases may also play important roles in myeloid leukemias, and future studies aimed at deciphering the molecular mechanisms regulating these phosphatases and their downstream targets is sure to identify further targets for novel therapies.

#### **9. Acknowledgements**

This work was supported by grants from the Cancer Council NSW, the Anthony Rothe Memorial Foundation, and the Hunter Medical Research Institute (HMRI). AMS was supported by an Australian Postgraduate Award (APA) and an Arrow Bone Marrow Trust scholarship, KGR by an APA and Cancer Institute NSW scholarship, and NMV by a National Health and Medical Research Council (NHMRC) Peter Doherty Fellowship.

#### **10. References**


expression of constitutively active mutant calcineurin favored leukemia progression, while treatment with Cyclosporin A or FK506 induced apoptosis of leukemic cells and rapid tumor clearance, and significantly improved mouse survival (Medyouf *et al.* 2007). Thus inhibiting calcineurin is a potential therapeutic strategy for lymphoid leukemias. In contrast, a specific role for calcineurin in myeloid leukemias has not been reported, however one study investigating its activity in AML patients of different FAB subtype found that calcineurin activity is relatively low in leukemic blasts arresting at the stage of early pluripotent stem cells, and increases during the course of myelomonocytic commitment and

Like PP2B, little information exists regarding the role of PP2C in myeloid leukemia. One study revealed it had low expression in AML patient blood cells and its activity and expression were relatively constant in various leukemic cell types from AML, ALL, and CLL patients (Yamamoto *et al.* 1999), suggesting it may not play a remarkable role in

There is no doubt that PP2A acts as a tumor suppressor in myeloid leukemias, and targeting its re-activation, either directly or via inhibition of an endogenous inhibitory protein such as SET, is a promising therapeutic strategy. Whether the pre-clinical promise will translate to improved survival of myeloid leukemia patients is currently unknown, but clinical trials of PP2A activators are eagerly awaited. Emerging studies indicate that other phosphatases may also play important roles in myeloid leukemias, and future studies aimed at deciphering the molecular mechanisms regulating these phosphatases and their

This work was supported by grants from the Cancer Council NSW, the Anthony Rothe Memorial Foundation, and the Hunter Medical Research Institute (HMRI). AMS was supported by an Australian Postgraduate Award (APA) and an Arrow Bone Marrow Trust scholarship, KGR by an APA and Cancer Institute NSW scholarship, and NMV by a

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**7.3 PP2C** 

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**8. Conclusions** 

**9. Acknowledgements** 

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myeloid leukaemia and associated with HL60 cells proliferation and

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*Oncol,* 235: 1263-1268 1019-6439 (Print).

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12974-12979 1091-6490 (Electronic).

serine/threonine protein phosphatase 2A in human breast cancer MCF-7 cells."*Int J* 

response by chronic myelogenous leukaemia granulocyte/macrophage


**8** 

*USA* 

**Role of JAK2 Beyond Myeloproliferative** 

**Malignancies and Solid Tumors** 

Theresa M. McDevitt and Matthew V. Lorenzi

*Oncology Drug Discovery, Bristol-Myers Squibb* 

**Neoplasms (MPNs): Rationale for Targeting the** 

Janus kinases (JAKs) are a family of receptor-associated tyrosine kinases which are crucial for the survival and proliferation of immune and hematopoietic-derived cells. There are four mammalian JAKs identified to date: JAK1, JAK2, JAK3 and TYK2 (Lopez et al., 2010). JAKs are responsible for mediating the intracellular signaling of numerous growth factors and cytokines (Murray et al., 2007 & O'Shea et al., 2002). Once activated JAKs directly phosphorylate and activate the transcription factors signal transducers and activators of transcriptions (STATs) which transduce JAK signaling by translocating to the nucleus to modulate a subset of genes that are critical for cell proliferation and survival (Aaronson et al., 2002 & Levy et al., 2002). Dysregulated JAK signaling has been implicated in the pathogenesis of several blood-borne cancers but most notably in myeloproliferative neoplasms (MPNs) (Nelson et al., 2006; Lucia et al., 2011 & Patnaik et al., 2009). MPNs are hematological malignancies defined by the excessive proliferation of one or more myeloidderived cells. MPNs are classified into two clinical categories either BCR-ABL-positive (BCR-ABL (+)) or BCR-ABL-negative (BCR-ABL (-)) based on the presence of the BCR-ABL fusion protein. The major BCR-ABL (-) MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). The specific pathogenic role that JAK2 has in driving some of these MPNs is highlighted by the recent seminal discovery of the JAK2V617F mutation (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; & Levine et al., 2005). The JAK2V617F mutation is a somatic, gain-of function point mutation which leads to constitutive activation of JAK2 and subsequent downstream activation of STATs (Baxter et al., 2005 & James et al., 2005). Current data demonstrates that JAK2 is a disease associated gene involved in the pathogenesis of BCR-ABL (-) MPNs. However, it remains unclear if JAK2 pathway mutations are the primary drivers of sustained disease progression. New data is emerging that suggests that targeting the JAK-STAT pathway may have implications beyond the treatment of MPN and can be useful for the treatment of other hematological and non-hematological cancers. This review will summarize the pathogenic role of JAK-STAT signaling in BCR-ABL (-) MPNs and introduce the potential broader implications by

**1. Introduction** 

**JAK-STAT Pathway in Other Hematological** 


## **Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs): Rationale for Targeting the JAK-STAT Pathway in Other Hematological**

Theresa M. McDevitt and Matthew V. Lorenzi *Oncology Drug Discovery, Bristol-Myers Squibb USA* 

**Malignancies and Solid Tumors** 

#### **1. Introduction**

148 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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dephosphorylation by protein phosphatases."*J Biol Chem,* 27735: 31818-31825 0021-

isoforms of protein phosphatase 2A: differences in expression, subunit interaction,

phosphatase 2A: identification of a novel isoform highly expressed in

Janus kinases (JAKs) are a family of receptor-associated tyrosine kinases which are crucial for the survival and proliferation of immune and hematopoietic-derived cells. There are four mammalian JAKs identified to date: JAK1, JAK2, JAK3 and TYK2 (Lopez et al., 2010). JAKs are responsible for mediating the intracellular signaling of numerous growth factors and cytokines (Murray et al., 2007 & O'Shea et al., 2002). Once activated JAKs directly phosphorylate and activate the transcription factors signal transducers and activators of transcriptions (STATs) which transduce JAK signaling by translocating to the nucleus to modulate a subset of genes that are critical for cell proliferation and survival (Aaronson et al., 2002 & Levy et al., 2002). Dysregulated JAK signaling has been implicated in the pathogenesis of several blood-borne cancers but most notably in myeloproliferative neoplasms (MPNs) (Nelson et al., 2006; Lucia et al., 2011 & Patnaik et al., 2009). MPNs are hematological malignancies defined by the excessive proliferation of one or more myeloidderived cells. MPNs are classified into two clinical categories either BCR-ABL-positive (BCR-ABL (+)) or BCR-ABL-negative (BCR-ABL (-)) based on the presence of the BCR-ABL fusion protein. The major BCR-ABL (-) MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). The specific pathogenic role that JAK2 has in driving some of these MPNs is highlighted by the recent seminal discovery of the JAK2V617F mutation (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; & Levine et al., 2005). The JAK2V617F mutation is a somatic, gain-of function point mutation which leads to constitutive activation of JAK2 and subsequent downstream activation of STATs (Baxter et al., 2005 & James et al., 2005). Current data demonstrates that JAK2 is a disease associated gene involved in the pathogenesis of BCR-ABL (-) MPNs. However, it remains unclear if JAK2 pathway mutations are the primary drivers of sustained disease progression. New data is emerging that suggests that targeting the JAK-STAT pathway may have implications beyond the treatment of MPN and can be useful for the treatment of other hematological and non-hematological cancers. This review will summarize the pathogenic role of JAK-STAT signaling in BCR-ABL (-) MPNs and introduce the potential broader implications by

Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs): Rationale

conserved throughout the JAK kinase family.

**FERM** 

**2.3 JAK-STAT signaling** 

promote cell growth and survival.

**No Ligand** 

**Y Y** 

**JAK2 JAK2** 

Fig. 2. Ligand-Induced JAK-STAT Activation

for Targeting the JAK-STAT Pathway in Other Hematological Malignancies and Solid Tumors 151

**SH-2-like Pseudokinase**

**PI3K** 

**S T A T**

**PY YP** 

**JAK2 JAK2** 

 **Ligand** 

**PY YP SH2** 

**S T A T**

**MAPK** 

**Kinase**

Fig. 1. **JAK Protein Domain Structure.** The seven homologous JAK domains (JH1-JH7)

**Dimerization** 

**Bcl-2, Cyclin D** 

JH7 JH6 JH5 JH4 JH2 JH1

JH3 **- -**

In the absence of ligand, JAKs are pre-associated with receptor monomers, upon ligand binding receptor dimerzation ensues leading to trans-phosphorylation and auto-activation of JAKs. Once activated, JAKs proceed to phosphorylate specific tyrosine residues within the cytoplasmic domains of growth factor receptors creating docking sites for downstream mediators of JAK signaling. JAKs can activate classical signaling pathways such as PI-3 kinase and MAPK but JAKs also directly activate STATs as described above. STATs are latent transcription factors that are activated and phosphorylated by JAKs at specific tyrosine residues (Darnell J, 1997 & Benekli et al., 2003). Once phosphorylated, STAT monomers can then homo/hetero-dimerize through reciprocal Src homology 2 (SH2) interactions and translocate to the nucleus to modulate the transcription of key genes which

which JAK-STAT pathway inhibition may have in treating hematological malignancies as well as solid tumors.

#### **2. Janus Kinase Family (JAKs)**

#### **2.1 Function**

Janus kinases are a family of cytoplasmic receptor associated tyrosine kinases responsible for integrating the signaling of numerous growth factors and cytokines that are critical for hematopoiesis and immune function. There are four JAK family members; JAK1, JAK2, JAK3 and TYK2. JAK1, JAK2 and TYK2 are ubiquitously expressed while JAK3 expression is restricted to myeloid and lymphoid tissue (Valentino et al., 2006 & Ward et al., 2000 ). Various cytokines and growth factor receptors rely on JAKs to propagate their intracellular signaling cascades since many of these receptors lack intrinsic kinase activity. JAKs can be differentially or coordinately activated in response to various growth factor or cytokine stimuli (Murray, 2007). Targeted disruption of individual JAKs have been generated in an effort to better understand the biological non-redundant roles that JAKs have in development. JAK1 knock-out mice have been generated and present with a perinatal lethal phenotype that is characterized by severe defects in lymphopoiesis and neuronal development (Rodig et al., 1998). JAK3 mutations in humans have been described in patients with severe combined immunodeficiency (SCID), an immune disorder where major defects in T- and B- cell development are observed. This clinical phenotype is recapitulated in JAK3 knock-out models where these mice manifest with SCID-like features characterized by impaired lymphocyte production (Nosaka et al., 1995 & Park et al., 2000). TYK2 deficiencies although rare have been reported, and these patients usually present with severe dermatitis and hyper-IgE syndrome-like symptoms (HIES). Genetic disruption studies involving TYK2 have presented with severe defects in IL-12 signaling and impaired Th1 and Th2 mediated immune responses (Karaghiosoff et al., 2003). Genetic inactivation of JAK2 in mice is embryonically lethal due to severe anemia, confirming the critical role that JAK2 is thought to have in erythropoiesis (Parganas et al., 1998).

#### **2.2 Structure**

JAK proteins share seven homologous structural domains (JH1-JH7) each of which elicit distinct functional features within JAK proteins. The JAK homology domains consist of the C-terminus JH1 domain which is the kinase-active domain, the auto-regulatory JH2 pseudokinase domain, the SH2-like JH3 domain and the N-terminus FERM (**f**our-point-one, **e**zrin, **r**adixin, and **m**oesin) domain (JH4-JH7). Although the JH3 domain possesses a SH2 like sequence it is unable to bind and interact with phospho-tyrosine residues and therefore its precise function remains unclear while the FERM domain is responsible for mediating the interactions of JAKs with their cognate receptors (Huang et al., 2001 & Radtke et al., 2005). The JH2 pseudokinase domain is a unique structural feature of the JAKs that is otherwise absent in other kinase families. The JH2 pseudokinase domain bears high sequence identity to the JH1 kinase domain but is devoid of any catalytic activity and is thought to act as a regulatory domain through catalytic auto-inhbition (Saharinen et al., 2000). Moreover, key activating point mutations have been identified in the JH2 pseudokinase domain of JAK2 that are critically involved in the pathogenesis of some myeloproliferative neoplasms (MPNs).

Fig. 1. **JAK Protein Domain Structure.** The seven homologous JAK domains (JH1-JH7) conserved throughout the JAK kinase family.

#### **2.3 JAK-STAT signaling**

150 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

which JAK-STAT pathway inhibition may have in treating hematological malignancies as

Janus kinases are a family of cytoplasmic receptor associated tyrosine kinases responsible for integrating the signaling of numerous growth factors and cytokines that are critical for hematopoiesis and immune function. There are four JAK family members; JAK1, JAK2, JAK3 and TYK2. JAK1, JAK2 and TYK2 are ubiquitously expressed while JAK3 expression is restricted to myeloid and lymphoid tissue (Valentino et al., 2006 & Ward et al., 2000 ). Various cytokines and growth factor receptors rely on JAKs to propagate their intracellular signaling cascades since many of these receptors lack intrinsic kinase activity. JAKs can be differentially or coordinately activated in response to various growth factor or cytokine stimuli (Murray, 2007). Targeted disruption of individual JAKs have been generated in an effort to better understand the biological non-redundant roles that JAKs have in development. JAK1 knock-out mice have been generated and present with a perinatal lethal phenotype that is characterized by severe defects in lymphopoiesis and neuronal development (Rodig et al., 1998). JAK3 mutations in humans have been described in patients with severe combined immunodeficiency (SCID), an immune disorder where major defects in T- and B- cell development are observed. This clinical phenotype is recapitulated in JAK3 knock-out models where these mice manifest with SCID-like features characterized by impaired lymphocyte production (Nosaka et al., 1995 & Park et al., 2000). TYK2 deficiencies although rare have been reported, and these patients usually present with severe dermatitis and hyper-IgE syndrome-like symptoms (HIES). Genetic disruption studies involving TYK2 have presented with severe defects in IL-12 signaling and impaired Th1 and Th2 mediated immune responses (Karaghiosoff et al., 2003). Genetic inactivation of JAK2 in mice is embryonically lethal due to severe anemia, confirming the critical role that JAK2 is thought

JAK proteins share seven homologous structural domains (JH1-JH7) each of which elicit distinct functional features within JAK proteins. The JAK homology domains consist of the C-terminus JH1 domain which is the kinase-active domain, the auto-regulatory JH2 pseudokinase domain, the SH2-like JH3 domain and the N-terminus FERM (**f**our-point-one, **e**zrin, **r**adixin, and **m**oesin) domain (JH4-JH7). Although the JH3 domain possesses a SH2 like sequence it is unable to bind and interact with phospho-tyrosine residues and therefore its precise function remains unclear while the FERM domain is responsible for mediating the interactions of JAKs with their cognate receptors (Huang et al., 2001 & Radtke et al., 2005). The JH2 pseudokinase domain is a unique structural feature of the JAKs that is otherwise absent in other kinase families. The JH2 pseudokinase domain bears high sequence identity to the JH1 kinase domain but is devoid of any catalytic activity and is thought to act as a regulatory domain through catalytic auto-inhbition (Saharinen et al., 2000). Moreover, key activating point mutations have been identified in the JH2 pseudokinase domain of JAK2 that are critically involved in the pathogenesis of some

well as solid tumors.

**2.1 Function** 

**2.2 Structure** 

**2. Janus Kinase Family (JAKs)** 

to have in erythropoiesis (Parganas et al., 1998).

myeloproliferative neoplasms (MPNs).

In the absence of ligand, JAKs are pre-associated with receptor monomers, upon ligand binding receptor dimerzation ensues leading to trans-phosphorylation and auto-activation of JAKs. Once activated, JAKs proceed to phosphorylate specific tyrosine residues within the cytoplasmic domains of growth factor receptors creating docking sites for downstream mediators of JAK signaling. JAKs can activate classical signaling pathways such as PI-3 kinase and MAPK but JAKs also directly activate STATs as described above. STATs are latent transcription factors that are activated and phosphorylated by JAKs at specific tyrosine residues (Darnell J, 1997 & Benekli et al., 2003). Once phosphorylated, STAT monomers can then homo/hetero-dimerize through reciprocal Src homology 2 (SH2) interactions and translocate to the nucleus to modulate the transcription of key genes which promote cell growth and survival.

Fig. 2. Ligand-Induced JAK-STAT Activation

Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs): Rationale

**3.3 V617F and other JAK2-associated mutations in MPNs** 

signaling may be beneficial for the treatment of MPN.

**4. JAK inhibitors in clinical development** 

diseases (Wernig et al., 2006)

for Targeting the JAK-STAT Pathway in Other Hematological Malignancies and Solid Tumors 153

JAK2 including transgenic and retroviral transduction methods leads to a MPN-like phenotype *in vivo*, further supporting a causal role for JAK2 in the pathogenesis of these

In 2005, several independent laboratories identified a unique somatic activating point mutation in the JAK2 gene (V617F). The JAK2V617F mutation occurs in the JH2 pseudokinase domain of JAK2 in exon 14 at position 617 where a valine is replaced with a phenylalanine which leads to constitutive activation of JAK2. The mechanism of activation of the V617F mutation is believed to be due to the removal of the auto-inhibitory function of the JH2 pseudokinase domain present in JAK2. The incidence of the JAK2V617F mutation is highly prevalent within the BCR-ABL (-) MPN patient population particularly within PV cohorts where more than 95% (Wernig et al., 2006) of PV patients are positive for it while 50% of ET and IMF patients are positive for it (Pikman et al, 2006). Although the JAK2V617F mutation represents a major genetic event underlying the pathogenesis of these MPNs there is still a fraction of PV patients (~5%) and ET/IMF patients (~50%) that present with these MPNs but yet are negative for the V617F JAK2 mutation. The existence of V617F-negative MPNs highlights the need to identify additional JAK2 signaling components that may be involved in the pathogenesis of these V617F-independent MPNs. Several gain-of-function mutations comprised of deletions, insertions and frame-shifts have been indentified in exon 12 of JAK2 in a small subset of V617-negative PV patients (~3%) (Scott et al., 2007). Interestingly, exon 12 JAK2 mutations are mutually exclusive from V617F JAK2 mutations and to date have only been described in V617F-negative PV patients. Expression of exon 12 JAK2 mutations results in a PV-like phenotype *in vivo* that is characterized by erythrocytosis. Alternatively, activating point mutations in the thrombopoietin receptor (Tpo-R/c-MPL) have been identified in a subset of V617F-negative ET and IMF patients (5-10%). The mechanism of action for the MPLW515L mutation is similar to that of the V617F mutation since it also results in the removal of an auto-inhibitory sequence thought to be located within the transmembrane region of the Tpo receptor which causes spontaneous activation of downstream JAK2-STAT signaling (Pikman et al., 2006). The relative frequencies of these JAK-related mutations are summarized in figure 3 (Paradanani et al, 2011). The oncogenic potential of these mutations in MPN is further highlighted by their ability to induce MPNlike phenotypes in murine bone marrow reconstitution studies. JAK2V617F expression induces a PV-like phenotype that presents with erythrocytosis and splenomegaly while (Wernig et al., 2006) MPLW515L promotes a more aggressive MF-like phenotype characterized by marked thrombocytopenia and severe fibrosis of the bone marrow (Pikman et al., 2006). Collectively, these results highlight a strong selective pressure to activate JAK-STAT signaling in BCR-ABL-(-) MPNs and thus therapies designed to target the JAK-STAT

Since the discovery of the JAK2V617F mutation in 2004 remarkable scientific progress has been made from bench to beside with the first JAK inhibitors being tested in humans by mid 2007. Currently there are 36 clinical trials in the US underway evaluating various treatments for MPNs or investigating the molecular pathobiology underlying MPNs. The current JAK inhibitors are typically small molecule ATP competitive inhibitors of both wild-type and

### **3. Myeloproliferative Neoplasms (MPNs)**

#### **3.1 Clinical biology**

MPNs are hematopoietic malignancies characterized by excessive growth of one or more myeloid derived cell lineages such as erythrocytes, platelets and/or granulocytes. MPNs are clinically grouped into either BCR-ABL (+) or BCR-ABL (-) based on the presence of the BCR-ABL protein, as seen in CML patients. Dysregulated JAK-STAT signaling has been implicated in the pathogenesis of these BCR-ABL (-) MPNs. The three most common BCR-ABL (-) MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) with an estimated annual cumulative incidence of 130,000 patients in the United States which translates into an estimated prevalence of 6.2 per 100,000 cases (Ma et al., 2008; Mesa et al., 1999 & Ania B et al., 1994). The major clinical features of PV include uncontrolled growth of multiple myeloid-derived lineages with the dominating lineage being erythrocytes which is often reflected by an increased hematocrit and splenomegaly, while minor features include trilineage myeloproliferation of the bone marrow (Wadleigh & Tefferi, 2010). ET is characterized by an increase in both platelet size and number while hematocrit is unchanged. IMF is the most severe of the three MPNs and the major clinical criteria for diagnosis usually includes megakaryocytic proliferation accompanied by severe fibrosis of the bone marrow or hypercellularity of the bone marrow that is characterized by granulocyte proliferation (Wadleigh & Tefferi, 2010). IMF can manifest on its own or it can be preceded by PV or ET, post-PV or post-ET IMF has a variable evolution rate of roughly 25 evolutions per 1000 patients ET/PV patients (Passamonti et al., 2008 & Gangat et al., 2007). Furthermore, each one of these MPNs has the potential to progress to acute myelogenous leukemia (AML). In an unselected BCR-ABL (-) MPN population the overall leukemic transformation (LT) rate registered at 4.0% with MF having the highest transformation rate: 11.4%-MF, 4.3%-PV & 1.75% for ET (Cervantes et al., 1991). Aberrant tyrosine kinase signaling has been implicated in the pathogenesis in the majority of MPNs, most notably with CML where the product of a chromosomal translocation results in the production of constitutively active tyrosine kinase known as the BCR-ABL (Druker et al., 1996). The discovery of the BCR-ABL tyrosine kinase led to the development of the clinically successful small molecule inhibitor, imatinib, which predominately targets BCR-ABL in addition to PDGF-R and KIT kinases (O'Brien et al., 2003). The initial observation that altered tyrosine kinase signaling could single-handedly drive the entire pathogenesis of a disease as seen with BCR-ABL and CML hinted at the possibility that other altered tyrosine kinase signaling could be involved in the pathogenesis of these BCR-ABL (-) MPNs.

#### **3.2 Pathogenic role of JAK2 in BCR-ABL-negative MPNs**

Mutations in JAK2 have been implicated as a key genetic factor responsible for driving BCR-ABL (-) MPNs and several lines of evidence highlight this finding. The major hematopoietic growth factors erythropoietin (Epo) and thrombopoietin (Tpo) that give rise to the myeloid derived cells that often over-populate PV and ET patients such as erythrocytes and platelets signal exclusively through JAK2. Loss of heterozygosity (LOH) on chromosome 9, where JAK2 is localized is observed in roughly 30% of PV patients (Kralovics et al., 2002), moreover, progenitor cells isolated from PV patients have routinely demonstrated an increased sensitivity to hematopoietic growth factors such as Epo and Tpo in ex vivo colony growth assays. Additionally, multiple animal models involving targeting expression of JAK2 including transgenic and retroviral transduction methods leads to a MPN-like phenotype *in vivo*, further supporting a causal role for JAK2 in the pathogenesis of these diseases (Wernig et al., 2006)

#### **3.3 V617F and other JAK2-associated mutations in MPNs**

152 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

MPNs are hematopoietic malignancies characterized by excessive growth of one or more myeloid derived cell lineages such as erythrocytes, platelets and/or granulocytes. MPNs are clinically grouped into either BCR-ABL (+) or BCR-ABL (-) based on the presence of the BCR-ABL protein, as seen in CML patients. Dysregulated JAK-STAT signaling has been implicated in the pathogenesis of these BCR-ABL (-) MPNs. The three most common BCR-ABL (-) MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) with an estimated annual cumulative incidence of 130,000 patients in the United States which translates into an estimated prevalence of 6.2 per 100,000 cases (Ma et al., 2008; Mesa et al., 1999 & Ania B et al., 1994). The major clinical features of PV include uncontrolled growth of multiple myeloid-derived lineages with the dominating lineage being erythrocytes which is often reflected by an increased hematocrit and splenomegaly, while minor features include trilineage myeloproliferation of the bone marrow (Wadleigh & Tefferi, 2010). ET is characterized by an increase in both platelet size and number while hematocrit is unchanged. IMF is the most severe of the three MPNs and the major clinical criteria for diagnosis usually includes megakaryocytic proliferation accompanied by severe fibrosis of the bone marrow or hypercellularity of the bone marrow that is characterized by granulocyte proliferation (Wadleigh & Tefferi, 2010). IMF can manifest on its own or it can be preceded by PV or ET, post-PV or post-ET IMF has a variable evolution rate of roughly 25 evolutions per 1000 patients ET/PV patients (Passamonti et al., 2008 & Gangat et al., 2007). Furthermore, each one of these MPNs has the potential to progress to acute myelogenous leukemia (AML). In an unselected BCR-ABL (-) MPN population the overall leukemic transformation (LT) rate registered at 4.0% with MF having the highest transformation rate: 11.4%-MF, 4.3%-PV & 1.75% for ET (Cervantes et al., 1991). Aberrant tyrosine kinase signaling has been implicated in the pathogenesis in the majority of MPNs, most notably with CML where the product of a chromosomal translocation results in the production of constitutively active tyrosine kinase known as the BCR-ABL (Druker et al., 1996). The discovery of the BCR-ABL tyrosine kinase led to the development of the clinically successful small molecule inhibitor, imatinib, which predominately targets BCR-ABL in addition to PDGF-R and KIT kinases (O'Brien et al., 2003). The initial observation that altered tyrosine kinase signaling could single-handedly drive the entire pathogenesis of a disease as seen with BCR-ABL and CML hinted at the possibility that other altered tyrosine kinase signaling could be involved in the pathogenesis

**3. Myeloproliferative Neoplasms (MPNs)** 

**3.1 Clinical biology** 

of these BCR-ABL (-) MPNs.

**3.2 Pathogenic role of JAK2 in BCR-ABL-negative MPNs** 

Mutations in JAK2 have been implicated as a key genetic factor responsible for driving BCR-ABL (-) MPNs and several lines of evidence highlight this finding. The major hematopoietic growth factors erythropoietin (Epo) and thrombopoietin (Tpo) that give rise to the myeloid derived cells that often over-populate PV and ET patients such as erythrocytes and platelets signal exclusively through JAK2. Loss of heterozygosity (LOH) on chromosome 9, where JAK2 is localized is observed in roughly 30% of PV patients (Kralovics et al., 2002), moreover, progenitor cells isolated from PV patients have routinely demonstrated an increased sensitivity to hematopoietic growth factors such as Epo and Tpo in ex vivo colony growth assays. Additionally, multiple animal models involving targeting expression of In 2005, several independent laboratories identified a unique somatic activating point mutation in the JAK2 gene (V617F). The JAK2V617F mutation occurs in the JH2 pseudokinase domain of JAK2 in exon 14 at position 617 where a valine is replaced with a phenylalanine which leads to constitutive activation of JAK2. The mechanism of activation of the V617F mutation is believed to be due to the removal of the auto-inhibitory function of the JH2 pseudokinase domain present in JAK2. The incidence of the JAK2V617F mutation is highly prevalent within the BCR-ABL (-) MPN patient population particularly within PV cohorts where more than 95% (Wernig et al., 2006) of PV patients are positive for it while 50% of ET and IMF patients are positive for it (Pikman et al, 2006). Although the JAK2V617F mutation represents a major genetic event underlying the pathogenesis of these MPNs there is still a fraction of PV patients (~5%) and ET/IMF patients (~50%) that present with these MPNs but yet are negative for the V617F JAK2 mutation. The existence of V617F-negative MPNs highlights the need to identify additional JAK2 signaling components that may be involved in the pathogenesis of these V617F-independent MPNs. Several gain-of-function mutations comprised of deletions, insertions and frame-shifts have been indentified in exon 12 of JAK2 in a small subset of V617-negative PV patients (~3%) (Scott et al., 2007). Interestingly, exon 12 JAK2 mutations are mutually exclusive from V617F JAK2 mutations and to date have only been described in V617F-negative PV patients. Expression of exon 12 JAK2 mutations results in a PV-like phenotype *in vivo* that is characterized by erythrocytosis. Alternatively, activating point mutations in the thrombopoietin receptor (Tpo-R/c-MPL) have been identified in a subset of V617F-negative ET and IMF patients (5-10%). The mechanism of action for the MPLW515L mutation is similar to that of the V617F mutation since it also results in the removal of an auto-inhibitory sequence thought to be located within the transmembrane region of the Tpo receptor which causes spontaneous activation of downstream JAK2-STAT signaling (Pikman et al., 2006). The relative frequencies of these JAK-related mutations are summarized in figure 3 (Paradanani et al, 2011). The oncogenic potential of these mutations in MPN is further highlighted by their ability to induce MPNlike phenotypes in murine bone marrow reconstitution studies. JAK2V617F expression induces a PV-like phenotype that presents with erythrocytosis and splenomegaly while (Wernig et al., 2006) MPLW515L promotes a more aggressive MF-like phenotype characterized by marked thrombocytopenia and severe fibrosis of the bone marrow (Pikman et al., 2006). Collectively, these results highlight a strong selective pressure to activate JAK-STAT signaling in BCR-ABL-(-) MPNs and thus therapies designed to target the JAK-STAT signaling may be beneficial for the treatment of MPN.

#### **4. JAK inhibitors in clinical development**

Since the discovery of the JAK2V617F mutation in 2004 remarkable scientific progress has been made from bench to beside with the first JAK inhibitors being tested in humans by mid 2007. Currently there are 36 clinical trials in the US underway evaluating various treatments for MPNs or investigating the molecular pathobiology underlying MPNs. The current JAK inhibitors are typically small molecule ATP competitive inhibitors of both wild-type and

Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs): Rationale

Table 1. Current JAK Inhibitors in the Clinic

signaling, these are summarized in table 2.

**cancers and solid tumors** 

for Targeting the JAK-STAT Pathway in Other Hematological Malignancies and Solid Tumors 155

**Compound Company Indication Phase NCT Trial #**  INCB18424 Incyte PV/ET/MF I/II/III NCT01445769 TG101348 TargeGen MF I/II NCT00631462 SB1518 S\*BIO PV/ET/MF I/II NCT00745550 CYT387 YM BIOSCIENCES MF I/II NCT00935987 AZD1480 Astra Zeneca MF I/II NCT00910728 LY2784544 Eli Lilly PV/ET/MF I NCT01134120 BMS-911543 Bristol Myers-Squibb MF I NCT01236352

**5. Implications for targeting JAK-STAT signaling in additional hematological** 

Activating mutations in JAK2 (V617F and exon 12) in BCR-ABL (-) MPNs appear to be the primary mechanism by which these cells preferentially activate JAK-STAT signaling, however there are additional mechanisms available for cells to activate JAKs. Cells can also undergo chromosomal rearrangements which also lead to constitutive activation of JAKs. Chromosomal translocations activate JAKs by causing constitutive dimerization through replacement of amino terminal sequences with a fusion partner. In non-MPN hematological malignancies such as acute leukemias and lymphomas chromosomal rearrangements involving JAK2 represent another mechanism for activating JAK2. There have been seven fusion partners identified for JAK2, most of which lead to constitutive activation of JAK2

**Fusion Partner Cancer Oncogenic Reference**  TEL (ETV6) T-ALL, aCML,B-ALL Yes Lacronique, 1997 PCM1 ALL, aCML Yes Reiter, 2005 BCR aCML Yes Greisinger, 2005 SSBP2 B-ALL Yes Poitras, 2008 STRN3 ALL Yes Mullighan, 2009 PAX5 ALL No Bousquet, 2007 SEC31-A cHL Yes VanRoosbroeck, 2011 Table 2. **Summary of JAK2 fusion partners due to chromosomal translocations involving chromosome 9p24**. Abbreviations include-: T-ALL = T-cell acute lymphoblastic leukemia, aCML= atypical chronic myelogenous leukemia, B-ALL = B-cell acute lymphoblastic leukemia, ALL = acute lymphoblastic leukemia and cHL = classic Hodgkin lymphoma.

**5.1 Role of JAK-STAT signaling in non-MPN hematological malignancies** 

Evidence is accumulating indicating dysregulated JAK-STAT signaling in various types of lymphomas. In classical Hodgkin lymphoma (cHL) and primary mediastinal B cell lymphoma (PMBL) several different mechanisms appear to be involved in the preferential activation of the JAK-STAT signaling network. An estimated 30% cHL patients and 35%- 45% of PMBL patients (Joos et al., 2003, 2000; Meier et al.,2009) present with genetic gains in chromosome band 9p24, the region where JAK2 is localized. Recently, several tumorgenic functions have been associated with this amplicon including JAK2-mediated increases in

Fig. 3. **Allele Frequency in IMF Cohort,** adapted from Chaligne R, et al. *Leukemia* (2008). Relative mutation frequency in a IMF cohort (n =603), V617F; 58%, MPLW515L; 8.1% and MPLW515L/V617F <1%

mutant JAK enzymes that can be either selective JAK2 inhibitors or non-selective JAK2 inhibitors with the latter having additional activity against other kinases besides JAK2. Initial clinical trials with JAK inhibitors were designed to recruit only IMF patients based on the biological severity of IMF compared to ET or PV, however more recently, clinical trials have expanded to include testing of JAK inhibitors in both PV and ET cohorts. The most advanced JAK inhibitor in the clinic to date is ruxolitinib (INCB018424), a pan-JAK inhibitor which demonstrates potent activity against JAK2 and JAK1 (Quintas-Cardama et al., 2010). A recent phase I/II trial (n=153) has commenced evaluating ruxolitinib for the treatment of IMF. While on ruxolitinib, a significant number of patients demonstrated a favorable clinical response including a reduction in spleen size, improvement in constitutional symptoms and normalization of pro-inflammatory cytokine levels. The suppression of inflammatory cytokines is thought to be linked to the pan inhibitory effects of ruxolitinib on both JAK1 and JAK2 (Verstovsek et al., 2010). Given the clinical response of ruxolitinib in the treatment of IMF, trials have begun to evaluate the safety and efficacy of ruxolitinib in the treatment of PV and ET. Interestingly, these clinical responses did not correlate with JAK2V617F status and molecular remission of the mutant allele was absent even after two years of continued therapy (Verstovsek et al., 2008). The lack of effect on mutant allele burden could be due to direct loss of the mutant allele itself also known as "V617F-independence" that is reported to occur in some patients throughout the evolution of MPN (Passamonti et al., 2010). TG101348 is small molecule inhibitor of JAK 2 in clinical development. Results from a phase I/II multicenter study in IMF patients revealed that >45% of patients on TG101348 observed a >50% reduction in spleen size which was evident by five months but could be detected early two months and more than 70% of patients that presented with elevated white blood cell counts prior to treatment normalized (Pardanani et al., 2009). Unlike ruxolinitib, there was no significant decrease in serum levels of inflammatory cytokines while on therapy although the majority of patients did report a resolution of IMF-associated constitutional symptoms.


Table 1. Current JAK Inhibitors in the Clinic

154 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

 **V617F +** 

Fig. 3. **Allele Frequency in IMF Cohort,** adapted from Chaligne R, et al. *Leukemia* (2008). Relative mutation frequency in a IMF cohort (n =603), V617F; 58%, MPLW515L; 8.1% and

mutant JAK enzymes that can be either selective JAK2 inhibitors or non-selective JAK2 inhibitors with the latter having additional activity against other kinases besides JAK2. Initial clinical trials with JAK inhibitors were designed to recruit only IMF patients based on the biological severity of IMF compared to ET or PV, however more recently, clinical trials have expanded to include testing of JAK inhibitors in both PV and ET cohorts. The most advanced JAK inhibitor in the clinic to date is ruxolitinib (INCB018424), a pan-JAK inhibitor which demonstrates potent activity against JAK2 and JAK1 (Quintas-Cardama et al., 2010). A recent phase I/II trial (n=153) has commenced evaluating ruxolitinib for the treatment of IMF. While on ruxolitinib, a significant number of patients demonstrated a favorable clinical response including a reduction in spleen size, improvement in constitutional symptoms and normalization of pro-inflammatory cytokine levels. The suppression of inflammatory cytokines is thought to be linked to the pan inhibitory effects of ruxolitinib on both JAK1 and JAK2 (Verstovsek et al., 2010). Given the clinical response of ruxolitinib in the treatment of IMF, trials have begun to evaluate the safety and efficacy of ruxolitinib in the treatment of PV and ET. Interestingly, these clinical responses did not correlate with JAK2V617F status and molecular remission of the mutant allele was absent even after two years of continued therapy (Verstovsek et al., 2008). The lack of effect on mutant allele burden could be due to direct loss of the mutant allele itself also known as "V617F-independence" that is reported to occur in some patients throughout the evolution of MPN (Passamonti et al., 2010). TG101348 is small molecule inhibitor of JAK 2 in clinical development. Results from a phase I/II multicenter study in IMF patients revealed that >45% of patients on TG101348 observed a >50% reduction in spleen size which was evident by five months but could be detected early two months and more than 70% of patients that presented with elevated white blood cell counts prior to treatment normalized (Pardanani et al., 2009). Unlike ruxolinitib, there was no significant decrease in serum levels of inflammatory cytokines while on therapy although the majority of patients did report a resolution of IMF-associated constitutional

MPLW515L/V617F <1%

 **MPLW515L+V617F+** 

 **MPLW515L +** 

**?** 

symptoms.

#### **5. Implications for targeting JAK-STAT signaling in additional hematological cancers and solid tumors**

Activating mutations in JAK2 (V617F and exon 12) in BCR-ABL (-) MPNs appear to be the primary mechanism by which these cells preferentially activate JAK-STAT signaling, however there are additional mechanisms available for cells to activate JAKs. Cells can also undergo chromosomal rearrangements which also lead to constitutive activation of JAKs. Chromosomal translocations activate JAKs by causing constitutive dimerization through replacement of amino terminal sequences with a fusion partner. In non-MPN hematological malignancies such as acute leukemias and lymphomas chromosomal rearrangements involving JAK2 represent another mechanism for activating JAK2. There have been seven fusion partners identified for JAK2, most of which lead to constitutive activation of JAK2 signaling, these are summarized in table 2.


Table 2. **Summary of JAK2 fusion partners due to chromosomal translocations involving chromosome 9p24**. Abbreviations include-: T-ALL = T-cell acute lymphoblastic leukemia, aCML= atypical chronic myelogenous leukemia, B-ALL = B-cell acute lymphoblastic leukemia, ALL = acute lymphoblastic leukemia and cHL = classic Hodgkin lymphoma.

#### **5.1 Role of JAK-STAT signaling in non-MPN hematological malignancies**

Evidence is accumulating indicating dysregulated JAK-STAT signaling in various types of lymphomas. In classical Hodgkin lymphoma (cHL) and primary mediastinal B cell lymphoma (PMBL) several different mechanisms appear to be involved in the preferential activation of the JAK-STAT signaling network. An estimated 30% cHL patients and 35%- 45% of PMBL patients (Joos et al., 2003, 2000; Meier et al.,2009) present with genetic gains in chromosome band 9p24, the region where JAK2 is localized. Recently, several tumorgenic functions have been associated with this amplicon including JAK2-mediated increases in

Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs): Rationale

**5.3 Implications for abnormal JAK-STAT signaling in solid tumors** 

of MM.

**6. Conclusion** 

inhibitors.

for Targeting the JAK-STAT Pathway in Other Hematological Malignancies and Solid Tumors 157

available for MM, in tumor models of myeloma. In summary, JAK inhibition may not be sufficient as a mono-therapy for the treatment of MM but studies that combine JAK inhibitors with the current treatments available for MM like bortezomib may prove beneficial for MM patients. Of note, the pan-JAK inhibitor, INCB18424, currently being tested for the treatment of MPN is also being evaluated in a phase I study for the treatment

Dysregulated JAK-STAT signaling has also been implicated in the pathogenesis of solid tumors primarily due to elevated levels of constitutively activated STATs. Among the STATs, STAT3 and STAT5 are the most studied and well characterized regarding their oncogenic potential. Activated STATs possess multiple oncogenic traits which promote tumorgenesis including tumor cell proliferation, induction of anti-apoptotic and immune cell responses. A defined role has been established for STAT5 in driving some hematological malignancies, including some MPNs where STAT5 expression leads to a MPN-like phenotype, while STAT3 appears to be more involved in solid tumor progression (Germain, 2007). In solid tumors, increased STAT activation is thought to be due to sustained cytokine stimulation via autocrine/paracrine signaling loops rather than increased activity in upstream activators of STATs such as JAKs. Elevated levels of activated STAT3 are observed in multiple solid tumors including breast, prostate, colon, pancreatic, head and neck and ovarian cancers (Song et al, 2006 & Frank., 2003) Recently, an extensive profiling study was done that identified a subset of solid tumor cell lines that expressed high levels of constitutive and IL-6 inducible activated STAT3 and when these cells were treated with specific JAK inhibitors both constitutive and inducible STAT3 activation was suppressed (Hedvat et al., 2009). In a panel of pancreatic cell lines, elevated activated STAT3 levels were found to correlate with gp130 expression, an IL-6 receptor, reinforcing the idea that STAT activation in solid tumors is related to sustained cytokine stimulation (Corcoran et al., 2011)

Emerging clinical data on a variety of JAK2 inhibitors support a role of these agents in the symptomatic relief of MPN patients. However, there are still unresolved issues associated with these inhibitors including the inability of most inhibitors to reduce the amount of mutant allele burden. The numerous mechanisms that blood-derived cancer cells have acquired to preferentially activate JAK-STAT signaling (point mutations, translocations & chromosomal gains) demonstrates the importance of this pathway in promoting hematological malignancies. The use of JAK inhibitors for the treatment of other non-MPN hematological cancers such as lymphomas and multiple myelomas are intriguing but require additional pre-clinical studies to determine which JAKs underlie these findings. In this regard, lymphomas with a high frequency of the 9p24 gain (30%) coupled with the recent identification of the novel JAK2-SEC31-A fusion protein implicates a preference for these cells to also activate JAK-STAT signaling. In multiple myeloma, a reliance of these cells on IL-6 for their survival and growth suggests that these patients could benefit from JAK inhibition therapies. For the role of JAK2-STAT signaling in solid tumors, it remains unclear of the potential of pathway inhibition but is likely to require rational combination strategies with other agents in clinical development to unmask the full therapeutic potential of JAK

programmed cell death 1 ligand 1 (PD-L1) expression. PD-L1 is also localized to band 9p24 and therefore cells positive for this genetic gain can also demonstrate increased PD-L1 expression in addition to elevated JAK 2 content. PD-L1 inhibits antitumor cytotoxic T lymphocyte (CTL) responses by activating its cognate inhibitor PD-1 receptor located on Tcells, therefore PD-L1-/PD-1 interactions can promote tumorgenesis by mediating tumor evasiveness. A recent study showed elevated expression of PD-L1 and JAK2 in a subset of cHL and PMBL cell lines that were positive for the 9p24 amplicon and went on to show that PD-L1 expression could be augmented upon JAK2 inhibition suggesting a regulatory role for JAK2 in PD-L1 expression (Green et al., 2010 & Rui et al., 2010). In another study, alternative oncogenic functions were implicated for the 9p24 amplicon based on STATindependent epigenetic functions of JAK2. In this study, JAK2 was shown to cooperate with the epigenetic modifier JMJDC2 in several lymphoma cell lines positive for the 9p24 gain to induce epigenetic remodeling of the oncogene MYC locus leading to altered expression of MYC (Rui et al., 2010). The cooperative interplay between JAK2 and JMJDC2 was further demonstrated when the anti-proliferative effects induced by loss of JMJD2C were exaggerated upon JAK inhibition. In addition, inactivating mutations in SOCS-1, a negative regulator of JAK-STAT signaling are observed in 40% of cHL patients (Weniger et al., 2006 & Mottok et al., 2007). Increased expression of activated STATs, namely STAT 3 and STAT6 are often observed in cHL patients and this is thought to be due to sustained signaling loops perpetuated by chronic IL-13 stimulation (Skinnider et al., 2002) furthermore, loss of STAT3 or STAT6 in cHL cell lines results in diminished proliferative capacity and induction of apoptosis (Baus, 2009 & Kube et al., 2005). To further validate a role for abnormal JAK-STAT signaling in lymphoma-type cancers is the more recent study in which an inverse relationship between the inhibitory microRNA-135a and JAK2 was observed in cHL patients. Interestingly, low levels of miR-135a expression were strongly correlated with disease relapse and shorter disease-free survival in a cohort of cHL patients. The regulatory role of miR-135a on JAK2 was confirmed when over- expression of miR-135a led to an increase in JAK2 expression (25%-55%) in lymphoma cell lines (Navarro et al., 2009).

#### **5.2 Role of dysregulated JAK-STAT signaling in multiple myeloma**

Altered JAK-STAT signaling has also been implicated in the progression of multiple myeloma (MM). MM is an aggressive hematological malignancy characterized by excessive proliferation of clonal plasma B-cells that accumulate in the bone marrow (Anderson et al., 1999). The maintenance of MM is highly dependent on the interaction of myeloma cells with resident bone marrow stromal cells (BMSC) both of which secrete various cytokines and growth factors that promote myeloma cell growth. IL-6 is recognized as a critical cytokine that's essential for the survival and proliferation of myeloma cells (Bommert et al., 2006). Elevated serum levels of IL-6 are frequently observed in MM patients who fail to respond to conventional chemotherapies and IL-6 levels also correlate with poor prognosis of MM (Niesvizky et al., 1995). IL-6 signals through JAK1/JAK2/TYK2 leading to downstream activation of STAT3 to promote proliferation and survival of myeloma cells (Murray et al., 2007). Interestingly, elevated levels of activated STAT3 have been observed in more than 50% of myeloma patient samples (Bharti AC et al., 2004). Pre-clinical studies have shown that JAK inhibition can induce apoptosis, inhibit proliferation and block constitutive and IL-6 induced activation of STAT3 in several MM cell-based models. Furthermore, JAK inhibition also enhanced the anti-tumorgenic effects of bortezomib, a current therapy available for MM, in tumor models of myeloma. In summary, JAK inhibition may not be sufficient as a mono-therapy for the treatment of MM but studies that combine JAK inhibitors with the current treatments available for MM like bortezomib may prove beneficial for MM patients. Of note, the pan-JAK inhibitor, INCB18424, currently being tested for the treatment of MPN is also being evaluated in a phase I study for the treatment of MM.

#### **5.3 Implications for abnormal JAK-STAT signaling in solid tumors**

Dysregulated JAK-STAT signaling has also been implicated in the pathogenesis of solid tumors primarily due to elevated levels of constitutively activated STATs. Among the STATs, STAT3 and STAT5 are the most studied and well characterized regarding their oncogenic potential. Activated STATs possess multiple oncogenic traits which promote tumorgenesis including tumor cell proliferation, induction of anti-apoptotic and immune cell responses. A defined role has been established for STAT5 in driving some hematological malignancies, including some MPNs where STAT5 expression leads to a MPN-like phenotype, while STAT3 appears to be more involved in solid tumor progression (Germain, 2007). In solid tumors, increased STAT activation is thought to be due to sustained cytokine stimulation via autocrine/paracrine signaling loops rather than increased activity in upstream activators of STATs such as JAKs. Elevated levels of activated STAT3 are observed in multiple solid tumors including breast, prostate, colon, pancreatic, head and neck and ovarian cancers (Song et al, 2006 & Frank., 2003) Recently, an extensive profiling study was done that identified a subset of solid tumor cell lines that expressed high levels of constitutive and IL-6 inducible activated STAT3 and when these cells were treated with specific JAK inhibitors both constitutive and inducible STAT3 activation was suppressed (Hedvat et al., 2009). In a panel of pancreatic cell lines, elevated activated STAT3 levels were found to correlate with gp130 expression, an IL-6 receptor, reinforcing the idea that STAT activation in solid tumors is related to sustained cytokine stimulation (Corcoran et al., 2011)

#### **6. Conclusion**

156 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

programmed cell death 1 ligand 1 (PD-L1) expression. PD-L1 is also localized to band 9p24 and therefore cells positive for this genetic gain can also demonstrate increased PD-L1 expression in addition to elevated JAK 2 content. PD-L1 inhibits antitumor cytotoxic T lymphocyte (CTL) responses by activating its cognate inhibitor PD-1 receptor located on Tcells, therefore PD-L1-/PD-1 interactions can promote tumorgenesis by mediating tumor evasiveness. A recent study showed elevated expression of PD-L1 and JAK2 in a subset of cHL and PMBL cell lines that were positive for the 9p24 amplicon and went on to show that PD-L1 expression could be augmented upon JAK2 inhibition suggesting a regulatory role for JAK2 in PD-L1 expression (Green et al., 2010 & Rui et al., 2010). In another study, alternative oncogenic functions were implicated for the 9p24 amplicon based on STATindependent epigenetic functions of JAK2. In this study, JAK2 was shown to cooperate with the epigenetic modifier JMJDC2 in several lymphoma cell lines positive for the 9p24 gain to induce epigenetic remodeling of the oncogene MYC locus leading to altered expression of MYC (Rui et al., 2010). The cooperative interplay between JAK2 and JMJDC2 was further demonstrated when the anti-proliferative effects induced by loss of JMJD2C were exaggerated upon JAK inhibition. In addition, inactivating mutations in SOCS-1, a negative regulator of JAK-STAT signaling are observed in 40% of cHL patients (Weniger et al., 2006 & Mottok et al., 2007). Increased expression of activated STATs, namely STAT 3 and STAT6 are often observed in cHL patients and this is thought to be due to sustained signaling loops perpetuated by chronic IL-13 stimulation (Skinnider et al., 2002) furthermore, loss of STAT3 or STAT6 in cHL cell lines results in diminished proliferative capacity and induction of apoptosis (Baus, 2009 & Kube et al., 2005). To further validate a role for abnormal JAK-STAT signaling in lymphoma-type cancers is the more recent study in which an inverse relationship between the inhibitory microRNA-135a and JAK2 was observed in cHL patients. Interestingly, low levels of miR-135a expression were strongly correlated with disease relapse and shorter disease-free survival in a cohort of cHL patients. The regulatory role of miR-135a on JAK2 was confirmed when over- expression of miR-135a led to an

increase in JAK2 expression (25%-55%) in lymphoma cell lines (Navarro et al., 2009).

Altered JAK-STAT signaling has also been implicated in the progression of multiple myeloma (MM). MM is an aggressive hematological malignancy characterized by excessive proliferation of clonal plasma B-cells that accumulate in the bone marrow (Anderson et al., 1999). The maintenance of MM is highly dependent on the interaction of myeloma cells with resident bone marrow stromal cells (BMSC) both of which secrete various cytokines and growth factors that promote myeloma cell growth. IL-6 is recognized as a critical cytokine that's essential for the survival and proliferation of myeloma cells (Bommert et al., 2006). Elevated serum levels of IL-6 are frequently observed in MM patients who fail to respond to conventional chemotherapies and IL-6 levels also correlate with poor prognosis of MM (Niesvizky et al., 1995). IL-6 signals through JAK1/JAK2/TYK2 leading to downstream activation of STAT3 to promote proliferation and survival of myeloma cells (Murray et al., 2007). Interestingly, elevated levels of activated STAT3 have been observed in more than 50% of myeloma patient samples (Bharti AC et al., 2004). Pre-clinical studies have shown that JAK inhibition can induce apoptosis, inhibit proliferation and block constitutive and IL-6 induced activation of STAT3 in several MM cell-based models. Furthermore, JAK inhibition also enhanced the anti-tumorgenic effects of bortezomib, a current therapy

**5.2 Role of dysregulated JAK-STAT signaling in multiple myeloma** 

Emerging clinical data on a variety of JAK2 inhibitors support a role of these agents in the symptomatic relief of MPN patients. However, there are still unresolved issues associated with these inhibitors including the inability of most inhibitors to reduce the amount of mutant allele burden. The numerous mechanisms that blood-derived cancer cells have acquired to preferentially activate JAK-STAT signaling (point mutations, translocations & chromosomal gains) demonstrates the importance of this pathway in promoting hematological malignancies. The use of JAK inhibitors for the treatment of other non-MPN hematological cancers such as lymphomas and multiple myelomas are intriguing but require additional pre-clinical studies to determine which JAKs underlie these findings. In this regard, lymphomas with a high frequency of the 9p24 gain (30%) coupled with the recent identification of the novel JAK2-SEC31-A fusion protein implicates a preference for these cells to also activate JAK-STAT signaling. In multiple myeloma, a reliance of these cells on IL-6 for their survival and growth suggests that these patients could benefit from JAK inhibition therapies. For the role of JAK2-STAT signaling in solid tumors, it remains unclear of the potential of pathway inhibition but is likely to require rational combination strategies with other agents in clinical development to unmask the full therapeutic potential of JAK inhibitors.

Role of JAK2 Beyond Myeloproliferative Neoplasms (MPNs): Rationale

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Passamonti, F., Rumi, E & Arcaini, L et al. (2005). Leukemic transformation of polycytehemia vera: a single center study of 23 patients. Cancer. 104(5):1032-1036. Passamonti, F., Rumi, E & Caramella, M et al (2008). A dynamic prognostic marker to predict survival in post-cytothemia vera myelofibrosis. Blood. 111(7):3383-3387. Patnaik, M & Tefferi A. (2009). Molecular diagnosis of myeloproliferative neoplasms. Expert

Peeters, A., Raynaud S., Cools, J et al. (1997) Fusion of TEL, the ETS-variant gene 6 (ETV6),

Pikman Y., Lee, b & Mercher, T et al. (2006). MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 3:1140-1151. Poitras, J., Dal Cin P & Aster, J et al. (2008). Novel SSBP2-JAK2 fusion gene resulting from a

Quintas-Cardama, A., Vaddi K & Liu, P et al. (2010). Pre-clinical characterization of a

Radtke, S., Haan, S & Jorissen, A et al. (2005). The Jak1 SH2 domain does not fulfill a

Reiter, A., Walz, C & Watmore, A et al. (2005) The t(8;9)(p22;p24) is recurrent abnormality in

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Rui, L., Emre, T & Kruhlak M et al. (2010). Cooperative epigenetic modulation by cancer

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selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment

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JAK2 in polycythemia vera, essential thrombocythemia and myeloid metaplasia


**9** 

*Taiwan* 

**Genetic Alterations and Their Clinical** 

Hsin-An Hou,Wen-Chien Chou and Hwei-Fang Tien

*Hospital, College of Medicine, National Taiwan University, Taipei,* 

**Implications in Acute Myeloid Leukemia** 

*Division of Hematology, Department of Internal Medicine, National Taiwan University* 

Acute myeloid leukemia (AML) is a hematologic malignancy with great variability in the pathogenesis, clinical features, and treatment outcomes. Advances in molecular research have greatly improved our understanding of the leukemogenesis in AML. A two-hit model proposes that the development of AML requires the cooperation between at least two classes of gene mutations.(Frohling*, et al* 2005, Gilliland 2002) Class I mutations, such as *RAS, FLT3, KIT, PTPN11* and *JAK2* mutations, involve genes in the kinase signaling pathways leading to cell survival and proliferation and Class II mutations, such as t(15;17)/*PML-RARA*, inv(16)/*CBFB-MYH11* and t(8;21)/*RUNX1-RUNX1T1* fusions, and *MLL/*PTD*,* and *CEBPA* and *AML1/RUNX1* mutations, involve transcription factors or cofactors resulting in impaired hematopoietic differentiation. In addition to genetic abnormalities, increasing evidences show that epigenetic deregulations are also critical to the pathogenesis of AML.(Chen*, et al* 2010) Compatible with these findings, several novel mutations involving genes related to epigenetic modifications, such as *isocitrate dehydrogenase 1* (*IDH1*), *IDH2*, *ten-eleven translocation* 2 (*TET2)*, *additional sex comb-like 1* (*ASXL1)*, and DNA methyltransferase 3A *(DNMT3A)* were detected in AML recently.(Chou*, et al* 2010b, Delhommeau*, et al* 2009, Gelsi-Boyer*, et al* 2009, Ley*, et al* 2010, Mardis*, et al* 2009,

Risk-adapted treatment may not only improve the prognosis, but also reduce the toxicity from the therapy in patients with AML. In addition to the conventional risk factors, such as age, white blood cell (WBC) counts and cytogenetics, molecular genetic alterations, such as mutations of *NPM1, CEBPA, AML1/RUNX1, WT1, FLT3, TET2*, and *DNMT3A* etc., are also important prognostic factors in AML patients. Furthermore, the gene mutations which are stable during treatment courses can also be used as biomarkers to monitor minimal residual disease (MRD). Herein, we will review the gene mutations in AML and discuss their clinical

FMS-like tyrosine kinase 3 (FLT3), mapped at 13q12, encodes a receptor tyrosine kinase.(Kiyoi*, et al* 1998) *FLT3*-internal tandem duplication (*FLT3*-ITD) mutation, one of the

**2. Class I mutations that lead to cell survival and proliferation** 

**1. Introduction** 

Metzeler*, et al* 2011)

implications.

**2.1** *FLT3* **mutations** 


### **Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia**

Hsin-An Hou,Wen-Chien Chou and Hwei-Fang Tien *Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan* 

#### **1. Introduction**

162 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Scott, L., Tong, W & Levine R et al. (2007). JAK2 exon 12 mutations in PV and idiopathic

Skinnider B., Elia, A & Gascoyne L et al. (2002) Signal transducer an activator of

Song, L., Bhattacharya S & Yunus A et al. (2006). Stat1 and SUMO modification. Blood.

Valentino, L & Pierre J. (2006). JAK/STAT signal transduction: regulators and implication in hematological malignancies. Biochemical Pharmacology. 71(6): 713-721. Van Roosbroeck, K., Cox, L & Tousseyn T et al. (2011). JAK2 rearrangements, including the

Verstovsek, S., Kantajian, H & Pardanani, A et al. (2008) Characterization of allele

Verstovsek, S., Kantarjian, H & Mesa, R et al. (2010). Safety and Efficacy of INCB018424, a

Wadleigh, M & Tefferi A. (2010). Classification and diagnosis of myeloproliferation

Ward, A., Touw, I & Yoshimura, A. (2000). The JAK-Stat pathway in normal and perturbed

Weniger, M., Melzner, C & Menz S et al. (2006). Mutations of the suppressor gene SOCS-1 in

Wernig G., Mercher, T & Okabe R et al. (2006). Expression of JAKV617F causes a

transcription 6 is frequently in Hodgkin and Reed-Sternberg cells of Hodgkin

novel SEC31-A-JAK2 fusion, are recurrent in classical Hodgkin lymphoma. Blood.

JAK2V617F allele burden in advanced myelofibrosis patients: no change in V617F:WT JAK2 ratio in patients with high allele burdens despite profound clinical improvement following treatment with the JAK inhibitor. Blood (Abstract) 112:

JAK1 and JAK2 inhibitor in myelofibrosis. New England Journal of Medicine. 363:

neoplasms according to the 2008 World Health Organization criteria. International

classical Hodgkin lymphoma are frequent and associated with nuclear phospho-

polycytothemia vera–like disease associated with myelofibrosis in a murine bone

erythrocytosis. N Engl. J Med, 356(5): 459-468.

lymphoma. Blood 99(2): 618-626.

Journal of Hematology. 91:174-179.

hematopoiesis. Blood. 95(1):19-29

STAT5 accumulation. Oncogene. (25):2679-2684.

marrow transplant model. Blood. 107(11):4274-4281.

108(10) 3237:3244.

117(15):4056-4064.

2802.

1117-1127.

Acute myeloid leukemia (AML) is a hematologic malignancy with great variability in the pathogenesis, clinical features, and treatment outcomes. Advances in molecular research have greatly improved our understanding of the leukemogenesis in AML. A two-hit model proposes that the development of AML requires the cooperation between at least two classes of gene mutations.(Frohling*, et al* 2005, Gilliland 2002) Class I mutations, such as *RAS, FLT3, KIT, PTPN11* and *JAK2* mutations, involve genes in the kinase signaling pathways leading to cell survival and proliferation and Class II mutations, such as t(15;17)/*PML-RARA*, inv(16)/*CBFB-MYH11* and t(8;21)/*RUNX1-RUNX1T1* fusions, and *MLL/*PTD*,* and *CEBPA* and *AML1/RUNX1* mutations, involve transcription factors or cofactors resulting in impaired hematopoietic differentiation. In addition to genetic abnormalities, increasing evidences show that epigenetic deregulations are also critical to the pathogenesis of AML.(Chen*, et al* 2010) Compatible with these findings, several novel mutations involving genes related to epigenetic modifications, such as *isocitrate dehydrogenase 1* (*IDH1*), *IDH2*, *ten-eleven translocation* 2 (*TET2)*, *additional sex comb-like 1* (*ASXL1)*, and DNA methyltransferase 3A *(DNMT3A)* were detected in AML recently.(Chou*, et al* 2010b, Delhommeau*, et al* 2009, Gelsi-Boyer*, et al* 2009, Ley*, et al* 2010, Mardis*, et al* 2009, Metzeler*, et al* 2011)

Risk-adapted treatment may not only improve the prognosis, but also reduce the toxicity from the therapy in patients with AML. In addition to the conventional risk factors, such as age, white blood cell (WBC) counts and cytogenetics, molecular genetic alterations, such as mutations of *NPM1, CEBPA, AML1/RUNX1, WT1, FLT3, TET2*, and *DNMT3A* etc., are also important prognostic factors in AML patients. Furthermore, the gene mutations which are stable during treatment courses can also be used as biomarkers to monitor minimal residual disease (MRD). Herein, we will review the gene mutations in AML and discuss their clinical implications.

#### **2. Class I mutations that lead to cell survival and proliferation**

#### **2.1** *FLT3* **mutations**

FMS-like tyrosine kinase 3 (FLT3), mapped at 13q12, encodes a receptor tyrosine kinase.(Kiyoi*, et al* 1998) *FLT3*-internal tandem duplication (*FLT3*-ITD) mutation, one of the

Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia 165

Hunter 2001, Bowen*, et al* 2005, Radich*, et al* 1990) High expression of *KIT* (CD117) is a common finding in AML,(Ikeda*, et al* 1991, Reuss-Borst*, et al* 1994) and activation mutations of *KIT*, most commonly affecting exons 8 and 17 can be identified in 20–45% of AML with inv (16) and 12.8–46.8% of AML with t(8;21), but infrequently found in other AML types.(Beghini*, et al* 2000, Beghini*, et al* 2004, Care*, et al* 2003) Most, though not all, studies,(Boissel*, et al* 2006, Care*, et al* 2003, Schnittger*, et al* 2006) showed *KIT* mutation was associated with inferior outcome in the core binding factor (CBF) AML, especially *KIT*-D816

JAK2 is a nonreceptor tyrosine kinase. The *JAK2* V617F mutation induces the activation of JAK2-STAT5 signal transduction pathway and then substantially alters the proliferation and self-renewal of hematopoietic precursors.(Liu*, et al* 1999, Walz*, et al* 2006) Although the *JAK2* V617F mutation is a common genetic event in the patients with myeloproliferative neoplasms (MPN),(Baxter*, et al* 2005, Goldman 2005, Kralovics*, et al* 2005, Levine*, et al* 2005b) it is seldom found (<1%-2%) in *de novo* AML patients.(Frohling*, et al* 2006, Illmer*, et al* 2007, Lee*, et al* 2006, Levine*, et al* 2005a) Illmer et al showed 3.6% of patients with CBF AML had *JAK2* V617F mutation and these patients had an aggressive clinical course and poor

SHP-2 is encoded by *PTPN11* which is located on chromosome 12q24. The protein is a nonreceptor tyrosine phosphatase participating in intracellular signaling elicited by a number of growth factors, cytokines, hormones and adhesion molecules.(Neel*, et al* 2003, Tartaglia*, et al* 2004) Germline *PTPN11* mutations were first reported by Tartaglia *et al* in patients afflicted with Noonan syndrome.(Tartaglia*, et al* 2002, Tartaglia*, et al* 2001) Subsequently, somatic *PTPN11* mutations were also found in patients with juvenile myelomonocytic leukemia, and myelodysplastic syndrome (MDS).(Chen*, et al* 2006, Loh*, et al* 2004b, Tartaglia*, et al* 2003) The *PTPN11* mutation is not a frequent molecular event (4-5%) in AML.(Hou*, et al* 2008, Loh*, et al* 2004a, Tartaglia*, et al* 2005) In a study of 272 primary AML patients, we found this gene mutation was closely associated with older age, French-American-British (FAB) M4/M5 subtype, CD14 expression, normal karyotype and *NPM1* mutation.(Hou*, et al* 2008) Loh *et al* and Tartaglia *et al* revealed that the *PTPN11* mutation had no prognostic implication for pediatric patients with AML;(Loh*, et al* 2004a, Tartaglia*, et al* 2005) however, this mutation may be a poor-risk factor for OS in adult AML patients without *NPM1* mutations.(Hou*, et al*

**3. Class II mutations that impair hematopoietic differentiation** 

CCAAT/enhancer binding protein (C/EBP) is a 42-kDa transcription factor that possesses a DNA-binding basic leucine zipper domain (bZIP) in the COOH terminus and two transactivation domains TAD 1 and TAD 2 in the NH2 terminus.(Friedman and McKnight 1990) As a transcription factor, it plays a crucial role in granulocytic differentiation and diminished C/EBP activity contributes to myeloid progenitor transformation.(Cammenga*, et al* 2003, Oelgeschlager*, et al* 1996, Smith*, et al* 1996) *CEBPA*

mutations in t(8;21)/*RUNX1-RUNX1T1*–positive AML.

**2.4** *JAK2* **mutations** 

outcome.(Illmer*, et al* 2007)

**2.5** *PTPN 11* **mutations** 

2008)

**3.1** *CEBPA* **mutations** 

most common mutations in AML, was found by Nakao et al in 1996.(Nakao*, et al* 1996) The mutation occurs as a duplication of nucleotide sequences of variable lengths in exons 14 and 15, leading to addition of repeated peptide in the juxtamembrane domain in the cytoplasm. Another activating *FLT3* mutation occurs in tyrosine kinase domain (*FLT3*-TKD), causing point mutations, small deletions or insertions mainly at codon 835 or 836 within the activation loop of the second kinase domain.(Bacher*, et al* 2008, Yamamoto*, et al* 2001) The FLT3 mutant protein constitutively activates the cascade of FLT3 signaling in the absence of FLT3 ligand promoting cell proliferation and decreased apoptosis.

*FLT3*-ITD occurs in about 25% of adult AML and shows association with normal karyotype and *NPM1* mutation. The patients with this mutation have higher WBC counts, shorter disease-free survival (DFS) and overall survival (OS), and increased relapse rate.(Kottaridis*, et al* 2001, Kottaridis*, et al* 2002) While mutant size may not be related to prognosis, higher mutant levels are associated with higher relapse rate and shorter survival.(Gale*, et al* 2008) Absence of *FLT3*-ITD combined with *NPM1* mutation is regarded as a favorable prognostic genotype.(Gale*, et al* 2008, Schlenk*, et al* 2008) Up to one third of AML patients with *FLT3*-ITD can lose the mutation at disease relapse, indicating that this mutation is much less stable than *NPM1* mutation, and is not a good marker for disease monitoring.(Chou*, et al* 2011b, Kottaridis*, et al* 2002, Palmisano*, et al* 2007, Shih*, et al* 2002) *FLT3*-TKD occurred in about 4%- 10% of AML patients.(Yamamoto*, et al* 2001, Bacher*, et al* 2008) AML with this mutation also shows specific clinical and biologic features, such as elevated WBC counts at diagnosis, higher frequency of normal karyotype and mutations in *NPM1*, *CEBPA*, and *NRAS*. However, the prognostic significance is still inconclusive.(Bacher*, et al* 2008, Whitman*, et al* 2008)

#### **2.2** *RAS* **mutations**

The RAS proteins are a large superfamily of low molecular-weight guanine nucleotidebinding proteins, which are activated by cytokine receptors in response to ligand stimulation and therefore control cell proliferation and survival of hematopoietic progenitors.(Downward 2003, Reuther and Der 2000, Shields*, et al* 2000, Wittinghofer 1998) Three members of the RAS family, HRAS, KRAS and NRAS, are found to be activated by mutations in human cancers.(Bos 1989, Downward 2003) Almost all RAS mutations occur by single nucleotide substitutions in codons 12, 13 and 61.(Bos*, et al* 1987, Farr*, et al* 1988, Senn*, et al* 1988, Toksoz*, et al* 1989) *NRAS* and *KRAS* mutations are found in approximately 12-30% and 9-14%, respectively, of AML patients. In a large cohort study of 2502 AML patients, the mutations were found much prevalent in patients with inv(16)/t(16;16) and inv(3)/t(3;3), but seldom found in those with t(15;17) and complex karyotype.(Bacher*, et al* 2006)

The prognostic relevance of *RAS* mutation in AML has not been firmly established. Some studies showed *RAS* mutation predicted poor prognosis(De Melo*, et al* 1997, Kiyoi*, et al* 1999, Meshinchi*, et al* 2003), some showed no impact on clinical outcome,(Bacher*, et al* 2006, Bowen*, et al* 2005, Radich*, et al* 1990, Ritter*, et al* 2004) whereas others found *RAS* mutations were associated with a favorable prognosis.(Coghlan*, et al* 1994, Neubauer*, et al* 1994) However, higher dose of cytarabine may decrease the relapse rate in *RAS*-mutated AML patients.(Neubauer*, et al* 2008)

#### **2.3** *KIT* **mutations**

KIT, a member of type III receptor tyrosine kinase family, is important for the development of hematopoietic progenitor cells and also crucial in leukemogenesis.(Blume-Jensen and Hunter 2001, Bowen*, et al* 2005, Radich*, et al* 1990) High expression of *KIT* (CD117) is a common finding in AML,(Ikeda*, et al* 1991, Reuss-Borst*, et al* 1994) and activation mutations of *KIT*, most commonly affecting exons 8 and 17 can be identified in 20–45% of AML with inv (16) and 12.8–46.8% of AML with t(8;21), but infrequently found in other AML types.(Beghini*, et al* 2000, Beghini*, et al* 2004, Care*, et al* 2003) Most, though not all, studies,(Boissel*, et al* 2006, Care*, et al* 2003, Schnittger*, et al* 2006) showed *KIT* mutation was associated with inferior outcome in the core binding factor (CBF) AML, especially *KIT*-D816 mutations in t(8;21)/*RUNX1-RUNX1T1*–positive AML.

#### **2.4** *JAK2* **mutations**

164 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

most common mutations in AML, was found by Nakao et al in 1996.(Nakao*, et al* 1996) The mutation occurs as a duplication of nucleotide sequences of variable lengths in exons 14 and 15, leading to addition of repeated peptide in the juxtamembrane domain in the cytoplasm. Another activating *FLT3* mutation occurs in tyrosine kinase domain (*FLT3*-TKD), causing point mutations, small deletions or insertions mainly at codon 835 or 836 within the activation loop of the second kinase domain.(Bacher*, et al* 2008, Yamamoto*, et al* 2001) The FLT3 mutant protein constitutively activates the cascade of FLT3 signaling in the absence of

*FLT3*-ITD occurs in about 25% of adult AML and shows association with normal karyotype and *NPM1* mutation. The patients with this mutation have higher WBC counts, shorter disease-free survival (DFS) and overall survival (OS), and increased relapse rate.(Kottaridis*, et al* 2001, Kottaridis*, et al* 2002) While mutant size may not be related to prognosis, higher mutant levels are associated with higher relapse rate and shorter survival.(Gale*, et al* 2008) Absence of *FLT3*-ITD combined with *NPM1* mutation is regarded as a favorable prognostic genotype.(Gale*, et al* 2008, Schlenk*, et al* 2008) Up to one third of AML patients with *FLT3*-ITD can lose the mutation at disease relapse, indicating that this mutation is much less stable than *NPM1* mutation, and is not a good marker for disease monitoring.(Chou*, et al* 2011b, Kottaridis*, et al* 2002, Palmisano*, et al* 2007, Shih*, et al* 2002) *FLT3*-TKD occurred in about 4%- 10% of AML patients.(Yamamoto*, et al* 2001, Bacher*, et al* 2008) AML with this mutation also shows specific clinical and biologic features, such as elevated WBC counts at diagnosis, higher frequency of normal karyotype and mutations in *NPM1*, *CEBPA*, and *NRAS*. However, the

prognostic significance is still inconclusive.(Bacher*, et al* 2008, Whitman*, et al* 2008)

but seldom found in those with t(15;17) and complex karyotype.(Bacher*, et al* 2006)

The prognostic relevance of *RAS* mutation in AML has not been firmly established. Some studies showed *RAS* mutation predicted poor prognosis(De Melo*, et al* 1997, Kiyoi*, et al* 1999, Meshinchi*, et al* 2003), some showed no impact on clinical outcome,(Bacher*, et al* 2006, Bowen*, et al* 2005, Radich*, et al* 1990, Ritter*, et al* 2004) whereas others found *RAS* mutations were associated with a favorable prognosis.(Coghlan*, et al* 1994, Neubauer*, et al* 1994) However, higher dose of cytarabine may decrease the relapse rate in *RAS*-mutated AML

KIT, a member of type III receptor tyrosine kinase family, is important for the development of hematopoietic progenitor cells and also crucial in leukemogenesis.(Blume-Jensen and

The RAS proteins are a large superfamily of low molecular-weight guanine nucleotidebinding proteins, which are activated by cytokine receptors in response to ligand stimulation and therefore control cell proliferation and survival of hematopoietic progenitors.(Downward 2003, Reuther and Der 2000, Shields*, et al* 2000, Wittinghofer 1998) Three members of the RAS family, HRAS, KRAS and NRAS, are found to be activated by mutations in human cancers.(Bos 1989, Downward 2003) Almost all RAS mutations occur by single nucleotide substitutions in codons 12, 13 and 61.(Bos*, et al* 1987, Farr*, et al* 1988, Senn*, et al* 1988, Toksoz*, et al* 1989) *NRAS* and *KRAS* mutations are found in approximately 12-30% and 9-14%, respectively, of AML patients. In a large cohort study of 2502 AML patients, the mutations were found much prevalent in patients with inv(16)/t(16;16) and inv(3)/t(3;3),

**2.2** *RAS* **mutations** 

patients.(Neubauer*, et al* 2008)

**2.3** *KIT* **mutations** 

FLT3 ligand promoting cell proliferation and decreased apoptosis.

JAK2 is a nonreceptor tyrosine kinase. The *JAK2* V617F mutation induces the activation of JAK2-STAT5 signal transduction pathway and then substantially alters the proliferation and self-renewal of hematopoietic precursors.(Liu*, et al* 1999, Walz*, et al* 2006) Although the *JAK2* V617F mutation is a common genetic event in the patients with myeloproliferative neoplasms (MPN),(Baxter*, et al* 2005, Goldman 2005, Kralovics*, et al* 2005, Levine*, et al* 2005b) it is seldom found (<1%-2%) in *de novo* AML patients.(Frohling*, et al* 2006, Illmer*, et al* 2007, Lee*, et al* 2006, Levine*, et al* 2005a) Illmer et al showed 3.6% of patients with CBF AML had *JAK2* V617F mutation and these patients had an aggressive clinical course and poor outcome.(Illmer*, et al* 2007)

#### **2.5** *PTPN 11* **mutations**

SHP-2 is encoded by *PTPN11* which is located on chromosome 12q24. The protein is a nonreceptor tyrosine phosphatase participating in intracellular signaling elicited by a number of growth factors, cytokines, hormones and adhesion molecules.(Neel*, et al* 2003, Tartaglia*, et al* 2004) Germline *PTPN11* mutations were first reported by Tartaglia *et al* in patients afflicted with Noonan syndrome.(Tartaglia*, et al* 2002, Tartaglia*, et al* 2001) Subsequently, somatic *PTPN11* mutations were also found in patients with juvenile myelomonocytic leukemia, and myelodysplastic syndrome (MDS).(Chen*, et al* 2006, Loh*, et al* 2004b, Tartaglia*, et al* 2003) The *PTPN11* mutation is not a frequent molecular event (4-5%) in AML.(Hou*, et al* 2008, Loh*, et al* 2004a, Tartaglia*, et al* 2005) In a study of 272 primary AML patients, we found this gene mutation was closely associated with older age, French-American-British (FAB) M4/M5 subtype, CD14 expression, normal karyotype and *NPM1* mutation.(Hou*, et al* 2008) Loh *et al* and Tartaglia *et al* revealed that the *PTPN11* mutation had no prognostic implication for pediatric patients with AML;(Loh*, et al* 2004a, Tartaglia*, et al* 2005) however, this mutation may be a poor-risk factor for OS in adult AML patients without *NPM1* mutations.(Hou*, et al* 2008)

#### **3. Class II mutations that impair hematopoietic differentiation**

#### **3.1** *CEBPA* **mutations**

CCAAT/enhancer binding protein (C/EBP) is a 42-kDa transcription factor that possesses a DNA-binding basic leucine zipper domain (bZIP) in the COOH terminus and two transactivation domains TAD 1 and TAD 2 in the NH2 terminus.(Friedman and McKnight 1990) As a transcription factor, it plays a crucial role in granulocytic differentiation and diminished C/EBP activity contributes to myeloid progenitor transformation.(Cammenga*, et al* 2003, Oelgeschlager*, et al* 1996, Smith*, et al* 1996) *CEBPA*

Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia 167

The *AML1/RUNX1* gene(Ito 2008), consisting of 10 exons (exons 1-6, 7A, 7B, 7C and 8), is one of the most frequently deregulated genes in leukemia through chromosomal translocations and point mutations.(Friedman 2009, Niebuhr*, et al* 2008, Osato 2004, Yamagata*, et al* 2005) Monoallelic germ-line mutation of the *RUNX1* gene occurs in rare cases of familial platelet disorder with predisposition to AML (FPD/AML).(Michaud*, et al* 2002) Acquired *RUNX1*  mutation was frequently reported in therapy-related MDS or MDS/AML.(Harada*, et al* 2004) The incidence of *RUNX1* mutation in AML varies from 2.9% to 46% depending on the population selected, the regions of *RUNX1* screened, and the methods used.(Dicker*, et al* 2007, Preudhomme*, et al* 2000, Tang*, et al* 2009) In a large cohort study of 470 adult patients with *de novo* non-M3 AML, we detected *RUNX1* mutation in 13.2% of cases. The *RUNX1*  mutation is closely associated with older age, immature FAB subtypes (M0/M1) and specific cytogenetic abnormalities such as trisomy 8 (+8), +13, or +21.(Dicker*, et al* 2007, Schnittger*, et al* 2011, Tang*, et al* 2009) None of the patients with t(8;21), inv(16), t(15;17) or 11q23 translocation shows *RUNX1* mutation.(Tang*, et al* 2009) One half of *RUNX1*-mutated patients have concurrently other gene mutations, mostly (83.9%) Class I mutations, especially *FLT3/*ITD*, FLT3/*TKD and *N-RAS* mutations(Tang*, et al* 2009) which all result in hyperactivation of the receptor tyrosine kinase (RTK)-RAS signalling pathways.(Niimi*, et al* 2006) This finding is consistent with the two-hit model of leukemogenesis.(Frohling*, et al* 2005, Gilliland 2002) Further, the *RUNX1* mutation is mutually exclusive with *CEBPA* and *NPM1* mutations, but closely associated with *MLL/*PTD.(Schnittger*, et al* 2011, Tang*, et al* 2009) The mutation may be lost at relapse in *RUNX1*-mutated patients, but none of the patients who do not harbor *RUNX1* mutation at diagnosis acquire novel mutation at relapse.(Tang*, et al* 2009) *RUNX1* mutation is an independent poor-risk factor for DFS and OS in *de novo* AML patients.(Gaidzik*, et al* 2011, Schnittger*, et al* 2011, Tang*, et al* 2009) In addition, HSCT seems to ameliorate the poor survival impact of *RUNX1* 

*NPM1* mutation in AML was first identified by Dr. Falini's group, who noticed that some AML patients' leukemia cells exhibited aberrant cytoplasmic localization of NPM1 protein, which normally located in nucleoli in non-mitotic cells.(Falini*, et al* 2005) Subsequent investigation revealed a tetra-nucleotide insertion near the C-terminal end of the coding sequence of *NPM1*. The most frequent form of mutation is duplication of TCTG (type A, c.860\_863dupTCTG), resulting in alteration of the peptide sequence from DLWQWRKSL\* to DLCL AVEEVSLRK\*. *NPM1* mutation occurs in about 30% of AML, more frequently in elder patients,(Falini*, et al* 2011, Falini*, et al* 2005) and is highly associated with normal karyotype, and *FLT*3/ITD, but significantly exclusive with *CEBPA* mutation, favorable karyotype, and expression of CD34 and HLA-DR.(Boissel*, et al* 2005, Chou*, et al* 2006, Dohner*, et al* 2005, Falini*, et al* 2005, Schnittger*, et al* 2005, Suzuki*, et al* 2005, Verhaak*, et al* 2005) *NPM1* mutation generally renders better prognosis,(Falini*, et al* 2005) especially when *FLT3*-ITD is absent.(Schlenk*, et al* 2008, Thiede*, et al* 2006) Further refinement of patient

), intermediate

), and poor (*NPM1*-/*FLT3*-ITD+).(Gale*, et al* 2008)

groups disclosed 3 groups with distinct prognosis: good (*NPM1*+/*FLT3*-ITD-

*NPM1* mutation seems quite consistent with disease status.(Chou*, et al* 2007, Schnittger*, et al*

/*FLT3*-ITD-

**3.3** *AML1/RUNX1* 

mutations.(Gaidzik*, et al* 2011, Tang*, et al* 2009)

**4. Other mutations 4.1** *NPM1* **mutations** 

(*NPM1*+/*FLT3*-ITD+ or *NPM1*-

mutations are observed in 7% to 18% of patients with AML.(Frohling*, et al* 2004, Lin*, et al* 2005, Pabst*, et al* 2001b, Preudhomme*, et al* 2002) Two major types of *CEBPA* mutations have been identified; one alters COOH terminal bZIP of *CEBPA*, resulting in decreased DNAbinding and/or dimerization activity and the other disrupts translation of the C/EBP NH2 terminus, thereby up-regulates an alternative 30-kDa isoform with dominant-negative effect on the full-length wild-type C/EBP.(Koschmieder*, et al* 2009, Lin*, et al* 1993, Pabst*, et al* 2001b) Most patients with *CEBPA* mutations harbored biallelic mutations involving both the NH2-terminal TAD region and the COOH-terminal bZIP domain.(Hou*, et al* 2009, Lin*, et al* 2005, Pabst*, et al* 2009, Renneville*, et al* 2009a) *CEBPA* mutations occur most frequently in patients with FAB subtype M2, and are closely associated with CD7, CD15, CD34, and HLA-DR expression on the leukemic cells, higher circulatory blasts and normal cytogenetics.(Frohling*, et al* 2004, Lin*, et al* 2005, Pabst*, et al* 2001a, Zhang*, et al* 1997) This mutation seems quite stable during AML evolution and may be a potential marker to monitor MRD. The fact that none of the AML patients who do not have *CEBPA* mutations at diagnosis acquire the mutation at relapse suggests that this mutation may not play a major role in the progression of AML.(Lin*, et al* 2005, Tiesmeier*, et al* 2003) Several studies have shown mutant *CEBPA* predicts favorable outcome in AML patients with intermediate or normal cytogenetics.(Barjesteh van Waalwijk van Doorn-Khosrovani*, et al* 2003, Bienz*, et al* 2005, Frohling*, et al* 2004, Preudhomme*, et al* 2002) The favorable impact of *CEBPA* mutations in the AML patients is only observed in the absence of *FLT3/*ITD or other associated cytogenetic abnormalities.(Renneville*, et al* 2009a) Moreover, only double *CEBPA* mutations, but not single *CEBPA* mutation, are associated with better prognosis and define a distinct genetic entity.(Dufour*, et al* 2010, Hou*, et al* 2009, Pabst*, et al* 2009, Wouters*, et al* 2009)

#### **3.2** *MLL* **-PTD**

The *MLL* partial tandem duplication (*MLL*-PTD) most commonly results from a duplication of a genomic region encompassing exon 5 through exon 11/12 and insertion of the duplicated segment into intron 4 of the full-length *MLL* gene.(Caligiuri*, et al* 1994, Schichman*, et al* 1994, Whitman*, et al* 2005) The duplication involves a portion of the gene corresponding to the amino terminus of the MLL protein which contains the AT hook and a region of homology to DNA methyltransferase motifs.(Schichman*, et al* 1995) The mechanism by which *MLL-*PTD contributes to leukemic phenotype is not clear, but may be through silencing of the wild-type *MLL* by epigenetic mechanisms.(Dimartino and Cleary 1999, Whitman*, et al* 2005) This mutation is found in 5-12% of patients with cytogenetically normal AML (CN-AML),(Dohner*, et al* 2002, Munoz*, et al* 2003, Schnittger*, et al* 2000, Shiah*, et al* 2002) and up to 54% of AML patients with trisomy 11.(Rege-Cambrin*, et al* 2005) Compared with patients without *MLL-*PTD*,* patients with this mutation more often have FAB M2 subtype, CD11b expression, wild-type *NPM1* and high *BAALC* expression, but lower WBC counts, less frequently extramedullary involvement and FAB M4/M5 subtype at diagnosis.(Shiah*, et al* 2002, Whitman*, et al* 2007) The presence of *MLL*-PTD predicts shorter remission duration and worse OS;(Dohner*, et al* 2002, Munoz*, et al* 2003, Shiah*, et al* 2002) however, more intensive consolidation therapy that includes hematopoietic stem cell transplantation (HSCT) during first complete remission (CR) may reverse the poor prognosis conferred by this mutation.(Whitman*, et al* 2007)

#### **3.3** *AML1/RUNX1*

166 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

mutations are observed in 7% to 18% of patients with AML.(Frohling*, et al* 2004, Lin*, et al* 2005, Pabst*, et al* 2001b, Preudhomme*, et al* 2002) Two major types of *CEBPA* mutations have been identified; one alters COOH terminal bZIP of *CEBPA*, resulting in decreased DNAbinding and/or dimerization activity and the other disrupts translation of the C/EBP NH2 terminus, thereby up-regulates an alternative 30-kDa isoform with dominant-negative effect on the full-length wild-type C/EBP.(Koschmieder*, et al* 2009, Lin*, et al* 1993, Pabst*, et al* 2001b) Most patients with *CEBPA* mutations harbored biallelic mutations involving both the NH2-terminal TAD region and the COOH-terminal bZIP domain.(Hou*, et al* 2009, Lin*, et al* 2005, Pabst*, et al* 2009, Renneville*, et al* 2009a) *CEBPA* mutations occur most frequently in patients with FAB subtype M2, and are closely associated with CD7, CD15, CD34, and HLA-DR expression on the leukemic cells, higher circulatory blasts and normal cytogenetics.(Frohling*, et al* 2004, Lin*, et al* 2005, Pabst*, et al* 2001a, Zhang*, et al* 1997) This mutation seems quite stable during AML evolution and may be a potential marker to monitor MRD. The fact that none of the AML patients who do not have *CEBPA* mutations at diagnosis acquire the mutation at relapse suggests that this mutation may not play a major role in the progression of AML.(Lin*, et al* 2005, Tiesmeier*, et al* 2003) Several studies have shown mutant *CEBPA* predicts favorable outcome in AML patients with intermediate or normal cytogenetics.(Barjesteh van Waalwijk van Doorn-Khosrovani*, et al* 2003, Bienz*, et al* 2005, Frohling*, et al* 2004, Preudhomme*, et al* 2002) The favorable impact of *CEBPA* mutations in the AML patients is only observed in the absence of *FLT3/*ITD or other associated cytogenetic abnormalities.(Renneville*, et al* 2009a) Moreover, only double *CEBPA* mutations, but not single *CEBPA* mutation, are associated with better prognosis and define a distinct genetic entity.(Dufour*, et al* 2010, Hou*, et al* 2009, Pabst*, et al* 2009, Wouters*, et al*

The *MLL* partial tandem duplication (*MLL*-PTD) most commonly results from a duplication of a genomic region encompassing exon 5 through exon 11/12 and insertion of the duplicated segment into intron 4 of the full-length *MLL* gene.(Caligiuri*, et al* 1994, Schichman*, et al* 1994, Whitman*, et al* 2005) The duplication involves a portion of the gene corresponding to the amino terminus of the MLL protein which contains the AT hook and a region of homology to DNA methyltransferase motifs.(Schichman*, et al* 1995) The mechanism by which *MLL-*PTD contributes to leukemic phenotype is not clear, but may be through silencing of the wild-type *MLL* by epigenetic mechanisms.(Dimartino and Cleary 1999, Whitman*, et al* 2005) This mutation is found in 5-12% of patients with cytogenetically normal AML (CN-AML),(Dohner*, et al* 2002, Munoz*, et al* 2003, Schnittger*, et al* 2000, Shiah*, et al* 2002) and up to 54% of AML patients with trisomy 11.(Rege-Cambrin*, et al* 2005) Compared with patients without *MLL-*PTD*,* patients with this mutation more often have FAB M2 subtype, CD11b expression, wild-type *NPM1* and high *BAALC* expression, but lower WBC counts, less frequently extramedullary involvement and FAB M4/M5 subtype at diagnosis.(Shiah*, et al* 2002, Whitman*, et al* 2007) The presence of *MLL*-PTD predicts shorter remission duration and worse OS;(Dohner*, et al* 2002, Munoz*, et al* 2003, Shiah*, et al* 2002) however, more intensive consolidation therapy that includes hematopoietic stem cell transplantation (HSCT) during first complete remission (CR) may reverse the poor

prognosis conferred by this mutation.(Whitman*, et al* 2007)

2009)

**3.2** *MLL* **-PTD**

The *AML1/RUNX1* gene(Ito 2008), consisting of 10 exons (exons 1-6, 7A, 7B, 7C and 8), is one of the most frequently deregulated genes in leukemia through chromosomal translocations and point mutations.(Friedman 2009, Niebuhr*, et al* 2008, Osato 2004, Yamagata*, et al* 2005) Monoallelic germ-line mutation of the *RUNX1* gene occurs in rare cases of familial platelet disorder with predisposition to AML (FPD/AML).(Michaud*, et al* 2002) Acquired *RUNX1*  mutation was frequently reported in therapy-related MDS or MDS/AML.(Harada*, et al* 2004) The incidence of *RUNX1* mutation in AML varies from 2.9% to 46% depending on the population selected, the regions of *RUNX1* screened, and the methods used.(Dicker*, et al* 2007, Preudhomme*, et al* 2000, Tang*, et al* 2009) In a large cohort study of 470 adult patients with *de novo* non-M3 AML, we detected *RUNX1* mutation in 13.2% of cases. The *RUNX1*  mutation is closely associated with older age, immature FAB subtypes (M0/M1) and specific cytogenetic abnormalities such as trisomy 8 (+8), +13, or +21.(Dicker*, et al* 2007, Schnittger*, et al* 2011, Tang*, et al* 2009) None of the patients with t(8;21), inv(16), t(15;17) or 11q23 translocation shows *RUNX1* mutation.(Tang*, et al* 2009) One half of *RUNX1*-mutated patients have concurrently other gene mutations, mostly (83.9%) Class I mutations, especially *FLT3/*ITD*, FLT3/*TKD and *N-RAS* mutations(Tang*, et al* 2009) which all result in hyperactivation of the receptor tyrosine kinase (RTK)-RAS signalling pathways.(Niimi*, et al* 2006) This finding is consistent with the two-hit model of leukemogenesis.(Frohling*, et al* 2005, Gilliland 2002) Further, the *RUNX1* mutation is mutually exclusive with *CEBPA* and *NPM1* mutations, but closely associated with *MLL/*PTD.(Schnittger*, et al* 2011, Tang*, et al* 2009) The mutation may be lost at relapse in *RUNX1*-mutated patients, but none of the patients who do not harbor *RUNX1* mutation at diagnosis acquire novel mutation at relapse.(Tang*, et al* 2009) *RUNX1* mutation is an independent poor-risk factor for DFS and OS in *de novo* AML patients.(Gaidzik*, et al* 2011, Schnittger*, et al* 2011, Tang*, et al* 2009) In addition, HSCT seems to ameliorate the poor survival impact of *RUNX1*  mutations.(Gaidzik*, et al* 2011, Tang*, et al* 2009)

#### **4. Other mutations**

#### **4.1** *NPM1* **mutations**

*NPM1* mutation in AML was first identified by Dr. Falini's group, who noticed that some AML patients' leukemia cells exhibited aberrant cytoplasmic localization of NPM1 protein, which normally located in nucleoli in non-mitotic cells.(Falini*, et al* 2005) Subsequent investigation revealed a tetra-nucleotide insertion near the C-terminal end of the coding sequence of *NPM1*. The most frequent form of mutation is duplication of TCTG (type A, c.860\_863dupTCTG), resulting in alteration of the peptide sequence from DLWQWRKSL\* to DLCL AVEEVSLRK\*. *NPM1* mutation occurs in about 30% of AML, more frequently in elder patients,(Falini*, et al* 2011, Falini*, et al* 2005) and is highly associated with normal karyotype, and *FLT*3/ITD, but significantly exclusive with *CEBPA* mutation, favorable karyotype, and expression of CD34 and HLA-DR.(Boissel*, et al* 2005, Chou*, et al* 2006, Dohner*, et al* 2005, Falini*, et al* 2005, Schnittger*, et al* 2005, Suzuki*, et al* 2005, Verhaak*, et al* 2005) *NPM1* mutation generally renders better prognosis,(Falini*, et al* 2005) especially when *FLT3*-ITD is absent.(Schlenk*, et al* 2008, Thiede*, et al* 2006) Further refinement of patient groups disclosed 3 groups with distinct prognosis: good (*NPM1*+/*FLT3*-ITD- ), intermediate (*NPM1*+/*FLT3*-ITD+ or *NPM1*-/*FLT3*-ITD- ), and poor (*NPM1*-/*FLT3*-ITD+).(Gale*, et al* 2008) *NPM1* mutation seems quite consistent with disease status.(Chou*, et al* 2007, Schnittger*, et al*

Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia 169

hybridization array, which revealed common deletion of this gene in chromosome 4q.(Delhommeau*, et al* 2009) Subsequent studies confirmed this mutation in MDS, MPN, MDS/MPN, and secondary AML, with frequencies around 10% to 26%, 7% to 13%, 22% to 58% and 24% to 32%, respectively.(Bacher*, et al* 2010, Couronne*, et al* 2010, Flach*, et al* 2010, Jankowska*, et al* 2009, Kosmider*, et al* 2009a, Kosmider*, et al* 2009b, Langemeijer*, et al* 2009, Saint-Martin*, et al* 2009, Schaub*, et al* 2010, Smith*, et al* 2010, Tefferi*, et al* 2009a, Tefferi*, et al* 2009b) *TET2* mutation occurs in 18.0% to 23% of CN-AML patients.(Chou*, et al* 2011a, Metzeler*, et al* 2011) It is closely associated with older age, higher WBC count, but mutually exclusive with *IDH* mutation.(Chou*, et al* 2011a, Metzeler*, et al* 2011) In our study of AML patients with and without chromosomal abnormalities, *TET2* mutation was also found to be positively associated with normal karyotype, intermediate-risk cytogenetics, isolated trisomy 8, *NPM1* mutation, and *ASXL1* mutation.(Chou*, et al* 2011a) In European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML with mutated *CEBPA* and/or mutated *NPM1* without *FLT3*-ITD),(Dohner*, et al* 2010) but not intermediate-1 risk group (CN-AML with wild-type *CEBPA* and wild-type *NPM1* and/or FLT3-ITD), *TET2* mutated patients were found to have a lower CR rate, shorter DFS and OS, compared with *TET2*-wild type patients.(Metzeler*, et al* 2011) However, we did not have the same finding, but found that *TET2* mutation was an unfavorable prognostic factor in patients with intermediate-risk cytogenetics, and its negative impact was further enhanced when the mutation was combined with *FLT3*-ITD, *NPM1*-wild, or unfavorable genotypes (other than ELN favorable-risk group).(Chou*, et al* 2011a) More studies are needed to clarify the

*IDH1* and *IDH2* genes encode two isoforms of isocitrate dehydrogenase which catalyzes the carboxylation of isocitrate to α-KG. *IDH1* and *IDH2* mutations were first detected in patients with brain tumors.(Parsons*, et al* 2008) Later, *IDH1* mutations (Mardis*, et al* 2009) and then *IDH2* mutations were discovered in AML patients, too.(Abbas*, et al* 2010, Marcucci*, et al* 2010, Ward*, et al* 2010) *IDH1* mutations affect arginine residue in position 132 (R132) and *IDH2* mutations, in R140 and R172 of exon 4. *IDH* mutations occur at low frequencies (3.6% to 5%) in MDS,(Kosmider*, et al* 2010) and in chronic-phase MPN (about 1.8%)(Pardanani*, et al* 2010), but obviously increased as these diseases progress to AML (7.5% to 21%),(Kosmider*, et al* 2010, Pardanani*, et al* 2010) indicating a role of *IDH* mutations in leukemogenesis. In *de novo* AML, *IDH2* mutations occur more frequently than *IDH1*  mutations, with frequencies of 11% vs. 6% in patients younger than 60 years,(Abbas*, et al* 2010) 15.4% vs. 7.7% in total patients,(Ward*, et al* 2010) and 19% vs. 14% in adults with normal karyotype.(Marcucci*, et al* 2010) The underlying mechanism of *IDH* mutations in the leukemogenesis of AML remains to be determined, but several implications of *IDH1/2* mutations in AML have been generated. First, *IDH* mutations are loss-of-function mutations, as mutant IDH proteins show decreased enzyme activities,(Zhao*, et al* 2009) and have dominant-negative effects on wild type IDH upon homodimerization.(Zhao*, et al* 2009) Secondly, *IDH* mutations are also gain-of-function mutations because the mutant proteins can convert α-KG to 2-hydroxyglutarate (2-HG), a metabolite that may contribute to tumor growth through activating hypoxia-inducing factor-1α (HIF-1α).(Dang*, et al* 2009, Reitman*, et al* 2010, Ward*, et al* 2010) Thirdly, *IDH* mutations reduce production of α-KG, a cofactor of TET2, thus impair catalytic function of TET2 resulting in global DNA hypermethylation,

prognostic implication of *TET2* mutations in AML.

**5.2** *IDH* **mutations** 

2009) Serial analyses of *NPM1* mutations showed the mutation disappeared at CR, but the same mutation usually reappeared at relapse. This feature makes *NPM1* mutation an ideal marker for MRD monitoring. Studies have shown *NPM1* mutant levels reflect disease status, predict impending relapse, and bring prognostic implication.(Chou*, et al* 2007, Gorello*, et al* 2006, Kronke*, et al* 2011, Schnittger*, et al* 2009)

#### **4.2** *WT1* **mutations**

The *Wilms' Tumor 1* (*WT1*) gene, encoding a zinc-finger transcription factor, is physiologically expressed in hematopoietic stem cells and involved in regulation of cellular growth and differentiation.(Baird and Simmons 1997, Ellisen*, et al* 2001) *WT1* was initially identified as a tumor suppressor gene,(Haber*, et al* 1990) but was later found to be overexpressed in AML as well as other cancers and thus was suggested to be an oncogene.(Bergmann*, et al* 1997, King-Underwood*, et al* 1996, Miwa*, et al* 1992) Mutations in *WT1* gene are found in about 7-13% of CN-AML patients with hotspots in the four Cys-His zinc finger domains on exons 7 and 9.(Gaidzik*, et al* 2009, Hou*, et al* 2010, King-Underwood*, et al* 1996, Paschka*, et al* 2008, Virappane*, et al* 2008) The precise role of WT1 mutations in the leukemogenesis remains to be defined. The majority of *WT1* mutations are frame-shift mutations occurring in exon 7, followed by single amino acid substitutions in exon 9; whereas frame-shift mutations in exon 9 are rare. *WT1* mutations occur with similar frequencies in patients with normal karyotype and those with abnormal cytogenetics.(Hou*, et al* 2010) Chromosomal abnormality t(7;11)(p15;15), a translocation resulting in *NUP98/HOXA9* fusion, is closely associated with *WT1* mutation.(Hou*, et al* 2010) *WT1* mutations are positively associated with *FLT3/*ITD and *CEBPA* mutations.(Gaidzik*, et al* 2009, Renneville*, et al* 2009b) Paschka *et al* showed patients with *WT1* mutations had higher expression of *ERG* and *BAALC* than patients without.(Paschka*, et al* 2008) *WT1* mutation is an independent poor prognostic factor in CN-AML as well as total AML patients.(Hou*, et al* 2010, Paschka*, et al* 2008, Renneville*, et al* 2009b, Virappane*, et al* 2008), though different results have been reported.(Gaidzik*, et al* 2009, Santamaria*, et al* 2009)

#### **5. Mutations of genes that involve epigenetic modifications**

Different from genetic abnormalities which result in DNA sequence changes, epigenetic dysregulation causes aberrant gene expression without alteration of gene sequences.(Baylin and Ohm 2006, Chen*, et al* 2010, Jones and Baylin 2002) Epigenetic regulation includes DNA methylation, histone modifications, such as methylation, acetylation and phosphorylation, etc, and microRNA expression. (Baylin and Ohm 2006, Chen*, et al* 2010, Jones and Baylin 2002) The recent findings that mutations of genes related to epigenetic modifications, such as *IDH1, IDH2, TET2, ASXL1* and *DNMT3A*, are detected in AML patients provide new insights into mechanisms of epigenetic deregulation in the leukemogenesis.

#### **5.1** *TET2* **mutations**

TET2 protein can catalyze the conversion of 5-methylcytosine (5-mC) of DNA to 5 hydroxymethylcytosine (5-hmC), with ferrous iron and α-ketoglutarate (α-KG) as cofactors, indicating a role of TET2 in DNA methylation.(Ito*, et al* 2010) Mutations of *TET2* result in global DNA hypermethylation.(Figueroa*, et al* 2010) *TET2* mutation was originally identified in myeloid malignancies via single nucleotide polymorphism and comparative genomichybridization array, which revealed common deletion of this gene in chromosome 4q.(Delhommeau*, et al* 2009) Subsequent studies confirmed this mutation in MDS, MPN, MDS/MPN, and secondary AML, with frequencies around 10% to 26%, 7% to 13%, 22% to 58% and 24% to 32%, respectively.(Bacher*, et al* 2010, Couronne*, et al* 2010, Flach*, et al* 2010, Jankowska*, et al* 2009, Kosmider*, et al* 2009a, Kosmider*, et al* 2009b, Langemeijer*, et al* 2009, Saint-Martin*, et al* 2009, Schaub*, et al* 2010, Smith*, et al* 2010, Tefferi*, et al* 2009a, Tefferi*, et al* 2009b) *TET2* mutation occurs in 18.0% to 23% of CN-AML patients.(Chou*, et al* 2011a, Metzeler*, et al* 2011) It is closely associated with older age, higher WBC count, but mutually exclusive with *IDH* mutation.(Chou*, et al* 2011a, Metzeler*, et al* 2011) In our study of AML patients with and without chromosomal abnormalities, *TET2* mutation was also found to be positively associated with normal karyotype, intermediate-risk cytogenetics, isolated trisomy 8, *NPM1* mutation, and *ASXL1* mutation.(Chou*, et al* 2011a) In European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML with mutated *CEBPA* and/or mutated *NPM1* without *FLT3*-ITD),(Dohner*, et al* 2010) but not intermediate-1 risk group (CN-AML with wild-type *CEBPA* and wild-type *NPM1* and/or FLT3-ITD), *TET2* mutated patients were found to have a lower CR rate, shorter DFS and OS, compared with *TET2*-wild type patients.(Metzeler*, et al* 2011) However, we did not have the same finding, but found that *TET2* mutation was an unfavorable prognostic factor in patients with intermediate-risk cytogenetics, and its negative impact was further enhanced when the mutation was combined with *FLT3*-ITD, *NPM1*-wild, or unfavorable genotypes (other than ELN favorable-risk group).(Chou*, et al* 2011a) More studies are needed to clarify the prognostic implication of *TET2* mutations in AML.

#### **5.2** *IDH* **mutations**

168 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

2009) Serial analyses of *NPM1* mutations showed the mutation disappeared at CR, but the same mutation usually reappeared at relapse. This feature makes *NPM1* mutation an ideal marker for MRD monitoring. Studies have shown *NPM1* mutant levels reflect disease status, predict impending relapse, and bring prognostic implication.(Chou*, et al* 2007, Gorello*, et al*

The *Wilms' Tumor 1* (*WT1*) gene, encoding a zinc-finger transcription factor, is physiologically expressed in hematopoietic stem cells and involved in regulation of cellular growth and differentiation.(Baird and Simmons 1997, Ellisen*, et al* 2001) *WT1* was initially identified as a tumor suppressor gene,(Haber*, et al* 1990) but was later found to be overexpressed in AML as well as other cancers and thus was suggested to be an oncogene.(Bergmann*, et al* 1997, King-Underwood*, et al* 1996, Miwa*, et al* 1992) Mutations in *WT1* gene are found in about 7-13% of CN-AML patients with hotspots in the four Cys-His zinc finger domains on exons 7 and 9.(Gaidzik*, et al* 2009, Hou*, et al* 2010, King-Underwood*, et al* 1996, Paschka*, et al* 2008, Virappane*, et al* 2008) The precise role of WT1 mutations in the leukemogenesis remains to be defined. The majority of *WT1* mutations are frame-shift mutations occurring in exon 7, followed by single amino acid substitutions in exon 9; whereas frame-shift mutations in exon 9 are rare. *WT1* mutations occur with similar frequencies in patients with normal karyotype and those with abnormal cytogenetics.(Hou*, et al* 2010) Chromosomal abnormality t(7;11)(p15;15), a translocation resulting in *NUP98/HOXA9* fusion, is closely associated with *WT1* mutation.(Hou*, et al* 2010) *WT1* mutations are positively associated with *FLT3/*ITD and *CEBPA* mutations.(Gaidzik*, et al* 2009, Renneville*, et al* 2009b) Paschka *et al* showed patients with *WT1* mutations had higher expression of *ERG* and *BAALC* than patients without.(Paschka*, et al* 2008) *WT1* mutation is an independent poor prognostic factor in CN-AML as well as total AML patients.(Hou*, et al* 2010, Paschka*, et al* 2008, Renneville*, et al* 2009b, Virappane*, et al* 2008), though different

results have been reported.(Gaidzik*, et al* 2009, Santamaria*, et al* 2009)

**5. Mutations of genes that involve epigenetic modifications** 

insights into mechanisms of epigenetic deregulation in the leukemogenesis.

Different from genetic abnormalities which result in DNA sequence changes, epigenetic dysregulation causes aberrant gene expression without alteration of gene sequences.(Baylin and Ohm 2006, Chen*, et al* 2010, Jones and Baylin 2002) Epigenetic regulation includes DNA methylation, histone modifications, such as methylation, acetylation and phosphorylation, etc, and microRNA expression. (Baylin and Ohm 2006, Chen*, et al* 2010, Jones and Baylin 2002) The recent findings that mutations of genes related to epigenetic modifications, such as *IDH1, IDH2, TET2, ASXL1* and *DNMT3A*, are detected in AML patients provide new

TET2 protein can catalyze the conversion of 5-methylcytosine (5-mC) of DNA to 5 hydroxymethylcytosine (5-hmC), with ferrous iron and α-ketoglutarate (α-KG) as cofactors, indicating a role of TET2 in DNA methylation.(Ito*, et al* 2010) Mutations of *TET2* result in global DNA hypermethylation.(Figueroa*, et al* 2010) *TET2* mutation was originally identified in myeloid malignancies via single nucleotide polymorphism and comparative genomic-

2006, Kronke*, et al* 2011, Schnittger*, et al* 2009)

**4.2** *WT1* **mutations** 

**5.1** *TET2* **mutations** 

*IDH1* and *IDH2* genes encode two isoforms of isocitrate dehydrogenase which catalyzes the carboxylation of isocitrate to α-KG. *IDH1* and *IDH2* mutations were first detected in patients with brain tumors.(Parsons*, et al* 2008) Later, *IDH1* mutations (Mardis*, et al* 2009) and then *IDH2* mutations were discovered in AML patients, too.(Abbas*, et al* 2010, Marcucci*, et al* 2010, Ward*, et al* 2010) *IDH1* mutations affect arginine residue in position 132 (R132) and *IDH2* mutations, in R140 and R172 of exon 4. *IDH* mutations occur at low frequencies (3.6% to 5%) in MDS,(Kosmider*, et al* 2010) and in chronic-phase MPN (about 1.8%)(Pardanani*, et al* 2010), but obviously increased as these diseases progress to AML (7.5% to 21%),(Kosmider*, et al* 2010, Pardanani*, et al* 2010) indicating a role of *IDH* mutations in leukemogenesis. In *de novo* AML, *IDH2* mutations occur more frequently than *IDH1*  mutations, with frequencies of 11% vs. 6% in patients younger than 60 years,(Abbas*, et al* 2010) 15.4% vs. 7.7% in total patients,(Ward*, et al* 2010) and 19% vs. 14% in adults with normal karyotype.(Marcucci*, et al* 2010) The underlying mechanism of *IDH* mutations in the leukemogenesis of AML remains to be determined, but several implications of *IDH1/2* mutations in AML have been generated. First, *IDH* mutations are loss-of-function mutations, as mutant IDH proteins show decreased enzyme activities,(Zhao*, et al* 2009) and have dominant-negative effects on wild type IDH upon homodimerization.(Zhao*, et al* 2009) Secondly, *IDH* mutations are also gain-of-function mutations because the mutant proteins can convert α-KG to 2-hydroxyglutarate (2-HG), a metabolite that may contribute to tumor growth through activating hypoxia-inducing factor-1α (HIF-1α).(Dang*, et al* 2009, Reitman*, et al* 2010, Ward*, et al* 2010) Thirdly, *IDH* mutations reduce production of α-KG, a cofactor of TET2, thus impair catalytic function of TET2 resulting in global DNA hypermethylation,

Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia 171

premature stop codon after another 11 amino acid).(Chou*, et al* 2010b) The mutation was closely associated with older age, male gender, isolated trisomy 8, *RUNX1* mutation, and expression of HLA-DR and CD34, but inversely associated with t(15;17), complex cytogenetics, *FLT3-*ITD, *NPM1* mutations, *WT1* mutations, and expression of CD33 and CD15.(Chou*, et al* 2010b) Patients with *ASXL1* mutations had a shorter OS than those without, but the mutation was not an independent adverse prognostic factor in multivariate analysis. Sequential analyses showed that the original *ASXL1* mutations could disappear at relapse and/or refractory status in some patients. Moreover, two out of the 109 *ASXL1-*wild patients acquired a novel *ASXL1* mutation at relapse.(Chou*, et al* 2010b) Thus, the *ASXL1* 

By whole genome sequencing on a single patient with normal cytogenetics, Ley and his colleagues found a mutation in *DNMT3A* gene, which encodes the enzyme DNA methyltransferase 3A which belongs to the family of DNMTs that catalyze the addition of methyl group to cytosine of CpG dinucleotide.(Ley*, et al* 2010) In this seminal study, *DNMT3A* mutation was detected in 22.1% of AML patients. Most of the mutations occurred at R882 amino acid. Others included mis-sense, non-sense and frame-shift mutations. Although DNMT3A is directly related to DNA methylation, the real significance of this mutation to leukemogenesis remains unknown. First, the wide spreading of mutation spots in *DNMT3A* suggests a loss-of-function mutation, but the remarkable aggregate of mutation at R882 implies a gain of function. Reduction of DNA methylation in 182 genomic areas was noted in R882 mutation-harboring AML cells, however, the methylation patterns of vast

majority of cytosine methylation regions are the same as wild type.(Ley*, et al* 2010)

*DNMT3A* mutations are associated with intermediate or normal cytogenetics, higher WBC counts, FAB M4/M5 subtypes, and *FLT3*-ITD, *NPM1*, and *IDH1* mutations but mutually exclusive with favorable karyotypes.(Ley*, et al* 2010, Thol*, et al* 2011) In our study of 500 AML patients, *DNMT3A* mutations were identified in 14% of total patients and 22.9% of patients with CN-AML. (Hou*, et al* 2011) In addition to the findings shown in previous reports, (Ley*, et al* 2010, Thol*, et al* 2011) we for the first time identified the *DNMT3A* mutation was positively associated with *PTPN11* and *IDH2* mutations, but negatively associated with *CEBPA* mutation.(Hou*, et al* 2011) Intriguingly, the majority (97.1%) of the *DNMT3A*-mutated patients showed additional molecular alterations at diagnosis. This mutation renders poor OS among all AML patients, patients with a normal karyotype, and those with *FLT3*-ITD.(Hou*, et al* 2011, Ley*, et al* 2010, Thol*, et al* 2011) Importantly, *DNMT3A* mutation is an independent poor prognostic factor. Further, *DNMT3A* mutation is rather stable during disease progression and can be a potential biomarker for monitoring of

**6. Gene mutations as markers to monitor Minimal Residual Disease (MRD)** 

Since gene mutations are theoretically absent in healthy people and restricted in leukemia cells, it is reasonably to monitor MRD by detection of gene mutations. This is an advantage of leukemia over solid tumors in that leukemia cells are indigenous to blood and marrow, which are easy for access. There are two critical considerations of MRD monitoring by gene mutations: one is the stability of the mutations, and the other is the pattern of mutation. An

mutation status can change during disease evolution in a few patients.

**5.4** *DNMT3A* **mutations** 

MRD.(Hou*, et al* 2011)

similar to the effect of *TET2* mutations. 2-HG converted from α-KG in *IDH*-mutated cells is also shown to inhibit TET2-mediated hydroxymethylation of cytosine, indicating overlapping effects of these two mutations.(Xu*, et al* 2011) Compatible with this, *IDH* and *TET2* mutations are mutually exclusive in AML patients.(Figueroa*, et al* 2010, Metzeler*, et al* 2011)

Studies have shown similar clinical features between AML with *IDH1* and *IDH2* mutations, including strong association of both mutations with normal karyotype and isolated monosomy 8, but inverse correlation with expression of HLA-DR. However, some differences exist. *IDH1* mutation shows strong correlation with *NPM1* mutation, and FAB M1 subtype, but is inversely associated with FAB M4 subtype and expression of CD13 and CD14. On the other hand, mutation of *IDH2* is associated with higher platelet counts, but is inversely correlated with expression of CD34, CD15, CD7, and CD56, and is mutually exclusive with *WT1* mutation and chromosomal translocations involving CBF. While there is no impact of *IDH1* mutation on patient survival, multivariate analysis reveals *IDH2* mutation as an independent favorable prognostic factor,(Chou*, et al* 2010a, Chou*, et al* 2011c,) but different results have also been reported.(Marcucci*, et al* 2010, Thol*, et al* 2010) More intriguing are the differences of clinical presentations between patients with R140 and R172 mutations. Compared with *IDH2* R140 mutation, *IDH2* R172 mutation is associated with younger age, lower WBC count and LDH level, and is mutually exclusive with *NPM1* mutation. Recent studies also reported worse prognosis in AML patients bearing *IDH2* R172Q,(Boissel*, et al* 2010, Marcucci*, et al* 2010) while *IDH2* R140Q, in the contrary, conferred a better prognosis.(Green*, et al* 2010) Why mutations in different isoforms or loci of the same gene render distinct clinical and prognostic features remains to be investigated. Serial analyses of *IDH1/2* mutations at both diagnosis and relapse confirmed high stability of these two mutations.(Chou*, et al* 2010a, Chou*, et al* 2011c)

#### **5.3** *ASXL1* **mutations**

Recently, mutations in exon 12 of *Additional sex comb-like 1* (*ASXL1*) gene were found in various types of myeloid malignances, including MDS, MPN, MDS/MPN, and AML.(Abdel-Wahab*, et al* 2010, Boultwood*, et al* 2010, Carbuccia*, et al* 2009, Carbuccia*, et al* 2010, Gelsi-Boyer*, et al* 2009) *ASXL1,* a human homologue of the *Additional sex combs* (*Asx*) gene of *Drosophila*, mapped to chromosome 20q11, a region commonly involved in cancers.(Fisher*, et al* 2003) It consists of an N-terminal *ASX* Homology (ASXH) domain and a C-terminal plant homeodomain (PHD) zinc finger region.(Fisher*, et al* 2003, Fisher*, et al* 2006) In human, the exact function of *ASXL1* mutation remains to be defined, but it is involved in regulation of histone methylation by cooperation with heterochromatin protein-1 (HP1) to modulate the activity of LSD1, a histone demethylase for H3K4 and H3K9.(Dange and Colman 2010, Wang*, et al* 2009) *ASXL1* mutations in exon 12 are found with an incidence of 10.8%, 8.9% and 12.9% among total cohort of patients with *de novo* AML, those with normal karyotype and abnormal cytogenetics, respectively.(Chou*, et al* 2010b) Most of the mutations appear to be either non-sense or frame-shift mutations, leading to disruption of the plant hoemodomain (PHD) at the C terminal of ASXL1, which is well conserved among different species and can recognize methylated H3K4.(Abdel-Wahab*, et al* 2010, Fisher*, et al* 2003, Pena*, et al* 2006, Shi*, et al* 2006, Wysocka*, et al* 2006) Up to two thirds of mutations occurred at c.1934dupG (an extra G after 1934th nucleotide of the coding sequence of *ASXL1*) causing G646WfsX12 (change of glycine to tryptophan at amino acid 646, with a premature stop codon after another 11 amino acid).(Chou*, et al* 2010b) The mutation was closely associated with older age, male gender, isolated trisomy 8, *RUNX1* mutation, and expression of HLA-DR and CD34, but inversely associated with t(15;17), complex cytogenetics, *FLT3-*ITD, *NPM1* mutations, *WT1* mutations, and expression of CD33 and CD15.(Chou*, et al* 2010b) Patients with *ASXL1* mutations had a shorter OS than those without, but the mutation was not an independent adverse prognostic factor in multivariate analysis. Sequential analyses showed that the original *ASXL1* mutations could disappear at relapse and/or refractory status in some patients. Moreover, two out of the 109 *ASXL1-*wild patients acquired a novel *ASXL1* mutation at relapse.(Chou*, et al* 2010b) Thus, the *ASXL1*  mutation status can change during disease evolution in a few patients.

#### **5.4** *DNMT3A* **mutations**

170 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

similar to the effect of *TET2* mutations. 2-HG converted from α-KG in *IDH*-mutated cells is also shown to inhibit TET2-mediated hydroxymethylation of cytosine, indicating overlapping effects of these two mutations.(Xu*, et al* 2011) Compatible with this, *IDH* and *TET2* mutations are mutually exclusive in AML patients.(Figueroa*, et al* 2010, Metzeler*, et al*

Studies have shown similar clinical features between AML with *IDH1* and *IDH2* mutations, including strong association of both mutations with normal karyotype and isolated monosomy 8, but inverse correlation with expression of HLA-DR. However, some differences exist. *IDH1* mutation shows strong correlation with *NPM1* mutation, and FAB M1 subtype, but is inversely associated with FAB M4 subtype and expression of CD13 and CD14. On the other hand, mutation of *IDH2* is associated with higher platelet counts, but is inversely correlated with expression of CD34, CD15, CD7, and CD56, and is mutually exclusive with *WT1* mutation and chromosomal translocations involving CBF. While there is no impact of *IDH1* mutation on patient survival, multivariate analysis reveals *IDH2* mutation as an independent favorable prognostic factor,(Chou*, et al* 2010a, Chou*, et al* 2011c,) but different results have also been reported.(Marcucci*, et al* 2010, Thol*, et al* 2010) More intriguing are the differences of clinical presentations between patients with R140 and R172 mutations. Compared with *IDH2* R140 mutation, *IDH2* R172 mutation is associated with younger age, lower WBC count and LDH level, and is mutually exclusive with *NPM1* mutation. Recent studies also reported worse prognosis in AML patients bearing *IDH2* R172Q,(Boissel*, et al* 2010, Marcucci*, et al* 2010) while *IDH2* R140Q, in the contrary, conferred a better prognosis.(Green*, et al* 2010) Why mutations in different isoforms or loci of the same gene render distinct clinical and prognostic features remains to be investigated. Serial analyses of *IDH1/2* mutations at both diagnosis and relapse confirmed high stability of these

Recently, mutations in exon 12 of *Additional sex comb-like 1* (*ASXL1*) gene were found in various types of myeloid malignances, including MDS, MPN, MDS/MPN, and AML.(Abdel-Wahab*, et al* 2010, Boultwood*, et al* 2010, Carbuccia*, et al* 2009, Carbuccia*, et al* 2010, Gelsi-Boyer*, et al* 2009) *ASXL1,* a human homologue of the *Additional sex combs* (*Asx*) gene of *Drosophila*, mapped to chromosome 20q11, a region commonly involved in cancers.(Fisher*, et al* 2003) It consists of an N-terminal *ASX* Homology (ASXH) domain and a C-terminal plant homeodomain (PHD) zinc finger region.(Fisher*, et al* 2003, Fisher*, et al* 2006) In human, the exact function of *ASXL1* mutation remains to be defined, but it is involved in regulation of histone methylation by cooperation with heterochromatin protein-1 (HP1) to modulate the activity of LSD1, a histone demethylase for H3K4 and H3K9.(Dange and Colman 2010, Wang*, et al* 2009) *ASXL1* mutations in exon 12 are found with an incidence of 10.8%, 8.9% and 12.9% among total cohort of patients with *de novo* AML, those with normal karyotype and abnormal cytogenetics, respectively.(Chou*, et al* 2010b) Most of the mutations appear to be either non-sense or frame-shift mutations, leading to disruption of the plant hoemodomain (PHD) at the C terminal of ASXL1, which is well conserved among different species and can recognize methylated H3K4.(Abdel-Wahab*, et al* 2010, Fisher*, et al* 2003, Pena*, et al* 2006, Shi*, et al* 2006, Wysocka*, et al* 2006) Up to two thirds of mutations occurred at c.1934dupG (an extra G after 1934th nucleotide of the coding sequence of *ASXL1*) causing G646WfsX12 (change of glycine to tryptophan at amino acid 646, with a

two mutations.(Chou*, et al* 2010a, Chou*, et al* 2011c)

**5.3** *ASXL1* **mutations** 

2011)

By whole genome sequencing on a single patient with normal cytogenetics, Ley and his colleagues found a mutation in *DNMT3A* gene, which encodes the enzyme DNA methyltransferase 3A which belongs to the family of DNMTs that catalyze the addition of methyl group to cytosine of CpG dinucleotide.(Ley*, et al* 2010) In this seminal study, *DNMT3A* mutation was detected in 22.1% of AML patients. Most of the mutations occurred at R882 amino acid. Others included mis-sense, non-sense and frame-shift mutations. Although DNMT3A is directly related to DNA methylation, the real significance of this mutation to leukemogenesis remains unknown. First, the wide spreading of mutation spots in *DNMT3A* suggests a loss-of-function mutation, but the remarkable aggregate of mutation at R882 implies a gain of function. Reduction of DNA methylation in 182 genomic areas was noted in R882 mutation-harboring AML cells, however, the methylation patterns of vast majority of cytosine methylation regions are the same as wild type.(Ley*, et al* 2010)

*DNMT3A* mutations are associated with intermediate or normal cytogenetics, higher WBC counts, FAB M4/M5 subtypes, and *FLT3*-ITD, *NPM1*, and *IDH1* mutations but mutually exclusive with favorable karyotypes.(Ley*, et al* 2010, Thol*, et al* 2011) In our study of 500 AML patients, *DNMT3A* mutations were identified in 14% of total patients and 22.9% of patients with CN-AML. (Hou*, et al* 2011) In addition to the findings shown in previous reports, (Ley*, et al* 2010, Thol*, et al* 2011) we for the first time identified the *DNMT3A* mutation was positively associated with *PTPN11* and *IDH2* mutations, but negatively associated with *CEBPA* mutation.(Hou*, et al* 2011) Intriguingly, the majority (97.1%) of the *DNMT3A*-mutated patients showed additional molecular alterations at diagnosis. This mutation renders poor OS among all AML patients, patients with a normal karyotype, and those with *FLT3*-ITD.(Hou*, et al* 2011, Ley*, et al* 2010, Thol*, et al* 2011) Importantly, *DNMT3A* mutation is an independent poor prognostic factor. Further, *DNMT3A* mutation is rather stable during disease progression and can be a potential biomarker for monitoring of MRD.(Hou*, et al* 2011)

#### **6. Gene mutations as markers to monitor Minimal Residual Disease (MRD)**

Since gene mutations are theoretically absent in healthy people and restricted in leukemia cells, it is reasonably to monitor MRD by detection of gene mutations. This is an advantage of leukemia over solid tumors in that leukemia cells are indigenous to blood and marrow, which are easy for access. There are two critical considerations of MRD monitoring by gene mutations: one is the stability of the mutations, and the other is the pattern of mutation. An

Genetic Alterations and Their Clinical Implications in Acute Myeloid Leukemia 173

Recently, ELN proposed a new classification to stratify AML patients into different risk groups according to cytogenetics and genetic alterations.(Dohner*, et al* 2010) In addition to CBF AML, CN-AML with mutated *NPM1* without *FLT3*-ITD and those with mutated *CEBPA* are categorized as favorable-risk groups; the regimen using repetitive cycles of HDAC as postremission therapy is considered beneficial for this group of patients. Allogeneic HSCT in first CR is not beneficial for CN-AML patients with mutated *NPM1* without *FLT3-*ITD*,*(Schlenk*, et al* 2008) and probably neither for those with mutated *CEBPA*. Allogeneic HSCT is generally not considered in patients with CBF AML in first CR, but may be indicated in those who harbor *KIT* mutations because such patients did poorly with chemotherapy. For the patients with adverse-risk genotype (other than mutated *NPM1* without *FLT3*/ITD or mutated *CEBPA*), an allogeneic HSCT from a matched related donor or even unrelated donor in first CR is suggested.(Basara*, et al* 2009, Cornelissen*, et al* 2007, Slovak*, et al* 2000, Suciu*, et al* 2003, Tallman*, et al* 2007) Recent studies showed that allogeneic HSCT may be considered in patients with *FLT3-*ITD even if definite results of prospective trials are not available.(Bornhauser*, et al* 2007, Gale*, et al* 2005, Schlenk*, et al* 2008) Besides, allogeneic HSCT also ameliorates the poor survival impact of *RUNX1* mutations on AML patients.(Gaidzik*, et al* 2011, Tang*, et al* 2009) The treatment of choice for patients with other recently documented poor-risk mutations, such as *WT1, TET2* and *DNMT3A* mutations is

In addition to chemotherapy and transplantation, targeted therapies aiming to specific molecular pathway are evolving as an adjunctive treatment in AML patients. *FLT3*/ITD and *FLT3*/TKD occur in about 20-35% of AML patients. Since FLT3 is a receptor tyrosine kinase and promote cancer phenotypes, it is an ideal target for therapy. Several FLT3 inhibitors, such as sorafenib, PKC-412 (midostaurin), sunitinib, semaxanib, tandutinib, AC220, KW-2449, and CEP701 (lestaurtinib) have been used in clinical trials and some effects were noticed in relapse/refractory setting.(Levis*, et al* 2002, Metzelder*, et al* 2009, Stone*, et al* 2005, Zhang*, et al* 2008) An ongoing international intergroup trial (10603 RATIFY), incorporating midostaurin into induction, consolidation or maintenance setting is currently underway. All-trans retinoic acid in combination with chemotherapy was found to be beneficial for *NPM1*-mutated patients (Burnett*, et al* 2010); however this preliminary result was not confirmed by the other study done on younger patients.(Schlenk*, et al* 2009) Tyrosine kinase inhibitor, such as imatinib, might be of clinical value in treatment of patients with *KIT* mutations.(Kindler*, et al* 2004, Kindler*, et al* 2003, Kohl*, et al* 2005) Epigenetic modification through demethylation agent azacitidine or decitibine may play a role in the treatment of patients with *MLL* rearrangement,(Altucci and Minucci 2009) and those with genetic alterations relating to epigenetic changes, such as *TET2* mutations.(Itzykson*, et al* 2011) Besides, recent report demonstrated that inhibition of glutaminase preferentially killed *IDH1*-mutated glial cells, which were more dependent on glutaminolysis pathway to supply α-KG, so glutaminase itself could be a potential therapeutical target.(Seltzer*, et al* 2010) Eventually, it may be reasonable to use combinations of molecularly targeted therapies and

This work was partially sponsored by grants from the National Science Council (NSC 97- 2314-B002-015-MY3, 98-2314-B-002-033-MY3, 100-2325-B-002-032, 100-2325-B-002-033 and 100-2628-B-002-003-MY3), National Health Research Institute (NHRI-EX97-9731BI),

chemotherapy to improve the clinical outcome in AML patients.

currently unclear.

**8. Acknowledgment** 

ideal MRD marker should be very consistent with disease status, while those that may disappear after disease evolution are not suitable for this purpose. Also, if the mutation appears as a point mutation, probably only qualitative rather than absolute quantitative measurement can be achieved because of inevitable background signals due to minimal sequence differences between wild-type and mutant alleles. Moreover, if the mutation occurs sporadically across the whole coding sequence without a hot spot, the absolute quantification techniques (usually fluorescence-based real-time PCR) would become very cumbersome.

Among the mutations in AML, *NPM1* mutation is perhaps the most useful and intensively studied marker of MRD because this mutation is quite stable, relatively prevalent, highly concentrated at a hot spot, and has 4 nucleotide insertion, which can be clearly discriminated from the wild-type allele in quantitative real-time PCR.(Chou*, et al* 2007, Schnittger*, et al* 2009) Studies have shown *NPM1* mutant levels reflect disease status, predict impending relapse, and bring prognostic implication.(Chou*, et al* 2007, Gorello*, et al* 2006, Kronke*, et al* 2011, Schnittger*, et al* 2009) Another stable marker is *IDH* mutation. *IDH1* and *IDH2* mutations are stable and highly consistent with disease status.(Chou*, et al* 2010a, Chou*, et al* 2011c) We have developed a single-tube, highly sensitive and specific PCR method to detect all *IDH1* mutations at R132 residue.(Chou*, et al* 2010c) However, the *IDH* mutation is not a good marker for MRD monitoring because the minimal difference between the point mutation and normal allele.

Other gene mutations are not readily applicable in MRD monitoring. *FLT3*-ITD is not stable. This mutation can disappear at disease relapse in a significant proportion of patients,(Chou*, et al* 2011b, Shih*, et al* 2002) although this length mutation can be readily and sensitively detected by GeneScan-based method.(Stirewalt and Radich 2003) *DNMT3A* mutation at R882, which occurs at a frequency of up to 60% of all *DNMT3A* mutation, can be a potential marker for qualitative assessment of MRD, but awaits for further testing.(Ley*, et al* 2010, Thol*, et al* 2011, Hou, *et al* 2011) *ASXL1* and *TET2* mutations do not have hot spots and are not stable during AML evolution. Other mutations have lower incidences and have not been well investigated in MRD monitoring.

#### **7. Risk-adapted treatment according to gene mutations in AML patients**

The ultimate goal of risk stratification according to molecular alterations is to explore personalized therapy, thereby reduce the risk of relapse and treatment-related side effects. How to integrate gene mutations into clinical management is a crucial issue. The choice between high-dose Cytarabine (HDAC) and allogeneic HSCT as the post-remission therapy is traditionally based on the cytogenetic risks and the patients' condition. The meta-analysis showed that allogeneic HSCT resulted in better clinical outcome in younger AML patients with intermediate- and unfavorable-risk cytogenetics in first CR.(Cornelissen*, et al* 2007, Koreth*, et al* 2009) Although allogeneic HSCT reduces the risk of relapse and is a curative approach for AML patients, the higher rate of transplantation related morbidity and mortality counterbalances its beneficial effect. Thus, allogeneic HSCT is currently recommended only in those patients with acceptable benefit-risk ratio. Given that AML is a heterogenous disease especially in intermediate-risk cytogenetics and CN-AML, increasing understanding of novel molecular genetic markers in AML leukemogenesis can further help to reassess the value of HSCT in different prognostic groups.

ideal MRD marker should be very consistent with disease status, while those that may disappear after disease evolution are not suitable for this purpose. Also, if the mutation appears as a point mutation, probably only qualitative rather than absolute quantitative measurement can be achieved because of inevitable background signals due to minimal sequence differences between wild-type and mutant alleles. Moreover, if the mutation occurs sporadically across the whole coding sequence without a hot spot, the absolute quantification techniques (usually fluorescence-based real-time PCR) would become very

Among the mutations in AML, *NPM1* mutation is perhaps the most useful and intensively studied marker of MRD because this mutation is quite stable, relatively prevalent, highly concentrated at a hot spot, and has 4 nucleotide insertion, which can be clearly discriminated from the wild-type allele in quantitative real-time PCR.(Chou*, et al* 2007, Schnittger*, et al* 2009) Studies have shown *NPM1* mutant levels reflect disease status, predict impending relapse, and bring prognostic implication.(Chou*, et al* 2007, Gorello*, et al* 2006, Kronke*, et al* 2011, Schnittger*, et al* 2009) Another stable marker is *IDH* mutation. *IDH1* and *IDH2* mutations are stable and highly consistent with disease status.(Chou*, et al* 2010a, Chou*, et al* 2011c) We have developed a single-tube, highly sensitive and specific PCR method to detect all *IDH1* mutations at R132 residue.(Chou*, et al* 2010c) However, the *IDH* mutation is not a good marker for MRD monitoring because the minimal difference between

Other gene mutations are not readily applicable in MRD monitoring. *FLT3*-ITD is not stable. This mutation can disappear at disease relapse in a significant proportion of patients,(Chou*, et al* 2011b, Shih*, et al* 2002) although this length mutation can be readily and sensitively detected by GeneScan-based method.(Stirewalt and Radich 2003) *DNMT3A* mutation at R882, which occurs at a frequency of up to 60% of all *DNMT3A* mutation, can be a potential marker for qualitative assessment of MRD, but awaits for further testing.(Ley*, et al* 2010, Thol*, et al* 2011, Hou, *et al* 2011) *ASXL1* and *TET2* mutations do not have hot spots and are not stable during AML evolution. Other mutations have lower incidences and have not

**7. Risk-adapted treatment according to gene mutations in AML patients** 

The ultimate goal of risk stratification according to molecular alterations is to explore personalized therapy, thereby reduce the risk of relapse and treatment-related side effects. How to integrate gene mutations into clinical management is a crucial issue. The choice between high-dose Cytarabine (HDAC) and allogeneic HSCT as the post-remission therapy is traditionally based on the cytogenetic risks and the patients' condition. The meta-analysis showed that allogeneic HSCT resulted in better clinical outcome in younger AML patients with intermediate- and unfavorable-risk cytogenetics in first CR.(Cornelissen*, et al* 2007, Koreth*, et al* 2009) Although allogeneic HSCT reduces the risk of relapse and is a curative approach for AML patients, the higher rate of transplantation related morbidity and mortality counterbalances its beneficial effect. Thus, allogeneic HSCT is currently recommended only in those patients with acceptable benefit-risk ratio. Given that AML is a heterogenous disease especially in intermediate-risk cytogenetics and CN-AML, increasing understanding of novel molecular genetic markers in AML leukemogenesis can further help

cumbersome.

the point mutation and normal allele.

been well investigated in MRD monitoring.

to reassess the value of HSCT in different prognostic groups.

Recently, ELN proposed a new classification to stratify AML patients into different risk groups according to cytogenetics and genetic alterations.(Dohner*, et al* 2010) In addition to CBF AML, CN-AML with mutated *NPM1* without *FLT3*-ITD and those with mutated *CEBPA* are categorized as favorable-risk groups; the regimen using repetitive cycles of HDAC as postremission therapy is considered beneficial for this group of patients. Allogeneic HSCT in first CR is not beneficial for CN-AML patients with mutated *NPM1* without *FLT3-*ITD*,*(Schlenk*, et al* 2008) and probably neither for those with mutated *CEBPA*. Allogeneic HSCT is generally not considered in patients with CBF AML in first CR, but may be indicated in those who harbor *KIT* mutations because such patients did poorly with chemotherapy. For the patients with adverse-risk genotype (other than mutated *NPM1* without *FLT3*/ITD or mutated *CEBPA*), an allogeneic HSCT from a matched related donor or even unrelated donor in first CR is suggested.(Basara*, et al* 2009, Cornelissen*, et al* 2007, Slovak*, et al* 2000, Suciu*, et al* 2003, Tallman*, et al* 2007) Recent studies showed that allogeneic HSCT may be considered in patients with *FLT3-*ITD even if definite results of prospective trials are not available.(Bornhauser*, et al* 2007, Gale*, et al* 2005, Schlenk*, et al* 2008) Besides, allogeneic HSCT also ameliorates the poor survival impact of *RUNX1* mutations on AML patients.(Gaidzik*, et al* 2011, Tang*, et al* 2009) The treatment of choice for patients with other recently documented poor-risk mutations, such as *WT1, TET2* and *DNMT3A* mutations is currently unclear.

In addition to chemotherapy and transplantation, targeted therapies aiming to specific molecular pathway are evolving as an adjunctive treatment in AML patients. *FLT3*/ITD and *FLT3*/TKD occur in about 20-35% of AML patients. Since FLT3 is a receptor tyrosine kinase and promote cancer phenotypes, it is an ideal target for therapy. Several FLT3 inhibitors, such as sorafenib, PKC-412 (midostaurin), sunitinib, semaxanib, tandutinib, AC220, KW-2449, and CEP701 (lestaurtinib) have been used in clinical trials and some effects were noticed in relapse/refractory setting.(Levis*, et al* 2002, Metzelder*, et al* 2009, Stone*, et al* 2005, Zhang*, et al* 2008) An ongoing international intergroup trial (10603 RATIFY), incorporating midostaurin into induction, consolidation or maintenance setting is currently underway. All-trans retinoic acid in combination with chemotherapy was found to be beneficial for *NPM1*-mutated patients (Burnett*, et al* 2010); however this preliminary result was not confirmed by the other study done on younger patients.(Schlenk*, et al* 2009) Tyrosine kinase inhibitor, such as imatinib, might be of clinical value in treatment of patients with *KIT* mutations.(Kindler*, et al* 2004, Kindler*, et al* 2003, Kohl*, et al* 2005) Epigenetic modification through demethylation agent azacitidine or decitibine may play a role in the treatment of patients with *MLL* rearrangement,(Altucci and Minucci 2009) and those with genetic alterations relating to epigenetic changes, such as *TET2* mutations.(Itzykson*, et al* 2011) Besides, recent report demonstrated that inhibition of glutaminase preferentially killed *IDH1*-mutated glial cells, which were more dependent on glutaminolysis pathway to supply α-KG, so glutaminase itself could be a potential therapeutical target.(Seltzer*, et al* 2010) Eventually, it may be reasonable to use combinations of molecularly targeted therapies and chemotherapy to improve the clinical outcome in AML patients.

#### **8. Acknowledgment**

This work was partially sponsored by grants from the National Science Council (NSC 97- 2314-B002-015-MY3, 98-2314-B-002-033-MY3, 100-2325-B-002-032, 100-2325-B-002-033 and 100-2628-B-002-003-MY3), National Health Research Institute (NHRI-EX97-9731BI),

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**10** 

Olga Blau

*Germany* 

**Bone Marrow Microenvironment** 

Acute myeloid leukemia (AML) arises from a series of genetic abnormalities in a stem or progenitor cell that lead to uncontrolled growth. Data from the past few decades have implicated the hematopoietic microenvironment (HM) in the pathogenesis of hematologic malignancies (Ramakrishnan et al., 2009). Hematopoietic stem cells (HSCs) live in a highly specialized complex microenvironment, also known as a niche (Scadden et al., 2007; Konopleva et al., 2009). Two distinct microenvironmental niches defined: "osteoblastic (endosteal)" and "vascular" niches (Perry and Li, 2007). Recent studies suggest that these niches work together. Coordination between the osteoblastic and vascular niches regulates HSC selfrenewal, proliferation, differentiation and mobilization in and out of the bone marrow (BM). HSCs leave the osteoblastic niche, mobilize to the vascular niche, and enter the blood vessel. They subsequently may undergo transendothelial migration from the peripheral circulation and return first to the vascular niche and then to the osteoblastic niche (Lapidot et al., 2005; Cancelas and Williams, 2006). Within the niche, there are critical bidirectional signals that ensure the regulation of normal HSCs (Calvi et al., 2003) and maintenance of the quiescent long-term HSC pool (Fleming et al., 2008). The quiescent fraction of immunophenotypically defined HSCs has been previously demonstrated to correlate with long-term repopulating ability of BM (Passegue et al., 2005) and loss of this fraction is associated with inability to sustain serial transplantation, the most stringent *in*

The HM consists of a complex structure of both non-hematopoietic and hematopoietic cells, extracellular matrix as well as soluble and membrane bound factors that cooperate to support normal hematopoiesis. It was known as early as the 1960s, based on experiments on mice, that normal hematopoiesis could not occur without a supportive environment (Russell et al., 1979). In vitro studies of the HM over the last several decades have mostly relied on

The key component of the HM is mesenchymal stromal cells (MSC). These plastic-adherent cells currently described as mesenchymal stem cells are termed multipotent mesenchymal stromal cells, while the term mesenchymal stem cell should be reserved for a subset of these cells that demonstrate stem cell activity by clearly stated criteria (Horowitz et al., 2005). MSCs are primitive cells originating from the mesodermal germ layer and were classically described to give rise to connective tissues, skeletal muscle cells, and cells of the vascular system. Friedenstein and colleagues (1974) first described MSC as fibroblast-like cells that

the long-term marrow culture system, first reported by Dexter (1977).

**1. Introduction** 

*vivo* assay of self-renewal (Fleming et al., 2008).

**in the Pathogenesis of AML** 

*Charité University School of Medicine* 

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### **Bone Marrow Microenvironment in the Pathogenesis of AML**

Olga Blau

*Charité University School of Medicine Germany* 

#### **1. Introduction**

184 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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3754.

Acute myeloid leukemia (AML) arises from a series of genetic abnormalities in a stem or progenitor cell that lead to uncontrolled growth. Data from the past few decades have implicated the hematopoietic microenvironment (HM) in the pathogenesis of hematologic malignancies (Ramakrishnan et al., 2009). Hematopoietic stem cells (HSCs) live in a highly specialized complex microenvironment, also known as a niche (Scadden et al., 2007; Konopleva et al., 2009). Two distinct microenvironmental niches defined: "osteoblastic (endosteal)" and "vascular" niches (Perry and Li, 2007). Recent studies suggest that these niches work together. Coordination between the osteoblastic and vascular niches regulates HSC selfrenewal, proliferation, differentiation and mobilization in and out of the bone marrow (BM). HSCs leave the osteoblastic niche, mobilize to the vascular niche, and enter the blood vessel. They subsequently may undergo transendothelial migration from the peripheral circulation and return first to the vascular niche and then to the osteoblastic niche (Lapidot et al., 2005; Cancelas and Williams, 2006). Within the niche, there are critical bidirectional signals that ensure the regulation of normal HSCs (Calvi et al., 2003) and maintenance of the quiescent long-term HSC pool (Fleming et al., 2008). The quiescent fraction of immunophenotypically defined HSCs has been previously demonstrated to correlate with long-term repopulating ability of BM (Passegue et al., 2005) and loss of this fraction is associated with inability to sustain serial transplantation, the most stringent *in vivo* assay of self-renewal (Fleming et al., 2008).

The HM consists of a complex structure of both non-hematopoietic and hematopoietic cells, extracellular matrix as well as soluble and membrane bound factors that cooperate to support normal hematopoiesis. It was known as early as the 1960s, based on experiments on mice, that normal hematopoiesis could not occur without a supportive environment (Russell et al., 1979). In vitro studies of the HM over the last several decades have mostly relied on the long-term marrow culture system, first reported by Dexter (1977).

The key component of the HM is mesenchymal stromal cells (MSC). These plastic-adherent cells currently described as mesenchymal stem cells are termed multipotent mesenchymal stromal cells, while the term mesenchymal stem cell should be reserved for a subset of these cells that demonstrate stem cell activity by clearly stated criteria (Horowitz et al., 2005). MSCs are primitive cells originating from the mesodermal germ layer and were classically described to give rise to connective tissues, skeletal muscle cells, and cells of the vascular system. Friedenstein and colleagues (1974) first described MSC as fibroblast-like cells that

Bone Marrow Microenvironment in the Pathogenesis of AML 187

(Sugiyama et al. 2006). These reticular cells, defined as CXCL12-abundant reticular cells, may serve as a transit pathway for shuttling HSC between the osteoblastic and vascular niches, where essential but different maintenance signals are provided (Perry and Li, 2007). The molecular interactions between HC and MSC involve ligand-receptor relationship between adhesion molecules on the surface of HC and stromal cells or between such molecules on the cells surface with specific domains within certain extracellular matrix molecules. BM engraftment involves subsequent cell-to-cell interactions through the MSCproduced complex extracellular matrix (ECM) (Zuckerman and Wicha, 1983; Wight et al., 1986). Vascular cell-adhesion molecule-1 (VCAM-1) or fibronectin is critical for adhesion to the MSC (Miyake et al., 1991; Garcia-Gila et al., 2002). One very important type of interaction between the MSC and the HSC is the synthesis and presentation by MSC of hematopoietic growth factors. Interactions of HSC with stromal elements of BM play a role

Whether MSC alterations influence hematological disorders and how such alterations contribute to the progression of the disease remains controversial. The molecular mechanisms for maintaining quiescence of normal stem cells may also facilitate leukemia stem cells (LSC) survival. Whereas LSC share certain features of self-renewal and differentiation with HSC, LSC differ in their deregulated proliferation and ability to invade and spread. LSC exhibit the capacity for long-term self-renewal (Holyoake et al., 2002; Warner et al., 2004; Liesveld et al., 2004) within the BM microenvironment, which is required for maintenance of the malignant clone (Braun and Shannon, 2008). LSCs are able to generate leukemic blasts, and the leukemic clone is organized as a hierarchy (Zhang et al., 2003). LSCs may steal the homeostatic mechanisms, take refuge within the HM during chemotherapy, and consequently contribute to eventual disease relapse (Warner et al., 2004; Lane et al., 2009). Consecutively, LSC are believed to arise through transforming events targeting HSC, which allow growth-independent survival and proliferation. MSC are capable of promoting the growth, survival and drug resistance of leukemic cells by providing the necessary cytokines and cell contact-mediated signals to LSC (Dazzi et al., 2006; Ramasamy et al., 2007). There is increasing evidence that microenvironment alterations may be important and pathogenic in leukemia leading to enhanced stem cell mobilization and the creation of alternate niches (Lataillade et al., 2008). Recent data indicate that, in parallel with leukemogenic events in the hematopoietic system, the niche is converted into an environment with dominant signals favoring cell proliferation and growth. In some cases, a combination of these events may be required (Li and Neaves, 2006). Therefore, LSC may receive the support of a BM niche for their survival and may in turn influence deregulation of the BM niche by their dominant proliferation-promoting signals. AML may arise in an abnormal HM, resulting in the generation of multiple populations with varying initiation events. Ninomiya et al. (2007) modeled the homing, proliferation, and survival sites of human leukemia cells and of cord blood CD34+ cells. The transplanted leukemia cells initially localized on the surface of osteoblasts in the epiphysial region and then expanded to the inner vascular and diaphysial regions. 8 weeks after transplantation, the number of leukemia cells transiently increased by as much as 50%, predominantly in the epiphysial region. After administration of high-dose cytarabine, residual leukemia cells clustered and adhered to the blood vessels as well as to the endosteum, suggesting that leukemia cells receive anti-apoptotic signals not only from osteoblasts but also from

in the egress of mature blood cells from the BM (Chamberlain et al., 2007).

vascular endothelium (Ninomiya et al., 2007).

could be isolated from BM via inherent adherence to plastic in culture. He defined a population of cells as multipotential stromal precursor cells that were spindle-shaped and clonogenic in culture conditions, defining them as colony-forming unit fibroblasts. MSCs, in the traditional view, should refer to stem cells that are also capable of producing blood cells; however, blood cells are actually derived from a distinct cell population called the hematopoietic stem cells. This allows classified MSC as nonhematopoietic, multipotential stem cells that are capable of differentiating into mesenchymal and non-mesenchymal cell lineages (Chamberlain et al., 2007). These cells were able to differentiate into adipocytes, chondrocytes, osteocytes, and myoblasts, both in vitro and in vivo. In addition, it has also been demonstrated that MSCs are capable of differentiating into cardiomyocytes, neurons, and astrocytes in vitro and in vivo (Pittenger et al., 1999; Jori et al., 2005; Beyer Nardi et al., 2006; Tokcaer-Keskin et al., 2009). By generating functionally distinct cell types and structures, MSC play a crucial role in supporting hematopoiesis as key components of the HM (Sacchetti et al., 2007).

Phenotypically MSCs express a number of markers, none of which are specific only to MSCs. It is generally agreed that adult human MSCs do not express the hematopoietic markers CD45, CD34, CD14, or CD11. They also do not express the costimulatory molecules CD80, CD86, or CD40 or the adhesion molecules CD31, CD18, or CD56, but they can express CD105 (SH2), CD73 (SH3/4), CD44, CD90 (Thy-1), CD71, and Stro-1 as well as the adhesion molecules CD106, CD166, intercellular adhesion molecule, and CD29 (Sordi et al., 2005; Chamberlain et al., 2007). Although there are no unique cell surface markers for the identification of MSCs, minimal criteria to define human MSC have been published. According to such criteria, MSC must be plastic-adherent; and have to express CD105, CD90 and CD73; they must lack expression of CD45, CD34 and CD14; and they must show in vitro differentiation capabilities into osteoblasts, adipocytes and chondroblasts (Horowitz et al., 2005; Chamberlain et al., 2007). This *in vitro* system has allowed for the dissection of the components of the microenvironment and the study of the complex contact dependent and contact independent interactions that occur between the stromal compartment and hematopoietic stem cells that regulate stem cell fate decisions.

Normal hematopoiesis requires complex bidirectional interactions between the HM and HSCs. The HM can regulate hematopoiesis by interacting directly with HC and/or by secreting regulatory molecules that exert a positive or negative influence on the growth of HC. These interactions influence HSC self-renewal. HM controls the formation of blood cells through the production and secretion of cytokines, chemokines, and intracellular signals initiated by cellular adhesion (Konopleva et al., 2009). Chemokines are a large superfamily of small glycoproteins that are required in a various series of biological processes, including leukocyte trafficking, hematopoiesis, angiogenesis, and organogenesis. MSCs have the ability to migrate into tissues from the circulation, possibly in response to signals that are upregulated under injury conditions. Although the mechanisms by which MSCs are recruited to tissues and cross the endothelial cell layer are not yet fully understood, it is probable that chemokines and their receptors are involved, as they are important factors known to control cell migration (Chamberlain et al., 2007).

CXCL12/stromal cell-derived factor-1alpha (SDF-1α) and its receptor CXCR4 are involved in homing of HSC into BM (Abkowitz et al., 2003; Broxmeyer et al., 2005; Morrison and Spradling, 2008). Perivascular reticular cells secrete much higher levels of CXCL12 than other constitutive sources of CXCL12, such as osteoblasts, fibroblasts, and endothelial cells

could be isolated from BM via inherent adherence to plastic in culture. He defined a population of cells as multipotential stromal precursor cells that were spindle-shaped and clonogenic in culture conditions, defining them as colony-forming unit fibroblasts. MSCs, in the traditional view, should refer to stem cells that are also capable of producing blood cells; however, blood cells are actually derived from a distinct cell population called the hematopoietic stem cells. This allows classified MSC as nonhematopoietic, multipotential stem cells that are capable of differentiating into mesenchymal and non-mesenchymal cell lineages (Chamberlain et al., 2007). These cells were able to differentiate into adipocytes, chondrocytes, osteocytes, and myoblasts, both in vitro and in vivo. In addition, it has also been demonstrated that MSCs are capable of differentiating into cardiomyocytes, neurons, and astrocytes in vitro and in vivo (Pittenger et al., 1999; Jori et al., 2005; Beyer Nardi et al., 2006; Tokcaer-Keskin et al., 2009). By generating functionally distinct cell types and structures, MSC play a crucial role in supporting hematopoiesis as key components of the

Phenotypically MSCs express a number of markers, none of which are specific only to MSCs. It is generally agreed that adult human MSCs do not express the hematopoietic markers CD45, CD34, CD14, or CD11. They also do not express the costimulatory molecules CD80, CD86, or CD40 or the adhesion molecules CD31, CD18, or CD56, but they can express CD105 (SH2), CD73 (SH3/4), CD44, CD90 (Thy-1), CD71, and Stro-1 as well as the adhesion molecules CD106, CD166, intercellular adhesion molecule, and CD29 (Sordi et al., 2005; Chamberlain et al., 2007). Although there are no unique cell surface markers for the identification of MSCs, minimal criteria to define human MSC have been published. According to such criteria, MSC must be plastic-adherent; and have to express CD105, CD90 and CD73; they must lack expression of CD45, CD34 and CD14; and they must show in vitro differentiation capabilities into osteoblasts, adipocytes and chondroblasts (Horowitz et al., 2005; Chamberlain et al., 2007). This *in vitro* system has allowed for the dissection of the components of the microenvironment and the study of the complex contact dependent and contact independent interactions that occur between the stromal compartment and

Normal hematopoiesis requires complex bidirectional interactions between the HM and HSCs. The HM can regulate hematopoiesis by interacting directly with HC and/or by secreting regulatory molecules that exert a positive or negative influence on the growth of HC. These interactions influence HSC self-renewal. HM controls the formation of blood cells through the production and secretion of cytokines, chemokines, and intracellular signals initiated by cellular adhesion (Konopleva et al., 2009). Chemokines are a large superfamily of small glycoproteins that are required in a various series of biological processes, including leukocyte trafficking, hematopoiesis, angiogenesis, and organogenesis. MSCs have the ability to migrate into tissues from the circulation, possibly in response to signals that are upregulated under injury conditions. Although the mechanisms by which MSCs are recruited to tissues and cross the endothelial cell layer are not yet fully understood, it is probable that chemokines and their receptors are involved, as they are important factors

CXCL12/stromal cell-derived factor-1alpha (SDF-1α) and its receptor CXCR4 are involved in homing of HSC into BM (Abkowitz et al., 2003; Broxmeyer et al., 2005; Morrison and Spradling, 2008). Perivascular reticular cells secrete much higher levels of CXCL12 than other constitutive sources of CXCL12, such as osteoblasts, fibroblasts, and endothelial cells

hematopoietic stem cells that regulate stem cell fate decisions.

known to control cell migration (Chamberlain et al., 2007).

HM (Sacchetti et al., 2007).

(Sugiyama et al. 2006). These reticular cells, defined as CXCL12-abundant reticular cells, may serve as a transit pathway for shuttling HSC between the osteoblastic and vascular niches, where essential but different maintenance signals are provided (Perry and Li, 2007).

The molecular interactions between HC and MSC involve ligand-receptor relationship between adhesion molecules on the surface of HC and stromal cells or between such molecules on the cells surface with specific domains within certain extracellular matrix molecules. BM engraftment involves subsequent cell-to-cell interactions through the MSCproduced complex extracellular matrix (ECM) (Zuckerman and Wicha, 1983; Wight et al., 1986). Vascular cell-adhesion molecule-1 (VCAM-1) or fibronectin is critical for adhesion to the MSC (Miyake et al., 1991; Garcia-Gila et al., 2002). One very important type of interaction between the MSC and the HSC is the synthesis and presentation by MSC of hematopoietic growth factors. Interactions of HSC with stromal elements of BM play a role in the egress of mature blood cells from the BM (Chamberlain et al., 2007).

Whether MSC alterations influence hematological disorders and how such alterations contribute to the progression of the disease remains controversial. The molecular mechanisms for maintaining quiescence of normal stem cells may also facilitate leukemia stem cells (LSC) survival. Whereas LSC share certain features of self-renewal and differentiation with HSC, LSC differ in their deregulated proliferation and ability to invade and spread. LSC exhibit the capacity for long-term self-renewal (Holyoake et al., 2002; Warner et al., 2004; Liesveld et al., 2004) within the BM microenvironment, which is required for maintenance of the malignant clone (Braun and Shannon, 2008). LSCs are able to generate leukemic blasts, and the leukemic clone is organized as a hierarchy (Zhang et al., 2003). LSCs may steal the homeostatic mechanisms, take refuge within the HM during chemotherapy, and consequently contribute to eventual disease relapse (Warner et al., 2004; Lane et al., 2009). Consecutively, LSC are believed to arise through transforming events targeting HSC, which allow growth-independent survival and proliferation. MSC are capable of promoting the growth, survival and drug resistance of leukemic cells by providing the necessary cytokines and cell contact-mediated signals to LSC (Dazzi et al., 2006; Ramasamy et al., 2007). There is increasing evidence that microenvironment alterations may be important and pathogenic in leukemia leading to enhanced stem cell mobilization and the creation of alternate niches (Lataillade et al., 2008). Recent data indicate that, in parallel with leukemogenic events in the hematopoietic system, the niche is converted into an environment with dominant signals favoring cell proliferation and growth. In some cases, a combination of these events may be required (Li and Neaves, 2006). Therefore, LSC may receive the support of a BM niche for their survival and may in turn influence deregulation of the BM niche by their dominant proliferation-promoting signals.

AML may arise in an abnormal HM, resulting in the generation of multiple populations with varying initiation events. Ninomiya et al. (2007) modeled the homing, proliferation, and survival sites of human leukemia cells and of cord blood CD34+ cells. The transplanted leukemia cells initially localized on the surface of osteoblasts in the epiphysial region and then expanded to the inner vascular and diaphysial regions. 8 weeks after transplantation, the number of leukemia cells transiently increased by as much as 50%, predominantly in the epiphysial region. After administration of high-dose cytarabine, residual leukemia cells clustered and adhered to the blood vessels as well as to the endosteum, suggesting that leukemia cells receive anti-apoptotic signals not only from osteoblasts but also from vascular endothelium (Ninomiya et al., 2007).

Bone Marrow Microenvironment in the Pathogenesis of AML 189

and the HM (Matsuoka et al., 2008) demonstrated that the tumor suppressor Fbxw7, which negatively regulates cyclin E, Notch, and c-Myc protein levels, plays a role in maintaining HSC quiescence and repressing potential oncogenic activity of HSC. Notably, Notch ligand Jagged is expressed by the HSC niche, and Jagged/Notch activation results in increased

Evidence from research conducted over the last few decades has clearly implicated abnormalities of the marrow microenvironment in the pathophysiology of hematologic malignancies. Marcondes et al. (2008) demonstrated that MSC derived from patients with MDS, in contrast to that from more advanced stages of MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls, and this constitutive IL-32 expression promoted apoptosis in MDS cells, reproducing the inefficient hematopoiesis and extensive apoptosis in MDS marrow. These findings indicate that stroma-produced IL-32 could contribute to the pathophysiology of MDS, and serve as a therapeutic target. Furthermore, this modified microenvironment phenotype was reproduced when the MSC were exposed to TNFα,

There are significant data to support mechanism, in which the malignant hematopoietic clone induces reversible functional changes in the HM that result in improved growth conditions for the malignant cells. Gene expression changes occurred in the stroma cell lines, HS5 and HS27a, derived from normal marrow in response to TNFα exposure (Stirewalt et al., 2008), known to be up-regulated in the bone marrow of patients with MDS. Previous experiments showed that interactions between MSC and HSC were required for TNFα to

Recent discoveries utilizing mouse models have provided the first experimental evidence for genetic changes in the HM contributing to or required for leukemogenesis. Raaijmakers et al. (2010) using transgenic mice showed that genetic alteration of HM can induce MDS with ineffective hematopoiesis and dysmorphic HCs, and with occasional transformation to AML. The authors used *Dicer1* deletion as a means of altering several gene products in subsets of mesenchymal osteolineage cells. *Dicer1* is an RNase III endonuclease essential for microRNA biogenesis (Bartel, 2004) and RNA processing (Krol et al., 2007), that regulates haematopoietic cell fate (Lu et al., 2008). Global repression of microRNA maturation by *Dicer1* deletion promotes cellular transformation and tumorigenesis (Kumar et al., 2007). Raaijmakers et al. (2010) show that deletion of *Dicer1* in HM cells of mouse may be sufficient to initiate a complex change of homeostasis with similarities to myelodysplasia. The authors demonstrated that the ability of HM abnormality to result in the emergence of a clonal neoplasm in a cell type of clearly distinct lineage with distinct secondary genetic changes

Previously, Walkley et al (2007a, 2007b) demonstrated that conditional deletion of the Retinoblastoma gene (RB) in the BM microenvironment can contribute to the development of pre-leukemic myeloproliferative disease in mice. They showed that this was a result of interactions between myeloid cells and the microenvironment. The defect had to be present in both hematopoietic cells and the microenvironment to initiate disease. Widespread inactivation of RB, a central regulator of the cell cycle and a tumor suppressor, resulted in extramedullary hematopoiesis and myeloproliferative disease in the murine hematopoietic system. However, myeloid-specific loss of RB did not induce myeloproliferative disease or HSC abnormalities. Therefore, the myeloproliferative-like disorder in the RB mutants is the result of perturbed interactions between hematopoietic cells and the BM microenvironment

HSC number and niche expansion (Calvi et al., 2003).

known to be produced at high levels by MDS cells.

trigger apoptosis in hematopoietic cells (Goda et al., 2006).

(Raaijmakers et al., 2010).

Several studies have proposed that important quantitative and functional alterations occur in MSCs of patients with different hematological disorders (Borojevic et al., 2004; Flores-Figueroa et al., 2008). In some disorders, such as multiple myeloma, MSC show alterations in the expression of some cell adhesion molecules and cytokines, and reduced immunosuppressive efficiency (Wallace et al., 2001; Arnulf et al., 2007; Corre et al., 2007). Neoplastic plasma cells communicate with the environment through cell/cell contact as well cytokines to induce functional changes that support the malignant population (Mitsiades et al., 2006; Podar et al., 2007). In myeloproliferative disorders, has been shown, that megakaryocytes and macrophages play a principle role in the pathogenesis of the fibrotic reaction by secreting PDGF, FGF and TGFα cytokines (Chagraoui et al., 2006). In chronic myeloid leukemia (CML), Bhatia (1995) showed that MSC did not provide optimal support for normal hematopoietic cells. In contrast, growth of CML cells on CML-derived stroma was significantly better, suggesting that the microenvironment in CML was more supportive for the malignant clone. Using fluorescent activated cell sorting (FACS) and fluorescent in situ hybridization (FISH), it was determined that stromal macrophages were all bcr-abl positive and were directly responsible for the selective advantage of clonal bcr-abl cells to proliferate through a contact-dependent mechanism (Bhatia et al. 1995). Interestingly, other researches estimated that CML-derived MSC do not express the bcr-abl gene (Zhao et al., 2006; Jootar et al., 2006). In myelodysplastic syndrome (MDS) MSC show alterations in the levels of TNFα (Deeg et al., 2000). Furthermore, the MDS-derived monocytes respond abnormally to stromal signals, MDS monocytes fail to upregulate matrix metalloproteinase-9 (MMP-9) expression when exposed to stromal signals (Iwata et al., 2007). MMP-9 has been implicated in the cleavage of SDF1 from the microenvironment and may facilitate the egress of HCs from the BM to the peripheral blood (Heissig et al., 2002). In the solid tumors, tumor-derived MSC shown acquire aberrant methylation patterns due either to direct contact with or via factors secreted by the malignant cells (Hanson et al., 2006; Fiegl et al., 2006).

Dysfunction of a BM niche may contribute to leukemogenesis by supplying abundant growth factors that promote proliferation and/or inhibit apoptosis (Jones and Wagers, 2008). MSCs seem to have a relevant role in AML as they prevent spontaneous and induced apoptosis and may attenuate chemotherapy-induced cell death. This possibility has been confirmed by the finding that co-cultivation of a leukemic cell line with the murine stroma cell line MS-5 can block apoptosis (Konopleva et al., 2002).

The significance role of the HM in initiation of leukemia has been suggested by studies with mice deficient in phosphatase and tensin homolog (PTEN) (Yilmaz et al., 2006). PTEN deficiency in both HSC and the HM resulted in myeloproliferation that progressed to overt leukemia/lymphoma. However, inducible PTEN deletion in HSC in the presence of a wild type HM promoted HSC depletion without evidence of myeloproliferation or leukemic development. These results suggest that PTEN deficiency in HSC alone is not sufficient for malignant transformation. Rupec et al. (2005) reported that activation of NF-kB in myelopoietic cells and the absence of its inhibitor IκBα are not sufficient for induction of hypergranulopoiesis, but these changes in the non-hematopoietic compartment, such as fetal liver, resulted in increased numbers of dysplastic hematopoietic cells with progression into secondary AML. These results indicate that non-hematopoietic cells with inactive IκBα can initiate premalignant hematopoietic disorder, conceivably via activation of the Notch pathway. Additional studies indicate the role of Notch signaling in the interactions of HSC

Several studies have proposed that important quantitative and functional alterations occur in MSCs of patients with different hematological disorders (Borojevic et al., 2004; Flores-Figueroa et al., 2008). In some disorders, such as multiple myeloma, MSC show alterations in the expression of some cell adhesion molecules and cytokines, and reduced immunosuppressive efficiency (Wallace et al., 2001; Arnulf et al., 2007; Corre et al., 2007). Neoplastic plasma cells communicate with the environment through cell/cell contact as well cytokines to induce functional changes that support the malignant population (Mitsiades et al., 2006; Podar et al., 2007). In myeloproliferative disorders, has been shown, that megakaryocytes and macrophages play a principle role in the pathogenesis of the fibrotic reaction by secreting PDGF, FGF and TGFα cytokines (Chagraoui et al., 2006). In chronic myeloid leukemia (CML), Bhatia (1995) showed that MSC did not provide optimal support for normal hematopoietic cells. In contrast, growth of CML cells on CML-derived stroma was significantly better, suggesting that the microenvironment in CML was more supportive for the malignant clone. Using fluorescent activated cell sorting (FACS) and fluorescent in situ hybridization (FISH), it was determined that stromal macrophages were all bcr-abl positive and were directly responsible for the selective advantage of clonal bcr-abl cells to proliferate through a contact-dependent mechanism (Bhatia et al. 1995). Interestingly, other researches estimated that CML-derived MSC do not express the bcr-abl gene (Zhao et al., 2006; Jootar et al., 2006). In myelodysplastic syndrome (MDS) MSC show alterations in the levels of TNFα (Deeg et al., 2000). Furthermore, the MDS-derived monocytes respond abnormally to stromal signals, MDS monocytes fail to upregulate matrix metalloproteinase-9 (MMP-9) expression when exposed to stromal signals (Iwata et al., 2007). MMP-9 has been implicated in the cleavage of SDF1 from the microenvironment and may facilitate the egress of HCs from the BM to the peripheral blood (Heissig et al., 2002). In the solid tumors, tumor-derived MSC shown acquire aberrant methylation patterns due either to direct contact with or via factors secreted by the malignant cells (Hanson et al.,

Dysfunction of a BM niche may contribute to leukemogenesis by supplying abundant growth factors that promote proliferation and/or inhibit apoptosis (Jones and Wagers, 2008). MSCs seem to have a relevant role in AML as they prevent spontaneous and induced apoptosis and may attenuate chemotherapy-induced cell death. This possibility has been confirmed by the finding that co-cultivation of a leukemic cell line with the murine stroma

The significance role of the HM in initiation of leukemia has been suggested by studies with mice deficient in phosphatase and tensin homolog (PTEN) (Yilmaz et al., 2006). PTEN deficiency in both HSC and the HM resulted in myeloproliferation that progressed to overt leukemia/lymphoma. However, inducible PTEN deletion in HSC in the presence of a wild type HM promoted HSC depletion without evidence of myeloproliferation or leukemic development. These results suggest that PTEN deficiency in HSC alone is not sufficient for malignant transformation. Rupec et al. (2005) reported that activation of NF-kB in myelopoietic cells and the absence of its inhibitor IκBα are not sufficient for induction of hypergranulopoiesis, but these changes in the non-hematopoietic compartment, such as fetal liver, resulted in increased numbers of dysplastic hematopoietic cells with progression into secondary AML. These results indicate that non-hematopoietic cells with inactive IκBα can initiate premalignant hematopoietic disorder, conceivably via activation of the Notch pathway. Additional studies indicate the role of Notch signaling in the interactions of HSC

cell line MS-5 can block apoptosis (Konopleva et al., 2002).

2006; Fiegl et al., 2006).

and the HM (Matsuoka et al., 2008) demonstrated that the tumor suppressor Fbxw7, which negatively regulates cyclin E, Notch, and c-Myc protein levels, plays a role in maintaining HSC quiescence and repressing potential oncogenic activity of HSC. Notably, Notch ligand Jagged is expressed by the HSC niche, and Jagged/Notch activation results in increased HSC number and niche expansion (Calvi et al., 2003).

Evidence from research conducted over the last few decades has clearly implicated abnormalities of the marrow microenvironment in the pathophysiology of hematologic malignancies. Marcondes et al. (2008) demonstrated that MSC derived from patients with MDS, in contrast to that from more advanced stages of MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls, and this constitutive IL-32 expression promoted apoptosis in MDS cells, reproducing the inefficient hematopoiesis and extensive apoptosis in MDS marrow. These findings indicate that stroma-produced IL-32 could contribute to the pathophysiology of MDS, and serve as a therapeutic target. Furthermore, this modified microenvironment phenotype was reproduced when the MSC were exposed to TNFα, known to be produced at high levels by MDS cells.

There are significant data to support mechanism, in which the malignant hematopoietic clone induces reversible functional changes in the HM that result in improved growth conditions for the malignant cells. Gene expression changes occurred in the stroma cell lines, HS5 and HS27a, derived from normal marrow in response to TNFα exposure (Stirewalt et al., 2008), known to be up-regulated in the bone marrow of patients with MDS. Previous experiments showed that interactions between MSC and HSC were required for TNFα to trigger apoptosis in hematopoietic cells (Goda et al., 2006).

Recent discoveries utilizing mouse models have provided the first experimental evidence for genetic changes in the HM contributing to or required for leukemogenesis. Raaijmakers et al. (2010) using transgenic mice showed that genetic alteration of HM can induce MDS with ineffective hematopoiesis and dysmorphic HCs, and with occasional transformation to AML. The authors used *Dicer1* deletion as a means of altering several gene products in subsets of mesenchymal osteolineage cells. *Dicer1* is an RNase III endonuclease essential for microRNA biogenesis (Bartel, 2004) and RNA processing (Krol et al., 2007), that regulates haematopoietic cell fate (Lu et al., 2008). Global repression of microRNA maturation by *Dicer1* deletion promotes cellular transformation and tumorigenesis (Kumar et al., 2007). Raaijmakers et al. (2010) show that deletion of *Dicer1* in HM cells of mouse may be sufficient to initiate a complex change of homeostasis with similarities to myelodysplasia. The authors demonstrated that the ability of HM abnormality to result in the emergence of a clonal neoplasm in a cell type of clearly distinct lineage with distinct secondary genetic changes (Raaijmakers et al., 2010).

Previously, Walkley et al (2007a, 2007b) demonstrated that conditional deletion of the Retinoblastoma gene (RB) in the BM microenvironment can contribute to the development of pre-leukemic myeloproliferative disease in mice. They showed that this was a result of interactions between myeloid cells and the microenvironment. The defect had to be present in both hematopoietic cells and the microenvironment to initiate disease. Widespread inactivation of RB, a central regulator of the cell cycle and a tumor suppressor, resulted in extramedullary hematopoiesis and myeloproliferative disease in the murine hematopoietic system. However, myeloid-specific loss of RB did not induce myeloproliferative disease or HSC abnormalities. Therefore, the myeloproliferative-like disorder in the RB mutants is the result of perturbed interactions between hematopoietic cells and the BM microenvironment

Bone Marrow Microenvironment in the Pathogenesis of AML 191

conditions, there may be agents that rather than drive hematopoiesis, might affect the

Understanding the niche has ramifications beyond simple biological interest. Niche biology and function has relevance not only in bone marrow transplantation, but in developing agents that may impact on the ability to generate a larger number of stem cells or increase the efficiency of stem cells to engraft in the transplant setting. By elucidating the role of the BM microenvironment in the pathogenesis of hematologic tumors, recent studies have provided the framework for identifying and validating novel therapies that target both leukemic cells and cells in their surrounding microenvironment (Konopleva et al., 2009). Thus in general, treatment strategies have been focused on the eradication of the stem or progenitor cell from which the malignancy arose. However, recent evidence suggests that focusing therapeutic strategies on the microenvironmental abnormalities can be extremely effective. The Imid family of agents has changed the treatment paradigm in diseases such as myeloma and MDS and highlighted the importance of targeting the microenvironment

If primary stromal defects are identified in humans and implicated in the initiation of malignancy, this clearly will have great impact on the treatment strategies offered to patients. By explanation the role of the MSC in the pathogenesis of AML, recent studies have provided novel therapies that target both leukemic cells and cells of microenvironment. Studies of MSC can also aid in potentially modifying the relative abundance of normal versus malignant cells in the context of the post chemotherapy setting in AML. The underlying molecular mechanisms implicated in stem cell activation and homing to the niche will provide important insight into the precise mechanisms involved in interactions between leukemic and normal cells that contribute to drug resistance. This understanding will provide a framework for the rational combination of agents in clinical trials to overcome drug resistance and improve patient outcomes. Detection of alterations in MSCs suggests that unstable MSCs may facilitate the expansion of malignant cells. In view of these data, alterations in MSCs may be a particular mechanism of leukemogenesis. Especially, further understanding of the contribution of the BM niche to the process of leukemogenesis may provide new targets aimed at destroying LSC without adversely

Abkowitz JL, Robinson AE, Kale S, Long MW, Chen J. Mobilization of hematopoietic stem cells during homeostasis and after cytokine exposure. *Blood* 2003;102:1249–1253. Arnulf B, Lecourt S, Soulier J et al. Phenotypic and functional characterization of bone

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(Walkley et al., 2007a). The final model, reported by the same group, may be the most compelling. In this report, deletion of the Retinoic Acid Receptor γ (RARγ) in mice resulted in a chronic myeloproliferative disorder. Transplant studies revealed that RARγhematopoietic cells functioned normally when transplanted into normal mice. However, transplantation of normal hematopoietic cells into the RARγ-microenvironment resulted in a myeloproliferative disorder in the transplanted cells. TNFα was implicated in the pathogenesis of this MPD as the disorder was partially abrogated when TNFα null stem cells were transplanted into the RARγ-microenvironment (Walkley et al., 2007b). These studies showed that a defect in HM could be sufficient to generate a myeloproliferative disorder.

Until recently, there has been little evidence to support the role of primary stromal abnormalities in the pathogenesis of hematologic neoplasms. Some independent studies have documented the existence of genomic alterations in the stroma of leukemia patients (Flores-Figueroa et al., 2005; Blau et al., 2007; Lopez-Villar et al., 2009; Klaus et al., 2010). Different groups have shown the extensive variability of the aberrations, such as hypodiploidy, balanced and unbalanced translocations, whole chromosome gains, and deletions. All cytogenetic markers in MSCs never repeated aberrations identified in HCs. Since there were no associations between chromosomal aberrations in HCs and MSCs, we can state that MSCs were devoid of residue HCs. These findings suggest enhanced genetic instability of MSC in leukemia, and indicate the potential involvement of MSC in the pathophysiology of these conditions (Blau et al., 2007). Recently, Lopez-Villar et al. (2009) reported the presence of cytogenetic aberrations on MSC from MDS patients by array-based comparative genomic hybridization and fluorescence in situ hybridization, some of them specially linked to a particular MDS subtype, the 5q-syndrome.

These date indicate that there are significant functional abnormalities, genetic aberrations, and epigenetic changes in MSC in leukemia patients. Also of interest are the recent reports of abnormalities in the stroma that lead to malignancies of the hematopoietic compartment. Although historically, hematologic malignancies are thought to arise from a stem or progenitor cell abnormality, there may be groups of patients that have a primary stromal defect leading to the hematologic malignancy. Moreover, although a series of genetic and epigenetic events in a single cell may be necessary for oncogenesis, they may not be sufficient, and a permissive microenvironment has been suggested to be required for frank malignancy to emerge (Hanahan and Weinberg, 2007).

It is known that even years after allogeneic stem cell transplantation (alloSCT), and despite successful engraftment of donor-derived hematopoiesis, MSC are in general of host origin (Rieger et al., 2005). Some patients after alloSCT do not recover their stem cells despite receiving high levels of CD34+ progenitor cells. The presumed basis for this is that the preparation regimen has in some way affected the niche, so it no longer has the same nurturing capability. It was shown that transplanted HSCs migrate to the endosteal surfaces of bone within hours of intravenous injection (Nilsson et al., 1997). Endochondral ossification has been shown to be an essential prerequisite for the development of normal haematopoiesis in the BM (Zhou et al., 1995), indicating a possible fundamental interrelationship of ossification to the mature haematopoietic process in mammals. Recent reports have identified that a key cellular component of the HSC niche is cells of the osteoblast lineage, the cell type responsible for the formation of bone (Calvi et al., 2003; Zhang et al., 2003). Additionally, these studies raise the issue that under transplant

(Walkley et al., 2007a). The final model, reported by the same group, may be the most compelling. In this report, deletion of the Retinoic Acid Receptor γ (RARγ) in mice resulted in a chronic myeloproliferative disorder. Transplant studies revealed that RARγhematopoietic cells functioned normally when transplanted into normal mice. However, transplantation of normal hematopoietic cells into the RARγ-microenvironment resulted in a myeloproliferative disorder in the transplanted cells. TNFα was implicated in the pathogenesis of this MPD as the disorder was partially abrogated when TNFα null stem cells were transplanted into the RARγ-microenvironment (Walkley et al., 2007b). These studies showed that a defect in HM could be sufficient to generate a myeloproliferative

Until recently, there has been little evidence to support the role of primary stromal abnormalities in the pathogenesis of hematologic neoplasms. Some independent studies have documented the existence of genomic alterations in the stroma of leukemia patients (Flores-Figueroa et al., 2005; Blau et al., 2007; Lopez-Villar et al., 2009; Klaus et al., 2010). Different groups have shown the extensive variability of the aberrations, such as hypodiploidy, balanced and unbalanced translocations, whole chromosome gains, and deletions. All cytogenetic markers in MSCs never repeated aberrations identified in HCs. Since there were no associations between chromosomal aberrations in HCs and MSCs, we can state that MSCs were devoid of residue HCs. These findings suggest enhanced genetic instability of MSC in leukemia, and indicate the potential involvement of MSC in the pathophysiology of these conditions (Blau et al., 2007). Recently, Lopez-Villar et al. (2009) reported the presence of cytogenetic aberrations on MSC from MDS patients by array-based comparative genomic hybridization and fluorescence in situ hybridization, some of them

These date indicate that there are significant functional abnormalities, genetic aberrations, and epigenetic changes in MSC in leukemia patients. Also of interest are the recent reports of abnormalities in the stroma that lead to malignancies of the hematopoietic compartment. Although historically, hematologic malignancies are thought to arise from a stem or progenitor cell abnormality, there may be groups of patients that have a primary stromal defect leading to the hematologic malignancy. Moreover, although a series of genetic and epigenetic events in a single cell may be necessary for oncogenesis, they may not be sufficient, and a permissive microenvironment has been suggested to be required for frank

It is known that even years after allogeneic stem cell transplantation (alloSCT), and despite successful engraftment of donor-derived hematopoiesis, MSC are in general of host origin (Rieger et al., 2005). Some patients after alloSCT do not recover their stem cells despite receiving high levels of CD34+ progenitor cells. The presumed basis for this is that the preparation regimen has in some way affected the niche, so it no longer has the same nurturing capability. It was shown that transplanted HSCs migrate to the endosteal surfaces of bone within hours of intravenous injection (Nilsson et al., 1997). Endochondral ossification has been shown to be an essential prerequisite for the development of normal haematopoiesis in the BM (Zhou et al., 1995), indicating a possible fundamental interrelationship of ossification to the mature haematopoietic process in mammals. Recent reports have identified that a key cellular component of the HSC niche is cells of the osteoblast lineage, the cell type responsible for the formation of bone (Calvi et al., 2003; Zhang et al., 2003). Additionally, these studies raise the issue that under transplant

specially linked to a particular MDS subtype, the 5q-syndrome.

malignancy to emerge (Hanahan and Weinberg, 2007).

disorder.

conditions, there may be agents that rather than drive hematopoiesis, might affect the osteoblast component.

Understanding the niche has ramifications beyond simple biological interest. Niche biology and function has relevance not only in bone marrow transplantation, but in developing agents that may impact on the ability to generate a larger number of stem cells or increase the efficiency of stem cells to engraft in the transplant setting. By elucidating the role of the BM microenvironment in the pathogenesis of hematologic tumors, recent studies have provided the framework for identifying and validating novel therapies that target both leukemic cells and cells in their surrounding microenvironment (Konopleva et al., 2009). Thus in general, treatment strategies have been focused on the eradication of the stem or progenitor cell from which the malignancy arose. However, recent evidence suggests that focusing therapeutic strategies on the microenvironmental abnormalities can be extremely effective. The Imid family of agents has changed the treatment paradigm in diseases such as myeloma and MDS and highlighted the importance of targeting the microenvironment (Sokol et al., 2007; Melchert and List, 2007).

If primary stromal defects are identified in humans and implicated in the initiation of malignancy, this clearly will have great impact on the treatment strategies offered to patients. By explanation the role of the MSC in the pathogenesis of AML, recent studies have provided novel therapies that target both leukemic cells and cells of microenvironment. Studies of MSC can also aid in potentially modifying the relative abundance of normal versus malignant cells in the context of the post chemotherapy setting in AML. The underlying molecular mechanisms implicated in stem cell activation and homing to the niche will provide important insight into the precise mechanisms involved in interactions between leukemic and normal cells that contribute to drug resistance. This understanding will provide a framework for the rational combination of agents in clinical trials to overcome drug resistance and improve patient outcomes. Detection of alterations in MSCs suggests that unstable MSCs may facilitate the expansion of malignant cells. In view of these data, alterations in MSCs may be a particular mechanism of leukemogenesis. Especially, further understanding of the contribution of the BM niche to the process of leukemogenesis may provide new targets aimed at destroying LSC without adversely affecting normal stem cell self-renewal.

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**11** 

*USA* 

Xiao Tang Hu

**Distinct Inhibitory Effect of TGFβ on the** 

**Growth of Human Myeloid Leukemia Cells** 

*Department of Biology, College of Arts & Sciences, Barry University, Miami Shores, FL,* 

Hematopoiesis is the formation and development of blood cells. In a healthy adult person, approximately 1011–1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation. Disruption of the steady state may cause the development of hematological diseases, such as acute (AML) or chronic myelogenous leukemia (CML). All mature blood cells ultimately originate from a rare population of primitive pluripotent (multipotential) stem cells. The pluripotent stem cells possess selfrenewal and multi-lineage differentiation potential. The latter process leads to two major multipotent progenitors: myeloid progenitors and lymphoid progenitors. The myeloid progenitors have the ability to differentiate into mature white cells, erythrocytes, megakaryocytes/platelets and mast cells; whereas the lymphoid progenitors cells mainly produce mature T, NK, and B lymphocytes. Production of myeloid lineage cells is a highly complex process involving the balance between cell proliferation and differentiation, which is regulated by growth stimulating or inhibiting signals. Transforming Growth Factor-beta (TGFβ) is a pleiotropic growth factor and is one of the major regulators of hematopoiesis (Fortunel NO et al. 2000). A major effect of TGFβ on hematopoietic cells is inhibitory (Hu X et al 1999, Batard P et al. 2000, Ducos K et al. 2000, Fortunel NO 2000) although its stimulatory effect has also been observed in some types of blood cells (Keller JR 1990, 1992). Due to its importance in the understanding human diseases including leukemia, over past 20 years, the mechanisms responsible for TGFβ-induced growth inhibition or stimulation have been a major area of investigation and considerable insights into its action mechanisms have been learned. This review highlights recent progress in understanding regulatory role of TGFβ and its signaling pathways in various types of cells with a focus on human myeloid

**2. Myeloid leukemia and myelogenous leukemia cell lines** 

Myeloid leukemia is a type of blood and bone marrow cancers, which includes AML and CML. AML is a characterized by the rapid accumulation of abnormal, immature white blood cells in both borrow and blood, which is caused by defects in the progenitors and/or stem cells population. Unlike AML, myeloid cell differentiation is preserved during the chronic phase of CML, a disease which originates from the presence of a genetic abnormality in stem cells/progenitors and the nuclei show an abnormal chromosome called the Philadelphia

**1. Introduction** 

leukemia cells.


### **Distinct Inhibitory Effect of TGFβ on the Growth of Human Myeloid Leukemia Cells**

Xiao Tang Hu

*Department of Biology, College of Arts & Sciences, Barry University, Miami Shores, FL, USA* 

#### **1. Introduction**

196 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Zhou H, Choong PFM, Henderson S et al. Marrow development and its relationship to bone

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term murine bone marrow cultures. *Blood* 1983;61:540–547.

rabbits. *Bone* 1995; 17:407–415.

formation *in vivo*: a histological study using an implantable titanium device in

Hematopoiesis is the formation and development of blood cells. In a healthy adult person, approximately 1011–1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation. Disruption of the steady state may cause the development of hematological diseases, such as acute (AML) or chronic myelogenous leukemia (CML). All mature blood cells ultimately originate from a rare population of primitive pluripotent (multipotential) stem cells. The pluripotent stem cells possess selfrenewal and multi-lineage differentiation potential. The latter process leads to two major multipotent progenitors: myeloid progenitors and lymphoid progenitors. The myeloid progenitors have the ability to differentiate into mature white cells, erythrocytes, megakaryocytes/platelets and mast cells; whereas the lymphoid progenitors cells mainly produce mature T, NK, and B lymphocytes. Production of myeloid lineage cells is a highly complex process involving the balance between cell proliferation and differentiation, which is regulated by growth stimulating or inhibiting signals. Transforming Growth Factor-beta (TGFβ) is a pleiotropic growth factor and is one of the major regulators of hematopoiesis (Fortunel NO et al. 2000). A major effect of TGFβ on hematopoietic cells is inhibitory (Hu X et al 1999, Batard P et al. 2000, Ducos K et al. 2000, Fortunel NO 2000) although its stimulatory effect has also been observed in some types of blood cells (Keller JR 1990, 1992). Due to its importance in the understanding human diseases including leukemia, over past 20 years, the mechanisms responsible for TGFβ-induced growth inhibition or stimulation have been a major area of investigation and considerable insights into its action mechanisms have been learned. This review highlights recent progress in understanding regulatory role of TGFβ and its signaling pathways in various types of cells with a focus on human myeloid leukemia cells.

#### **2. Myeloid leukemia and myelogenous leukemia cell lines**

Myeloid leukemia is a type of blood and bone marrow cancers, which includes AML and CML. AML is a characterized by the rapid accumulation of abnormal, immature white blood cells in both borrow and blood, which is caused by defects in the progenitors and/or stem cells population. Unlike AML, myeloid cell differentiation is preserved during the chronic phase of CML, a disease which originates from the presence of a genetic abnormality in stem cells/progenitors and the nuclei show an abnormal chromosome called the Philadelphia

Distinct Inhibitory Effect of TGFβ on the Growth of Human Myeloid Leukemia Cells 199

induced biological activities are achieved through the binding of the ligand to its cell surface receptor, a serine-threonine kinase complex, known as type I, II, and III (adaptor) receptors. TGFβ binds to and activates the type II receptor. Then the type II receptor recruits and phosphorylates the type I receptor, which in turn activates Smad family proteins that carry signals from the receptors to the nucleus to control transcription of target genes. These gene products may stimulate or inhibit cell cycle progression. TGF plays an essential regulatory role in the control of hematopoiesis (Fortunel NO et al. 2000). First, the development of hematopoietic cells from mesoderm in embryos is regulated by TGFβ signaling. Park et al (Park C et al. 2004) demonstrated that BMP-4, a member of TGFβ superfamily, is able to induce hematopoietic differentiation from mouse embryonic stem cells. The differentiated cells contain all types of myeloid and lymphoid progeny (Olsen AL et al. 2006). Similar roles of TGFβ in human hematopoietic stem cells have been reported (Kaufman DS et al. 2001, Chadwick K et al. 2003, Cerdan C et al. 2004). Second, it has also been found that TGF is required for maintaining hematopoietic stem/progenitor cells in quiescent states (Batard P et al. 2000); in contrast, application of anti-endogenous TGF antibody induces release of these cells from G1 phase (Ducos K et al. 2000). In TGFβ-/- mice, enhanced numbers of circulating granulocytes, monocytes, platelets, as well as colony forming units, were observed (Kulkarni AB et al 1993, Shull MM et al 1992). In addition, inactivation of the TGF signaling pathway leads to malignant transformation of early hematopoietic cells (Le Bousse-Kerdiles MC et al. 1996). These data indicate that TGFβ is inhibitory to hematopoietic cells. However, studies from several groups suggest that the response of myeloid stem/progenitor cells to the inhibitory effect of TGFβ appears to be inversely correlated with their maturation stage (Sing GK 1988, Keller JR 1990, Ottmann OG & Pellus LM 1991, Snoeck H-W 1996). Long-term repopulating stem cells, CD34+ or CD34+/CD38 stem/progenitor cells, early myeloid and erythroid progenitors are markedly inhibited in the presence of TGF, whereas more differentiated myeloid progenitors forming granulocyte-macrophage colonies, and late erythroid progenitors forming CFU-e are less inhibited or even stimulated by TGF (Keller JR 1990, 1992, 1994, Fortunel NO 2000, Ruscetti FW 2005, Sitnicka E 1996, McNiece IK 1992, Goey H 1989, Morita N 2003, Classen S 2007). The inhibitory or stimulatory role by TGFβ in some cases seems to correlate with the cytokines used to promote cell growth. TGFβ inhibits SCF-, IL-3- and CSF-1-induced colony formation from mouse and human hematopoietic progenitors (McNiece IK 1992, Sitnicka E et al 1996). In contrast, TGFβ enhances GM-CSF-induced colony formation derived from

normal bone marrow cells and Lin- progenitors (Keller JR 1991).

of transcription factor, FOXO, in a CML mouse model (Naka K et al. 2010).

For leukemia cells, both inhibitory and stimulatory effects have been reported (Tessier N & Hoang T 1988, Otten J 2011, Aglietta M 1989, Cashman JD 1992). TGF has been shown to inhibit the growth of clonogenic blast cells from patients with acute myeloblastic leukemia (AML) (Tessier N & Hoang T 1988). Mutations in the components of TGFβ signal pathways may relate to the disease development (Imai Y et al. 2001). In contrast, high expression of ALK-1 and ALK-5, two members of TGFβ type I receptors, was reported to be associated with AML (Otten J 2011). An inhibitory effect of TGFβ was suggested by the finding of low levels of TGFβ type II receptors in CML (Rooke HM 1999) and lymphoid neoplasms (Inman GJ et al 2000); whereas, by using TGFβ inhibitors, Naka et al found that TGFβ signal is required for maintaining Leukemia Initiating Cells (LIC) by controlling nuclear localization

The data described above suggest that TGF has both negative and positive effects on the proliferation of normal hematopoietic and leukemia cells. However, some of the data appear

chromosome. In a healthy person, bone marrow makes the blood stem cells and progenitors that mature into mature white blood cells, red blood cells and blood platelets.

In order to simplify the study of leukemia and basic research, a number of human AML cell lines were established. These lines provide model systems to study the control of proliferation and differentiation in human myelogenous leukemia and, in a broader framework, the controls of normal myeloid development. More than 10 leukemic cell lines have been used in our lab to study Transforming Growth Factor-β (TGFβ)-mediated cell cycle control and transformed events, among which K562 is the first myelogenous leukemia cell line established. The cell line was isolated from a 53 year old female CML patient in blast crisis (Lozzio CB and Lozzio BB 1975). K652 composed of undifferentiated blast cells that are rich in glycophorin and may be induced to produce fetal and embryonic hemoglobin in the presence of hemin. The rest nine cell lines were isolated from AML patients as described below. MV4-11 cell line was derived from blast cells of a 10-year-old male with biphenotypic myelomonocytic leukemia (Lange B et al. 1987). This cell line expresses mutated FLT3, a member of the type III receptor tyrosine kinase (RTK) family (Quentmeier 2003). KG-1 cell line is composed predominantly of myeloblasts and promyelocytes ( Koeffler HP et al. 1978). Recent studies identified a fusion of two genes encoding fibroblast growth factor receptors, making this cell line is a good model for studying gene fusion-linked leukemia (Gu TL et al. 2006). KG-1a ( Koeffler HP et al. 1980) is a subline isolated from KG-1. TF-1 is a growth factor-dependent human cell line that was originally isolated from the bone marrow cells of a patient with erythroleukemia (Kitamura T et al. 1989). TF-1 cells express a wide range of cytokine receptors and respond to a variety of cytokines. Thus, this cell line is a useful tool for studying cytokine signaling network (Rosas M et al 2005). TF-1a (Hu X et al. 1998) is growth factor-independent subline isolated from TF-1. A unique characteristic of the KG-1 and TF-1 cells is their dependence on colony-stimulating factor (CSF) or GM-CSF/IL-3 for proliferation, respectively, whereas KG-1a and TF-1a are factor-independent cell lines for their growth, although TF-1a retains its ability to respond to several growth factors. HL-60 is a promyelocytic cell line derived from peripheral blood leukocytes from a female patient with acute promyelocytic leukemia, which can be induced into granulocytes in the presence of DMSO (Gallagher R et al.1979). HL-60 cells differentiate into mononuclear phagocytes when exposed to phorbol esters (Xu YZ et al. 2011). HEL is an erythroleukemia cell line isolated from the bone marrow cells of a patient (Martin P and Papayannopoulou 1982). A mutation in *Janus kinase 2* gene (JAK2) in HEL cells was identified in 2006 (Quentmeier H et al 2006). Mo7e (Avancd GC et al. 1988) is a subline of the M-07 human megakaryoblastic leukemia cell line. The cells express platelet glycoprotein-2b-3a. The growth of Mo7e is dependent on IL-3 or GM-CSF. U937 is a cell line established from a diffuse histiocytic lymphoma of 37 year old male patient (Sundstrom C et al. 1976). A histocyte is a cell that is part of the mononuclear phagocytic system that takes part in the immune responses. Since U937 displays many monocytic characteristics and has thus served as a model for the differentiation of monocytes and macrophage in vitro (Aida J, et al. 2011). This cell line is also a good model for induction of apoptosis (Piedfer M et al. 2011).

#### **3. Effect of TGFβ on the growth of human myeloid leukemia cell lines in culture**

Transforming Growth Factor-β (TGF) has three isoforms in mammals: TGFβ1, TGFβ2, and TGFβ3. The β1 isoform is the most abundant, universally expressed, which has been intensively studied. This review focuses on TGFβ1 unless indicated otherwise. The TGFβ-

chromosome. In a healthy person, bone marrow makes the blood stem cells and progenitors

In order to simplify the study of leukemia and basic research, a number of human AML cell lines were established. These lines provide model systems to study the control of proliferation and differentiation in human myelogenous leukemia and, in a broader framework, the controls of normal myeloid development. More than 10 leukemic cell lines have been used in our lab to study Transforming Growth Factor-β (TGFβ)-mediated cell cycle control and transformed events, among which K562 is the first myelogenous leukemia cell line established. The cell line was isolated from a 53 year old female CML patient in blast crisis (Lozzio CB and Lozzio BB 1975). K652 composed of undifferentiated blast cells that are rich in glycophorin and may be induced to produce fetal and embryonic hemoglobin in the presence of hemin. The rest nine cell lines were isolated from AML patients as described below. MV4-11 cell line was derived from blast cells of a 10-year-old male with biphenotypic myelomonocytic leukemia (Lange B et al. 1987). This cell line expresses mutated FLT3, a member of the type III receptor tyrosine kinase (RTK) family (Quentmeier 2003). KG-1 cell line is composed predominantly of myeloblasts and promyelocytes ( Koeffler HP et al. 1978). Recent studies identified a fusion of two genes encoding fibroblast growth factor receptors, making this cell line is a good model for studying gene fusion-linked leukemia (Gu TL et al. 2006). KG-1a ( Koeffler HP et al. 1980) is a subline isolated from KG-1. TF-1 is a growth factor-dependent human cell line that was originally isolated from the bone marrow cells of a patient with erythroleukemia (Kitamura T et al. 1989). TF-1 cells express a wide range of cytokine receptors and respond to a variety of cytokines. Thus, this cell line is a useful tool for studying cytokine signaling network (Rosas M et al 2005). TF-1a (Hu X et al. 1998) is growth factor-independent subline isolated from TF-1. A unique characteristic of the KG-1 and TF-1 cells is their dependence on colony-stimulating factor (CSF) or GM-CSF/IL-3 for proliferation, respectively, whereas KG-1a and TF-1a are factor-independent cell lines for their growth, although TF-1a retains its ability to respond to several growth factors. HL-60 is a promyelocytic cell line derived from peripheral blood leukocytes from a female patient with acute promyelocytic leukemia, which can be induced into granulocytes in the presence of DMSO (Gallagher R et al.1979). HL-60 cells differentiate into mononuclear phagocytes when exposed to phorbol esters (Xu YZ et al. 2011). HEL is an erythroleukemia cell line isolated from the bone marrow cells of a patient (Martin P and Papayannopoulou 1982). A mutation in *Janus kinase 2* gene (JAK2) in HEL cells was identified in 2006 (Quentmeier H et al 2006). Mo7e (Avancd GC et al. 1988) is a subline of the M-07 human megakaryoblastic leukemia cell line. The cells express platelet glycoprotein-2b-3a. The growth of Mo7e is dependent on IL-3 or GM-CSF. U937 is a cell line established from a diffuse histiocytic lymphoma of 37 year old male patient (Sundstrom C et al. 1976). A histocyte is a cell that is part of the mononuclear phagocytic system that takes part in the immune responses. Since U937 displays many monocytic characteristics and has thus served as a model for the differentiation of monocytes and macrophage in vitro (Aida J, et al. 2011). This cell line is also a

that mature into mature white blood cells, red blood cells and blood platelets.

good model for induction of apoptosis (Piedfer M et al. 2011).

**culture** 

**3. Effect of TGFβ on the growth of human myeloid leukemia cell lines in** 

Transforming Growth Factor-β (TGF) has three isoforms in mammals: TGFβ1, TGFβ2, and TGFβ3. The β1 isoform is the most abundant, universally expressed, which has been intensively studied. This review focuses on TGFβ1 unless indicated otherwise. The TGFβinduced biological activities are achieved through the binding of the ligand to its cell surface receptor, a serine-threonine kinase complex, known as type I, II, and III (adaptor) receptors. TGFβ binds to and activates the type II receptor. Then the type II receptor recruits and phosphorylates the type I receptor, which in turn activates Smad family proteins that carry signals from the receptors to the nucleus to control transcription of target genes. These gene products may stimulate or inhibit cell cycle progression. TGF plays an essential regulatory role in the control of hematopoiesis (Fortunel NO et al. 2000). First, the development of hematopoietic cells from mesoderm in embryos is regulated by TGFβ signaling. Park et al (Park C et al. 2004) demonstrated that BMP-4, a member of TGFβ superfamily, is able to induce hematopoietic differentiation from mouse embryonic stem cells. The differentiated cells contain all types of myeloid and lymphoid progeny (Olsen AL et al. 2006). Similar roles of TGFβ in human hematopoietic stem cells have been reported (Kaufman DS et al. 2001, Chadwick K et al. 2003, Cerdan C et al. 2004). Second, it has also been found that TGF is required for maintaining hematopoietic stem/progenitor cells in quiescent states (Batard P et al. 2000); in contrast, application of anti-endogenous TGF antibody induces release of these cells from G1 phase (Ducos K et al. 2000). In TGFβ-/- mice, enhanced numbers of circulating granulocytes, monocytes, platelets, as well as colony forming units, were observed (Kulkarni AB et al 1993, Shull MM et al 1992). In addition, inactivation of the TGF signaling pathway leads to malignant transformation of early hematopoietic cells (Le Bousse-Kerdiles MC et al. 1996). These data indicate that TGFβ is inhibitory to hematopoietic cells. However, studies from several groups suggest that the response of myeloid stem/progenitor cells to the inhibitory effect of TGFβ appears to be inversely correlated with their maturation stage (Sing GK 1988, Keller JR 1990, Ottmann OG & Pellus LM 1991, Snoeck H-W 1996). Long-term repopulating stem cells, CD34+ or CD34+/CD38 stem/progenitor cells, early myeloid and erythroid progenitors are markedly inhibited in the presence of TGF, whereas more differentiated myeloid progenitors forming granulocyte-macrophage colonies, and late erythroid progenitors forming CFU-e are less inhibited or even stimulated by TGF (Keller JR 1990, 1992, 1994, Fortunel NO 2000, Ruscetti FW 2005, Sitnicka E 1996, McNiece IK 1992, Goey H 1989, Morita N 2003, Classen S 2007). The inhibitory or stimulatory role by TGFβ in some cases seems to correlate with the cytokines used to promote cell growth. TGFβ inhibits SCF-, IL-3- and CSF-1-induced colony formation from mouse and human hematopoietic progenitors (McNiece IK 1992, Sitnicka E et al 1996). In contrast, TGFβ enhances GM-CSF-induced colony formation derived from normal bone marrow cells and Lin- progenitors (Keller JR 1991).

For leukemia cells, both inhibitory and stimulatory effects have been reported (Tessier N & Hoang T 1988, Otten J 2011, Aglietta M 1989, Cashman JD 1992). TGF has been shown to inhibit the growth of clonogenic blast cells from patients with acute myeloblastic leukemia (AML) (Tessier N & Hoang T 1988). Mutations in the components of TGFβ signal pathways may relate to the disease development (Imai Y et al. 2001). In contrast, high expression of ALK-1 and ALK-5, two members of TGFβ type I receptors, was reported to be associated with AML (Otten J 2011). An inhibitory effect of TGFβ was suggested by the finding of low levels of TGFβ type II receptors in CML (Rooke HM 1999) and lymphoid neoplasms (Inman GJ et al 2000); whereas, by using TGFβ inhibitors, Naka et al found that TGFβ signal is required for maintaining Leukemia Initiating Cells (LIC) by controlling nuclear localization of transcription factor, FOXO, in a CML mouse model (Naka K et al. 2010).

The data described above suggest that TGF has both negative and positive effects on the proliferation of normal hematopoietic and leukemia cells. However, some of the data appear

Distinct Inhibitory Effect of TGFβ on the Growth of Human Myeloid Leukemia Cells 201

affinity for pRb is not the only determinant of the E2F1 activity (Sahin F and Sladek TL 2010a). Of the seven E2Fs well documented, E2F1, E2F2, and E2F3 are considered as "activator" E2Fs, whereas, E2F4, E2F5, E2F6, and E2F7 are considered to be "repressor" E2Fs (Attwooll C et al. 2004, Sahin F and Sladek TL 2010b). pRb and two pRb-related proteins, p107 and p130, show a common structure, which are collectively termed the "pocket proteins". E2F1-3 bind to pRb, and E2F5 binds to p130. E2F4 can interact with the all three

Dephosphorylated pRb preferentially interacts with E2F1-3 (Weinberg RA et al. 1995), and the pRb/E2F complexes are found primarily during the G1 phase of the cell cycle (Shirodkar S et al. 1992, Schwarz JK et al. 1993), during which time E2F-responsive promoters are repressed. The dissociation of the pRb/E2F-repressor complex and liberation of free E2F allows for the activation of these promoters (Hiebert SW et al. 1993, Qin X-Q et al. 1995, Helin K et al. 1993). It was demonstrated that E2F-1 responsive gene activation was inhibited by overexpression of wild-type pRb, but not by a mutant pRb, whereas transactivation mediated by mutants of E2F1, which do not bind to pRb, was not affected by overexpression of wild-type pRb. Loss of E2F1-3 resulted in an acute cell cycle arrest (Wu L et al. 2001). It has been found that Rb gene was often mutated in patients with various cancers, whereas p107 and p130 genes appear to be less frequently mutated in human cancers, suggesting that pRb is a tumor suppressor, whereas p107 and p130 are not. However, recent studies from several labs have shown that p130 and p107 also actively involved in E2F-dependent transcription activities. For example, the major E2F complexes in quiescent fibroblasts were E2F4-p130. As cells enter the cell cycle, E2F4-p130 was replaced by E2F4-p107 and E2F4-pRb (Ikeda M et al. 1996, Moberg K et al. 1996). Subsequent investigators, by performing chromatin immunoprecipitation (ChIP) experiments, were able to locate E2F complexes in a gene promoter. They have demonstrated that in serumdeprived cells, predominantly E2F4, p130, and a histone deacetylase (HDAC) are found to bind to corresponding E2F-responsive promoters (Rayman JB et al. 2002, Ren B et al. 2002, Takahashi Y et al. 2000). Since histones are generally hypoacetylated when a gene is turned off, which is regulated by HDAC, the results described above suggest that p130-E2F4 DNA binding activity is required for the gene inactivity and for arresting cells in quiescent state. As cells progress through mid-G1, E2F4 and p130 are replaced by activators E2F1, 2, and 3. Unexpectedly, loss of pRB had no effect on HDAC recruitment to the promoter or pRb was not detectable in the promoters containing the E2F responsive site in quiescent cells (Rayman JB et al. 2002, Takahashi Y et al. 2000). Thus, these data suggest that pRb may not be a primary negative regulator of E2F activity in control of cell cycle progression in these

As indicated in the previous section, TGF dramatically inhibits the growth of several human myeloid leukemia cell lines in culture with a maximal growth inhibition being observed in MV4-11 cells. We asked how pRb-E2F complexes were regulated in MV4-11 cells. We found that in actively proliferating MV4-11 cells, p130 and pRb were expressed as peptides that migrated in multiple bands in SDS-PAGE, whereas in the cells treated with TGFβ, p130 and pRb were dephosphorylated. However, TGFβ did not dephosphrylate p107 but inhibited expression of the molecule (Hu X et al. 2000). Subsequently, we found that TGFβ-treated MV4-11 cells had very low levels of pRb-associated E2F1 and E2F3. The low levels of pRb-E2Fs are resulted from a low expression of E2F1 and E2F3 in response to TGF that arrested cells in G1 phase. Unexpectedly, proliferating cells without TGFβ contained

pocket proteins.

cells.

discordant, which may be due to the complexity of the network in vivo. In the presence of other growth factors, TGF-induced stimulation or inhibition could be an indirect result or affected by other factors. Therefore, using a cell line as a research model may provide a direct effect of TGFβ and produce more consistent results. A TGF knock out hematopoietic cell line exhibited increased colony formation in response to IL-3, IL-6, and G-CSF (Kim S-J & Letterio J 2003), suggesting that TGFβ is inhibitory to the cells. TGF has been reported to inhibit proliferation of mouse myeloid leukemic M1 cells and induce adherence of M1 myeloid leukemia cells to plastic dishes (Okabe-Kado J 1989). In Josk-1 monoblastic leukemia cells, dephosphorylation of pRb and inhibition of c-myc were responsible for TGF1-induced G1 arrest (Furukawa Y 1992). In U937 cells, it identified that the inhibition of the cells by TGF1 was linked to low levels of transcription factors, C-jun, C-fos and Jun B (Kanatni Y 1996). In order to delineate the effect of TGF on more myeloid leukemia cell lines and to reach a general conclusion, we examined more than 10 human myeloid leukemia cell lines, among which MV4-11 and TF-1a are factor-independent but response cell lines; TF-1 and Mo7e are factor-dependent cell lines; and KG-1, KG-1a, K562, U937, HL60, and HEL are factor-independent and insensitive to any growth factors. We demonstrated that TGF is inhibitory for all these human myeloid leukemia cell lines in culture, with heterogeneous inhibitory responses being observed (Hu X et al 1999, Hu X & Zuckerman KS 2000). Inhibition of TF-1, KG-1, and K562, Mo7e, and HEL by TGFβ has also been reported by other groups recently (Moller GM et al. 2007, Koutoulaki A et al 2010, Fukuchi Y et al 2001, Kalina U et al. 2001). Our data and some recent discoveries, clearly indicate that the responses of the cell lines to TGFβ are not related to the apparent differentiation stages of the cells (Hu X et al 1999) but could result from differences in the amount of TGF receptors expressed or the degrees of the mutations (Quentmeier et al. 2003, 2006, Gu TL et al. 2006) in TGFβ signal components in these cells. The mechanisms by which TGFβ inhibits hematopoietic cells involves in the regulation of pRb pocket proteins-E2F complexes, downregulation of a number of cell cycle regulatory molecules, and upregulation of p21 (Ducos K et al 2000) and p57 (Scandura JM et al 2004) as described in following sections**.**

#### **4. Regulation of pRb-E2F complexes in human myeloid leukemia cells**

pRb acts as a signal transducer collecting signals from cell cycle regulatory molecules to transcriptional machinery. G1 protein kinases have been reported to modify pRb by phosphorylation, thereby promoting cell cycle progression toward DNA replication (Suryadinata R et al. 2011, Takaki T et al. 2004). The growth inhibition of human myeloid leukemia cells by TGFβ is linked to suppression of pRb phosphorylation on multiple serine and threonine residues, including Ser249/Thr252, Thr373, Ser780, Ser795, and Ser807/811 (Chen P et al. 1989, Mihara K et al. 1989, Hu X et al. 1999). Dephosphorylated pRb interacts with E2F transcription factors. E2F-binding sites have been found in the promoters of many genes that control cell cycle progression and cell proliferation, thereby E2Fs play a critical role in cell growth control (Iaquinta PJ et al. 2007). Previous studies identified that E2F binds to the pocket domain of pRB. In E2F1, the pRB-binding domain interacts with the Cterminus of the E2F molecule. As a consequence of the pRB-E2F interaction, E2F activity is repressed. pRB, therefore, exerts its growth suppression function mainly through its sequestration of E2F activity. However, one recent report suggests that E2F-1 binding

discordant, which may be due to the complexity of the network in vivo. In the presence of other growth factors, TGF-induced stimulation or inhibition could be an indirect result or affected by other factors. Therefore, using a cell line as a research model may provide a direct effect of TGFβ and produce more consistent results. A TGF knock out hematopoietic cell line exhibited increased colony formation in response to IL-3, IL-6, and G-CSF (Kim S-J & Letterio J 2003), suggesting that TGFβ is inhibitory to the cells. TGF has been reported to inhibit proliferation of mouse myeloid leukemic M1 cells and induce adherence of M1 myeloid leukemia cells to plastic dishes (Okabe-Kado J 1989). In Josk-1 monoblastic leukemia cells, dephosphorylation of pRb and inhibition of c-myc were responsible for TGF1-induced G1 arrest (Furukawa Y 1992). In U937 cells, it identified that the inhibition of the cells by TGF1 was linked to low levels of transcription factors, C-jun, C-fos and Jun B (Kanatni Y 1996). In order to delineate the effect of TGF on more myeloid leukemia cell lines and to reach a general conclusion, we examined more than 10 human myeloid leukemia cell lines, among which MV4-11 and TF-1a are factor-independent but response cell lines; TF-1 and Mo7e are factor-dependent cell lines; and KG-1, KG-1a, K562, U937, HL60, and HEL are factor-independent and insensitive to any growth factors. We demonstrated that TGF is inhibitory for all these human myeloid leukemia cell lines in culture, with heterogeneous inhibitory responses being observed (Hu X et al 1999, Hu X & Zuckerman KS 2000). Inhibition of TF-1, KG-1, and K562, Mo7e, and HEL by TGFβ has also been reported by other groups recently (Moller GM et al. 2007, Koutoulaki A et al 2010, Fukuchi Y et al 2001, Kalina U et al. 2001). Our data and some recent discoveries, clearly indicate that the responses of the cell lines to TGFβ are not related to the apparent differentiation stages of the cells (Hu X et al 1999) but could result from differences in the amount of TGF receptors expressed or the degrees of the mutations (Quentmeier et al. 2003, 2006, Gu TL et al. 2006) in TGFβ signal components in these cells. The mechanisms by which TGFβ inhibits hematopoietic cells involves in the regulation of pRb pocket proteins-E2F complexes, downregulation of a number of cell cycle regulatory molecules, and upregulation of p21 (Ducos K et al 2000) and p57 (Scandura JM et al 2004) as described in

**4. Regulation of pRb-E2F complexes in human myeloid leukemia cells** 

pRb acts as a signal transducer collecting signals from cell cycle regulatory molecules to transcriptional machinery. G1 protein kinases have been reported to modify pRb by phosphorylation, thereby promoting cell cycle progression toward DNA replication (Suryadinata R et al. 2011, Takaki T et al. 2004). The growth inhibition of human myeloid leukemia cells by TGFβ is linked to suppression of pRb phosphorylation on multiple serine and threonine residues, including Ser249/Thr252, Thr373, Ser780, Ser795, and Ser807/811 (Chen P et al. 1989, Mihara K et al. 1989, Hu X et al. 1999). Dephosphorylated pRb interacts with E2F transcription factors. E2F-binding sites have been found in the promoters of many genes that control cell cycle progression and cell proliferation, thereby E2Fs play a critical role in cell growth control (Iaquinta PJ et al. 2007). Previous studies identified that E2F binds to the pocket domain of pRB. In E2F1, the pRB-binding domain interacts with the Cterminus of the E2F molecule. As a consequence of the pRB-E2F interaction, E2F activity is repressed. pRB, therefore, exerts its growth suppression function mainly through its sequestration of E2F activity. However, one recent report suggests that E2F-1 binding

following sections**.**

affinity for pRb is not the only determinant of the E2F1 activity (Sahin F and Sladek TL 2010a). Of the seven E2Fs well documented, E2F1, E2F2, and E2F3 are considered as "activator" E2Fs, whereas, E2F4, E2F5, E2F6, and E2F7 are considered to be "repressor" E2Fs (Attwooll C et al. 2004, Sahin F and Sladek TL 2010b). pRb and two pRb-related proteins, p107 and p130, show a common structure, which are collectively termed the "pocket proteins". E2F1-3 bind to pRb, and E2F5 binds to p130. E2F4 can interact with the all three pocket proteins.

Dephosphorylated pRb preferentially interacts with E2F1-3 (Weinberg RA et al. 1995), and the pRb/E2F complexes are found primarily during the G1 phase of the cell cycle (Shirodkar S et al. 1992, Schwarz JK et al. 1993), during which time E2F-responsive promoters are repressed. The dissociation of the pRb/E2F-repressor complex and liberation of free E2F allows for the activation of these promoters (Hiebert SW et al. 1993, Qin X-Q et al. 1995, Helin K et al. 1993). It was demonstrated that E2F-1 responsive gene activation was inhibited by overexpression of wild-type pRb, but not by a mutant pRb, whereas transactivation mediated by mutants of E2F1, which do not bind to pRb, was not affected by overexpression of wild-type pRb. Loss of E2F1-3 resulted in an acute cell cycle arrest (Wu L et al. 2001). It has been found that Rb gene was often mutated in patients with various cancers, whereas p107 and p130 genes appear to be less frequently mutated in human cancers, suggesting that pRb is a tumor suppressor, whereas p107 and p130 are not. However, recent studies from several labs have shown that p130 and p107 also actively involved in E2F-dependent transcription activities. For example, the major E2F complexes in quiescent fibroblasts were E2F4-p130. As cells enter the cell cycle, E2F4-p130 was replaced by E2F4-p107 and E2F4-pRb (Ikeda M et al. 1996, Moberg K et al. 1996). Subsequent investigators, by performing chromatin immunoprecipitation (ChIP) experiments, were able to locate E2F complexes in a gene promoter. They have demonstrated that in serumdeprived cells, predominantly E2F4, p130, and a histone deacetylase (HDAC) are found to bind to corresponding E2F-responsive promoters (Rayman JB et al. 2002, Ren B et al. 2002, Takahashi Y et al. 2000). Since histones are generally hypoacetylated when a gene is turned off, which is regulated by HDAC, the results described above suggest that p130-E2F4 DNA binding activity is required for the gene inactivity and for arresting cells in quiescent state. As cells progress through mid-G1, E2F4 and p130 are replaced by activators E2F1, 2, and 3. Unexpectedly, loss of pRB had no effect on HDAC recruitment to the promoter or pRb was not detectable in the promoters containing the E2F responsive site in quiescent cells (Rayman JB et al. 2002, Takahashi Y et al. 2000). Thus, these data suggest that pRb may not be a primary negative regulator of E2F activity in control of cell cycle progression in these cells.

As indicated in the previous section, TGF dramatically inhibits the growth of several human myeloid leukemia cell lines in culture with a maximal growth inhibition being observed in MV4-11 cells. We asked how pRb-E2F complexes were regulated in MV4-11 cells. We found that in actively proliferating MV4-11 cells, p130 and pRb were expressed as peptides that migrated in multiple bands in SDS-PAGE, whereas in the cells treated with TGFβ, p130 and pRb were dephosphorylated. However, TGFβ did not dephosphrylate p107 but inhibited expression of the molecule (Hu X et al. 2000). Subsequently, we found that TGFβ-treated MV4-11 cells had very low levels of pRb-associated E2F1 and E2F3. The low levels of pRb-E2Fs are resulted from a low expression of E2F1 and E2F3 in response to TGF that arrested cells in G1 phase. Unexpectedly, proliferating cells without TGFβ contained

Distinct Inhibitory Effect of TGFβ on the Growth of Human Myeloid Leukemia Cells 203

2001). Our recent studies, using TF-1 and MV4-11 human myeloid leukemia cells, demonstrated that TGF1-induced inhibition of leukemia cell growth is not due solely to downregulation of G1 cyclins and cdks but also G2 check point kinases (Hu X 2007). In MV4- 11 cells, TGF caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216), with 60% and 70% inhibition of phosphorylation of the both proteins by 2-3 hours, respectively, followed by returning to near normal by 12 hours, and 70-100% inhibitions again by 24-72 hours, repectively. The dephosphorylation of cdc2 and cdc25c was followed by a dramatic decrease of total cdc25c (100% decrease at 48 and 72 hours) and cylcin B1 and cdc2 (70-90% reduction at 24 to 72 hours). Consistent with these data, cdc2 kinase activity, as determined by ability to phosphorylate histone H1 and GST-Rb in vitro, was markedly reduced in the leukemia cells treated with TGF for 24-48 h. Cyclin B1 and cdc25c were degraded in a ubiquitin/26S proteosome assay system in vitro, which suggests strongly that ubiquitination and proteasomal degradation may be the key mechanism of degradation that leads to block of G2-M transition. The downregulation of cdc2, cdc25c, and cyclin B1 was not a result of G1 arrest but a direct effect of TGFβ, because the time required for the inhibition of the cells released from G2 is much shorter than the cells released from G1. Using immunoprecipitation and Western blotting we detected that the levels of cyclin B1-cdc2 complexes were much lower in extracts of TGF-treated cells than in control cells. Based on our data, we suggest that TGFβ suppresses growth of human myeloid leukemia cells through multiple pathways, by inhibiting both G1 and G2 checkpoint kinases. Since the G1 and G2 checkpoint kinases are able to phosphorylate pRb in proliferating cells, the down regulation of G1 and G2 checkpoint kinases would prevent pRb phospharylation, leading to G1 arrest. The data also suggest that cdc2, a traditional G2 regulator in mammalian cells, may participate in G1 regulation. Fig. 1 illustrates diagrammatically the negative roles of

TGFβ in the cell cycle control in myeloid leukemic MV4-11 cells.

Fig. 1. TGFβ inhibits both G! and G@ cdks in MV4-11 cells

substantial quantities of pRb-E2F1 and pRb-E2F3 complexes. In contrast, the cells treated with TGFβ (i.e. G1 cells) showed increased levels of pRb-E2F4 and p130-E2F4 and low level of p107-E2F4, as detected by immunoprecipitation and Western blotting. Since TGFβ treatment inhibited the expression p107 but not E2F4, thus, the low level of p107-E2F4 complexes is a result of the inhibition of p107. Our data indicate that E2F4 was switched from p107 to pRb and p130 when p107 was inhibited in response to TGF treatment. By performing electrophoretic gel mobility shift assay (EMSA), we were not able to detect pRb-E2F4 DNA binding activities either in proliferating cells or in G1 cells but exhibited the existence of p107-E2F4 and p130-E2F4 DNA binding complexes in G1 cells. which is consistent with the reports from the ChIP experiments described above. Taking together, our data suggest that p107 and p130, but not pRb, and the repressor E2F, but not activator E2Fs, play a critical role in regulating E2F-responsive gene expression in TGFβ-mediated cell cycle control in human myeloid leukemia cells. This conclusion is consistent with several studies that have failed to detect pRb at E2F-responsive genes, whereas p107 and p130 are readily detectable (Aslanian A 2004, Rayman JB 2002, Takahashi Y 2000, Wells J 2000). In addition, many cell cycle-regulated E2F targets are depressed in p107-/- and p130- /- cells but are not depressed in Rb-/- cells (Herrera RE 1996, Hurford Jr RK 1997, Mulligan GJ 1998). It is not clear if traditional activator E2Fs have any significant impact on cell cycle progression of MV4-11 cells, because pRb-E2F1 and pRb-E2F3 complexes have been found in the proliferating but not in G1 phase cells. Previous studies suggest that pRb may bind other regulatory regions of DNA or uses other mechanisms (Sun H et al. 2011), directly or indirectly, for its inhibitory function as a tumor repressor. pRb may also have both positive or negative role on cell growth, depending on the transcription factors it interacted (Calo E et al. 2010).

#### **5. TGFβ inhibits both G1 and G2 protein kinases in human myeloid leukemia cells**

In mammalian cells, the cell cycle progression is regulated by a group of cyclin dependent kinases (cdks) and their regulatory subunits in sequential order: cyclin D-cdk4/cdk6 and cyclin E-cdk2 complexes act on G1 and G1-S transition, respectively, followed by cyclin Acdk2 on S and cyclin B-cdc2 at G2-M transition. TGF is well known to be a negative regulator of G1 cyclin/cdk activity in many types of cells. This has been reported to be a major mechanism that is responsible for TGF-induced growth inhibition of cells. In epithelial and fibroblasts, the negative role of TGFβ includes inhibition of cdk4 and disruption of cyclin E-cdk2 assembly (Ewen ME et al. 1993, Koff A et al 1993). Although TGFβ is known to be a G1 cdk inhibitor, studies from several groups suggest potential effect of TGFβ on G2 checkpoint kinase. For instance, the mouse epithelial cells treated with TGFβ expressed a lower level of cdc2 as compared with the cells without TGFβ ( Fautsch MP et al 1995); in a hepatoma cell line, TGFβ activates cdc2 and cdk2 followed by phosphorylation of pRb which in turn triggers apoptosis of the cells (Choi KS et al 1999). A new report published in 2008 presented evidence that disruption of Smad 3, a key component of TGFβinduced signal pathway, elevated cdk 1(cdc2) activity via upregulating the expression of cyclin B. As a result, the uncontrolled cdk1 delayed mitotic progression (Fujita T 2008). In human myeloid leukemia cells, we found that inhibition of multiple cdks including cdc2 and cdc25C is a dominant event in TGFβ-induced growth inhibition of the cells (Hu X et al.

substantial quantities of pRb-E2F1 and pRb-E2F3 complexes. In contrast, the cells treated with TGFβ (i.e. G1 cells) showed increased levels of pRb-E2F4 and p130-E2F4 and low level of p107-E2F4, as detected by immunoprecipitation and Western blotting. Since TGFβ treatment inhibited the expression p107 but not E2F4, thus, the low level of p107-E2F4 complexes is a result of the inhibition of p107. Our data indicate that E2F4 was switched from p107 to pRb and p130 when p107 was inhibited in response to TGF treatment. By performing electrophoretic gel mobility shift assay (EMSA), we were not able to detect pRb-E2F4 DNA binding activities either in proliferating cells or in G1 cells but exhibited the existence of p107-E2F4 and p130-E2F4 DNA binding complexes in G1 cells. which is consistent with the reports from the ChIP experiments described above. Taking together, our data suggest that p107 and p130, but not pRb, and the repressor E2F, but not activator E2Fs, play a critical role in regulating E2F-responsive gene expression in TGFβ-mediated cell cycle control in human myeloid leukemia cells. This conclusion is consistent with several studies that have failed to detect pRb at E2F-responsive genes, whereas p107 and p130 are readily detectable (Aslanian A 2004, Rayman JB 2002, Takahashi Y 2000, Wells J 2000). In addition, many cell cycle-regulated E2F targets are depressed in p107-/- and p130- /- cells but are not depressed in Rb-/- cells (Herrera RE 1996, Hurford Jr RK 1997, Mulligan GJ 1998). It is not clear if traditional activator E2Fs have any significant impact on cell cycle progression of MV4-11 cells, because pRb-E2F1 and pRb-E2F3 complexes have been found in the proliferating but not in G1 phase cells. Previous studies suggest that pRb may bind other regulatory regions of DNA or uses other mechanisms (Sun H et al. 2011), directly or indirectly, for its inhibitory function as a tumor repressor. pRb may also have both positive or negative role on cell growth, depending on the transcription factors it interacted (Calo E

**5. TGFβ inhibits both G1 and G2 protein kinases in human myeloid leukemia** 

In mammalian cells, the cell cycle progression is regulated by a group of cyclin dependent kinases (cdks) and their regulatory subunits in sequential order: cyclin D-cdk4/cdk6 and cyclin E-cdk2 complexes act on G1 and G1-S transition, respectively, followed by cyclin Acdk2 on S and cyclin B-cdc2 at G2-M transition. TGF is well known to be a negative regulator of G1 cyclin/cdk activity in many types of cells. This has been reported to be a major mechanism that is responsible for TGF-induced growth inhibition of cells. In epithelial and fibroblasts, the negative role of TGFβ includes inhibition of cdk4 and disruption of cyclin E-cdk2 assembly (Ewen ME et al. 1993, Koff A et al 1993). Although TGFβ is known to be a G1 cdk inhibitor, studies from several groups suggest potential effect of TGFβ on G2 checkpoint kinase. For instance, the mouse epithelial cells treated with TGFβ expressed a lower level of cdc2 as compared with the cells without TGFβ ( Fautsch MP et al 1995); in a hepatoma cell line, TGFβ activates cdc2 and cdk2 followed by phosphorylation of pRb which in turn triggers apoptosis of the cells (Choi KS et al 1999). A new report published in 2008 presented evidence that disruption of Smad 3, a key component of TGFβinduced signal pathway, elevated cdk 1(cdc2) activity via upregulating the expression of cyclin B. As a result, the uncontrolled cdk1 delayed mitotic progression (Fujita T 2008). In human myeloid leukemia cells, we found that inhibition of multiple cdks including cdc2 and cdc25C is a dominant event in TGFβ-induced growth inhibition of the cells (Hu X et al.

et al. 2010).

**cells** 

2001). Our recent studies, using TF-1 and MV4-11 human myeloid leukemia cells, demonstrated that TGF1-induced inhibition of leukemia cell growth is not due solely to downregulation of G1 cyclins and cdks but also G2 check point kinases (Hu X 2007). In MV4- 11 cells, TGF caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216), with 60% and 70% inhibition of phosphorylation of the both proteins by 2-3 hours, respectively, followed by returning to near normal by 12 hours, and 70-100% inhibitions again by 24-72 hours, repectively. The dephosphorylation of cdc2 and cdc25c was followed by a dramatic decrease of total cdc25c (100% decrease at 48 and 72 hours) and cylcin B1 and cdc2 (70-90% reduction at 24 to 72 hours). Consistent with these data, cdc2 kinase activity, as determined by ability to phosphorylate histone H1 and GST-Rb in vitro, was markedly reduced in the leukemia cells treated with TGF for 24-48 h. Cyclin B1 and cdc25c were degraded in a ubiquitin/26S proteosome assay system in vitro, which suggests strongly that ubiquitination and proteasomal degradation may be the key mechanism of degradation that leads to block of G2-M transition. The downregulation of cdc2, cdc25c, and cyclin B1 was not a result of G1 arrest but a direct effect of TGFβ, because the time required for the inhibition of the cells released from G2 is much shorter than the cells released from G1. Using immunoprecipitation and Western blotting we detected that the levels of cyclin B1-cdc2 complexes were much lower in extracts of TGF-treated cells than in control cells. Based on our data, we suggest that TGFβ suppresses growth of human myeloid leukemia cells through multiple pathways, by inhibiting both G1 and G2 checkpoint kinases. Since the G1 and G2 checkpoint kinases are able to phosphorylate pRb in proliferating cells, the down regulation of G1 and G2 checkpoint kinases would prevent pRb phospharylation, leading to G1 arrest. The data also suggest that cdc2, a traditional G2 regulator in mammalian cells, may participate in G1 regulation. Fig. 1 illustrates diagrammatically the negative roles of TGFβ in the cell cycle control in myeloid leukemic MV4-11 cells.

Fig. 1. TGFβ inhibits both G! and G@ cdks in MV4-11 cells

Distinct Inhibitory Effect of TGFβ on the Growth of Human Myeloid Leukemia Cells 205

response to TGFβ. The downregulation is a result of inhibition of total cdk2 by TGFβ. However, TGF had no effect on the expression of cyclin E. By carrying out p27 immunodepletion and transfection experiments, we further demonstrated that that at least some cdk2 kinase activity is associated with p27 in MV4-11cells. Additional support for this possibility was the observation that cells transfected with anti-sense p27 cDNA but not empty plasmid, downregulated cdk2 kinase activity by more than 60%. Although p27 also may act as an adapter of cyclin E activity, as suggested by expression of cyclin E- p27 complexes in proliferating MV4-11 cells, p27 mainly acts as an inhibitor of cyclin E activity in response to TGF. This is indicated by the fact that TGF1 upregulated cyclin Eassociated p27 and was accompanied by the inhibition of cyclin E-dependent kinase activity. Moreover, p27 immunodepletion dramatically upregulated cyclin E-dependent kinase activity in TGF1 treated cells. The effect of p27 on cyclin E-dependent kinase activity is dose-dependent, with only high concentrations of p27 being inhibitory to cyclin E activity. This may explain the observation that immunodepletion of p27 or anti-sense p27 cDNA did not significantly increase cyclin E-dependent kinase activity in proliferating MV4-11 cells. These results are against the notion that p27 is a negative regulator of cdk2 kinase *in vivo,* at least in these human myeloid leukemia cells. Our data are consistent with previous findings, in which cell cycle arrest mediated by TGF remained intact in p27-/- cells (Nakayama et al., 1996) and p21 and p27 has dual roles in cell cycle control (Rowell EA et al. 2005, LaBaer et al. 1997, Zhang H et al. 1994). The difference is that the negative or positive role of p27 in our model is related to the molecules p27 interacted but not the concentrations of p27 as reported in the case of p21 (Matsushime et al., 1992). Taken together, we interpret our results to suggest that in proliferating cells p27 is associated with cdk2 and cdk4 as an adapter. TGF1 downregulates cdk2, cdk4, and D-type cyclins, which leaves p27kip1 free to bind and saturate cyclin E, inhibiting cyclin E-cdk2 assembly, rendering cyclin E-dependent kinase inactive. By using these two different regulatory mechanisms, TGF1 magnifies its

cell cycle inhibitory efficiency in human myeloid leukemia cells (Hu X et al 2001).

and p21 have both positive and negative effect on G1 cdk activities.

New findings in recent years have provided insight into the complex roles and action mechanisms of TGFβ in various types of cells. TGFβ exhibits its distinct effect on the growth of human myeloid leukemia cells. Although both positive and negative roles of TGFβ in hematopoiesis have been reported, the major role of TGFβ appears to be inhibitory with heterogeneous responses being observed in human myeloid leukemia cells. The heterogeneous responses of the cells to TGFβ are not related to the apparent differentiation stages of the cells but could result from differences in the expression of TGF receptors or degrees of the mutations in the components of TGFβ signal pathways. TGFβ, a traditional G1 cdk inhibitor, represses not only G1 but also G2 protein kinases in human myeloid leukemia cells. pRb has been reported to be a tumor suppressor in various types of cells. The new studies suggest that p130, whose gene is less mutated in cancer patients, plays a critical role in maintaining cells in quiescent status and in TGFβ-mediated growth inhibition. Traditionally, p27 and p21 are G1 cdk inhibitors. However, new evidence suggests that p27

Based on the data described in this review, it is clear that TGFβ signals have a defined role in regulating normal hematopoiesis, and disruption of the signaling components could lead

**7. Conclusion** 

#### **6. Dual roles of cdk inhibitors in human myeloid leukemia cells**

It is well known that activity of cdks is counterbalanced by cdk inhibitors (ckis). The cdk inhibitors can be classified into two different families: Ink4 (p16, p15, p18 and p19) and cip/kip (p21, p27, and p57), based on their structure similarity. Among these cdk inhibitors, p27 and p21 have been intensively studied in various types of cells. In quiescent cells or cells treated with TGFβ show high level of p27, whereas proliferating cells or cells treated with mitogens express low level of p27 (Moller MB 2000). Overexpression of p27 in cultured cells arrests the cells in G1 phase and antisense inhibition of p27 reverses cell cycle arrest induced by p27 (Coats S et al 1996; Rivard N et al. 1996). In vivo, p27 is a critical regulator of cellular proliferation. Mice lacking p27 gene are 15–30% larger than their wild-type or hemizygous (*p27*+/−) littermates (Fero et al. 1996; Kiyokawa et al. 1996). p27 was initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. The crystal structure of p27 bound to cyclin-cdk2 reveals that a p27 fragment inserts itself within the cdk2 catalytic site, blocking ATP access (Russo et al., 1996). These data lead to a popular model in which anti-proliferative stimuli up-regulate p27, followed by inhibition of cyclinD-cdk4, cyclinE-cdk2, cyclin A-cdk2 activities, and by G1 arrest/growth inhibition. Similar to p27, as a negative regulator of the cell cycle progression, p21 is thought to bind and inhibit cdk2/cyclin E and/or cdk4/cyclin D complexes, thereby arresting cells at the G1 phase. The induction of p21 is regulated by the tumor suppressor gene, p53, in response to DNA damage.

The traditional model of p27 or p21 as a cdk inhibitor has been challenged by a number of studies from several labs in which p21 or p27 were demonstrated to be required for a cdk/cyclin kinase activity or to be a dual player in cell cycle control (Zhang H et al 1994, LaBaer et al 1997). It was reported that p21, at low concentration, promotes the assembly of active kinase complexes, whereas a high concentration of p21 causes the opposite effect in SAOS-2 cells derived from osteosarcoma (LaBaer et al., 1997). In an animal model, it was found that low concentration of p21 could promote tumor progression in the presence of oncogenic factors (Jones JM 1999). Primary mouse fibroblasts that are p21-/- and p27-/ were failed to assemble cylcin D-CDK complexes and were unable to direct cyclin D nuclear translocation (Cheng M 1999). Consistent with this report, p27 was demonstrated to be essential for mouse mammary gland morphogenesis and function (Muraoka RS et al. 2001); an increased level of p21 was found to be parallel with S-phase entry of oligodendrocytes (Bansal R et al. 2005). In another report published in the same year, p27 in mouse T cells was found to acts as a promoter during the early stage of the cell cycle (Rowell EA et al 2005). In addition, it has been reported that in MCF-7 cells, cyclin D1 promotes cellular migration through inducing the expression of p27 and by physically associating with p27 to promote cellular migration (Li Z et al. 2006). These dual roles of p21 and p27 in non-hematopoietic cells are consistent with what we found in human hematopoietic cells a few years ago. By using several human myeloid leukemia cell lines as research models, we demonstrated that p27 has differential effect on cdk2 and cyclin E activities, which provides supplementary information to the evidence of p27 and p21 having dual roles in cell cycle control described above (Hu X et al. 2001). Although TGFβ greatly upregulated p27 in both cytosol and nucleus in MV4-11 cells, the association of p27 with cylcin E and cdk2 showed different patterns with increased amount of cyclin E-p27 and decreased levels of cdk2-p27 being detected by Western blot and immunoprecipitation. Surprised, a significant amount of p27 was found to associate with cdk2 in proliferating cells, which was downregulated in response to TGFβ. The downregulation is a result of inhibition of total cdk2 by TGFβ. However, TGF had no effect on the expression of cyclin E. By carrying out p27 immunodepletion and transfection experiments, we further demonstrated that that at least some cdk2 kinase activity is associated with p27 in MV4-11cells. Additional support for this possibility was the observation that cells transfected with anti-sense p27 cDNA but not empty plasmid, downregulated cdk2 kinase activity by more than 60%. Although p27 also may act as an adapter of cyclin E activity, as suggested by expression of cyclin E- p27 complexes in proliferating MV4-11 cells, p27 mainly acts as an inhibitor of cyclin E activity in response to TGF. This is indicated by the fact that TGF1 upregulated cyclin Eassociated p27 and was accompanied by the inhibition of cyclin E-dependent kinase activity. Moreover, p27 immunodepletion dramatically upregulated cyclin E-dependent kinase activity in TGF1 treated cells. The effect of p27 on cyclin E-dependent kinase activity is dose-dependent, with only high concentrations of p27 being inhibitory to cyclin E activity. This may explain the observation that immunodepletion of p27 or anti-sense p27 cDNA did not significantly increase cyclin E-dependent kinase activity in proliferating MV4-11 cells. These results are against the notion that p27 is a negative regulator of cdk2 kinase *in vivo,* at least in these human myeloid leukemia cells. Our data are consistent with previous findings, in which cell cycle arrest mediated by TGF remained intact in p27-/- cells (Nakayama et al., 1996) and p21 and p27 has dual roles in cell cycle control (Rowell EA et al. 2005, LaBaer et al. 1997, Zhang H et al. 1994). The difference is that the negative or positive role of p27 in our model is related to the molecules p27 interacted but not the concentrations of p27 as reported in the case of p21 (Matsushime et al., 1992). Taken together, we interpret our results to suggest that in proliferating cells p27 is associated with cdk2 and cdk4 as an adapter. TGF1 downregulates cdk2, cdk4, and D-type cyclins, which leaves p27kip1 free to bind and saturate cyclin E, inhibiting cyclin E-cdk2 assembly, rendering cyclin E-dependent kinase inactive. By using these two different regulatory mechanisms, TGF1 magnifies its cell cycle inhibitory efficiency in human myeloid leukemia cells (Hu X et al 2001).

#### **7. Conclusion**

204 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

It is well known that activity of cdks is counterbalanced by cdk inhibitors (ckis). The cdk inhibitors can be classified into two different families: Ink4 (p16, p15, p18 and p19) and cip/kip (p21, p27, and p57), based on their structure similarity. Among these cdk inhibitors, p27 and p21 have been intensively studied in various types of cells. In quiescent cells or cells treated with TGFβ show high level of p27, whereas proliferating cells or cells treated with mitogens express low level of p27 (Moller MB 2000). Overexpression of p27 in cultured cells arrests the cells in G1 phase and antisense inhibition of p27 reverses cell cycle arrest induced by p27 (Coats S et al 1996; Rivard N et al. 1996). In vivo, p27 is a critical regulator of cellular proliferation. Mice lacking p27 gene are 15–30% larger than their wild-type or hemizygous (*p27*+/−) littermates (Fero et al. 1996; Kiyokawa et al. 1996). p27 was initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. The crystal structure of p27 bound to cyclin-cdk2 reveals that a p27 fragment inserts itself within the cdk2 catalytic site, blocking ATP access (Russo et al., 1996). These data lead to a popular model in which anti-proliferative stimuli up-regulate p27, followed by inhibition of cyclinD-cdk4, cyclinE-cdk2, cyclin A-cdk2 activities, and by G1 arrest/growth inhibition. Similar to p27, as a negative regulator of the cell cycle progression, p21 is thought to bind and inhibit cdk2/cyclin E and/or cdk4/cyclin D complexes, thereby arresting cells at the G1 phase. The induction of p21 is regulated by the tumor suppressor gene, p53, in response to DNA

The traditional model of p27 or p21 as a cdk inhibitor has been challenged by a number of studies from several labs in which p21 or p27 were demonstrated to be required for a cdk/cyclin kinase activity or to be a dual player in cell cycle control (Zhang H et al 1994, LaBaer et al 1997). It was reported that p21, at low concentration, promotes the assembly of active kinase complexes, whereas a high concentration of p21 causes the opposite effect in SAOS-2 cells derived from osteosarcoma (LaBaer et al., 1997). In an animal model, it was found that low concentration of p21 could promote tumor progression in the presence of oncogenic factors (Jones JM 1999). Primary mouse fibroblasts that are p21-/- and p27-/ were failed to assemble cylcin D-CDK complexes and were unable to direct cyclin D nuclear translocation (Cheng M 1999). Consistent with this report, p27 was demonstrated to be essential for mouse mammary gland morphogenesis and function (Muraoka RS et al. 2001); an increased level of p21 was found to be parallel with S-phase entry of oligodendrocytes (Bansal R et al. 2005). In another report published in the same year, p27 in mouse T cells was found to acts as a promoter during the early stage of the cell cycle (Rowell EA et al 2005). In addition, it has been reported that in MCF-7 cells, cyclin D1 promotes cellular migration through inducing the expression of p27 and by physically associating with p27 to promote cellular migration (Li Z et al. 2006). These dual roles of p21 and p27 in non-hematopoietic cells are consistent with what we found in human hematopoietic cells a few years ago. By using several human myeloid leukemia cell lines as research models, we demonstrated that p27 has differential effect on cdk2 and cyclin E activities, which provides supplementary information to the evidence of p27 and p21 having dual roles in cell cycle control described above (Hu X et al. 2001). Although TGFβ greatly upregulated p27 in both cytosol and nucleus in MV4-11 cells, the association of p27 with cylcin E and cdk2 showed different patterns with increased amount of cyclin E-p27 and decreased levels of cdk2-p27 being detected by Western blot and immunoprecipitation. Surprised, a significant amount of p27 was found to associate with cdk2 in proliferating cells, which was downregulated in

**6. Dual roles of cdk inhibitors in human myeloid leukemia cells** 

damage.

New findings in recent years have provided insight into the complex roles and action mechanisms of TGFβ in various types of cells. TGFβ exhibits its distinct effect on the growth of human myeloid leukemia cells. Although both positive and negative roles of TGFβ in hematopoiesis have been reported, the major role of TGFβ appears to be inhibitory with heterogeneous responses being observed in human myeloid leukemia cells. The heterogeneous responses of the cells to TGFβ are not related to the apparent differentiation stages of the cells but could result from differences in the expression of TGF receptors or degrees of the mutations in the components of TGFβ signal pathways. TGFβ, a traditional G1 cdk inhibitor, represses not only G1 but also G2 protein kinases in human myeloid leukemia cells. pRb has been reported to be a tumor suppressor in various types of cells. The new studies suggest that p130, whose gene is less mutated in cancer patients, plays a critical role in maintaining cells in quiescent status and in TGFβ-mediated growth inhibition. Traditionally, p27 and p21 are G1 cdk inhibitors. However, new evidence suggests that p27 and p21 have both positive and negative effect on G1 cdk activities.

Based on the data described in this review, it is clear that TGFβ signals have a defined role in regulating normal hematopoiesis, and disruption of the signaling components could lead

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**12** 

*Frederick, Maryland* 

*Frederick, Maryland* 

*USA* 

**Novel Targets in Myelogenous Leukemia:** 

**The Id Family of Proteins** 

Kimberly D. Klarmann1, Ming Ji1, Huajie Li1,

Ande Satyanarayana2, Wonil Kim1, Emily Bowers2, Bjorg Gudmundsdottir2 and Jonathan R. Keller1

*1Laboratory of Cancer Prevention, SAIC-Frederick, Inc., NCI-Frederick,* 

*2Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute,* 

The successful treatment of myelogenous leukemia depends upon the continuing coordinate efforts of the medical and research communities and patients to understand the complex nature of leukemia development and progression, and to battle drug resistant cells. Novel drug therapies, like Imatinib, have been developed which target the mutant BCR-ABL (Breakpoint Cluster Region-Abelson Kinase) protein, and have significantly changed the treatment regimens for chronic myelogenous leukemia (CML). However, while Imatinib is frequently effective against the majority of the leukemic cells, and patients often undergo remission after treatment, these patients also relapse due to a leukemic drug-resistant population (Schemionek et al., 2010). In comparison to CML, the treatment for acute myelogenous leukemia (AML) is predominantly induction therapy using strong anti-mitotic agents, followed by consolidation therapy to kill residual diseased cells (Venditti et al., 2000). These therapies are not specifically targeted to mutated proteins in the cells, and therefore can be more toxic to the patient's normal cells. Researchers have attempted to target mutated proteins in AML. However, unlike BCR-ABL, targeting FLT3-ITD (FMS-like tyrosine kinase 3-Internal Tandem Duplication) and FLT3-TKD (FMS-like tyrosine kinase 3- Tyrosine Kinase Domain) mutations have not been as successful as CML-Imatinib therapy (Kindler et al., 2010). Therefore, challenges facing patients with AML remain significant, and

To identify novel drug targets, researchers will have to gain a better understanding of the molecular mechanisms that regulate normal hematopoietic development. Hematopoiesis depends upon the activity of transcription factors and their regulators to function at the correct time and place during differentiation. Failure to control these processes can lead to deregulated proliferation and impaired normal differentiation. Ultimately, these errors can

**1.1 Myelogenous leukemia and the need for novel drug targets** 

**1.2 Transcription factor deregulation in myelogenous leukemia** 

**1. Introduction** 

drug-resistant relapses remain a threat.


### **Novel Targets in Myelogenous Leukemia: The Id Family of Proteins**

Kimberly D. Klarmann1, Ming Ji1, Huajie Li1, Ande Satyanarayana2, Wonil Kim1, Emily Bowers2, Bjorg Gudmundsdottir2 and Jonathan R. Keller1 *1Laboratory of Cancer Prevention, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 2Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, Frederick, Maryland USA* 

#### **1. Introduction**

214 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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#### **1.1 Myelogenous leukemia and the need for novel drug targets**

The successful treatment of myelogenous leukemia depends upon the continuing coordinate efforts of the medical and research communities and patients to understand the complex nature of leukemia development and progression, and to battle drug resistant cells. Novel drug therapies, like Imatinib, have been developed which target the mutant BCR-ABL (Breakpoint Cluster Region-Abelson Kinase) protein, and have significantly changed the treatment regimens for chronic myelogenous leukemia (CML). However, while Imatinib is frequently effective against the majority of the leukemic cells, and patients often undergo remission after treatment, these patients also relapse due to a leukemic drug-resistant population (Schemionek et al., 2010). In comparison to CML, the treatment for acute myelogenous leukemia (AML) is predominantly induction therapy using strong anti-mitotic agents, followed by consolidation therapy to kill residual diseased cells (Venditti et al., 2000). These therapies are not specifically targeted to mutated proteins in the cells, and therefore can be more toxic to the patient's normal cells. Researchers have attempted to target mutated proteins in AML. However, unlike BCR-ABL, targeting FLT3-ITD (FMS-like tyrosine kinase 3-Internal Tandem Duplication) and FLT3-TKD (FMS-like tyrosine kinase 3- Tyrosine Kinase Domain) mutations have not been as successful as CML-Imatinib therapy (Kindler et al., 2010). Therefore, challenges facing patients with AML remain significant, and drug-resistant relapses remain a threat.

#### **1.2 Transcription factor deregulation in myelogenous leukemia**

To identify novel drug targets, researchers will have to gain a better understanding of the molecular mechanisms that regulate normal hematopoietic development. Hematopoiesis depends upon the activity of transcription factors and their regulators to function at the correct time and place during differentiation. Failure to control these processes can lead to deregulated proliferation and impaired normal differentiation. Ultimately, these errors can

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 217

marrow, testis and brain. Id4 is expressed during embryogenesis, but is not detectable in the fetal liver. Id4 expression is present in the adult bone marrow, testis, kidney, brain, and spleen. These data suggest that the Id proteins may be critical during development, as well as having important functions in the adult. In this regard, simultaneous deletion of any two Id gene family members results in early embryonic lethality, demonstrating that these genes are required for development (Fraidenraich et al., 2004)**.** Therefore, in summary, as a family the Id proteins are present in many different tissues, and are expressed during early

**2.2 Id protein function: Regulation of transcription factors and cell cycle regulators**  The Id proteins function by blocking the DNA binding activity of three types of essential transcription factors: **(1)** The basic Helix-Loop-Helix (bHLH) proteins (E proteins), **(2)** Helix-Turn-Helix ETS transcription factors, and **(3)** Pax transcription factors (Pax-5 in B cells) **(Figure 2)**. In addition, specific members of the Id family decrease the activity of Rb

Fig. 2. Id proteins bind bHLH, ETS, and Pax transcription factors and block their function. **(A)** bHLH, ETS, and Pax factors dimerize and bind to and activate the promoter regions of

**(B)** When Id proteins are present, they bind to bHLH, ETS and Pax transcription factors,

blocking their dimerization and inhibiting their ability to activate transcription.

genes essential for differentiation and cell cycle arrest.

development as well as in adult tissues.

tumor suppressors (Iavarone et al., 1994; Lasorella et al., 1996).

lead to myeloproliferative diseases (MPD), myelodysplastic syndromes (MDS), and chronic or acute myelogenous leukemia (Rosenbauer et al., 2005). Current research efforts are focused on the molecular mechanism(s) that contribute to transcription factor deregulation with a goal of designing better, safer and less toxic therapies, to restore normal differentiation or initiate apoptosis of leukemic cell populations. Deregulation can result from many different types of mutations including translocations, deletions, and point mutations, which can result in loss of function, gain of function, overexpression or underexpression. The Id (Inhibitor of DNA Binding) proteins are key downstream transcriptional regulators of multiple neoplastic mutations and are deregulated (have increased expression) in many types of cancer, and therefore may be good therapeutic targets particularly for drug-resistant forms of leukemia.

#### **2. Id proteins inhibit transcription factors that regulate cell cycle and differentiation**

#### **2.1 Tissue specific expression of Id proteins**

The Id proteins belong to a subclass of the helix-loop-helix (HLH) family of proteins. There are four family members (Id1, Id2, Id3, Id4), which range in size from 13 to 20 kDa **(Figure 1)**. In normal tissues, Id1 and Id3 are ubiquitously expressed; they are detectable throughout embryonic development and are present in the bone marrow, testis, kidney, brain, liver and spleen. The expression of Id2 and Id4 are more restricted (Riechmann et al., 1994). Id2, is not detectable until day 13.5 of fetal liver development. Id2 is most highly expressed in the bone

Fig. 1. Schematic representation of the Id family

Id2, Id3, and Id4 have Cdk2 phosphorylation sites, although only Id2 and Id3 are known to be phosphorylated by cyclinA/E-Cdk2. Cdk2-dependent phosphorylation of Id2 (Hara et al., 1997) and Id3 (Deed et al., 1997) inhibits binding to E proteins and Ets transcription factors. Id proteins are ubiquitinated and undergo rapid turnover during the cell cycle mediated by ubiquitination and proteosomal degredation, and two different targeting mechanisms have been reported. Id1, Id2, and Id4 have "D box" destruction motifs (RxxLxxxN) which is recognized by the anaphase promoting complex (APC) which targets the proteins for ubiquitination (Harper et al., 2002; Lasorella et al., 2006).Degradation of Id1 and Id3, is mediated by the COP9 signalosome (Berse et al., 2004).

lead to myeloproliferative diseases (MPD), myelodysplastic syndromes (MDS), and chronic or acute myelogenous leukemia (Rosenbauer et al., 2005). Current research efforts are focused on the molecular mechanism(s) that contribute to transcription factor deregulation with a goal of designing better, safer and less toxic therapies, to restore normal differentiation or initiate apoptosis of leukemic cell populations. Deregulation can result from many different types of mutations including translocations, deletions, and point mutations, which can result in loss of function, gain of function, overexpression or underexpression. The Id (Inhibitor of DNA Binding) proteins are key downstream transcriptional regulators of multiple neoplastic mutations and are deregulated (have increased expression) in many types of cancer, and therefore may be good therapeutic

**2. Id proteins inhibit transcription factors that regulate cell cycle and** 

The Id proteins belong to a subclass of the helix-loop-helix (HLH) family of proteins. There are four family members (Id1, Id2, Id3, Id4), which range in size from 13 to 20 kDa **(Figure 1)**. In normal tissues, Id1 and Id3 are ubiquitously expressed; they are detectable throughout embryonic development and are present in the bone marrow, testis, kidney, brain, liver and spleen. The expression of Id2 and Id4 are more restricted (Riechmann et al., 1994). Id2, is not detectable until day 13.5 of fetal liver development. Id2 is most highly expressed in the bone

Id2, Id3, and Id4 have Cdk2 phosphorylation sites, although only Id2 and Id3 are known to be phosphorylated by cyclinA/E-Cdk2. Cdk2-dependent phosphorylation of Id2 (Hara et al., 1997) and Id3 (Deed et al., 1997) inhibits binding to E proteins and Ets transcription factors. Id proteins are ubiquitinated and undergo rapid turnover during the cell cycle mediated by ubiquitination and proteosomal degredation, and two different targeting mechanisms have been reported. Id1, Id2, and Id4 have "D box" destruction motifs

(RxxLxxxN) which is recognized by the anaphase promoting complex (APC) which targets the proteins for ubiquitination (Harper et al., 2002; Lasorella et al., 2006).Degradation of Id1

and Id3, is mediated by the COP9 signalosome (Berse et al., 2004).

targets particularly for drug-resistant forms of leukemia.

**2.1 Tissue specific expression of Id proteins** 

Fig. 1. Schematic representation of the Id family

**differentiation** 

marrow, testis and brain. Id4 is expressed during embryogenesis, but is not detectable in the fetal liver. Id4 expression is present in the adult bone marrow, testis, kidney, brain, and spleen. These data suggest that the Id proteins may be critical during development, as well as having important functions in the adult. In this regard, simultaneous deletion of any two Id gene family members results in early embryonic lethality, demonstrating that these genes are required for development (Fraidenraich et al., 2004)**.** Therefore, in summary, as a family the Id proteins are present in many different tissues, and are expressed during early development as well as in adult tissues.

#### **2.2 Id protein function: Regulation of transcription factors and cell cycle regulators**

The Id proteins function by blocking the DNA binding activity of three types of essential transcription factors: **(1)** The basic Helix-Loop-Helix (bHLH) proteins (E proteins), **(2)** Helix-Turn-Helix ETS transcription factors, and **(3)** Pax transcription factors (Pax-5 in B cells) **(Figure 2)**. In addition, specific members of the Id family decrease the activity of Rb tumor suppressors (Iavarone et al., 1994; Lasorella et al., 1996).

Fig. 2. Id proteins bind bHLH, ETS, and Pax transcription factors and block their function. **(A)** bHLH, ETS, and Pax factors dimerize and bind to and activate the promoter regions of genes essential for differentiation and cell cycle arrest.

**(B)** When Id proteins are present, they bind to bHLH, ETS and Pax transcription factors, blocking their dimerization and inhibiting their ability to activate transcription.

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 219

**Retinoblastoma (Rb) and p53 tumor suppressors.** The retinoblastoma protein (Rb), is an important part of normal cell cycle control. When Rb is hypo-phosphorylated, it is active and bound to E2F, causing cell cycle arrest by preventing E2F from inducing the expression of genes necessary for cell proliferation (Sherr and McCormick, 2002). When Rb is phosphorylated by the cyclin dependent kinases (Cdks), it dissociates from E2F, and this allows E2F to activate the expression of genes necessary for DNA replication. Rb is frequently mutated in many different malignancies, and finding ways to restore Rb function remains a goal in cancer research. Interestingly, Id2 alone of the Id family binds to the hypophosphorylated Rb, as well as related proteins p107 and p130 and inhibits their growth suppressive activities. The Id proteins do not affect the expression levels or phosphorylation state of Rb (Lasorella et al., 1996). However, when Id2 binds in the pocket-binding of Rb site it prevents it from arresting the cell cycle (Iavarone et al., 1994; Lasorella et al., 1996). There are also reports that Id1 can decrease p53 activity, although it is not known whether this is a direct or an indirect effect. The reduction of p53 activity results in decreased PTEN tumor suppressor expression, and ultimately results in increased cellular proliferation (Lee et al.,

In summary, the Id family of proteins affects both differentiation and cell cycle progression by inhibiting the DNA binding activity of the E and ETS protein transcription factors, by binding to and disrupting the activity of Rb and related proteins, and by decreasing p53

Id proteins were first identified in a mouse leukemic cell line in the search for novel factors containing HLH domains. Benezra et al. identified the first Id protein in murine erythroleukemia (MEL) cells in 1990 (Benezra et al., 1990). They observed that Id1 was present in less-differentiated cell types, and found that Id expression decreased after differentiation was induced in erythroid, muscle, and endothelial cell lines. Subsequently, using a myeloid progenitor cell line, 32DCl3, it was found that endogenous Id1 expression rapidly decreased in cells induced to differentiate to neutrophilic granulocytes by granulocyte colony stimulating factor (G-CSF), then rose again during the later stages of differentiation. Id2 expression also decreased following G-CSF treatment of 32DCl3 cells (Leeanansaksiri et al., 2005), as well as in multiple other myeloid cell lines (Ishiguro et al., 1996). When Id1 concentration was artificially elevated in the 32DCl3 cells, the cells failed to differentiate in response to G-CSF (Kreider et al., 1992). Similarly, overexpression of Id1 in MEL cells also resulted in a block in differentiation (Shoji et al., 1994). Therefore, in normal cells, Id1 expression needs to decrease for a defined period after the induction of differentiation. If Id proteins are overexpressed during this phase, the cells do not

In mouse bone marrow, moderate levels of Id1 are present in the hematopoietic stem cells (HSC) and common lymphoid progenitors (CLP). Id1 expression is increased in the more differentiated common myeloid progenitors (CMP), and further increased in the granulocyte macrophage progenitors (GMP), however Id1 levels do not increase in megakaryocyte erythroid progenitors (MEP). The dynamic levels of Id1 expression during these early stages of hematopoiesis suggest that Id1 may regulate cell fate decisions during myeloid, erythroid, and lymphoid development (Leeanansaksiri et al., 2005). In support of this

differentiate in spite of the extracellular signals instructing them to mature.

**3. The role of Id proteins in normal myelopoiesis** 

**3.1 Id protein levels during myelopoiesis** 

2009).

activity.

**E proteins and regulation of differentiation.** The HLH transcription factors are essential for regulating the differentiation programs of multiple tissues types. The E proteins, also known as "Class A" HLH family members, include *E2A* (encoding E12 and E47 proteins), *HEB*, and *E2-2*. E proteins homodimerize or heterodimerize with "Class B" bHLH family members, tissue specific proteins such as SCL/tal1 (hematopoiesis), HAND, MyoD, myogenin (myogenesis), NeuroD/BETA2 (pancreatic development and neurogenesis), HES-1, and MASH-1 (neurogenesis). When dimerized, these bHLH proteins bind to E box consensus sequence (CANNTG, where "N" is any nucleotide) through their basic N-terminal regions. When Id proteins are present, they bind to the E proteins and disrupt their dimerization with the Class B binding partners (Langlands et al., 1997), and block the differentiation of muscle, neuronal, pancreatic and hematopoietic cells. The Id proteins homodimerize poorly with each other and preferentially bind to the Class A HLH factors (Sun et al., 1991). In summary, the effect of Id proteins, prevents or arrests differentiation by blocking the E protein transcription activating function in many different cell types.

**E proteins and cell cycle control.** The E proteins also induce the expression of cell cycle inhibitors, such as p16INK4a and p21Cip1. The cell cycle inhibitors cyclin dependent kinases 4 and 6 (Cdk4, Cdk6), are necessary for the cell to progress to S phase. Therefore, Id proteins inhibit the transcriptional activity of the E proteins, which decrease the expression of cell cycle inhibitors, and ultimately results in increased cellular proliferation (Alani et al., 2001; Zheng et al., 2004; Prabhu et al., 1997). In support of this, when Id1 is knocked-out in mice, mouse embryo fibroblasts that lack Id1 (Id1-/-) undergo premature senescence, due in part to increased expression of p16INK4a (Alani et al., 2001). Thus, increased expression of Id1 proteins promotes cell proliferation by inhibiting the function of E protein transcriptional activity, by blocking their ability to induce the expression of key cell cycle inhibitors.

**Regulation of ETS proteins by Ids.** The Id proteins also regulate the activity of the ETS (E twenty-six) helix-turn-helix family of transcription factors. The ETS proteins comprise one of the largest families of transcription factors, and they bind the canonical sequence GGAA/T. The ETS proteins are characterized by the presence of the ETS DNA binding domain, and the family includes PU.1 (SPI1), Ets1, Ets2, TEL, and TEL2 as well as the ternary complex factors (TCF): Elk1, Elk3 (Net/SAP2), and Elk4 (SAP1). The Id family members interact with the ETS protein DNA binding domain, blocking the ability of the ETS proteins to bind DNA, and repressing their ability to activate transcription. Id1, Id2 and Id3 have all been shown to bind to the TCF proteins Elk-1 and Elk-4, and Id1 and Id3 both suppress the expression of the cell cycle inhibitor p27Kip1 by blocking Elk-1's ability to activate transcription (Yates et al., 1999; Chassot et al., 2007). As previously described, Ids can decrease p16INK4a protein expression by repressing E protein activity, in addition, Id1 has also been shown to reduce p16INK4a expression by the inhibiting the activation of its expression by Ets1 and Ets2 (Ohtani et al., 2001). Interestingly, the ability of the Id proteins to bind to the ETS transcription factors can be inhibited by phosphorylation of the Ids. Within the Id family, only Id2 and Id3 undergo phosphorylation, and phosphorylation of these Ids by cyclin A/E-Cdk2 decreases their ability to bind to ETS proteins (specifically, Elk-1), resulting in cell cycle arrest (Stinson et al., 2003). This mechanism may compose part of a normal cell cycle regulatory feedback mechanism in cells that permits cell populations to expand and then return to a growth arrested state. In summary, Id proteins inhibit the function of the ETS proteins, blocking their ability to activate transcription of cell cycle inhibitors, which results in increased proliferation.

**E proteins and regulation of differentiation.** The HLH transcription factors are essential for regulating the differentiation programs of multiple tissues types. The E proteins, also known as "Class A" HLH family members, include *E2A* (encoding E12 and E47 proteins), *HEB*, and *E2-2*. E proteins homodimerize or heterodimerize with "Class B" bHLH family members, tissue specific proteins such as SCL/tal1 (hematopoiesis), HAND, MyoD, myogenin (myogenesis), NeuroD/BETA2 (pancreatic development and neurogenesis), HES-1, and MASH-1 (neurogenesis). When dimerized, these bHLH proteins bind to E box consensus sequence (CANNTG, where "N" is any nucleotide) through their basic N-terminal regions. When Id proteins are present, they bind to the E proteins and disrupt their dimerization with the Class B binding partners (Langlands et al., 1997), and block the differentiation of muscle, neuronal, pancreatic and hematopoietic cells. The Id proteins homodimerize poorly with each other and preferentially bind to the Class A HLH factors (Sun et al., 1991). In summary, the effect of Id proteins, prevents or arrests differentiation by blocking the E

**E proteins and cell cycle control.** The E proteins also induce the expression of cell cycle inhibitors, such as p16INK4a and p21Cip1. The cell cycle inhibitors cyclin dependent kinases 4 and 6 (Cdk4, Cdk6), are necessary for the cell to progress to S phase. Therefore, Id proteins inhibit the transcriptional activity of the E proteins, which decrease the expression of cell cycle inhibitors, and ultimately results in increased cellular proliferation (Alani et al., 2001; Zheng et al., 2004; Prabhu et al., 1997). In support of this, when Id1 is knocked-out in mice, mouse embryo fibroblasts that lack Id1 (Id1-/-) undergo premature senescence, due in part to increased expression of p16INK4a (Alani et al., 2001). Thus, increased expression of Id1 proteins promotes cell proliferation by inhibiting the function of E protein transcriptional activity, by blocking their ability to induce the expression of

**Regulation of ETS proteins by Ids.** The Id proteins also regulate the activity of the ETS (E twenty-six) helix-turn-helix family of transcription factors. The ETS proteins comprise one of the largest families of transcription factors, and they bind the canonical sequence GGAA/T. The ETS proteins are characterized by the presence of the ETS DNA binding domain, and the family includes PU.1 (SPI1), Ets1, Ets2, TEL, and TEL2 as well as the ternary complex factors (TCF): Elk1, Elk3 (Net/SAP2), and Elk4 (SAP1). The Id family members interact with the ETS protein DNA binding domain, blocking the ability of the ETS proteins to bind DNA, and repressing their ability to activate transcription. Id1, Id2 and Id3 have all been shown to bind to the TCF proteins Elk-1 and Elk-4, and Id1 and Id3 both suppress the expression of the cell cycle inhibitor p27Kip1 by blocking Elk-1's ability to activate transcription (Yates et al., 1999; Chassot et al., 2007). As previously described, Ids can decrease p16INK4a protein expression by repressing E protein activity, in addition, Id1 has also been shown to reduce p16INK4a expression by the inhibiting the activation of its expression by Ets1 and Ets2 (Ohtani et al., 2001). Interestingly, the ability of the Id proteins to bind to the ETS transcription factors can be inhibited by phosphorylation of the Ids. Within the Id family, only Id2 and Id3 undergo phosphorylation, and phosphorylation of these Ids by cyclin A/E-Cdk2 decreases their ability to bind to ETS proteins (specifically, Elk-1), resulting in cell cycle arrest (Stinson et al., 2003). This mechanism may compose part of a normal cell cycle regulatory feedback mechanism in cells that permits cell populations to expand and then return to a growth arrested state. In summary, Id proteins inhibit the function of the ETS proteins, blocking their ability to activate transcription of cell cycle inhibitors, which results

protein transcription activating function in many different cell types.

key cell cycle inhibitors.

in increased proliferation.

**Retinoblastoma (Rb) and p53 tumor suppressors.** The retinoblastoma protein (Rb), is an important part of normal cell cycle control. When Rb is hypo-phosphorylated, it is active and bound to E2F, causing cell cycle arrest by preventing E2F from inducing the expression of genes necessary for cell proliferation (Sherr and McCormick, 2002). When Rb is phosphorylated by the cyclin dependent kinases (Cdks), it dissociates from E2F, and this allows E2F to activate the expression of genes necessary for DNA replication. Rb is frequently mutated in many different malignancies, and finding ways to restore Rb function remains a goal in cancer research. Interestingly, Id2 alone of the Id family binds to the hypophosphorylated Rb, as well as related proteins p107 and p130 and inhibits their growth suppressive activities. The Id proteins do not affect the expression levels or phosphorylation state of Rb (Lasorella et al., 1996). However, when Id2 binds in the pocket-binding of Rb site it prevents it from arresting the cell cycle (Iavarone et al., 1994; Lasorella et al., 1996). There are also reports that Id1 can decrease p53 activity, although it is not known whether this is a direct or an indirect effect. The reduction of p53 activity results in decreased PTEN tumor suppressor expression, and ultimately results in increased cellular proliferation (Lee et al., 2009).

In summary, the Id family of proteins affects both differentiation and cell cycle progression by inhibiting the DNA binding activity of the E and ETS protein transcription factors, by binding to and disrupting the activity of Rb and related proteins, and by decreasing p53 activity.

#### **3. The role of Id proteins in normal myelopoiesis**

#### **3.1 Id protein levels during myelopoiesis**

Id proteins were first identified in a mouse leukemic cell line in the search for novel factors containing HLH domains. Benezra et al. identified the first Id protein in murine erythroleukemia (MEL) cells in 1990 (Benezra et al., 1990). They observed that Id1 was present in less-differentiated cell types, and found that Id expression decreased after differentiation was induced in erythroid, muscle, and endothelial cell lines. Subsequently, using a myeloid progenitor cell line, 32DCl3, it was found that endogenous Id1 expression rapidly decreased in cells induced to differentiate to neutrophilic granulocytes by granulocyte colony stimulating factor (G-CSF), then rose again during the later stages of differentiation. Id2 expression also decreased following G-CSF treatment of 32DCl3 cells (Leeanansaksiri et al., 2005), as well as in multiple other myeloid cell lines (Ishiguro et al., 1996). When Id1 concentration was artificially elevated in the 32DCl3 cells, the cells failed to differentiate in response to G-CSF (Kreider et al., 1992). Similarly, overexpression of Id1 in MEL cells also resulted in a block in differentiation (Shoji et al., 1994). Therefore, in normal cells, Id1 expression needs to decrease for a defined period after the induction of differentiation. If Id proteins are overexpressed during this phase, the cells do not differentiate in spite of the extracellular signals instructing them to mature.

In mouse bone marrow, moderate levels of Id1 are present in the hematopoietic stem cells (HSC) and common lymphoid progenitors (CLP). Id1 expression is increased in the more differentiated common myeloid progenitors (CMP), and further increased in the granulocyte macrophage progenitors (GMP), however Id1 levels do not increase in megakaryocyte erythroid progenitors (MEP). The dynamic levels of Id1 expression during these early stages of hematopoiesis suggest that Id1 may regulate cell fate decisions during myeloid, erythroid, and lymphoid development (Leeanansaksiri et al., 2005). In support of this

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 221

growth factors they produce, reducing the amount of SCF and SDF produced, and increasing the amount of GM-CSF, G-CSF, and M-CSF produced by the stromal cells (Suh et al., 2009). These changes in the stromal microenvironment may explain the myeloid-priming effect reported in the Id1-null LT-HSC (Jankovic et al., 2007). In summary, the loss of Id1 can disrupt normal hematopoiesis, causing significant changes in the bone marrow

While Id1 expression is important for maintenance of the hematopoietic microenvironment, and affects the competitive repopulation ability of LT-HSC, Id2 is essential for normal hematopoietic development as demonstrated by deletion of Id2 in mouse models. Id2 knockout mouse models have defects in natural killer (NK) and dendritic cell development, as well as impaired lymphopoiesis and erythroid defects (Yokota et al., 1999). Id2 regulates fate determination in myeloid development by interaction with the ETS transcription factor PU.1. Id2 relieves PU.1 repression of the GATA-1 transcription factor, which shifts progenitor cell development from a monocyte/granulocyte differentiation program to an erythroid program (Ji et al., 2008). In summary, Id2 is essential for normal hematopoietic development and is a key regulatory factor in the fate determination of B and erythroid cell

In contrast to its family members Id1 and Id2, the normal physiological role of Id3 appears to be regulation of lymphoid development. Specifically Id3 has a major role in supporting the proper maturation of B cells, as mice that are Id3 null have defects in B cell proliferation and maturation (Pan et al., 1999; Rivera et al., 2000). However, Id3 is not normally expressed in myeloid development, nor is Id4 (Ishiguro et al., 1995; Ishiguro et al., 1996). The levels at which the Id proteins are expressed in the cells and the stage in differentiation at which they are expressed as well as the type of cell in which they are expressed affect how Id proteins function "normally" in the cells. Altogether, the Id family of proteins has two functions in normal cells: to regulate the process of fate determination and differentiation, and to control

*Id proteins have oncogenic properties in myeloid cells.* Overexpression of the Id genes is associated with malignancy and poor prognosis in many different tissues **(Table 1).**  Whether increased expression of Id proteins are a by-product of deregulated growth or are causative is still under investigation. Id gene overexpression in cell lines results in hyperproliferation and blocked differentiation. For example, Shoji et al., demonstrated that Id1 overexpression inhibits differentiation of erythroleukemia cell lines (Shoji et al., 1994) and Kreider et al. demonstrated Id1 blocks 32DCl3 myeloid progenitor differentiation

When Id protein expression is deregulated, normal progenitor expansion and differentiation becomes unbalanced and shifts towards progenitor hyperproliferation. In the myeloid lineage, forced expression of Id1 or Id2 can impair monocyte, granulocyte, and erythroid maturation (Jen et al., 1996; Kreider et al., 1992; Leeanansaksiri et al., 2005; Lister et al., 1995). Furthermore, in mouse bone marrow cells, overexpression of either Id1 or Id3 can immortalize growth factor-dependent hematopoietic progenitors *in vitro*, resulting in cells with an AML-like morphology. Immunophenotypic and gene expression analyses of these

microenvironment as well as in the hematopoietic stem and progenitor populations.

development.

cell proliferation.

(Kreider et al., 1992).

**4. Id proteins and leukemia 4.1 Ids and leukemogenesis** 

hypothesis, culture of mouse bone marrow cells in the presence of interleukin-3 (IL-3) or IL-3 with stem cell factor (SCF), or GM-CSF, which promote granulocyte and macrophage development, all increased Id1 expression. Correspondingly, culture of human CD34+ bone marrow cells with myeloid growth factors SCF and IL-3, SCF and macrophage colony stimulating factor (M-CSF), SCF and granulocyte macrophage colony stimulating factor (GM-CSF), or with GM-CSF alone also induced Id1 expression (Suh et al., 2008). However, SCF alone, SCF and erythropoietin (EPO), or SCF and thrombopoietin (TPO), which promote erythroid and megakaryocyte development, did not induce Id1 expression in murine or human hematopoietic progenitors. Furthermore, initial reports showed that Id2 expression increases during differentiation of myeloid cells (Ishiguro et al., 1996), although the onset of its expression in myelopoiesis is later in hematopoietic development than Id1 (Cooper et al., 1997). In summary, in normal mouse and human progenitor cells, hematopoietic growth factors that promote granulocyte and macrophage development induce the expression of Id1.

#### **3.2 Id protein levels rise in later stages of differentiation**

The study by Kreider et al., which examined Id1 expression during induction of differentiation in 32DCl3 cells by G-CSF showed that Id1 levels also increased during terminal neutrophil differentiation of 32DCl3 (Kreider et al., 1992). In agreement with this, Shoji et al. demonstrated that if Id1 is overexpressed in later stages of myeloid differentiation, that Id1 did not block myeloid maturation, as in the early stages of myeloid differentiation (Shoji et al., 1994). These reports suggest that Id protein expression may have an additional role in terminal myeloid differentiation. This model is supported by studies in human CD34+ cord blood cells that show ectopic expression of Id1 can enhance neutrophil development; and expression of Id2 accelerates final maturation of eosinophils and neutrophils (Buitenhuis et al., 2005). In addition, restoring Id1 expression (along with C/EBPα expression) in cells isolated from patients with low-risk myelodysplastic syndrome (MDS) restores neutrophil maturation *in vitro* (Geest et al., 2009). Thus, the expression of the Id proteins is biphasic during normal hematopoietic development, appearing first in the stem and progenitor stages, decreases during the intermediate developmental stages, and then rises again during terminal differentiation.

#### **3.3 Loss of Id proteins disrupts normal hematopoietic development**

The Id proteins are necessary for proper hematopoietic development, including progenitor expansion and fate determination. While Id1 knockout mice are viable and show no overt developmental defects, several groups have reported that hematopoietic stem and progenitor cells in these mice show impaired development (Yan et al., 1997; Perry et al., 2007; Suh et al., 2009). Specifically, the hematopoietic stem cells responsible for long term engraftment and repopulation (LT-HSC) fail to compete against co-transplanted bone marrow cells as well as normal control bone marrow LT-HSC (Jankovic et al., 2007; Perry et al., 2007). However, four serial transplantations of Id1-/- bone marrow, without the addition of co-transplanted competitor bone marrow, supported equal engraftment and viability of the transplant recipients as wild type mouse bone marrow cells, suggesting the intrinsic loss of Id1 does not impair the fundamental long-term self-renewal ability of LT-HSC (Suh et al., 2009). In addition, these studies also showed that the loss of Id1 also significantly alters the bone marrow microenvironment, specifically the stromal cells and the hematopoietic

hypothesis, culture of mouse bone marrow cells in the presence of interleukin-3 (IL-3) or IL-3 with stem cell factor (SCF), or GM-CSF, which promote granulocyte and macrophage development, all increased Id1 expression. Correspondingly, culture of human CD34+ bone marrow cells with myeloid growth factors SCF and IL-3, SCF and macrophage colony stimulating factor (M-CSF), SCF and granulocyte macrophage colony stimulating factor (GM-CSF), or with GM-CSF alone also induced Id1 expression (Suh et al., 2008). However, SCF alone, SCF and erythropoietin (EPO), or SCF and thrombopoietin (TPO), which promote erythroid and megakaryocyte development, did not induce Id1 expression in murine or human hematopoietic progenitors. Furthermore, initial reports showed that Id2 expression increases during differentiation of myeloid cells (Ishiguro et al., 1996), although the onset of its expression in myelopoiesis is later in hematopoietic development than Id1 (Cooper et al., 1997). In summary, in normal mouse and human progenitor cells, hematopoietic growth factors that promote granulocyte and macrophage development

The study by Kreider et al., which examined Id1 expression during induction of differentiation in 32DCl3 cells by G-CSF showed that Id1 levels also increased during terminal neutrophil differentiation of 32DCl3 (Kreider et al., 1992). In agreement with this, Shoji et al. demonstrated that if Id1 is overexpressed in later stages of myeloid differentiation, that Id1 did not block myeloid maturation, as in the early stages of myeloid differentiation (Shoji et al., 1994). These reports suggest that Id protein expression may have an additional role in terminal myeloid differentiation. This model is supported by studies in human CD34+ cord blood cells that show ectopic expression of Id1 can enhance neutrophil development; and expression of Id2 accelerates final maturation of eosinophils and neutrophils (Buitenhuis et al., 2005). In addition, restoring Id1 expression (along with C/EBPα expression) in cells isolated from patients with low-risk myelodysplastic syndrome (MDS) restores neutrophil maturation *in vitro* (Geest et al., 2009). Thus, the expression of the Id proteins is biphasic during normal hematopoietic development, appearing first in the stem and progenitor stages, decreases during the intermediate developmental stages, and

The Id proteins are necessary for proper hematopoietic development, including progenitor expansion and fate determination. While Id1 knockout mice are viable and show no overt developmental defects, several groups have reported that hematopoietic stem and progenitor cells in these mice show impaired development (Yan et al., 1997; Perry et al., 2007; Suh et al., 2009). Specifically, the hematopoietic stem cells responsible for long term engraftment and repopulation (LT-HSC) fail to compete against co-transplanted bone marrow cells as well as normal control bone marrow LT-HSC (Jankovic et al., 2007; Perry et al., 2007). However, four serial transplantations of Id1-/- bone marrow, without the addition of co-transplanted competitor bone marrow, supported equal engraftment and viability of the transplant recipients as wild type mouse bone marrow cells, suggesting the intrinsic loss of Id1 does not impair the fundamental long-term self-renewal ability of LT-HSC (Suh et al., 2009). In addition, these studies also showed that the loss of Id1 also significantly alters the bone marrow microenvironment, specifically the stromal cells and the hematopoietic

induce the expression of Id1.

**3.2 Id protein levels rise in later stages of differentiation** 

then rises again during terminal differentiation.

**3.3 Loss of Id proteins disrupts normal hematopoietic development** 

growth factors they produce, reducing the amount of SCF and SDF produced, and increasing the amount of GM-CSF, G-CSF, and M-CSF produced by the stromal cells (Suh et al., 2009). These changes in the stromal microenvironment may explain the myeloid-priming effect reported in the Id1-null LT-HSC (Jankovic et al., 2007). In summary, the loss of Id1 can disrupt normal hematopoiesis, causing significant changes in the bone marrow microenvironment as well as in the hematopoietic stem and progenitor populations.

While Id1 expression is important for maintenance of the hematopoietic microenvironment, and affects the competitive repopulation ability of LT-HSC, Id2 is essential for normal hematopoietic development as demonstrated by deletion of Id2 in mouse models. Id2 knockout mouse models have defects in natural killer (NK) and dendritic cell development, as well as impaired lymphopoiesis and erythroid defects (Yokota et al., 1999). Id2 regulates fate determination in myeloid development by interaction with the ETS transcription factor PU.1. Id2 relieves PU.1 repression of the GATA-1 transcription factor, which shifts progenitor cell development from a monocyte/granulocyte differentiation program to an erythroid program (Ji et al., 2008). In summary, Id2 is essential for normal hematopoietic development and is a key regulatory factor in the fate determination of B and erythroid cell development.

In contrast to its family members Id1 and Id2, the normal physiological role of Id3 appears to be regulation of lymphoid development. Specifically Id3 has a major role in supporting the proper maturation of B cells, as mice that are Id3 null have defects in B cell proliferation and maturation (Pan et al., 1999; Rivera et al., 2000). However, Id3 is not normally expressed in myeloid development, nor is Id4 (Ishiguro et al., 1995; Ishiguro et al., 1996). The levels at which the Id proteins are expressed in the cells and the stage in differentiation at which they are expressed as well as the type of cell in which they are expressed affect how Id proteins function "normally" in the cells. Altogether, the Id family of proteins has two functions in normal cells: to regulate the process of fate determination and differentiation, and to control cell proliferation.

#### **4. Id proteins and leukemia**

#### **4.1 Ids and leukemogenesis**

*Id proteins have oncogenic properties in myeloid cells.* Overexpression of the Id genes is associated with malignancy and poor prognosis in many different tissues **(Table 1).**  Whether increased expression of Id proteins are a by-product of deregulated growth or are causative is still under investigation. Id gene overexpression in cell lines results in hyperproliferation and blocked differentiation. For example, Shoji et al., demonstrated that Id1 overexpression inhibits differentiation of erythroleukemia cell lines (Shoji et al., 1994) and Kreider et al. demonstrated Id1 blocks 32DCl3 myeloid progenitor differentiation (Kreider et al., 1992).

When Id protein expression is deregulated, normal progenitor expansion and differentiation becomes unbalanced and shifts towards progenitor hyperproliferation. In the myeloid lineage, forced expression of Id1 or Id2 can impair monocyte, granulocyte, and erythroid maturation (Jen et al., 1996; Kreider et al., 1992; Leeanansaksiri et al., 2005; Lister et al., 1995). Furthermore, in mouse bone marrow cells, overexpression of either Id1 or Id3 can immortalize growth factor-dependent hematopoietic progenitors *in vitro*, resulting in cells with an AML-like morphology. Immunophenotypic and gene expression analyses of these

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 223

immortalized cells suggest they are developmentally arrested at the CMP/GMP phase of maturation (Suh et al., 2008). The bone marrow cells immortalized by Id1 overexpression showed decreased levels of p15INK, p16INK4a, p19ARF and p21Cip1 during culture (Suh et al., 2008), indicating that Id1 expression reduced the expression of multiple cell cycle inhibitors

These results showed that overexpression of Id1 immortalized normal bone marrow cells *in vitro*, and led researchers to investigate if deregulated Id1 expression cells would cause a myeloproliferative disease *in vivo*. Transplantation of Id1 overexpressing mouse HSPC resulted in the development of a myeloproliferative disease in mice, causing the animals to become moribund within a year. Complete blood cell counts showed the mice had monocytosis, and low levels of hemoglobin and hematocrit, as well as the presence of cells with leukemic-blast morphology. Pathology revealed that the mice had myeloid and erythroid hyperplasia in their bone marrow and spleens which lead to splenomegaly (Suh et al., 2008). Therefore, overexpression of Id1 is sufficient to immortalize mouse progenitor

Similar to Id1, overexpression of Id2 has also been shown to cause the development of leukemia in mouse models. Transgenic overexpression of Id2 in mouse models leads to a block in lymphoid differentiation, aberrant apoptosis, and development of T cell lymphoma (Morrow et al., 1999). Id2 also blocks differentiation in human cells, specifically, ectopic expression of Id2 in human CD34+ myeloid progenitor cells inhibits the acquisition of monocyte characteristics, suggesting that Id2 can block myeloid as well as lymphoid differentiation (Heinz et al., 2006). It is important to note, that the effects of Id1 or Id2 overexpression depend upon the developmental stage and lineage in which overexpression occurs. In summary, overexpression of both Id1 and Id2 can block differentiation, over-ride senescence, and lead to the development of myelo- and lymphoproliferative disease, and

**Increased Id1 and Id2 expression is associated with human AML.** The finding that Ids immortalize primary mouse bone marrow cells *in vitro*, and promote MPD *in vivo*, led investigators to ask if Id expression levels are elevated in myelogenous leukemia. A microarray performed on 285 AML patient samples demonstrated an increase in Id1 and Id2 in AML subsets (Suh et al., 2008). Specifically, 17.5% of the mRNA samples from AML patients had increased Id1 expression, and 19.2% had increased Id2 mRNA levels. Surprisingly, most of the patients with elevated Id1 and Id2 levels had a normal karyotype, and were evenly distributed across the French-American-British (FAB) subtype classifications: M0-M6. 61% and 50% of patients with 5q and/or 7q deletion and 28 and 22%of patients with t(15;17) showed increased expression of Id1 and Id2, suggesting that elevated Id expression levels contribute to a block in granulocyte development. Seventy-eight patients had high levels of FLT3-ITD mutations, and 23 of those (29%) had elevated levels of Id1 expression. Twenty-three patients had EVI1 deregulation, of which 8 patients (35%) had increased Id1 expression. Id2 expression was found in patients with deregulated NRAS (30%), KRAS (44%), C/EBPα (6%) and EVI1 (30%) expression. Altogether, the analysis suggests that Id gene expression may be induced downstream of multiple signal transduction pathways of activated oncogenes. A separate analysis of a different cohort of AML samples showed that patients with the highest levels of Id1 had a poor prognosis compared to those with lower levels of Id1 (Tang et al., 2009). The causes of Id1 and Id2 overexpression in AML are still under investigation. Thus, elevated levels of Id gene expression could be used diagnostically to elucidate the severity and

to cause increased growth in deregulated myelopoiesis.

ultimately leukemogenesis.

prognosis of AML patients.

cells *in vitro*, and leads to a lethal myeloproliferative disease *in vivo*.


Table 1. Id Protein Expression in Cancer

**Member Tumor Type Reference** 

Id4 Testicular Seminoma (Sablitzky et al., 1998)

Id1 Thyroid Cancer (Kebebew et al., 2000,

Id1 Endometrial Cancer (Takai et al., 2001) Id1 Cervical Cancer (Schindl et al., 2001) Id1 Melanoma (Polsky et al., 2001) Id2 Neuroblastoma (Lasorella et al., 2002;

Id1, Id2, Id3 Astrocytic Tumor (Vandeputte et al.,

Id1 Basal Cell Carcinoma (Chaturvedi et al.,

Id1, Id2, Id3 Colorectal Carcinoma (Norton, 2000; Wilson

Id1 Hepatocellular Carcinoma (Lee et al., 2003) Id1 Kaposi's Sarcoma (Tang et al., 2003) Id1 Ovarian Cancer (Schindl et al., 2003) Id1 Prostate Cancer (Ouyang et al., 2002) Id1 AML subsets (Suh et al., 2008; Tang

Neuroblastoma (Lyden et al., 1999)

(Kleeff et al., 1998; Maruyama et al., 1999)

2003)

(Hu et al., 2001; Langlands et al., 2000; Nishimine et al., 2003; Wang et al., 2002)

(Lin et al., 2000; Schoppmann et al., 2003)

Lasorella et al., 2000)

(Fukuma et al., 2003; Nishimori et al., 2002)

2002)

2003)

et al., 2001)

et al., 2009)

Id1, Id3 Glioblastoma, Medulloblastoma,

Id1, Id2, Id3 Pancreatic Cancer

Id1, Id2, Id3 Squamous Cell Carcinoma

Id1 Breast Cancer

Id2 Ewing Sarcoma

Table 1. Id Protein Expression in Cancer

**Id Family** 

Id1, Id2, Id3,

immortalized cells suggest they are developmentally arrested at the CMP/GMP phase of maturation (Suh et al., 2008). The bone marrow cells immortalized by Id1 overexpression showed decreased levels of p15INK, p16INK4a, p19ARF and p21Cip1 during culture (Suh et al., 2008), indicating that Id1 expression reduced the expression of multiple cell cycle inhibitors to cause increased growth in deregulated myelopoiesis.

These results showed that overexpression of Id1 immortalized normal bone marrow cells *in vitro*, and led researchers to investigate if deregulated Id1 expression cells would cause a myeloproliferative disease *in vivo*. Transplantation of Id1 overexpressing mouse HSPC resulted in the development of a myeloproliferative disease in mice, causing the animals to become moribund within a year. Complete blood cell counts showed the mice had monocytosis, and low levels of hemoglobin and hematocrit, as well as the presence of cells with leukemic-blast morphology. Pathology revealed that the mice had myeloid and erythroid hyperplasia in their bone marrow and spleens which lead to splenomegaly (Suh et al., 2008). Therefore, overexpression of Id1 is sufficient to immortalize mouse progenitor cells *in vitro*, and leads to a lethal myeloproliferative disease *in vivo*.

Similar to Id1, overexpression of Id2 has also been shown to cause the development of leukemia in mouse models. Transgenic overexpression of Id2 in mouse models leads to a block in lymphoid differentiation, aberrant apoptosis, and development of T cell lymphoma (Morrow et al., 1999). Id2 also blocks differentiation in human cells, specifically, ectopic expression of Id2 in human CD34+ myeloid progenitor cells inhibits the acquisition of monocyte characteristics, suggesting that Id2 can block myeloid as well as lymphoid differentiation (Heinz et al., 2006). It is important to note, that the effects of Id1 or Id2 overexpression depend upon the developmental stage and lineage in which overexpression occurs. In summary, overexpression of both Id1 and Id2 can block differentiation, over-ride senescence, and lead to the development of myelo- and lymphoproliferative disease, and ultimately leukemogenesis.

**Increased Id1 and Id2 expression is associated with human AML.** The finding that Ids immortalize primary mouse bone marrow cells *in vitro*, and promote MPD *in vivo*, led investigators to ask if Id expression levels are elevated in myelogenous leukemia. A microarray performed on 285 AML patient samples demonstrated an increase in Id1 and Id2 in AML subsets (Suh et al., 2008). Specifically, 17.5% of the mRNA samples from AML patients had increased Id1 expression, and 19.2% had increased Id2 mRNA levels. Surprisingly, most of the patients with elevated Id1 and Id2 levels had a normal karyotype, and were evenly distributed across the French-American-British (FAB) subtype classifications: M0-M6. 61% and 50% of patients with 5q and/or 7q deletion and 28 and 22%of patients with t(15;17) showed increased expression of Id1 and Id2, suggesting that elevated Id expression levels contribute to a block in granulocyte development. Seventy-eight patients had high levels of FLT3-ITD mutations, and 23 of those (29%) had elevated levels of Id1 expression. Twenty-three patients had EVI1 deregulation, of which 8 patients (35%) had increased Id1 expression. Id2 expression was found in patients with deregulated NRAS (30%), KRAS (44%), C/EBPα (6%) and EVI1 (30%) expression. Altogether, the analysis suggests that Id gene expression may be induced downstream of multiple signal transduction pathways of activated oncogenes. A separate analysis of a different cohort of AML samples showed that patients with the highest levels of Id1 had a poor prognosis compared to those with lower levels of Id1 (Tang et al., 2009). The causes of Id1 and Id2 overexpression in AML are still under investigation. Thus, elevated levels of Id gene expression could be used diagnostically to elucidate the severity and prognosis of AML patients.

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 225

addition, Id1 overexpression in prostate cancer cells actually protects malignant cells from apoptosis (Ling et al., 2003; Wong et al., 2004). Furthermore, decreasing the activity of the Id proteins promotes cell cycle arrest and apoptosis in both breast and ovarian cancer cells (Mern et al., 2010a; Mern et al., 2010b). In summary, the Id proteins can have variable affects on apoptosis, depending upon the developmental stage and microenvironment of the cell. **Id4 functions as a tumor suppressor in myeloid leukemia.** Id4 is interesting because of reports that suggest it may function *not* as an oncogene, but as a tumor suppressor in leukemia (in contrast to its siblings). Methylation of the Id4 promoter is associated with poor prognosis in high risk MDS patients, who have a greater probability of developing leukemia (Wang et al., 2010b). In addition, high risk myelodysplastic patients whose Id4 promoters are methylated have an increased risk of leukemic transformation, an obersvation which would support for the use of demethylating agents in MDS/AML treatment (Chen et al., 2011; Wang et al., 2010b). Id4 methylation also correlates with CML progression. Specifically, Id4 is un-methylated in the chronic phase of CML, but is frequently methylated

Reports also show that Id4 promoter is methylated in AML cell lines and in AML primary patient samples (Yu et al., 2005). This study also demonstrated that Id4 promoter methylation resulted in decreased Id4 mRNA expression. At the same time, the role of Id4 as a tumor suppressor in myelodysplasia appears to be cell type specific, as Id4 overexpression increases the proliferation of other cell types (breast cancer) (Dell'Orso et al., 2010). Therefore, of all of the Id family members, Id4 is the only one that is consistently reported to be a tumor suppressor in myeloid cells, although how it functions

Altogether, the reports described above suggest a strong link between Id expression levels and malignancy. It is possible, therefore, that Ids may be part of a mechanism that helps the malignant cells evade standard chemotherapeutic treatments. A search of the Gene Expression Omnibus (GEO; (Barrett and Edgar, 2006)) database reveals studies, which show a correlation between increased Id expression and drug resistance in AML and CML cell lines. Specifically, these analyses show that Id1 expression is increased in cytarabine resistant AML cell lines **(Figure 3A)**(GDS 1907), and that Id1 is also increased in cyclophosphamide resistant CML **(Figure 3B)** (GDS2729) (Bao et al., 2007). In addition, Id3 mRNA levels are increased in cyclophosphamide resistant CML lines **(Figure 3C)** (GDS2729) (Bao et al., 2007). Altogether, these analyses indicate that high levels of Id1 and/or Id3 expression may correlate with a drug resistant phenotype in myelogenous

These analyses agree with many other studies that show Id1 functions as an anti-apoptotic factor in cancerous cells from non-hematopoietic tissues. Id1 expression has been shown to prevent malignant cells from undergoing programmed cell death when treated with chemotherapeutic agents (Summarized in (Zhang et al., 2007)). Decreasing Id1 expression with Id1-targeted siRNA restored drug sensitivity in many of these malignant cell lines, however, it has not yet been tested in hematopoietic cells (Zhang et al., 2007). In summary, the data from these studies suggest that high levels of Id1 expression may be indicative of drug resistance in leukemic cells, or may possibly be part of the mechanism underlying drug resistance, which will likely be a focus of future studies with both AML and CML.

in patients in the accelerated and blast crisis stages (Wang et al., 2010a).

mechanistically in that role is not yet understood.

**4.2 Ids and drug resistance in leukemia** 

leukemia.

**Expression of Id1 and Id2 are increased by known leukemic oncogenes.** It is possible that in many cases Id1 and Id2 mRNA levels increase due to mutations in upstream regulatory pathways. Id1 is a downstream target of multiple leukemia-associated oncogenic tyrosine kinases, including BCR-ABL and FLT3-ITD (Tam et al., 2008). In addition, the JAK2V617F— STAT5, and AML-ETO mutations can also increase Id1 expression (Cammenga et al., 2003; Wood et al., 2009). PLZF and the PLZF translocation products, associated with APL (acute promyelocytic leukemia), activate Id1 and Id3 expression (Bernardo et al., 2007; Doulatov et al., 2009; Rice et al., 2009). In addition, overexpression of HOXA9 and HOXA10, both of which are associated with myeloid hyperplasia, directly activate expression of Id2 (Nagel et al., 2010). Furthermore, viruses also induce Id expression, for example, Id1 is upregulated by Epstein-Barr viral protein LMP1 (NH) (Li et al., 2004). Therefore, the Id proteins are likely to be part of the mechanism by which several of the known oncogenic mutations impair growth control.

**Id2 has both oncogenic and tumor suppressor properties.** Interestingly, although Id2 has been shown to be upregulated in AML patient samples (as described above), Id2 may have have both oncogenic and tumor suppressor properties. Specifically, there are data demonstrating that the loss of Id2 *in vivo* results in the development of a lethal myeloproliferative disorder (Ko et al., 2008). This effect was seen in Id2 knockout mice backcrossed onto a C57Bl/6 background, but not in 129/sv Id2 knockout, although the reasons for this difference are not yet known. Altogether, the data suggest that Id2 is important for functional hematopoiesis, but that its function as an oncogene or as a tumor suppressor depend upon the cellular environment in which it is expressed.

**Id1 causes genomic instability.** Of the Id family of proteins, overexpression of Id1 is most consistently reported as associated with oncogenesis. A possible explanation for this may be that Id1 has been associated with genomic instability. Interestingly, it was shown that Id1 contributes to the acquisition of secondary mutations by causing centrosome abnormalities. One mechanism by which this been shown to occur is by Id1 binding to the S5A protein. Loss of S5A has been associated with mitotic defects resulting in abnormal chromosome segregation (Szlanka et al., 2003). When Id1 is overexpressed it interacts with S5A, and this interaction causes overduplication of the centrosomes (Hasskarl et al., 2004; Hasskarl et al., 2008). Leukemogenic translocation mutations such as BCR-ABL can induce genomic instability (Dierov et al., 2009). Based on the data showing that BCR-ABL can induce expression of Id1, it is possible that part of the mechanism by which BCR-ABL causes genomic instability is by inducing overexpression of Id1 (Dierov et al., 2009). Thus, the presence of overexpressed Id1 can not only block differentiation and override senescence; it can actively contribute to the acquisition of additional mutations.

**Id family overexpression is associated with disruption of apoptosis.** In addition to causing genomic instability, another mechanism by which cancer cells survive is by avoiding programmed cell death. Multiple reports have shown that Id1, Id2, and Id3 can influence apoptosis. However, whether the Id proteins behave as pro-apoptotic or anti-apoptotic factors depends upon the the cell context. Transgenic expression of Id1 in developing T cells in a mouse model resulted in a large percentage of the developing thymocytes to undergo apoptosis. The authors of the study speculated that this was because the thymocytes were blocked in maturation and apoptosis was initiated during the process of V(D)J recombination (Kim et al., 1999). Interestingly, lymphomas developed from the surviving thymocytes, suggesting that overexpression of Id1 alone did not drive apoptosis. In

**Expression of Id1 and Id2 are increased by known leukemic oncogenes.** It is possible that in many cases Id1 and Id2 mRNA levels increase due to mutations in upstream regulatory pathways. Id1 is a downstream target of multiple leukemia-associated oncogenic tyrosine kinases, including BCR-ABL and FLT3-ITD (Tam et al., 2008). In addition, the JAK2V617F— STAT5, and AML-ETO mutations can also increase Id1 expression (Cammenga et al., 2003; Wood et al., 2009). PLZF and the PLZF translocation products, associated with APL (acute promyelocytic leukemia), activate Id1 and Id3 expression (Bernardo et al., 2007; Doulatov et al., 2009; Rice et al., 2009). In addition, overexpression of HOXA9 and HOXA10, both of which are associated with myeloid hyperplasia, directly activate expression of Id2 (Nagel et al., 2010). Furthermore, viruses also induce Id expression, for example, Id1 is upregulated by Epstein-Barr viral protein LMP1 (NH) (Li et al., 2004). Therefore, the Id proteins are likely to be part of the mechanism by which several of the known oncogenic mutations impair

**Id2 has both oncogenic and tumor suppressor properties.** Interestingly, although Id2 has been shown to be upregulated in AML patient samples (as described above), Id2 may have have both oncogenic and tumor suppressor properties. Specifically, there are data demonstrating that the loss of Id2 *in vivo* results in the development of a lethal myeloproliferative disorder (Ko et al., 2008). This effect was seen in Id2 knockout mice backcrossed onto a C57Bl/6 background, but not in 129/sv Id2 knockout, although the reasons for this difference are not yet known. Altogether, the data suggest that Id2 is important for functional hematopoiesis, but that its function as an oncogene or as a tumor

**Id1 causes genomic instability.** Of the Id family of proteins, overexpression of Id1 is most consistently reported as associated with oncogenesis. A possible explanation for this may be that Id1 has been associated with genomic instability. Interestingly, it was shown that Id1 contributes to the acquisition of secondary mutations by causing centrosome abnormalities. One mechanism by which this been shown to occur is by Id1 binding to the S5A protein. Loss of S5A has been associated with mitotic defects resulting in abnormal chromosome segregation (Szlanka et al., 2003). When Id1 is overexpressed it interacts with S5A, and this interaction causes overduplication of the centrosomes (Hasskarl et al., 2004; Hasskarl et al., 2008). Leukemogenic translocation mutations such as BCR-ABL can induce genomic instability (Dierov et al., 2009). Based on the data showing that BCR-ABL can induce expression of Id1, it is possible that part of the mechanism by which BCR-ABL causes genomic instability is by inducing overexpression of Id1 (Dierov et al., 2009). Thus, the presence of overexpressed Id1 can not only block differentiation and override senescence; it

**Id family overexpression is associated with disruption of apoptosis.** In addition to causing genomic instability, another mechanism by which cancer cells survive is by avoiding programmed cell death. Multiple reports have shown that Id1, Id2, and Id3 can influence apoptosis. However, whether the Id proteins behave as pro-apoptotic or anti-apoptotic factors depends upon the the cell context. Transgenic expression of Id1 in developing T cells in a mouse model resulted in a large percentage of the developing thymocytes to undergo apoptosis. The authors of the study speculated that this was because the thymocytes were blocked in maturation and apoptosis was initiated during the process of V(D)J recombination (Kim et al., 1999). Interestingly, lymphomas developed from the surviving thymocytes, suggesting that overexpression of Id1 alone did not drive apoptosis. In

suppressor depend upon the cellular environment in which it is expressed.

can actively contribute to the acquisition of additional mutations.

growth control.

addition, Id1 overexpression in prostate cancer cells actually protects malignant cells from apoptosis (Ling et al., 2003; Wong et al., 2004). Furthermore, decreasing the activity of the Id proteins promotes cell cycle arrest and apoptosis in both breast and ovarian cancer cells (Mern et al., 2010a; Mern et al., 2010b). In summary, the Id proteins can have variable affects on apoptosis, depending upon the developmental stage and microenvironment of the cell.

**Id4 functions as a tumor suppressor in myeloid leukemia.** Id4 is interesting because of reports that suggest it may function *not* as an oncogene, but as a tumor suppressor in leukemia (in contrast to its siblings). Methylation of the Id4 promoter is associated with poor prognosis in high risk MDS patients, who have a greater probability of developing leukemia (Wang et al., 2010b). In addition, high risk myelodysplastic patients whose Id4 promoters are methylated have an increased risk of leukemic transformation, an obersvation which would support for the use of demethylating agents in MDS/AML treatment (Chen et al., 2011; Wang et al., 2010b). Id4 methylation also correlates with CML progression. Specifically, Id4 is un-methylated in the chronic phase of CML, but is frequently methylated in patients in the accelerated and blast crisis stages (Wang et al., 2010a).

Reports also show that Id4 promoter is methylated in AML cell lines and in AML primary patient samples (Yu et al., 2005). This study also demonstrated that Id4 promoter methylation resulted in decreased Id4 mRNA expression. At the same time, the role of Id4 as a tumor suppressor in myelodysplasia appears to be cell type specific, as Id4 overexpression increases the proliferation of other cell types (breast cancer) (Dell'Orso et al., 2010). Therefore, of all of the Id family members, Id4 is the only one that is consistently reported to be a tumor suppressor in myeloid cells, although how it functions mechanistically in that role is not yet understood.

#### **4.2 Ids and drug resistance in leukemia**

Altogether, the reports described above suggest a strong link between Id expression levels and malignancy. It is possible, therefore, that Ids may be part of a mechanism that helps the malignant cells evade standard chemotherapeutic treatments. A search of the Gene Expression Omnibus (GEO; (Barrett and Edgar, 2006)) database reveals studies, which show a correlation between increased Id expression and drug resistance in AML and CML cell lines. Specifically, these analyses show that Id1 expression is increased in cytarabine resistant AML cell lines **(Figure 3A)**(GDS 1907), and that Id1 is also increased in cyclophosphamide resistant CML **(Figure 3B)** (GDS2729) (Bao et al., 2007). In addition, Id3 mRNA levels are increased in cyclophosphamide resistant CML lines **(Figure 3C)** (GDS2729) (Bao et al., 2007). Altogether, these analyses indicate that high levels of Id1 and/or Id3 expression may correlate with a drug resistant phenotype in myelogenous leukemia.

These analyses agree with many other studies that show Id1 functions as an anti-apoptotic factor in cancerous cells from non-hematopoietic tissues. Id1 expression has been shown to prevent malignant cells from undergoing programmed cell death when treated with chemotherapeutic agents (Summarized in (Zhang et al., 2007)). Decreasing Id1 expression with Id1-targeted siRNA restored drug sensitivity in many of these malignant cell lines, however, it has not yet been tested in hematopoietic cells (Zhang et al., 2007). In summary, the data from these studies suggest that high levels of Id1 expression may be indicative of drug resistance in leukemic cells, or may possibly be part of the mechanism underlying drug resistance, which will likely be a focus of future studies with both AML and CML.

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 227

The results shown above lead to the question of whether or not the Ids might be expressed in the "Leukemia Initiating Cell" (LIC) population that is resistant to drug treatment. These quiescent leukemic stem cells have been termed the "Holy Grail of Leukemia Therapy" (Misaghian et al., 2009). However, identifying and understanding the regulation of these cells depends on a better understanding of normal transcription factors in stem cells; and how they may be deregulated, and become leukemia stem cells (Rosenbauer et al. 2005). The process of normal stem-cell self-renewal must be tightly regulated to prevent the creation of leukemic stem cells. Therefore, the proteins that regulate the HSC self-renewal process are currently under intense investigation. E proteins maintain the stem cell pool and promote myeloid progenitor differentiation, and because Id proteins control E protein function, variations in Id protein levels can significantly affect HSC self-renewal (Semerad et al., 2009). In support of this concept, Id proteins were shown to have a role in maintaining the self-renewal capacity of stem cells. Specifically, BMP induction of Id proteins suppresses differentiation and sustains ES cell self-renewal in collaboration with STAT3 (Ying et al., 2003). In addition, in glioma cancer stem cells, decreasing expression of Id1 and Id3 effectively targeted and reduced the malignant stem cell population (Anido et al., 2010). There are also reports that loss of Id1 significantly decreases hematopoietic stem cell function, although reports vary on whether this is an environmental effect or a result of changes in the bone marrow microenvironment (Jankovic et al., 2007; Perry et al., 2007; Suh et al., 2009). In addition, it has been observed that loss of Id2 significantly decreases the long-term self-renewal capacity of hematopoietic stem cells (Ming Ji et al., unpublished). All of these studies suggest that Id genes contribute to the control of self-renewal of normal HSC and deregulation of the Id proteins may contribute to the generation of leukemic stem cells. Future studies will likely focus on this important aspect of Id protein function, as they

**4.3 Ids and Leukemia Initiating Cells** 

may provide a novel approach to target leukemic stem cells.

**5. Id proteins and the bone marrow microenvironment** 

Chemotherapy-resistant human leukemia stem cells home to and engraft within the bonemarrow endosteal region (Ishikawa et al., 2007), and reports indicate that the Id proteins may also have an effect on this microenvironment. As previously described, the loss of Id1 in the bone marrow niche leads to increased progenitor cycling caused by a change in the cytokine milieu (Suh et al., 2009). Thus, Id genes may additionally contribute to the initiation or progression of AML by affecting the stromal cells that constitute the hematopoietic niche. Interestingly, in addition to affecting the local cytokine milieu, the Ids regulate the fundamental structure of the bones by controlling osteoblast and osteoclast activity. For example, ectopic Id4 expression promotes osteoblast differentiation and was suggested as a possible preventative treatment for senile osteoporosis (Tokuzawa et al., 2010). Id1, Id2, and Id3 are all upregulated in response to BMP9 treatment of mesenchymal progenitor cells (via Smad4), which results in increased proliferation and a block in differentiation of osteogenic progenitors (Peng et al., 2004). In addition, Id proteins have been shown to regulate osteogenic transcription factor activity (Ogata and Noda, 1991; Tamura and Noda, 1994; Zhang et al., 2008). Id2, and Id4 genes also regulate adipogenesis by regulating the proliferation pre-adipoctye progenitors and their ability to undergo differentiation (Murad et al., 2010; Park et al., 2008). Altogether, these results suggest that the Id family members

Fig. 3. Increased Id levels associated with drug resistance in leukemic cell lines **(A)** Id1 mRNA expression is increased in cytarabine resistant AML cell lines (GDS 1907); **(B)** Id1 is also increased in cyclophosphamide resistant CML (GDS2729), and **(C)** Id3 mRNA is increased in cyclophosphamide resistant CML lines (GDS2729)

#### **4.3 Ids and Leukemia Initiating Cells**

226 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Fig. 3. Increased Id levels associated with drug resistance in leukemic cell lines

is increased in cyclophosphamide resistant CML lines (GDS2729)

**(A)** Id1 mRNA expression is increased in cytarabine resistant AML cell lines (GDS 1907); **(B)** Id1 is also increased in cyclophosphamide resistant CML (GDS2729), and **(C)** Id3 mRNA The results shown above lead to the question of whether or not the Ids might be expressed in the "Leukemia Initiating Cell" (LIC) population that is resistant to drug treatment. These quiescent leukemic stem cells have been termed the "Holy Grail of Leukemia Therapy" (Misaghian et al., 2009). However, identifying and understanding the regulation of these cells depends on a better understanding of normal transcription factors in stem cells; and how they may be deregulated, and become leukemia stem cells (Rosenbauer et al. 2005).

The process of normal stem-cell self-renewal must be tightly regulated to prevent the creation of leukemic stem cells. Therefore, the proteins that regulate the HSC self-renewal process are currently under intense investigation. E proteins maintain the stem cell pool and promote myeloid progenitor differentiation, and because Id proteins control E protein function, variations in Id protein levels can significantly affect HSC self-renewal (Semerad et al., 2009). In support of this concept, Id proteins were shown to have a role in maintaining the self-renewal capacity of stem cells. Specifically, BMP induction of Id proteins suppresses differentiation and sustains ES cell self-renewal in collaboration with STAT3 (Ying et al., 2003). In addition, in glioma cancer stem cells, decreasing expression of Id1 and Id3 effectively targeted and reduced the malignant stem cell population (Anido et al., 2010). There are also reports that loss of Id1 significantly decreases hematopoietic stem cell function, although reports vary on whether this is an environmental effect or a result of changes in the bone marrow microenvironment (Jankovic et al., 2007; Perry et al., 2007; Suh et al., 2009). In addition, it has been observed that loss of Id2 significantly decreases the long-term self-renewal capacity of hematopoietic stem cells (Ming Ji et al., unpublished). All of these studies suggest that Id genes contribute to the control of self-renewal of normal HSC and deregulation of the Id proteins may contribute to the generation of leukemic stem cells. Future studies will likely focus on this important aspect of Id protein function, as they may provide a novel approach to target leukemic stem cells.

#### **5. Id proteins and the bone marrow microenvironment**

Chemotherapy-resistant human leukemia stem cells home to and engraft within the bonemarrow endosteal region (Ishikawa et al., 2007), and reports indicate that the Id proteins may also have an effect on this microenvironment. As previously described, the loss of Id1 in the bone marrow niche leads to increased progenitor cycling caused by a change in the cytokine milieu (Suh et al., 2009). Thus, Id genes may additionally contribute to the initiation or progression of AML by affecting the stromal cells that constitute the hematopoietic niche. Interestingly, in addition to affecting the local cytokine milieu, the Ids regulate the fundamental structure of the bones by controlling osteoblast and osteoclast activity. For example, ectopic Id4 expression promotes osteoblast differentiation and was suggested as a possible preventative treatment for senile osteoporosis (Tokuzawa et al., 2010). Id1, Id2, and Id3 are all upregulated in response to BMP9 treatment of mesenchymal progenitor cells (via Smad4), which results in increased proliferation and a block in differentiation of osteogenic progenitors (Peng et al., 2004). In addition, Id proteins have been shown to regulate osteogenic transcription factor activity (Ogata and Noda, 1991; Tamura and Noda, 1994; Zhang et al., 2008). Id2, and Id4 genes also regulate adipogenesis by regulating the proliferation pre-adipoctye progenitors and their ability to undergo differentiation (Murad et al., 2010; Park et al., 2008). Altogether, these results suggest that the Id family members

Novel Targets in Myelogenous Leukemia: The Id Family of Proteins 229

in a human embryonic kidney cell line (293) and in neuroblastoma cell lines restored the ability of the E protein E47 to bind and activate the promoters of cell cycle inhibitors. In addition, 13I was also able to induce differentiation in neuroblastoma cells suggesting it can over-ride both the cell cycle promotion and differentiation blocking functions of the Id proteins (Ciarapica et al., 2009). To date, 13I has not yet been tested on normal or

Each of these inhibitors represents a hopeful step forward to reducing aberrant Id levels and restoring proper differentiation to hyperproliferative cells. The attractive feature of the Ids as potential therapeutic targets is that their expression does not need to be completely reduced, just suppressed to low enough levels to restore differentiation, as seen in normal

In summary, the Id proteins are potential targets in myelogenous leukemia. Much of the work that has gone into characterizing this family of small proteins indicates that if the expression of Id1, Id2, and Id3 is maintained at high levels in progenitor cells, and if their expression is not reduced at the correct time during maturation or differentiation that this could contribute to hyperplasia or neoplasia. Currently there are three targeting molecules

The discovery of novel mediators of oncogenesis, and biomarkers for disease identification and progression, such as the Id family of proteins, will contribute to the design of better

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN26120080001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This Research was supported [in part] by the Intramural Research Program of the NIH, National Cancer

Alani, R.M., Young, A.Z., and Shifflett, C.B. (2001). Id1 regulation of cellular senescence

Anido, J., Saez-Borderias, A., Gonzalez-Junca, A., Rodon, L., Folch, G., Carmona, M.A.,

Bao, F., Polk, P., Nordberg, M.L., Veillon, D.M., Sun, A., Deininger, M., Murray, D.,

Academy of Sciences of the United States of America *98*, 7812-7816.

Population in Human Glioblastoma. Cancer cell *18*, 655-668.

through transcriptional repression of p16/Ink4a. Proceedings of the National

Prieto-Sanchez, R.M., Barba, I., Martinez-Saez, E., Prudkin, L.*, et al.* (2010). TGFbeta Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell

Andersson, B.S., and Munker, R. (2007). Comparative gene expression analysis of a chronic myelogenous leukemia cell line resistant to cyclophosphamide using

available that are undergoing testing at the basic research level.

drugs and more effective therapies for myelogenous leukemia.

hematopoietic cells.

tissues.

**7. Summary** 

**8. Acknowledgements** 

**9. References** 

Institute, Center for Cancer Research.

not only regulate hematopoietic growth factor expression, but the fundamental structure of the niche itself.

#### **6. Targeting Id proteins**

The identification of Id1 as a common downstream effector of oncogenic mutations such as BCR-ABL and FLT3-ITD in CML and AML, as well as their upreglation in multiple types of cancer, suggest that the Id proteins represent potential targets for therapeutic intervention (Suh et al., 2008; Tam et al., 2008). Inhibiting Id1 protein expression in human induced pluripotential stem cells using with small inhibitory RNAs (siRNA) increases differentiation into committed progenitors suggesting that decreasing Id expression can support cell maturation (Hong et al., 2011)*.* To date, there are three novel inhibitors of Id function currently under investigation.

#### **6.1 Peptide conjugated antisense oligonucleotides**

Henke et al. generated an anti-sense oligonucleotide that is covalently coupled to an "address-peptide," Id1-PCAO (peptide-conjugated anti-sense oligonucleotide) (Henke et al., 2008). The peptide used in this study was designed specifically to target endothelial cells; however, other targeting peptides are available and could be linked to the Id1 anti-sense molecule to direct the anti-sense molecule to hematopoietic cells. While the Id1-PCAO strategy has not been tested in hematopoietic cells, it is a feasible approach for use in the hematopoietic malignancies. Targeting peptides have been identified which home to bone marrow and bind primitive hematopoietic stem cells (Nowakowski et al., 2004). The benefit of this procedure would be that it does not require the use of any viral transduction procedures, and the targeting peptide should limit potential toxicity by localizing the antisense molecule to the diseased area.

#### **6.2 Peptide aptamer**

Another small molecule which has recently been developed is a peptide that binds both Id1 and Id3. Mern et al. identified a peptide aptamer (a short peptide), Id1/Id3-PA7, from a randomized combinatorial expression library using yeast and mammalian two-hybrid systems. When the aptamer is fused to a "cell-penetrating protein transduction domain" or PTD (truncated VP22 ORF), and tested on ovarian cancer cell lines, it causes increased expression of p16INK4a, and it induced apoptosis (as indicated by PARP cleavage) (Mern et al., 2010a). The aptamer colocalized with Id1 and Id3 staining based on immunohistochemistry, suggesting that its effects result from its direct interaction with Id1 and/or Id3. To date, the authors have tested this molecule on ovarian and breast cancer cell lines *in vitro* and demonstrated growth inhibition, but have not yet examined its effects on normal cells nor in hematopoiesis (Mern et al., 2010a; Mern et al., 2010b).

#### **6.3 Dominant interfering molecule 13I**

In an effort to better understand the rules of HLH protein interaction, Ciarapica and collegues screened a phage display library to identify and isolate mutant domains with could interfere with HLH domain interactions. They discovered a dominant interfering HLH domain, 13I, which selectively binds to Ids (Id1, Id2, Id3) as opposed to their E protein HLH binding partners, and also impairs complex formation with Rb (Id2). Expression of 13I in a human embryonic kidney cell line (293) and in neuroblastoma cell lines restored the ability of the E protein E47 to bind and activate the promoters of cell cycle inhibitors. In addition, 13I was also able to induce differentiation in neuroblastoma cells suggesting it can over-ride both the cell cycle promotion and differentiation blocking functions of the Id proteins (Ciarapica et al., 2009). To date, 13I has not yet been tested on normal or hematopoietic cells.

Each of these inhibitors represents a hopeful step forward to reducing aberrant Id levels and restoring proper differentiation to hyperproliferative cells. The attractive feature of the Ids as potential therapeutic targets is that their expression does not need to be completely reduced, just suppressed to low enough levels to restore differentiation, as seen in normal tissues.

#### **7. Summary**

228 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

not only regulate hematopoietic growth factor expression, but the fundamental structure of

The identification of Id1 as a common downstream effector of oncogenic mutations such as BCR-ABL and FLT3-ITD in CML and AML, as well as their upreglation in multiple types of cancer, suggest that the Id proteins represent potential targets for therapeutic intervention (Suh et al., 2008; Tam et al., 2008). Inhibiting Id1 protein expression in human induced pluripotential stem cells using with small inhibitory RNAs (siRNA) increases differentiation into committed progenitors suggesting that decreasing Id expression can support cell maturation (Hong et al., 2011)*.* To date, there are three novel inhibitors of Id function

Henke et al. generated an anti-sense oligonucleotide that is covalently coupled to an "address-peptide," Id1-PCAO (peptide-conjugated anti-sense oligonucleotide) (Henke et al., 2008). The peptide used in this study was designed specifically to target endothelial cells; however, other targeting peptides are available and could be linked to the Id1 anti-sense molecule to direct the anti-sense molecule to hematopoietic cells. While the Id1-PCAO strategy has not been tested in hematopoietic cells, it is a feasible approach for use in the hematopoietic malignancies. Targeting peptides have been identified which home to bone marrow and bind primitive hematopoietic stem cells (Nowakowski et al., 2004). The benefit of this procedure would be that it does not require the use of any viral transduction procedures, and the targeting peptide should limit potential toxicity by localizing the anti-

Another small molecule which has recently been developed is a peptide that binds both Id1 and Id3. Mern et al. identified a peptide aptamer (a short peptide), Id1/Id3-PA7, from a randomized combinatorial expression library using yeast and mammalian two-hybrid systems. When the aptamer is fused to a "cell-penetrating protein transduction domain" or PTD (truncated VP22 ORF), and tested on ovarian cancer cell lines, it causes increased expression of p16INK4a, and it induced apoptosis (as indicated by PARP cleavage) (Mern et al., 2010a). The aptamer colocalized with Id1 and Id3 staining based on immunohistochemistry, suggesting that its effects result from its direct interaction with Id1 and/or Id3. To date, the authors have tested this molecule on ovarian and breast cancer cell lines *in vitro* and demonstrated growth inhibition, but have not yet examined its effects on

In an effort to better understand the rules of HLH protein interaction, Ciarapica and collegues screened a phage display library to identify and isolate mutant domains with could interfere with HLH domain interactions. They discovered a dominant interfering HLH domain, 13I, which selectively binds to Ids (Id1, Id2, Id3) as opposed to their E protein HLH binding partners, and also impairs complex formation with Rb (Id2). Expression of 13I

normal cells nor in hematopoiesis (Mern et al., 2010a; Mern et al., 2010b).

the niche itself.

**6. Targeting Id proteins** 

currently under investigation.

sense molecule to the diseased area.

**6.3 Dominant interfering molecule 13I** 

**6.2 Peptide aptamer** 

**6.1 Peptide conjugated antisense oligonucleotides** 

In summary, the Id proteins are potential targets in myelogenous leukemia. Much of the work that has gone into characterizing this family of small proteins indicates that if the expression of Id1, Id2, and Id3 is maintained at high levels in progenitor cells, and if their expression is not reduced at the correct time during maturation or differentiation that this could contribute to hyperplasia or neoplasia. Currently there are three targeting molecules available that are undergoing testing at the basic research level.

The discovery of novel mediators of oncogenesis, and biomarkers for disease identification and progression, such as the Id family of proteins, will contribute to the design of better drugs and more effective therapies for myelogenous leukemia.

#### **8. Acknowledgements**

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN26120080001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This Research was supported [in part] by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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**13** 

 *Japan* 

Shinichiro Takahashi

*Sagamihara, Kanagawa,* 

**PU.1, a Versatile Transcription Factor and** 

*Division of Hematology, Kitasato University School of Allied Health Sciences,* 

PU.1 is a member of the Ezb transformation-specific sequence (Ets) family of transcription factors, and is expressed in granulocytic, monocytic and B-lymphoid cells (Chen et al., 1995b; Klemsz et al., 1990). PU.1 expression levels increase during the differentiation of granulocytes (Chen et al., 1995b). PU.1-deficient mice exhibit defects in the development of neutrophils, macrophages and B cells (McKercher et al., 1996; Scott et al., 1994). Therefore, PU.1 is indispensable for myeloid differentiation during normal hematopoiesis. Moreover, mice carrying hypomorphic PU.1 alleles that reduce PU.1 expression to 20% of its normal levels were reported to develop acute myeloid leukemia (AML) (Rosenbauer et al., 2004), suggesting that disruption of the function of this transcription factor plays a pivotal role in myeloid leukemia biology through the control of numerous target genes. Binding sites for PU.1 are found on almost all myeloid-specific promoters. Notable target genes are receptors for the cytokines, *macrophage colony-stimulating factor* (*M-CSF*), *granulocyte colony-stimulating factor* (*G-CSF*) and *granulocyte macrophage colony-stimulating factor* (*GM-CSF*) (Hohaus et al., 1995; Smith et al., 1996; Zhang et al., 1994), the characteristic antigens *CD11b*/*CD18*  (Rosmarin et al., 1995), primary granule enzymes (Iwama et al., 1998) and the transcription factors c-Jun and JunB (Steidl et al., 2006). In addition, a transcriptional repressor function of PU.1 toward a certain set of genes has been demonstrated. Although the importance of the PU.1 function as a positive regulator of these myeloid genes has been widely examined and reviewed, studies on the role of PU.1 as a transcriptional repressor are relatively scarce. The author's group and others demonstrated that loss of PU.1 function resulted in upregulation of several drug-resistance or growth-regulating genes, including *metallothionein* (*MT*), *vimentin* (*VIM*), *c-myc* and *Flt3,* through disruption of the transcriptional repressor function (Imoto et al., 2010; Inomata et al., 2006; Iseki et al., 2009). This review summarizes the current understanding of the molecular functions of PU.1. Moreover, this review has a particular focus on the transcriptional function of PU.1 as an activator as well as a repressor.

Furthermore, the role of PU.1 in the biology of myeloid leukemia is discussed.

The Ets family of transcription factors is so named because the first gene was identified in the E26 avian retrovirus. Ets factors constitute a relatively large gene family, with 27

**2. PU.1, a member of the ETS transcription factor family** 

**1. Introduction** 

**a Suppressor of Myeloid Leukemia** 


### **PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia**

Shinichiro Takahashi

*Division of Hematology, Kitasato University School of Allied Health Sciences, Sagamihara, Kanagawa, Japan* 

#### **1. Introduction**

238 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Zhang, Y., Hassan, M.Q., Li, Z.Y., Stein, J.L., Lian, J.B., van Wijnen, A.J., and Stein, G.S.

Zheng, W., Wang, H., Xue, L., Zhang, Z., and Tong, T. (2004). Regulation of cellular

fibroblast. The Journal of biological chemistry *279*, 31524-31532.

Journal of cellular biochemistry *105*, 487-496.

(2008). Intricate gene regulatory networks of helix-loop-helix (HLH) proteins support regulation of bone-tissue related genes during osteoblast differentiation.

senescence and p16(INK4a) expression by Id1 and E47 proteins in human diploid

PU.1 is a member of the Ezb transformation-specific sequence (Ets) family of transcription factors, and is expressed in granulocytic, monocytic and B-lymphoid cells (Chen et al., 1995b; Klemsz et al., 1990). PU.1 expression levels increase during the differentiation of granulocytes (Chen et al., 1995b). PU.1-deficient mice exhibit defects in the development of neutrophils, macrophages and B cells (McKercher et al., 1996; Scott et al., 1994). Therefore, PU.1 is indispensable for myeloid differentiation during normal hematopoiesis. Moreover, mice carrying hypomorphic PU.1 alleles that reduce PU.1 expression to 20% of its normal levels were reported to develop acute myeloid leukemia (AML) (Rosenbauer et al., 2004), suggesting that disruption of the function of this transcription factor plays a pivotal role in myeloid leukemia biology through the control of numerous target genes. Binding sites for PU.1 are found on almost all myeloid-specific promoters. Notable target genes are receptors for the cytokines, *macrophage colony-stimulating factor* (*M-CSF*), *granulocyte colony-stimulating factor* (*G-CSF*) and *granulocyte macrophage colony-stimulating factor* (*GM-CSF*) (Hohaus et al., 1995; Smith et al., 1996; Zhang et al., 1994), the characteristic antigens *CD11b*/*CD18*  (Rosmarin et al., 1995), primary granule enzymes (Iwama et al., 1998) and the transcription factors c-Jun and JunB (Steidl et al., 2006). In addition, a transcriptional repressor function of PU.1 toward a certain set of genes has been demonstrated. Although the importance of the PU.1 function as a positive regulator of these myeloid genes has been widely examined and reviewed, studies on the role of PU.1 as a transcriptional repressor are relatively scarce. The author's group and others demonstrated that loss of PU.1 function resulted in upregulation of several drug-resistance or growth-regulating genes, including *metallothionein* (*MT*), *vimentin* (*VIM*), *c-myc* and *Flt3,* through disruption of the transcriptional repressor function (Imoto et al., 2010; Inomata et al., 2006; Iseki et al., 2009). This review summarizes the current understanding of the molecular functions of PU.1. Moreover, this review has a particular focus on the transcriptional function of PU.1 as an activator as well as a repressor. Furthermore, the role of PU.1 in the biology of myeloid leukemia is discussed.

#### **2. PU.1, a member of the ETS transcription factor family**

The Ets family of transcription factors is so named because the first gene was identified in the E26 avian retrovirus. Ets factors constitute a relatively large gene family, with 27

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 241

corresponds to the DNA-binding domain and recognizes sequences harboring the core GGAA motif. This domain is also involved in the protein-protein interactions between PU.1 and other factors such as GATA-1 (Nerlov et al., 2000), c-Jun (Behre et al., 1999), Runx 1 or

The activation domain of PU.1 is located within the N-terminus and consists of several regions rich in either acidic amino acids or glutamines (designated the Gln-rich domain) (Klemsz & Maki, 1996). The activation domain at the N-terminal 75 amino acids of PU.1 has been shown to interact *in vitro* with the basal transcription factor IID (TFIID) and the tumor suppressor retinoblastoma (Rb) protein (Hagemeier et al., 1993). Yamamoto *et al.* (Yamamoto et al., 1999) previously found that this transcriptional activation domain of amino acids 74–122 binds to a transcriptional coactivator, cAMP response element-binding protein (CREB)-binding protein (CBP). CBP potentiated PU.1-mediated transcription of a reporter gene driven by multimerized PU.1-binding sites, suggesting that CBP functions as a coactivator for PU.1 (Yamamoto et al., 1999). A region from amino acids 118–160, which has large numbers of prolines, glutamic acids, serines and threonines, is called the PEST domain (Klemsz & Maki, 1996). This domain plays important roles in the protein-protein interactions, particularly those with interferon (IFN) consensus sequence-binding protein

The C-terminal region of PU.1 has been reported to interact with the leucine zipper transcription factor CCAAT/enhancer-binding protein (C/EBP) , DNA-binding protein HMG I/Y, multifunctional phosphatase MKP-1 and chaperone protein HSP90 (Nagulapalli et al., 1995) as well as the basic transcription factor TATA box-binding protein (TBP)

The PU.1 protein is also the target of phosphorylation events, notably on serine 148, which are regulated by various extracellular signals and play critical roles in modifying the activity

**4. Role of PU.1 in normal hematopoiesis, especially in the myeloid lineage,** 

differentiation, overexpression of PU.1 results in erythroid differentiation blockade.

relatively low PU.1 levels induce B-cell differentiation (DeKoter & Singh, 2000).

Expression of PU.1 is observed in CD34+ hematopoietic progenitors and differentiation commitment toward the myeloid and lymphoid lineages appears to be determined by the expression levels of PU.1, since high PU.1 levels promote macrophage differentiation and

Collectively, PU.1 functions in multiple hematopoietic lineages including myeloid, lymphoid and erythroid cells. Many reviews describing the roles of PU.1 in hematopoiesis

As described in the introduction, PU.1 is expressed in B-lymphoid, granulocytic and monocytic cells (Chen et al., 1995b; Klemsz et al., 1990). PU.1 expression levels increase during the differentiation of granulocytes (Chen et al., 1995b). PU.1-deficient mice exhibit defects in the development of neutrophils, macrophages and B cells, although the generation of erythroid and megakaryocytic cells is intact (McKercher et al., 1996; Scott et al., 1994). In the erythroid lineage, the expression of PU.1 is downregulated during terminal differentiation of murine erythroleukemia (MEL) cells by treatment with dimethylsulfoxide (Hensold et al., 1996). Enforced expression of PU.1 in MEL cells inhibits erythroid differentiation (Oikawa et al., 1999; Yamada et al., 2001). In contrast to the lack of PU.1 expression in the myelomonocytic lineage, which results in impairment of the

of this factor (Joo et al., 2004). These aspects will be discussed later in section 7.

(ICSBP, also called IFN regulatory factor-8) (Nakano et al., 2005).

C/EBP (Petrovick et al., 1998).

(Kihara-Negishi et al., 2001).

**through the regulation of its target genes** 

members encoded by the human genome (Sharrocks, 2001). As precisely reviewed by Oikawa and Yamada (Oikawa & Yamada, 2003), Ets family proteins can be divided into several subfamilies on the basis of their structural compositions and the similarities in their DNA-binding Ets domains. Most of them have the Ets domains in their C-terminal regions. However, several Ets family proteins, such as the ternary complex factor (TCF) subfamily members, have the Ets domains in their N-terminal regions. In addition, besides the conserved Ets domain, a subset of Ets family proteins (e.g. Ets-1, Ets-2, ERG, TEL) have another evolutionarily conserved domain called the pointed domain at their N-terminal regions, which forms a helix-loop-helix (HLH) structure for protein-protein interactions (Oikawa & Yamada, 2003). PU.1 is the most distantly related member of the Ets family proteins having only about 40% identity with Ets-1 in its DNA-binding domain (Oikawa & Yamada, 2003).

#### **3. Structure of PU.1**

*Spi-1*, the gene name for PU.1, was first identified by Moreau-Gachelin and colleagues as the product of a gene targeted by recurrent insertions of the Spleen Focus Forming Virus (SFFV) in Friend's erythroleukemia. An important and unusual feature of the genomic locus *Spi-1* for SFFV proviral integration is that rearrangements caused by SFFV integration were found in 95% of the erythroid tumors examined (Moreau-Gachelin et al., 1988). They subsequently isolated PU.1 (for purine-rich box 1) as a factor that binds to a purine-rich motif in the MHC class II gene promoter. Basically, monomeric PU.1 binds to the consensus DNA site 5'- AAAG(A/C/G)GGAAG-3' via its C-terminal Ets domain and activates transcription via its N-terminal glutamine-rich and acidic domains (Klemsz et al., 1990) (Figure 1).

Fig. 1. Schematic presentation of PU.1 and its interacting partners. Proteins involved in transcription are shown in ovals, with transcriptional repressors shown in light gray and basic transcription factors shown in dark gray.

PU.1 is encoded by the *Spi-1 (sfpi1)* gene located on chromosome 11 in humans (chromosome 2 in mice). PU.1 transcripts are 1333 nucleotides long and are produced from the splicing of five exons (Kastner & Chan, 2008). The PU.1 protein is 272 amino acids long (predicted MW: 31 kDa). The structure of PU.1 is shown in Figure 1. The DNA-binding Ets domain shows sequence similarity with other members of the Ets family and is contained within amino acids 171–267 of the C-terminus (Klemsz et al., 1990). The Ets domain

members encoded by the human genome (Sharrocks, 2001). As precisely reviewed by Oikawa and Yamada (Oikawa & Yamada, 2003), Ets family proteins can be divided into several subfamilies on the basis of their structural compositions and the similarities in their DNA-binding Ets domains. Most of them have the Ets domains in their C-terminal regions. However, several Ets family proteins, such as the ternary complex factor (TCF) subfamily members, have the Ets domains in their N-terminal regions. In addition, besides the conserved Ets domain, a subset of Ets family proteins (e.g. Ets-1, Ets-2, ERG, TEL) have another evolutionarily conserved domain called the pointed domain at their N-terminal regions, which forms a helix-loop-helix (HLH) structure for protein-protein interactions (Oikawa & Yamada, 2003). PU.1 is the most distantly related member of the Ets family proteins having only about 40% identity with Ets-1 in its DNA-binding domain (Oikawa &

*Spi-1*, the gene name for PU.1, was first identified by Moreau-Gachelin and colleagues as the product of a gene targeted by recurrent insertions of the Spleen Focus Forming Virus (SFFV) in Friend's erythroleukemia. An important and unusual feature of the genomic locus *Spi-1* for SFFV proviral integration is that rearrangements caused by SFFV integration were found in 95% of the erythroid tumors examined (Moreau-Gachelin et al., 1988). They subsequently isolated PU.1 (for purine-rich box 1) as a factor that binds to a purine-rich motif in the MHC class II gene promoter. Basically, monomeric PU.1 binds to the consensus DNA site 5'- AAAG(A/C/G)GGAAG-3' via its C-terminal Ets domain and activates transcription via its

N-terminal glutamine-rich and acidic domains (Klemsz et al., 1990) (Figure 1).

Fig. 1. Schematic presentation of PU.1 and its interacting partners. Proteins involved in transcription are shown in ovals, with transcriptional repressors shown in light gray and

PU.1 is encoded by the *Spi-1 (sfpi1)* gene located on chromosome 11 in humans (chromosome 2 in mice). PU.1 transcripts are 1333 nucleotides long and are produced from the splicing of five exons (Kastner & Chan, 2008). The PU.1 protein is 272 amino acids long (predicted MW: 31 kDa). The structure of PU.1 is shown in Figure 1. The DNA-binding Ets domain shows sequence similarity with other members of the Ets family and is contained within amino acids 171–267 of the C-terminus (Klemsz et al., 1990). The Ets domain

basic transcription factors shown in dark gray.

Yamada, 2003).

**3. Structure of PU.1** 

corresponds to the DNA-binding domain and recognizes sequences harboring the core GGAA motif. This domain is also involved in the protein-protein interactions between PU.1 and other factors such as GATA-1 (Nerlov et al., 2000), c-Jun (Behre et al., 1999), Runx 1 or C/EBP (Petrovick et al., 1998).

The activation domain of PU.1 is located within the N-terminus and consists of several regions rich in either acidic amino acids or glutamines (designated the Gln-rich domain) (Klemsz & Maki, 1996). The activation domain at the N-terminal 75 amino acids of PU.1 has been shown to interact *in vitro* with the basal transcription factor IID (TFIID) and the tumor suppressor retinoblastoma (Rb) protein (Hagemeier et al., 1993). Yamamoto *et al.* (Yamamoto et al., 1999) previously found that this transcriptional activation domain of amino acids 74–122 binds to a transcriptional coactivator, cAMP response element-binding protein (CREB)-binding protein (CBP). CBP potentiated PU.1-mediated transcription of a reporter gene driven by multimerized PU.1-binding sites, suggesting that CBP functions as a coactivator for PU.1 (Yamamoto et al., 1999). A region from amino acids 118–160, which has large numbers of prolines, glutamic acids, serines and threonines, is called the PEST domain (Klemsz & Maki, 1996). This domain plays important roles in the protein-protein interactions, particularly those with interferon (IFN) consensus sequence-binding protein (ICSBP, also called IFN regulatory factor-8) (Nakano et al., 2005).

The C-terminal region of PU.1 has been reported to interact with the leucine zipper transcription factor CCAAT/enhancer-binding protein (C/EBP) , DNA-binding protein HMG I/Y, multifunctional phosphatase MKP-1 and chaperone protein HSP90 (Nagulapalli et al., 1995) as well as the basic transcription factor TATA box-binding protein (TBP) (Kihara-Negishi et al., 2001).

The PU.1 protein is also the target of phosphorylation events, notably on serine 148, which are regulated by various extracellular signals and play critical roles in modifying the activity of this factor (Joo et al., 2004). These aspects will be discussed later in section 7.

#### **4. Role of PU.1 in normal hematopoiesis, especially in the myeloid lineage, through the regulation of its target genes**

As described in the introduction, PU.1 is expressed in B-lymphoid, granulocytic and monocytic cells (Chen et al., 1995b; Klemsz et al., 1990). PU.1 expression levels increase during the differentiation of granulocytes (Chen et al., 1995b). PU.1-deficient mice exhibit defects in the development of neutrophils, macrophages and B cells, although the generation of erythroid and megakaryocytic cells is intact (McKercher et al., 1996; Scott et al., 1994).

In the erythroid lineage, the expression of PU.1 is downregulated during terminal differentiation of murine erythroleukemia (MEL) cells by treatment with dimethylsulfoxide (Hensold et al., 1996). Enforced expression of PU.1 in MEL cells inhibits erythroid differentiation (Oikawa et al., 1999; Yamada et al., 2001). In contrast to the lack of PU.1 expression in the myelomonocytic lineage, which results in impairment of the differentiation, overexpression of PU.1 results in erythroid differentiation blockade.

Expression of PU.1 is observed in CD34+ hematopoietic progenitors and differentiation commitment toward the myeloid and lymphoid lineages appears to be determined by the expression levels of PU.1, since high PU.1 levels promote macrophage differentiation and relatively low PU.1 levels induce B-cell differentiation (DeKoter & Singh, 2000).

Collectively, PU.1 functions in multiple hematopoietic lineages including myeloid, lymphoid and erythroid cells. Many reviews describing the roles of PU.1 in hematopoiesis

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 243

The M-CSF receptor (M-CSFR) (Zhang et al., 1994), GM-CSF receptor (GM-CSFR) (Hohaus et al., 1995) and G-CSF receptor (G-CSFR) (Smith et al., 1996) gene promoters were reported to be directly regulated by PU.1. These promoters are regulated by PU.1 in combination with C/EBP (Hohaus et al., 1995; Li et al., 2005; Smith et al., 1996). By employing sense or antisense PU.1 expression, Celada *et al.* (Celada et al., 1996) showed that PU.1 is necessary for M-CSF-dependent proliferation of macrophages, through the control of M-CSFR expression. Another group demonstrated that PU.1-deficient myeloid progenitors can proliferate *in vitro* in response to the multilineage cytokines interleukin (IL)-3, IL-6 and stem cell factor, but are unresponsive to the myeloid-specific cytokines GM-CSF, G-CSF and M-CSF (DeKoter et al., 1998). Expression of M-CSFR or G-CSFR in PU.1-deficient marrow cells did not rescue myeloid development, indicating that PU.1 is required beyond the induction of these cytokine receptors (DeKoter et al., 1998; Henkel et

The CD11b (or macrophage-1 antigen; MAC-1) leukocyte integrin subunit exists on the surface of human granulocytes and monocytes/macrophages coupled with the CD18 () subunit in a heterodimer. This heterodimer mediates multifaceted adherence reactivity of these myeloid cells, including the ability to adhere to endothelial cells (Arnaout, 1990). The CD11b promoter was reported to be positively regulated by both Sp1 and PU.1 (Chen et al., 1993). CD18 promoter activity is also regulated by PU.1 (Bottinger et al., 1994; Rosmarin et al., 1995). Consistently, CD11b/CD18 are undetectable on the surfaces of cells lacking PU.1 (Henkel et al., 1996; Olson et al., 1995). These molecules are from the 2 integrin chain, otherwise known as complement receptor 3 (CR3) (Ehlers, 2000). The CD11b subunit of CR3 promotes phagocytosis and macrophage migration by binding to extracellular matrix proteins, coagulation proteins and a variety of microbial antigens

Primary granule enzymes are a group of serine proteases or closely related molecules found in cells of the granulocyte series. PU.1-deficient myeloid promoters lack or exhibit low expression of primary granule enzymes, including myeloperoxidase (MPO), neutrophil elastase (NE), proteinase 3 (PR3) and lysozyme (Iwama et al., 1998). Ford *et al.* (Ford et al., 1996) demonstrated that the MPO upstream enhancer is accessible in multipotent cell chromatin, but functionally incompetent before granulocyte commitment. Multipotent cells contain both PU.1 and C/EBP as enhancer-binding activities. It was reported that a 220-bp portion of the second intron of the *PR3* gene, which contains an Sp1 consensus site flanked by two Ets family consensus sequences that bind PU.1, functions as an enhancer of NE expression (Nuchprayoon et al., 1999). In addition, PU.1 cooperates with C/EBP and c-Myb to regulate the NE promoter (Oelgeschlager et al., 1996). PR3 expression has also been shown to be regulated by PU.1 (Sturrock et al., 1996). PU.1-deficient neutrophils do not express secondary granule components, including collagenase, lysozyme and lactoferrin, and have limited ability to ingest and kill bacteria (Anderson et al., 1998; Iwama et al., 1998). The activity of the lysozyme gene enhancer was demonstrated to be controlled by PU.1 in avian macrophages (Ahne & Stratling, 1994). In addition, the expression of gp91phox and p47phox, which are both components of nicotinamide adenine dinucleotide phosphate (NADPH) phagocyte oxidase, is positively regulated by PU.1 (Li et al., 1997; Suzuki et al.,

In addition to these myeloid-specific genes, several transcription factors play crucial roles in the phenotype of PU.1-deficient mice. Steidl *et al.* (Steidl et al., 2006) examined the

al., 1999).

(Ehlers, 2000).

1998).

have been published (Gallant & Gilkeson, 2006; Kastner & Chan, 2008; Oikawa et al., 1999; Oikawa & Yamada, 2003). Therefore, this section of the present review particularly focuses on the roles of PU.1 in myeloid lineage development through the regulation of its target genes.

Early myeloid differentiation is controlled by PU.1 and other families of transcription factors, such as C/EBPs, AML1/Runx1, retinoic acid receptor (RAR), c-Myb and others (Friedman, 2002). C/EBP and C/EBP knockout mice also have defects in the development of mature macrophages and neutrophils (Yamanaka et al., 1997; Zhang et al., 1999). Hence, PU.1, as well as the C/EBP family members, is a critical master regulator for the determination of the developmental pathway toward the myeloid lineage in hematopoietic cells.


Table 1. PU.1 target genes play crucial roles in myeloid lineage development

Binding sites for PU.1 are found on almost all myeloid-specific promoters. A large collection of PU.1-dependent promoters have been identified, which control an array of genes encoding predominantly growth factor receptors, adhesion molecules and characteristic enzymes. Notable target genes are receptors for cytokines, adhesion molecules, primary granule enzymes and transcription factors.

have been published (Gallant & Gilkeson, 2006; Kastner & Chan, 2008; Oikawa et al., 1999; Oikawa & Yamada, 2003). Therefore, this section of the present review particularly focuses on the roles of PU.1 in myeloid lineage development through the regulation of its target

Early myeloid differentiation is controlled by PU.1 and other families of transcription factors, such as C/EBPs, AML1/Runx1, retinoic acid receptor (RAR), c-Myb and others (Friedman, 2002). C/EBP and C/EBP knockout mice also have defects in the development of mature macrophages and neutrophils (Yamanaka et al., 1997; Zhang et al., 1999). Hence, PU.1, as well as the C/EBP family members, is a critical master regulator for the determination of the

developmental pathway toward the myeloid lineage in hematopoietic cells.

**Gene Level of regulation References** 

M-CSFR Promoter (Zhang et al., 1994)

GM-CSFR Promoter (Hohaus et al., 1995)

G-CSFR Promoter (Smith et al., 1996)

CD11b Promoter (Chen et al., 1993)

MPO Enhancer (Ford et al., 1996)

CD18 Promoter (Rosmarin et al., 1995)

NE Enhancer (Nuchprayoon et al., 1999)

NE Promoter (Oelgeschlager et al., 1996)

PR3 Promoter (Sturrock et al., 1996)

gp91phox Promoter (Suzuki et al., 1998)

c-Jun Unknown (Steidl et al., 2006)

JunB Promoter (Steidl et al., 2006)

PU.1 Promoter (Chen et al., 1995a)

Binding sites for PU.1 are found on almost all myeloid-specific promoters. A large collection of PU.1-dependent promoters have been identified, which control an array of genes encoding predominantly growth factor receptors, adhesion molecules and characteristic enzymes. Notable target genes are receptors for cytokines, adhesion molecules, primary

Table 1. PU.1 target genes play crucial roles in myeloid lineage development

granule enzymes and transcription factors.

p47phox Promoter (Li et al., 1997)

Lysozyme Enhancer (Ahne & Stratling, 1994)

(Li et al., 2005)

(Bottinger et al., 1994)

genes.

The M-CSF receptor (M-CSFR) (Zhang et al., 1994), GM-CSF receptor (GM-CSFR) (Hohaus et al., 1995) and G-CSF receptor (G-CSFR) (Smith et al., 1996) gene promoters were reported to be directly regulated by PU.1. These promoters are regulated by PU.1 in combination with C/EBP (Hohaus et al., 1995; Li et al., 2005; Smith et al., 1996). By employing sense or antisense PU.1 expression, Celada *et al.* (Celada et al., 1996) showed that PU.1 is necessary for M-CSF-dependent proliferation of macrophages, through the control of M-CSFR expression. Another group demonstrated that PU.1-deficient myeloid progenitors can proliferate *in vitro* in response to the multilineage cytokines interleukin (IL)-3, IL-6 and stem cell factor, but are unresponsive to the myeloid-specific cytokines GM-CSF, G-CSF and M-CSF (DeKoter et al., 1998). Expression of M-CSFR or G-CSFR in PU.1-deficient marrow cells did not rescue myeloid development, indicating that PU.1 is required beyond the induction of these cytokine receptors (DeKoter et al., 1998; Henkel et al., 1999).

The CD11b (or macrophage-1 antigen; MAC-1) leukocyte integrin subunit exists on the surface of human granulocytes and monocytes/macrophages coupled with the CD18 () subunit in a heterodimer. This heterodimer mediates multifaceted adherence reactivity of these myeloid cells, including the ability to adhere to endothelial cells (Arnaout, 1990). The CD11b promoter was reported to be positively regulated by both Sp1 and PU.1 (Chen et al., 1993). CD18 promoter activity is also regulated by PU.1 (Bottinger et al., 1994; Rosmarin et al., 1995). Consistently, CD11b/CD18 are undetectable on the surfaces of cells lacking PU.1 (Henkel et al., 1996; Olson et al., 1995). These molecules are from the 2 integrin chain, otherwise known as complement receptor 3 (CR3) (Ehlers, 2000). The CD11b subunit of CR3 promotes phagocytosis and macrophage migration by binding to extracellular matrix proteins, coagulation proteins and a variety of microbial antigens (Ehlers, 2000).

Primary granule enzymes are a group of serine proteases or closely related molecules found in cells of the granulocyte series. PU.1-deficient myeloid promoters lack or exhibit low expression of primary granule enzymes, including myeloperoxidase (MPO), neutrophil elastase (NE), proteinase 3 (PR3) and lysozyme (Iwama et al., 1998). Ford *et al.* (Ford et al., 1996) demonstrated that the MPO upstream enhancer is accessible in multipotent cell chromatin, but functionally incompetent before granulocyte commitment. Multipotent cells contain both PU.1 and C/EBP as enhancer-binding activities. It was reported that a 220-bp portion of the second intron of the *PR3* gene, which contains an Sp1 consensus site flanked by two Ets family consensus sequences that bind PU.1, functions as an enhancer of NE expression (Nuchprayoon et al., 1999). In addition, PU.1 cooperates with C/EBP and c-Myb to regulate the NE promoter (Oelgeschlager et al., 1996). PR3 expression has also been shown to be regulated by PU.1 (Sturrock et al., 1996). PU.1-deficient neutrophils do not express secondary granule components, including collagenase, lysozyme and lactoferrin, and have limited ability to ingest and kill bacteria (Anderson et al., 1998; Iwama et al., 1998). The activity of the lysozyme gene enhancer was demonstrated to be controlled by PU.1 in avian macrophages (Ahne & Stratling, 1994). In addition, the expression of gp91phox and p47phox, which are both components of nicotinamide adenine dinucleotide phosphate (NADPH) phagocyte oxidase, is positively regulated by PU.1 (Li et al., 1997; Suzuki et al., 1998).

In addition to these myeloid-specific genes, several transcription factors play crucial roles in the phenotype of PU.1-deficient mice. Steidl *et al.* (Steidl et al., 2006) examined the

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 245

In contrast to PU.1 or C/EBPs, the other transcription factors are widely expressed outside of the hematopoietic system, indicating that myeloid-specific expression may be principally caused by PU.1 or unique combinations of adjoining transcription factors working in concert with PU.1 (Fisher & Scott., 1998). In this group of myeloid promoters, as depicted in Figure 2, binding of PU.1 close to the start point of transcription may facilitate recruitment to the TFIID complex and promote the binding of adjoining transcription factors. TFIID is a protein complex consisting of TBP and a set of TBP-associated factors (TAFs) (Pugh, 1996). Binding of TFIID to the promoter region is the first step in the assembly process that forms the transcription initiation complex. Not only does the N-terminal of PU.1 interact with TFIID, but its C-terminal was reported to interact with TBP (Kihara-Negishi et al., 2001) (Figures 1 and 2). Even though PU.1 may make the initial contact with TFIID, other transcriptional factors (e.g. C/EBPs, Sp1) are likely to help stabilize the TFIID complex through TAF interactions. The PU.1-initiated cooperative stabilization of TFIID may be

As described in the previous section, expression of PU.1 is critical for hematopoiesis. Therefore, the regulatory mechanisms of PU.1 expression have been extensively studied. To date, most aspects of PU.1 regulation have been explained by invoking two regulatory elements, the promoter and an upstream regulatory element (URE) at –14 kb upstream of

The *Spi-1* promoter contains octamer-binding sites that affect B cell expression (Chen et al., 1996). As described in the previous section, PU.1 was demonstrated to bind to its own promoter to regulate itself in myeloid cells (Chen et al., 1995a). *Spi-1* promoter activity can also be directed in myeloid cells by C/EBP and AP-1 (Cai et al., 2008) (Figure 3). Although the promoter alone cannot drive reporter expression in a chromatin context, a search for added regulatory function yielded the conserved URE at around –14 kb, reported to be a myeloid-specific enhancer, that enhanced the promoter activity in a myeloid cell line (Li et al., 2001). Huang *et al.* (Huang et al., 2008) demonstrated that AML1/Runx1 binds to functionally important sites within the URE and regulates PU.1 expression at both embryonic and adult stages of development. As described later in section 8, NF-B was reported to regulate PU.1 through a novel site within the upstream URE (Bonadies et al., 2010). Zarnegar *et al.* (Zarnegar et al., 2010) recently uncovered a set of conserved *cis*regulatory regions for *Spi-1*. One is a novel enhancer (–10 kb) that can amplify myeloid cellspecific expression of PU.1. The other is a separate pro-T-cell-specific silencer element (–9 kb). The enhancer element at –9 kb functions as a weak silencer in myeloid cells. The schematic layout is presented in Figure 3. Using a combination of transgenic studies, global chromatin assays and detailed molecular analyses, Leddin *et al.* (Leddin et al., 2011) presented evidence that PU.1 is regulated by a novel mechanism involving crosstalk between different *cis*-elements together with lineage-restricted autoregulation. They revealed that myeloid progenitors express C/EBP, which binds to the URE and induces activation of an enhancer at –12 kb to allow the formation of a second PU.1 autoregulatory loop and binding of additional PU.1 driven-transcription factors, such as early growth response 2 (EGR2). In this model, PU.1 regulates its expression in B cells and macrophages by differently associating with cell type-specific transcription factors at one of its *cis*-

regulatory elements to establish differential activity patterns at other elements.

sufficient to evoke myeloid-specific expression for this group of genes.

the transcription start site of the *Spi-1* gene encoding PU.1.

**5. Regulation of PU.1** 

transcriptome of preleukemic hematopoietic stem cells (HSCs) from mice in which PU.1 was knocked down to identify transcriptional changes. They demonstrated that the transcription factors c-Jun and JunB were among the top downregulated targets. They concluded that the decreased levels of c-Jun and especially JunB contribute to the development of PU.1 knockdown-induced AML by blocking differentiation and increasing self-renewal. It was previously reported that PU.1 autoregulates its expression in myeloid cells (Chen et al., 1995a). However, autoregulation by PU.1 remains uncertain, since the expression of GFP reporters driven by the endogenous PU.1 promoter remains high in PU.1-null hematopoietic cells (Back et al., 2005; Dakic et al., 2005).

Fig. 2. PU.1 as a transcriptional activator. The known myeloid promoters regulated by a set of transcription factors in combination with PU.1 are depicted.

Table 1 shows a list of myeloid genes that are known to be positively regulated by PU.1. Collectively, PU.1 plays a pivotal role in myeloid development through the regulation of cytokines, receptors, transcription factors and various myeloid specific genes.

The majority of the PU.1-dependent promoters in the myeloid lineage share several structural features, including a PU.1-binding site close to the site(s) of transcriptional initiation, no TATA box and a cluster of binding sites for Sp1 and members of the C/EBP or core-binding factor (CBF) families (Fisher & Scott, 1998; Tenen et al., 1997). A schematic layout of the transcription factors binding to the proximal promoter regions is shown in Figure 2. The G-CSFR, M-CSFR and GM-CSFR promoters are regulated by PU.1 and C/EBPs, whereas the CD11b and NE promoters are regulated by PU.1 in combination with Sp1.

In contrast to PU.1 or C/EBPs, the other transcription factors are widely expressed outside of the hematopoietic system, indicating that myeloid-specific expression may be principally caused by PU.1 or unique combinations of adjoining transcription factors working in concert with PU.1 (Fisher & Scott., 1998). In this group of myeloid promoters, as depicted in Figure 2, binding of PU.1 close to the start point of transcription may facilitate recruitment to the TFIID complex and promote the binding of adjoining transcription factors. TFIID is a protein complex consisting of TBP and a set of TBP-associated factors (TAFs) (Pugh, 1996). Binding of TFIID to the promoter region is the first step in the assembly process that forms the transcription initiation complex. Not only does the N-terminal of PU.1 interact with TFIID, but its C-terminal was reported to interact with TBP (Kihara-Negishi et al., 2001) (Figures 1 and 2). Even though PU.1 may make the initial contact with TFIID, other transcriptional factors (e.g. C/EBPs, Sp1) are likely to help stabilize the TFIID complex through TAF interactions. The PU.1-initiated cooperative stabilization of TFIID may be sufficient to evoke myeloid-specific expression for this group of genes.

#### **5. Regulation of PU.1**

244 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

transcriptome of preleukemic hematopoietic stem cells (HSCs) from mice in which PU.1 was knocked down to identify transcriptional changes. They demonstrated that the transcription factors c-Jun and JunB were among the top downregulated targets. They concluded that the decreased levels of c-Jun and especially JunB contribute to the development of PU.1 knockdown-induced AML by blocking differentiation and increasing self-renewal. It was previously reported that PU.1 autoregulates its expression in myeloid cells (Chen et al., 1995a). However, autoregulation by PU.1 remains uncertain, since the expression of GFP reporters driven by the endogenous PU.1 promoter remains high in PU.1-null hematopoietic

Fig. 2. PU.1 as a transcriptional activator. The known myeloid promoters regulated by a set

Table 1 shows a list of myeloid genes that are known to be positively regulated by PU.1. Collectively, PU.1 plays a pivotal role in myeloid development through the regulation of

The majority of the PU.1-dependent promoters in the myeloid lineage share several structural features, including a PU.1-binding site close to the site(s) of transcriptional initiation, no TATA box and a cluster of binding sites for Sp1 and members of the C/EBP or core-binding factor (CBF) families (Fisher & Scott, 1998; Tenen et al., 1997). A schematic layout of the transcription factors binding to the proximal promoter regions is shown in Figure 2. The G-CSFR, M-CSFR and GM-CSFR promoters are regulated by PU.1 and C/EBPs, whereas the CD11b and NE promoters are regulated by PU.1 in combination with

cytokines, receptors, transcription factors and various myeloid specific genes.

of transcription factors in combination with PU.1 are depicted.

Sp1.

cells (Back et al., 2005; Dakic et al., 2005).

As described in the previous section, expression of PU.1 is critical for hematopoiesis. Therefore, the regulatory mechanisms of PU.1 expression have been extensively studied. To date, most aspects of PU.1 regulation have been explained by invoking two regulatory elements, the promoter and an upstream regulatory element (URE) at –14 kb upstream of the transcription start site of the *Spi-1* gene encoding PU.1.

The *Spi-1* promoter contains octamer-binding sites that affect B cell expression (Chen et al., 1996). As described in the previous section, PU.1 was demonstrated to bind to its own promoter to regulate itself in myeloid cells (Chen et al., 1995a). *Spi-1* promoter activity can also be directed in myeloid cells by C/EBP and AP-1 (Cai et al., 2008) (Figure 3). Although the promoter alone cannot drive reporter expression in a chromatin context, a search for added regulatory function yielded the conserved URE at around –14 kb, reported to be a myeloid-specific enhancer, that enhanced the promoter activity in a myeloid cell line (Li et al., 2001). Huang *et al.* (Huang et al., 2008) demonstrated that AML1/Runx1 binds to functionally important sites within the URE and regulates PU.1 expression at both embryonic and adult stages of development. As described later in section 8, NF-B was reported to regulate PU.1 through a novel site within the upstream URE (Bonadies et al., 2010). Zarnegar *et al.* (Zarnegar et al., 2010) recently uncovered a set of conserved *cis*regulatory regions for *Spi-1*. One is a novel enhancer (–10 kb) that can amplify myeloid cellspecific expression of PU.1. The other is a separate pro-T-cell-specific silencer element (–9 kb). The enhancer element at –9 kb functions as a weak silencer in myeloid cells. The schematic layout is presented in Figure 3. Using a combination of transgenic studies, global chromatin assays and detailed molecular analyses, Leddin *et al.* (Leddin et al., 2011) presented evidence that PU.1 is regulated by a novel mechanism involving crosstalk between different *cis*-elements together with lineage-restricted autoregulation. They revealed that myeloid progenitors express C/EBP, which binds to the URE and induces activation of an enhancer at –12 kb to allow the formation of a second PU.1 autoregulatory loop and binding of additional PU.1 driven-transcription factors, such as early growth response 2 (EGR2). In this model, PU.1 regulates its expression in B cells and macrophages by differently associating with cell type-specific transcription factors at one of its *cis*regulatory elements to establish differential activity patterns at other elements.

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 247

Borras *et al.* (Borras et al., 1995) first identified PU.1 as a transcriptional repressor. They examined the ability of PU.1 to regulate the expression of the *major histocompatibility complex I-Ab* gene. Their results indicated that PU.1 represses *I-Ab* expression, possibly by binding to a PU.1 box that is located between the Y-box and the transcription start site (Borras et al., 1995). The *c-myc* promoter (–138 to +517) was found to be a target for downregulation by PU.1 in a dose-dependent manner by luciferase assays (Kihara-Negishi et al., 2001). These

*myb* promoter activity through the PU.1-binding site (Bellon et al., 1997).

complex I-Ab Promoter (Borras et al., 1995)

p16 (INK4A) Promoter (Suzuki et al., 2006)

c-Myb Promoter (Bellon et al., 1997)

MT-1s, VIM Promoter (Imoto et al., 2010)

Annexin 1 Unknown (Iseki et al., 2009)

Table 2. Suppressive gene targets regulated by PU.1

Flt3 Promoter (Inomata et al., 2006)

c-myc Promoter (Kihara-Negishi et al., 2001)

PCNA Promoter (Kihara-Negishi et al., 2001)

CD11c Promoter (Lopez-Rodriguez & Corbi, 1997)

It was reported that PU.1 negatively regulates the *CD11c integrin* gene promoter through recognition of the PU.1-binding site at the major transcriptional start site (Lopez-Rodriguez

**Gene Level of regulation References** 

expression of PU.1, without binding to a PU.1-binding site. They further showed that the *cmyc* minimal promoter including a TATA box is sufficient to exert PU.1 repression. Using luciferase assays, they demonstrated that a restricted region of PU.1 including the PEST domain is responsible for PU.1-mediated transcriptional repression. They clearly demonstrated that C-terminal amino acids 101–272 of PU.1 formed a complex with mSin3A and HDAC1 *in vivo* (Kihara-Negishi et al., 2001) (Figure 1). By affinity purification assays, Suzuki *et al.* (Suzuki et al., 2003) found that PU.1 bound directly to methyl-CpG-binding protein (MeCP) 2 via the C-terminal Ets domain. They revealed that MeCP2 was integrated into the PU.1–mSin3A–HDAC complex. The same group clearly revealed an interaction between PU.1 and DNA methyltransferase (Dnmt) 3a and Dnmt3b via the Ets domain (Suzuki et al., 2006). They further showed that the CpG sites in the *p16(INK4A)* promoter were methylated by PU.1 overexpression in NIH3T3 cells, accompanied by downregulation of *p16* (*INK4A*) gene expression (Suzuki et al., 2006). Moreover, hematopoietic precursor cells expressing PU.1 showed downregulation of the c-Myb protein levels. Transient expression of PU.1 in cotransfection assays in different cell lines resulted in inhibition of *c-*

*-actin* and *PCNA* promoters are suppressed by

(Kihara-Negishi et al., 2005)

authors also found that the *c-fos*, *SV-40*,

Major histocompatibility

c-fos, SV-40, -actin,

Other mechanisms underlying the regulation of PU.1 expression have been demonstrated. Ebralidze *et al.* (Ebralidze et al., 2008) identified naturally occurring antisense transcripts overlapping the PU.1 coding region. They demonstrated that noncoding antisense RNAs are regulated by shared evolutionarily conserved *cis*-regulatory elements, and further showed that antisense RNAs inhibit PU.1 expression by modulating RNA translation. Vigorito *et al.* (Vigorito et al., 2007) carried out gene expression profiling of activated B cells and validated PU.1 as a direct target of microRNA-155 (miR-155)-mediated inhibition. Using mouse bone marrow (BM) cells, Hu *et al.* (Hu et al., 2010) revealed HOXA9-mediated upregulation, which is frequently observed in AML, of miR-155 in fractionated bone marrow progenitors. Furthermore, they revealed that ectopic expression of miR-155 resulted in a decrease in PU.1 protein (Hu et al., 2010). These findings suggest that the expression of PU.1 is controlled both transcriptionally and post-transcriptionally.

Fig. 3. Regulation of PU.1 expression.

#### **6. PU.1 suppressive targets**

Stiedl *et al.* (Steidl et al., 2006) revealed that ~21,500 of 45,000 transcripts were expressed in PU.1-knockdown HSCs, among which 225 transcripts were downregulated and 97 were upregulated in PU.1-knockdown HSCs. These findings indicate that PU.1 not only activates but also represses a substantial number of genes in HSCs. Consistent with this notion, several PU.1 suppressive targets have been identified and reported to date. Table 2 shows a list of the known PU.1 suppressive targets.

Other mechanisms underlying the regulation of PU.1 expression have been demonstrated. Ebralidze *et al.* (Ebralidze et al., 2008) identified naturally occurring antisense transcripts overlapping the PU.1 coding region. They demonstrated that noncoding antisense RNAs are regulated by shared evolutionarily conserved *cis*-regulatory elements, and further showed that antisense RNAs inhibit PU.1 expression by modulating RNA translation. Vigorito *et al.* (Vigorito et al., 2007) carried out gene expression profiling of activated B cells and validated PU.1 as a direct target of microRNA-155 (miR-155)-mediated inhibition. Using mouse bone marrow (BM) cells, Hu *et al.* (Hu et al., 2010) revealed HOXA9-mediated upregulation, which is frequently observed in AML, of miR-155 in fractionated bone marrow progenitors. Furthermore, they revealed that ectopic expression of miR-155 resulted in a decrease in PU.1 protein (Hu et al., 2010). These findings suggest that the expression of PU.1 is controlled

Stiedl *et al.* (Steidl et al., 2006) revealed that ~21,500 of 45,000 transcripts were expressed in PU.1-knockdown HSCs, among which 225 transcripts were downregulated and 97 were upregulated in PU.1-knockdown HSCs. These findings indicate that PU.1 not only activates but also represses a substantial number of genes in HSCs. Consistent with this notion, several PU.1 suppressive targets have been identified and reported to date. Table 2 shows a

both transcriptionally and post-transcriptionally.

Fig. 3. Regulation of PU.1 expression.

list of the known PU.1 suppressive targets.

**6. PU.1 suppressive targets** 

Borras *et al.* (Borras et al., 1995) first identified PU.1 as a transcriptional repressor. They examined the ability of PU.1 to regulate the expression of the *major histocompatibility complex I-Ab* gene. Their results indicated that PU.1 represses *I-Ab* expression, possibly by binding to a PU.1 box that is located between the Y-box and the transcription start site (Borras et al., 1995). The *c-myc* promoter (–138 to +517) was found to be a target for downregulation by PU.1 in a dose-dependent manner by luciferase assays (Kihara-Negishi et al., 2001). These authors also found that the *c-fos*, *SV-40*, *-actin* and *PCNA* promoters are suppressed by expression of PU.1, without binding to a PU.1-binding site. They further showed that the *cmyc* minimal promoter including a TATA box is sufficient to exert PU.1 repression. Using luciferase assays, they demonstrated that a restricted region of PU.1 including the PEST domain is responsible for PU.1-mediated transcriptional repression. They clearly demonstrated that C-terminal amino acids 101–272 of PU.1 formed a complex with mSin3A and HDAC1 *in vivo* (Kihara-Negishi et al., 2001) (Figure 1). By affinity purification assays, Suzuki *et al.* (Suzuki et al., 2003) found that PU.1 bound directly to methyl-CpG-binding protein (MeCP) 2 via the C-terminal Ets domain. They revealed that MeCP2 was integrated into the PU.1–mSin3A–HDAC complex. The same group clearly revealed an interaction between PU.1 and DNA methyltransferase (Dnmt) 3a and Dnmt3b via the Ets domain (Suzuki et al., 2006). They further showed that the CpG sites in the *p16(INK4A)* promoter were methylated by PU.1 overexpression in NIH3T3 cells, accompanied by downregulation of *p16* (*INK4A*) gene expression (Suzuki et al., 2006). Moreover, hematopoietic precursor cells expressing PU.1 showed downregulation of the c-Myb protein levels. Transient expression of PU.1 in cotransfection assays in different cell lines resulted in inhibition of *cmyb* promoter activity through the PU.1-binding site (Bellon et al., 1997).


Table 2. Suppressive gene targets regulated by PU.1

It was reported that PU.1 negatively regulates the *CD11c integrin* gene promoter through recognition of the PU.1-binding site at the major transcriptional start site (Lopez-Rodriguez

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 249

Fig. 4. PU.1 as a transcriptional repressor. This is a schematic picture about the hypothetical formation of the PU.1 repressor complexes, because some of these informations are putative. Since the *Flt3 gene* promoter has a CpG-rich region without a functional PU.1-binding site (Inomata et al., 2006), Flt3 may be classified into the upper group, although direct binding of

enhances the transcriptional activity of PU.1 (Yamamoto et al., 1999). Of particular interest is the regulatory mechanism of the selection of the interacting partners, CBP and mSin3A-HDAC, which possess opposite functions in the regulation of gene expression. Posttranslational modification of PU.1, such as phosphorylation or acetylation, may be

In general, the expression of PU.1 is usually unaffected by most stimuli that regulate gene activation and its expression levels remain relatively constant (Yordy & Muise-Helmericks 2000). PU.1 is primarily regulated post-transcriptionally by phosphorylation (Lloberas et al., 1999; Yordy & Muise-Helmericks, 2000). PU.1 is potentially phosphorylated on five separate serine residues (Ser41, Ser45, Ser132, Ser133 and Ser148) and is an *in vitro* substrate for casein kinase II (CKII) (Lodie et al., 1997). Lodie *et al.* (Lodie et al., 1997) revealed that lipopolysaccharide stimulation induces phosphorylation of PU.1 at Ser148 (Figure 1), located within a CKII consensus motif, which increases the transactivation

PU.1 to the *Flt3 gene* promoter has not been examined.

involved in the preferential recruitment of coactivators or corepressors.

& Corbi, 1997). The inhibitory action of PU.1 on CD11c is in contrast to its positive regulatory effect on the other integrin-related *CD11b*/*CD18* gene promoters (Rosmarin et al., 1995), as described in section 4. The opposite changes in CD11b and CD11c expression that take place in extravasating and maturing blood monocytes (Prieto et al., 1994) might be explained by the differential actions of PU.1 on the regulatory regions of the *CD11b* and *CD11c* genes.

The author's group recently revealed that *MT-1* and *VIM* gene expression is directly regulated by PU.1 (Imoto et al., 2010). The MT proteins comprise a group of low molecular weight cysteine-rich intracellular proteins that can be activated by a variety of stimuli, including metal ions, oxidative stress, cytokines, glucocorticoids and growth factors (Cherian et al., 2003). MT was reported to be a potential negative regulator of apoptosis (Shimoda et al., 2003). VIM is a cytoskeletal protein that belongs to the intermediate filament family (Lazarides, 1982). VIM suppresses the apoptotic effect of chemotherapeutic treatment (Belichenko et al., 2001). These findings imply that induced expression of MT, as well as VIM, may function in anti-apoptosis mechanisms in leukemia cells. We demonstrated that MeCP2 and PU.1 bind to the CpG-rich regions in the *MT-1* and *VIM* promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites are tightly correlated with the PU.1 expression levels (Imoto et al., 2010). Importantly, the mRNA expression levels of the *MT-1* and *VIM* genes were inversely correlated with PU.1 mRNA expression in 43 primary AML specimens (MT-1G: R=–0.50, p<0.001; MT-1A: R=–0.58, p<0.0005; VIM: R=–0.39, p<0.01) (Imoto et al., 2010).

The author's group also demonstrated an inverse correlation between the mRNA expression of Annexin 1 and PU.1 in AML specimens (Iseki et al., 2009). In addition, the author and colleagues have reported that the *Flt3* gene promoter is suppressed by overexpression of PU.1, and that there is a significant negative correlation between FLT3 and PU.1 (r=–0.43, p<0.05) (Inomata et al., 2006). Therefore, PU.1 downregulation, which is important in the pathogenesis of AML as described in section 8, may impact on the overexpression of FLT3, which is frequently observed in AML, leading to activation of the FLT3 pathway (Ozeki et al., 2004; Takahashi, 2011).

Together with these findings, PU.1 represses several genes through its epigenetic activity under certain circumstances. The repression of PU.1 is directed in at least two ways. The author's group demonstrated that PU.1 binds to the methylated CpG-rich region with MeCP2, where no consensus PU.1-binding sites were found (Imoto et al., 2010). The other way is direct binding of PU.1 to PU.1-binding sites, as demonstrated in the promoters of the *CD11c*, *p16 (INK4A)*, *major histocompatibility complex I-Ab, c-myb* and other genes (Borras et al., 1995; Lopez-Rodriguez & Corbi, 1997; Suzuki et al., 2006). In addition, some suppressive target genes of PU.1 may be indirect effects (e.g. Iseki et al., 2009), because direct binding to their promoters has not been demonstrated. A schematic layout of the complex of PU.1 and its corepressors binding to these promoters is shown in Figure 4.

#### **7. Putative regulatory mechanisms for PU.1 as a transcriptional activator or repressor**

As mentioned above, Kihara-Negishi *et al.* (Kihara-Negishi et al., 2001) showed a direct interaction of PU.1 with mSin3A. On the other hand, the same group previously demonstrated a direct interaction of PU.1 with the transcriptional coactivator CBP, which

& Corbi, 1997). The inhibitory action of PU.1 on CD11c is in contrast to its positive regulatory effect on the other integrin-related *CD11b*/*CD18* gene promoters (Rosmarin et al., 1995), as described in section 4. The opposite changes in CD11b and CD11c expression that take place in extravasating and maturing blood monocytes (Prieto et al., 1994) might be explained by the differential actions of PU.1 on the regulatory regions of the *CD11b* and

The author's group recently revealed that *MT-1* and *VIM* gene expression is directly regulated by PU.1 (Imoto et al., 2010). The MT proteins comprise a group of low molecular weight cysteine-rich intracellular proteins that can be activated by a variety of stimuli, including metal ions, oxidative stress, cytokines, glucocorticoids and growth factors (Cherian et al., 2003). MT was reported to be a potential negative regulator of apoptosis (Shimoda et al., 2003). VIM is a cytoskeletal protein that belongs to the intermediate filament family (Lazarides, 1982). VIM suppresses the apoptotic effect of chemotherapeutic treatment (Belichenko et al., 2001). These findings imply that induced expression of MT, as well as VIM, may function in anti-apoptosis mechanisms in leukemia cells. We demonstrated that MeCP2 and PU.1 bind to the CpG-rich regions in the *MT-1* and *VIM* promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites are tightly correlated with the PU.1 expression levels (Imoto et al., 2010). Importantly, the mRNA expression levels of the *MT-1* and *VIM* genes were inversely correlated with PU.1 mRNA expression in 43 primary AML specimens (MT-1G: R=–0.50, p<0.001; MT-1A: R=–0.58, p<0.0005; VIM: R=–0.39, p<0.01) (Imoto et al., 2010). The author's group also demonstrated an inverse correlation between the mRNA expression of Annexin 1 and PU.1 in AML specimens (Iseki et al., 2009). In addition, the author and colleagues have reported that the *Flt3* gene promoter is suppressed by overexpression of PU.1, and that there is a significant negative correlation between FLT3 and PU.1 (r=–0.43, p<0.05) (Inomata et al., 2006). Therefore, PU.1 downregulation, which is important in the pathogenesis of AML as described in section 8, may impact on the overexpression of FLT3, which is frequently observed in AML, leading to activation of the FLT3 pathway (Ozeki et

Together with these findings, PU.1 represses several genes through its epigenetic activity under certain circumstances. The repression of PU.1 is directed in at least two ways. The author's group demonstrated that PU.1 binds to the methylated CpG-rich region with MeCP2, where no consensus PU.1-binding sites were found (Imoto et al., 2010). The other way is direct binding of PU.1 to PU.1-binding sites, as demonstrated in the promoters of the *CD11c*, *p16 (INK4A)*, *major histocompatibility complex I-Ab, c-myb* and other genes (Borras et al., 1995; Lopez-Rodriguez & Corbi, 1997; Suzuki et al., 2006). In addition, some suppressive target genes of PU.1 may be indirect effects (e.g. Iseki et al., 2009), because direct binding to their promoters has not been demonstrated. A schematic layout of the complex of PU.1 and

**7. Putative regulatory mechanisms for PU.1 as a transcriptional activator or** 

As mentioned above, Kihara-Negishi *et al.* (Kihara-Negishi et al., 2001) showed a direct interaction of PU.1 with mSin3A. On the other hand, the same group previously demonstrated a direct interaction of PU.1 with the transcriptional coactivator CBP, which

its corepressors binding to these promoters is shown in Figure 4.

*CD11c* genes.

al., 2004; Takahashi, 2011).

**repressor** 

Fig. 4. PU.1 as a transcriptional repressor. This is a schematic picture about the hypothetical formation of the PU.1 repressor complexes, because some of these informations are putative. Since the *Flt3 gene* promoter has a CpG-rich region without a functional PU.1-binding site (Inomata et al., 2006), Flt3 may be classified into the upper group, although direct binding of PU.1 to the *Flt3 gene* promoter has not been examined.

enhances the transcriptional activity of PU.1 (Yamamoto et al., 1999). Of particular interest is the regulatory mechanism of the selection of the interacting partners, CBP and mSin3A-HDAC, which possess opposite functions in the regulation of gene expression. Posttranslational modification of PU.1, such as phosphorylation or acetylation, may be involved in the preferential recruitment of coactivators or corepressors.

In general, the expression of PU.1 is usually unaffected by most stimuli that regulate gene activation and its expression levels remain relatively constant (Yordy & Muise-Helmericks 2000). PU.1 is primarily regulated post-transcriptionally by phosphorylation (Lloberas et al., 1999; Yordy & Muise-Helmericks, 2000). PU.1 is potentially phosphorylated on five separate serine residues (Ser41, Ser45, Ser132, Ser133 and Ser148) and is an *in vitro* substrate for casein kinase II (CKII) (Lodie et al., 1997). Lodie *et al.* (Lodie et al., 1997) revealed that lipopolysaccharide stimulation induces phosphorylation of PU.1 at Ser148 (Figure 1), located within a CKII consensus motif, which increases the transactivation

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 251

For many years, it has been known that irradiated mice frequently develop AML after a long latent period, and that nearly all of their tumors contain an interstitial deletion in one copy of chromosome 2 that includes the *PU.1* gene (Hayata et al., 1983; Silver et al., 1999; Trakhtenbrot et al., 1988). Subsequent studies revealed that mice with -irradiation-induced myeloid leukemia acquired both a deletion in one copy of chromosome 2 and a point mutation in the ETS domain of PU.1, which impaired DNA binding of the other PU.1 allele (Cook et al., 2004; Suraweera et al., 2005). These single amino acid substitutions were shown to alter the function of PU.1 by reducing its transactivation activity. Grisolano *et al* (Grisolano et al., 1997) previously showed that 10–20% of transgenic mice expressing PML-RAR in their early myeloid cells develop acute promyelocytic leukemia (APL) after a long latent period. The penetrance of APL development increases substantially when RAR-PML is coexpressed in early myeloid cells (Pollock et al., 1999) or when young mice are sublethally irradiated (Walter et al., 2004). In both scenarios, the vast majority of APL tumors contain a large (>20 Mb) interstitial deletion in one copy of chromosome 2 that invariably includes the *PU.1* gene (Walter et al., 2004; Zimonjic et al., 2000). These data strongly suggest that PU.1 acts in a similar manner to a tumor suppressor in myeloid progenitors, and that deletion of one allele, followed by a reduction in the function of the other allele, is relevant for AML pathogenesis. Indeed, PU.1 is expressed at low levels in

PU.1 expression and/or function appear to be suppressed by several leukemogenic fusion products, such as RUNX1-ETO (Vangala et al., 2003), FLT3-ITD (Mizuki et al., 2003) and PML-RAR (Mueller et al., 2006a). Following these initial reports of suppressed PU.1 function in human AML, the leukemic potential of tumor progenitors with critically reduced PU.1 function has been illustrated by a series of mouse models. The level of PU.1 expression is critical for specifying cell fate and, if perturbed, even modest decreases in PU.1 can lead to leukemias and lymphomas (DeKoter & Singh, 2000a; Rosenbauer et al., 2004). Mice with an engineered deletion of the URE of PU.1 show decreased PU.1 expression and subsequently develop AML, through suppression of the AP-1 transcription factors JunB and c-Jun, by blocking differentiation and increasing self-renewal as described in the previous section. However, while these observations indicate that low but detectable amounts of PU.1 might favor the malignant potential of leukemic cells, this view was challenged by a report

suggesting that complete loss of PU.1 can also lead to AML (Metcalf et al., 2006).

As described in section 5, the URE is important for the regulation of PU.1. Several reports have described associations between genetic abnormalities of this region and AML biology. In an analysis of 209 AML patient specimens, Steidl *et al.* (Steidl et al., 2007) revealed that a distal single nucleotide polymorphism (SNP) alters the long-range regulation of the *PU.1* gene in AML. The homozygous SNP within a highly conserved distal enhancer element in humans, which is 2.4-fold more frequent in AML with a complex karyotype (24.3%) than in AML with a normal karyotype (10.0%), leads to decreased enhancer activity and reduces PU.1 expression in myeloid progenitors in a development-dependent manner (Steidl et al., 2007). Bonadies *et al.* (Bonadies et al., 2010a) examined the URE sequence in 120 AML patients. They identified one AML patient with heterozygous loss of the entire URE sequence with markedly reduced PU.1 expression. The link between PU.1 URE mutations and AML pathogenesis might be explained by another model. The same group also identified NF-B as an activator of PU.1 expression through a novel site within the upstream

**8. Role of PU.1 in the pathogenesis of AML** 

most cases of human AML (Steidl et al., 2006).

function of PU.1. PU.1 is also phosphorylated at Ser142 by stimulation of IL-3-induced p38 MAPK-mediated pathways (Wang et al., 2003). Mutation of Ser142 to alanine attenuates the IL-3-enhanced transactivation activity of PU.1. In contrast, Breig *et al.* (Breig et al., 2010) recently described that Ser41, but not Ser148, is necessary for Spi-1/PU.1 mediated repression of hemoglobin expression in Friend erythroleukemia cells. Phosphorylation of critical residues in PU.1 may switch the function of this transcription factor from an activator to a repressor. Kihara-Negishi *et al.* (Kihara-Negishi et al., 2005) examined whether acetylation regulates the physical and functional activities of PU.1 in MEL cells. They found that PU.1 is acetylated *in vivo* and that its repressor activity is reduced when the putative acetylation motifs in the Ets domain are mutated. The mutant cooperates with the coactivator CBP, similar to the case for wild-type PU.1, but shows insufficient cooperation with the corepressor mSin3A, which binds to the Ets domain of PU.1. These observations are summarized in Table 3.

The modifications of each functional domain (as depicted in Figure 1), which are required for transcriptional activation or repression, may result in the dissociation of cofactors, leading to changes in the transcriptional ability of PU.1.

However, a question still remains. As described in the previous section, in a same-cell population like HSCs (Steidl et al., 2006), some genes are suppressed while other genes are upregulated by PU.1. How are these genes differently regulated even in the same cells? It is possible that the context of the promoter may be important, and that promoter-specific cofactors may modify PU.1 and regulate the activity of this protein. These mechanisms remain to be elucidated in future studies.


Table 3. List of the post-translational modifications of PU.1

#### **8. Role of PU.1 in the pathogenesis of AML**

250 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

function of PU.1. PU.1 is also phosphorylated at Ser142 by stimulation of IL-3-induced p38 MAPK-mediated pathways (Wang et al., 2003). Mutation of Ser142 to alanine attenuates the IL-3-enhanced transactivation activity of PU.1. In contrast, Breig *et al.* (Breig et al., 2010) recently described that Ser41, but not Ser148, is necessary for Spi-1/PU.1 mediated repression of hemoglobin expression in Friend erythroleukemia cells. Phosphorylation of critical residues in PU.1 may switch the function of this transcription factor from an activator to a repressor. Kihara-Negishi *et al.* (Kihara-Negishi et al., 2005) examined whether acetylation regulates the physical and functional activities of PU.1 in MEL cells. They found that PU.1 is acetylated *in vivo* and that its repressor activity is reduced when the putative acetylation motifs in the Ets domain are mutated. The mutant cooperates with the coactivator CBP, similar to the case for wild-type PU.1, but shows insufficient cooperation with the corepressor mSin3A, which binds to the Ets domain of

The modifications of each functional domain (as depicted in Figure 1), which are required for transcriptional activation or repression, may result in the dissociation of cofactors,

However, a question still remains. As described in the previous section, in a same-cell population like HSCs (Steidl et al., 2006), some genes are suppressed while other genes are upregulated by PU.1. How are these genes differently regulated even in the same cells? It is possible that the context of the promoter may be important, and that promoter-specific cofactors may modify PU.1 and regulate the activity of this protein. These mechanisms

> Casein Kinase II

> p38MAP kinase

PI3 kinase /Akt

Unknown

**Signaling Effect Ref.** 

148 increases transactivation function.

142 increases

resulted in the stimulation of mcl-1 gene transcription.

site impairs its repression activity.

Phosphorylation of Ser

Phosphorylation of Ser

transactivation of PU.1,

Ser 41 is responsible for the PU.1 mediated repression, and

phosphorylation of this

The repressor activity of PU.1 was impaired by mutations of both two acetylation motifs (Lodie et al., 1997)

(Wang et al., 2003)

(Breig et al., 2010)

(Kihara -Negishi et al., 2005)

PU.1. These observations are summarized in Table 3.

leading to changes in the transcriptional ability of PU.1.

**Site Cells Responsible** 

Raw264.7 (macrophage)

Ba/F3 (IL-3 dependent pro B

leukemia cells

Zinc inducible PU.1 transgenic MELB8/3 cells, 293T cells

cells

cells)

Ser 41 Friend erythro-

Table 3. List of the post-translational modifications of PU.1

remain to be elucidated in future studies.

Ser41, Ser45, Ser132, Ser133 and Ser 148

Ser 142

Lysine


For many years, it has been known that irradiated mice frequently develop AML after a long latent period, and that nearly all of their tumors contain an interstitial deletion in one copy of chromosome 2 that includes the *PU.1* gene (Hayata et al., 1983; Silver et al., 1999; Trakhtenbrot et al., 1988). Subsequent studies revealed that mice with -irradiation-induced myeloid leukemia acquired both a deletion in one copy of chromosome 2 and a point mutation in the ETS domain of PU.1, which impaired DNA binding of the other PU.1 allele (Cook et al., 2004; Suraweera et al., 2005). These single amino acid substitutions were shown to alter the function of PU.1 by reducing its transactivation activity. Grisolano *et al* (Grisolano et al., 1997) previously showed that 10–20% of transgenic mice expressing PML-RAR in their early myeloid cells develop acute promyelocytic leukemia (APL) after a long latent period. The penetrance of APL development increases substantially when RAR-PML is coexpressed in early myeloid cells (Pollock et al., 1999) or when young mice are sublethally irradiated (Walter et al., 2004). In both scenarios, the vast majority of APL tumors contain a large (>20 Mb) interstitial deletion in one copy of chromosome 2 that invariably includes the *PU.1* gene (Walter et al., 2004; Zimonjic et al., 2000). These data strongly suggest that PU.1 acts in a similar manner to a tumor suppressor in myeloid progenitors, and that deletion of one allele, followed by a reduction in the function of the other allele, is relevant for AML pathogenesis. Indeed, PU.1 is expressed at low levels in most cases of human AML (Steidl et al., 2006).

PU.1 expression and/or function appear to be suppressed by several leukemogenic fusion products, such as RUNX1-ETO (Vangala et al., 2003), FLT3-ITD (Mizuki et al., 2003) and PML-RAR (Mueller et al., 2006a). Following these initial reports of suppressed PU.1 function in human AML, the leukemic potential of tumor progenitors with critically reduced PU.1 function has been illustrated by a series of mouse models. The level of PU.1 expression is critical for specifying cell fate and, if perturbed, even modest decreases in PU.1 can lead to leukemias and lymphomas (DeKoter & Singh, 2000a; Rosenbauer et al., 2004). Mice with an engineered deletion of the URE of PU.1 show decreased PU.1 expression and subsequently develop AML, through suppression of the AP-1 transcription factors JunB and c-Jun, by blocking differentiation and increasing self-renewal as described in the previous section. However, while these observations indicate that low but detectable amounts of PU.1 might favor the malignant potential of leukemic cells, this view was challenged by a report suggesting that complete loss of PU.1 can also lead to AML (Metcalf et al., 2006).

As described in section 5, the URE is important for the regulation of PU.1. Several reports have described associations between genetic abnormalities of this region and AML biology. In an analysis of 209 AML patient specimens, Steidl *et al.* (Steidl et al., 2007) revealed that a distal single nucleotide polymorphism (SNP) alters the long-range regulation of the *PU.1* gene in AML. The homozygous SNP within a highly conserved distal enhancer element in humans, which is 2.4-fold more frequent in AML with a complex karyotype (24.3%) than in AML with a normal karyotype (10.0%), leads to decreased enhancer activity and reduces PU.1 expression in myeloid progenitors in a development-dependent manner (Steidl et al., 2007). Bonadies *et al.* (Bonadies et al., 2010a) examined the URE sequence in 120 AML patients. They identified one AML patient with heterozygous loss of the entire URE sequence with markedly reduced PU.1 expression. The link between PU.1 URE mutations and AML pathogenesis might be explained by another model. The same group also identified NF-B as an activator of PU.1 expression through a novel site within the upstream

PU.1, a Versatile Transcription Factor and a Suppressor of Myeloid Leukemia 253

part, by functional impairment of PU.1 epigenetic activities as a transcriptional repressor, mainly through downregulation of its expression. It is possible that this may also play an

Fig. 5. Putative mechanisms of deregulated PU.1 repressor activity in AML. A, Normal cells. B, AML cells. A reduction in PU.1 expression (left panel), as well as mutations in Dnmt3a

Collectively, not only deregulation of proper myeloid-specific gene expression by downregulation of PU.1, but also insufficient recruitment of such epigenetic factors may play important roles in leukemia pathogenesis. Further clarification of the functions of this versatile transcription factor, with a particular focus on its function as a transcriptional

I thank Dr. Alison Sherwin (Edanz Editing) for helpful advice on the English language and editing of this manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (No. 23590687) from the Ministry of Education, Science and Culture, Japan, Mitsubishi Pharma Research Foundation, Nakayama Foundation for Human Science, Takeda Science Foundation, and a foundation from Kitasato University School of Allied

Ahne, B. & Stratling, W.H. (1994) Characterization of a myeloid-specific enhancer of the

chicken lysozyme gene. Major role for an Ets transcription factor-binding site. *J Biol* 

Health Sciences (Grant-in-Aid for Research Project, No. 2010-1004, No. 2011-1001).

(right panel), result in aberrant target gene expression, which plays a role in the

repressor, may reveal the molecular biology of myeloid leukemia.

*Chem,* Vol. 269, No.27, (July 1994), pp. 17794-801.

important role in AML pathogenesis (Figure 5).

pathogenesis of AML.

**10. Acknowledgments** 

**11. References** 

URE (Bonadies et al., 2010b). They found sequence variations of this particular NF-B site in four of 120 AML patients. These variant NF-B sequences failed to mediate activation of PU.1, and AML patients with such variant sequences had suppressed PU.1 mRNA expression. These findings suggest that the change of a single base pair in a distal element critically affects the regulation of the tumor suppressor gene *PU.1*, thereby contributing to the development of AML.

Mutations of PU.1 have previously been examined by several groups. Mueller *et al*. (Mueller et al., 2002; Mueller et al., 2003) identified 10 mutant alleles of the *PU.1* gene in 9 of 126 AML patients. However, these findings could not be confirmed in two subsequent studies. Vegesna *et al.* (Vegesna et al., 2002) analyzed 381 samples of hematopoietic and solid malignancies, including 60 cases of *de novo* AML and 60 cases of myelodysplastic syndromes (MDS), and were not able to identify *PU.1* coding region mutations. Similarly, Lamandin *et al*. (Lamandin et al., 2002) failed to detect *PU.1* gene mutations in 77 primary AML samples. Collectively, direct genetic inactivation of the *PU.1* gene in myeloid leukemia has only been found in very rare cases. Although there is no doubt that a reduction in PU.1 expression plays a very important role in the pathogenesis of AML, its mutations in human myeloid leukemia biology remain unclear.

#### **9. Perspectives and conclusions**

The author has summarized the recent advances in the molecular biology of the PU.1 transcription factor, with a focus on its functions as both a transcriptional activator and repressor. In addition, the role of PU.1 in AML biology has been described.

In 2009, inactivating mutations of the *Ten-Eleven-Translocation oncogene family member 2*  (*TET2*) gene were identified in about 15% of patients with various myeloid malignancies, such as MDS (19%), myeloproliferative disorders (12%), secondary AML (24%) and chronic myelomonocytic leukemia (22%) (Delhommeau et al., 2009). TET2 can convert 5 methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) (Ko et al., 2010), which is hypothesized to be an intermediate in the demethylation of DNA. Ko *et al.* (Ko et al., 2010) demonstrated that disruption of TET2 enzymatic activity favors myeloid tumorigenesis.

Consistent with this observation, it was reported that Dnmt3a mutations were detected in 62 of 281 (22.1%) AML patients (Ley et al., 2010). These mutations were highly enriched in a group of patients with an intermediate-risk cytogenetic profile, as well as in FLT3 receptor mutations (25 of 61 (41.0%) patients; p<0.003) (Ley et al., 2010). Walter *et al.* (Walter et al., 2011) also described relatively frequent mutations of Dnmt3a in MDS. They performed sequencing in patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12 of 150 (8.0%) patients. Yan *et al.* (Yan et al., 2011) discovered mutations in DNMT3a in 23 of 112 (20.5%) cases with the M5 subtype of AML. The DNMT mutants showed reduced enzymatic activity or aberrant affinity for histone H3 *in vitro*. Notably, there were alterations in the DNA methylation patterns and/or gene expression profiles, such as *HOXB* genes, in samples with DNMT3a mutations compared with those without such mutations (Yan et al., 2011). These recent findings strongly suggest a link between recurrent genetic alterations and aberrant DNA methylation status in myeloid malignancies.

As shown in Figures 1 and 4, PU.1 interacts with DNMT3a and DNMT3b (Suzuki et al., 2006) and suppresses its target genes through Dnmt activity (Imoto et al., 2010; Suzuki et al., 2006). Since the expression of DNMT3s is constant in AML specimens (Ley et al., 2010), it is tempting to speculate that DNMT3a mutations exert their leukemogenic potential, at least in part, by functional impairment of PU.1 epigenetic activities as a transcriptional repressor, mainly through downregulation of its expression. It is possible that this may also play an important role in AML pathogenesis (Figure 5).

Fig. 5. Putative mechanisms of deregulated PU.1 repressor activity in AML. A, Normal cells. B, AML cells. A reduction in PU.1 expression (left panel), as well as mutations in Dnmt3a (right panel), result in aberrant target gene expression, which plays a role in the pathogenesis of AML.

Collectively, not only deregulation of proper myeloid-specific gene expression by downregulation of PU.1, but also insufficient recruitment of such epigenetic factors may play important roles in leukemia pathogenesis. Further clarification of the functions of this versatile transcription factor, with a particular focus on its function as a transcriptional repressor, may reveal the molecular biology of myeloid leukemia.

#### **10. Acknowledgments**

252 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

URE (Bonadies et al., 2010b). They found sequence variations of this particular NF-B site in four of 120 AML patients. These variant NF-B sequences failed to mediate activation of PU.1, and AML patients with such variant sequences had suppressed PU.1 mRNA expression. These findings suggest that the change of a single base pair in a distal element critically affects the regulation of the tumor suppressor gene *PU.1*, thereby contributing to

Mutations of PU.1 have previously been examined by several groups. Mueller *et al*. (Mueller et al., 2002; Mueller et al., 2003) identified 10 mutant alleles of the *PU.1* gene in 9 of 126 AML patients. However, these findings could not be confirmed in two subsequent studies. Vegesna *et al.* (Vegesna et al., 2002) analyzed 381 samples of hematopoietic and solid malignancies, including 60 cases of *de novo* AML and 60 cases of myelodysplastic syndromes (MDS), and were not able to identify *PU.1* coding region mutations. Similarly, Lamandin *et al*. (Lamandin et al., 2002) failed to detect *PU.1* gene mutations in 77 primary AML samples. Collectively, direct genetic inactivation of the *PU.1* gene in myeloid leukemia has only been found in very rare cases. Although there is no doubt that a reduction in PU.1 expression plays a very important role in the pathogenesis of AML, its mutations in human myeloid

The author has summarized the recent advances in the molecular biology of the PU.1 transcription factor, with a focus on its functions as both a transcriptional activator and

In 2009, inactivating mutations of the *Ten-Eleven-Translocation oncogene family member 2*  (*TET2*) gene were identified in about 15% of patients with various myeloid malignancies, such as MDS (19%), myeloproliferative disorders (12%), secondary AML (24%) and chronic myelomonocytic leukemia (22%) (Delhommeau et al., 2009). TET2 can convert 5 methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) (Ko et al., 2010), which is hypothesized to be an intermediate in the demethylation of DNA. Ko *et al.* (Ko et al., 2010) demonstrated that disruption of TET2 enzymatic activity favors myeloid tumorigenesis. Consistent with this observation, it was reported that Dnmt3a mutations were detected in 62 of 281 (22.1%) AML patients (Ley et al., 2010). These mutations were highly enriched in a group of patients with an intermediate-risk cytogenetic profile, as well as in FLT3 receptor mutations (25 of 61 (41.0%) patients; p<0.003) (Ley et al., 2010). Walter *et al.* (Walter et al., 2011) also described relatively frequent mutations of Dnmt3a in MDS. They performed sequencing in patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12 of 150 (8.0%) patients. Yan *et al.* (Yan et al., 2011) discovered mutations in DNMT3a in 23 of 112 (20.5%) cases with the M5 subtype of AML. The DNMT mutants showed reduced enzymatic activity or aberrant affinity for histone H3 *in vitro*. Notably, there were alterations in the DNA methylation patterns and/or gene expression profiles, such as *HOXB* genes, in samples with DNMT3a mutations compared with those without such mutations (Yan et al., 2011). These recent findings strongly suggest a link between recurrent genetic alterations

As shown in Figures 1 and 4, PU.1 interacts with DNMT3a and DNMT3b (Suzuki et al., 2006) and suppresses its target genes through Dnmt activity (Imoto et al., 2010; Suzuki et al., 2006). Since the expression of DNMT3s is constant in AML specimens (Ley et al., 2010), it is tempting to speculate that DNMT3a mutations exert their leukemogenic potential, at least in

repressor. In addition, the role of PU.1 in AML biology has been described.

and aberrant DNA methylation status in myeloid malignancies.

the development of AML.

leukemia biology remain unclear.

**9. Perspectives and conclusions** 

I thank Dr. Alison Sherwin (Edanz Editing) for helpful advice on the English language and editing of this manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (No. 23590687) from the Ministry of Education, Science and Culture, Japan, Mitsubishi Pharma Research Foundation, Nakayama Foundation for Human Science, Takeda Science Foundation, and a foundation from Kitasato University School of Allied Health Sciences (Grant-in-Aid for Research Project, No. 2010-1004, No. 2011-1001).

#### **11. References**

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N. & Oikawa, T. (2006) Site-specific DNA methylation by a complex of PU.1 and

association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression. *Oncogene*, Vol. 22, No.54, (November

& Nakamura, M. (1998) PU.1 as an essential activator for the expression of gp91(phox) gene in human peripheral neutrophils, monocytes, and B lymphocytes.

myeloid leukemia: biology and therapeutic implications. *J Hematol Oncol*, Vol. 4,

chromosome 2 is an early event in the development of radiation-induced myeloid


**14** 

*Italy* 

**Vav1: A Key Player in Agonist-Induced** 

**Differentiation of Promyelocytes from** 

Valeria Bertagnolo, Federica Brugnoli and Silvano Capitani

Acute promyelocytic leukemia (APL) is the M3 subtype of acute myeloid leukemia (AML), characterized by aberrant hyperproliferation of progenitors originally committed to terminal differentiation into granulocytes but blocked at the promyelocytic stage. Although clinical studies have introduced treatments employing arsenic trioxide, anthracyclines and anti-CD33 monoclonal antibodies, all-*trans* retinoic acid (ATRA)-based therapy represents, until today, the standard cure of APL patients (Lo-Coco & Ammatuna, 2006; Tallman, 2007). ATRA treatment of APL constitutes, at present, the only example of successful differentiation therapy of a human cancer, in which tumor cells are induced to complete their maturation to neutrophils. Studies on both APL blasts and APL-derived cell lines have elucidated that ATRA acts throughout a complex network that includes the degradation of the PML/RAR fusion protein and the activation of RAR-mediated gene transcription (Breitman et al., 1980; Lanotte et al., 1991; Yang et al., 2003). In addition, it has been reported that ATRA- and phorbol 12-myristate 13-acetate (PMA)-mediated differentiation of human myeloid leukemia cell lines results in changes of their sensitivity to chemotherapeutic drugs, suggesting that advantages in the cure of APL and other malignancies could be obtained by combining differentiating agents and conventional anticancer drugs (Jasek et al., 2008;

Even if the mechanism by which ATRA interacts with its receptor located on specific DNA sequences is well known, the events mediated by the ATRA target genes, able to elicit the integrated signaling networks that promote maturation of tumoral promyelocytes, have not been fully clarified and are currently under study to also identify specific molecular targets

One of the proteins up-regulated by ATRA in APL-derived cells and that resulted chiefly involved in the maturation program of tumoral promyelocytes is Vav1, the sole member of the Vav family of proteins physiologically expressed only in haematopoietic cells, where it works as an important signal transducer in immune response (Katzav, 2009; Tybulewicz, 2005). Relevant insights into the function of Vav1 in hematopoietic cells have been provided by studies with knockout mice, demonstrating that the targeted down-modulation of Vav1 compromises maturation of both lymphoid and myeloid cells (Zhang et al., 1994). In

**1. Introduction** 

Kogan, 2009; Nasr et al., 2008).

for new therapies of APL.

**Acute Myeloid Leukemia (APL)** 

*University of Ferrara, Section of Human Anatomy, Department of Morphology and Embryology,* 

GATA proteins repress PU.1. *Proc Natl Acad Sci U S A*, Vol. 96, No.15, (July 1999), pp. 8705-10.

Zimonjic, D.B.; Pollock, J.L.; Westervelt, P.; Popescu, N.C. & Ley, T.J. (2000) Acquired, nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice. *Proc Natl Acad Sci U S A*, Vol. 97, No.24, (November 2000), pp. 13306-11.

### **Vav1: A Key Player in Agonist-Induced Differentiation of Promyelocytes from Acute Myeloid Leukemia (APL)**

Valeria Bertagnolo, Federica Brugnoli and Silvano Capitani *University of Ferrara, Section of Human Anatomy, Department of Morphology and Embryology, Italy* 

#### **1. Introduction**

262 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Zimonjic, D.B.; Pollock, J.L.; Westervelt, P.; Popescu, N.C. & Ley, T.J. (2000) Acquired,

pp. 8705-10.

(November 2000), pp. 13306-11.

GATA proteins repress PU.1. *Proc Natl Acad Sci U S A*, Vol. 96, No.15, (July 1999),

nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice. *Proc Natl Acad Sci U S A*, Vol. 97, No.24,

> Acute promyelocytic leukemia (APL) is the M3 subtype of acute myeloid leukemia (AML), characterized by aberrant hyperproliferation of progenitors originally committed to terminal differentiation into granulocytes but blocked at the promyelocytic stage. Although clinical studies have introduced treatments employing arsenic trioxide, anthracyclines and anti-CD33 monoclonal antibodies, all-*trans* retinoic acid (ATRA)-based therapy represents, until today, the standard cure of APL patients (Lo-Coco & Ammatuna, 2006; Tallman, 2007). ATRA treatment of APL constitutes, at present, the only example of successful differentiation therapy of a human cancer, in which tumor cells are induced to complete their maturation to neutrophils. Studies on both APL blasts and APL-derived cell lines have elucidated that ATRA acts throughout a complex network that includes the degradation of the PML/RAR fusion protein and the activation of RAR-mediated gene transcription (Breitman et al., 1980; Lanotte et al., 1991; Yang et al., 2003). In addition, it has been reported that ATRA- and phorbol 12-myristate 13-acetate (PMA)-mediated differentiation of human myeloid leukemia cell lines results in changes of their sensitivity to chemotherapeutic drugs, suggesting that advantages in the cure of APL and other malignancies could be obtained by combining differentiating agents and conventional anticancer drugs (Jasek et al., 2008; Kogan, 2009; Nasr et al., 2008).

> Even if the mechanism by which ATRA interacts with its receptor located on specific DNA sequences is well known, the events mediated by the ATRA target genes, able to elicit the integrated signaling networks that promote maturation of tumoral promyelocytes, have not been fully clarified and are currently under study to also identify specific molecular targets for new therapies of APL.

> One of the proteins up-regulated by ATRA in APL-derived cells and that resulted chiefly involved in the maturation program of tumoral promyelocytes is Vav1, the sole member of the Vav family of proteins physiologically expressed only in haematopoietic cells, where it works as an important signal transducer in immune response (Katzav, 2009; Tybulewicz, 2005). Relevant insights into the function of Vav1 in hematopoietic cells have been provided by studies with knockout mice, demonstrating that the targeted down-modulation of Vav1 compromises maturation of both lymphoid and myeloid cells (Zhang et al., 1994). In

Vav1: A Key Player in Agonist-Induced Differentiation of

acquire the mature phenotype of differentiated neutrophils.

morphology (Bertagnolo et al, 2005).

**2.1 Tyrosine phosphorylation of Vav1** 

2005, 2008).

Promyelocytes from Acute Myeloid Leukemia (APL) 265

neutrophils. Treatments with differentiating doses of ATRA induce a significant increase of Vav1 expression in primary blasts obtained from the bone marrow of APL patients as well as in the APL-derived cell lines HL-60 and NB4. A similar increase of Vav1 is observed when normal CD34+ hematopoietic progenitors are treated with a cytokine cocktail promoting granulocytic differentiation, clearly indicating that an adequate expression of Vav1 has to be achieved along with neutrophil maturation of both normal precursors and poorly differentiated neoplastic cells (Bertagnolo et al., 2005). These evidence, obtained in a variety of normal and neoplastic cells under different experimental conditions, ascribe to Vav1 the potential role of a ubiquitous key player in the path leading myeloid precursors to

The issue of whether the increase of Vav1 observed in differentiation of tumoral promyelocytes is merely designed to the function of the protein in mature cells or, more intriguingly, it is functionally relevant to the maturation mechanism, has been addressed by studies in which the expression of Vav1 was forcedly modulated. The experiments have been performed in ATRA treated HL-60 and NB4 cells, which are blocked at different levels of granulocytic differentiation, and thus constitute models well suited to better understand the role of Vav1 in the maturation process. As a consequence of Vav1 over-expression during ATRA treatment of both cell lines, the expression of the myeloid surface marker CD11b increases, indicating that Vav1 supports the role of ATRA in regulating the maturation process. On the other hand, the sole over-expression of Vav1 is capable to significantly induce the expression of CD11b only in HL-60 cells, that, compared to NB4 cells, are blocked to a less differentiated stage (Bertagnolo et al., 2005). This suggests the existence of a direct and ATRA-independent role of Vav1 in regulating the expression of CD11b, at least in cells that are blocked at early stages of the neutrophilic maturation. Neutrophils radically change in shape during development and functional life (Sanchez & Wangh, 1999). Accordingly, profound rearrangements of the cell morphology take place throughout differentiation of myeloid precursors along the granulocytic lineage, and the nucleus is the cell compartment that undergoes the biggest architectural changes by a mechanism still largely unknown. Modifications of the nuclear shape constitute one of the markers of neutrophil maturation of tumoral promyelocytes and are particularly evident after treatment with ATRA of HL-60 cells, according to the notion that this cell type is blocked at an early stage of maturation. On the other hand, the sole over-expression of Vav1 is unable to induce nuclear modifications in both HL-60 and NB4 cells, indicating that other ATRA-induced events are required to regulate the maturation-related rearrangements of cell

The use of siRNAs specific for Vav1 unequivocally demonstrates that Vav1 is not dispensable for the progression of tumoral promyelocytes along the granulocytic lineage. In fact, the down-modulation of Vav1 expression during ATRA treatment of both HL-60 and NB4 cells counteracts the agonist-induced increase of CD11b expression and prevents the maturation-related modifications of cell/nucleus morphology, definitely assigning to Vav1 a crucial role in regulating phenotypical maturation of APL-derived cells (Bertagnolo et al,

In parallel with the increase of Vav1 expression, ATRA treatment of HL-60 and NB4 cells also induces Vav1 tyrosine phosphorylation. Since, in the whole cell, the rise of the

particular, a severe impairment of IL-2 production and calcium mobilization in response to external stimuli has been found in T and B cells (Fujikawa et al., 2003; Haubert & Weckbecker, 2010) and the defective motility observed in the Vav1-/- neutrophils, concomitant with the decrease in migration, has been demonstrated to reduce the capacity for an innate immune response (Phillipson et al., 2009). Vav1 has a critical role also in regulating the acquisition by macrophages of maturation-related competence, as demonstrated by the smaller adhesive area, the reduced motility and the lower migration speed of macrophages from Vav1-deficient mice (Hall et al., 2006; Wells et al., 2005). More recently, Vav1 activity has been demonstrated to be required specifically for SDF1αdependent perivascular homing and subsequent engraftment of hematopoietic stem cells (Sanchez-Aguilera et al., 2011). In both lymphoid and myeloid cells, Vav1 is involved in the dynamic regulation of the filamentous actin cytoskeleton, critical to numerous physical cellular processes, including adhesion, migration and phagocytosis (Cougoule et al., 2006; Stricker et al., 2010).

Vav1 contains an array of structural motifs that enable it to play a role in several distinct cell functions, like cytoskeletal reorganization and regulation of gene expression during proliferation, maturation, and apoptosis of hematopoietic cells (Clevenger et al., 1995; Fischer et al., 1998; Kong et al., 1998). The Vav1 domains include a DH region which exhibits a GDP/GTP exchange activity for the RhoA, Rac1 and CDC42 small GTPases, a PH domain interacting with phosphoinositides, two SH3 domains and one SH2 domain mediating protein-protein interactions, a CH domain that functions as an actin-binding motif and an AC region that contains 3 regulatory tyrosines. Vav1 also possesses 2 putative nuclear localization signals, indicative for a role of the protein also inside the nuclear compartment (Bustelo, 2001).

In both myeloid and lymphoid cells, the best known function of Vav proteins is the guanosine exchange factor (GEF) for small G proteins which is modulated, at variance with the other exchange factors for Rho/Rac in humans, by phosphorylation on tyrosine residues (Bustelo, 2002). However, some functions of Vav1 in hematopoietic cells are independent of its GEF activity and are attributed to its ability to interact with a number of signalling molecules, in both cytoplasm and nucleus. In particular, inside the nuclear compartment Vav1 seems to play its most intriguing role as part of transcriptionally active complexes (Houlard et al., 2002) and by interacting with components of the DNA-dependent protein kinase complex as well as with hnRNP proteins (Romero et al., 1996, 1998).

In addition to the role played in the acquisition of a mature phenotype by normal hematopoietic cells, Vav1 has been found to promote the agonist-induced completion of the differentiation program of tumoral myeloid precursors. In cell lines derived from APL patiens treated with differentiating agents Vav1 plays indeed multiple roles aimed to regulate different aspects of maturation along the neutrophilic and the monocytic/macrophagic lineages. Since Vav1 may be recruited by various differentiating agents and plays a central role in the completion of the differentiation program of leukemic promyelocytes along diverse hematopoietic lineages, it might be considered a common target for developing new therapeutic strategies for the different subtypes of myeloid leukemias.

#### **2. Vav1 and netrophil-like phenotypical maturation**

Promyelocytes derived from APL, which are blocked at different steps of their neutrophil differentiation, contain levels of Vav1 variably lower than those found in mature

particular, a severe impairment of IL-2 production and calcium mobilization in response to external stimuli has been found in T and B cells (Fujikawa et al., 2003; Haubert & Weckbecker, 2010) and the defective motility observed in the Vav1-/- neutrophils, concomitant with the decrease in migration, has been demonstrated to reduce the capacity for an innate immune response (Phillipson et al., 2009). Vav1 has a critical role also in regulating the acquisition by macrophages of maturation-related competence, as demonstrated by the smaller adhesive area, the reduced motility and the lower migration speed of macrophages from Vav1-deficient mice (Hall et al., 2006; Wells et al., 2005). More recently, Vav1 activity has been demonstrated to be required specifically for SDF1αdependent perivascular homing and subsequent engraftment of hematopoietic stem cells (Sanchez-Aguilera et al., 2011). In both lymphoid and myeloid cells, Vav1 is involved in the dynamic regulation of the filamentous actin cytoskeleton, critical to numerous physical cellular processes, including adhesion, migration and phagocytosis (Cougoule et al., 2006;

Vav1 contains an array of structural motifs that enable it to play a role in several distinct cell functions, like cytoskeletal reorganization and regulation of gene expression during proliferation, maturation, and apoptosis of hematopoietic cells (Clevenger et al., 1995; Fischer et al., 1998; Kong et al., 1998). The Vav1 domains include a DH region which exhibits a GDP/GTP exchange activity for the RhoA, Rac1 and CDC42 small GTPases, a PH domain interacting with phosphoinositides, two SH3 domains and one SH2 domain mediating protein-protein interactions, a CH domain that functions as an actin-binding motif and an AC region that contains 3 regulatory tyrosines. Vav1 also possesses 2 putative nuclear localization signals, indicative for a role of the protein also inside the nuclear compartment

In both myeloid and lymphoid cells, the best known function of Vav proteins is the guanosine exchange factor (GEF) for small G proteins which is modulated, at variance with the other exchange factors for Rho/Rac in humans, by phosphorylation on tyrosine residues (Bustelo, 2002). However, some functions of Vav1 in hematopoietic cells are independent of its GEF activity and are attributed to its ability to interact with a number of signalling molecules, in both cytoplasm and nucleus. In particular, inside the nuclear compartment Vav1 seems to play its most intriguing role as part of transcriptionally active complexes (Houlard et al., 2002) and by interacting with components of the DNA-dependent protein

In addition to the role played in the acquisition of a mature phenotype by normal hematopoietic cells, Vav1 has been found to promote the agonist-induced completion of the differentiation program of tumoral myeloid precursors. In cell lines derived from APL patiens treated with differentiating agents Vav1 plays indeed multiple roles aimed to regulate different aspects of maturation along the neutrophilic and the monocytic/macrophagic lineages. Since Vav1 may be recruited by various differentiating agents and plays a central role in the completion of the differentiation program of leukemic promyelocytes along diverse hematopoietic lineages, it might be considered a common target for developing new

Promyelocytes derived from APL, which are blocked at different steps of their neutrophil differentiation, contain levels of Vav1 variably lower than those found in mature

kinase complex as well as with hnRNP proteins (Romero et al., 1996, 1998).

therapeutic strategies for the different subtypes of myeloid leukemias.

**2. Vav1 and netrophil-like phenotypical maturation** 

Stricker et al., 2010).

(Bustelo, 2001).

neutrophils. Treatments with differentiating doses of ATRA induce a significant increase of Vav1 expression in primary blasts obtained from the bone marrow of APL patients as well as in the APL-derived cell lines HL-60 and NB4. A similar increase of Vav1 is observed when normal CD34+ hematopoietic progenitors are treated with a cytokine cocktail promoting granulocytic differentiation, clearly indicating that an adequate expression of Vav1 has to be achieved along with neutrophil maturation of both normal precursors and poorly differentiated neoplastic cells (Bertagnolo et al., 2005). These evidence, obtained in a variety of normal and neoplastic cells under different experimental conditions, ascribe to Vav1 the potential role of a ubiquitous key player in the path leading myeloid precursors to acquire the mature phenotype of differentiated neutrophils.

The issue of whether the increase of Vav1 observed in differentiation of tumoral promyelocytes is merely designed to the function of the protein in mature cells or, more intriguingly, it is functionally relevant to the maturation mechanism, has been addressed by studies in which the expression of Vav1 was forcedly modulated. The experiments have been performed in ATRA treated HL-60 and NB4 cells, which are blocked at different levels of granulocytic differentiation, and thus constitute models well suited to better understand the role of Vav1 in the maturation process. As a consequence of Vav1 over-expression during ATRA treatment of both cell lines, the expression of the myeloid surface marker CD11b increases, indicating that Vav1 supports the role of ATRA in regulating the maturation process. On the other hand, the sole over-expression of Vav1 is capable to significantly induce the expression of CD11b only in HL-60 cells, that, compared to NB4 cells, are blocked to a less differentiated stage (Bertagnolo et al., 2005). This suggests the existence of a direct and ATRA-independent role of Vav1 in regulating the expression of CD11b, at least in cells that are blocked at early stages of the neutrophilic maturation.

Neutrophils radically change in shape during development and functional life (Sanchez & Wangh, 1999). Accordingly, profound rearrangements of the cell morphology take place throughout differentiation of myeloid precursors along the granulocytic lineage, and the nucleus is the cell compartment that undergoes the biggest architectural changes by a mechanism still largely unknown. Modifications of the nuclear shape constitute one of the markers of neutrophil maturation of tumoral promyelocytes and are particularly evident after treatment with ATRA of HL-60 cells, according to the notion that this cell type is blocked at an early stage of maturation. On the other hand, the sole over-expression of Vav1 is unable to induce nuclear modifications in both HL-60 and NB4 cells, indicating that other ATRA-induced events are required to regulate the maturation-related rearrangements of cell morphology (Bertagnolo et al, 2005).

The use of siRNAs specific for Vav1 unequivocally demonstrates that Vav1 is not dispensable for the progression of tumoral promyelocytes along the granulocytic lineage. In fact, the down-modulation of Vav1 expression during ATRA treatment of both HL-60 and NB4 cells counteracts the agonist-induced increase of CD11b expression and prevents the maturation-related modifications of cell/nucleus morphology, definitely assigning to Vav1 a crucial role in regulating phenotypical maturation of APL-derived cells (Bertagnolo et al, 2005, 2008).

#### **2.1 Tyrosine phosphorylation of Vav1**

In parallel with the increase of Vav1 expression, ATRA treatment of HL-60 and NB4 cells also induces Vav1 tyrosine phosphorylation. Since, in the whole cell, the rise of the

Vav1: A Key Player in Agonist-Induced Differentiation of

Promyelocytes from Acute Myeloid Leukemia (APL) 267

expression of Vav1 is down-modulated during the differentiation treatment (Bertagnolo et al., 2008). However, since Piceatannol fails to abrogate completely the ATRA-induced tyrosine phosphorylation of Vav1 in both HL-60 and NB4 cells (Bertagnolo et al., 2008), other kinase/s,

The results from these inhibition studies support the hypothesized action model that requires tyrosine phosphorylated Vav1 in maturation of tumoral myeloid precursors. Nevertheless, the issue needs further investigations, in order to identify the kinase/s involved. In fact, by extending the analysis to other tyrosine kinase inhibitors, it has been found that PP1 and AG490, inhibitors of Src and Jak tyrosine kinase families, respectively, did not affect to any significant extent the tyrosine phosphorylation of Vav1 (Bertagnolo et al., 2004), leaving open the question of which other tyrosine kinases, in addition to Syk,

In addition to the obvious interaction with tyrosine kinases, the optimal phosphorylation of Vav1 seems to require the association with adaptor molecules that facilitate the spatial proximity between Vav1 and the upstream kinases. These associations also depend on the tyrosine phosphorylation of the involved proteins and often require the engagement of either the SH3 or the SH2 domains of Vav1 as interacting motifs (Bustelo, 2002). In this context, SLP-76, an adaptor protein predominantly expressed in T cells and myeloid cells and which is a substrate for ZAP-70 and Syk tyrosine kinases, has been reported to associate, via tyrosinephosphorylated residues in its NH2-terminal domain, with the SH2 domain of Vav1 after ligation of the T-cell antigen receptor (Tuosto et al., 1996; Pauker & Barda-Saad,, 2011). SLP-76 was also described as an important adaptor molecule that is regulated by Syk in C-reactive protein-stimulated platelets (Gross et. al, 1999) and that plays a critical role in FcRI-mediated

Some of the Vav1-interacting molecules play a role in down modulation of Vav1 signals. A potential negative regulator of Vav1 is Cbl, which down-modulates Syk/ZAP-70 and other protein tyrosine kinases (Lupher et al., 1999). Cbl associates with Vav1 upon T-cell receptor stimulation of primary murine lymphocytes and Jurkat T cells. This interaction appears to require the whole SH3-SH2-SH3 COOH-terminal domain of Vav1 and a proline-rich sequence of Cbl and seems to inhibit the Vav1-dependent signal transduction (Bustelo et al., 1997). Very recently, Chiang & Hodes (2011) have demonstrated a role of Cbl in repressing signaling events that can mediate thymic differentiation in the absence of Vav1, since Cbl

The molecules involved in the recruitment and phosphorylation of Vav1 during the ATRAdependent granulocytic differentiation of tumoral promyelocytes were investigated in HL-60 cells by using GST-fusion NH2-terminal and COOH-terminal Vav1-SH3 and GST-fusion Vav1-SH3-SH2-SH3 proteins. One of the proteins interacting with Vav1 in HL-60 cells is the adaptor molecule Cbl, present only in the cytoplasm and strongly phosphorylated in response to ATRA treatment. The Vav1/Cbl interaction in HL-60 cells occurs also in control conditions and requires the entire SH3-SH2-SH3 domain of Vav1 (Bertagnolo et al., 2001). The adaptor protein SLP-76 has been also identified as a phosphorylated protein interacting with the SH3-SH2-SH3 fragment of Vav1 in both cells and nuclei of HL-60 after ATRA treatment. Similarly to Cbl, SLP-76 associates with Vav1 also in control conditions, without quantitative changes due to the differentiation process. Vav1-associated SLP-76 was more

in addition to Syk, are probably recruited by ATRA in these cell models.

phosphorylate Vav1 during maturation of tumoral promyelocytes.

activation of mast cells *in vivo* and *in vitro* (Pivniouk et al., 1999).

inactivation rescued defective T cell development in Vav1-/- mice.

**2.1.1 Participation to protein complexes with signaling molecules** 

phosphosphorylation level is almost proportional to the increase in total Vav1 amount, ATRA seems to ensure to differentiating cells the achievement of an adequate amount of tyrosine phosphorylated Vav1, according with the functions of Vav1 in mature neutrophils. On the other hand, the accumulation of tyrosine-phosphorylated Vav1 inside the nuclear compartment seems to be a distinctive feature of the differentiation process induced by ATRA in APL-derived cells (Bertagnolo et al., 2005). In fact, a progressive increase of tyrosine phosphorylated Vav1 inside the nucleus accompanies the agonist-induced maturation (Bertagnolo et al., 1998), indicative of a specific role of tyrosine phosphorylated Vav1 inside the nuclear compartment. The tyrosine phosphorylation level of nuclear Vav1 reaches a maximum in ATRA-treated NB4 cells, which constitute the cell model with the most advanced level of neutrophil maturation among APL-derived precursors (Bertagnolo et al., 2005), clearly correlating tyrosine phosphorylated Vav1 with the maturation-related events that occur inside the nuclear compartment and opening the question of which kinase/s is/are involved in this process.

In both myeloid and lymphoid cells, Vav1 is phosphorylated in tyrosine/s by receptors with intrinsic tyrosine kinase activity or by membrane and/or cytoplasmic tyrosine kinases of the Syk/Zap70, Src and Jak families (Bustelo, 2002). More recently, also the c-Abl kinase has been reported to be specifically involved in regulating the activity of Vav1 in integrinmediated neutrophil adhesion (Cui et al., 2009). Recruitment and phosphorylation of Vav1 depend on its ability to interact with a number of signalling proteins by means of its various domains. In particular, the interaction between the SH2 domain of Vav1 and phosphorylated proteins is thought to serve for recruitment of activated kinases, which in turn can phosphorylate Vav1 (Bustelo, 2002). The Syk/ZAP-70 family of tyrosine kinases constitutes an example of proteins that contain two SH2 domains, a tandem sequence that might confer high specificity in tyrosine kinase- mediated signalling. In addition, both ZAP-70 and Syk contain a consensus binding sequence for the Vav1 SH2 domain that seems to be critical for antigen receptor-mediated signal transduction (Ottinger et al., 1998).

Experiments performed with HL-60 cells have demonstrated the association of tyrosine phosphorylated Syk with the Vav1-SH2 domain, in both whole cell and nuclear compartment, as a consequence of ATRA treatment (Bertagnolo et al., 2001). These data are in agreement with the notion that activation of Syk occurs in differentiating HL-60 cells (Qin & Yamamura, 1997) and mature neutrophils, in which it regulates migration (Schymeinsky et al., 2006) and the formation of lamellipodia during phagocytosis (Shi et al., 2006). While in whole HL-60 cells the Vav1/Syk association takes place regardless of their phosphorylation level and of ATRA treatment, the formation of Vav1/Syk complexes inside the nuclear compartment strongly increases during the differentiation process, suggesting a specific role for this tyrosine kinase in the nucleus. The role of Syk in phosphorylating Vav1 has been demonstrated by means of *in vitro* assays (Bertagnolo et al., 2001) and confirmed by the use of a pharmacological model of Syk inhibition, in which both HL-60 and NB4 cells were treated with Piceatannol (Bertagnolo et al., 2001, 2008), a tyrosine kinase inhibitor with a reported selectivity for Syk (Law et al, 1999; Seow et al., 2002).

The Syk-dependent tyrosine phosphorylation of Vav1 during the ATRA-induced phenotypical differentiation is not relevant for the expression of the surface marker CD11b, as indicated by the use of Piceatannol in both HL-60 and NB4 cells, but seems to play a crucial role in regulating the reorganization of cell architecture. In fact, when Piceatannol is administered in combination with ATRA, the modifications of nuclear morphology typical of granulocytic differentiation are almost completely abrogated, similarly to what observed when the

phosphosphorylation level is almost proportional to the increase in total Vav1 amount, ATRA seems to ensure to differentiating cells the achievement of an adequate amount of tyrosine phosphorylated Vav1, according with the functions of Vav1 in mature neutrophils. On the other hand, the accumulation of tyrosine-phosphorylated Vav1 inside the nuclear compartment seems to be a distinctive feature of the differentiation process induced by ATRA in APL-derived cells (Bertagnolo et al., 2005). In fact, a progressive increase of tyrosine phosphorylated Vav1 inside the nucleus accompanies the agonist-induced maturation (Bertagnolo et al., 1998), indicative of a specific role of tyrosine phosphorylated Vav1 inside the nuclear compartment. The tyrosine phosphorylation level of nuclear Vav1 reaches a maximum in ATRA-treated NB4 cells, which constitute the cell model with the most advanced level of neutrophil maturation among APL-derived precursors (Bertagnolo et al., 2005), clearly correlating tyrosine phosphorylated Vav1 with the maturation-related events that occur inside the nuclear compartment and opening the question of which

In both myeloid and lymphoid cells, Vav1 is phosphorylated in tyrosine/s by receptors with intrinsic tyrosine kinase activity or by membrane and/or cytoplasmic tyrosine kinases of the Syk/Zap70, Src and Jak families (Bustelo, 2002). More recently, also the c-Abl kinase has been reported to be specifically involved in regulating the activity of Vav1 in integrinmediated neutrophil adhesion (Cui et al., 2009). Recruitment and phosphorylation of Vav1 depend on its ability to interact with a number of signalling proteins by means of its various domains. In particular, the interaction between the SH2 domain of Vav1 and phosphorylated proteins is thought to serve for recruitment of activated kinases, which in turn can phosphorylate Vav1 (Bustelo, 2002). The Syk/ZAP-70 family of tyrosine kinases constitutes an example of proteins that contain two SH2 domains, a tandem sequence that might confer high specificity in tyrosine kinase- mediated signalling. In addition, both ZAP-70 and Syk contain a consensus binding sequence for the Vav1 SH2 domain that seems to be

critical for antigen receptor-mediated signal transduction (Ottinger et al., 1998).

reported selectivity for Syk (Law et al, 1999; Seow et al., 2002).

Experiments performed with HL-60 cells have demonstrated the association of tyrosine phosphorylated Syk with the Vav1-SH2 domain, in both whole cell and nuclear compartment, as a consequence of ATRA treatment (Bertagnolo et al., 2001). These data are in agreement with the notion that activation of Syk occurs in differentiating HL-60 cells (Qin & Yamamura, 1997) and mature neutrophils, in which it regulates migration (Schymeinsky et al., 2006) and the formation of lamellipodia during phagocytosis (Shi et al., 2006). While in whole HL-60 cells the Vav1/Syk association takes place regardless of their phosphorylation level and of ATRA treatment, the formation of Vav1/Syk complexes inside the nuclear compartment strongly increases during the differentiation process, suggesting a specific role for this tyrosine kinase in the nucleus. The role of Syk in phosphorylating Vav1 has been demonstrated by means of *in vitro* assays (Bertagnolo et al., 2001) and confirmed by the use of a pharmacological model of Syk inhibition, in which both HL-60 and NB4 cells were treated with Piceatannol (Bertagnolo et al., 2001, 2008), a tyrosine kinase inhibitor with a

The Syk-dependent tyrosine phosphorylation of Vav1 during the ATRA-induced phenotypical differentiation is not relevant for the expression of the surface marker CD11b, as indicated by the use of Piceatannol in both HL-60 and NB4 cells, but seems to play a crucial role in regulating the reorganization of cell architecture. In fact, when Piceatannol is administered in combination with ATRA, the modifications of nuclear morphology typical of granulocytic differentiation are almost completely abrogated, similarly to what observed when the

kinase/s is/are involved in this process.

expression of Vav1 is down-modulated during the differentiation treatment (Bertagnolo et al., 2008). However, since Piceatannol fails to abrogate completely the ATRA-induced tyrosine phosphorylation of Vav1 in both HL-60 and NB4 cells (Bertagnolo et al., 2008), other kinase/s, in addition to Syk, are probably recruited by ATRA in these cell models.

The results from these inhibition studies support the hypothesized action model that requires tyrosine phosphorylated Vav1 in maturation of tumoral myeloid precursors. Nevertheless, the issue needs further investigations, in order to identify the kinase/s involved. In fact, by extending the analysis to other tyrosine kinase inhibitors, it has been found that PP1 and AG490, inhibitors of Src and Jak tyrosine kinase families, respectively, did not affect to any significant extent the tyrosine phosphorylation of Vav1 (Bertagnolo et al., 2004), leaving open the question of which other tyrosine kinases, in addition to Syk, phosphorylate Vav1 during maturation of tumoral promyelocytes.

#### **2.1.1 Participation to protein complexes with signaling molecules**

In addition to the obvious interaction with tyrosine kinases, the optimal phosphorylation of Vav1 seems to require the association with adaptor molecules that facilitate the spatial proximity between Vav1 and the upstream kinases. These associations also depend on the tyrosine phosphorylation of the involved proteins and often require the engagement of either the SH3 or the SH2 domains of Vav1 as interacting motifs (Bustelo, 2002). In this context, SLP-76, an adaptor protein predominantly expressed in T cells and myeloid cells and which is a substrate for ZAP-70 and Syk tyrosine kinases, has been reported to associate, via tyrosinephosphorylated residues in its NH2-terminal domain, with the SH2 domain of Vav1 after ligation of the T-cell antigen receptor (Tuosto et al., 1996; Pauker & Barda-Saad,, 2011). SLP-76 was also described as an important adaptor molecule that is regulated by Syk in C-reactive protein-stimulated platelets (Gross et. al, 1999) and that plays a critical role in FcRI-mediated activation of mast cells *in vivo* and *in vitro* (Pivniouk et al., 1999).

Some of the Vav1-interacting molecules play a role in down modulation of Vav1 signals. A potential negative regulator of Vav1 is Cbl, which down-modulates Syk/ZAP-70 and other protein tyrosine kinases (Lupher et al., 1999). Cbl associates with Vav1 upon T-cell receptor stimulation of primary murine lymphocytes and Jurkat T cells. This interaction appears to require the whole SH3-SH2-SH3 COOH-terminal domain of Vav1 and a proline-rich sequence of Cbl and seems to inhibit the Vav1-dependent signal transduction (Bustelo et al., 1997). Very recently, Chiang & Hodes (2011) have demonstrated a role of Cbl in repressing signaling events that can mediate thymic differentiation in the absence of Vav1, since Cbl inactivation rescued defective T cell development in Vav1-/- mice.

The molecules involved in the recruitment and phosphorylation of Vav1 during the ATRAdependent granulocytic differentiation of tumoral promyelocytes were investigated in HL-60 cells by using GST-fusion NH2-terminal and COOH-terminal Vav1-SH3 and GST-fusion Vav1-SH3-SH2-SH3 proteins. One of the proteins interacting with Vav1 in HL-60 cells is the adaptor molecule Cbl, present only in the cytoplasm and strongly phosphorylated in response to ATRA treatment. The Vav1/Cbl interaction in HL-60 cells occurs also in control conditions and requires the entire SH3-SH2-SH3 domain of Vav1 (Bertagnolo et al., 2001).

The adaptor protein SLP-76 has been also identified as a phosphorylated protein interacting with the SH3-SH2-SH3 fragment of Vav1 in both cells and nuclei of HL-60 after ATRA treatment. Similarly to Cbl, SLP-76 associates with Vav1 also in control conditions, without quantitative changes due to the differentiation process. Vav1-associated SLP-76 was more

Vav1: A Key Player in Agonist-Induced Differentiation of

an important role in Vav1 function (Lazer et al., 2010).

2011).

physiological roles of Vav1.

**2.1.3 GEF activity** 

Promyelocytes from Acute Myeloid Leukemia (APL) 269

several of the tyrosine residues at the carboxyl terminus of Vav1 have been shown to be phosphorylated in cancer cells, raising the possibility that also these tyrosine residues play

The residue Tyr174, phosphorylated by members of Syk/Zap70 and Src tyrosine kinase families, plays a central role in regulating GEF activity of Vav1 in mature neutrophils, including 2 integrin-mediated neutrophil migration *in vitro* and neutrophil recruitment during the inflammatory response *in vivo (*Schymeinsky et al., 2006). In APL-derived cells, ATRA induces the phosphorylation of Tyr174 in NB4 but not in HL-60 cells and independently of the activity of Syk (Bertagnolo et al., 2011). Both cytofluorimetrical analysis of CD11b expression and migration assays on NB4 cells over-expressing the Tyr174Phemutated Vav1 have ruled out any relevant role for this tyrosine residue in supporting the activity of ATRA in this cell line (Bertagnolo et al., 2010). Since phosphorylation of Tyr174 in neutrophils has been mainly associated to the GEF activity of Vav1 in mature cells, these findings suggest that the ATRA-induced phosphorylation of Tyr174 occurs in parallel with differentiation and may constitute a marker of the acquisition of a mature phenotype. This hypothesis is confirmed by the failure of ATRA in inducing the phosphorylation of Tyr174 in HL-60 cells, that reach indeed only a partially differentiatiated phenotype (Bertagnolo et al.,

Since the phosphorylation of Vav1 on the Tyr174 residue seems unrelated to the path by which tyrosine phosphorylated Vav1 affects the ATRA-induced maturation of APL-derived cells, proteomic studies have been undertaken to identify other tyrosine residues phosphorylated after ATRA treament in both HL-60 and NB4 cells. Mass spectra analysis performed on Vav1 immunoprecipitated from NB4 whole cells identified Tyr745 as an ATRA-induced phosphorylated residue, within a highly conserved Vav1 sequence. The analysis of maturation-related features in differentiating NB4 cells over-expressing the Tyr745Phe-mutated form of Vav1 have clearly shown that phosphorylation of this tyrosine residue is crucial in regulating CD11b expression as well as in promoting the acquisition of migratory capabilities (Bertagnolo et al., 2010). Even if Tyr745 has never been correlated with the known roles of Vav1, multiple sequence alignment analysis of proteins from different species indicates that this is a highly conserved aminoacid, likely involved in

Inhibition studies have ruled out the role of Syk in phosphorylating Tyr745 as a consequence of ATRA treatment and, at present, no data are available about the involved tyrosine kinase. This is in part due to the fact that analysis performed with softwares designed to predict cell signaling interactions using short sequence motifs failed to recognize the Tyr745 of Vav1 as a putative phosphorylation site by the known tyrosine kinases. However, some tyrosine residues of Vav1 are not surface exposed and/or may be involved in intramolecular interactions, thereby precluding their tyrosine phosphorylation and impairing their recognition by the data base analysis. Since Tyr745 is located inside a short helix on the SH2 domain of Vav1, its phosphorylation could be an event secondary to phosphorylation of other tyrosine residues, which may induce conformational changes of

The best known function of tyrosine phosphorylated Vav1 is a catalytic role as a GEF towards the Rho family of GTPases, in which Tyr174 is crucial. Tyr174 lies within an α-helix

Vav1 allowing Tyr745 to become accessible to a specific tyrosine kinase.

abundant in nuclei than in whole cell lysates, indicating a preferential association into the nucleus of these two molecules, in contrast with an exclusive cytoplasmic distribution of Vav1/Cbl complexes (Bertagnolo et al., 2001).

The interaction of Vav1 with Cbl and SLP-76 in HL-60 cells may be correlated to the transmembrane signaling mediated by CD38, an early biomarker of ATRA-induced differentiation in the HL-60 cell line, in which it may play a causal role in myeloid differentiation (Lamkin et al., 2006). A correlation between Vav1 and CD38-activated signaling has been recently demonstrated by experiments in which the expression of a cytosolic deletion mutant of CD38 caused failure to up-regulate ATRA-induced proteins such as CD11b, Vav1 and Fgr, this latter able to phosphorylate Vav1 after ATRA treatment of HL-60 cells (Congleton et al., 2011).

Exclusive of the ATRA treatment of HL-60 seems to be the compartmentalized association between Vav1 and interacting proteins during ATRA treatment, since Cbl/Vav1 complexes are located in the cytoplasm while SLP-76/Vav1 complexes reside in the inner nuclear compartment. This suggests that Vav1 is recruited by one or more signal transduction cascades, starting from cell membrane and directed to the nucleus and involving the two adaptor proteins Cbl and SLP-76, which may then discretely regulate the amount of Vav1 in the cytoplasmic and nuclear compartments (Fig. 1).

The Vav1-associated protein complexes identified in HL-60 cells also contain the tyrosine kinase Syk. In particular, Vav1/Cbl/Syk complexes have been found in cytoplasm whereas Vav1/SLP-76/Syk complexes have been demonstrated inside the nuclear compartment. These associations are present in control conditions and result strongly increased after ATRA treatment (Bertagnolo et al., 2001). This suggests that, during the maturation of APLderived myeloid precursors, a sequence of signals originated from membrane receptors and directed to the nuclear compartment is ended to regulate the amount of tyrosine phosphorylated Vav1 inside the nucleus (Fig. 1) and that this pathway may involve the negative regulation of Cbl on Vav1 activity.

In both cells and nuclei of HL-60 cells, other signalling molecules associate with Vav1 as a consequence of ATRA treatment. They include the 1 isoform of PI-PLC and the p85 regulatory subunit of PI3K. In particular, ATRA treatment increases the binding of tyrosinephosphorylated Vav1 to both N-terminal and C-terminal SH2 domains of p85 (Bertagnolo et al., 1998). Since Vav1 is the only member of the Vav1/PLC-1/PI3K complex to possess a nuclear localization sequence (Bustelo, 2001), it is conceivable that Vav1 is directly involved in regulating the amount of PLC-1 and PI3K inside the nuclear compartment.

#### **2.1.2 Phosphorylation of Vav1 on Tyr745**

Vav1 contains 31 tyrosine residues whose phosphorylation was originally investigated almost exclusively in relation to the function of Vav1 as a GEF. A crucial role in this context seems to be played by Tyr174 in both lymphoid and myeloid cells, even if other mechanisms have emerged in the last few years as regulators of Vav1 GEF activity, and recent data suggest that Tyr174 is coversely involved in roles of Vav1 not mediated by GEF activity (Katzav, 2009). In addition to Tyr174, other conserved residues, Tyr142 and Tyr160, have been described to be phosphorylated in activated Vav1. It has also been suggested that phosphorylation of the tyrosines located inside the acidic region of Vav1 may allow Tyr142, Tyr160, and Tyr174 to become docking sites for kinases, which can then phosphorylate additional tyrosine residues in Vav proteins (Miletic et al., 2006; Yu et al., 2010). Recently,

abundant in nuclei than in whole cell lysates, indicating a preferential association into the nucleus of these two molecules, in contrast with an exclusive cytoplasmic distribution of

The interaction of Vav1 with Cbl and SLP-76 in HL-60 cells may be correlated to the transmembrane signaling mediated by CD38, an early biomarker of ATRA-induced differentiation in the HL-60 cell line, in which it may play a causal role in myeloid differentiation (Lamkin et al., 2006). A correlation between Vav1 and CD38-activated signaling has been recently demonstrated by experiments in which the expression of a cytosolic deletion mutant of CD38 caused failure to up-regulate ATRA-induced proteins such as CD11b, Vav1 and Fgr, this latter able to phosphorylate Vav1 after ATRA treatment

Exclusive of the ATRA treatment of HL-60 seems to be the compartmentalized association between Vav1 and interacting proteins during ATRA treatment, since Cbl/Vav1 complexes are located in the cytoplasm while SLP-76/Vav1 complexes reside in the inner nuclear compartment. This suggests that Vav1 is recruited by one or more signal transduction cascades, starting from cell membrane and directed to the nucleus and involving the two adaptor proteins Cbl and SLP-76, which may then discretely regulate the amount of Vav1 in

The Vav1-associated protein complexes identified in HL-60 cells also contain the tyrosine kinase Syk. In particular, Vav1/Cbl/Syk complexes have been found in cytoplasm whereas Vav1/SLP-76/Syk complexes have been demonstrated inside the nuclear compartment. These associations are present in control conditions and result strongly increased after ATRA treatment (Bertagnolo et al., 2001). This suggests that, during the maturation of APLderived myeloid precursors, a sequence of signals originated from membrane receptors and directed to the nuclear compartment is ended to regulate the amount of tyrosine phosphorylated Vav1 inside the nucleus (Fig. 1) and that this pathway may involve the

In both cells and nuclei of HL-60 cells, other signalling molecules associate with Vav1 as a consequence of ATRA treatment. They include the 1 isoform of PI-PLC and the p85 regulatory subunit of PI3K. In particular, ATRA treatment increases the binding of tyrosinephosphorylated Vav1 to both N-terminal and C-terminal SH2 domains of p85 (Bertagnolo et al., 1998). Since Vav1 is the only member of the Vav1/PLC-1/PI3K complex to possess a nuclear localization sequence (Bustelo, 2001), it is conceivable that Vav1 is directly involved

Vav1 contains 31 tyrosine residues whose phosphorylation was originally investigated almost exclusively in relation to the function of Vav1 as a GEF. A crucial role in this context seems to be played by Tyr174 in both lymphoid and myeloid cells, even if other mechanisms have emerged in the last few years as regulators of Vav1 GEF activity, and recent data suggest that Tyr174 is coversely involved in roles of Vav1 not mediated by GEF activity (Katzav, 2009). In addition to Tyr174, other conserved residues, Tyr142 and Tyr160, have been described to be phosphorylated in activated Vav1. It has also been suggested that phosphorylation of the tyrosines located inside the acidic region of Vav1 may allow Tyr142, Tyr160, and Tyr174 to become docking sites for kinases, which can then phosphorylate additional tyrosine residues in Vav proteins (Miletic et al., 2006; Yu et al., 2010). Recently,

in regulating the amount of PLC-1 and PI3K inside the nuclear compartment.

Vav1/Cbl complexes (Bertagnolo et al., 2001).

of HL-60 cells (Congleton et al., 2011).

the cytoplasmic and nuclear compartments (Fig. 1).

negative regulation of Cbl on Vav1 activity.

**2.1.2 Phosphorylation of Vav1 on Tyr745** 

several of the tyrosine residues at the carboxyl terminus of Vav1 have been shown to be phosphorylated in cancer cells, raising the possibility that also these tyrosine residues play an important role in Vav1 function (Lazer et al., 2010).

The residue Tyr174, phosphorylated by members of Syk/Zap70 and Src tyrosine kinase families, plays a central role in regulating GEF activity of Vav1 in mature neutrophils, including 2 integrin-mediated neutrophil migration *in vitro* and neutrophil recruitment during the inflammatory response *in vivo (*Schymeinsky et al., 2006). In APL-derived cells, ATRA induces the phosphorylation of Tyr174 in NB4 but not in HL-60 cells and independently of the activity of Syk (Bertagnolo et al., 2011). Both cytofluorimetrical analysis of CD11b expression and migration assays on NB4 cells over-expressing the Tyr174Phemutated Vav1 have ruled out any relevant role for this tyrosine residue in supporting the activity of ATRA in this cell line (Bertagnolo et al., 2010). Since phosphorylation of Tyr174 in neutrophils has been mainly associated to the GEF activity of Vav1 in mature cells, these findings suggest that the ATRA-induced phosphorylation of Tyr174 occurs in parallel with differentiation and may constitute a marker of the acquisition of a mature phenotype. This hypothesis is confirmed by the failure of ATRA in inducing the phosphorylation of Tyr174 in HL-60 cells, that reach indeed only a partially differentiatiated phenotype (Bertagnolo et al., 2011).

Since the phosphorylation of Vav1 on the Tyr174 residue seems unrelated to the path by which tyrosine phosphorylated Vav1 affects the ATRA-induced maturation of APL-derived cells, proteomic studies have been undertaken to identify other tyrosine residues phosphorylated after ATRA treament in both HL-60 and NB4 cells. Mass spectra analysis performed on Vav1 immunoprecipitated from NB4 whole cells identified Tyr745 as an ATRA-induced phosphorylated residue, within a highly conserved Vav1 sequence. The analysis of maturation-related features in differentiating NB4 cells over-expressing the Tyr745Phe-mutated form of Vav1 have clearly shown that phosphorylation of this tyrosine residue is crucial in regulating CD11b expression as well as in promoting the acquisition of migratory capabilities (Bertagnolo et al., 2010). Even if Tyr745 has never been correlated with the known roles of Vav1, multiple sequence alignment analysis of proteins from different species indicates that this is a highly conserved aminoacid, likely involved in physiological roles of Vav1.

Inhibition studies have ruled out the role of Syk in phosphorylating Tyr745 as a consequence of ATRA treatment and, at present, no data are available about the involved tyrosine kinase. This is in part due to the fact that analysis performed with softwares designed to predict cell signaling interactions using short sequence motifs failed to recognize the Tyr745 of Vav1 as a putative phosphorylation site by the known tyrosine kinases. However, some tyrosine residues of Vav1 are not surface exposed and/or may be involved in intramolecular interactions, thereby precluding their tyrosine phosphorylation and impairing their recognition by the data base analysis. Since Tyr745 is located inside a short helix on the SH2 domain of Vav1, its phosphorylation could be an event secondary to phosphorylation of other tyrosine residues, which may induce conformational changes of Vav1 allowing Tyr745 to become accessible to a specific tyrosine kinase.

#### **2.1.3 GEF activity**

The best known function of tyrosine phosphorylated Vav1 is a catalytic role as a GEF towards the Rho family of GTPases, in which Tyr174 is crucial. Tyr174 lies within an α-helix

Vav1: A Key Player in Agonist-Induced Differentiation of

and nucleoskeleton during maturation of tumoral promyelocytes.

cytoskeletal regulators, has also been demonstrated (Gomez et al., 2005).

**2.2 GEF-independent activity of phosphorylated Vav1** 

**2.2.1 Regulation of actin cytoskeleton** 

(Han et al., 1998).

Promyelocytes from Acute Myeloid Leukemia (APL) 271

In APL-derived HL-60 cells, ATRA treatment induces an increase of total GEF activity, not attributable to Vav1, as deduced by *in vitro* assays on Ras/Rac small G proteins performed on Vav1 immunoprecipitated from both whole cells and isolated nuclei (Bertagnolo et al., 2001). This implies that, in this cell model, the Syk-dependent tyrosine phosphorylation of Vav1 is not ended to regulate its GEF activity and that alternative pathways have to be considered to explain the mechanism by which Vav1 affects the organization of cytoskeleton

In addition to act as a GEF, Vav1 may mediate actin reorganization through other, GEFindependent mechanisms. The presence in its structure of a number of tyrosines and domains potentially involved in protein-protein interactions suggests for Vav1 a role in actin polymerization as an adapter protein that links signaling and cytoskeletal molecules. In T cells, Vav1 binds constitutively Talin and Vinculin, anchoring the actin cytoskeleton to the plasma membrane, and the cytoskeletal protein Zyxin (Hornstein et al., 2004). In the same cell model, a direct link between Vav1 and dynamin 2 (Dyn2), a component of the

A mechanism by means of which tyrosine-phosphorylated Vav1 regulates cytoskeleton of ATRA-treated tumoral promyelocytes, identified in HL-60 cells, implies the interaction of Vav1 with the p85 regulatory subunit of PI3K. Studies aimed to establish the functional meaning of this interaction have demonstrated that, in maturating myeloid precursors, PI3K activity closely depends on its association with tyrosine phosphorylated Vav1 and that when Vav1/PI3K interaction and/or PI3K activity are abrogated, the phenotypic differentiation of ATRA-treated HL-60 is compromised (Bertagnolo et al., 1999, 2004). These evidence assign to Vav1/PI3K interaction a prominent function in the regulation of cytoskeleton alternative to the described role of 3-phosphoinositides on GEF activity of Vav1

Also actin participates in the ATRA-induced protein complexes containing Vav1 and PI3K in HL-60 cells. Remarkably, when the association between Vav1 and PI3K is inhibited, the formation of PI3K/actin complexes is reduced, suggesting that the interaction of PI3K with Vav1 is essential for its association with actin (Bertagnolo et al., 2004). Since the recovery of 3-phosphoinositides is strongly reduced when the Vav1-dependent PI3K/actin interaction is abrogated, it can be concluded that Vav1 regulates the physical contact of PI3K with their cytoskeleton-associated substrates. These observations suggest that in addition to playing a

PI3K is likely to play essential roles in granulocytic differentiation of tumoral myeloid precursors, considering that both down-modulation of its expression and pharmacological inhibition of its activity during ATRA treatment significantly reduce the tendency of HL-60 cells to acquire the differentiated phenotype (Bertagnolo et al., 1999). The response to ATRA and the downstream effects of PI3K observed during the induced differentiation support the notion that PI3K is recruited in the path controling cytoskeleton in mature granulocytes. In fact, PI3K is activated in response to chemotactic factors in murine and human neutrophils (Cicchetti et al., 2002; Niggli & Keller, 1997; Stephens et al., 2002) in which newly produced PIP3 is involved in determining the localization and possibly the crosslinking/stabilization of actin filaments (Chen et al., 2003; Hannigan et al., 2002; Wang et al., 2002). In vitro experiments

regulatory role in Vav1 activation, PI3K activity may itself be regulated by Vav1.

and binds directly with the GTPase interaction pocket of the DH domain, blocking access to substrate and inhibiting Vav1 GEF activity. Phosphorylation releases Tyr174 from the binding pocket, relieving the auto-inhibition (Bustelo et al., 2001). The activity of the DH domain is also regulated by the CH domain, as deletion of this domain results in constitutively active GEF activity. It has been suggested that the Vav1 CH domain can bind to the C1 region, occluding the DH domain and blocking access to GTPases. CH–C1 interaction apparently stabilizes the inhibitory Tyr174–DH interaction. In addition, the PH domain regulates Vav1 catalytic activity by interaction with two inositol lipids: phosphatidylinositol 4, 5- bisphosphate (PIP2) and phosphatidylinositol 3, 4, 5-trisphosphate (PIP3). Whereas the binding of PIP3 moderately enhances the in vitro GEF activity of Vav1, binding to PIP2 has an inhibitory effect. Consistent with this model, Vav1 carrying a single mutation in its PH domain is constitutively active and induces cytoskeleton rearrangements as a consequence of Rac activation. Deletions of C1 domain or mutations that disrupt its structural integrity inhibit Vav1 GEF activity. High resolution X-ray structure of DH–PH–C1 domains suggests that PH and C1 domains contribute to GEF activity by stabilizing the DH domain structure and not through direct contacts with GTPases (Bustelo, 2002).

GEF activity of Vav1 has long been regarded as the key for transferring the signal from activated receptors to the cytoskeleton. Among the molecules constituting the cytoskeleton architecture, actin seems to be a preferred target of the Vav1-dependent GEF activity. Several partners are involved in the pathway by which Vav1 affects actin cytoskeleton. It has been reported that Vav1 is a preferential exchange factor for Rac1, which in turn may activate phosphatidylinositol-4-phosphate (PIP) 5-kinase which phosphorylates PIP to PIP2. PIP2 may function as an activator of actin-binding proteins, like talin and vinculin, that attach the cytoskeleton to the cell membrane. Another potential target for the GEF activity of Vav1, Cdc42, may activate the WASP protein, a key mediator of actin polymerization (Hornstein et al., 2004). Finally, Vav1-activated small G proteins play an essential role in regulating actin cytoskeleton dynamics by also interacting with the p21-activated serinethreonine kinase (PAK) family of actin-regulatory enzymes (Daniels & Bokoch, 1999).

In both lymphoid and myeloid cells, like other proteins with a GEF activity, Vav1 mediates a number of cytoskeletal-associated cellular processes, being an essential part of the molecular link connecting activated receptors to the actin cytoskeleton. A consistent number of studies (reviewed in Hornstein et al., 2004) have reported the role of Vav1 in the formation of immunological synapse and in phagocytosis of T cells. In non adherent neutrophils, stimulation of chemoattractant receptors induces a complex sequence of events: actin reorganization, shape changes, development of polarity and reversible adhesion, all culminating in chemotaxis. The complex signaling mechanisms that regulate neutrophil migration are well studied, and Vav1 appears to be a major point of the inhibitory crosstalk between adhesion receptors and cytokine receptors (Gakidis et al., 2004). In particular, the activity of Vav1 as GEF for Rac2 is inhibited in adherent cells, as a possible consequence of the activation by adhesion of one or more tyrosine phosphatases responsible of dephosphorylating Vav1. On the other hand, experiments performed with Vav1-/- mice have demonstrated that motility and mobilization into peripheral blood induced in neutrophils by FMLP are significantly reduced, as well as the generation of filamentous actin (Kim et al., 2003). Studies performed in a rat model have demonstrated that the M-CSF-induced chemotaxis of bone marrow macrophages is initiated by the 3 phosphoinositide-dependent GEF activity of Vav1 on Rac (Vedham et al., 2005).

and binds directly with the GTPase interaction pocket of the DH domain, blocking access to substrate and inhibiting Vav1 GEF activity. Phosphorylation releases Tyr174 from the binding pocket, relieving the auto-inhibition (Bustelo et al., 2001). The activity of the DH domain is also regulated by the CH domain, as deletion of this domain results in constitutively active GEF activity. It has been suggested that the Vav1 CH domain can bind to the C1 region, occluding the DH domain and blocking access to GTPases. CH–C1 interaction apparently stabilizes the inhibitory Tyr174–DH interaction. In addition, the PH domain regulates Vav1 catalytic activity by interaction with two inositol lipids: phosphatidylinositol 4, 5- bisphosphate (PIP2) and phosphatidylinositol 3, 4, 5-trisphosphate (PIP3). Whereas the binding of PIP3 moderately enhances the in vitro GEF activity of Vav1, binding to PIP2 has an inhibitory effect. Consistent with this model, Vav1 carrying a single mutation in its PH domain is constitutively active and induces cytoskeleton rearrangements as a consequence of Rac activation. Deletions of C1 domain or mutations that disrupt its structural integrity inhibit Vav1 GEF activity. High resolution X-ray structure of DH–PH–C1 domains suggests that PH and C1 domains contribute to GEF activity by stabilizing the DH

domain structure and not through direct contacts with GTPases (Bustelo, 2002).

phosphoinositide-dependent GEF activity of Vav1 on Rac (Vedham et al., 2005).

GEF activity of Vav1 has long been regarded as the key for transferring the signal from activated receptors to the cytoskeleton. Among the molecules constituting the cytoskeleton architecture, actin seems to be a preferred target of the Vav1-dependent GEF activity. Several partners are involved in the pathway by which Vav1 affects actin cytoskeleton. It has been reported that Vav1 is a preferential exchange factor for Rac1, which in turn may activate phosphatidylinositol-4-phosphate (PIP) 5-kinase which phosphorylates PIP to PIP2. PIP2 may function as an activator of actin-binding proteins, like talin and vinculin, that attach the cytoskeleton to the cell membrane. Another potential target for the GEF activity of Vav1, Cdc42, may activate the WASP protein, a key mediator of actin polymerization (Hornstein et al., 2004). Finally, Vav1-activated small G proteins play an essential role in regulating actin cytoskeleton dynamics by also interacting with the p21-activated serinethreonine kinase (PAK) family of actin-regulatory enzymes (Daniels & Bokoch, 1999). In both lymphoid and myeloid cells, like other proteins with a GEF activity, Vav1 mediates a number of cytoskeletal-associated cellular processes, being an essential part of the molecular link connecting activated receptors to the actin cytoskeleton. A consistent number of studies (reviewed in Hornstein et al., 2004) have reported the role of Vav1 in the formation of immunological synapse and in phagocytosis of T cells. In non adherent neutrophils, stimulation of chemoattractant receptors induces a complex sequence of events: actin reorganization, shape changes, development of polarity and reversible adhesion, all culminating in chemotaxis. The complex signaling mechanisms that regulate neutrophil migration are well studied, and Vav1 appears to be a major point of the inhibitory crosstalk between adhesion receptors and cytokine receptors (Gakidis et al., 2004). In particular, the activity of Vav1 as GEF for Rac2 is inhibited in adherent cells, as a possible consequence of the activation by adhesion of one or more tyrosine phosphatases responsible of dephosphorylating Vav1. On the other hand, experiments performed with Vav1-/- mice have demonstrated that motility and mobilization into peripheral blood induced in neutrophils by FMLP are significantly reduced, as well as the generation of filamentous actin (Kim et al., 2003). Studies performed in a rat model have demonstrated that the M-CSF-induced chemotaxis of bone marrow macrophages is initiated by the 3In APL-derived HL-60 cells, ATRA treatment induces an increase of total GEF activity, not attributable to Vav1, as deduced by *in vitro* assays on Ras/Rac small G proteins performed on Vav1 immunoprecipitated from both whole cells and isolated nuclei (Bertagnolo et al., 2001). This implies that, in this cell model, the Syk-dependent tyrosine phosphorylation of Vav1 is not ended to regulate its GEF activity and that alternative pathways have to be considered to explain the mechanism by which Vav1 affects the organization of cytoskeleton and nucleoskeleton during maturation of tumoral promyelocytes.

#### **2.2 GEF-independent activity of phosphorylated Vav1 2.2.1 Regulation of actin cytoskeleton**

In addition to act as a GEF, Vav1 may mediate actin reorganization through other, GEFindependent mechanisms. The presence in its structure of a number of tyrosines and domains potentially involved in protein-protein interactions suggests for Vav1 a role in actin polymerization as an adapter protein that links signaling and cytoskeletal molecules. In T cells, Vav1 binds constitutively Talin and Vinculin, anchoring the actin cytoskeleton to the plasma membrane, and the cytoskeletal protein Zyxin (Hornstein et al., 2004). In the same cell model, a direct link between Vav1 and dynamin 2 (Dyn2), a component of the cytoskeletal regulators, has also been demonstrated (Gomez et al., 2005).

A mechanism by means of which tyrosine-phosphorylated Vav1 regulates cytoskeleton of ATRA-treated tumoral promyelocytes, identified in HL-60 cells, implies the interaction of Vav1 with the p85 regulatory subunit of PI3K. Studies aimed to establish the functional meaning of this interaction have demonstrated that, in maturating myeloid precursors, PI3K activity closely depends on its association with tyrosine phosphorylated Vav1 and that when Vav1/PI3K interaction and/or PI3K activity are abrogated, the phenotypic differentiation of ATRA-treated HL-60 is compromised (Bertagnolo et al., 1999, 2004). These evidence assign to Vav1/PI3K interaction a prominent function in the regulation of cytoskeleton alternative to the described role of 3-phosphoinositides on GEF activity of Vav1 (Han et al., 1998).

Also actin participates in the ATRA-induced protein complexes containing Vav1 and PI3K in HL-60 cells. Remarkably, when the association between Vav1 and PI3K is inhibited, the formation of PI3K/actin complexes is reduced, suggesting that the interaction of PI3K with Vav1 is essential for its association with actin (Bertagnolo et al., 2004). Since the recovery of 3-phosphoinositides is strongly reduced when the Vav1-dependent PI3K/actin interaction is abrogated, it can be concluded that Vav1 regulates the physical contact of PI3K with their cytoskeleton-associated substrates. These observations suggest that in addition to playing a regulatory role in Vav1 activation, PI3K activity may itself be regulated by Vav1.

PI3K is likely to play essential roles in granulocytic differentiation of tumoral myeloid precursors, considering that both down-modulation of its expression and pharmacological inhibition of its activity during ATRA treatment significantly reduce the tendency of HL-60 cells to acquire the differentiated phenotype (Bertagnolo et al., 1999). The response to ATRA and the downstream effects of PI3K observed during the induced differentiation support the notion that PI3K is recruited in the path controling cytoskeleton in mature granulocytes. In fact, PI3K is activated in response to chemotactic factors in murine and human neutrophils (Cicchetti et al., 2002; Niggli & Keller, 1997; Stephens et al., 2002) in which newly produced PIP3 is involved in determining the localization and possibly the crosslinking/stabilization of actin filaments (Chen et al., 2003; Hannigan et al., 2002; Wang et al., 2002). In vitro experiments

Vav1: A Key Player in Agonist-Induced Differentiation of

**2.2.2 Regulation of gene expression** 

maturation of tumoral myeloid precursors.

Promyelocytes from Acute Myeloid Leukemia (APL) 273

Microarray analysis performed on APL-derived cell lines has identified several genes whose expression is modified by ATRA treatment, including genes for a number of cytokines, in turn involved in the differentiative program of tumoral promyelocytes (Hsu et al., 1999; Visani et al., 1996). As a consequence of ATRA administration, tyrosine-phosphorylated Vav1 accumulates inside the nuclear compartment of APL-derived cells and becomes involved in the changes of nuclear morphology. Since local reorganization of nuclear architecture is required for both transcription and post-transcriptional events, it is

An array analysis performed on HL-60 cells focussed on genes coding for cytokines and cytokine receptors indicates that the inhibition of the Syk-dependent tyrosinephosphorylation of Vav1 during ATRA treatment prevents the ATRA-induced expression of 8 genes (Bertagnolo et al., 2005). Among them, the thymosin beta-10 (TMSB10) gene has been found, encoding for a small G-actin binding protein that induces depolymerization of intracellular F-actin pools and thus deeply affects actin architecture (Liu et al., 2004; Rho et al., 2004). Tyrosine-phosphorylated Vav1 is also involved in regulating the ATRA-induced expression of the gene for Notch homolog, that codifies for a molecule playing a role in mediating cell fate decisions during hematopoiesis (Ohishi et al., 2003) and whose signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein (Li et al., 2010). The involvement of Vav1 in regulating ATRA-dependent expression of cytokines and/or growth factors has been ascertained by silencing Vav1 during ATRA admistration, further confirming that the increase of Vav1 expression is not an epiphenomenon but constitutes a key event able to actually promote the granulocytic

The evidence that Vav1 has a role in regulating ATRA-dependent gene expression in APLderived cells suggests the participation of Vav1 to transcriptional complexes activated by ATRA, also considering that, in both myeloid and lymphoid cells, Vav1 seems to be involved in regulating DNA transcription, by direct interaction with, or as a facilitator of, transcription factors (Katzav, 2004). In particular, Vav1 regulates Nuclear Factor of Activated T-cells (NFAT), Activator Protein-1 (AP-1) and Nuclear Factor κB (NF-κB) in Tcells in response to TCR stimulation, and exerts a specific role in regulating the CREBdependent gene transcription (Haubert & Weckbecker, 2010; Schneider & Rudd, 2008). Direct evidence for the presence of Vav1 as a component of an active transcriptional complex has been reported by Houlard et al. (2002) demonstrating the participation of Vav1 in complexes with NFAT and NF-kB-like, as facilitator of their transcriptional activity. In APL-derived cells, nuclear Vav1 associates with PU.1 (Brugnoli et al., 2010), a transcription factor induced by ATRA and able to play a crucial role in the completion of granulocytic differentiation of APL-derived myeloid precursors (Mueller et al., 2006). In particular, the down-modulation of PU.1 by means of specific siRNAs has allowed to establish that, like in other tumoral myeloid precursors (Denkinger et al., 2002), PU.1 regulates the expression of Vav1 induced by ATRA in NB4 cells (Brugnoli et al., 2010). In AML-derived myeloid precursors, PU.l represents a major determinant of the myeloid expression of CD11b (Kastner & Chan, 2008; Pahl et al., 1993), an integrin receptor whose surface expression increases concurrently with CD11b mRNA levels during myeloid differentiation of APL-derived cell lines (Barber et al., 2008). Chromatin immunoprecipitation (ChIP) experiments performed on NB4 cells treated with ATRA have

conceivable that Vav1 plays a role in regulating ATRA-related gene expression.

have demonstrated that PI3K may affect actin-related modifications of cytoskeleton also by directly affecting PAK kinase activity (Menard & Mattingly, 2004). PI3K has a more general influence on cytoskeleton by determining the amount of the inositol-containing lipids, that have emerged as major players in regulating actin assembly at several levels and with different mechanisms, including the direct interaction with cytoskeletal proteins, such as vinculin and gelsolin (Janmey et al., 1999, Takenawa & Itoh, 2001).

Fig. 1. Schematic representation of the recruitment and phosphorylation of Vav1 in ATRAtreated promyelocytes.

Since, in ATRA-treated promyelocytes, both PI3K activity and the modifications of the nucleus architecture depend on the formation of Vav1/PI3K complexes, Vav1 may be important for targeting PI3K to its nuclear substrates. The association of Vav1 with other lipid modifying enzymes, including specific PI-PLC isoforms (Bertagnolo et al., 1998;), suggests a more general role of Vav1 in determining the composition of the actin-associated phosphoinositide pool and, ultimately, in regulating actin polymerization in differentiating HL-60 cells.

#### **2.2.2 Regulation of gene expression**

272 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

have demonstrated that PI3K may affect actin-related modifications of cytoskeleton also by directly affecting PAK kinase activity (Menard & Mattingly, 2004). PI3K has a more general influence on cytoskeleton by determining the amount of the inositol-containing lipids, that have emerged as major players in regulating actin assembly at several levels and with different mechanisms, including the direct interaction with cytoskeletal proteins, such as

Fig. 1. Schematic representation of the recruitment and phosphorylation of Vav1 in ATRA-

Since, in ATRA-treated promyelocytes, both PI3K activity and the modifications of the nucleus architecture depend on the formation of Vav1/PI3K complexes, Vav1 may be important for targeting PI3K to its nuclear substrates. The association of Vav1 with other lipid modifying enzymes, including specific PI-PLC isoforms (Bertagnolo et al., 1998;), suggests a more general role of Vav1 in determining the composition of the actin-associated phosphoinositide pool and, ultimately, in regulating actin polymerization in differentiating

treated promyelocytes.

HL-60 cells.

vinculin and gelsolin (Janmey et al., 1999, Takenawa & Itoh, 2001).

Microarray analysis performed on APL-derived cell lines has identified several genes whose expression is modified by ATRA treatment, including genes for a number of cytokines, in turn involved in the differentiative program of tumoral promyelocytes (Hsu et al., 1999; Visani et al., 1996). As a consequence of ATRA administration, tyrosine-phosphorylated Vav1 accumulates inside the nuclear compartment of APL-derived cells and becomes involved in the changes of nuclear morphology. Since local reorganization of nuclear architecture is required for both transcription and post-transcriptional events, it is conceivable that Vav1 plays a role in regulating ATRA-related gene expression.

An array analysis performed on HL-60 cells focussed on genes coding for cytokines and cytokine receptors indicates that the inhibition of the Syk-dependent tyrosinephosphorylation of Vav1 during ATRA treatment prevents the ATRA-induced expression of 8 genes (Bertagnolo et al., 2005). Among them, the thymosin beta-10 (TMSB10) gene has been found, encoding for a small G-actin binding protein that induces depolymerization of intracellular F-actin pools and thus deeply affects actin architecture (Liu et al., 2004; Rho et al., 2004). Tyrosine-phosphorylated Vav1 is also involved in regulating the ATRA-induced expression of the gene for Notch homolog, that codifies for a molecule playing a role in mediating cell fate decisions during hematopoiesis (Ohishi et al., 2003) and whose signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein (Li et al., 2010). The involvement of Vav1 in regulating ATRA-dependent expression of cytokines and/or growth factors has been ascertained by silencing Vav1 during ATRA admistration, further confirming that the increase of Vav1 expression is not an epiphenomenon but constitutes a key event able to actually promote the granulocytic maturation of tumoral myeloid precursors.

The evidence that Vav1 has a role in regulating ATRA-dependent gene expression in APLderived cells suggests the participation of Vav1 to transcriptional complexes activated by ATRA, also considering that, in both myeloid and lymphoid cells, Vav1 seems to be involved in regulating DNA transcription, by direct interaction with, or as a facilitator of, transcription factors (Katzav, 2004). In particular, Vav1 regulates Nuclear Factor of Activated T-cells (NFAT), Activator Protein-1 (AP-1) and Nuclear Factor κB (NF-κB) in Tcells in response to TCR stimulation, and exerts a specific role in regulating the CREBdependent gene transcription (Haubert & Weckbecker, 2010; Schneider & Rudd, 2008). Direct evidence for the presence of Vav1 as a component of an active transcriptional complex has been reported by Houlard et al. (2002) demonstrating the participation of Vav1 in complexes with NFAT and NF-kB-like, as facilitator of their transcriptional activity.

In APL-derived cells, nuclear Vav1 associates with PU.1 (Brugnoli et al., 2010), a transcription factor induced by ATRA and able to play a crucial role in the completion of granulocytic differentiation of APL-derived myeloid precursors (Mueller et al., 2006). In particular, the down-modulation of PU.1 by means of specific siRNAs has allowed to establish that, like in other tumoral myeloid precursors (Denkinger et al., 2002), PU.1 regulates the expression of Vav1 induced by ATRA in NB4 cells (Brugnoli et al., 2010).

In AML-derived myeloid precursors, PU.l represents a major determinant of the myeloid expression of CD11b (Kastner & Chan, 2008; Pahl et al., 1993), an integrin receptor whose surface expression increases concurrently with CD11b mRNA levels during myeloid differentiation of APL-derived cell lines (Barber et al., 2008). Chromatin immunoprecipitation (ChIP) experiments performed on NB4 cells treated with ATRA have

Vav1: A Key Player in Agonist-Induced Differentiation of

and, possibly, the expression of this surface antigen.

**3. Vav1 and protein expression** 

al., 2004; Wan et al., 2001; Wang et al., 2004;).

expression.

in APL.

also targeting metabolic pathways.

Promyelocytes from Acute Myeloid Leukemia (APL) 275

2010). It is then conceivable that Vav1, and in particular tyrosine-phosphorylated Vav1, regulates the recruitment of PU.1 to its consensus sequence on the *CD11b* promoter region

Proteome analysis currently provides the opportunity to identify global changes in gene expression by directly measuring protein amount. A number of recent studies have used this approach to evaluate protein expression during differentiation/apoptosis induced by different agonists in APL-derived cells. In particular, it has been reported that ATRA modulates the expression level of structural and signal transduction proteins as well as of molecules involved in the different phases of protein synthesis (Dong et al, 2006; Harris et

On the basis of the evidence that inside the nucleus of a number of different cell lines, including APL-derived cells, Vav1 participates to molecular complexes with DNA-related proteins (Brugnoli et al., 2010; Houlard et al., 2002; Romero et al., 1998) a more general role of Vav1 in regulating events ended to control protein expression can be hypothesized. 2D electrophoresis followed by mass spectrometry have established that, in both HL-60 and NB4 cells, the down-modulation of Vav1 abrogates the capacity of ATRA of modulating the expression of proteins associated to cytoskeleton and involved in proliferation and of apoptosis-related proteins, as well as of molecules implicated in metabolism, synthesis, folding and degradation of proteins (Bertagnolo et al., 2008). The majority of the identified proteins are affected by Vav1 down-modulation only in one of the two analyzed cell lines, according to the notion that HL-60 and NB4 cells, even if both derived from patients with APL, show peculiar genotypic and phenotypic profiles (Barber et al., 2008). Interestingly, in NB4 cells, the lack of Vav1 affects the ATRA-dependent expression of the Splicing factor, arg/ser rich 3 (Sfrs3), a member of SR proteins, known as non-snRNP splicing factors, that may affect both constitutive and alternative splicing of mRNA (Sanford et al., 2005). This evidence further supports the role of Vav1 in regulating the ATRA-dependent gene

Some of the identified proteins are differentially expressed, as a consequence of Vav1-downmodulation during ATRA treatment, in both cell lines, suggesting that they may constitute a common part of the signalling activated by ATRA in APL-derived promyelocytes. Notably, this group of proteins includes the isoform of the 14-3-3 family of proteins, specifically involved in the caspase networks (Liou et al., 2007). The increased expression of 14-3-3 in both HL-60 and NB4 cells when Vav1 is down-modulated during ATRA treatment suggests that the amount of Vav1 may be critical in determining the mechanism of caspase activation

Vav1 also affects the ATRA-dependent expression of -enolase, a multifunction protein involved in glycolysis and up-regulated in the sera of a number of cancer patients, in which it seems to have a role in tumorigenesis (Zou et al., 2005). -enolase is expressed at high levels in most AML subtypes in which it might contribute to the adverse evolution of the disease (Lopez-Pedrera et al., 2006). Since down-modulation of Vav1 during ATRA treatment of APL-derived cells reduces the expression of -enolase (Bertagnolo et al., 2008), it has been suggested that Vav1 promotes the differentiation of tumoral promyelocytes by

demonstrated that PU.1 is recruited to its consensus sequence within the *CD11b* promoter (Brugnoli et al., 2010). Since the over-expression of PU.1 might influence phenotype and restore differentiation of primary myeloid leukemic blasts (Durual et al., 2007), and its silencing counteracts the ATRA ability to induce the expression of the granulocytic marker CD11b (Mueller et al., 2006), PU.1 may be used by ATRA to promote CD11b expression during the late stages of the maturation of APL-derived cells. This is confirmed by *in vitro* experiments demonstrating the formation of PU.1-containing complexes on the *CD11b* promoter (Brugnoli et al., 2010).

Fig. 2. Schematic representation of the involvement of Vav1 in regulating gene expression in differentiating promyelocytes.

Also Vav1 is recruited to the PU.1 consensus sequence on the *CD11b* promoter in untreated NB4 cells. ATRA treatment, by inducing an increase in Syk-dependent tyrosine phosphorylation of Vav1, displaces this protein from existing molecular complexes on the *CD11b* promoter. Accordingly, the specific inhibition of Syk activity is accompanied by the appearance of a Vav1-containing complex (Brugnoli et al., 2010). The participation of Vav1 to molecular complexes including PU.1 has been ruled out by EMSA experiments. On the other hand, both expression and tyrosine phosphorylation levels of Vav1 seem to play a role in regulating the formation of PU.1-containing complexes. In fact, when the amount of Vav1 is forcedly reduced or its tyrosine phosphorylation is inhibited during the differentiation treatment, the formation of a PU.1-containing complex is negatively affected (Brugnoli et al., 2010). It is then conceivable that Vav1, and in particular tyrosine-phosphorylated Vav1, regulates the recruitment of PU.1 to its consensus sequence on the *CD11b* promoter region and, possibly, the expression of this surface antigen.

### **3. Vav1 and protein expression**

274 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

demonstrated that PU.1 is recruited to its consensus sequence within the *CD11b* promoter (Brugnoli et al., 2010). Since the over-expression of PU.1 might influence phenotype and restore differentiation of primary myeloid leukemic blasts (Durual et al., 2007), and its silencing counteracts the ATRA ability to induce the expression of the granulocytic marker CD11b (Mueller et al., 2006), PU.1 may be used by ATRA to promote CD11b expression during the late stages of the maturation of APL-derived cells. This is confirmed by *in vitro* experiments demonstrating the formation of PU.1-containing complexes on the *CD11b*

Fig. 2. Schematic representation of the involvement of Vav1 in regulating gene expression in

**Transcriptional activity** 

**Vav1** 

**Soluble molecules Cell surface receptors** 

**Signal transduction pathways** 

**Syk** 

**Vav1 P**

> **Vav1 <sup>P</sup> PU.1**

> > **PU.1**

**CD11b** 

**CD11b** 

 **CD11b** 

Also Vav1 is recruited to the PU.1 consensus sequence on the *CD11b* promoter in untreated NB4 cells. ATRA treatment, by inducing an increase in Syk-dependent tyrosine phosphorylation of Vav1, displaces this protein from existing molecular complexes on the *CD11b* promoter. Accordingly, the specific inhibition of Syk activity is accompanied by the appearance of a Vav1-containing complex (Brugnoli et al., 2010). The participation of Vav1 to molecular complexes including PU.1 has been ruled out by EMSA experiments. On the other hand, both expression and tyrosine phosphorylation levels of Vav1 seem to play a role in regulating the formation of PU.1-containing complexes. In fact, when the amount of Vav1 is forcedly reduced or its tyrosine phosphorylation is inhibited during the differentiation treatment, the formation of a PU.1-containing complex is negatively affected (Brugnoli et al.,

promoter (Brugnoli et al., 2010).

**Signalling proteins** 

differentiating promyelocytes.

**ATRA**

Proteome analysis currently provides the opportunity to identify global changes in gene expression by directly measuring protein amount. A number of recent studies have used this approach to evaluate protein expression during differentiation/apoptosis induced by different agonists in APL-derived cells. In particular, it has been reported that ATRA modulates the expression level of structural and signal transduction proteins as well as of molecules involved in the different phases of protein synthesis (Dong et al, 2006; Harris et al., 2004; Wan et al., 2001; Wang et al., 2004;).

On the basis of the evidence that inside the nucleus of a number of different cell lines, including APL-derived cells, Vav1 participates to molecular complexes with DNA-related proteins (Brugnoli et al., 2010; Houlard et al., 2002; Romero et al., 1998) a more general role of Vav1 in regulating events ended to control protein expression can be hypothesized. 2D electrophoresis followed by mass spectrometry have established that, in both HL-60 and NB4 cells, the down-modulation of Vav1 abrogates the capacity of ATRA of modulating the expression of proteins associated to cytoskeleton and involved in proliferation and of apoptosis-related proteins, as well as of molecules implicated in metabolism, synthesis, folding and degradation of proteins (Bertagnolo et al., 2008). The majority of the identified proteins are affected by Vav1 down-modulation only in one of the two analyzed cell lines, according to the notion that HL-60 and NB4 cells, even if both derived from patients with APL, show peculiar genotypic and phenotypic profiles (Barber et al., 2008). Interestingly, in NB4 cells, the lack of Vav1 affects the ATRA-dependent expression of the Splicing factor, arg/ser rich 3 (Sfrs3), a member of SR proteins, known as non-snRNP splicing factors, that may affect both constitutive and alternative splicing of mRNA (Sanford et al., 2005). This evidence further supports the role of Vav1 in regulating the ATRA-dependent gene expression.

Some of the identified proteins are differentially expressed, as a consequence of Vav1-downmodulation during ATRA treatment, in both cell lines, suggesting that they may constitute a common part of the signalling activated by ATRA in APL-derived promyelocytes. Notably, this group of proteins includes the isoform of the 14-3-3 family of proteins, specifically involved in the caspase networks (Liou et al., 2007). The increased expression of 14-3-3 in both HL-60 and NB4 cells when Vav1 is down-modulated during ATRA treatment suggests that the amount of Vav1 may be critical in determining the mechanism of caspase activation in APL.

Vav1 also affects the ATRA-dependent expression of -enolase, a multifunction protein involved in glycolysis and up-regulated in the sera of a number of cancer patients, in which it seems to have a role in tumorigenesis (Zou et al., 2005). -enolase is expressed at high levels in most AML subtypes in which it might contribute to the adverse evolution of the disease (Lopez-Pedrera et al., 2006). Since down-modulation of Vav1 during ATRA treatment of APL-derived cells reduces the expression of -enolase (Bertagnolo et al., 2008), it has been suggested that Vav1 promotes the differentiation of tumoral promyelocytes by also targeting metabolic pathways.

Vav1: A Key Player in Agonist-Induced Differentiation of

expression of the microtubule component -tubulin.

macrophages in response to CSF-1 (Wells et al., 2005).

adhesion efficiency (Wells et al., 2005).

**4.1 Regulation of actin** 

expression.

Promyelocytes from Acute Myeloid Leukemia (APL) 277

Also cell adhesion is affected by down-modulation of Vav1 during PMA treatment of HL-60 and NB4 cells, in terms of both number of adherent cells and of adhesion area of cells that remain attached to the flask bottom. These results are in agreement with the data obtained with macrophages from Vav1\_/\_ mice, showing a smaller adhesive area or a decreased

As above reported, the main known functional role of tyrosine phosphorylated Vav1 is to regulate cytoskeleton reorganization, a phenomenon at the basis of both adhesion and migration of monocytes/macrophages. Vav1 regulates cell architecture not only by means of its GEF activity but also by interacting with proteins in turn involved in cytoskeleton reorganization. In addition, in both HL-60 and NB4 cells, Vav1 affects the ATRA-induced

Contrarily to what observed during granulocytic differentiation, Vav1 down-modulation does not have any effect on expression and architectural organization of -tubulin during PMA-induced monocytic/macrophagic maturation of NB4 cells (Bertagnolo et al., 2011). This indicates that, during the maturation process of APL-derived cells, Vav1 exerts an agonist- and lineage-specific role in regulating -tubulin. From a more general point of view, concerning microtubule organization, it can be speculated that the role of Vav1 is restricted to the control of the motility of mature cells, as also suggested by the evidence that changes in microtubule dynamics contribute to the reduced migration speed of Vav1\_/\_

In living cells, the F-actin cytoskeleton encompasses a variety of different structures that are essential for many different aspects of cell physiology. In particular, dynamic modulation of the filamentous actin cytoskeleton is critical to numerous physical cellular processes, including adhesion, migration and phagocytosis, all requiring precise regulation of cell shape (Stricker et al., 2010). Recent data demonstrate that Vav proteins, including Vav1, are required for actin cytoskeleton reorganization during migration of macrophages, by coupling RhoA and Rac1 activity to adhesion receptors (Bhavsar et al., 2009). Also in ATRAinduced maturation of cells derived from APL Vav1 seems to regulate actin organization. In HL-60 and NB4 cells treated with PMA, an unprecedented involvement of Vav1 in regulating the increase of actin expression has been shown very recently (Bertagnolo et al., 2011), that constitutes a further confirmation that Vav1, besides being involved in the formation of filaments, takes part to cytoskeleton reorganization as a modulator of protein

The modifications of cell shape in the different cell processes seem to be regulated by the existence of the F-actin cortex, a thin, membrane-bound F-actin network (Stricker et al., 2010). Defective actin-cap formation has been found in lymphocytes from a Vav-deficient mice, clearly correlating Vav1 activity with the regulation of cell shape (Holsinger et al., 1998). Furthermore, a recent work in which adhesive micropatterned surfaces have been used to control the overall shape of fibroblasts, has demonstrated that the shape of the nucleus is tightly regulated through a perinuclear actin cap, which is located above and around the interphase nucleus (Khatau et al., 2009). A wide variety of contractile F-actin networks with different architectures and polarity have also been found near cell adhesion surfaces, correlated with the migratory capability of adherent cells (Stricker et al., 2010). By means of confocal analysis of PMA-treated adherent NB4 cells, it has been demonstrated the

The proteasome component "splice isoform 2 subunit 3" is down-modulated as well in HL-60 and NB4 cells under the same experimental conditions (Bertagnolo et al., 2008). Since proteasome is the major cellular proteolytic machinery responsible for degradation of normal and damaged proteins (Von Mikecz, 2006), Vav1 may also be involved in regulating protein degradation during ATRA dependent maturation of tumoral promyelocytes.

Also the component of microtubules -tubulin is affected by down-modulation of Vav1 during ATRA treatment (Bertagnolo et al., 2008), indicating that Vav1, in addition to regulate cytoskeleton reorganization, takes part to the profound architectural changes of differentiating promyelocytes by regulating the expression of cytoskeleton components.

#### **4. Vav1 and monocytic/macrophagic differentiation**

The human promyelocytic leukemia cell lines HL-60 and NB4 can be differentiated either toward neutrophils by ATRA or to monocytes/macrophages by PMA (Murao et al., 1983; Song & Norman, 1998). PMA is a stable analogue of 2, 3-diacylglycerol that induces, even if with dynamics not identical in HL-60 and NB4 cells, morphological and functional changes related to monocyte maturation, accompanied by a loss of proliferative capacity (Jasek et al., 2008). Immunochemical and immunocytochemical analysis demonstrate that the expression of Vav1 increases also during the PMA-induced acquisition of a monocyte-like phenotype of HL-60 and NB4 cells (Bertagnolo et al., 2011). This is consistent with the notion that also mature monocytes express Vav1 and that proper amounts of the protein are necessary for their inflammation related functions (Bhavsar et al., 2009; Hall et al., 2006). In the same cell types, PMA also induces a relevant increase of tyrosine phosphorylation of Vav1. On the other hand, and in constrast to what observed in the ATRA-treatment of the same cell line, no role for Syk was demonstrated in this event (Bertagnolo et al., 2011), consistent with the notion that, at least in HL-60 cells, Syk might exert a narrower role, restricted to directing cells toward granulocyte differentiation (Qin & Yamamura, 1997). In both HL-60 and NB4 cells, PMA induces a relevant increase of tyrosine phosphorylation of Vav1 on the Tyr174 residue (Bertagnolo et al., 2011), according to the GEF role played by Vav1 in myeloid cells. These results are also in agreement with other data indicating that, in macrophage-like differentiated HL-60 cells, the activity of Syk is ended to regulate the roles played by mature cells in immune response, including their complement-mediated phagocytosis, in which the kinase regulates both actin dynamics and the Vav1-RhoA activation pathway (Shi et al., 2006).

Also in differentiation of APL-derived cells to monocytes/macrophages, a crucial role for Vav1 in determining the acquisition of maturation-related features has been demonstrated by silencing the expression of Vav1 induced by PMA (Bertagnolo et al., 2011). Under these conditions, the expression of CD11b, which is induced by PMA and constitutes a marker also for monocyte differentiation, is significantly reduced, similarly to what demonstrated during the treatment with ATRA of HL-60 and NB4 cells (Bertagnolo et al. 2011). This suggests that, in differentiating APL-derived cells, Vav1 plays a role in regulating the expression of the CD11b surface antigen regardless the agonist employed and the maturation lineage. Since in NB4 cells treated with ATRA Vav1 is recuited to protein/DNA complexes on the *CD11b* promoter (Brugnoli et al., 2010), it can be speculated that Vav1 plays a specific role in driving the expression of CD11b as part of the transcriptional machinery also during the differentiation of NB4 cells along the monocytic/macrophagic lineage.

Also cell adhesion is affected by down-modulation of Vav1 during PMA treatment of HL-60 and NB4 cells, in terms of both number of adherent cells and of adhesion area of cells that remain attached to the flask bottom. These results are in agreement with the data obtained with macrophages from Vav1\_/\_ mice, showing a smaller adhesive area or a decreased adhesion efficiency (Wells et al., 2005).

#### **4.1 Regulation of actin**

276 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

The proteasome component "splice isoform 2 subunit 3" is down-modulated as well in HL-60 and NB4 cells under the same experimental conditions (Bertagnolo et al., 2008). Since proteasome is the major cellular proteolytic machinery responsible for degradation of normal and damaged proteins (Von Mikecz, 2006), Vav1 may also be involved in regulating

Also the component of microtubules -tubulin is affected by down-modulation of Vav1 during ATRA treatment (Bertagnolo et al., 2008), indicating that Vav1, in addition to regulate cytoskeleton reorganization, takes part to the profound architectural changes of differentiating promyelocytes by regulating the expression of cytoskeleton components.

The human promyelocytic leukemia cell lines HL-60 and NB4 can be differentiated either toward neutrophils by ATRA or to monocytes/macrophages by PMA (Murao et al., 1983; Song & Norman, 1998). PMA is a stable analogue of 2, 3-diacylglycerol that induces, even if with dynamics not identical in HL-60 and NB4 cells, morphological and functional changes related to monocyte maturation, accompanied by a loss of proliferative capacity (Jasek et al., 2008). Immunochemical and immunocytochemical analysis demonstrate that the expression of Vav1 increases also during the PMA-induced acquisition of a monocyte-like phenotype of HL-60 and NB4 cells (Bertagnolo et al., 2011). This is consistent with the notion that also mature monocytes express Vav1 and that proper amounts of the protein are necessary for their inflammation related functions (Bhavsar et al., 2009; Hall et al., 2006). In the same cell types, PMA also induces a relevant increase of tyrosine phosphorylation of Vav1. On the other hand, and in constrast to what observed in the ATRA-treatment of the same cell line, no role for Syk was demonstrated in this event (Bertagnolo et al., 2011), consistent with the notion that, at least in HL-60 cells, Syk might exert a narrower role, restricted to directing cells toward granulocyte differentiation (Qin & Yamamura, 1997). In both HL-60 and NB4 cells, PMA induces a relevant increase of tyrosine phosphorylation of Vav1 on the Tyr174 residue (Bertagnolo et al., 2011), according to the GEF role played by Vav1 in myeloid cells. These results are also in agreement with other data indicating that, in macrophage-like differentiated HL-60 cells, the activity of Syk is ended to regulate the roles played by mature cells in immune response, including their complement-mediated phagocytosis, in which the kinase regulates both actin dynamics and the Vav1-RhoA activation pathway (Shi et al.,

Also in differentiation of APL-derived cells to monocytes/macrophages, a crucial role for Vav1 in determining the acquisition of maturation-related features has been demonstrated by silencing the expression of Vav1 induced by PMA (Bertagnolo et al., 2011). Under these conditions, the expression of CD11b, which is induced by PMA and constitutes a marker also for monocyte differentiation, is significantly reduced, similarly to what demonstrated during the treatment with ATRA of HL-60 and NB4 cells (Bertagnolo et al. 2011). This suggests that, in differentiating APL-derived cells, Vav1 plays a role in regulating the expression of the CD11b surface antigen regardless the agonist employed and the maturation lineage. Since in NB4 cells treated with ATRA Vav1 is recuited to protein/DNA complexes on the *CD11b* promoter (Brugnoli et al., 2010), it can be speculated that Vav1 plays a specific role in driving the expression of CD11b as part of the transcriptional machinery also during the differentiation of NB4 cells along the monocytic/macrophagic

protein degradation during ATRA dependent maturation of tumoral promyelocytes.

**4. Vav1 and monocytic/macrophagic differentiation** 

2006).

lineage.

As above reported, the main known functional role of tyrosine phosphorylated Vav1 is to regulate cytoskeleton reorganization, a phenomenon at the basis of both adhesion and migration of monocytes/macrophages. Vav1 regulates cell architecture not only by means of its GEF activity but also by interacting with proteins in turn involved in cytoskeleton reorganization. In addition, in both HL-60 and NB4 cells, Vav1 affects the ATRA-induced expression of the microtubule component -tubulin.

Contrarily to what observed during granulocytic differentiation, Vav1 down-modulation does not have any effect on expression and architectural organization of -tubulin during PMA-induced monocytic/macrophagic maturation of NB4 cells (Bertagnolo et al., 2011). This indicates that, during the maturation process of APL-derived cells, Vav1 exerts an agonist- and lineage-specific role in regulating -tubulin. From a more general point of view, concerning microtubule organization, it can be speculated that the role of Vav1 is restricted to the control of the motility of mature cells, as also suggested by the evidence that changes in microtubule dynamics contribute to the reduced migration speed of Vav1\_/\_ macrophages in response to CSF-1 (Wells et al., 2005).

In living cells, the F-actin cytoskeleton encompasses a variety of different structures that are essential for many different aspects of cell physiology. In particular, dynamic modulation of the filamentous actin cytoskeleton is critical to numerous physical cellular processes, including adhesion, migration and phagocytosis, all requiring precise regulation of cell shape (Stricker et al., 2010). Recent data demonstrate that Vav proteins, including Vav1, are required for actin cytoskeleton reorganization during migration of macrophages, by coupling RhoA and Rac1 activity to adhesion receptors (Bhavsar et al., 2009). Also in ATRAinduced maturation of cells derived from APL Vav1 seems to regulate actin organization.

In HL-60 and NB4 cells treated with PMA, an unprecedented involvement of Vav1 in regulating the increase of actin expression has been shown very recently (Bertagnolo et al., 2011), that constitutes a further confirmation that Vav1, besides being involved in the formation of filaments, takes part to cytoskeleton reorganization as a modulator of protein expression.

The modifications of cell shape in the different cell processes seem to be regulated by the existence of the F-actin cortex, a thin, membrane-bound F-actin network (Stricker et al., 2010). Defective actin-cap formation has been found in lymphocytes from a Vav-deficient mice, clearly correlating Vav1 activity with the regulation of cell shape (Holsinger et al., 1998). Furthermore, a recent work in which adhesive micropatterned surfaces have been used to control the overall shape of fibroblasts, has demonstrated that the shape of the nucleus is tightly regulated through a perinuclear actin cap, which is located above and around the interphase nucleus (Khatau et al., 2009). A wide variety of contractile F-actin networks with different architectures and polarity have also been found near cell adhesion surfaces, correlated with the migratory capability of adherent cells (Stricker et al., 2010). By means of confocal analysis of PMA-treated adherent NB4 cells, it has been demonstrated the

Vav1: A Key Player in Agonist-Induced Differentiation of

Promyelocytes from Acute Myeloid Leukemia (APL) 279

Fig. 3. Overall role of Vav1 in regulating maturation of APL-derived promyelocytes.

MAE (Italy-Croatia bilateral project 2009-2011) and by University of Ferrara (Italy).

*Leukemia Research,* Vol.32, No.2, (February 2008), pp. 315-322.

The authors are supported by MIUR (Cofin 2008 and FIRB Accordi di Programma 2010),

Barber, N., Belov, & L., Christopherson, R.I. (2008). All-trans retinoic acid induces different

immunophenotypic changes on human HL60 and NB4 myeloid leukaemias.

**6. Acknowledgments** 

**7. References** 

existence of an agonist-induced F-actin network, in which F-actin colocalyzes with Vav1, that sharply defines the cytoplasmatic cell border, accumulates inside thin and long cell processes and surrounds the nuclear compartment (Bertagnolo et al., 2011). Since the existence of cytoplasmatic processes in PMA-treated adherent cells are indicative of migratory activity (Stricker et al., 2010), Vav1/F-actin co-localization in cytoplasm protrusions is suggestive of a synergy of the two molecules in controling cell motility. This is in agreement with the role described for Vav proteins in the maintenance of normal morphology and migratory behaviour in macrophages (Bhavsar et al., 2009). The strong Vav1/F-actin co-localization observed at the nuclear periphery and, in particular, in the region above the nucleus, suggests that the two proteins may cooperate in regulating the shape of the nucleus through an actin filament structure similar to the perinuclear actin cap described by Khatau et al. (2009). On the other hand, the role of Vav1 in modulating cell adhesion of PMA-treated cells seems to be related to its ability to regulate expression of integrins, like CD11b, rather than to a direct effect on actin-based cytoskeleton.

#### **5. Conclusion**

The present review focuses on the role of the multidomain protein Vav1 in promoting and sustaining the completion of the differentiation program of tumoral promyelocytes. Vav1 is a key protein in the ATRA- and PMA-induced maturation of APL-derived cells, since either its down-modulation or over-expression respectively prevents or potentiates the ability of these agonists to induce the acquisition of a mature phenotype. Alternatively to the best known function of Vav1 as a GEF for small G proteins, ended to regulate cell shape by affecting actin assembly, other mechanisms by which Vav1 affects myeloid differentiation have been described, reflecting the great interactive and regulatory potential of Vav1, which make the full understanding of its functions a very difficult, yet fascinating story.

An example of the complex role played by Vav1 during myeloid differentiation of APLderived cells is the interaction of Vav1 with various lipid-modifying enzymes ended to regulate the pool of phosphoinositides associated to cytoskeleton. The resulting modifications of actin cytoskeleton contribute to the changes of cellular and nuclear shape occurring in differentiating tumoral promyelocytes (Fig. 3).

The participation of Vav1 to molecular complexes with other adaptor proteins differently distributed in the cytoplasm and in the nucleus suggests the existence of a signal sequence originated from membrane receptors and directed to the nuclear compartment. Inside the nucleus of APL-derived cells, Vav1 seems to play its most intriguing role by regulating the expression of CD11b, a surface marker of both granulocyte and monocyte differentiation, and of a number of ATRA-modulated proteins (Fig. 3). The nuclear issue assumes thus great relevance, confering to Vav1 compartimentalized strategic roles in regulating the maturation process of tumoral promyelocytes.

The bulk of the studies reviewed here are mostly concerned with two cell lines, HL-60 and NB4, derived from APL patients, driven to achieve differentiation by treatment with drugs of the retinoids or phorbol esters families. Even though a better understanding of the functional engagement of Vav1 will be required before converting scientific achievements into clinical advances, Vav1 might be considered a common target for developing new therapeutic strategies for the different subtypes of myeloid leukemias.

In addition, it can be speculated that the identified pathways involving Vav1 are of more general interest and may be potentially extended also outside the haemopoietic/immunological systems.

existence of an agonist-induced F-actin network, in which F-actin colocalyzes with Vav1, that sharply defines the cytoplasmatic cell border, accumulates inside thin and long cell processes and surrounds the nuclear compartment (Bertagnolo et al., 2011). Since the existence of cytoplasmatic processes in PMA-treated adherent cells are indicative of migratory activity (Stricker et al., 2010), Vav1/F-actin co-localization in cytoplasm protrusions is suggestive of a synergy of the two molecules in controling cell motility. This is in agreement with the role described for Vav proteins in the maintenance of normal morphology and migratory behaviour in macrophages (Bhavsar et al., 2009). The strong Vav1/F-actin co-localization observed at the nuclear periphery and, in particular, in the region above the nucleus, suggests that the two proteins may cooperate in regulating the shape of the nucleus through an actin filament structure similar to the perinuclear actin cap described by Khatau et al. (2009). On the other hand, the role of Vav1 in modulating cell adhesion of PMA-treated cells seems to be related to its ability to regulate expression of

integrins, like CD11b, rather than to a direct effect on actin-based cytoskeleton.

make the full understanding of its functions a very difficult, yet fascinating story.

occurring in differentiating tumoral promyelocytes (Fig. 3).

therapeutic strategies for the different subtypes of myeloid leukemias.

process of tumoral promyelocytes.

haemopoietic/immunological systems.

The present review focuses on the role of the multidomain protein Vav1 in promoting and sustaining the completion of the differentiation program of tumoral promyelocytes. Vav1 is a key protein in the ATRA- and PMA-induced maturation of APL-derived cells, since either its down-modulation or over-expression respectively prevents or potentiates the ability of these agonists to induce the acquisition of a mature phenotype. Alternatively to the best known function of Vav1 as a GEF for small G proteins, ended to regulate cell shape by affecting actin assembly, other mechanisms by which Vav1 affects myeloid differentiation have been described, reflecting the great interactive and regulatory potential of Vav1, which

An example of the complex role played by Vav1 during myeloid differentiation of APLderived cells is the interaction of Vav1 with various lipid-modifying enzymes ended to regulate the pool of phosphoinositides associated to cytoskeleton. The resulting modifications of actin cytoskeleton contribute to the changes of cellular and nuclear shape

The participation of Vav1 to molecular complexes with other adaptor proteins differently distributed in the cytoplasm and in the nucleus suggests the existence of a signal sequence originated from membrane receptors and directed to the nuclear compartment. Inside the nucleus of APL-derived cells, Vav1 seems to play its most intriguing role by regulating the expression of CD11b, a surface marker of both granulocyte and monocyte differentiation, and of a number of ATRA-modulated proteins (Fig. 3). The nuclear issue assumes thus great relevance, confering to Vav1 compartimentalized strategic roles in regulating the maturation

The bulk of the studies reviewed here are mostly concerned with two cell lines, HL-60 and NB4, derived from APL patients, driven to achieve differentiation by treatment with drugs of the retinoids or phorbol esters families. Even though a better understanding of the functional engagement of Vav1 will be required before converting scientific achievements into clinical advances, Vav1 might be considered a common target for developing new

In addition, it can be speculated that the identified pathways involving Vav1 are of more general interest and may be potentially extended also outside the

**5. Conclusion** 

Fig. 3. Overall role of Vav1 in regulating maturation of APL-derived promyelocytes.

#### **6. Acknowledgments**

The authors are supported by MIUR (Cofin 2008 and FIRB Accordi di Programma 2010), MAE (Italy-Croatia bilateral project 2009-2011) and by University of Ferrara (Italy).

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**15** 

 *Washington DC,* 

*USA* 

**p15INK4b, a Tumor Suppressor** 

*1Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, Maryland,* 

*p15INK4b* expression is lost in a striking 80% of all patients suffering from acute myeloid leukemia (AML). Specific inactivation of the gene by aberrant promoter hypermethylation is also detected in about 50% of patients diagnosed with myelodysplastic syndromes (MDS) and almost 60% of patients with myeloproliferative disorders (MPD). More importantly, a strong correlation between the methylation levels of *p15INK4b* and poor prognosis is now well established in these patients. Hypermethylation levels also provide a marker for subsequent transformation and progression of the disease to a more aggressive phenotype. These clinical observations establish the repression of *p15INK4b* expression by promoter hypermethylation as the most prevalent genetic abnormality in myeloid leukemia. The *p15INK4b* gene (also referred to as *CDKN2B* and *MTS2*) encodes a 15kDa cyclin dependent kinase inhibitor (CDKI). Specific and preferential epigenetic targeting of *p15INK4b* for silencing over other CDKIs such as *p16INK4a* and *p21WAF/CIP* in AML, MDS and MPD patients strongly supports a role for this protein as a tumor suppressor in hematological

This chapter provides a review of the literature outlining the high prevalence of *p15INK4b* loss of expression in human myeloid malignancies, as well as the latest research carried out in mice which supports a role for p15Ink4b as a tumor suppressor. It also focuses on the well established function of p15INK4b in the control of the cell cycle, as well as its role during early and late myeloid cells development. Finally, this chapter discusses the multiple mechanisms by which *p15INK4b* is silenced and presents a few examples of clinical studies of drugs that target *p15INK4b* for re-expression. These include treatments for reversing aberrant DNA methylation, and are currently being tested and used for the therapy of MDS

*p15INK4b* silencing by promoter hypermethylation occurs almost exclusively in cancers of the hematopoietic system, and is observed in acute leukemias of myeloid (AML) and lymphoid (ALL) origins (Drexler, 1998). Aberrant hypermethylation occurs at the gene's

**1. Introduction** 

and AML.

malignancies of the myeloid lineage.

**2. Role of p15INK4b in myeloid malignancies** 

**2.1 Inactivation in human AML and MDS** 

*2Georgetown University, Department of Biochemistry and Molecular Biology,* 

**in Acute Myeloid Leukemia** 

Joanna Fares1,2, Linda Wolff1 and Juraj Bies1


### **p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia**

Joanna Fares1,2, Linda Wolff1 and Juraj Bies1 *1Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, Maryland, 2Georgetown University, Department of Biochemistry and Molecular Biology, Washington DC, USA* 

#### **1. Introduction**

288 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Zhang, R., Tsai, F.Y., & Orkin, S.H. (1994). Hematopoietic development of vav-/- mouse

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pp. 1387-1391.

embryonic stem cells. *Proceedings of the National Academy of Sciences of the United* 

Y., Zhi, Y. (2005). Identification of leukemia-associated antigens in chronic myeloid leukemia by proteomic analysis. *Leukemia Research,* Vol.29, No.12, (December 2005),

> *p15INK4b* expression is lost in a striking 80% of all patients suffering from acute myeloid leukemia (AML). Specific inactivation of the gene by aberrant promoter hypermethylation is also detected in about 50% of patients diagnosed with myelodysplastic syndromes (MDS) and almost 60% of patients with myeloproliferative disorders (MPD). More importantly, a strong correlation between the methylation levels of *p15INK4b* and poor prognosis is now well established in these patients. Hypermethylation levels also provide a marker for subsequent transformation and progression of the disease to a more aggressive phenotype. These clinical observations establish the repression of *p15INK4b* expression by promoter hypermethylation as the most prevalent genetic abnormality in myeloid leukemia. The *p15INK4b* gene (also referred to as *CDKN2B* and *MTS2*) encodes a 15kDa cyclin dependent kinase inhibitor (CDKI). Specific and preferential epigenetic targeting of *p15INK4b* for silencing over other CDKIs such as *p16INK4a* and *p21WAF/CIP* in AML, MDS and MPD patients strongly supports a role for this protein as a tumor suppressor in hematological malignancies of the myeloid lineage.

> This chapter provides a review of the literature outlining the high prevalence of *p15INK4b* loss of expression in human myeloid malignancies, as well as the latest research carried out in mice which supports a role for p15Ink4b as a tumor suppressor. It also focuses on the well established function of p15INK4b in the control of the cell cycle, as well as its role during early and late myeloid cells development. Finally, this chapter discusses the multiple mechanisms by which *p15INK4b* is silenced and presents a few examples of clinical studies of drugs that target *p15INK4b* for re-expression. These include treatments for reversing aberrant DNA methylation, and are currently being tested and used for the therapy of MDS and AML.

#### **2. Role of p15INK4b in myeloid malignancies**

#### **2.1 Inactivation in human AML and MDS**

*p15INK4b* silencing by promoter hypermethylation occurs almost exclusively in cancers of the hematopoietic system, and is observed in acute leukemias of myeloid (AML) and lymphoid (ALL) origins (Drexler, 1998). Aberrant hypermethylation occurs at the gene's

p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia 291

stages of MDS such as refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) (Uchida et al., 1997; Christiansen et al., 2003). However, more recently, it was found that even in patients with the RARS subtype, which falls within the lower risk of MDS, *p15INK4b* was found to be the most frequently methylated gene (>20% of cases) of 25 known tumor suppressors that were evaluated in the study (Valencia et al., 2011). Although most studies have been conducted in MDS in adults, comparable levels of aberrant methylation patterns have been observed in pediatric MDS patients as well (Hasegawa et al., 2005), and a similar correlation with the disease subtypes has been

In chronic myelomonocytic leukemia (CMML), aberrant methylation of *p15INK4b* is found in about 60% of cases and is associated with a high proportion of blastic transformation (Tessema et al., 2003). CMML is a disease that was originally categorized under myelodysplastic disorders, but is now classified by the WHO as a disorder that bridges MDS with myeloproliferative features (Harris et al., 1999). In these patients, genomic sequencing techniques have revealed that hypermethylation spans a wide area in the 5' region of the gene and is correlated with reduced expression of the mRNA levels. High variability between and within individual patients, consistent with observations in AML patients, were also reported (Tessema et al., 2003, Aggerholm et al., 1999, Cameron et al., 1999). In the pediatric form of the disease, juvenile myelomonocytic leukemia (JMML), *p15INK4b* hypermethylation is found to be a less frequent, however, still significant event

With regards to cytogenetic abnormalities, *p15INK4b* methylation levels have been found to occur at higher frequencies in AML/MDS patients with an unfavorable karyotype (Wong et al., 2000; Galm et al., 2005; Shimamoto et al., 2005; Markus et al., 2007). Cases with unmethylated or low levels of hypermethylated *p15INK4b* were associated with normal karyotype or with those karyotypic abnormalities that are associated with a favorable prognosis (Wong et al., 2000; Markus et al., 2007). Studies have consistently reported an increased tendency for *p15INK4b* hypermethylation in unfavorable cytogenetics (Shimamoto et al., 2005). These results suggest interplay between *p15INK4b* loss of expression and the frequent chromosomal translocations, inversions and deletions observed in AML and MDS. The mechanisms underlying *p15INK4b* hypermethylation are not completely understood, but a few theories involving maintaining and de-novo DNA methylation through action of DNA methyltransferases (DNMT), as well as histone modification pathways have been suggested to play a role (Paul et al., 2010). Specifically in t-AML and therapy-induced MDS (t-MDS), deletion or loss of chromosome arm 7q, which is the most common cytogenetic abnormality in those categories, has been found to be closely associated with

*p15INK4b* is now used as an independent prognosticator in AML and MDS (Chim et al., 2001b; Teofili et al., 2003; Christiansen et al., 2003; Shimamoto et al., 2005; Chim et al., 2006). In the many categories of the diseases, aberrant *p15INK4b* methylation levels have been associated with a generally poor prognosis. In studies that monitor patients across all AML FAB subtypes, patients without *p15INK4b* hypermethylation at diagnosis had increased complete remission rates which also correlated with increased survival times (Shimamoto et al., 2005; Deneberg et al., 2010). Consistent with these observations, in APL patients, abnormal *p15INK4b* methylation was associated with a shorter disease-free survival (DFS) period and a higher incidence of relapse during the 5-year follow up period (Teofili et al.,

established (Rodrigues et al., 2010).

(17% of cases) (Hasegawa et al., 2005).

hypermethylation of *p15INK4b* (Christiansen et al., 2003).

CpG islands which extend throughout the promoter region, exon 1 and part of intron 1 (Herman et al., 1996). DNA methylation is the addition of methyl groups on cytosine bases on the DNA molecules of mammalian cells which affects gene expression (Deaton et al., 2011). Methylation is carried out by the enzymes DNA methyltransferases (DNMT) which catalyze the reaction converting cytosine to 5-methylcytosine (Bird et al., 2002). In the earlier studies DNA methylation was assessed by southern blotting and methylationspecific PCR technique and more recently by more sensitive assays including bisulfate pyrosequencing and genome-wide sequencing methods (Deaton et al., 2011).

Despite the broad clinical diversity of AML, with more than a hundred cytogenetic alterations described (Vardiman et al., 2002; Trost et al., 2006), aberrant methylation of *p15INK4b* has been reported in up to 80% of patients with primary and secondary AML. Hypermethylation levels have been shown to correlate with a reduction in the mRNA and the protein expression levels of p15INK4b (Cameron et al., 1999; Matsuno et al., 2005). Furthermore, density of the methylation has been shown to vary greatly between and within AML patients and its levels closely correlate with the degree of transcriptional repression (Aggerholm et al., 1999, Cameron et al., 1999). AML classification into ten different subtypes was originally defined by the French-American-British (FAB) cooperative group (Bennett et al., 1976, 1985). Numerous studies have been conducted to assess the methylation levels of *p15INK4b* on samples of patients with AML across the different FAB subtypes (Herman et al., 1996, 1997; Aggerholm et al., 1999; Guo et al., 2000; Chim et al., 2001a, 2001b; Christiansen et al., 2003; Garcia-Manero et al., 2003; Teofili et al., 2003; Shimamoto et al., 2005). In patients with adult and childhood AML, hypermethylation of *p15INK4b* in cells isolated from bone marrow and peripheral blood is observed in nearly all morphological FAB subtypes. Higher frequencies are generally observed in the M1, M2, M3 and M4 subtypes than in the M5, M6 or M7 subtypes and are found to occur in the vast majority of the patients' leukemic cells (Aggerholm et al., 1999; Wong et al., 2000; Shimamoto et al., 2005; Tsellou et al., 2005). In patients with therapy-induced AML (t-AML), aberrant methylation of *p15INK4b* (in over 90% of patients) is found to be independent from the patient's type of previous therapy which ranges from alkylating agents, topoisomerase II inhibitors to radiotherapy (Christiansen et al., 2003).

A similar pattern of aberrant methylation is also well documented in patients with MDS (Uchida et al., 1997; Quesnel et al., 1998; Aoki et al., 2000; Tien et al., 2001; Christiansen et al., 2003; Teofili et al., 2003). The FAB classification system for MDS is mainly based on the percentage of blast cells in the bone marrow and the peripheral blood and the degree of cytopenia (Bennet et al., 1982). Methylation levels have also been shown to increase during follow-up and in conversion to overt AML (Tien et al., 2001, Christiansen et al., 2003). Importantly, aberrant DNA methylation of *p15INK4b* was found to be one of the most dominant molecular events in MDS progression to AML (Jiang et al., 2009). Similar to AML, cytogenetics of MDS is also a crucial factor in the prognosis and development of the disease (Haase et al., 2007). The World Health Organization (WHO) classification system for myeloid neoplasms was developed and takes into consideration both morphology and cytogenetic abnormalities (Harris et al., 2000). In MDS patients, methylation of *p15INK4b* is associated with an increased percentage of immature myeloblasts in the bone marrow (Christiansen et al., 2003). The presence of DNA hypermethylation at the *p15INK4b* promoter is found predominantly in high risk MDS patients with increased levels being reported in the subtypes characterized by advanced stages of the disease such as refractory anemia with excess blasts (RAEB). Reduced levels are reported in patients with the early

CpG islands which extend throughout the promoter region, exon 1 and part of intron 1 (Herman et al., 1996). DNA methylation is the addition of methyl groups on cytosine bases on the DNA molecules of mammalian cells which affects gene expression (Deaton et al., 2011). Methylation is carried out by the enzymes DNA methyltransferases (DNMT) which catalyze the reaction converting cytosine to 5-methylcytosine (Bird et al., 2002). In the earlier studies DNA methylation was assessed by southern blotting and methylationspecific PCR technique and more recently by more sensitive assays including bisulfate

Despite the broad clinical diversity of AML, with more than a hundred cytogenetic alterations described (Vardiman et al., 2002; Trost et al., 2006), aberrant methylation of *p15INK4b* has been reported in up to 80% of patients with primary and secondary AML. Hypermethylation levels have been shown to correlate with a reduction in the mRNA and the protein expression levels of p15INK4b (Cameron et al., 1999; Matsuno et al., 2005). Furthermore, density of the methylation has been shown to vary greatly between and within AML patients and its levels closely correlate with the degree of transcriptional repression (Aggerholm et al., 1999, Cameron et al., 1999). AML classification into ten different subtypes was originally defined by the French-American-British (FAB) cooperative group (Bennett et al., 1976, 1985). Numerous studies have been conducted to assess the methylation levels of *p15INK4b* on samples of patients with AML across the different FAB subtypes (Herman et al., 1996, 1997; Aggerholm et al., 1999; Guo et al., 2000; Chim et al., 2001a, 2001b; Christiansen et al., 2003; Garcia-Manero et al., 2003; Teofili et al., 2003; Shimamoto et al., 2005). In patients with adult and childhood AML, hypermethylation of *p15INK4b* in cells isolated from bone marrow and peripheral blood is observed in nearly all morphological FAB subtypes. Higher frequencies are generally observed in the M1, M2, M3 and M4 subtypes than in the M5, M6 or M7 subtypes and are found to occur in the vast majority of the patients' leukemic cells (Aggerholm et al., 1999; Wong et al., 2000; Shimamoto et al., 2005; Tsellou et al., 2005). In patients with therapy-induced AML (t-AML), aberrant methylation of *p15INK4b* (in over 90% of patients) is found to be independent from the patient's type of previous therapy which ranges from alkylating agents, topoisomerase II

A similar pattern of aberrant methylation is also well documented in patients with MDS (Uchida et al., 1997; Quesnel et al., 1998; Aoki et al., 2000; Tien et al., 2001; Christiansen et al., 2003; Teofili et al., 2003). The FAB classification system for MDS is mainly based on the percentage of blast cells in the bone marrow and the peripheral blood and the degree of cytopenia (Bennet et al., 1982). Methylation levels have also been shown to increase during follow-up and in conversion to overt AML (Tien et al., 2001, Christiansen et al., 2003). Importantly, aberrant DNA methylation of *p15INK4b* was found to be one of the most dominant molecular events in MDS progression to AML (Jiang et al., 2009). Similar to AML, cytogenetics of MDS is also a crucial factor in the prognosis and development of the disease (Haase et al., 2007). The World Health Organization (WHO) classification system for myeloid neoplasms was developed and takes into consideration both morphology and cytogenetic abnormalities (Harris et al., 2000). In MDS patients, methylation of *p15INK4b* is associated with an increased percentage of immature myeloblasts in the bone marrow (Christiansen et al., 2003). The presence of DNA hypermethylation at the *p15INK4b* promoter is found predominantly in high risk MDS patients with increased levels being reported in the subtypes characterized by advanced stages of the disease such as refractory anemia with excess blasts (RAEB). Reduced levels are reported in patients with the early

pyrosequencing and genome-wide sequencing methods (Deaton et al., 2011).

inhibitors to radiotherapy (Christiansen et al., 2003).

stages of MDS such as refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) (Uchida et al., 1997; Christiansen et al., 2003). However, more recently, it was found that even in patients with the RARS subtype, which falls within the lower risk of MDS, *p15INK4b* was found to be the most frequently methylated gene (>20% of cases) of 25 known tumor suppressors that were evaluated in the study (Valencia et al., 2011). Although most studies have been conducted in MDS in adults, comparable levels of aberrant methylation patterns have been observed in pediatric MDS patients as well (Hasegawa et al., 2005), and a similar correlation with the disease subtypes has been established (Rodrigues et al., 2010).

In chronic myelomonocytic leukemia (CMML), aberrant methylation of *p15INK4b* is found in about 60% of cases and is associated with a high proportion of blastic transformation (Tessema et al., 2003). CMML is a disease that was originally categorized under myelodysplastic disorders, but is now classified by the WHO as a disorder that bridges MDS with myeloproliferative features (Harris et al., 1999). In these patients, genomic sequencing techniques have revealed that hypermethylation spans a wide area in the 5' region of the gene and is correlated with reduced expression of the mRNA levels. High variability between and within individual patients, consistent with observations in AML patients, were also reported (Tessema et al., 2003, Aggerholm et al., 1999, Cameron et al., 1999). In the pediatric form of the disease, juvenile myelomonocytic leukemia (JMML), *p15INK4b* hypermethylation is found to be a less frequent, however, still significant event (17% of cases) (Hasegawa et al., 2005).

With regards to cytogenetic abnormalities, *p15INK4b* methylation levels have been found to occur at higher frequencies in AML/MDS patients with an unfavorable karyotype (Wong et al., 2000; Galm et al., 2005; Shimamoto et al., 2005; Markus et al., 2007). Cases with unmethylated or low levels of hypermethylated *p15INK4b* were associated with normal karyotype or with those karyotypic abnormalities that are associated with a favorable prognosis (Wong et al., 2000; Markus et al., 2007). Studies have consistently reported an increased tendency for *p15INK4b* hypermethylation in unfavorable cytogenetics (Shimamoto et al., 2005). These results suggest interplay between *p15INK4b* loss of expression and the frequent chromosomal translocations, inversions and deletions observed in AML and MDS. The mechanisms underlying *p15INK4b* hypermethylation are not completely understood, but a few theories involving maintaining and de-novo DNA methylation through action of DNA methyltransferases (DNMT), as well as histone modification pathways have been suggested to play a role (Paul et al., 2010). Specifically in t-AML and therapy-induced MDS (t-MDS), deletion or loss of chromosome arm 7q, which is the most common cytogenetic abnormality in those categories, has been found to be closely associated with hypermethylation of *p15INK4b* (Christiansen et al., 2003).

*p15INK4b* is now used as an independent prognosticator in AML and MDS (Chim et al., 2001b; Teofili et al., 2003; Christiansen et al., 2003; Shimamoto et al., 2005; Chim et al., 2006). In the many categories of the diseases, aberrant *p15INK4b* methylation levels have been associated with a generally poor prognosis. In studies that monitor patients across all AML FAB subtypes, patients without *p15INK4b* hypermethylation at diagnosis had increased complete remission rates which also correlated with increased survival times (Shimamoto et al., 2005; Deneberg et al., 2010). Consistent with these observations, in APL patients, abnormal *p15INK4b* methylation was associated with a shorter disease-free survival (DFS) period and a higher incidence of relapse during the 5-year follow up period (Teofili et al.,

p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia 293

Although hypermethylation is the most common mode of inactivation of *p15INK4b* in myeloid neoplasms, other silencing mechanisms have also been described. In AML with chromosome 16 inversion (*inv16*), the overall *p15INK4b* methylation levels are found to be very low and almost comparable to levels in normal patients. However, expression of the gene is severely suppressed. In this type of AML, the inversion (16) results in a fusion protein between the core binding factor (CBFβ) and the smooth myosin heavy chain gene (SMMHC). This chimeric transcription factor CBFβ-SMMHC binds directly to the promoter of *p15INK4b* and represses its expression (Markus et al., 2007). These results further emphasize an important role of *p15INK4b* silencing in leukemogenesis of the myeloid lineage, and suggest, that in the absence of a repressive epigenetic event, other mechanisms

In B and T cell acute lymphoblastic leukemias (B-ALL, T-ALL), *p15INK4b* hypermethylation as well as deletion of the entire 9p21 locus which includes *p15INK4b*, *p16INK4a* and *ARF* genes has been reported (Roussel, 1999; Ruas et al., 1998). Homozygous deletions of *p16INK4a* and *p15INK4b* are found in approximately 30% of childhood acute lymphoblastic leukemia at first presentation, with striking rates in T-ALL (60 to 80%), and lower rates in Bcell precursor ALL (5 to 20%) (Drexler HG, 1998; Chim et al., 2001a). Further studies have analyzed methylation levels of the two genes in these disease categories specifically in terms of overall survival and absence of relapse at 6 years of follow-up. *p15INK4b* and *p16INK4a* methylation levels were found to occur at similar rates (35%) in adults and children with mature B-ALL (Graf-Einsiedel et al., 2002). Deletion of the entire locus was observed in 12% and 30% of children and adults, respectively. Interestingly, results show that deletion is associated with poor overall survival (OS) in adults only, but not in children (Van Zutven et al., 2005; Mirebeau et al., 2006; Kim et al., 2009). Furthermore, it did not affect the type of relapse or DFS time in children. In untreated adult patients with precursor B-ALL, high methylation levels of *p15INK4b* (found in 43% of patients) were significantly associated with decreased DFS at 4 years (Hoshino et al., 2002). Recent methylation profiles in 95 children with ALL supported older studies showing that methylation of *p15INK4b* occurred predominantly in T-ALL as opposed to B-ALL, and *p15INK4b* is one of the most commonly methylated genes among the 14 genes analyzed (Takeuchi et al., 2011). A clear correlation

between increased methylation and prognoses in T-ALL has not been established.

p15INK4b function plays an important role in the development of myeloid leukemia.

To define the role of p15INK4b as a tumor suppressor in AML, mouse models have been developed and characterized (Latres et al., 2000; Wolff et al., 2003a, 2004; Bies et al., 2010). These have provided strong experimental evidence to support the hypothesis that loss of

A p15Ink4b-/- mouse model was first described by Latres et al. (2000). Mice were generated by genetic targeting with elimination of the second coding exon of the p15Ink4b gene. Knockout mice were viable, fertile, and did not exhibit any behavioral abnormalities. Mouse embryonic fibroblasts were found to have a higher proliferation rate and plating efficiency when compared to their wild type counterparts. More importantly, they were more susceptible to transformation with *c-myc* and *ras* oncogenes, confirming the reported results that p15Ink4b participates in the tumor suppressor activity triggered after inappropriate

may result in inhibition of *p15INK4b* expression (Markus et al., 2007).

**2.2 Inactivation in other types of human leukemias** 

**2.3 p15Ink4b as a tumor suppressor in mice** 

2003). In the relapsed patients, the *p15INK4b* hypermethylation levels remained persistent following treatment. In contrast, the patients without detectable hypermethylation displayed prolonged survival (Chim et al., 2001b). Additionally, as previously stated, patients with MDS with high methylation levels at diagnosis had a significantly higher chance of the disease progressing to AML (Tien et al., 2001; Jiang et al., 2009). It was also reported that in early stage of MDS, the *p15INK4b* hypermethylation is a negative risk factor for patients, closely correlating with leukemic transformation (Aggerholm et al., 2006). The same correlation has been shown in patients with t-MDS, in which methylation resulted in significantly shorter survival (Christiansen et al., 2003). A recent study showed that the high levels of methylation in lower risk MDS categories suggest a poor prognosis in those patients as well (Valencia et al, 2011). In JMML p15INK4b hypermethylation was associated with reduced overall survival rates and higher relapse of the disease following hematopoietic stem cell transplantation (Olk-Batz et al., 2011). All these results suggest that lack of *p15INK4b* expression, mediated by promoter hypermethylation, not only affects the prognosis in patients with AML and MDS, but can be used to predict the outcome of the diseases.

The studies described above confirm that aberrant hypermethylation levels of *p15INK4b* have important prognostic implications for clinical monitoring in MDS and assessment of risk of progression into AML. However, its potential use as a biomarker in leukemia excluded estimation of minimal residual disease in patients who have achieved clinical remission, and its implications in terms of subsequent relapse. A study aiming at addressing this issue, evaluated *p15INK4b* methylation levels in AML patients in complete clinical remission (Agrawal et al., 2007). The study reported that even in remission, leukemia patients that harbored a significant amount of methylation in the bone marrow cells had a higher risk for leukemia relapse. Moreover, the time of disease-free survival was found to be significantly reduced in correlation with the amount of residual hypermethylation of the *p15Ink4b* gene. Concurrently, low levels of *p15INK4b* methylation during complete remission were associated with reduced relapse rates during the 12 month follow-up. It was suggested that analysis of *p15INK4b* methylation levels during clinical remission can be potentially used as a prognosticator for the occurrence of relapse (Agrawal et al., 2007).

In recent years, it has been suggested in a number of studies that DNA methylation of *p15INK4b* could also help predict response to therapy (Grovdal et al., 2007; Shen et al., 2009). Grövdal et al. (2007) studied DNA methylation patterns in older patients with high risk MDS and AML following MDS. Patients were treated with conventional induction therapy. Methylation levels of *p15INK4b*, *E-cadherin*, and *HIC1* (hypermethylated in cancer 1), were assessed prior to initiation of treatment. Abnormal levels of methylation of *p15INK4b* alone did not correlate with decreased complete remission (CR), but all patients with all three genes methylated did not achieve CR. Another study, in which patients with MDS and AML were treated with the DNA methyltransferase inhibitor 5-azacytidine (5-aza-C), reported consistent results with these observations (Raj et al., 2007; Tran et al., 2011). Patients with levels exceeding 24% methylation in the *p15Ink4b* promoter region did not respond to treatment (Raj et al., 2007). The possibility of using methylation of *p15INK4b* as an indicator for treatment outcome is still under investigation. However, results suggest that studying *p15INK4b* methylation density in conjunction with other altered genes at diagnosis and monitoring its levels following treatment might have predictive information with respect to the patient's response to treatment.

2003). In the relapsed patients, the *p15INK4b* hypermethylation levels remained persistent following treatment. In contrast, the patients without detectable hypermethylation displayed prolonged survival (Chim et al., 2001b). Additionally, as previously stated, patients with MDS with high methylation levels at diagnosis had a significantly higher chance of the disease progressing to AML (Tien et al., 2001; Jiang et al., 2009). It was also reported that in early stage of MDS, the *p15INK4b* hypermethylation is a negative risk factor for patients, closely correlating with leukemic transformation (Aggerholm et al., 2006). The same correlation has been shown in patients with t-MDS, in which methylation resulted in significantly shorter survival (Christiansen et al., 2003). A recent study showed that the high levels of methylation in lower risk MDS categories suggest a poor prognosis in those patients as well (Valencia et al, 2011). In JMML p15INK4b hypermethylation was associated with reduced overall survival rates and higher relapse of the disease following hematopoietic stem cell transplantation (Olk-Batz et al., 2011). All these results suggest that lack of *p15INK4b* expression, mediated by promoter hypermethylation, not only affects the prognosis in patients with AML and MDS, but can be used to predict the outcome of the

The studies described above confirm that aberrant hypermethylation levels of *p15INK4b* have important prognostic implications for clinical monitoring in MDS and assessment of risk of progression into AML. However, its potential use as a biomarker in leukemia excluded estimation of minimal residual disease in patients who have achieved clinical remission, and its implications in terms of subsequent relapse. A study aiming at addressing this issue, evaluated *p15INK4b* methylation levels in AML patients in complete clinical remission (Agrawal et al., 2007). The study reported that even in remission, leukemia patients that harbored a significant amount of methylation in the bone marrow cells had a higher risk for leukemia relapse. Moreover, the time of disease-free survival was found to be significantly reduced in correlation with the amount of residual hypermethylation of the *p15Ink4b* gene. Concurrently, low levels of *p15INK4b* methylation during complete remission were associated with reduced relapse rates during the 12 month follow-up. It was suggested that analysis of *p15INK4b* methylation levels during clinical remission can be potentially

In recent years, it has been suggested in a number of studies that DNA methylation of *p15INK4b* could also help predict response to therapy (Grovdal et al., 2007; Shen et al., 2009). Grövdal et al. (2007) studied DNA methylation patterns in older patients with high risk MDS and AML following MDS. Patients were treated with conventional induction therapy. Methylation levels of *p15INK4b*, *E-cadherin*, and *HIC1* (hypermethylated in cancer 1), were assessed prior to initiation of treatment. Abnormal levels of methylation of *p15INK4b* alone did not correlate with decreased complete remission (CR), but all patients with all three genes methylated did not achieve CR. Another study, in which patients with MDS and AML were treated with the DNA methyltransferase inhibitor 5-azacytidine (5-aza-C), reported consistent results with these observations (Raj et al., 2007; Tran et al., 2011). Patients with levels exceeding 24% methylation in the *p15Ink4b* promoter region did not respond to treatment (Raj et al., 2007). The possibility of using methylation of *p15INK4b* as an indicator for treatment outcome is still under investigation. However, results suggest that studying *p15INK4b* methylation density in conjunction with other altered genes at diagnosis and monitoring its levels following treatment might have predictive information with respect to

used as a prognosticator for the occurrence of relapse (Agrawal et al., 2007).

the patient's response to treatment.

diseases.

Although hypermethylation is the most common mode of inactivation of *p15INK4b* in myeloid neoplasms, other silencing mechanisms have also been described. In AML with chromosome 16 inversion (*inv16*), the overall *p15INK4b* methylation levels are found to be very low and almost comparable to levels in normal patients. However, expression of the gene is severely suppressed. In this type of AML, the inversion (16) results in a fusion protein between the core binding factor (CBFβ) and the smooth myosin heavy chain gene (SMMHC). This chimeric transcription factor CBFβ-SMMHC binds directly to the promoter of *p15INK4b* and represses its expression (Markus et al., 2007). These results further emphasize an important role of *p15INK4b* silencing in leukemogenesis of the myeloid lineage, and suggest, that in the absence of a repressive epigenetic event, other mechanisms may result in inhibition of *p15INK4b* expression (Markus et al., 2007).

#### **2.2 Inactivation in other types of human leukemias**

In B and T cell acute lymphoblastic leukemias (B-ALL, T-ALL), *p15INK4b* hypermethylation as well as deletion of the entire 9p21 locus which includes *p15INK4b*, *p16INK4a* and *ARF* genes has been reported (Roussel, 1999; Ruas et al., 1998). Homozygous deletions of *p16INK4a* and *p15INK4b* are found in approximately 30% of childhood acute lymphoblastic leukemia at first presentation, with striking rates in T-ALL (60 to 80%), and lower rates in Bcell precursor ALL (5 to 20%) (Drexler HG, 1998; Chim et al., 2001a). Further studies have analyzed methylation levels of the two genes in these disease categories specifically in terms of overall survival and absence of relapse at 6 years of follow-up. *p15INK4b* and *p16INK4a* methylation levels were found to occur at similar rates (35%) in adults and children with mature B-ALL (Graf-Einsiedel et al., 2002). Deletion of the entire locus was observed in 12% and 30% of children and adults, respectively. Interestingly, results show that deletion is associated with poor overall survival (OS) in adults only, but not in children (Van Zutven et al., 2005; Mirebeau et al., 2006; Kim et al., 2009). Furthermore, it did not affect the type of relapse or DFS time in children. In untreated adult patients with precursor B-ALL, high methylation levels of *p15INK4b* (found in 43% of patients) were significantly associated with decreased DFS at 4 years (Hoshino et al., 2002). Recent methylation profiles in 95 children with ALL supported older studies showing that methylation of *p15INK4b* occurred predominantly in T-ALL as opposed to B-ALL, and *p15INK4b* is one of the most commonly methylated genes among the 14 genes analyzed (Takeuchi et al., 2011). A clear correlation between increased methylation and prognoses in T-ALL has not been established.

#### **2.3 p15Ink4b as a tumor suppressor in mice**

To define the role of p15INK4b as a tumor suppressor in AML, mouse models have been developed and characterized (Latres et al., 2000; Wolff et al., 2003a, 2004; Bies et al., 2010). These have provided strong experimental evidence to support the hypothesis that loss of p15INK4b function plays an important role in the development of myeloid leukemia.

A p15Ink4b-/- mouse model was first described by Latres et al. (2000). Mice were generated by genetic targeting with elimination of the second coding exon of the p15Ink4b gene. Knockout mice were viable, fertile, and did not exhibit any behavioral abnormalities. Mouse embryonic fibroblasts were found to have a higher proliferation rate and plating efficiency when compared to their wild type counterparts. More importantly, they were more susceptible to transformation with *c-myc* and *ras* oncogenes, confirming the reported results that p15Ink4b participates in the tumor suppressor activity triggered after inappropriate

p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia 295

p15Ink4bfl/flLysMcre mice had a significant increase in both mature myeloid (Gr-1+/Mac-1+) and monocytic (Gr-1-/lo/Mac-1+) cells. This increase correlated with a significantly higher proportion of immature myeloid (Mac-1+/lo/c-Kit+) cells in the BM. Inactivation of *p15Ink4b* in myeloid cells promoted a mild preleukemic myeloproliferative-like disease (Bies et al., 2010). A small percentage of the targeted mice spontaneously progressed to a form of leukemia featuring an increased number of mature circulating myeloid cells in the peripheral blood, as well as an increase in the number of progenitors in the BM. The disease observed in mice most closely resembled an advanced form of CMML (Bies et al., 2010). However, the disease did not progress to an acute form of leukemia over the period of 15 months in any of the mice. These results were in agreement with studies carried on in the embryonal p15Ink4b-/- mice, and suggested that inactivation of the *p15Ink4b* gene without an additional genetic/epigenetic hit is not sufficient to cause acute leukemia. Retrovirusinduced mutagenesis in p15Ink4bfl/flLysMcre mice was used to identify genetic changes that

Fig. 1. Cell cycle regulation by the CDKI families. CDKs are switched on and off at different times during the cell cycle; the cyclins Ds-dependent kinases 4 and 6, and CDK2/cyclin E, CDK2/cyclin A regulate G1 progression and entry into the S phase. CDK1/cyclin B regulates entry and exit from mitosis. The CDK/cyclin complexes phosphorylate pRb to allow for the transcription of genes under the control of E2Fs which include factors necessary for cell cycle progression. CIP/KIP family members of CDKI can be either

activators or inhibitors of cyclin/CDK assembly (Sherr & Roberts, 1999).

oncogenic *ras* activation of the Raf-Mek-Erk pathway (Malumbres et al., 2000). However, with respect to AML, deleting p15Ink4b did not result in leukemogenesis in these mice. Of note, extramedullary hematopoiesis and lymphoid hyperplasia in the spleen were observed in mice aged less than 9 months and resulted in death of over 75% of the mice at an older age. Taken together, these experimental results were the first to suggest that p15Ink4b might be playing a tumor suppressor role in AML (Latres et al., 2000).

A role for p15Ink4b in myeloid neoplasia in mice was first supported by the finding that retrovirus-induced AML had hypermethylation of the CpG promoter region of the *p15Ink4b* gene. Based upon this, further mouse models were developed to determine if loss of p15Ink4b increases susceptibility to myeloid leukemia when additional oncogenic events were provided by retroviral insertional mutagenesis. For these studies, a specific retrovirus with a broad tropism and the capability of inducing a high incidence of myeloid leukemia was constructed (Wolff et al., 2003b and 2004). It consists of a recombinant virus incorporating Moloney murine leukemia virus (Mo-MuLV) sequences, and regulatory LTR sequences of retrovirus 4070A. The recombinant virus, named MOL4070LTR, combines the capacity of 4070A to accelerate myeloid disease with the wide tropism of Mo-MuLV, and successfully produced myeloid disease when inoculated intraperitoneally into wild-type FVB and BALB/c mice as neonates (Wolff et al., 2003b). The *p15Ink4b* knockout mice were developed using the same targeting vector as described by Latres et al. (2000), and MOL4070LTR was inoculated into neonates. While there was no incidence of disease in control wild-type mice (p15Ink4b+/+), a significant percentage of heterozygous mice (p15Ink4b+/-) developed myeloid leukemia within a year. Surprisingly, a smaller percentage of homozygous knockout mice (p15Ink4b-/-) developed myeloid tumors (Wolff et al., 2003a). Further experiments demonstrated that in heterozygous p15Ink4b+/- mice, the second remaining *p15Ink4b* allele was actually hypermethylated, with a reduction of its mRNA expression. This data supported the fact that p15Ink4b functions as a tumor suppressor for myeloid leukemia, however, it was difficult to explain why mice heterozygous for the null allele were more susceptible than homozygous null mice. One explanation might be that in the homozygous null mice, *p15Ink4b* is lost in all the tissues and loss of expression in one tissue may have compensating effects on loss in another tissue.

A new mouse system in which deletion of the gene is restricted to the myeloid lineage was developed to mimic more closely myeloid lineage disease in man (Bies et al., 2010). The mouse strain utilizes a Cre-loxP system for conditional deletion of the *p15Ink4b* gene through action of Cre recombinase exclusively expressed in blood cells of the myeloid lineage (Clausen et al., 1999). In this model, Cre recombinase specifically recognizes loxP sites to mediate efficient excision of exon 2 of the *p15Ink4b* gene in myeloid cells. In order to monitor disease development in mice with targeted *p15Ink4b* deletion (p15Ink4bfl/fl LysMcre), white cell counts were performed in circulating blood from targeted and wildtype animals from different age groups. Interestingly, p15Ink4bfl/flLysMcre mice showed a significant increase in the number of circulating monocytes compared to control mice (p15Ink4bwt/wtLysMcre), whereas neutrophils, lymphocytes, platelets and red blood cell counts were not affected. Monocytosis remained in targeted mice beyond 8 months of age, while wild-type mice showed a marked decrease in monocytes resulting in an even greater significance in the statistical comparisons (Figure 3A). Expansion of myelomonocytic cells in the bone marrow (BM) of p15Ink4bfl/flLysMcre mice was also observed. Analysis of BM cells for the cell surface markers Gr-1, Mac-1 and c-Kit revealed that BM cells from

oncogenic *ras* activation of the Raf-Mek-Erk pathway (Malumbres et al., 2000). However, with respect to AML, deleting p15Ink4b did not result in leukemogenesis in these mice. Of note, extramedullary hematopoiesis and lymphoid hyperplasia in the spleen were observed in mice aged less than 9 months and resulted in death of over 75% of the mice at an older age. Taken together, these experimental results were the first to suggest that p15Ink4b might

A role for p15Ink4b in myeloid neoplasia in mice was first supported by the finding that retrovirus-induced AML had hypermethylation of the CpG promoter region of the *p15Ink4b* gene. Based upon this, further mouse models were developed to determine if loss of p15Ink4b increases susceptibility to myeloid leukemia when additional oncogenic events were provided by retroviral insertional mutagenesis. For these studies, a specific retrovirus with a broad tropism and the capability of inducing a high incidence of myeloid leukemia was constructed (Wolff et al., 2003b and 2004). It consists of a recombinant virus incorporating Moloney murine leukemia virus (Mo-MuLV) sequences, and regulatory LTR sequences of retrovirus 4070A. The recombinant virus, named MOL4070LTR, combines the capacity of 4070A to accelerate myeloid disease with the wide tropism of Mo-MuLV, and successfully produced myeloid disease when inoculated intraperitoneally into wild-type FVB and BALB/c mice as neonates (Wolff et al., 2003b). The *p15Ink4b* knockout mice were developed using the same targeting vector as described by Latres et al. (2000), and MOL4070LTR was inoculated into neonates. While there was no incidence of disease in control wild-type mice (p15Ink4b+/+), a significant percentage of heterozygous mice (p15Ink4b+/-) developed myeloid leukemia within a year. Surprisingly, a smaller percentage of homozygous knockout mice (p15Ink4b-/-) developed myeloid tumors (Wolff et al., 2003a). Further experiments demonstrated that in heterozygous p15Ink4b+/- mice, the second remaining *p15Ink4b* allele was actually hypermethylated, with a reduction of its mRNA expression. This data supported the fact that p15Ink4b functions as a tumor suppressor for myeloid leukemia, however, it was difficult to explain why mice heterozygous for the null allele were more susceptible than homozygous null mice. One explanation might be that in the homozygous null mice, *p15Ink4b* is lost in all the tissues and loss of expression in one

A new mouse system in which deletion of the gene is restricted to the myeloid lineage was developed to mimic more closely myeloid lineage disease in man (Bies et al., 2010). The mouse strain utilizes a Cre-loxP system for conditional deletion of the *p15Ink4b* gene through action of Cre recombinase exclusively expressed in blood cells of the myeloid lineage (Clausen et al., 1999). In this model, Cre recombinase specifically recognizes loxP sites to mediate efficient excision of exon 2 of the *p15Ink4b* gene in myeloid cells. In order to monitor disease development in mice with targeted *p15Ink4b* deletion (p15Ink4bfl/fl LysMcre), white cell counts were performed in circulating blood from targeted and wildtype animals from different age groups. Interestingly, p15Ink4bfl/flLysMcre mice showed a significant increase in the number of circulating monocytes compared to control mice (p15Ink4bwt/wtLysMcre), whereas neutrophils, lymphocytes, platelets and red blood cell counts were not affected. Monocytosis remained in targeted mice beyond 8 months of age, while wild-type mice showed a marked decrease in monocytes resulting in an even greater significance in the statistical comparisons (Figure 3A). Expansion of myelomonocytic cells in the bone marrow (BM) of p15Ink4bfl/flLysMcre mice was also observed. Analysis of BM cells for the cell surface markers Gr-1, Mac-1 and c-Kit revealed that BM cells from

be playing a tumor suppressor role in AML (Latres et al., 2000).

tissue may have compensating effects on loss in another tissue.

p15Ink4bfl/flLysMcre mice had a significant increase in both mature myeloid (Gr-1+/Mac-1+) and monocytic (Gr-1-/lo/Mac-1+) cells. This increase correlated with a significantly higher proportion of immature myeloid (Mac-1+/lo/c-Kit+) cells in the BM. Inactivation of *p15Ink4b* in myeloid cells promoted a mild preleukemic myeloproliferative-like disease (Bies et al., 2010). A small percentage of the targeted mice spontaneously progressed to a form of leukemia featuring an increased number of mature circulating myeloid cells in the peripheral blood, as well as an increase in the number of progenitors in the BM. The disease observed in mice most closely resembled an advanced form of CMML (Bies et al., 2010). However, the disease did not progress to an acute form of leukemia over the period of 15 months in any of the mice. These results were in agreement with studies carried on in the embryonal p15Ink4b-/- mice, and suggested that inactivation of the *p15Ink4b* gene without an additional genetic/epigenetic hit is not sufficient to cause acute leukemia. Retrovirusinduced mutagenesis in p15Ink4bfl/flLysMcre mice was used to identify genetic changes that

Fig. 1. Cell cycle regulation by the CDKI families. CDKs are switched on and off at different times during the cell cycle; the cyclins Ds-dependent kinases 4 and 6, and CDK2/cyclin E, CDK2/cyclin A regulate G1 progression and entry into the S phase. CDK1/cyclin B regulates entry and exit from mitosis. The CDK/cyclin complexes phosphorylate pRb to allow for the transcription of genes under the control of E2Fs which include factors necessary for cell cycle progression. CIP/KIP family members of CDKI can be either activators or inhibitors of cyclin/CDK assembly (Sherr & Roberts, 1999).

p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia 297

sequence homology or overlapping roles in the cell (Ozenne et al., 2010; Sherr CJ, 2006). Furthermore, p14ARF bears little or no structural similarities with the INK4 family members, and is unable to bind or inhibit CDKs. It is not considered to be part of the INK4 family of inhibitors, but it still participates in the negative regulation of the cell cycle by antagonizing the effects of MDM2, a ubiquitin ligase that targets the tumor suppressor

Fig. 2. 9p21 chromosomal locus showing the promoters and exons that are involved in the transcription of *p15INK4b*, *p14ARF* and *p16INK4a* genes. The CpG island is depicted for the *p15INK4b* gene only and extends throughout the promoter region, exon 1 and part of intron

INK4 family members are highly conserved among species, with over 90% identity between the human and the corresponding mouse proteins. In human, the four proteins share roughly 40% sequence homology with each other and have a very closely related structure, characterized by the presence of ankyrin motif tandem repeats. Four ankyrin repeats are found in p15INK4b and p16INK4a, and five repeats in p18INK4c and p19INK4d (Yuan et al., 2000). Ankyrin motif repeats consist of a helix-loop-helix structure that promotes protein-protein interaction (Li et al., 1999). Structural studies involving programmed mutations and generation of chimeric proteins have revealed that the third ankyrin repeat is necessary for the interaction with CDK4 and CDK6, and is responsible for the inhibitory activity of these CDKIs. Crystallography work on the CDK6 bound p16INK4a, p18INK4c and p19INK4d suggested that the INK4 proteins bind to one side of the catalytic cleft, opposite to the cyclin binding site, and that binding and recognition are mediated mostly via hydrogen bonds (Brotherton et al., 1998; Noh et al., 1999; Russo et al., 1998). INK4 proteins were found not to interfere with the cyclin binding site which is consistent with the presence of INK4s/cyclin Ds/CDK4 or CDK6 ternary complexes. When bound in the absence of cyclin D, they cause a conformation switch in CDK4 and CDK6 which distorts the cyclin and the ATP-binding sites leading to rapid recycling of unbound D-type cyclins by the ubiquitin-dependent 26S proteasome. The specificity towards CDK 4 and CDK6 was found to be due primarily to the critical residues involved in the hydrogen bonds with INK4 proteins which are conserved exclusively in CDK4 and CDK6, but not in the other CDKs (Russo et al., 1998). Interestingly, several of the residues necessary for recognition and

The structural similarities observed between the INK4 proteins are consistent with the shared biological and biochemical properties of these molecules. However, the expression

protein p53 for degradation by the 26S proteasome (Ruas et al., 1998).

binding have been reported to be mutated in cancer (Li et al., 1999).

1 (Herman et al., 1996).

could cooperate with the loss of p15Ink4b in leukemia development. Mice inoculated with the MOL4070LTR retrovirus were monitored for 15 months for signs of disease. Control mice developed leukemia with low penetrance, whereas the incidence of retrovirus-induced leukemia was statistically highly significant for the p15Ink4bfl/flLysMcre animals. Additionally, phenotypic analyses of tumor cells demonstrated a strong bias towards the development of AML in the knockout animals. Myeloid–specific inactivation of *p15Ink4b* results in retrovirus-induced development of tumors mostly monocytic (F4/80+, F4/80+/Mac1+) and myelomonocytic (F4/80+/Mac1+/Gr-1+), whereas there was an equal distribution of lymphoid and monocytic tumors in the control mice (Figure 3B). Results generated using this model complement the embryonal *p15nk4b*-/- studies and demonstrate an active role for *p15Ink4b* silencing in promoting the establishment of preleukemic conditions. These results also provided strong experimental evidence that p15Ink4b functions as a tumor suppressor for myeloid leukemia development (Bies et al., 2010).

#### **3. Conventional and novel functions of p15INK4b**

#### **3.1 p15INK4B as a regulator of the cell cycle**

p15INK4b belongs to the INK4 family of cyclin-dependent kinases inhibitors (CDKIs). The family comprises three other members – p16INK4a, p18INK4c and p19INK4d and is one of two families of CDKIs that have been identified and defined based on their structure and CDK specificities. INK4 proteins all show a high and exclusive specificity towards the activity of cyclin-dependent kinase 4 (CDK 4) and cyclin-dependent kinase 6 (CDK6) during the early and mid-G1 phase of the cell cycle (Sherr & Roberts, 1999). The cell cycle is comprised of 4 phases, G1, S, G2 (also referred to as interphase), and M phase (mitosis). Entrance of cells from the quiescent G0/G1 phase into cycle is governed by the actions of kinases CDK4/CDK6 and CDK2 that are activated by cyclins Ds and Es, respectively (Figure 1).

During the transition to S phase, CDKs hyperphosphorylate pRb causing its dissociation from the nuclear transcription factors E2Fs. E2Fs regulate the transcription of genes which are required for the completion of the cell cycle and include cyclins A and E, thymidine synthetase and PCNA (Korenjak & Brehm, 2005). Throughout the S, G2 and M phases, pRb is kept in a hyperphosphorylated state by an orchestrated mechanism that involves sequential activities of multiple cyclins/CDKs (Sherr & Roberts, 1999). INK4 proteins inhibit CDKs/cyclin Ds complexes and, therefore, function in G1-S checkpoint control. When INK4 proteins block formation of these complexes, the pRb is in a hypophosphorylated, active state and interacts with E2F to inhibit its function (Korenjak & Brehm, 2005). Structural studies have demonstrated that INK4 proteins perform their inhibitory activity by allosteric competition with cyclins Ds to bind CDK4 and CDK6. CDK4/6-INK4s protein complexes have reduced affinity toward the D-type cyclins (Jeffrey et al., 2000; Yuan et al 2000).

*p15INK4b* and *p16INK4a* are tandemly linked on human chromosome 9p21 within a 40kb DNA region, whereas *p18INK4c* and *p19INK4d* are located in the chromosomal regions 1p32.3 and 19p13.2, respectively. The 9p21 chromosomal locus is referred to as the *INK4/ARF* locus has been tightly linked to the formation of many types of tumors (Nobori et al., 1994). In addition to *p15INK4b* and *p16INK4a*, it also encodes a third gene called *p14ARF*, originally identified as an alternative transcript of *p16INK4a* (Figure 2). *p14ARF* is transcribed from exon 1β and exons 2 and 3 of *p16INK4a*, but using a different reading frame (Figure 2). The p14ARF protein (p19Arf in mouse) is immunologically and functionally unrelated to the p16INK4a protein; they are not considered to be isoforms and do not share

could cooperate with the loss of p15Ink4b in leukemia development. Mice inoculated with the MOL4070LTR retrovirus were monitored for 15 months for signs of disease. Control mice developed leukemia with low penetrance, whereas the incidence of retrovirus-induced leukemia was statistically highly significant for the p15Ink4bfl/flLysMcre animals. Additionally, phenotypic analyses of tumor cells demonstrated a strong bias towards the development of AML in the knockout animals. Myeloid–specific inactivation of *p15Ink4b* results in retrovirus-induced development of tumors mostly monocytic (F4/80+, F4/80+/Mac1+) and myelomonocytic (F4/80+/Mac1+/Gr-1+), whereas there was an equal distribution of lymphoid and monocytic tumors in the control mice (Figure 3B). Results generated using this model complement the embryonal *p15nk4b*-/- studies and demonstrate an active role for *p15Ink4b* silencing in promoting the establishment of preleukemic conditions. These results also provided strong experimental evidence that p15Ink4b

functions as a tumor suppressor for myeloid leukemia development (Bies et al., 2010).

p15INK4b belongs to the INK4 family of cyclin-dependent kinases inhibitors (CDKIs). The family comprises three other members – p16INK4a, p18INK4c and p19INK4d and is one of two families of CDKIs that have been identified and defined based on their structure and CDK specificities. INK4 proteins all show a high and exclusive specificity towards the activity of cyclin-dependent kinase 4 (CDK 4) and cyclin-dependent kinase 6 (CDK6) during the early and mid-G1 phase of the cell cycle (Sherr & Roberts, 1999). The cell cycle is comprised of 4 phases, G1, S, G2 (also referred to as interphase), and M phase (mitosis). Entrance of cells from the quiescent G0/G1 phase into cycle is governed by the actions of kinases CDK4/CDK6 and

During the transition to S phase, CDKs hyperphosphorylate pRb causing its dissociation from the nuclear transcription factors E2Fs. E2Fs regulate the transcription of genes which are required for the completion of the cell cycle and include cyclins A and E, thymidine synthetase and PCNA (Korenjak & Brehm, 2005). Throughout the S, G2 and M phases, pRb is kept in a hyperphosphorylated state by an orchestrated mechanism that involves sequential activities of multiple cyclins/CDKs (Sherr & Roberts, 1999). INK4 proteins inhibit CDKs/cyclin Ds complexes and, therefore, function in G1-S checkpoint control. When INK4 proteins block formation of these complexes, the pRb is in a hypophosphorylated, active state and interacts with E2F to inhibit its function (Korenjak & Brehm, 2005). Structural studies have demonstrated that INK4 proteins perform their inhibitory activity by allosteric competition with cyclins Ds to bind CDK4 and CDK6. CDK4/6-INK4s protein complexes

have reduced affinity toward the D-type cyclins (Jeffrey et al., 2000; Yuan et al 2000).

*p15INK4b* and *p16INK4a* are tandemly linked on human chromosome 9p21 within a 40kb DNA region, whereas *p18INK4c* and *p19INK4d* are located in the chromosomal regions 1p32.3 and 19p13.2, respectively. The 9p21 chromosomal locus is referred to as the *INK4/ARF* locus has been tightly linked to the formation of many types of tumors (Nobori et al., 1994). In addition to *p15INK4b* and *p16INK4a*, it also encodes a third gene called *p14ARF*, originally identified as an alternative transcript of *p16INK4a* (Figure 2). *p14ARF* is transcribed from exon 1β and exons 2 and 3 of *p16INK4a*, but using a different reading frame (Figure 2). The p14ARF protein (p19Arf in mouse) is immunologically and functionally unrelated to the p16INK4a protein; they are not considered to be isoforms and do not share

**3. Conventional and novel functions of p15INK4b** 

CDK2 that are activated by cyclins Ds and Es, respectively (Figure 1).

**3.1 p15INK4B as a regulator of the cell cycle** 

sequence homology or overlapping roles in the cell (Ozenne et al., 2010; Sherr CJ, 2006). Furthermore, p14ARF bears little or no structural similarities with the INK4 family members, and is unable to bind or inhibit CDKs. It is not considered to be part of the INK4 family of inhibitors, but it still participates in the negative regulation of the cell cycle by antagonizing the effects of MDM2, a ubiquitin ligase that targets the tumor suppressor protein p53 for degradation by the 26S proteasome (Ruas et al., 1998).

Fig. 2. 9p21 chromosomal locus showing the promoters and exons that are involved in the transcription of *p15INK4b*, *p14ARF* and *p16INK4a* genes. The CpG island is depicted for the *p15INK4b* gene only and extends throughout the promoter region, exon 1 and part of intron 1 (Herman et al., 1996).

INK4 family members are highly conserved among species, with over 90% identity between the human and the corresponding mouse proteins. In human, the four proteins share roughly 40% sequence homology with each other and have a very closely related structure, characterized by the presence of ankyrin motif tandem repeats. Four ankyrin repeats are found in p15INK4b and p16INK4a, and five repeats in p18INK4c and p19INK4d (Yuan et al., 2000). Ankyrin motif repeats consist of a helix-loop-helix structure that promotes protein-protein interaction (Li et al., 1999). Structural studies involving programmed mutations and generation of chimeric proteins have revealed that the third ankyrin repeat is necessary for the interaction with CDK4 and CDK6, and is responsible for the inhibitory activity of these CDKIs. Crystallography work on the CDK6 bound p16INK4a, p18INK4c and p19INK4d suggested that the INK4 proteins bind to one side of the catalytic cleft, opposite to the cyclin binding site, and that binding and recognition are mediated mostly via hydrogen bonds (Brotherton et al., 1998; Noh et al., 1999; Russo et al., 1998). INK4 proteins were found not to interfere with the cyclin binding site which is consistent with the presence of INK4s/cyclin Ds/CDK4 or CDK6 ternary complexes. When bound in the absence of cyclin D, they cause a conformation switch in CDK4 and CDK6 which distorts the cyclin and the ATP-binding sites leading to rapid recycling of unbound D-type cyclins by the ubiquitin-dependent 26S proteasome. The specificity towards CDK 4 and CDK6 was found to be due primarily to the critical residues involved in the hydrogen bonds with INK4 proteins which are conserved exclusively in CDK4 and CDK6, but not in the other CDKs (Russo et al., 1998). Interestingly, several of the residues necessary for recognition and binding have been reported to be mutated in cancer (Li et al., 1999).

The structural similarities observed between the INK4 proteins are consistent with the shared biological and biochemical properties of these molecules. However, the expression

p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia 299

pituitary tumors and lymphomas (Bai et al., 2003). Loss of p16Ink4a expression was shown to be a necessary event in conjunction with loss of p18Ink4c for the mice to develop aggressive advanced stages of pituitary carcinoma (Morishita et al., 2004). Furthermore, it is now clear that p18INK4c is a tumor suppressor in human glioblastoma multiform and hepatocellular carcinoma (Solomon, 2008a and 2008b). Interestingly, studies in mice deficient for p19Ink4d have not revealed increased susceptibility to any cancer or other proliferative diseases, suggesting a limited or nonexistent role in carcinogenesis (Buchold et

Although these cell cycle regulators exhibit overlapping and surely compensatory activities mainly due to their structural similarities (Krimpenfort et al., 2007), their temporal and tissue-restricted expression patterns as well as their differential involvement in the pathogenesis of human cancers clearly suggest that they can harbor specific and distinct

The strikingly high prevalence of *p15INK4b* loss of expression during the development of myeloid disease in human patients has triggered scientists to explore alternate functions for this CDKI. As a cell cycle regulator, p15INK4b was suggested to be one of the players determining the fine balances in differentiation and proliferation of myeloid cells. To test this hypothesis, the previously described p15Ink4b germline knockout mice as well as murine transplant models were used. Results revealed a novel role for the protein during

Myelopoiesis is the process by which an undifferentiated progenitor cell gives rise to mature differentiated functional myeloid cells. The process takes place in the bone marrow and is driven by a pool of hematopoietic cytokines that have different binding specificities to cell surface markers in a stage and lineage dependent manner. Committed progenitors with a restricted myeloid lineage fate arise from earlier common myeloid progenitor cells (CMP),

In p15Ink4b knockout mice (p15Ink4b-/-), loss of p15Ink4b was found to favor the differentiation of CMPs into granulocyte macrophage progenitors (GMP) which results in an imbalance between the erythroid and myeloid compartments (Rosu-Myles et al 2007). This defect was in the bipotent differentiation capacity of the CMP, and did not affect the frequency of early long-term HSCs, or their ability to self-renew and proliferate. Therefore, this finding differs from the traditional role of p15Ink4b in regulating the cell cycle. As shown in Figure 3A, the increased number of myeloid progenitors was found to occur at the expense of differentiation of CMPs towards the erythroid progenitors .Furthermore, competitive repopulating assays have shown that the defect is intrinsic to the cells. Loss of p15Ink4b provided a competitive advantage over the wild-type cells within the myeloid

Interestingly, an earlier study carried out in p18Ink4c-/- mice, had shown that this CDKI also impacts cell fate but targets a different cell type. Deletion of p18Ink4c was found to result in a long-term engraftment advantage in HSCs. The observed effects were not due to increased proliferation capacity of the cells, but rather to an enhanced potential of self-renewal of these cells as opposed to differentiation. This lead to an efficient expansion of HSCs as well as hematopoietic progenitor pools, which fully retained their multi-lineage differentiation

early and late stages of myeloid cells development (Rosu-Myles et al., 2007, 2008).

who themselves originate from hematopoietic stem cells (HSC) (Akashi et al. 2000).

**3.2 Function as a regulator of cell fate during early myelopoiesis** 

al. 2007, Zindy et al, 2000).

functions during development.

compartment (Rosu-Myles et al., 2007).

potential (Yuan et al., 2004).

pattern of each of the human INK4 proteins appears to be highly specific to the cell type and tissue localization, as well as the differentiation stage of the cells (Shwaller et al., 1997; Thullberg et al., 2000). For instance, in normal hematopoietic cells, the expression of p15INK4b is shown to be lineage restricted, and is mainly detected in monocytes and lymphocytes, but not in any of the erythroid precursors (Teofili et al., 1998). Myeloidrestricted expression of *p15INK4b* is observed in peripheral blood and bone marrow and its levels are reported to be increased during megakaryocyte and monocyte/macrophage differentiation (Furukawa et al., 2000; Teofili et al., 2001; Haviernik et al., 2003).

p15INK4b, in particular, is an important downstream effector of anti-proliferative signaling by the transforming growth factor- β1 (TGF-β1) (Hannon & Beach, 1994). In different human and mouse cell lines, treatment with this negative growth factor, as well as interleukin 6 (IL-6) or Interferon β (IFN- β) leads to G0/G1 cell cycle arrest (Schmidt et al., 2004; Haviernik et al., 2003). Treatment with TGF-β1 induces a significant increase in the transcription levels of *p15INK4b*, but also induces a major increase in p15INK4b protein stability (Sandhu et al., 1997). Following treatment with TGF-β1, p15INK4b-CDK4/6 complexes are more abundant compared to the Cyclin D-Cdk4 complexes indicating strong inhibitor activity (Sandhu et al., 1997). In contrast to *p15INK4b*, whose expression is absent in hematopoietic stem cells, but increases as the cells mature along the myeloid lineage, *p16INK4a* is highly expressed in hematopoietic stem cells, and down-regulated with differentiation of all lineages (Furukawa et al., 2000). *p18INK4c* is found to be the most homogeneously and abundantly expressed member of the family, whereas *p19INK4d* is the most restricted, and its expression is limited to lymphoid cell, epithelial cells, seminiferous tubes and adrenal gland cells (Thullberg et al., 2000). The differential expression patterns of the INK4 proteins, suggest nonoverlapping physiological functions.

Despite their common function in regulation of the cell cycle, the four members are found to be differentially involved in tumorigenesis. Mouse knock-out models along with genetic screenings of human tumors and gene expression profiling of cell lines have been used to help elucidate the role of these cell cycle inhibitors in the establishment and progression of cancer (Cánepa et al., 2007). In concordance with the molecular analysis of human tumor tissues, mice deficient in different Ink4 proteins display an increased susceptibility to the development of various types of tumors with variable penetrance. *p16INK4a* is a family member that has a prominent role in carcinomas of the pancreas and the bladder, glioblastomas, leukemias and melanomas among others. Its expression is lost by several mechanisms including point mutations, small deletion and epigenetic modifications which have been reported in thousands of human cancers (Serrano et al., 1996; Krimpenfort et al., 2001). On the other hand, as previously described, *p15INK4b* is noted to be silenced primarily by an epigenetic mechanism in human cancers, and loss of its expression through hypermethylation of its promoter region is well documented in hematologic neoplasms in particular (Drexler HG, 1998; Roussel, 1999). In these types of cancers, inactivation of *p15INK4b* has been reported in the absence of aberrant modification or deletion of *p16INK4a*. In contrast with p15INK4b and p16INK4a, p18INK4c was originally found to play a more limited role as a human tumor suppressor, and p19INK4d is not thought to be involved in the pathogenesis of cancer (Thullberg et al., 2000). *p18Ink4c*-null mice are viable but display an unusual phenotype with pronounced gigantism, lymphomas and more importantly pituitary hyperplasia (Franklin et al., 1998). Later examination of these mice revealed that p18Ink4c is a haploinsufficient tumor suppressor for spontaneous and carcinogen-induced

pattern of each of the human INK4 proteins appears to be highly specific to the cell type and tissue localization, as well as the differentiation stage of the cells (Shwaller et al., 1997; Thullberg et al., 2000). For instance, in normal hematopoietic cells, the expression of p15INK4b is shown to be lineage restricted, and is mainly detected in monocytes and lymphocytes, but not in any of the erythroid precursors (Teofili et al., 1998). Myeloidrestricted expression of *p15INK4b* is observed in peripheral blood and bone marrow and its levels are reported to be increased during megakaryocyte and monocyte/macrophage

p15INK4b, in particular, is an important downstream effector of anti-proliferative signaling by the transforming growth factor- β1 (TGF-β1) (Hannon & Beach, 1994). In different human and mouse cell lines, treatment with this negative growth factor, as well as interleukin 6 (IL-6) or Interferon β (IFN- β) leads to G0/G1 cell cycle arrest (Schmidt et al., 2004; Haviernik et al., 2003). Treatment with TGF-β1 induces a significant increase in the transcription levels of *p15INK4b*, but also induces a major increase in p15INK4b protein stability (Sandhu et al., 1997). Following treatment with TGF-β1, p15INK4b-CDK4/6 complexes are more abundant compared to the Cyclin D-Cdk4 complexes indicating strong inhibitor activity (Sandhu et al., 1997). In contrast to *p15INK4b*, whose expression is absent in hematopoietic stem cells, but increases as the cells mature along the myeloid lineage, *p16INK4a* is highly expressed in hematopoietic stem cells, and down-regulated with differentiation of all lineages (Furukawa et al., 2000). *p18INK4c* is found to be the most homogeneously and abundantly expressed member of the family, whereas *p19INK4d* is the most restricted, and its expression is limited to lymphoid cell, epithelial cells, seminiferous tubes and adrenal gland cells (Thullberg et al., 2000). The differential expression patterns of the INK4 proteins, suggest non-

Despite their common function in regulation of the cell cycle, the four members are found to be differentially involved in tumorigenesis. Mouse knock-out models along with genetic screenings of human tumors and gene expression profiling of cell lines have been used to help elucidate the role of these cell cycle inhibitors in the establishment and progression of cancer (Cánepa et al., 2007). In concordance with the molecular analysis of human tumor tissues, mice deficient in different Ink4 proteins display an increased susceptibility to the development of various types of tumors with variable penetrance. *p16INK4a* is a family member that has a prominent role in carcinomas of the pancreas and the bladder, glioblastomas, leukemias and melanomas among others. Its expression is lost by several mechanisms including point mutations, small deletion and epigenetic modifications which have been reported in thousands of human cancers (Serrano et al., 1996; Krimpenfort et al., 2001). On the other hand, as previously described, *p15INK4b* is noted to be silenced primarily by an epigenetic mechanism in human cancers, and loss of its expression through hypermethylation of its promoter region is well documented in hematologic neoplasms in particular (Drexler HG, 1998; Roussel, 1999). In these types of cancers, inactivation of *p15INK4b* has been reported in the absence of aberrant modification or deletion of *p16INK4a*. In contrast with p15INK4b and p16INK4a, p18INK4c was originally found to play a more limited role as a human tumor suppressor, and p19INK4d is not thought to be involved in the pathogenesis of cancer (Thullberg et al., 2000). *p18Ink4c*-null mice are viable but display an unusual phenotype with pronounced gigantism, lymphomas and more importantly pituitary hyperplasia (Franklin et al., 1998). Later examination of these mice revealed that p18Ink4c is a haploinsufficient tumor suppressor for spontaneous and carcinogen-induced

differentiation (Furukawa et al., 2000; Teofili et al., 2001; Haviernik et al., 2003).

overlapping physiological functions.

pituitary tumors and lymphomas (Bai et al., 2003). Loss of p16Ink4a expression was shown to be a necessary event in conjunction with loss of p18Ink4c for the mice to develop aggressive advanced stages of pituitary carcinoma (Morishita et al., 2004). Furthermore, it is now clear that p18INK4c is a tumor suppressor in human glioblastoma multiform and hepatocellular carcinoma (Solomon, 2008a and 2008b). Interestingly, studies in mice deficient for p19Ink4d have not revealed increased susceptibility to any cancer or other proliferative diseases, suggesting a limited or nonexistent role in carcinogenesis (Buchold et al. 2007, Zindy et al, 2000).

Although these cell cycle regulators exhibit overlapping and surely compensatory activities mainly due to their structural similarities (Krimpenfort et al., 2007), their temporal and tissue-restricted expression patterns as well as their differential involvement in the pathogenesis of human cancers clearly suggest that they can harbor specific and distinct functions during development.

#### **3.2 Function as a regulator of cell fate during early myelopoiesis**

The strikingly high prevalence of *p15INK4b* loss of expression during the development of myeloid disease in human patients has triggered scientists to explore alternate functions for this CDKI. As a cell cycle regulator, p15INK4b was suggested to be one of the players determining the fine balances in differentiation and proliferation of myeloid cells. To test this hypothesis, the previously described p15Ink4b germline knockout mice as well as murine transplant models were used. Results revealed a novel role for the protein during early and late stages of myeloid cells development (Rosu-Myles et al., 2007, 2008).

Myelopoiesis is the process by which an undifferentiated progenitor cell gives rise to mature differentiated functional myeloid cells. The process takes place in the bone marrow and is driven by a pool of hematopoietic cytokines that have different binding specificities to cell surface markers in a stage and lineage dependent manner. Committed progenitors with a restricted myeloid lineage fate arise from earlier common myeloid progenitor cells (CMP), who themselves originate from hematopoietic stem cells (HSC) (Akashi et al. 2000).

In p15Ink4b knockout mice (p15Ink4b-/-), loss of p15Ink4b was found to favor the differentiation of CMPs into granulocyte macrophage progenitors (GMP) which results in an imbalance between the erythroid and myeloid compartments (Rosu-Myles et al 2007). This defect was in the bipotent differentiation capacity of the CMP, and did not affect the frequency of early long-term HSCs, or their ability to self-renew and proliferate. Therefore, this finding differs from the traditional role of p15Ink4b in regulating the cell cycle. As shown in Figure 3A, the increased number of myeloid progenitors was found to occur at the expense of differentiation of CMPs towards the erythroid progenitors .Furthermore, competitive repopulating assays have shown that the defect is intrinsic to the cells. Loss of p15Ink4b provided a competitive advantage over the wild-type cells within the myeloid compartment (Rosu-Myles et al., 2007).

Interestingly, an earlier study carried out in p18Ink4c-/- mice, had shown that this CDKI also impacts cell fate but targets a different cell type. Deletion of p18Ink4c was found to result in a long-term engraftment advantage in HSCs. The observed effects were not due to increased proliferation capacity of the cells, but rather to an enhanced potential of self-renewal of these cells as opposed to differentiation. This lead to an efficient expansion of HSCs as well as hematopoietic progenitor pools, which fully retained their multi-lineage differentiation potential (Yuan et al., 2004).

p15INK4b, a Tumor Suppressor in Acute Myeloid Leukemia 301

The M1 leukemia cell line, does not express p53, proliferates autonomously and is often used as a model for monocytic differentiation. When terminal differentiation is induced in these cells, upregulation of p15Ink4b expression is accompanied by inhibition of Cdk4 kinase activity as well as by a decrease in levels of phosphorylated Rb (Bergh et al., 1999). Furthermore, over-expression of p15Ink4b in M1 cells causes cell cycle arrest in the G1 phase, providing additional evidence for the implication of the protein in maturation and cell cycle inhibition of late stage progenitors (Haviernik et al., 2003). This function is further supported by studies in human CD34+ hematopoietic progenitor cells. When expression of p15INK4b is triggered, higher levels of the protein are associated with transcriptional upregulation of genes known to induce myeloid differentiation, such as the colony stimulating factor 1 receptor gene (c-fms), the myeloperoxidase gene (mpo) and lactoferrin (lf), among others (Furukawa et al 2000). Overexpression of p15INK4b is also linked with a dramatic decrease in early blast progenitor populations and an increase in the numbers of

This data suggests that p15INK4b has functions during both the early and late stages of myelopoiesis; in early progenitors, p15Ink4b influences cell fate by altering the balance between myeloid and erythroid progenitors, whereas during late myelopoiesis, p15INK4b

The reversible nature of epigenetic alterations makes them very attractive therapeutic targets for AML and MDS. In the case of *p15INK4b*, these epigenetic alterations consist of DNA methylation as well as chromatin remodeling by post-transcriptional histone modifications. Studying the association of *p15INK4b* promoter DNA methylation with histone modifications revealed important insight into the interplay of these two types of epigenetic mechanisms (Paul et al., 2010). Histones undergo post-transcriptional modifications that target primarily the N-terminal tail regions, and involve the attachment of phospho, acetyl, methyl, ribosyl, and small ubiquitin-like modifier or ubiquitin groups on the side chains of the different amino acids residues of histone molecules (Biancotto et al., 2010). Acetylation and de-acetylation of lysine residues are catalyzed by two groups of enzymes with opposing actions: histone acetyltransferases (HAT) and histone deacetylases (HDAC) (Jenuwein & Allis, 2001), whereas histone methyltransferases (HMT), and histone demethylases (HDM) control the balance of histone methylations. Similar to DNA methylation, histone modifications are fully biochemically reversible, result in changes in the protein structure and affect the affinity of histone tails to DNA molecules (Varier & Timmers, 2011). Paul et al. (2010) found that in AML cell lines with aberrant *p15INK4b* DNA hypermethylation, the histone 3 trimethylated at lysine 4 (H3K4me3), which is a transcriptional activation mark, was at lower levels than in AML cell lines without hypermethylation. Interestingly, irrespective of the methylation status of *p15INK4b*, this study also reported the presence of the repressive mark H3K27me3 (histone 3 trimethylated at lysine 27) at the 9p21 locus. Human AML blasts with hypermethylation of *p15INK4b* were

appears to be causing withdrawal from the cell-cycle in response to cytokines.

**4. Targeting p15INK4b for re-expression in AML and MDS** 

similarly found to have H3K27me3, but lacked H3K4me3 at the gene.

The tight collaboration of the different epigenetic alterations in silencing the *p15INK4b* gene makes combinatorial therapeutics a promising approach for its reexpression. Importantly, removal of methyl groups from hypermethylated CpG clusters associated with the gene promoter reverses the inhibitory effects and restores normal gene expression (Jones &

cells that adopt a myeloid morphology (Teofili et al., 2000).

#### **3.3 Function in cell cycle arrest in late myeloid cell development**

In addition to its hypothesized role in cell fate decision of early myeloid progenitors, p15Ink4b is implicated during the late stages of myelopoiesis. In this case its role appears to be the induction of cell-cycle arrest. p15Ink4b expression has been shown to increase specifically during myeloid differentiation in vivo both in human bone marrow and peripheral blood cells (Teofili et al. 2000); and in vitro, in murine M1 myeloblastic cells which undergo monocytic differentiation following treatment with IL-6 (Schmidt et al 2004).

Fig. 3. The loss of p15Ink4b in myeloid lineage results in: A. Imbalance in the myeloid progenitor pools and nonreactive monocytosis; B. Increased incidence of the retrovirusinduced leukemia with preference toward the myeloid phenotype.

In addition to its hypothesized role in cell fate decision of early myeloid progenitors, p15Ink4b is implicated during the late stages of myelopoiesis. In this case its role appears to be the induction of cell-cycle arrest. p15Ink4b expression has been shown to increase specifically during myeloid differentiation in vivo both in human bone marrow and peripheral blood cells (Teofili et al. 2000); and in vitro, in murine M1 myeloblastic cells which undergo monocytic differentiation following treatment with IL-6 (Schmidt et al 2004).

Fig. 3. The loss of p15Ink4b in myeloid lineage results in: A. Imbalance in the myeloid progenitor pools and nonreactive monocytosis; B. Increased incidence of the retrovirus-

induced leukemia with preference toward the myeloid phenotype.

**3.3 Function in cell cycle arrest in late myeloid cell development** 

The M1 leukemia cell line, does not express p53, proliferates autonomously and is often used as a model for monocytic differentiation. When terminal differentiation is induced in these cells, upregulation of p15Ink4b expression is accompanied by inhibition of Cdk4 kinase activity as well as by a decrease in levels of phosphorylated Rb (Bergh et al., 1999). Furthermore, over-expression of p15Ink4b in M1 cells causes cell cycle arrest in the G1 phase, providing additional evidence for the implication of the protein in maturation and cell cycle inhibition of late stage progenitors (Haviernik et al., 2003). This function is further supported by studies in human CD34+ hematopoietic progenitor cells. When expression of p15INK4b is triggered, higher levels of the protein are associated with transcriptional upregulation of genes known to induce myeloid differentiation, such as the colony stimulating factor 1 receptor gene (c-fms), the myeloperoxidase gene (mpo) and lactoferrin (lf), among others (Furukawa et al 2000). Overexpression of p15INK4b is also linked with a dramatic decrease in early blast progenitor populations and an increase in the numbers of cells that adopt a myeloid morphology (Teofili et al., 2000).

This data suggests that p15INK4b has functions during both the early and late stages of myelopoiesis; in early progenitors, p15Ink4b influences cell fate by altering the balance between myeloid and erythroid progenitors, whereas during late myelopoiesis, p15INK4b appears to be causing withdrawal from the cell-cycle in response to cytokines.

#### **4. Targeting p15INK4b for re-expression in AML and MDS**

The reversible nature of epigenetic alterations makes them very attractive therapeutic targets for AML and MDS. In the case of *p15INK4b*, these epigenetic alterations consist of DNA methylation as well as chromatin remodeling by post-transcriptional histone modifications. Studying the association of *p15INK4b* promoter DNA methylation with histone modifications revealed important insight into the interplay of these two types of epigenetic mechanisms (Paul et al., 2010). Histones undergo post-transcriptional modifications that target primarily the N-terminal tail regions, and involve the attachment of phospho, acetyl, methyl, ribosyl, and small ubiquitin-like modifier or ubiquitin groups on the side chains of the different amino acids residues of histone molecules (Biancotto et al., 2010). Acetylation and de-acetylation of lysine residues are catalyzed by two groups of enzymes with opposing actions: histone acetyltransferases (HAT) and histone deacetylases (HDAC) (Jenuwein & Allis, 2001), whereas histone methyltransferases (HMT), and histone demethylases (HDM) control the balance of histone methylations. Similar to DNA methylation, histone modifications are fully biochemically reversible, result in changes in the protein structure and affect the affinity of histone tails to DNA molecules (Varier & Timmers, 2011). Paul et al. (2010) found that in AML cell lines with aberrant *p15INK4b* DNA hypermethylation, the histone 3 trimethylated at lysine 4 (H3K4me3), which is a transcriptional activation mark, was at lower levels than in AML cell lines without hypermethylation. Interestingly, irrespective of the methylation status of *p15INK4b*, this study also reported the presence of the repressive mark H3K27me3 (histone 3 trimethylated at lysine 27) at the 9p21 locus. Human AML blasts with hypermethylation of *p15INK4b* were similarly found to have H3K27me3, but lacked H3K4me3 at the gene.

The tight collaboration of the different epigenetic alterations in silencing the *p15INK4b* gene makes combinatorial therapeutics a promising approach for its reexpression. Importantly, removal of methyl groups from hypermethylated CpG clusters associated with the gene promoter reverses the inhibitory effects and restores normal gene expression (Jones &

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HDAC inhibitors such as Trichostatin A (TSA) have been used for targeting of histone acetylations. In the case of AML samples with *p15INK4b* hypermethylation, it was shown that TSA treatment alone is insufficient to increase p15INK4b expression levels (Scott et al., 2007; Paul et al., 2010). However, studies that incorporated the use of both HDAC and DNMT inhibitors have proven successful at inducing higher levels of p15INK4b expression (Cameron et al., 1999; Scott et al., 2007; Paul et al., 2010).

#### **5. Conclusion**

p15INK4b is a CDK inhibitor whose expression is lost in a very high proportion of patients with MDS and AML. This implicates an important role for the loss of the protein expression in the development of myeloid disease in humans. Its role in myeloid leukemogenesis as a tumor suppressor is now confirmed by research carried out in several mouse models. In addition to its role in inhibiting CDKs to arrest the cell cycle during late myeloid development, it is now known to affect cell fate decisions during early myelopoiesis. In myeloid progenitors, loss of p15Ink4b results in an imbalance in the progenitor pools, and favors the expansion of GMPs at the expense of MEPs. Importantly, when deleted in the cells of the myeloid lineage only, mice develop nonreactive monocytosis and are strongly predisposed to succumb to retrovirus-induced leukemia, especially of myeloid origin. These results validate a tight link between loss of p15INK4b and human myeloid neoplasia. Clinical observations that associate *p15INK4b* demethylation and re-expression with improved prognosis and survival, further stress the importance of this gene in AML and MDS treatment.

#### **6. References**


Baylin, 2002). The DNMT inhibitor 5-azacytidine (5-aza-C) and its analogue 5-azadeoxycytidine (5-aza-dC, Decitabine) are powerful hypomethylating agents that are used in the therapy of high-risk MDS and AML. It has been demonstrated that they can lead to the reversal of hypermethylation and subsequent reexpression of the *p15INK4b* gene in patients with MDS (Daskalakis et al., 2002; Farinha et al., 2004; Gore et al., 2006; Santos et al., 2010). It was shown that treatment of patient blasts with hypomethylating agent Decitabine also affects histone modifications. Paul et al. (2010) provided evidence that the levels of H3K4me3 increased with retention of H3K27me3, thus inducing a state of bivalency. The use of other HMT inhibitors such as 3-Deazaneplanocin A (DZNep) have been reported to successfully decrease global DNA methylation levels, but not to induce re-expression of

HDAC inhibitors such as Trichostatin A (TSA) have been used for targeting of histone acetylations. In the case of AML samples with *p15INK4b* hypermethylation, it was shown that TSA treatment alone is insufficient to increase p15INK4b expression levels (Scott et al., 2007; Paul et al., 2010). However, studies that incorporated the use of both HDAC and DNMT inhibitors have proven successful at inducing higher levels of p15INK4b expression

p15INK4b is a CDK inhibitor whose expression is lost in a very high proportion of patients with MDS and AML. This implicates an important role for the loss of the protein expression in the development of myeloid disease in humans. Its role in myeloid leukemogenesis as a tumor suppressor is now confirmed by research carried out in several mouse models. In addition to its role in inhibiting CDKs to arrest the cell cycle during late myeloid development, it is now known to affect cell fate decisions during early myelopoiesis. In myeloid progenitors, loss of p15Ink4b results in an imbalance in the progenitor pools, and favors the expansion of GMPs at the expense of MEPs. Importantly, when deleted in the cells of the myeloid lineage only, mice develop nonreactive monocytosis and are strongly predisposed to succumb to retrovirus-induced leukemia, especially of myeloid origin. These results validate a tight link between loss of p15INK4b and human myeloid neoplasia. Clinical observations that associate *p15INK4b* demethylation and re-expression with improved prognosis and survival, further stress the importance of this gene in AML and

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**16** 

**New Molecular Markers in** 

Silvia de la Iglesia Iñigo1, María Teresa Gómez Casares1,

*1Servicio de Hematología, Hospital de Gran Canaria Doctor Negrín,* 

*3Secció de Citologia Hematològica, Servei d'Anatomia Patològica* 

Acute leukemia is characterized by abnormal proliferation, inhibition of differentiation and expansion of leukemic cells blocked at the early stage of hematopoiesis. Acute myeloid leukemia (AML) is a malignant hematological disease of hematopoietic precursor cells of non-lymphoid lineage. Recent insights into the molecular mechanisms of AML are challenging the traditional diagnostic classification, prognostic significance and clinical

AML is a clinically heterogeneous disorder with distinct clinical and biological features. Until the 1970s, diagnosis was based on morphological examination of bone marrow and peripheral blood samples. In 1976, French, American, and British hematologists (the FAB group) defined six subgroups of AML morphological variants (Bennett et al., French-American-British Cooperative Group [FAB], 1976). This classification allowed us to identify several types of AML: M1-M6. Blastic populations were identified using standard staining techniques and consequently classified depending on reactivity to certain cytochemicals, namely, myeloperoxidase and Sudan black B (markers of myeloid differentiation) and nonspecific esterase reactions such as alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase (for monocytic lineage). Later on, this group identified two new types of AML (M0 and M7) according to cytochemical and inmunophenotypical features (Bennett et al., FAB, 1985). Over the past decade, refinement in the diagnosis of subtypes of AML and advances in therapeutic approaches have improved the outlook for patients with AML

**1. Introduction** 

practice of this hematological disorder.

**1.1 Concept and classification** 

(Döhner et al., 2010).

Carmen Elsa López Jorge1,2, Jezabel López Brito1

*2Unit Research, Hospital de Gran Canaria Doctor Negrín,* 

**Acute Myeloid Leukemia** 

and Pedro Martin Cabrera3

*Hospital Universitari de Bellvitge,* 

*Las Palmas de GranCanaria* 

*Las Palmas de GranCanaria* 

*Barcelona, Spain* 

inhibitor, p18INK4C. *Nature Cell Biology,* Vol.6, No.5, (May 2004), pp. 436-442, ISSN 1465-7392


### **New Molecular Markers in Acute Myeloid Leukemia**

Silvia de la Iglesia Iñigo1, María Teresa Gómez Casares1, Carmen Elsa López Jorge1,2, Jezabel López Brito1 and Pedro Martin Cabrera3 *1Servicio de Hematología, Hospital de Gran Canaria Doctor Negrín, Las Palmas de GranCanaria 2Unit Research, Hospital de Gran Canaria Doctor Negrín, Las Palmas de GranCanaria 3Secció de Citologia Hematològica, Servei d'Anatomia Patològica Hospital Universitari de Bellvitge, Barcelona, Spain* 

#### **1. Introduction**

312 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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Acute leukemia is characterized by abnormal proliferation, inhibition of differentiation and expansion of leukemic cells blocked at the early stage of hematopoiesis. Acute myeloid leukemia (AML) is a malignant hematological disease of hematopoietic precursor cells of non-lymphoid lineage. Recent insights into the molecular mechanisms of AML are challenging the traditional diagnostic classification, prognostic significance and clinical practice of this hematological disorder.

#### **1.1 Concept and classification**

AML is a clinically heterogeneous disorder with distinct clinical and biological features. Until the 1970s, diagnosis was based on morphological examination of bone marrow and peripheral blood samples. In 1976, French, American, and British hematologists (the FAB group) defined six subgroups of AML morphological variants (Bennett et al., French-American-British Cooperative Group [FAB], 1976). This classification allowed us to identify several types of AML: M1-M6. Blastic populations were identified using standard staining techniques and consequently classified depending on reactivity to certain cytochemicals, namely, myeloperoxidase and Sudan black B (markers of myeloid differentiation) and nonspecific esterase reactions such as alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase (for monocytic lineage). Later on, this group identified two new types of AML (M0 and M7) according to cytochemical and inmunophenotypical features (Bennett et al., FAB, 1985). Over the past decade, refinement in the diagnosis of subtypes of AML and advances in therapeutic approaches have improved the outlook for patients with AML (Döhner et al., 2010).

New Molecular Markers in Acute Myeloid Leukemia 315

**Acute myeloid leukemia with recurrent genetic abnormalities**

 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); *RPN1-EVI1*  AML (megakaryoblastic) with t(1;22)(p13;q13); *RBM15-MKL1* 

**Acute myeloid leukemia with myelodysplasia-related changes** 

AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); *CBFB-MYH11* 

AML with t(8;21)(q22;q22); *RUNX1-RUNX1T1*

 APL with t(15;17)(q22;q12); *PML-RARA*  AML with t(9;11)(p22;q23); *MLLT3-MLL*  AML with t(6;9)(p23;q34); *DEK-NUP214* 

 *Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA* 

**Acute myeloid leukemia, not otherwise specified** 

**Myeloid proliferations related to Down syndrome** 

 Myeloid leukemia associated with Down syndrome **Blastic plasmacytoid dendritic cell neoplasm**

Table 2. WHO classification of acute myeloid leukemia and related neoplasms.

**1.2.1 Useful at diagnosis and to evaluate Minimal Residual Disease (MRD)** 

Molecular techniques allow us to diagnose and classify AML, establishing groups of patients with different disease behaviour and prognosis. In the same way, the development of more specific therapies and the application of risk-adjusted therapeutic approaches have been made possible. Disease relapse can be favored by persistent low numbers of leukemic cells undetectable by conventional techniques. Monitoring of AML patients has given rise to a higher insight into the effectiveness of treatment. At diagnosis, some types of myeloid leukemias display a molecular change that might be useful as a marker of neoplastic disease and MRD (Hilden et al., 1995; Nakao et al., 1996; Foroni et al., 1999; Lo Coco et al., 1999a). Specific molecular alterations of certain hematologic malignancies are useful in the detection of MRD. Translocations are the most widely involved chromosomal abnormalities in AML. These can give rise to an altered function or activity of oncogenes located at or near the translocated breakpoint. The first molecular cytogenetic marker described in AML was t(15;17), which originates a single morphologic phenotype (hyper- or micro-granular French-American-British [FAB] AML-M3 acute myeloid leukemia) (Bernstein et al., 1980). This rearrangement disappears with complete remission and is able to predict a relapse very

**Therapy-related myeloid neoplasms** 

AML with minimal differentiation

 Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia

 Erythroleukemia, erythroid/myeloid Acute megakaryoblastic leukemia Acute basophilic leukemia

Acute panmyelosis with myelofibrosis

Transient abnormal myelopoiesis

 AML without maturation AML with maturation

 Acute erythroid leukemia Pure erythroid leukemia

**Myeloid sarcoma** 

**1.2 Molecular markers** 


Table 1. FAB classification of acute myeloid leukemia (AML).

Since several specific cytogenetic and genetic abnormalities in AML are associated with a characteristic morphology and have distinctive clinical behavior (Harris et al., World Health Organization [WHO], 1999). The World Health Organization (WHO), classification of myeloid neoplasms and acute leukemias integrates genetic, clinical data, and morphological features.

Three prognostic groups have been described classically according to cytogenetic findings. The favorable prognostic group includes the following chromosomal abnormalities: t(15;17), t(8;21) and inv(16). Normal karyotype and cytogenetic abnormalities not classified as favorable or adverse comprise the intermediate group. The adverse prognosis group includes patients with complex karyotype, del (5q), 5 or 7 monosomy, 3q abnormalities and t(6;9) (Dohner et al., 2010).

Recently the WHO published a revised and updated edition of the 1998 WHO classification where the importance of gene mutations as diagnostic and prognostic markers in myeloid neoplasms was acknowledged. This group recommended that fluorescence in situ hybridization (FISH), reverse transcriptase–polymerase chain reaction (RT-PCR) and mutational status studies should be guided through clinical, laboratory, and morphologic information. Mutational studies for mutated *NPM1*, *CEBPA*, *FLT3*, *KIT*, *WT1*, and *MLL* are recommended in all cytogenetically normal AML. Table 2 lists the major subgroups of AML in the WHO classification (WHO, 2009).

The genes involved in the pathogenesis of leukemia are normal genes (proto-oncogenes) with either structural alterations or deregulated expression patterns, which generates in turn a novel gene (an oncogene) whose protein product acts on the host cell to enhance malignancy-related characteristics. Oncogene activation and the loss of tumor-suppressor genes are consistently associated with some types of leukemia (Cline, 1994*)*. Some of the molecular alterations involved in the pathogenesis of AML are: translocations, mutations and overexpression of normal genes, which often characterizes a particular subtype of AML. Therefore, there are also activating mutations which lead to increased proliferation or survival, or both, of haematopoietic progenitor cells through the stimulation of tyrosine kinases such as FLT3 or RAS family members. These are considered class I mutations (Haferlach et al., 2007; Schlenk et al., 2008). Class II mutations interfere with transcription and lead to a maturation arrest either through a direct alteration of transcription factors due to gene fusions (CBF-leukemias or PML-RARA positive leukemia) or by indirect interference with transcription (MLL-rearrangements). Cytogenetic and molecular analysis of leukemic blasts, provide critical diagnostic, therapeutic and prognostic information.

**FAB classification** 

Since several specific cytogenetic and genetic abnormalities in AML are associated with a characteristic morphology and have distinctive clinical behavior (Harris et al., World Health Organization [WHO], 1999). The World Health Organization (WHO), classification of myeloid neoplasms and acute leukemias integrates genetic, clinical data, and morphological

Three prognostic groups have been described classically according to cytogenetic findings. The favorable prognostic group includes the following chromosomal abnormalities: t(15;17), t(8;21) and inv(16). Normal karyotype and cytogenetic abnormalities not classified as favorable or adverse comprise the intermediate group. The adverse prognosis group includes patients with complex karyotype, del (5q), 5 or 7 monosomy, 3q abnormalities and

Recently the WHO published a revised and updated edition of the 1998 WHO classification where the importance of gene mutations as diagnostic and prognostic markers in myeloid neoplasms was acknowledged. This group recommended that fluorescence in situ hybridization (FISH), reverse transcriptase–polymerase chain reaction (RT-PCR) and mutational status studies should be guided through clinical, laboratory, and morphologic information. Mutational studies for mutated *NPM1*, *CEBPA*, *FLT3*, *KIT*, *WT1*, and *MLL* are recommended in all cytogenetically normal AML. Table 2 lists the major subgroups of AML

The genes involved in the pathogenesis of leukemia are normal genes (proto-oncogenes) with either structural alterations or deregulated expression patterns, which generates in turn a novel gene (an oncogene) whose protein product acts on the host cell to enhance malignancy-related characteristics. Oncogene activation and the loss of tumor-suppressor genes are consistently associated with some types of leukemia (Cline, 1994*)*. Some of the molecular alterations involved in the pathogenesis of AML are: translocations, mutations and overexpression of normal genes, which often characterizes a particular subtype of AML. Therefore, there are also activating mutations which lead to increased proliferation or survival, or both, of haematopoietic progenitor cells through the stimulation of tyrosine kinases such as FLT3 or RAS family members. These are considered class I mutations (Haferlach et al., 2007; Schlenk et al., 2008). Class II mutations interfere with transcription and lead to a maturation arrest either through a direct alteration of transcription factors due to gene fusions (CBF-leukemias or PML-RARA positive leukemia) or by indirect interference with transcription (MLL-rearrangements). Cytogenetic and molecular analysis of leukemic blasts, provide critical diagnostic, therapeutic and prognostic information.

M0: AML minimally differentiated M1: AML without maturation M2: AML with maturation

M3: Acute promyelocytic leukemia M4: Acute myelomonocytic leukemia M5: Acute monoblastic leukemia M6: Acute erythroleukemia

M7: Acute megakaryoblastic leukemia

Table 1. FAB classification of acute myeloid leukemia (AML).

features.

t(6;9) (Dohner et al., 2010).

in the WHO classification (WHO, 2009).


Table 2. WHO classification of acute myeloid leukemia and related neoplasms.

#### **1.2 Molecular markers**

#### **1.2.1 Useful at diagnosis and to evaluate Minimal Residual Disease (MRD)**

Molecular techniques allow us to diagnose and classify AML, establishing groups of patients with different disease behaviour and prognosis. In the same way, the development of more specific therapies and the application of risk-adjusted therapeutic approaches have been made possible. Disease relapse can be favored by persistent low numbers of leukemic cells undetectable by conventional techniques. Monitoring of AML patients has given rise to a higher insight into the effectiveness of treatment. At diagnosis, some types of myeloid leukemias display a molecular change that might be useful as a marker of neoplastic disease and MRD (Hilden et al., 1995; Nakao et al., 1996; Foroni et al., 1999; Lo Coco et al., 1999a). Specific molecular alterations of certain hematologic malignancies are useful in the detection of MRD. Translocations are the most widely involved chromosomal abnormalities in AML. These can give rise to an altered function or activity of oncogenes located at or near the translocated breakpoint. The first molecular cytogenetic marker described in AML was t(15;17), which originates a single morphologic phenotype (hyper- or micro-granular French-American-British [FAB] AML-M3 acute myeloid leukemia) (Bernstein et al., 1980). This rearrangement disappears with complete remission and is able to predict a relapse very

New Molecular Markers in Acute Myeloid Leukemia 317

In recent years, the availability of new genetic and molecular prognostic markers in AML has grown considerably. This is particularly important in the case of patients with normal cytogenetics who comprise the largest subgroup of AML patients (approximately 45%) where many new prognostic factors have been identified. These include gene mutations in FLT3 (Fms-like tyrosine kinase 3; generally FLT3-ITD has been associated with significantly worse survival (Sheikhha et al, 2003)), NPM1 (nucleophosmin 1) and CEBPA (CCAAT enhancer-binding protein-α; generally favorable in cases of biallelic mutations) and gene

Identifying alterations in these genes might provide independent prognostic value in predicting the outcome of acute leukemia, as in the case of the NPM1 mutation gene, which is a relatively frequent abnormality in AML patients and is useful in detecting MRD (Falini

Leukemias affecting CBF are characterized by rearrangements of genes that code for components of the heterodimeric transcription factor CBF, which plays an essential role in haematopoiesis (Gabert et al., 2006). CBF complex is a heterodimer composed of RUNX1 (also called AML1) and CBF and is the target of at least three common transloctions in AML: t(8;21)/RUNX1-RUNX1T1, t(3;21)/RUNX1-EVI1 and inv(16) or t(16;16) resulting in CBF-MYH11. Rearrangements of *AML1* and *CBFB* with other genes lead to chimeric

AML1, also called, RUNX1, is normally expressed in all hematopoietic lineages and regulates the expression of several genes specifically linked to hematopoiesis, including the granulocyte colony-stimulating factor receptor, interleukin 3, T-cell receptor, and myeloperoxidase (MPO) genes. The *AML1* gene (on 21q22) is one of the genes most frequently deregulated in leukemias, generally through translocations that produce chimeric messenger RNA. Chimeric protein AML1-ETO (*RUNX1-RUNX1T1)* results from the t(8;21)(q22;q22) involving the AMLI gene on chromosome 21 and the ETO gene on chromosome 8. This rearrangement is detected in approximately 8% of AML cases in children and young adults. *RUNX1-RUNX1T1* i*s* a marker for favourable outcome and an important PCR target for MRD detection. Most patients achieve a CR after induction therapy and those patients benefit most from a postremission therapy with high-dose cytarabin (Bloomfield et al., 1998; Perea et al., 2006). Hence, this marker permits to single out a relatively small subgroup of patients who are more likely to relapse (Gabert et al., 2003). *RUNX1-RUNX1T1* is also frequently associated with c-kit mutations which determine an adverse outcome within this group of patients with favorable prognosis. In addition, the loss of Y chromosome in male patients with t(8;21) leukemia is a negative prognostic factor for the overall survival (Schlenk et al., 2004). While the molecular diagnosis is performed by qualitative and quantitative Real Time RT-PCR (QRT-PCR), MRD monitoring is performed

proteins that disrupt the CBF complex, suppressing the activation of transcription.

**1.2.2 Genetic alterations with prognosis value** 

et al., 2007).

overexpression as BAALC, WT1, EVI1 and MN1 (Foran, 2010).

Next, we will describe the most common molecular markers in AML.

**2. Detailed description of molecular markers** 

**2.1 Core Binding Factor (CBF)** 

**2.1.1 AML1 (RUNX1) rearrangements** 

accurately. The presence of this translocation or its fusion gene (PML/ RAR) is thus a detectable tumor marker in leukemic cells, which allows assessment of the molecular response to therapy in AML-M3 patients (Lo Coco et al., 1999b). *RUNX1-RUNX1T1* and CBF-MYH11 are other genetic alterations less likely to predict relapse, since they may persist even when complete remission (CR) has been achieved. However, the applicability of this strategy has been limited to those leukemia subsets characterized by genetic markers. Recent interest has focused on identifying new molecular markers that might prove significant in the diagnosis and follow-up of MRD in AML patients. In recent years, a variety ot potential molecular markers have been identified (see table 2 and table 3) (Radich & Thomson, 1997; Inoue et al., 1994; Kreuzer et al., 2001; Alberta et al., 2003; Lin et al., 2005; Gilliland & Griffin, 2002; Morishita et al., 1992).


Table 3. Molecular markers in AML: prevalence, prognostic, associated mutations, genetic alterations and utility in MDR.

accurately. The presence of this translocation or its fusion gene (PML/ RAR) is thus a detectable tumor marker in leukemic cells, which allows assessment of the molecular response to therapy in AML-M3 patients (Lo Coco et al., 1999b). *RUNX1-RUNX1T1* and CBF-MYH11 are other genetic alterations less likely to predict relapse, since they may persist even when complete remission (CR) has been achieved. However, the applicability of this strategy has been limited to those leukemia subsets characterized by genetic markers. Recent interest has focused on identifying new molecular markers that might prove significant in the diagnosis and follow-up of MRD in AML patients. In recent years, a variety ot potential molecular markers have been identified (see table 2 and table 3) (Radich & Thomson, 1997; Inoue et al., 1994; Kreuzer et al., 2001; Alberta et al., 2003; Lin et al., 2005;

**Prevalence Prognostic value Associated**

Favorable (Poor with KIT in normal karyotype

FLT3 NRAS KIT

biallelic - Yes

(without FLT3) FLT3 Yes

65% Poor - Possible

10-22% Poor - Possible

10-15% Poor - Yes

Table 3. Molecular markers in AML: prevalence, prognostic, associated mutations, genetic

M0) Yes

26%) -

leukemia)

PML-RARA 10-15% Favorable FLT3 (40%) Yes MLL mutation 10-30% Poor - Yes

mutation 28-34% Poor Possible

KRAS 5% Not influence FLT3-ITD (24-

c-KIT mutation 6-48% Poor CBF-leukemias Yes

mutation 20% Poor FLT3-ITD - IDH1/2 mutation 15% Controversial NPM1, FLT3-ITD -

AML1 mutation 1-20% Poor FLT3 (in 20%

mutation 20-30% Controversial CBF, NPM1

**mutations Utility in MRD** 

Possible

Gilliland & Griffin, 2002; Morishita et al., 1992).

15%

CEBPA mutation 15-20% Favorable if

**Involvement of transcription** 

CBF-leukemias: Inv 16/t(16;16) CBFB/MYH11 t(8;21);*RUNX1- RUNX1T1*

**Activating mutations**

RAS mutation NRAS 11%

alterations and utility in MDR.

NPM1 mutation 35% Favorable

**Other genes alterations**

FLT3-ITD

FLT3-TKD

BAALC overexpression

EVI-1

WT1

overexpression

overexpression

DNMT3A

#### **1.2.2 Genetic alterations with prognosis value**

In recent years, the availability of new genetic and molecular prognostic markers in AML has grown considerably. This is particularly important in the case of patients with normal cytogenetics who comprise the largest subgroup of AML patients (approximately 45%) where many new prognostic factors have been identified. These include gene mutations in FLT3 (Fms-like tyrosine kinase 3; generally FLT3-ITD has been associated with significantly worse survival (Sheikhha et al, 2003)), NPM1 (nucleophosmin 1) and CEBPA (CCAAT enhancer-binding protein-α; generally favorable in cases of biallelic mutations) and gene overexpression as BAALC, WT1, EVI1 and MN1 (Foran, 2010).

Identifying alterations in these genes might provide independent prognostic value in predicting the outcome of acute leukemia, as in the case of the NPM1 mutation gene, which is a relatively frequent abnormality in AML patients and is useful in detecting MRD (Falini et al., 2007).

#### **2. Detailed description of molecular markers**

Next, we will describe the most common molecular markers in AML.

#### **2.1 Core Binding Factor (CBF)**

Leukemias affecting CBF are characterized by rearrangements of genes that code for components of the heterodimeric transcription factor CBF, which plays an essential role in haematopoiesis (Gabert et al., 2006). CBF complex is a heterodimer composed of RUNX1 (also called AML1) and CBF and is the target of at least three common transloctions in AML: t(8;21)/RUNX1-RUNX1T1, t(3;21)/RUNX1-EVI1 and inv(16) or t(16;16) resulting in CBF-MYH11. Rearrangements of *AML1* and *CBFB* with other genes lead to chimeric proteins that disrupt the CBF complex, suppressing the activation of transcription.

#### **2.1.1 AML1 (RUNX1) rearrangements**

AML1, also called, RUNX1, is normally expressed in all hematopoietic lineages and regulates the expression of several genes specifically linked to hematopoiesis, including the granulocyte colony-stimulating factor receptor, interleukin 3, T-cell receptor, and myeloperoxidase (MPO) genes. The *AML1* gene (on 21q22) is one of the genes most frequently deregulated in leukemias, generally through translocations that produce chimeric messenger RNA. Chimeric protein AML1-ETO (*RUNX1-RUNX1T1)* results from the t(8;21)(q22;q22) involving the AMLI gene on chromosome 21 and the ETO gene on chromosome 8. This rearrangement is detected in approximately 8% of AML cases in children and young adults. *RUNX1-RUNX1T1* i*s* a marker for favourable outcome and an important PCR target for MRD detection. Most patients achieve a CR after induction therapy and those patients benefit most from a postremission therapy with high-dose cytarabin (Bloomfield et al., 1998; Perea et al., 2006). Hence, this marker permits to single out a relatively small subgroup of patients who are more likely to relapse (Gabert et al., 2003). *RUNX1-RUNX1T1* is also frequently associated with c-kit mutations which determine an adverse outcome within this group of patients with favorable prognosis. In addition, the loss of Y chromosome in male patients with t(8;21) leukemia is a negative prognostic factor for the overall survival (Schlenk et al., 2004). While the molecular diagnosis is performed by qualitative and quantitative Real Time RT-PCR (QRT-PCR), MRD monitoring is performed

New Molecular Markers in Acute Myeloid Leukemia 319

C-terminal basic leucine zipper (bZIP) regions (Figure 1). The latter affect both DNA binding and homo and heterodimerization with other CEBP-proteins. The former introduce a premature stoppage of the translation of the p42 CEBPA protein, preserving a p30 isoform, which was shown to inhibit DNA binding and transactivation by C/EBPpCEBPA mutations are exclusively related to the intermediate risk group karyotype AML. CEBPA insertion, deletions and point mutations are detected usually by DNA sequencing (Wouters

Acute promyelocytic leukemia is a distinct subtype of AML (AML-M3) according to the FAB classification. AML-M3 is characterized by t(15;17) that involves the retinoic acid receptor a (RAR) gene on chromosome 17 and the promyelocytic leukemia gene (PML) on chromosome 15. At the molecular level, t(15;17) results in a hybrid PML/ RAR gene, which is easily identified by reverse transcriptase-polymerase chain reaction (RT-PCR). This test provides a rapid and refined diagnosis. The usefulness of minimal residual disease monitoring during follow up (Lo Coco et al., 1992; Lo Coco et al., 1999a) has been well established. The different breakpoints within the PML gene cluster can be located in three

The assessment of remission status at the molecular level by RT-PCR of PML-RAR represents a significant clinical advance with respect to other poorly sensitive methods (morphology, karyotype). The treatment of this disease (arsenicals, liposomal ATRA, other retinoid derivatives, etc) needs to be assessed taking into account the response at the RT-PCR level. The detection of residual PML-RAR transcripts during clinical remission predicts subsequent hematologic relapse. This determines the need for additional treatment given the benefits of anticipating salvage therapy in AML-M3. Figure 3 shows the statistically significant difference between patients treated for molecular relapse and the

Fig. 2. Kaplan-Meier estimates of overall survival from relapse in patients treated at the time

Around 40% of all cases of PML-RAR-positive AML show FLT3 mutations. FLT3-ITD mutations, but not *FLT3-D835*, are associated with characteristic diagnostic hematological

of molecular relapse (dotted line) and in the historical series of patients treated for

hematologic relapse (continuous line).

historical series treated for hematologic relapse (Lo Coco et al., 1999b).

et al., 2009; Pabst et al., 2001; Fuchs et al., 2008).

**2.2 PML-RAR**

regions: bcr1, 2 and 3.

by quantitative QRT-PCR. The rare balanced t(3;21)(q26;q22) was described in AML, mainly after treatment with topoisomerase II inhibitors. This translocation fuses the AML1 gene on 21q22 and the EVI-1 gene on 3q26 resulting in t(3;21)(q26;q22). This translocation is associated with higher WBC and platelets counts but is not, however, predictive for relapsefree or overall survival (Preudhomme et al., 2000; Meyers et al., 1993; Lutghart et al., 2010).

#### **2.1.2 AML1 mutations**

AML1 mutations can be found in *de novo* leukemia, particularly subtypes FAB M0 and M7, as well as in patients with trisomy 21 and myelodysplastic syndromes. Testing for mutations can be performed By real time PCR or PCR-single stranded conformational polymorphism (SSCP). A multivariate analysis carried out by Schnittger et al., showed an independent unfavourable prognostic significance of AML1 (or RUNX1) mutations for overall survival (Schnittger et al., 2011).

#### **2.1.3 CBFb-MYH11**

Inv(16)(p13;q22) or t(16;16)(p13;q22) are among the most frequent recurring chromosomal rearrangements detected in AML, generally observed in cases showing myelomonocytic differentiation and having abnormal bone marrow eosinophils (M4 Eo AML in the French American British (FAB) classification). Inv(16)(p13;q22) is found in approximately 10-12% of cases of AML. It can occur in all age groups but is predominantly seen in younger patients. This rearrangement results in the disruption of the myosin heavy chain (MYH) gene at 16p13 and the core binding factor (CBF) gene at 16q22 (FAB, 1976). Ten different CBF-MYH11 transcripts have been reported, but the frequency of each transcript is variable. CBFB-MYH11 positive patients are considered to have a favourable prognosis. This rearrangement is frequently associated with c-kit and FLT3 mutations which worsen the prognosis. Quantitative QRT-PCR allows monitoring of CR (Gabert et al., 2003; Perea et al., 2005).

#### **2.1.4 CEBPA**

The CCAAT/enhancer binding alpha protein (C/EBP) is the founding member of a family of related leucine zipper transcription factors. Mutations in CEBPA are found in 5-14% of AML and have been associated with a relatively favourable outcome only in biallelic mutations of this gene. There are two main classes of mutation situated at the N- terminal or

Fig. 1. Schematic representation of CEBPA (by Smith, 2004).

C-terminal basic leucine zipper (bZIP) regions (Figure 1). The latter affect both DNA binding and homo and heterodimerization with other CEBP-proteins. The former introduce a premature stoppage of the translation of the p42 CEBPA protein, preserving a p30 isoform, which was shown to inhibit DNA binding and transactivation by C/EBPpCEBPA mutations are exclusively related to the intermediate risk group karyotype AML. CEBPA insertion, deletions and point mutations are detected usually by DNA sequencing (Wouters et al., 2009; Pabst et al., 2001; Fuchs et al., 2008).

#### **2.2 PML-RAR**

318 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

by quantitative QRT-PCR. The rare balanced t(3;21)(q26;q22) was described in AML, mainly after treatment with topoisomerase II inhibitors. This translocation fuses the AML1 gene on 21q22 and the EVI-1 gene on 3q26 resulting in t(3;21)(q26;q22). This translocation is associated with higher WBC and platelets counts but is not, however, predictive for relapsefree or overall survival (Preudhomme et al., 2000; Meyers et al., 1993; Lutghart et al., 2010).

AML1 mutations can be found in *de novo* leukemia, particularly subtypes FAB M0 and M7, as well as in patients with trisomy 21 and myelodysplastic syndromes. Testing for mutations can be performed By real time PCR or PCR-single stranded conformational polymorphism (SSCP). A multivariate analysis carried out by Schnittger et al., showed an independent unfavourable prognostic significance of AML1 (or RUNX1) mutations for overall survival

Inv(16)(p13;q22) or t(16;16)(p13;q22) are among the most frequent recurring chromosomal rearrangements detected in AML, generally observed in cases showing myelomonocytic differentiation and having abnormal bone marrow eosinophils (M4 Eo AML in the French American British (FAB) classification). Inv(16)(p13;q22) is found in approximately 10-12% of cases of AML. It can occur in all age groups but is predominantly seen in younger patients. This rearrangement results in the disruption of the myosin heavy chain (MYH) gene at 16p13 and the core binding factor (CBF) gene at 16q22 (FAB, 1976). Ten different CBF-MYH11 transcripts have been reported, but the frequency of each transcript is variable. CBFB-MYH11 positive patients are considered to have a favourable prognosis. This rearrangement is frequently associated with c-kit and FLT3 mutations which worsen the prognosis. Quantitative

The CCAAT/enhancer binding alpha protein (C/EBP) is the founding member of a family of related leucine zipper transcription factors. Mutations in CEBPA are found in 5-14% of AML and have been associated with a relatively favourable outcome only in biallelic mutations of this gene. There are two main classes of mutation situated at the N- terminal or

QRT-PCR allows monitoring of CR (Gabert et al., 2003; Perea et al., 2005).

Fig. 1. Schematic representation of CEBPA (by Smith, 2004).

**2.1.2 AML1 mutations** 

(Schnittger et al., 2011).

**2.1.3 CBFb-MYH11** 

**2.1.4 CEBPA** 

Acute promyelocytic leukemia is a distinct subtype of AML (AML-M3) according to the FAB classification. AML-M3 is characterized by t(15;17) that involves the retinoic acid receptor a (RAR) gene on chromosome 17 and the promyelocytic leukemia gene (PML) on chromosome 15. At the molecular level, t(15;17) results in a hybrid PML/ RAR gene, which is easily identified by reverse transcriptase-polymerase chain reaction (RT-PCR). This test provides a rapid and refined diagnosis. The usefulness of minimal residual disease monitoring during follow up (Lo Coco et al., 1992; Lo Coco et al., 1999a) has been well established. The different breakpoints within the PML gene cluster can be located in three regions: bcr1, 2 and 3.

The assessment of remission status at the molecular level by RT-PCR of PML-RAR represents a significant clinical advance with respect to other poorly sensitive methods (morphology, karyotype). The treatment of this disease (arsenicals, liposomal ATRA, other retinoid derivatives, etc) needs to be assessed taking into account the response at the RT-PCR level. The detection of residual PML-RAR transcripts during clinical remission predicts subsequent hematologic relapse. This determines the need for additional treatment given the benefits of anticipating salvage therapy in AML-M3. Figure 3 shows the statistically significant difference between patients treated for molecular relapse and the historical series treated for hematologic relapse (Lo Coco et al., 1999b).

Fig. 2. Kaplan-Meier estimates of overall survival from relapse in patients treated at the time of molecular relapse (dotted line) and in the historical series of patients treated for hematologic relapse (continuous line).

Around 40% of all cases of PML-RAR-positive AML show FLT3 mutations. FLT3-ITD mutations, but not *FLT3-D835*, are associated with characteristic diagnostic hematological

New Molecular Markers in Acute Myeloid Leukemia 321

cohort. The global mortality was analyzed disregarding risk factors, with a mortality of 67 % in the NPM1+/FLT3-ITD- group standing in clear contrast to a 100% death rate in the NPM1+/FLT3-ITD+ group (Lopez Jorge et al., 2006). This was confirmed in another study where we analysed the incidence and prognostic relevance of CD34/CD7/DR surface markers in a group of forty two NPM1positive patients and related it to FLT3-ITD mutations. We found that 84% of the NPM1 positive patients had normal karyotypes. Mutations in FLT3-ITD were detected in 23.8% of the NPM1+ patients. The screening for this mutation could be very useful in the future as patients with normal karyotype and expression of this molecular marker is included in a better prognostic subgroup (Gomez

**Patients** (%) 55 17 (30.9) 38(69) **Median (range age) (years**) 60 (18-95) 58 (18-82) 60 (19-95)

> 5 8/10 12/4 1 15

**FLT3-ITD+ (%)** 16 (29) 7(41.2) 9(23.6) Table 4. Cytogenetics and demographic characteristics in a group of 55 AML patients (Lopez

*KIT* is a proto-oncogene located on chromosome band 4q11-12 and encodes a 145-kDa transmembrane glycoprotein member of the type III receptor tyrosine kinase family. Ligand independent activation of KIT results from mutations in the extracellular portion of the receptor (exon 8), transmembrane and juxtamembrane domains (exons 10 and 11, respectively), and activation loop of the tyrosine kinase domain (exon 17). *c-KIT* mutations have been found in a variable but relatively high frequency (up to 50%) in patients with CBF

**Nº patients NPM1+ NPM1-**

0 4/4 3/1 0 5

5 4/6 9/3 1 10

Casares et al., 2009; Dohner et al., 2010).

Age, r**FAB subtype** 

M0 M1/M2 M4/M5 M6

Not labeled

del(5) del(7) trisomy 8 t(4;11) t(8;21) t(10;11) t(9;11) t(12;13) t(3;10) +11 -Y

**Cytogenetic:**  Normal karyotype

Complex: >3 abnormalities

Non determinated

Jorge et al., 2006).

**2.5 c-KIT** 

features of acute promyelocytic leukemia, in particular with high WBC counts. Also, FLT3 mutations, especially ITD, can adversely affect overall survival and disease-free survival in PML-RAR-positive AML (Beitinjaneh et al., 2010). However, in a large series of 739 patients with acute promyelocytic leukemia treated with ATRA and anthracycline-based chemotherapy, we were unable to demonstrate an independent prognostic value of *FLT3*  mutations (Barragan et al., 2011).

#### **2.3 DEK-CAN**

AML defined by t(6;9) is a relatively rare disease, associated with specific clinical and morphological features (Garcon et al., 2005). Especially in young adults, the leukemic phase can be preceded by dysplastic features, conferring a bad prognosis. Morphological findings usually correlate with FAB M2 (60%), M4 (30%), or Ml (von Lindern et al., 1990). A chimeric protein is generated, resulting from the fusion between DEK and the 30- terminus of the CAN gene, also known as NUP214 (von Lindern et al., 1990). DEK is a component of metazoan chromatin capable of modifying the structure of DNA by introducing supercoils. CAN is a nuclear pore complex protein implicated in nucleocytoplasmic trafficking. The CAN gene is also involved in several fusion transcripts described in acute leukemia other than DEK in t(6;9)(p23;q34) AML, such as the SET gene and recently with ABL in T-cell acute lymphoblastic leukaemia (ALL). The DEK-CAN transcript can be used as a marker of t(6;9) AML which can be sensitively monitored by the polymerase chain reaction. This offers a great advantage in the diagnosis, monitoring of response to chemotherapy, and detection of minimal residual disease after bone marrow transplantation (von Lindern et al., 1992). DEK-CAN is related with an adverse prognosis (Dohner et al., 2010).

#### **2.4 NPM1**

*NPM1* gene is located in 5q35 and encodes a phosphoprotein, nucleophosmin, which moves between the nucleus and the cytoplasm. The gene product is thought to be involved in several processes including regulation of the ARF/p53 pathway. Mutations in exon 12 in this gene are associated with AML with normal karyotype (50%) and especially correlate with monocytic leukemias. Patients with *NPM1* mutations have a significantly higher rate of complete remissions (CRs) after standard induction chemotherapy except for cases associated with internal tandem duplications mutations of FLT3 (Falini et al., 2007; Gale et al., 2008). Gale et al. (2008) identified 3 prognostic groups among the NPM1+ AML patients: good in those with only a NPM1 mutation and absence of a FLT3-ITD; intermediate in those with either absence of FLT3-ITD or NPM1 mutations or mutations in both genes; and poor in those with only FLT3-ITD. Monitoring can be performed by quantitative PCR (Schnittger et al., 2009). In our group, the incidence of NPM1 mutation was 30% (17 of 55 patients with AML), being this prevalence similar to that found for FLT3-ITD in the same population (29 %). The 17 NPM1+ AML patients were distributed as follows: eight M1-M2 (47.1 %), four M4-M5 (23.5%), and five not labelled (29.4 %) because cytogenetic studies were not informative due to several reasons. 64.7% of the NPM1+ patients had a normal karyotype, while 5.8% of them had cytogenetic anomalies. FLT3-ITD mutations were found in 41.2 % of the NPM1+ AML cases. In contrast to what has been described in the literature our group did not find a higher incidence of M4 or M5 subtypes (Thiede et al., 2002). In our case, a higher incidence of M1 and M2 subtypes was detected (Table 4). However the differences are most likely attributable to the small sample size of our cohort as compared to the Thiede

features of acute promyelocytic leukemia, in particular with high WBC counts. Also, FLT3 mutations, especially ITD, can adversely affect overall survival and disease-free survival in PML-RAR-positive AML (Beitinjaneh et al., 2010). However, in a large series of 739 patients with acute promyelocytic leukemia treated with ATRA and anthracycline-based chemotherapy, we were unable to demonstrate an independent prognostic value of *FLT3* 

AML defined by t(6;9) is a relatively rare disease, associated with specific clinical and morphological features (Garcon et al., 2005). Especially in young adults, the leukemic phase can be preceded by dysplastic features, conferring a bad prognosis. Morphological findings usually correlate with FAB M2 (60%), M4 (30%), or Ml (von Lindern et al., 1990). A chimeric protein is generated, resulting from the fusion between DEK and the 30- terminus of the CAN gene, also known as NUP214 (von Lindern et al., 1990). DEK is a component of metazoan chromatin capable of modifying the structure of DNA by introducing supercoils. CAN is a nuclear pore complex protein implicated in nucleocytoplasmic trafficking. The CAN gene is also involved in several fusion transcripts described in acute leukemia other than DEK in t(6;9)(p23;q34) AML, such as the SET gene and recently with ABL in T-cell acute lymphoblastic leukaemia (ALL). The DEK-CAN transcript can be used as a marker of t(6;9) AML which can be sensitively monitored by the polymerase chain reaction. This offers a great advantage in the diagnosis, monitoring of response to chemotherapy, and detection of minimal residual disease after bone marrow transplantation (von Lindern et al., 1992).

*NPM1* gene is located in 5q35 and encodes a phosphoprotein, nucleophosmin, which moves between the nucleus and the cytoplasm. The gene product is thought to be involved in several processes including regulation of the ARF/p53 pathway. Mutations in exon 12 in this gene are associated with AML with normal karyotype (50%) and especially correlate with monocytic leukemias. Patients with *NPM1* mutations have a significantly higher rate of complete remissions (CRs) after standard induction chemotherapy except for cases associated with internal tandem duplications mutations of FLT3 (Falini et al., 2007; Gale et al., 2008). Gale et al. (2008) identified 3 prognostic groups among the NPM1+ AML patients: good in those with only a NPM1 mutation and absence of a FLT3-ITD; intermediate in those with either absence of FLT3-ITD or NPM1 mutations or mutations in both genes; and poor in those with only FLT3-ITD. Monitoring can be performed by quantitative PCR (Schnittger et al., 2009). In our group, the incidence of NPM1 mutation was 30% (17 of 55 patients with AML), being this prevalence similar to that found for FLT3-ITD in the same population (29 %). The 17 NPM1+ AML patients were distributed as follows: eight M1-M2 (47.1 %), four M4-M5 (23.5%), and five not labelled (29.4 %) because cytogenetic studies were not informative due to several reasons. 64.7% of the NPM1+ patients had a normal karyotype, while 5.8% of them had cytogenetic anomalies. FLT3-ITD mutations were found in 41.2 % of the NPM1+ AML cases. In contrast to what has been described in the literature our group did not find a higher incidence of M4 or M5 subtypes (Thiede et al., 2002). In our case, a higher incidence of M1 and M2 subtypes was detected (Table 4). However the differences are most likely attributable to the small sample size of our cohort as compared to the Thiede

DEK-CAN is related with an adverse prognosis (Dohner et al., 2010).

mutations (Barragan et al., 2011).

**2.3 DEK-CAN** 

**2.4 NPM1** 

cohort. The global mortality was analyzed disregarding risk factors, with a mortality of 67 % in the NPM1+/FLT3-ITD- group standing in clear contrast to a 100% death rate in the NPM1+/FLT3-ITD+ group (Lopez Jorge et al., 2006). This was confirmed in another study where we analysed the incidence and prognostic relevance of CD34/CD7/DR surface markers in a group of forty two NPM1positive patients and related it to FLT3-ITD mutations. We found that 84% of the NPM1 positive patients had normal karyotypes. Mutations in FLT3-ITD were detected in 23.8% of the NPM1+ patients. The screening for this mutation could be very useful in the future as patients with normal karyotype and expression of this molecular marker is included in a better prognostic subgroup (Gomez Casares et al., 2009; Dohner et al., 2010).


Table 4. Cytogenetics and demographic characteristics in a group of 55 AML patients (Lopez Jorge et al., 2006).

#### **2.5 c-KIT**

*KIT* is a proto-oncogene located on chromosome band 4q11-12 and encodes a 145-kDa transmembrane glycoprotein member of the type III receptor tyrosine kinase family. Ligand independent activation of KIT results from mutations in the extracellular portion of the receptor (exon 8), transmembrane and juxtamembrane domains (exons 10 and 11, respectively), and activation loop of the tyrosine kinase domain (exon 17). *c-KIT* mutations have been found in a variable but relatively high frequency (up to 50%) in patients with CBF

New Molecular Markers in Acute Myeloid Leukemia 323

implicated in the development or progress of myeloid disorders (Lugthart et al., 2008). Previous studies, applying microarray technology, indicate that high levels of EVI1 expression are detected in 10% -22% of patients with AML (Barjesteh et al., 2003). Although the higher expression of EVI1 gene was clearly associated with myeloid malignancies, is not restricted to this group. It is noteworthy that overexpression of EVI1 was also observed in 13.8% of patients with acute lymphoid leukemia. The correlation between overexpression of EVI1 in bone marrow and poor outcome in AML is a frequent issue of discussion in the

We analyzed the incidence of EVI1 overexpression in haematological malignancies and its value as a prognostic factor. EVI1 overexpression (EVI1+) was examined by RT-PCR in bone marrow and/or peripheral blood samples of 113 AML patients at diagnosis and during follow-up. We found that 23.8% of AML overexpressed EVI1, as established by our previously determined cut-off point. Correlation with FAB subtypes stands as follows: 2M0, 1 M1,3 M2, 6 M4, 7 M5, 1 M6, 4 secondary AML and 3 not labeled. Survival curves in the AML group didn´t show any significant differences in overall survival and disease free survival when comparing EVI1+ to EVI1- populations. In AML samples a greater than expected incidence of EVI1 expression was observed (22.8% vs 11% previously described). When survival curves were analyzed in the AML group with ages ranging from 14 to 60 years, all of them treated with similar chemotherapy schemes, no significant difference was observed. However, a recent collaboration with other groups, showed that *EVI1*  overexpression is a poor prognostic marker in patients <65 years in an independent large cohort, and showed that the total absence of *EVI1* expression has a prognostic impact in the

The Brain and Acute Leukemia Cytoplasmic *(BAALC)*, human gene located on chromosome 8q22.3, has also been found to be an important adverse prognostic factor if overexpressed in normal karyotype AML, suggesting a role for BAALC overexpression in acute leukemia. This gene is highly conserved in mammals. Normally, BAALC is almost exclusively expressed in neuroectoderm-derived tissues. Though little is known about the biological function of BAALC, it is highly expressed in hematopoietic precursor cells as well as leukemic blasts and is down-regulated during differentiation. BAALC has been postulated to play its role in the cytoskeleton network due to its cellular location. BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile. Recent studies revealed the prognostic impact of BAALC expression in AML and also as a marker in minimal residual disease. Currently, determination of BAALC expression is performed by qualitative and quantitative real-time PCR, although the lack of validation or standardization studies limits its utility (Tanner et al., 2001; Gregory et al.,

The meningioma 1 *(MN1)* gene is localized on human chromosome 22. *MN1* overexpression is a prognostic marker in patients with AML with normal karyotype characterized by an intermediate prognosis. Patients with high *MN1* expression had a significantly worse prognosis (the overall survival was shorter and relapse rate was higher in this group compared to low *MN1* expression group). High *MN1* expression has as well been associated with other AML characteristics like inv(16) or overexpression of *EVI-1* (Heuser et al, 2007). In addition, MN1 is able to induce myeloid leukemias in a murine model (Heuser et al, 2011). This suggested that MN1 may play a functional role in the pathogenesis of AML.

literature (Lugthart et al., 2008; Luzardo et al., 2007).

outcome of acute myeloid leukemia patients (Vazquez et al.,2011).

**2.8 BAALC AND MN1** 

2009; Baldus et al., 2003; Najima et al., 2010).

AML, including both t(8;21) and the other major type of CBF AML inv(16)(p13q22) or t(16;16)(p13;q22). This appears to confer a quite unfavourable prognosis with higher relapse risk (associated to inv(16) or t(8;21)). An adverse effect on OS in AML with inv(16) has been described, particularly those that occur in exon 17 (Paschka et al., 2006). Such patients may warrant more aggressive or alternative therapy. The presence of the *c-KIT* mutation would be also important because it provides a target for novel tyrosine kinase inhibitor (TKI) therapy (Pollard et al., *2010).* 

#### **2.6 FLT3**

Flt3 is a member of the class III tyrosine kinase receptor family that includes the c-kit, c-fms, and PDGF receptors. The Flt3 receptor is preferentially expressed on hematopoietic stem cells and mediates stem cell differentiation and proliferation. Interaction of the Flt3 receptor with Flt3 ligand causes receptor dimerization, leading to the activation of the receptor tyrosine kinase and receptor autophosphorylation. The phosphorylated Flt3 transduces activation signals through associations with various cytoplasmic proteins, including ras GTPase-activating protein, phospholipase C, and Src family tyrosine kinases. Activation of the Flt3 receptor by ligand-dependent phosphorylation induces cellular proliferation via activation of cytoplasmic mediators. Thus, constitutive activation of the Flt3 pathway may lead to disease proliferation and may block cellular apoptotic response to conventional chemotherapy. An internal tandem duplication of the juxtamembrane (JM) domain-coding sequence of the *FLT3 (FLT3-IT)* gene on chromosome 13 has been identified in a group of patients with AML. Constitutive activation of the Flt3 receptor tyrosine kinase, either by internal tandem duplication (ITD) mutations of the juxtamembrane domain or point mutations clustering in the second tyrosine kinase domain (TKD mutations as D835), has been found in 20% to 30% of patients with AML and in 30% to 45% of patients with normal karyotype (reviewed by Stirewalt and Radich (2003)). ITD mutations have been associated with an increased risk of treatment failure after conventional chemotherapy (overall survival and disease-free survival were worse for ITD positive patients versus *FLT3* wildtype patients), whereas the prognostic relevance of *FLT3* point mutations is less evident (D835 mutants did not appear to have a worse median overal suvival or disease-free survival compared with the wildtype group) (Sheikhha et al., 2003). Recently, Spassov et al analyzed for *WT1* and *FLT3*-internal tandem duplication (*FLT3-ITD*) expression in 30 samples of AML patients and determined that high *WT1* expression correlated with the presence of *FLT3-ITD* (P = 0·014) and with a lower rate of complete remissions (P = 0·023) (Spassov et al., 2011). The detection of both molecular markers (*WT1* and *FLT3-ITD*) may be helpful in defining high risk AML patients that need special therapeutic strategies.

 Several studies described that a higher mutational load as determined by calculation of *FLT3-ITD/FLT3* wildtype ratio indicates a worse prognosis in mutation carriers. Therefore, it was suggested that not the FLT3-ITD per se, but more likely loss of heterozygosity is associated with the unfavorable outcome in FLT3-ITD mutated AML (Meshinchi et al., 2001; Yanada et al., 2005; Thiede et al., 2002; Schnittger et al., 2011).

#### *2.7* **EVI1 overexpression**

The ectopic viral integration site 1 (EVI1), located in chromosome 3q26, has been recognized in the last years as one of the most aggressive oncogenes associated to human leukemia (Gröschel et al., 2010). The inappropriate expression of EVI1 in hematopoietic cells has been

AML, including both t(8;21) and the other major type of CBF AML inv(16)(p13q22) or t(16;16)(p13;q22). This appears to confer a quite unfavourable prognosis with higher relapse risk (associated to inv(16) or t(8;21)). An adverse effect on OS in AML with inv(16) has been described, particularly those that occur in exon 17 (Paschka et al., 2006). Such patients may warrant more aggressive or alternative therapy. The presence of the *c-KIT* mutation would be also important because it provides a target for novel tyrosine kinase inhibitor (TKI)

Flt3 is a member of the class III tyrosine kinase receptor family that includes the c-kit, c-fms, and PDGF receptors. The Flt3 receptor is preferentially expressed on hematopoietic stem cells and mediates stem cell differentiation and proliferation. Interaction of the Flt3 receptor with Flt3 ligand causes receptor dimerization, leading to the activation of the receptor tyrosine kinase and receptor autophosphorylation. The phosphorylated Flt3 transduces activation signals through associations with various cytoplasmic proteins, including ras GTPase-activating protein, phospholipase C, and Src family tyrosine kinases. Activation of the Flt3 receptor by ligand-dependent phosphorylation induces cellular proliferation via activation of cytoplasmic mediators. Thus, constitutive activation of the Flt3 pathway may lead to disease proliferation and may block cellular apoptotic response to conventional chemotherapy. An internal tandem duplication of the juxtamembrane (JM) domain-coding sequence of the *FLT3 (FLT3-IT)* gene on chromosome 13 has been identified in a group of patients with AML. Constitutive activation of the Flt3 receptor tyrosine kinase, either by internal tandem duplication (ITD) mutations of the juxtamembrane domain or point mutations clustering in the second tyrosine kinase domain (TKD mutations as D835), has been found in 20% to 30% of patients with AML and in 30% to 45% of patients with normal karyotype (reviewed by Stirewalt and Radich (2003)). ITD mutations have been associated with an increased risk of treatment failure after conventional chemotherapy (overall survival and disease-free survival were worse for ITD positive patients versus *FLT3* wildtype patients), whereas the prognostic relevance of *FLT3* point mutations is less evident (D835 mutants did not appear to have a worse median overal suvival or disease-free survival compared with the wildtype group) (Sheikhha et al., 2003). Recently, Spassov et al analyzed for *WT1* and *FLT3*-internal tandem duplication (*FLT3-ITD*) expression in 30 samples of AML patients and determined that high *WT1* expression correlated with the presence of *FLT3-ITD* (P = 0·014) and with a lower rate of complete remissions (P = 0·023) (Spassov et al., 2011). The detection of both molecular markers (*WT1* and *FLT3-ITD*) may be

helpful in defining high risk AML patients that need special therapeutic strategies.

Yanada et al., 2005; Thiede et al., 2002; Schnittger et al., 2011).

*2.7* **EVI1 overexpression** 

 Several studies described that a higher mutational load as determined by calculation of *FLT3-ITD/FLT3* wildtype ratio indicates a worse prognosis in mutation carriers. Therefore, it was suggested that not the FLT3-ITD per se, but more likely loss of heterozygosity is associated with the unfavorable outcome in FLT3-ITD mutated AML (Meshinchi et al., 2001;

The ectopic viral integration site 1 (EVI1), located in chromosome 3q26, has been recognized in the last years as one of the most aggressive oncogenes associated to human leukemia (Gröschel et al., 2010). The inappropriate expression of EVI1 in hematopoietic cells has been

therapy (Pollard et al., *2010).* 

**2.6 FLT3** 

implicated in the development or progress of myeloid disorders (Lugthart et al., 2008). Previous studies, applying microarray technology, indicate that high levels of EVI1 expression are detected in 10% -22% of patients with AML (Barjesteh et al., 2003). Although the higher expression of EVI1 gene was clearly associated with myeloid malignancies, is not restricted to this group. It is noteworthy that overexpression of EVI1 was also observed in 13.8% of patients with acute lymphoid leukemia. The correlation between overexpression of EVI1 in bone marrow and poor outcome in AML is a frequent issue of discussion in the literature (Lugthart et al., 2008; Luzardo et al., 2007).

We analyzed the incidence of EVI1 overexpression in haematological malignancies and its value as a prognostic factor. EVI1 overexpression (EVI1+) was examined by RT-PCR in bone marrow and/or peripheral blood samples of 113 AML patients at diagnosis and during follow-up. We found that 23.8% of AML overexpressed EVI1, as established by our previously determined cut-off point. Correlation with FAB subtypes stands as follows: 2M0, 1 M1,3 M2, 6 M4, 7 M5, 1 M6, 4 secondary AML and 3 not labeled. Survival curves in the AML group didn´t show any significant differences in overall survival and disease free survival when comparing EVI1+ to EVI1- populations. In AML samples a greater than expected incidence of EVI1 expression was observed (22.8% vs 11% previously described). When survival curves were analyzed in the AML group with ages ranging from 14 to 60 years, all of them treated with similar chemotherapy schemes, no significant difference was observed. However, a recent collaboration with other groups, showed that *EVI1*  overexpression is a poor prognostic marker in patients <65 years in an independent large cohort, and showed that the total absence of *EVI1* expression has a prognostic impact in the outcome of acute myeloid leukemia patients (Vazquez et al.,2011).

#### **2.8 BAALC AND MN1**

The Brain and Acute Leukemia Cytoplasmic *(BAALC)*, human gene located on chromosome 8q22.3, has also been found to be an important adverse prognostic factor if overexpressed in normal karyotype AML, suggesting a role for BAALC overexpression in acute leukemia. This gene is highly conserved in mammals. Normally, BAALC is almost exclusively expressed in neuroectoderm-derived tissues. Though little is known about the biological function of BAALC, it is highly expressed in hematopoietic precursor cells as well as leukemic blasts and is down-regulated during differentiation. BAALC has been postulated to play its role in the cytoskeleton network due to its cellular location. BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile. Recent studies revealed the prognostic impact of BAALC expression in AML and also as a marker in minimal residual disease. Currently, determination of BAALC expression is performed by qualitative and quantitative real-time PCR, although the lack of validation or standardization studies limits its utility (Tanner et al., 2001; Gregory et al., 2009; Baldus et al., 2003; Najima et al., 2010).

The meningioma 1 *(MN1)* gene is localized on human chromosome 22. *MN1* overexpression is a prognostic marker in patients with AML with normal karyotype characterized by an intermediate prognosis. Patients with high *MN1* expression had a significantly worse prognosis (the overall survival was shorter and relapse rate was higher in this group compared to low *MN1* expression group). High *MN1* expression has as well been associated with other AML characteristics like inv(16) or overexpression of *EVI-1* (Heuser et al, 2007). In addition, MN1 is able to induce myeloid leukemias in a murine model (Heuser et al, 2011). This suggested that MN1 may play a functional role in the pathogenesis of AML.

New Molecular Markers in Acute Myeloid Leukemia 325

The *MLL* (mixed lineage leukemia) gene located at 11q23, is fused to a variety of partner genes through chromosomal translocations in acute leukemias. Up to now, more than 40 different *MLL* partner genes have been identified. *MLL* gene contains 100 kb of DNA, but nearly all breakpoints are clustered within a 8.3 kb region. Molecular analysis shows that fusion of the amino terminus of *MLL* to the carboxy terminus of partner genes generates the critical leukemogenic fusion proteins. Abnormalities of the mixed-lineage leukemia (*MLL*) gene can be detected in *de novo* acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) as well as in therapy-related AML, particularly after treatment with DNA topoisomerase II inhibitors (generally less than 5–10% of patients carried this rearrangements).The most common translocation involving 11q23 in acute myeloid leukemia (AML) is t(9;11)(p22;q23), which results in the generation of a fusion transcript of *MLL*-*AF.* The other common translocations involving 11q23 in AML are t(6;11)(q27;q23), t(10;11)(p12;q23), t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3); these translocations result in the generation of fusion transcripts *MLL*-*AF6*, *MLL-AF10*, *MLL*-*ELL* or *MLL*-*ENL*, respectively. In AML, *MLL*-*AF9* has generally been associated with a more favourable outcome although this finding is controversial. This rearrangement is related to a superior event-free and overall survival as compared to patients with other chromosomal abnormalities or with no detectable rearrangements (Dimartino et al., 1999; Balgobind et al., 2005). In contrast, the other traslocation involving Cr 11q23 are asociated with poor prognostic in more cases (Dimartino et al., 1999; Balgobind et al., 2005). t(9;11)(p22;q23) is classified as intermediate and t(v;11)(v;q23) as adverse prognostic group (Döhner et al., 2010). Therefore, detection and identification of the different types of *MLL* rearrangements is of clinical importance. A recent study indicated that quantification by RQ-PCR of the fusion gene transcript levels at diagnosis may be of prognostic relevance (Shih et al., 2006; Jansen et al., 2005). One type of *MLL* rearrangement not detectable by classic cytogenetic is the partial tandem duplication of *MLL* (*MLL*-PTD). This rearrangement results most commonly from a duplication of a genomic region encompassing either *MLL* exons 5 through 11 or *MLL* exons 5 through 12 that is inserted into intron 4 of a full-length *MLL* gene, thus fusing introns 11 or 12 with intron 4. At a transcriptional level, this results in a unique in-frame fusion of exons 11 or 12 upstream of exon 5. In adult de novo AML with a normal karyotype, the presence of the *MLL*-PTD has been associated with a worse prognosis (ie, shorter duration of remission) when compared with normal karyotype AML without the *MLL*-PTD (Whitman et al., 2005).

**2.10 MLL rearrangement and MLL-PTD** 

**2.11 Other molecular markers in research: Renin expression** 

There have been reports of experimental findings that relate the renin-angiotensin system (RAS) with hematopoiesis. Some studies have identified RAS components located in the bone marrow (Haznedaroglu & Buyukasik, 1997) that might functionally affect cellular proliferation and differentiation in physiological or pathological states (Huckle & Earp, 1994; Comte et al., 1997). It has been demonstrated that the RAS component renin is expressed in the bone marrow microenvironment as well as in hematopoietic cells (Abali et al., 2002). Bone marrow blast cells of some types of AML (M4 and M5 FAB types) express renin (Wulf et al., 1998), however no expression has been detected in normal bone marrow from healthy donors (Gómez Casares et al., 2002). Our group also detected renin expression

We performed a retrospective analysis of MN1 expression in a group of 49 AML patients with a mean age of 52 years (43 de novo AML and 6 secondary AML). In order to analyze response to chemotherapy, overall survival and correlation to other molecular markers (NPM1, FLT3- ITD, EVI1 and BAALC), patients were further classified into three groups depending on prognosis (favourable with 11 patients, intermediate with 25 and poor with 12). We analysed by real-time PCR the expression of MN1 in patients samples. We used as positive control of expression RNA of KG1 cell line that overexpress MN1. The incidence of MN1 overespression was 65.3%. The study showed that MN1 overexpression correlated to BAALC expression. We did not find relation with other markers, such as EVI-1, NPM-1 and FLT3. MN1 and BAALC overexpression have been associated to induction treatment refractoriness. However, due to the limited sample size in our series, determining whether MN1 or BAALC were actually involved with refractoriness was not possible. The 2-year overall survival was 52% and 53% for MN1 overexpressed and MN1 not overexpressed patients respectively. The 5-year overall survival was 52% and 42% respectively, showing no significant differences. Survival analysis for the intermediate risk AML group did not show significant differences either. MN1 overexpression was not associated with a worse prognosis in any of the studied patients, probably due to a small sample size (Rodriguez et al., 2010).

#### **2.9 WT1**

The Wilms' tumor locus was identified as a tumor suppressor gene, which is inactivated in Wilms' tumor, a pediatric kidney cancer. This protein, displaying characteristic features of a transcription factor, and with an expression restricted to kidney and haematopoetic cells, was called Wilms' tumor gene 1 (*WT1*). This gene is located on chromosome 11p13 and encodes a zinc-finger transcription factor influencing the expression of several growth factors and their corresponding receptors. It is also known to be involved in the early stage of hematological cell differentiation. Aberrant expression may be one mechanism by which the normal function of *WT1* is disrupted. However, the exact role of *WT1* in hematopoiesis and leukemogenesis still remains unclear. The abundant overexpression of *WT1* in leukemia creates a very attractive target for quantitative MRD studies in AML, especially in those samples with no specific fusion gene available. The method used to determine the expression of *WT1* gene is the RT-PCR, which will reveal its value as a marker to detect minimal residual disease (Keilholz et al., 2005; Weisser et al., 2005; Inoue et al., 1994; Garg et al., 2003).

Mutations of the coding region (most frequenly in 7 and 9 exons) of the *WT1* gene have also been described and occur in 10–15% of AML (figure 3). Gaidzik et al. concluded that *WT1* mutation as a single molecular marker did not have an impact on outcome. On the other hand, Hou et al. demostrated that *WT1* mutations disappeared in *WT1*-mutated studied patients who achieved complete remission, suggesting its potential use as MRD marker (Gaidzik et al., 2009; Hou et al., 2010).

Fig. 3. Structure of the *WT1* gene and localization of the 87 mutations identified by Gaidzik et al.

#### **2.10 MLL rearrangement and MLL-PTD**

324 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

We performed a retrospective analysis of MN1 expression in a group of 49 AML patients with a mean age of 52 years (43 de novo AML and 6 secondary AML). In order to analyze response to chemotherapy, overall survival and correlation to other molecular markers (NPM1, FLT3- ITD, EVI1 and BAALC), patients were further classified into three groups depending on prognosis (favourable with 11 patients, intermediate with 25 and poor with 12). We analysed by real-time PCR the expression of MN1 in patients samples. We used as positive control of expression RNA of KG1 cell line that overexpress MN1. The incidence of MN1 overespression was 65.3%. The study showed that MN1 overexpression correlated to BAALC expression. We did not find relation with other markers, such as EVI-1, NPM-1 and FLT3. MN1 and BAALC overexpression have been associated to induction treatment refractoriness. However, due to the limited sample size in our series, determining whether MN1 or BAALC were actually involved with refractoriness was not possible. The 2-year overall survival was 52% and 53% for MN1 overexpressed and MN1 not overexpressed patients respectively. The 5-year overall survival was 52% and 42% respectively, showing no significant differences. Survival analysis for the intermediate risk AML group did not show significant differences either. MN1 overexpression was not associated with a worse prognosis in any of the studied patients,

The Wilms' tumor locus was identified as a tumor suppressor gene, which is inactivated in Wilms' tumor, a pediatric kidney cancer. This protein, displaying characteristic features of a transcription factor, and with an expression restricted to kidney and haematopoetic cells, was called Wilms' tumor gene 1 (*WT1*). This gene is located on chromosome 11p13 and encodes a zinc-finger transcription factor influencing the expression of several growth factors and their corresponding receptors. It is also known to be involved in the early stage of hematological cell differentiation. Aberrant expression may be one mechanism by which the normal function of *WT1* is disrupted. However, the exact role of *WT1* in hematopoiesis and leukemogenesis still remains unclear. The abundant overexpression of *WT1* in leukemia creates a very attractive target for quantitative MRD studies in AML, especially in those samples with no specific fusion gene available. The method used to determine the expression of *WT1* gene is the RT-PCR, which will reveal its value as a marker to detect minimal residual disease (Keilholz et al.,

Mutations of the coding region (most frequenly in 7 and 9 exons) of the *WT1* gene have also been described and occur in 10–15% of AML (figure 3). Gaidzik et al. concluded that *WT1* mutation as a single molecular marker did not have an impact on outcome. On the other hand, Hou et al. demostrated that *WT1* mutations disappeared in *WT1*-mutated studied patients who achieved complete remission, suggesting its potential use as MRD marker

Fig. 3. Structure of the *WT1* gene and localization of the 87 mutations identified by Gaidzik

probably due to a small sample size (Rodriguez et al., 2010).

2005; Weisser et al., 2005; Inoue et al., 1994; Garg et al., 2003).

(Gaidzik et al., 2009; Hou et al., 2010).

**2.9 WT1** 

et al.

The *MLL* (mixed lineage leukemia) gene located at 11q23, is fused to a variety of partner genes through chromosomal translocations in acute leukemias. Up to now, more than 40 different *MLL* partner genes have been identified. *MLL* gene contains 100 kb of DNA, but nearly all breakpoints are clustered within a 8.3 kb region. Molecular analysis shows that fusion of the amino terminus of *MLL* to the carboxy terminus of partner genes generates the critical leukemogenic fusion proteins. Abnormalities of the mixed-lineage leukemia (*MLL*) gene can be detected in *de novo* acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) as well as in therapy-related AML, particularly after treatment with DNA topoisomerase II inhibitors (generally less than 5–10% of patients carried this rearrangements).The most common translocation involving 11q23 in acute myeloid leukemia (AML) is t(9;11)(p22;q23), which results in the generation of a fusion transcript of *MLL*-*AF.* The other common translocations involving 11q23 in AML are t(6;11)(q27;q23), t(10;11)(p12;q23), t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3); these translocations result in the generation of fusion transcripts *MLL*-*AF6*, *MLL-AF10*, *MLL*-*ELL* or *MLL*-*ENL*, respectively. In AML, *MLL*-*AF9* has generally been associated with a more favourable outcome although this finding is controversial. This rearrangement is related to a superior event-free and overall survival as compared to patients with other chromosomal abnormalities or with no detectable rearrangements (Dimartino et al., 1999; Balgobind et al., 2005). In contrast, the other traslocation involving Cr 11q23 are asociated with poor prognostic in more cases (Dimartino et al., 1999; Balgobind et al., 2005). t(9;11)(p22;q23) is classified as intermediate and t(v;11)(v;q23) as adverse prognostic group (Döhner et al., 2010). Therefore, detection and identification of the different types of *MLL* rearrangements is of clinical importance. A recent study indicated that quantification by RQ-PCR of the fusion gene transcript levels at diagnosis may be of prognostic relevance (Shih et al., 2006; Jansen et al., 2005). One type of *MLL* rearrangement not detectable by classic cytogenetic is the partial tandem duplication of *MLL* (*MLL*-PTD). This rearrangement results most commonly from a duplication of a genomic region encompassing either *MLL* exons 5 through 11 or *MLL* exons 5 through 12 that is inserted into intron 4 of a full-length *MLL* gene, thus fusing introns 11 or 12 with intron 4. At a transcriptional level, this results in a unique in-frame fusion of exons 11 or 12 upstream of exon 5. In adult de novo AML with a normal karyotype, the presence of the *MLL*-PTD has been associated with a worse prognosis (ie, shorter duration of remission) when compared with normal karyotype AML without the *MLL*-PTD (Whitman et al., 2005).

#### **2.11 Other molecular markers in research: Renin expression**

There have been reports of experimental findings that relate the renin-angiotensin system (RAS) with hematopoiesis. Some studies have identified RAS components located in the bone marrow (Haznedaroglu & Buyukasik, 1997) that might functionally affect cellular proliferation and differentiation in physiological or pathological states (Huckle & Earp, 1994; Comte et al., 1997). It has been demonstrated that the RAS component renin is expressed in the bone marrow microenvironment as well as in hematopoietic cells (Abali et al., 2002). Bone marrow blast cells of some types of AML (M4 and M5 FAB types) express renin (Wulf et al., 1998), however no expression has been detected in normal bone marrow from healthy donors (Gómez Casares et al., 2002). Our group also detected renin expression

New Molecular Markers in Acute Myeloid Leukemia 327

proliferation in K562 cells with captopril (C) 10 mM and trandolapril (T) 2 mM (figure 6a) as well as an increase in the apoptotic rate in renin positive leukemia cell lines (K562) after the treatment with captopril (data not shown) and trandolapril (figure 6b) (De la Iglesia et

Fig. 6. **(a)** Proliferation in K562 after the treatment with captopril (C) and trandolapril (T). **(b)** C-myc gene expression in K562 after the treatment with the same drugs. **(c)** Apoptosis determined by annexin V binding in K562 and K562 transfected with bcl-2 and bcl-x (Kbcl2

The leukaemogenic role of renin gene expression and the implicated molecular pathways have not yet been elucidated but it may offer novel therapeutic approaches in pathological

and Kbclx) cells treated with 1mM trandolapril (T) (De la Iglesia et al., 2009). All

experiments were performed in triplicate.

or neoplastic conditions (Haznedaroglu & Öztürk, 2003).

al., 2009).

in AML patients, but it did not relate to monocytic dififerentiation or to the existence of other cytogenetic risk markers (Gómez Casares et al., 2002). Moreover, renin expression has been found to be related to disease activity, disappearing with AML remission and returning with relapse (De la Iglesia et al., 2006).

We did not found statistically significant differences in the outcome between renin-positive and renin-negative patients (De la Iglesia et al., 2006).

Fig. 4. Renin:glucuronidase ratios vs clinical state of the quantified patients. \*This data belong to third relapse, CR and fourth relapse, respectively. \*\*This patient did not reach CR, continuing the positivity of the renin gene expression. \*\*\*Patient 28 in morphological CR was presented with 1% blasts in BM that later disappeared (De la Iglesia et al., 2006).

Fig. 5. (a) Overall survival of renin-positive and renin-negative patients with AML. Kaplan-Meier plot showing the correlation between overall survival and renin expression in AML patients. (b)Disease-free survival of renin-positive and renin-negative patients with AML. Kaplan-Meier plot showing the correlation between disease-free survival and renin expression in AML patients. (De la Iglesia et al., 2006).

Renin expression is related to undifferentiated phenotypic cell lines (K562, KU812), since no expression of the renin gene was found in cellular lines that showed a highly differentiated phenotype like HL60 and U937 (Gómez Casares et al., 2002). ACE inhibitors (captopril and trandolapril) and AT1 receptor blocker (losartan) produce a stop in

in AML patients, but it did not relate to monocytic dififerentiation or to the existence of other cytogenetic risk markers (Gómez Casares et al., 2002). Moreover, renin expression has been found to be related to disease activity, disappearing with AML remission and

We did not found statistically significant differences in the outcome between renin-positive

Fig. 4. Renin:glucuronidase ratios vs clinical state of the quantified patients. \*This data belong to third relapse, CR and fourth relapse, respectively. \*\*This patient did not reach CR, continuing the positivity of the renin gene expression. \*\*\*Patient 28 in morphological CR was presented with 1% blasts in BM that later disappeared (De la Iglesia et al., 2006).

Fig. 5. (a) Overall survival of renin-positive and renin-negative patients with AML. Kaplan-Meier plot showing the correlation between overall survival and renin expression in AML patients. (b)Disease-free survival of renin-positive and renin-negative patients with AML. Kaplan-Meier plot showing the correlation between disease-free survival and renin

Renin expression is related to undifferentiated phenotypic cell lines (K562, KU812), since no expression of the renin gene was found in cellular lines that showed a highly differentiated phenotype like HL60 and U937 (Gómez Casares et al., 2002). ACE inhibitors (captopril and trandolapril) and AT1 receptor blocker (losartan) produce a stop in

expression in AML patients. (De la Iglesia et al., 2006).

returning with relapse (De la Iglesia et al., 2006).

and renin-negative patients (De la Iglesia et al., 2006).

proliferation in K562 cells with captopril (C) 10 mM and trandolapril (T) 2 mM (figure 6a) as well as an increase in the apoptotic rate in renin positive leukemia cell lines (K562) after the treatment with captopril (data not shown) and trandolapril (figure 6b) (De la Iglesia et al., 2009).

Fig. 6. **(a)** Proliferation in K562 after the treatment with captopril (C) and trandolapril (T). **(b)** C-myc gene expression in K562 after the treatment with the same drugs. **(c)** Apoptosis determined by annexin V binding in K562 and K562 transfected with bcl-2 and bcl-x (Kbcl2 and Kbclx) cells treated with 1mM trandolapril (T) (De la Iglesia et al., 2009). All experiments were performed in triplicate.

The leukaemogenic role of renin gene expression and the implicated molecular pathways have not yet been elucidated but it may offer novel therapeutic approaches in pathological or neoplastic conditions (Haznedaroglu & Öztürk, 2003).

New Molecular Markers in Acute Myeloid Leukemia 329

Mutated *CEBPA* (normal karyotype)

Intermediate-1 Mutated *NPM1* and *FLT3*-ITD (normal karyotype)

Adverse inv(3)(q21q26.2) or t(3;3)(q21;q26.2); *RPN1*-*EVI1* 

t(6;9)(p23;q34); *DEK*-*NUP214*  t(v;11)(v;q23); *MLL* rearranged

Table 5. Standardized reporting for correlation of cytogenetic and molecular genetic data in

In recent past, treatments had an empirical basis, lack of specificity and, therefore, limited effectiveness combined with high toxicity. The treatment of AML pioneered the search for specific therapies with the use of ATRA in APL. Currently, the ultimate goal of the understanding and classification of molecular aberrations in every AML subtype is to design a targeted therapy, which will reduce the risk of relapse and treatment side effects. That is because the future of AML therapy should be in the search and development of drugs that are directed against specific molecular or fusion proteins aberrations. On the other hand, due to the interaction between different molecular aberrations that arise in AML, the design of these new drugs has to be focused on the combined inhibition of several

Abali H, Haznedaroglu IC, Goker H, Celik I, Ozatli D, Koray Z & Caglar M. (2002).

Baldus, C. D., Tanner, S. M., Kusewitt, D. F., Liyanarachchi, S., Choi, C., Caligiuri, M. A.,

Hematopoiesis: Preliminary Evidences. Hematology, Vol. 7, No.75-82. Alberta, J. A., Springett, G. M., Rayburn, H., Natoli, T. A., Loring, J., Kreidberg, J. A., &

Circulating and Local Bone Marrow Renin-angiotensin System in Leukemic

Housman, D. (2003). Role of the WT1 tumor suppressor in murine hematopoiesis.

Bloomfield, C. D., & Chapelle, A. d. l. (2003). BAALC, a novel marker of human

inv(16)(p13.1q22) or t(16;16)(p13.1;q22); *CBFB*-*MYH11*  Mutated *NPM1* without *FLT3*-ITD (normal karyotype)

Wild-type *NPM1* and *FLT3*-ITD (normal karyotype) Wild-type *NPM1* without *FLT3*-ITD (normal karyotype)

Cytogenetic abnormalities not classified as favorable or


**GENETIC GROUP SUBSET** 

Favorable t(8;21)(q22;q22); *RUNX1*-*RUNX1T1* 

Intermediate-2 t(9;11)(p22;q23); *MLLT3*-*MLL* 

adverse

AML with clinical data (Döhner. European LeukemiaNet , 2010)

signalling pathways to achieve maximum clinical benefit.

Blood, Vol. 101, No.7, 2570-2574.

**4. References** 

### **3. Conclusion**

The AML embraces very heterogeneous entities with different clinical behaviours. Some of these leukemias have certain chromosomal alterations that are related to specific morphological and clinical subtypes. Their identification has had great impact on different management aspects of these diseases, either from the standpoint of diagnosis, prognosis or development of a treatment plan tailored to the risk of relapse. This has been largely possible thanks to the application of molecular biology studies, which have provided a better and deeper understanding of the pathogenesis of leukemia.

The genetic alterations that occur at different stages fall into two broad groups: those that activate signal transduction as c-KIT and FLT3 (type I) and those which alter transcription factors such as CBF, RAR-, CEBPA and NPM1 (type II). These two groups work together in leukemogenesis, being very frequent the coexistence of alterations of the two groups in the same patient.

The knowledge of these molecular markers helps us to classify patients within certain prognostic groups. In this way, in patients with normal cytogenetics, it is very important to know if there is a mutation of the FLT3 gene as well as its kind (ITD or TKD), because this information is of great relevance in order to select the appropriate treatment scheme. Moreover, the overexpression of the genes BAALC (under OS, DFS), MN1 (induction failure in patients with normal karyotype) or EVI1 (adverse impact) has been shown to have prognostic significance in AML.

In a laboratory setting there are different ways to classify leukemias. One of them is the rationale showed in the figure 5. According to the cytogenetic or molecular biology at diagnosis, it is possible to perform different determinations which will allow us to classify the patients within a favourable or unfavourable prognostic group. AML patients are classified at the time of diagnosis into three different prognostic groups based on their cytogenetic profiles.

There are plenty of algorithms which allow us to classify patients into different prognostic groups. They are undergoing continuous changes with the enrichment of new data. If we are in the presence of a CBF leukemia (by showing RUNX1-RUNXT1 or inv (16)), mutational analysis of c-kit should be performed, as this mutation translates into a higher risk of relapse. In patients with AML with normal karyotype, we recommend to analyze for FLT3- ITD first, as FLT-ITD3 is a well-known adverse prognostic factor. We then recommend screening for NPM1 mutations as a next step, because it is another mutation with clinical relevance of this subgroup of patients in the absence of FLT3-ITD. Then, given the favourable prognosis of NK-AML with biallelic CEBPA mutation, we recommend a CEBPA mutational analysis in those patients non-carriers of FLT3-ITD or NPM1. Also, FLT3-TKD an others mutations can be analyze in a context of clinical trials, even though there is no prognostic relevance.

There after, it is possible to perform other studies such as the detection of mutations in MLL/AF9 or BAALC, MN1 and EVI1 gene overexpression in order to make a better stratification of the prognosis. Besides working with prognostic and follow up markers with a recognized utility, we are also carrying out experimental work with new, not yet validated markers which will probably help us in the follow up, prognosis, classification and monitoring of AML patients. Renin, EVI1 and WT1 would be markers applicable to AML patients, especially in those with normal karyotype where they will facilitate the monitoring of minimal residual disease.

The AML embraces very heterogeneous entities with different clinical behaviours. Some of these leukemias have certain chromosomal alterations that are related to specific morphological and clinical subtypes. Their identification has had great impact on different management aspects of these diseases, either from the standpoint of diagnosis, prognosis or development of a treatment plan tailored to the risk of relapse. This has been largely possible thanks to the application of molecular biology studies, which have provided a

The genetic alterations that occur at different stages fall into two broad groups: those that activate signal transduction as c-KIT and FLT3 (type I) and those which alter transcription factors such as CBF, RAR-, CEBPA and NPM1 (type II). These two groups work together in leukemogenesis, being very frequent the coexistence of alterations of the two groups in the

The knowledge of these molecular markers helps us to classify patients within certain prognostic groups. In this way, in patients with normal cytogenetics, it is very important to know if there is a mutation of the FLT3 gene as well as its kind (ITD or TKD), because this information is of great relevance in order to select the appropriate treatment scheme. Moreover, the overexpression of the genes BAALC (under OS, DFS), MN1 (induction failure in patients with normal karyotype) or EVI1 (adverse impact) has been shown to have

In a laboratory setting there are different ways to classify leukemias. One of them is the rationale showed in the figure 5. According to the cytogenetic or molecular biology at diagnosis, it is possible to perform different determinations which will allow us to classify the patients within a favourable or unfavourable prognostic group. AML patients are classified at the time of diagnosis into three different prognostic groups based on their

There are plenty of algorithms which allow us to classify patients into different prognostic groups. They are undergoing continuous changes with the enrichment of new data. If we are in the presence of a CBF leukemia (by showing RUNX1-RUNXT1 or inv (16)), mutational analysis of c-kit should be performed, as this mutation translates into a higher risk of relapse. In patients with AML with normal karyotype, we recommend to analyze for FLT3- ITD first, as FLT-ITD3 is a well-known adverse prognostic factor. We then recommend screening for NPM1 mutations as a next step, because it is another mutation with clinical relevance of this subgroup of patients in the absence of FLT3-ITD. Then, given the favourable prognosis of NK-AML with biallelic CEBPA mutation, we recommend a CEBPA mutational analysis in those patients non-carriers of FLT3-ITD or NPM1. Also, FLT3-TKD an others mutations can be analyze in a context of clinical trials, even though there is no

There after, it is possible to perform other studies such as the detection of mutations in MLL/AF9 or BAALC, MN1 and EVI1 gene overexpression in order to make a better stratification of the prognosis. Besides working with prognostic and follow up markers with a recognized utility, we are also carrying out experimental work with new, not yet validated markers which will probably help us in the follow up, prognosis, classification and monitoring of AML patients. Renin, EVI1 and WT1 would be markers applicable to AML patients, especially in those with normal karyotype where they will facilitate the monitoring

better and deeper understanding of the pathogenesis of leukemia.

**3. Conclusion** 

same patient.

prognostic significance in AML.

cytogenetic profiles.

prognostic relevance.

of minimal residual disease.


Table 5. Standardized reporting for correlation of cytogenetic and molecular genetic data in AML with clinical data (Döhner. European LeukemiaNet , 2010)

In recent past, treatments had an empirical basis, lack of specificity and, therefore, limited effectiveness combined with high toxicity. The treatment of AML pioneered the search for specific therapies with the use of ATRA in APL. Currently, the ultimate goal of the understanding and classification of molecular aberrations in every AML subtype is to design a targeted therapy, which will reduce the risk of relapse and treatment side effects. That is because the future of AML therapy should be in the search and development of drugs that are directed against specific molecular or fusion proteins aberrations. On the other hand, due to the interaction between different molecular aberrations that arise in AML, the design of these new drugs has to be focused on the combined inhibition of several signalling pathways to achieve maximum clinical benefit.

#### **4. References**


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**17** 

*Germany* 

**Analysis of Leukemogenic Gene Products** 

*1Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt am Main* 

*2Institute of Transfusion Medicine, German Red Cross Blood Center, Frankfurt am Main* 

One major limitation in the development of effective and targeted cancer therapies is the incomplete understanding of the molecular mechanisms driving malignant cell growth and the resistance of residual cancer stem cells to standard therapies, such as chemotherapy and radiation. Consequently, numerous attempts have been made to elucidate the molecular

Among the different cancer entities, leukemias harbor only a few mutations and therefore represent a good model system for the study of oncogenesis. The cloning and subsequent analysis of leukemia-associated gene products have strongly facilitated the understanding of their biological function and the development of appropriate model systems. In early experiments, leukemia-associated oncogenes were ectopically expressed in fibroblasts, a cell type which is not optimal for modeling leukemia initiation and progression. Later, the development of cell separation methods for murine and human hematopoietic precursor cells allowed for the isolation of specific target cells, in large quantities and with high cell population purity (Belvedere et al., 1999). Together with the utilization of retroviral expression vectors, which enable stable integration and expression of a transgene of choice, these cell isolation procedures represent a significant methodological improvement for leukemia modeling approaches in the hematopoietic tissue. With these technologies in hand, the genetic lesions of leukemia can be better modeled in the appropriate cell compartment. By performing cell biology experiments and tumor sample deep sequencing it became clear that both solid tumors and hematological malignancies depend on multiple oncogenic alterations to ultimately result in cellular transformation (Hanahan & Weinberg, 2000, 2011; Kinzler & Vogelstein, 1996). Additionally, the overall number of mutated genes in solid tumors was found to be higher than in leukemias. These observations resulted in the formulation of the multi-hit model of tumorigenesis. Initiating mutations induce stem cell expansion and survival, thereby allowing for the occurrence of additional genetic alterations that result in the progression of malignant transformation. The later stages of this process are characterized by uncontrolled proliferation with overgrowth of the ´healthy´ cell compartment and metastasis, in the case of solid tumors. This multi-hit cancer model has been well described for colon cancer by Kenneth W. Kinzler and Bert Vogelstein (Kinzler &

circuits initiating and perpetuating malignant cell transformation.

**1. Introduction** 

**in Hematopoietic Progenitor Cells** 

Julia Schanda1, Reinhard Henschler2, Manuel Grez1 and Christian Wichmann1

expression profile that is uniquely associated with a favorable outcome. Blood, Vol. 113, No.13, 3088-3091.


### **Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells**

Julia Schanda1, Reinhard Henschler2,

Manuel Grez1 and Christian Wichmann1 *1Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt am Main 2Institute of Transfusion Medicine, German Red Cross Blood Center, Frankfurt am Main Germany* 

#### **1. Introduction**

338 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Wulf, G. G., Jahns-Streubel, G., Nobiling, R., Strutz, F., Hemmerlein, B., Hiddemann, W., &

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> One major limitation in the development of effective and targeted cancer therapies is the incomplete understanding of the molecular mechanisms driving malignant cell growth and the resistance of residual cancer stem cells to standard therapies, such as chemotherapy and radiation. Consequently, numerous attempts have been made to elucidate the molecular circuits initiating and perpetuating malignant cell transformation.

> Among the different cancer entities, leukemias harbor only a few mutations and therefore represent a good model system for the study of oncogenesis. The cloning and subsequent analysis of leukemia-associated gene products have strongly facilitated the understanding of their biological function and the development of appropriate model systems. In early experiments, leukemia-associated oncogenes were ectopically expressed in fibroblasts, a cell type which is not optimal for modeling leukemia initiation and progression. Later, the development of cell separation methods for murine and human hematopoietic precursor cells allowed for the isolation of specific target cells, in large quantities and with high cell population purity (Belvedere et al., 1999). Together with the utilization of retroviral expression vectors, which enable stable integration and expression of a transgene of choice, these cell isolation procedures represent a significant methodological improvement for leukemia modeling approaches in the hematopoietic tissue. With these technologies in hand, the genetic lesions of leukemia can be better modeled in the appropriate cell compartment. By performing cell biology experiments and tumor sample deep sequencing it became clear that both solid tumors and hematological malignancies depend on multiple oncogenic alterations to ultimately result in cellular transformation (Hanahan & Weinberg, 2000, 2011; Kinzler & Vogelstein, 1996). Additionally, the overall number of mutated genes in solid tumors was found to be higher than in leukemias. These observations resulted in the formulation of the multi-hit model of tumorigenesis. Initiating mutations induce stem cell expansion and survival, thereby allowing for the occurrence of additional genetic alterations that result in the progression of malignant transformation. The later stages of this process are characterized by uncontrolled proliferation with overgrowth of the ´healthy´ cell compartment and metastasis, in the case of solid tumors. This multi-hit cancer model has been well described for colon cancer by Kenneth W. Kinzler and Bert Vogelstein (Kinzler &

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 341

the chronic phase, the accelerated phase and the acute phase with typical features of AML (Sawyers, 1999). The t(9;22) chromosomal translocation, also called the Philadelphia chromosome, which generates the fusion protein BCR/ABL, is frequently observed in CML patient samples. The BCR-portion of the fusion protein triggers multimerization of the ABL portion, thereby locking the ABL kinase into a hyperactive state. BCR/ABL multimers induce constitutive intracellular proliferation and survival signaling, thereby triggering the development of leukemia (Hoelbl et al., 2010; Melo & Barnes, 2007). Over the last years, a breakthrough in the treatment of CML was achieved with the development of the ABL-kinase inhibitor, imatinib (Sawyers et al., 2002). BCR/ABL positive CML patients are now treated with one of several targeted, first-line therapies, including imatinib, dasatinib, and nilotinib, which have dramatically increased patient survival rates to nearly 90% since the advent of these therapies. These kinase inhibitors specifically inhibit the oncogenic tyrosine kinase activity of BCR/ABL. For the first time, an inhibitor has been developed that targets a driving oncogene with high efficiency, which is currently used in many clinical trials (Garcia-Manero et al., 2003). Later, it was found that imatinib also targets c-kit, a receptor tyrosine kinase frequently mutated in gastrointestinal stromal tumors (GIST), thereby widening the application spectrum of imatinib to include solid tumors (Demetri, 2002). Currently, large efforts are being made in the development of specific inhibitors for other oncoproteins. The elucidation of the molecular mechanisms controlling the process of leukemic transformation represents a prerequisite for the development of targeted oncogene inhibitors designed to improve

For both acute and chronic myeloid leukemia, 'leukemia stem cells' are most likely the origin of disease and are probably responsible for recurrence after chemotherapy (Trumpp & Wiestler, 2008). These leukemia stem cells share common features with normal stem cells. They are described as dormant or slowly cycling cells with an increased self-renewal capacity and a resistance to chemotherapeutic drugs. It has been proposed that they reside in the bone marrow niches and can fully regenerate the leukemia after standard chemotherapy. Perturbed self-renewal programs of leukemia stem cells represent a major problem for leukemia therapy. Even in BCR/ABL positive CML, for which a specific oncogene inhibitor is available, CML stem cells are resistant to imatinib treatment (Holyoake et al., 1999). Therefore, lifelong treatment of CML patients with tyrosine kinase

Most of the current understandings of leukemogenic fusion genes and mutations have resulted from overexpression experiments using either cell line models or primary murine and human hematopoietic stem cells, both *in vitro* and *in vivo*. In these studies, leukemic gene products were usually expressed with the help of retroviral expression vectors resulting in ectopic and stable expression. This represents a convenient experimental strategy to model leukemia in mammalian hematopoietic stem and progenitor cells. The following sections will recapitulate the different retroviral expression vector systems used for modeling leukemia development and will summarize exemplary results acquired from

When modeling leukemia in the mammalian hematopoietic system, there are two major strategies used to express a leukemia-associated gene product. The creation of knock-in or

current therapeutic strategies for myeloid leukemias.

inhibitors is necessary (Corbin et al., 2011; Diamond & Melo, 2011).

studies of both the murine and human progenitor cell systems.

**2. Retroviral vector systems and expression cassettes** 

Vogelstein, 1996). Similarly, it has been suggested for hematopoietic malignancies, that only a concerted signaling network resulting from the activity of several activated oncogenes leads to full transformation of preleukemic cell clones. However, the exact chronological order of these genetic events and the causality of their mutual cooperation are hardly understood (Schuringa et al., 2010).

Myeloid leukemia represents a well-depicted tumor entity in terms of molecular pathogenesis. It is characterized by an uncontrolled proliferation behavior based on the aberrant self-renewal capacity of the myeloid progenitor cell compartment including the erythroid, monocytic and granulocytic lineages. Depending on the morphology and genetics of the cells and its clinical progression, myeloid leukemia is classified as either acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). AML is distinguished by the rapid proliferation of immature myeloid progenitor cells, which overgrow the normal hematopoietic cells. AML is the most common acute leukemia, which predominantly affects adults. Its incidence strongly increases with age, with a median age at diagnosis of 65 years, suggesting that several oncogenic events are required for the onset and progression of the disease (Estey & Dohner, 2006). If left untreated, the disease proceeds rapidly and is typically fatal within weeks or months. To date, numerous AML-specific fusion genes and mutations have been described (Look, 1997). Chromosomal translocations and mutations involving the AML1 gene are amongst the most common alterations found in AML (Schnittger et al., 2011). AML1 is an essential transcription factor that controls the myeloid cell differentiation of progenitor cells into mature blood cells. Both point mutations and chromosomal translocations, such as t(8;21) and t(3;21), disrupt the physiological function of AML1 and hinder the normal maturation process of the progenitor cells (Niebuhr et al., 2008). IIn addition, mutations of the receptor tyrosine kinase, c-kit, are frequently detected in these cells and seem to be associated with poor prognosis due to their correlation with higher relapse rates after chemotherapy (Boissel et al., 2006; Paschka et al., 2006). Mutations of the nucleophosmin (NPM1) gene are also frequently observed in AML. This gene is involved in multiple cellular processes including protein shuttling between the nucleus and cytoplasm, RNA biogenesis, cell cycle control and transcriptional regulation. It is also described as a cellular stress sensor associated with the p53-dependent apoptosis pathway (Grisendi et al., 2006). Commonly identified NPM1 mutations render this protein mislocalized to the cytoplasm. The oncogenic mechanisms behind this phenomenon are currently under intense investigation (Falini et al., 2010; Falini et al., 2007). Among the mutated kinases in AML, mutations of the FLT3 cytokine receptor gene are common and are associated with poor prognosis (Gilliland & Griffin, 2002; Meshinchi et al., 2001). Hyperactive FLT3 induces a strong intracellular signal transduction response, mainly by activating STAT5, a latent transcription factor important for self-renewal and proliferation of hematopoietic progenitors. For this leukemia entity, FLT3-inhibitors blocking its kinase activity are currently being tested in clinical trials (Wiernik, 2010). Isocitrate dehydrogenases, IDH1 and IDH2, represent genes that were recently identified to be frequently mutated in AML with adverse prognosis in cytogenetically normal leukemias (Mardis et al., 2009; Paschka et al., 2010). Mutations in IDH genes have also been found in human brain tumors, including gliomas. Interestingly, the accumulation of an intracellular metabolite, 2-hydroxyglutarate, was discovered in these tumor cells (Dang et al., 2009). This so-called 'oncometabolite' alters the genome-wide histone and DNA methylation patterns, thereby exerting oncogenic activity (Xu et al., 2011).

Chronic myeloid leukemia is characterized by the increased proliferation rate of mature granulocytes and their precursors. The course of disease can be divided into three phases:

Vogelstein, 1996). Similarly, it has been suggested for hematopoietic malignancies, that only a concerted signaling network resulting from the activity of several activated oncogenes leads to full transformation of preleukemic cell clones. However, the exact chronological order of these genetic events and the causality of their mutual cooperation are hardly

Myeloid leukemia represents a well-depicted tumor entity in terms of molecular pathogenesis. It is characterized by an uncontrolled proliferation behavior based on the aberrant self-renewal capacity of the myeloid progenitor cell compartment including the erythroid, monocytic and granulocytic lineages. Depending on the morphology and genetics of the cells and its clinical progression, myeloid leukemia is classified as either acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). AML is distinguished by the rapid proliferation of immature myeloid progenitor cells, which overgrow the normal hematopoietic cells. AML is the most common acute leukemia, which predominantly affects adults. Its incidence strongly increases with age, with a median age at diagnosis of 65 years, suggesting that several oncogenic events are required for the onset and progression of the disease (Estey & Dohner, 2006). If left untreated, the disease proceeds rapidly and is typically fatal within weeks or months. To date, numerous AML-specific fusion genes and mutations have been described (Look, 1997). Chromosomal translocations and mutations involving the AML1 gene are amongst the most common alterations found in AML (Schnittger et al., 2011). AML1 is an essential transcription factor that controls the myeloid cell differentiation of progenitor cells into mature blood cells. Both point mutations and chromosomal translocations, such as t(8;21) and t(3;21), disrupt the physiological function of AML1 and hinder the normal maturation process of the progenitor cells (Niebuhr et al., 2008). IIn addition, mutations of the receptor tyrosine kinase, c-kit, are frequently detected in these cells and seem to be associated with poor prognosis due to their correlation with higher relapse rates after chemotherapy (Boissel et al., 2006; Paschka et al., 2006). Mutations of the nucleophosmin (NPM1) gene are also frequently observed in AML. This gene is involved in multiple cellular processes including protein shuttling between the nucleus and cytoplasm, RNA biogenesis, cell cycle control and transcriptional regulation. It is also described as a cellular stress sensor associated with the p53-dependent apoptosis pathway (Grisendi et al., 2006). Commonly identified NPM1 mutations render this protein mislocalized to the cytoplasm. The oncogenic mechanisms behind this phenomenon are currently under intense investigation (Falini et al., 2010; Falini et al., 2007). Among the mutated kinases in AML, mutations of the FLT3 cytokine receptor gene are common and are associated with poor prognosis (Gilliland & Griffin, 2002; Meshinchi et al., 2001). Hyperactive FLT3 induces a strong intracellular signal transduction response, mainly by activating STAT5, a latent transcription factor important for self-renewal and proliferation of hematopoietic progenitors. For this leukemia entity, FLT3-inhibitors blocking its kinase activity are currently being tested in clinical trials (Wiernik, 2010). Isocitrate dehydrogenases, IDH1 and IDH2, represent genes that were recently identified to be frequently mutated in AML with adverse prognosis in cytogenetically normal leukemias (Mardis et al., 2009; Paschka et al., 2010). Mutations in IDH genes have also been found in human brain tumors, including gliomas. Interestingly, the accumulation of an intracellular metabolite, 2-hydroxyglutarate, was discovered in these tumor cells (Dang et al., 2009). This so-called 'oncometabolite' alters the genome-wide histone

and DNA methylation patterns, thereby exerting oncogenic activity (Xu et al., 2011).

Chronic myeloid leukemia is characterized by the increased proliferation rate of mature granulocytes and their precursors. The course of disease can be divided into three phases:

understood (Schuringa et al., 2010).

the chronic phase, the accelerated phase and the acute phase with typical features of AML (Sawyers, 1999). The t(9;22) chromosomal translocation, also called the Philadelphia chromosome, which generates the fusion protein BCR/ABL, is frequently observed in CML patient samples. The BCR-portion of the fusion protein triggers multimerization of the ABL portion, thereby locking the ABL kinase into a hyperactive state. BCR/ABL multimers induce constitutive intracellular proliferation and survival signaling, thereby triggering the development of leukemia (Hoelbl et al., 2010; Melo & Barnes, 2007). Over the last years, a breakthrough in the treatment of CML was achieved with the development of the ABL-kinase inhibitor, imatinib (Sawyers et al., 2002). BCR/ABL positive CML patients are now treated with one of several targeted, first-line therapies, including imatinib, dasatinib, and nilotinib, which have dramatically increased patient survival rates to nearly 90% since the advent of these therapies. These kinase inhibitors specifically inhibit the oncogenic tyrosine kinase activity of BCR/ABL. For the first time, an inhibitor has been developed that targets a driving oncogene with high efficiency, which is currently used in many clinical trials (Garcia-Manero et al., 2003). Later, it was found that imatinib also targets c-kit, a receptor tyrosine kinase frequently mutated in gastrointestinal stromal tumors (GIST), thereby widening the application spectrum of imatinib to include solid tumors (Demetri, 2002). Currently, large efforts are being made in the development of specific inhibitors for other oncoproteins. The elucidation of the molecular mechanisms controlling the process of leukemic transformation represents a prerequisite for the development of targeted oncogene inhibitors designed to improve current therapeutic strategies for myeloid leukemias.

For both acute and chronic myeloid leukemia, 'leukemia stem cells' are most likely the origin of disease and are probably responsible for recurrence after chemotherapy (Trumpp & Wiestler, 2008). These leukemia stem cells share common features with normal stem cells. They are described as dormant or slowly cycling cells with an increased self-renewal capacity and a resistance to chemotherapeutic drugs. It has been proposed that they reside in the bone marrow niches and can fully regenerate the leukemia after standard chemotherapy. Perturbed self-renewal programs of leukemia stem cells represent a major problem for leukemia therapy. Even in BCR/ABL positive CML, for which a specific oncogene inhibitor is available, CML stem cells are resistant to imatinib treatment (Holyoake et al., 1999). Therefore, lifelong treatment of CML patients with tyrosine kinase inhibitors is necessary (Corbin et al., 2011; Diamond & Melo, 2011).

Most of the current understandings of leukemogenic fusion genes and mutations have resulted from overexpression experiments using either cell line models or primary murine and human hematopoietic stem cells, both *in vitro* and *in vivo*. In these studies, leukemic gene products were usually expressed with the help of retroviral expression vectors resulting in ectopic and stable expression. This represents a convenient experimental strategy to model leukemia in mammalian hematopoietic stem and progenitor cells. The following sections will recapitulate the different retroviral expression vector systems used for modeling leukemia development and will summarize exemplary results acquired from studies of both the murine and human progenitor cell systems.

#### **2. Retroviral vector systems and expression cassettes**

When modeling leukemia in the mammalian hematopoietic system, there are two major strategies used to express a leukemia-associated gene product. The creation of knock-in or

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 343

appropriate laboratory biosafety level. For safety reasons, genes required for the assembly of the viral particles, *gag*, *pol* and *env*, are expressed from separate, so-called helper plasmids. In contrast to the expression vector, which codes for the gene of interest, the helper plasmids do not contain coding sequences for the packaging signal. Therefore, transcribed RNAs from the helper plasmids are not incorporated into the virus particle. With these replication defective retroviral particles, only a single round of transduction is possible. Thus, the risk of generating replication competent virus particles during production is greatly reduced. Retroviral transduction efficacy can be improved by concentrating the viral particles via ultracentrifugation (Kanbe & Zhang, 2004; Naldini et al., 1996). Gamma-retroviral particles can be preloaded onto RetroNectin-coated surfaces. RetroNectin, a recombinant peptide consisting of a mixture of fragments of the fibronectin molecule, co-localizes the viral particles and target cells into close proximity, thereby strongly increasing the efficiency of transduction (Hanenberg et al., 1996). A standard protocol for routine gamma-retroviral particle production and transduction of suspension cells is provided in Table 1. Expression of the gene of interest is usually coupled to the expression of a marker gene. This allows for immediate determination of the viral transduction efficacy as well as for the identification of transduced cells for the measurement of proliferation, differentiation and cell death. EGFP is the most commonly used marker protein, which exhibits a bright green fluorescence after laser excitation at 488 nm. Enrichment of vector-transduced fluorescent-labeled cells can be performed by fluorescence activated cell sorting (FACS). The usage of drug-selectable marker genes, such as neomycin and puromycin, represents another method used to obtain pure populations of transduced cells. Cellular expression of more than one transgene by

retroviral vectors can be accomplished by a variety of strategies (Figure 1).

(Donnelly et al., 2001).

The inclusion of an internal promoter allows for the expression of a second transgene when one transgene is expressed from the vector 5´-LTR. However, this approach also carries the risk of promoter interference with the vector LTR during retroviral particle production, thereby reducing vector titers and expression levels of the GOI (de Felipe et al., 1999). Another commonly employed strategy is the expression of two separate genes from one single transcript using the IRES element derived from the picornavirus (Gallardo et al., 1997). The IRES element allows for the initiation of mRNA translation in a 5' cap-independent manner (Mountford & Smith, 1995). However, expression levels of the two genes usually differ significantly. Typically, the gene located downstream is expressed at a level ten times lower than that of the other gene (Mizuguchi et al., 2000). The gene of interest and the marker gene can be fused to increase their stability and to allow for expression from one promoter. However, fusing two transgenes can result in functional restriction of the proteins caused by the fusion. Therefore, a short, flexible linker should be used to connect the proteins (Robinson & Sauer, 1998). Repeats of small side chain amino acids, such as glycine, serine and alanine, are well suited for the creation of linker sequences. The expression of two separate transgenes in equimolar amounts can be accomplished using self-cleaving 2A peptides, originating from the foot-and-mouth disease virus (Ryan et al., 1991). The 2A peptide sequence disrupts the peptide bond formation via a ribosomal skip mechanism, which releases the polypeptide from the translational complex and allows the production of the downstream translation product

transgenic mice enables robust transgene expression *in vivo*. However, transgene expression is usually not restricted to a certain tissue and permanent oncogene expression often causes delirious side effects, e.g. embryonic lethality, thereby impeding accurate analysis. Alternatively, genes can be delivered by retroviral vectors directly into the hematopoietic system via bone marrow transplantation of hematopoietic progenitors, transduced with the gene of interest (GOI). Gene marking of few cells also more precisely resembles leukemia development from a single cell clone. In addition, this method provides the advantage of rapid testing of numerous gene expression constructs in contrast to the time-consuming gene-manipulation of mice. Therefore, gamma-retroviral and lentiviral expression vector systems represent attractive and efficient techniques for the stable expression of a gene of interest in hematopoietic progenitor cells. Retroviruses are enveloped RNA viruses. Viral RNA is linear, single-stranded and approximately 7-12 kb in size. The common characteristic of retroviruses is their strategy of replication: the viral RNA is reverse transcribed into linear double-stranded DNA followed by its integration into the genome of the cell (Coffin et al., 2007). The most commonly used delivery system is the gammaretroviral vector system. These vectors are derived from the Moloney murine leukemia virus (Mo-MLV) genome (Kohn et al., 1987). The murine stem cell virus (MSCV) expression vector is one of the most frequently employed vector systems, as it enables stable and high transgene expression in virtually all cell types (Hawley et al., 1994). This vector is available with several marker genes, such as the enhanced green fluorescent protein (eGFP) and derivatives thereof, which are co-expressed via the internal ribosomal entry site (IRES) elements. One of the disadvantages of gamma-retroviral vectors is their inability to target non-dividing cells. Successful cell transduction is completely dependent on the breakdown of the nuclear membrane during mitosis (Roe et al., 1993). In contrast, lentiviral vectors, which are mostly derived from the HIV-1 genome, are able to integrate into both dividing and non-dividing cells (Lewis et al., 1992; Lewis & Emerman, 1994). Compared with gamma-retroviral vectors, lentiviral vectors have the capacity to incorporate larger transgenes, up to 10 kilobases, although vector titers decrease when using larger inserts (Matrai et al., 2010). The integration of retroviral vectors into the cell genome results in the stable and permanent expression of the transgene, which is transferred to all daughter cells during cell division. Therefore, retrovirally transduced oncogene expressing cells resemble cancer cells in that they pass their oncogenic alterations down to all progeny. However, during integration into the genome there is a risk of insertional mutagenesis (Baum, 2007). This must be kept in mind while analyzing the potential oncogenic effects of a transgene delivered by retroviral vectors. In general, gamma-retro as well as lentiviral vectors integrate into transcriptional units. Gamma-retroviruses tend to integrate either upstream or downstream of the start of transcriptional units (Wu et al., 2003), while lentiviral vectors integrate randomly within a transcriptional unit (Naldini et al., 2006). During retroviral integration into the genome of a cell, there is a risk of proto-oncogene activation close to the integration site of the vector driven by either the enhancer element or the promoter sequences present in the U3 region of the viral long terminal repeats (LTRs). Deletion of the U3 region within the LTRs and the insertion of an internal enhancer/promoter result in the formation of self-inactivating (SIN) vectors with the reduced risk of cellular oncogene activation (Yu et al., 1986). Retroviral particle production and transduction procedures represent convenient routine methods, which can be carried out in any laboratory with the

transgenic mice enables robust transgene expression *in vivo*. However, transgene expression is usually not restricted to a certain tissue and permanent oncogene expression often causes delirious side effects, e.g. embryonic lethality, thereby impeding accurate analysis. Alternatively, genes can be delivered by retroviral vectors directly into the hematopoietic system via bone marrow transplantation of hematopoietic progenitors, transduced with the gene of interest (GOI). Gene marking of few cells also more precisely resembles leukemia development from a single cell clone. In addition, this method provides the advantage of rapid testing of numerous gene expression constructs in contrast to the time-consuming gene-manipulation of mice. Therefore, gamma-retroviral and lentiviral expression vector systems represent attractive and efficient techniques for the stable expression of a gene of interest in hematopoietic progenitor cells. Retroviruses are enveloped RNA viruses. Viral RNA is linear, single-stranded and approximately 7-12 kb in size. The common characteristic of retroviruses is their strategy of replication: the viral RNA is reverse transcribed into linear double-stranded DNA followed by its integration into the genome of the cell (Coffin et al., 2007). The most commonly used delivery system is the gammaretroviral vector system. These vectors are derived from the Moloney murine leukemia virus (Mo-MLV) genome (Kohn et al., 1987). The murine stem cell virus (MSCV) expression vector is one of the most frequently employed vector systems, as it enables stable and high transgene expression in virtually all cell types (Hawley et al., 1994). This vector is available with several marker genes, such as the enhanced green fluorescent protein (eGFP) and derivatives thereof, which are co-expressed via the internal ribosomal entry site (IRES) elements. One of the disadvantages of gamma-retroviral vectors is their inability to target non-dividing cells. Successful cell transduction is completely dependent on the breakdown of the nuclear membrane during mitosis (Roe et al., 1993). In contrast, lentiviral vectors, which are mostly derived from the HIV-1 genome, are able to integrate into both dividing and non-dividing cells (Lewis et al., 1992; Lewis & Emerman, 1994). Compared with gamma-retroviral vectors, lentiviral vectors have the capacity to incorporate larger transgenes, up to 10 kilobases, although vector titers decrease when using larger inserts (Matrai et al., 2010). The integration of retroviral vectors into the cell genome results in the stable and permanent expression of the transgene, which is transferred to all daughter cells during cell division. Therefore, retrovirally transduced oncogene expressing cells resemble cancer cells in that they pass their oncogenic alterations down to all progeny. However, during integration into the genome there is a risk of insertional mutagenesis (Baum, 2007). This must be kept in mind while analyzing the potential oncogenic effects of a transgene delivered by retroviral vectors. In general, gamma-retro as well as lentiviral vectors integrate into transcriptional units. Gamma-retroviruses tend to integrate either upstream or downstream of the start of transcriptional units (Wu et al., 2003), while lentiviral vectors integrate randomly within a transcriptional unit (Naldini et al., 2006). During retroviral integration into the genome of a cell, there is a risk of proto-oncogene activation close to the integration site of the vector driven by either the enhancer element or the promoter sequences present in the U3 region of the viral long terminal repeats (LTRs). Deletion of the U3 region within the LTRs and the insertion of an internal enhancer/promoter result in the formation of self-inactivating (SIN) vectors with the reduced risk of cellular oncogene activation (Yu et al., 1986). Retroviral particle production and transduction procedures represent convenient routine methods, which can be carried out in any laboratory with the appropriate laboratory biosafety level. For safety reasons, genes required for the assembly of the viral particles, *gag*, *pol* and *env*, are expressed from separate, so-called helper plasmids. In contrast to the expression vector, which codes for the gene of interest, the helper plasmids do not contain coding sequences for the packaging signal. Therefore, transcribed RNAs from the helper plasmids are not incorporated into the virus particle. With these replication defective retroviral particles, only a single round of transduction is possible. Thus, the risk of generating replication competent virus particles during production is greatly reduced. Retroviral transduction efficacy can be improved by concentrating the viral particles via ultracentrifugation (Kanbe & Zhang, 2004; Naldini et al., 1996). Gamma-retroviral particles can be preloaded onto RetroNectin-coated surfaces. RetroNectin, a recombinant peptide consisting of a mixture of fragments of the fibronectin molecule, co-localizes the viral particles and target cells into close proximity, thereby strongly increasing the efficiency of transduction (Hanenberg et al., 1996). A standard protocol for routine gamma-retroviral particle production and transduction of suspension cells is provided in Table 1. Expression of the gene of interest is usually coupled to the expression of a marker gene. This allows for immediate determination of the viral transduction efficacy as well as for the identification of transduced cells for the measurement of proliferation, differentiation and cell death. EGFP is the most commonly used marker protein, which exhibits a bright green fluorescence after laser excitation at 488 nm. Enrichment of vector-transduced fluorescent-labeled cells can be performed by fluorescence activated cell sorting (FACS). The usage of drug-selectable marker genes, such as neomycin and puromycin, represents another method used to obtain pure populations of transduced cells. Cellular expression of more than one transgene by retroviral vectors can be accomplished by a variety of strategies (Figure 1).

The inclusion of an internal promoter allows for the expression of a second transgene when one transgene is expressed from the vector 5´-LTR. However, this approach also carries the risk of promoter interference with the vector LTR during retroviral particle production, thereby reducing vector titers and expression levels of the GOI (de Felipe et al., 1999). Another commonly employed strategy is the expression of two separate genes from one single transcript using the IRES element derived from the picornavirus (Gallardo et al., 1997). The IRES element allows for the initiation of mRNA translation in a 5' cap-independent manner (Mountford & Smith, 1995). However, expression levels of the two genes usually differ significantly. Typically, the gene located downstream is expressed at a level ten times lower than that of the other gene (Mizuguchi et al., 2000). The gene of interest and the marker gene can be fused to increase their stability and to allow for expression from one promoter. However, fusing two transgenes can result in functional restriction of the proteins caused by the fusion. Therefore, a short, flexible linker should be used to connect the proteins (Robinson & Sauer, 1998). Repeats of small side chain amino acids, such as glycine, serine and alanine, are well suited for the creation of linker sequences. The expression of two separate transgenes in equimolar amounts can be accomplished using self-cleaving 2A peptides, originating from the foot-and-mouth disease virus (Ryan et al., 1991). The 2A peptide sequence disrupts the peptide bond formation via a ribosomal skip mechanism, which releases the polypeptide from the translational complex and allows the production of the downstream translation product (Donnelly et al., 2001).

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 345

Fig. 1. Retroviral co-expression vector constructs, including eGFP as a marker gene. The depicted co-expression vectors are based on a standard 5'-LTR driven retroviral expression vector (top). 2A elements allow for co-expression of several GOI at equimolar levels. For intracellular stabilization, the GOI can be fused to eGFP. An internal promoter, e.g. the CMV promoter, drives GOI 2 expression independently of GOI 1. LTR, long terminal repeats; GOI, gene of interest; IRES, internal ribosomal entry site; 2A, 2A peptide sequence; eGFP,

In the context of the MSCV-based retroviral vectors, it has been shown that coupling four transgenes via three different 2A peptide cleavage sequences can generate four separate proteins both *in vitro* and *in vivo*. One restriction with this system is that the small remaining 2A-tag at the N-terminal end of the protein may potentially affect protein function (Szymczak et al., 2004). Therefore, proper cleavage and function of the individual proteins should be carefully verified. Table 2 summarizes the advantages and disadvantages of the

The lentiviral gene ontology (LeGO) vectors represent a useful collection of state-of-the-art expression vectors for functional gene analysis. These vectors contain the retroviral enhancer/promoter of the spleen focus-forming virus, which has a broad and high expression pattern. LeGO expression vectors are available with a wide spectrum of different fluorescent markers, e.g. Cerulean, eGFP, tdTomato and mCherry, which are expressed in combination with the gene of interest via IRES elements. LeGO vectors that contain fluorescent marker genes linked via 2A peptides to different drug-selectable genes, e.g. blasticidin, hygromycin, neomycin, puromycin or zeocin, are also available. Furthermore, the expression cassettes of these LeGO vectors are flanked by loxP sites, which allows for displacement of the transgene after introduction of the CRE recombinase (Weber, 2007;

different expression cassettes used for the co-expression of two genes of interest.

enhanced green fluorescent protein; CMV, cytomegalovirus.

Weber et al., 2008; Weber et al., 2010).


Table 1. Vector particle production and transduction standard protocol for suspension cells with gamma-retroviral vectors.

plasmids

temperature

1xPBS

room temperature

stored in 4°C with PBS)

medium for the target cells

supernatant (0.22 m filter)

80°C for several months

For retroviral vector particle production subconfluent (80%)

adherent cell lines (e.g. HEK293T, HeLA) are co-transfected with the retroviral expression construct and the appropriate helper

6 hours (or overnight) after transfection, exchange the appropriate

 24 hours later, harvest the viral supernatant: centrifuge the viral supernatant at 1.500 rpm to pellet cell debris and filtrate the

Virus supernatant is ready for transduction or can be stored at –

RetroNectin (RN) solution (50 µg/ml) to each well of a 24-well plate (non-tissue culture treated) and incubate overnight at room

 The next day, discard the RN solution (reusable up to 8 times), add 1 ml stop solution (1xPBS+2% BSA) and incubate for 30 minutes at

Discard the stop solution, rinse 1x with HBSS solution and 1x with

Coated plates are ready for virus preload (RN coated plates can be

 Quickly defrost the virus supernatant and dilute as necessary Discard the 1xPBS and add the viral supernatant with the

 Discard the viral supernatant and repeat the preload *at least* 3 times, each with the appropriate volume (minimum: 500 l)

 Remove the remaining viral supernatant before adding the cells Count the cells and dilute the cells to a concentration of 7.5x105

 Add 500 µl (minimum) cell suspension to each well and incubate at 37°C and 5% CO2 for at least 8 hours (or overnight) Optional: The addition of protamine sulfate (4 µg/ml) might

 Prepare a second RN coated 24-well plate and repeat the preload and transduction procedure for a second, third or fourth

8 hours after the final transduction, exchange the medium and

Optional: Wash the plate 3x with 1x PBS to collect the remaining

transfer the transduced cells into a fresh tissue-coated 24-well plate

appropriate multiplicities of infection (MOI) Centrifuge at 3000 rpm at 4°C for 20-25 minutes

cells/ml in the appropriate culture medium

transduction (max. 2 transductions per day)

Table 1. Vector particle production and transduction standard protocol for suspension cells

increase the transduction efficiency

without RN

with gamma-retroviral vectors.

cells from the plate

One day before transduction (at least 6 hours), add 500 µl

**Step 1: Retroviral vector production** 

**Step 2:** 

**Step 3: Preload of viral particles** 

**Step 4: Transduction of target cells** 

**Preparation of RetroNectincoated plates** 

Fig. 1. Retroviral co-expression vector constructs, including eGFP as a marker gene. The depicted co-expression vectors are based on a standard 5'-LTR driven retroviral expression vector (top). 2A elements allow for co-expression of several GOI at equimolar levels. For intracellular stabilization, the GOI can be fused to eGFP. An internal promoter, e.g. the CMV promoter, drives GOI 2 expression independently of GOI 1. LTR, long terminal repeats; GOI, gene of interest; IRES, internal ribosomal entry site; 2A, 2A peptide sequence; eGFP, enhanced green fluorescent protein; CMV, cytomegalovirus.

In the context of the MSCV-based retroviral vectors, it has been shown that coupling four transgenes via three different 2A peptide cleavage sequences can generate four separate proteins both *in vitro* and *in vivo*. One restriction with this system is that the small remaining 2A-tag at the N-terminal end of the protein may potentially affect protein function (Szymczak et al., 2004). Therefore, proper cleavage and function of the individual proteins should be carefully verified. Table 2 summarizes the advantages and disadvantages of the different expression cassettes used for the co-expression of two genes of interest.

The lentiviral gene ontology (LeGO) vectors represent a useful collection of state-of-the-art expression vectors for functional gene analysis. These vectors contain the retroviral enhancer/promoter of the spleen focus-forming virus, which has a broad and high expression pattern. LeGO expression vectors are available with a wide spectrum of different fluorescent markers, e.g. Cerulean, eGFP, tdTomato and mCherry, which are expressed in combination with the gene of interest via IRES elements. LeGO vectors that contain fluorescent marker genes linked via 2A peptides to different drug-selectable genes, e.g. blasticidin, hygromycin, neomycin, puromycin or zeocin, are also available. Furthermore, the expression cassettes of these LeGO vectors are flanked by loxP sites, which allows for displacement of the transgene after introduction of the CRE recombinase (Weber, 2007; Weber et al., 2008; Weber et al., 2010).

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 347

and tibia preparation, bone marrow cells can be harvested by flushing the bones. Further purification of hematopoietic progenitor cells is typically carried out by 'lineage depletion'. Using this method, differentiated 'lineage positive' cells, such as monocytes and macrophages, T- and B- cells, erythrocytes and granulocytes, are separated from their committed precursors, the 'lineage negative' fraction. Freshly isolated, complete bone marrow suspension cells are incubated with a mixture of magnetically labeled antibodies against cell surface markers for mature blood cells, typically B220, CD4, CD8, Gr-1, Mac-1 and Ter-119. Separation of the lineage negative progenitor cell fraction is performed by passing the labeled and non-labeled cells through magnetic columns (Challen et al., 2009). Amongst all vertebrates, the murine hematopoietic system is the best characterized. For the subdivision of stem and progenitor cell populations, the LSK- and SLAM-markers have

and simultaneously express high levels of the stem cell markers, Sca1 and c-kit. As little as one hundred LSK cells have been shown to be sufficient for the long-term multilineage repopulation of lethally irradiated recipient mice (Okada et al., 1992). The LSK cell compartment can be further separated into hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) depending on their diverse expression pattern of the family of 'signaling lymphocyte activation molecule' (SLAM) receptors (Kiel et al., 2005). The SLAM cell surface marker, CD150, is exclusively expressed on HSCs, whereas CD244 is expressed by transiently reconstituting MPPs. CD34 and Flt3 expression patterns further allow for the discrimination between long term-HSC (LT-HSCs; CD34-/Flt3-) and short term HSCs (ST-HSCs; CD34+/Flt3+) (Adolfsson et al., 2001; Osawa et al., 1996). ST-HSCs have a reduced self-renewal capacity for a restricted time compared with the life-long self-renewal capacity of LT-HSCs (Adolfsson et al., 2001; Morrison & Weissman, 1994; Yang et al., 2005). Because the murine hematopoietic system has been well characterized, with precisely classified and characterized stem and progenitor cell compartments, and simple precursor cell purification procedures have been established and optimized, murine hematopoietic progenitor cells are well suited for the functional analysis of oncogenic fusion genes and mutations associated

The cell transforming activity of a given leukemia-associated oncogene can be measured by performing an *in vitro* colony forming unit (CFU) assay. This assay allows for the quantification of colony forming progenitors and their iterative replating capacity. Hematopoietic progenitor cells expressing the gene of interest are plated in methylcellulose at low density. In general, normal progenitor cells only form colonies in the first round of plating. However, oncogene-expressing cells are able to confer serial replating capacity for several rounds of plating. For example, cells expressing the t(8;21) translocation product, AML1/ETO, are able to confer serial replating for up to 13 rounds of plating (Rhoades et al., 2000). Transgene expressing murine progenitor cells can also be cultivated in liquid suspension cultures. Here, cytokines such as IL3, IL6 and SCF must be supplemented continuously. Under these *ex vivo* cultivation parameters specific for cell differentiation, proliferation and apoptosis can be measured under various cell culture conditions, such as serum or cytokine deprivation and hypoxic conditions, which imitate the hypoxic niches of

Gene modified precursor cells can be further examined *in vivo* in a mouse transplantation model. In this case, immediately after retroviral gene transfer, lineage depleted bone marrow cells stably expressing the oncogene of interest are transplanted into lethally irradiated syngeneic recipient mice. The transplantation is performed by injection of gene-

, Sca1+, c-kit+) cells lack the markers of mature blood cells

become widely accepted. LSK (lin-

with leukemia development.

the bone marrow.


Table 2. Overview of expression cassette elements for co-expression of two genes of interest.

To accurately characterize oncogene activity it is important to have the ability to selectively induce and terminate oncogene expression. This is particularly important if the delivered gene product is toxic at high concentrations. The regulation of gene expression can be achieved by using the tetracycline-controlled transactivator (tTA)-responsive promoter (Tetsystem), a prokaryotic inducible promoter system that is also applicable to mammalian cells (Gossen & Bujard, 1992). The Tet system comprises two components: one component that drives expression of the transactivator (tTA) and a second component that contains the (tTA)-responsive promoter element for transgene expression. In the first developed tet-off system, the transactivator binds to the promoter in the absence of tetracycline. The addition of tetracycline inhibits its binding, thereby resulting in inhibition of transgene expression (Gossen et al., 1995). To avoid constant tetracycline supplementation, a reverse system ('teton' system) allows for transgene expression only in the presence of doxycycline, a tetracycline derivate (Gossen et al., 1995). Traditionally, transactivator and promoter components had to be introduced by two separate transduction steps to obtain inducible gene expression. The development of an 'all-in-one' vector system circumvents this complication. In this system, all components required for tet-regulated transgene expression have been inserted as a bidirectional expression cassette (Heinz et al., 2011). Another means of intracellular oncogene dosage control is the regulation at the protein level. With this type of expression control, the gene of interest can be fused to a small destabilizing domain, thereby mediating the intracellular destruction of the protein by targeting it for proteasomal degradation. Degradation of the fusion protein can be specifically blocked by cell membrane-permeable small molecules, which bind to and inhibit the destabilizing domain mediated degradation, thereby resulting in the accumulation of the expressed transgene (Banaszynski et al., 2006).

#### **3. Murine progenitor cells**

Murine hematopoietic stem and progenitor cells have been well characterized. To study these cell types, progenitor cells can be simply isolated from the bone marrow. After femora

**(internal)** 

Low titer of viral particles

*Felipe et al., 1999 Rizo et al., 2010* 

Expression of 2 separate transgenes

**Co-transduction** 

Low rate of double transduced cells; increases risk of insertional mutagenesis

Independent expression; results in single and double transduced cells

**IRES 2A 2nd promoter** 

Equal expression levels of the 2

genes

*2004* 

Remaining 2A tag at the end of the N-terminal protein

*Szymczak et al.,* 

Table 2. Overview of expression cassette elements for co-expression of two genes of interest. To accurately characterize oncogene activity it is important to have the ability to selectively induce and terminate oncogene expression. This is particularly important if the delivered gene product is toxic at high concentrations. The regulation of gene expression can be achieved by using the tetracycline-controlled transactivator (tTA)-responsive promoter (Tetsystem), a prokaryotic inducible promoter system that is also applicable to mammalian cells (Gossen & Bujard, 1992). The Tet system comprises two components: one component that drives expression of the transactivator (tTA) and a second component that contains the (tTA)-responsive promoter element for transgene expression. In the first developed tet-off system, the transactivator binds to the promoter in the absence of tetracycline. The addition of tetracycline inhibits its binding, thereby resulting in inhibition of transgene expression (Gossen et al., 1995). To avoid constant tetracycline supplementation, a reverse system ('teton' system) allows for transgene expression only in the presence of doxycycline, a tetracycline derivate (Gossen et al., 1995). Traditionally, transactivator and promoter components had to be introduced by two separate transduction steps to obtain inducible gene expression. The development of an 'all-in-one' vector system circumvents this complication. In this system, all components required for tet-regulated transgene expression have been inserted as a bidirectional expression cassette (Heinz et al., 2011). Another means of intracellular oncogene dosage control is the regulation at the protein level. With this type of expression control, the gene of interest can be fused to a small destabilizing domain, thereby mediating the intracellular destruction of the protein by targeting it for proteasomal degradation. Degradation of the fusion protein can be specifically blocked by cell membrane-permeable small molecules, which bind to and inhibit the destabilizing domain mediated degradation, thereby resulting in the accumulation of the expressed transgene

Murine hematopoietic stem and progenitor cells have been well characterized. To study these cell types, progenitor cells can be simply isolated from the bone marrow. After femora

Advantage Expression of 2

Disadvantage 10x lower

(Banaszynski et al., 2006).

**3. Murine progenitor cells** 

*Literature Gallardo et al., 1997*

*2000* 

separate transgenes

expression of the downstream gene

*Mizuguchi et al.,* 

and tibia preparation, bone marrow cells can be harvested by flushing the bones. Further purification of hematopoietic progenitor cells is typically carried out by 'lineage depletion'. Using this method, differentiated 'lineage positive' cells, such as monocytes and macrophages, T- and B- cells, erythrocytes and granulocytes, are separated from their committed precursors, the 'lineage negative' fraction. Freshly isolated, complete bone marrow suspension cells are incubated with a mixture of magnetically labeled antibodies against cell surface markers for mature blood cells, typically B220, CD4, CD8, Gr-1, Mac-1 and Ter-119. Separation of the lineage negative progenitor cell fraction is performed by passing the labeled and non-labeled cells through magnetic columns (Challen et al., 2009). Amongst all vertebrates, the murine hematopoietic system is the best characterized. For the subdivision of stem and progenitor cell populations, the LSK- and SLAM-markers have become widely accepted. LSK (lin- , Sca1+, c-kit+) cells lack the markers of mature blood cells and simultaneously express high levels of the stem cell markers, Sca1 and c-kit. As little as one hundred LSK cells have been shown to be sufficient for the long-term multilineage repopulation of lethally irradiated recipient mice (Okada et al., 1992). The LSK cell compartment can be further separated into hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) depending on their diverse expression pattern of the family of 'signaling lymphocyte activation molecule' (SLAM) receptors (Kiel et al., 2005). The SLAM cell surface marker, CD150, is exclusively expressed on HSCs, whereas CD244 is expressed by transiently reconstituting MPPs. CD34 and Flt3 expression patterns further allow for the discrimination between long term-HSC (LT-HSCs; CD34-/Flt3-) and short term HSCs (ST-HSCs; CD34+/Flt3+) (Adolfsson et al., 2001; Osawa et al., 1996). ST-HSCs have a reduced self-renewal capacity for a restricted time compared with the life-long self-renewal capacity of LT-HSCs (Adolfsson et al., 2001; Morrison & Weissman, 1994; Yang et al., 2005). Because the murine hematopoietic system has been well characterized, with precisely classified and characterized stem and progenitor cell compartments, and simple precursor cell purification procedures have been established and optimized, murine hematopoietic progenitor cells are well suited for the functional analysis of oncogenic fusion genes and mutations associated with leukemia development.

The cell transforming activity of a given leukemia-associated oncogene can be measured by performing an *in vitro* colony forming unit (CFU) assay. This assay allows for the quantification of colony forming progenitors and their iterative replating capacity. Hematopoietic progenitor cells expressing the gene of interest are plated in methylcellulose at low density. In general, normal progenitor cells only form colonies in the first round of plating. However, oncogene-expressing cells are able to confer serial replating capacity for several rounds of plating. For example, cells expressing the t(8;21) translocation product, AML1/ETO, are able to confer serial replating for up to 13 rounds of plating (Rhoades et al., 2000). Transgene expressing murine progenitor cells can also be cultivated in liquid suspension cultures. Here, cytokines such as IL3, IL6 and SCF must be supplemented continuously. Under these *ex vivo* cultivation parameters specific for cell differentiation, proliferation and apoptosis can be measured under various cell culture conditions, such as serum or cytokine deprivation and hypoxic conditions, which imitate the hypoxic niches of the bone marrow.

Gene modified precursor cells can be further examined *in vivo* in a mouse transplantation model. In this case, immediately after retroviral gene transfer, lineage depleted bone marrow cells stably expressing the oncogene of interest are transplanted into lethally irradiated syngeneic recipient mice. The transplantation is performed by injection of gene-

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 349

expression of PML/RAR in murine progenitor cells efficiently induces leukemia development in transplanted lethally irradiated recipient mice (Minucci et al., 2002). The efficacy of arsenic trioxide and ATRA has been recapitulated in PML/RAR mouse models displaying the impressive anti-leukemia effects of both substances (Rego et al., 2000). Furthermore, in transplantation models based on ATRA-resistant APL cell lines, treatment with arsenic trioxide significantly decreases tumor formation (Kinjo et al., 2000). Zhang et al have further showed that arsenic trioxide functions by directly binding to PML/RAR. The researchers identified cysteine residues within the PML portion that are directly bound by arsenic trioxide, which triggered SUMOylation and subsequent degradation of the

Fig. 2. Functional analysis of gene-modified murine/human hematopoietic progenitor cells. Experimental procedures include cloning of the retroviral expression vector, viral particle production, hematopoietic progenitor cell isolation, transduction and functional analysis in

Other reports have described that oncogene expression experiments involving murine progenitor cells supported the clinical finding reporting a highly leukemogenic splice variant of the familiar AML1/ETO fusion gene resulting from the translocation t(8;21). Several groups have shown that the full length AML1/ETO protein does not lead to the development of leukemia when transplanting LSK bone marrow cells expressing AML1/ETO into syngeneic mice (de Guzman et al., 2002). However, in similar transplantation experiments performed by Yan and colleagues, one mouse developed leukemia 14 weeks after bone marrow transplantation. Further analysis showed that a Cterminal truncated AML1/ETO protein was expressed in this mouse due to a one-base pair insertion into the full length AML1/ETO gene. Subsequently, the cloning and creation of

methylcellulose assays, liquid cultures and murine syngeneic or xenogeneic mouse transplantation models. GOI, gene of interest; MSCV, murine stem cell virus; LTR, long

oncoprotein (Zhang et al., 2010).

terminal repeats; lin-, lineage negative.

modified cells into the tail vein or the retro-orbital sinus of recipient mice (Larochelle et al., 1995). For successful hematopoietic rescue, transplanted cells must reach the appropriate milieu in the bone marrow niches, which supports their proliferation and differentiation; this cell migration process is referred to as 'homing' (Szilvassy et al., 1999). To analyze disease progression in mice following the transplantation of progenitor cells body weight and blood profile values must be documented carefully over time. If transplanted mice develop any symptoms of leukemia, e.g. sudden body weight reduction, increasing peripheral leukocyte counts or poor general health conditions, they should be sacrificed and analyzed. Blood cells isolated from the periphery, spleen and bone marrow compartment can be studied separately. Flow cytometric analysis conveniently allows for the detection of oncogene expressing blood cells by discerning the expression of the fluorescence marker proteins. Gene marked cells can be examined for cellular differentiation (CD differentiation marker), the proliferation/cell cycle profile and apoptosis by AnnexinV-staining, for example. FACS based methods allow for the parallel analysis of transduced and nontransduced cells, using the latter as the internal experimental control. Transgene expression of leukemic cells must be analyzed by RT-PCR and western immunoblotting, which assesses mRNA and protein levels, respectively. There are highly specific antibodies that can detect epitope-tagged oncoproteins, e.g. Ha- or Flag-tagged proteins, even at very low expression levels (Terpe, 2003). To exclude the potential oncogenic effects of vector-mediated insertional mutagenesis, clonality and integration site analysis must be performed carefully. The oncogene activity of the expressed transgene can be accurately validated by analyzing the downstream signal transduction pathways of the cell. For example, phosphorylated STAT5 and CRKL are regarded as downstream indicators of oncogenic BCR/ABL signaling. Bone marrow and spleen, which are the target organs of leukemic origin, and peripheral blood must be analyzed for the presence of immature blast cells. This can be carried out by conventional blood smear and flow cytometric analysis. For organ histology, samples of each organ, including the spleen, liver, lymph nodes and thymus, are transferred into paraffin, sectioned and examined. Hematoxylin and eosin are the most commonly used staining solutions in histology for light microscopical analysis. A final validation of the leukemia must be demonstrated by re-transplantating the leukemic blood cells into secondary lethally irradiated recipient mice. In these secondary transplanted mice, leukemia onset should appear much earlier than in primary recipients. In 2002, Kogan et al published the 'Bethesda proposals for the classification of hematopoietic neoplasms in mice' (Kogan et al., 2002). As with the World Health Organization (WHO) classification of human disorders, theses proposals utilize morphologic, immunophenotypic, clinical, biological and genetic characteristics to classify neoplasms.

Both methods, the colony-forming assay and the mouse transplantation model are commonly used techniques for the investigation of the transforming potential of oncogenes in leukemia (Figure 2).

The following examples illustrate both the genetic and biochemical approaches utilizing murine hematopoietic progenitor cells for leukemia modeling.

The acute promyelocytic leukemia (APL) associated t(15;17) translocation product, PML/RAR, is one of the most studied fusion proteins associated with AML (Puccetti & Ruthardt, 2004). In the clinical setting, APL patients not only respond well to 'all trans retinoic acid' (ATRA) therapy, which has been well documented (Huang et al., 1988), but also benefit from treatment with arsenic trioxide (Chen et al., 1997), which exerts a profound anti-cancer effect by inducing the degradation of the PML/RAR oncoprotein. Retroviral

modified cells into the tail vein or the retro-orbital sinus of recipient mice (Larochelle et al., 1995). For successful hematopoietic rescue, transplanted cells must reach the appropriate milieu in the bone marrow niches, which supports their proliferation and differentiation; this cell migration process is referred to as 'homing' (Szilvassy et al., 1999). To analyze disease progression in mice following the transplantation of progenitor cells body weight and blood profile values must be documented carefully over time. If transplanted mice develop any symptoms of leukemia, e.g. sudden body weight reduction, increasing peripheral leukocyte counts or poor general health conditions, they should be sacrificed and analyzed. Blood cells isolated from the periphery, spleen and bone marrow compartment can be studied separately. Flow cytometric analysis conveniently allows for the detection of oncogene expressing blood cells by discerning the expression of the fluorescence marker proteins. Gene marked cells can be examined for cellular differentiation (CD differentiation marker), the proliferation/cell cycle profile and apoptosis by AnnexinV-staining, for example. FACS based methods allow for the parallel analysis of transduced and nontransduced cells, using the latter as the internal experimental control. Transgene expression of leukemic cells must be analyzed by RT-PCR and western immunoblotting, which assesses mRNA and protein levels, respectively. There are highly specific antibodies that can detect epitope-tagged oncoproteins, e.g. Ha- or Flag-tagged proteins, even at very low expression levels (Terpe, 2003). To exclude the potential oncogenic effects of vector-mediated insertional mutagenesis, clonality and integration site analysis must be performed carefully. The oncogene activity of the expressed transgene can be accurately validated by analyzing the downstream signal transduction pathways of the cell. For example, phosphorylated STAT5 and CRKL are regarded as downstream indicators of oncogenic BCR/ABL signaling. Bone marrow and spleen, which are the target organs of leukemic origin, and peripheral blood must be analyzed for the presence of immature blast cells. This can be carried out by conventional blood smear and flow cytometric analysis. For organ histology, samples of each organ, including the spleen, liver, lymph nodes and thymus, are transferred into paraffin, sectioned and examined. Hematoxylin and eosin are the most commonly used staining solutions in histology for light microscopical analysis. A final validation of the leukemia must be demonstrated by re-transplantating the leukemic blood cells into secondary lethally irradiated recipient mice. In these secondary transplanted mice, leukemia onset should appear much earlier than in primary recipients. In 2002, Kogan et al published the 'Bethesda proposals for the classification of hematopoietic neoplasms in mice' (Kogan et al., 2002). As with the World Health Organization (WHO) classification of human disorders, theses proposals utilize morphologic, immunophenotypic, clinical, biological and genetic

Both methods, the colony-forming assay and the mouse transplantation model are commonly used techniques for the investigation of the transforming potential of oncogenes

The following examples illustrate both the genetic and biochemical approaches utilizing

The acute promyelocytic leukemia (APL) associated t(15;17) translocation product, PML/RAR, is one of the most studied fusion proteins associated with AML (Puccetti & Ruthardt, 2004). In the clinical setting, APL patients not only respond well to 'all trans retinoic acid' (ATRA) therapy, which has been well documented (Huang et al., 1988), but also benefit from treatment with arsenic trioxide (Chen et al., 1997), which exerts a profound anti-cancer effect by inducing the degradation of the PML/RAR oncoprotein. Retroviral

characteristics to classify neoplasms.

murine hematopoietic progenitor cells for leukemia modeling.

in leukemia (Figure 2).

expression of PML/RAR in murine progenitor cells efficiently induces leukemia development in transplanted lethally irradiated recipient mice (Minucci et al., 2002). The efficacy of arsenic trioxide and ATRA has been recapitulated in PML/RAR mouse models displaying the impressive anti-leukemia effects of both substances (Rego et al., 2000). Furthermore, in transplantation models based on ATRA-resistant APL cell lines, treatment with arsenic trioxide significantly decreases tumor formation (Kinjo et al., 2000). Zhang et al have further showed that arsenic trioxide functions by directly binding to PML/RAR. The researchers identified cysteine residues within the PML portion that are directly bound by arsenic trioxide, which triggered SUMOylation and subsequent degradation of the oncoprotein (Zhang et al., 2010).

Fig. 2. Functional analysis of gene-modified murine/human hematopoietic progenitor cells. Experimental procedures include cloning of the retroviral expression vector, viral particle production, hematopoietic progenitor cell isolation, transduction and functional analysis in methylcellulose assays, liquid cultures and murine syngeneic or xenogeneic mouse transplantation models. GOI, gene of interest; MSCV, murine stem cell virus; LTR, long terminal repeats; lin-, lineage negative.

Other reports have described that oncogene expression experiments involving murine progenitor cells supported the clinical finding reporting a highly leukemogenic splice variant of the familiar AML1/ETO fusion gene resulting from the translocation t(8;21). Several groups have shown that the full length AML1/ETO protein does not lead to the development of leukemia when transplanting LSK bone marrow cells expressing AML1/ETO into syngeneic mice (de Guzman et al., 2002). However, in similar transplantation experiments performed by Yan and colleagues, one mouse developed leukemia 14 weeks after bone marrow transplantation. Further analysis showed that a Cterminal truncated AML1/ETO protein was expressed in this mouse due to a one-base pair insertion into the full length AML1/ETO gene. Subsequently, the cloning and creation of

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 351

Human blood progenitor cells are characterized by the high expression levels of the cell surface marker CD34 and the absence of CD38 expression (CD34+/CD38-). CD34 is a member of the single-pass transmembrane sialomucin protein family, which is expressed in early hematopoietic and vascular-associated tissues; however, little is known about its exact biological function (Furness & McNagny, 2006). There are a variety of ways to obtain hematopoietic CD34+ cells from the human body. The cells can be isolated from the peripheral blood by leukapheresis after stimulation and mobilization of the bone marrow precursors with G-CSF (granulocytic colony stimulating factor). Bone marrow aspiration represents the classical but invasive isolation method, which also allows for the isolation of large numbers of CD34+ cells. A third option involves the usage of placental derived cord blood cells of which the quality/stemness and engraftment potential are superior to that of adult CD34+ stem cells (Hao et al., 1995). The efficient enrichment of human hematopoietic CD34+ cells can be achieved by immunomagnetic cell sorting, which represents a standard and convenient method in laboratory work (Clarke & Davies, 2001). Magnetic labeled CD34 antibodies are used to recover high-purity populations of CD34+ cells by passing cells through magnetic columns. Isolated CD34+ cells can then be used for clinical and experimental purposes. For transgene expression studies, the cells can be manipulated by retroviral transduction immediately after recovery or after a short pre-cultivation in a cytokine cocktail, which usually contains IL-3, IL6 and SCF for cell cycle activation. Like the murine system, transduced human CD34+ cells can be propagated *in vitro* in methylcellulose assays for colony formation as well as in long-term liquid cultures used to assess self-renewal and proliferation/expansion capacities (Figure 1). Furthermore, human CD34+ cells expressing leukemic gene products can be transplanted into severe combined immunodeficient (NOD/SCID) mice (xenotransplants). Additionally, the NOG (NOD/Shiscid/IL-2Rγnull) mice accept heterologous transplanted cells more easily than any other type of immunodeficient nude or NOD/SCID mice. Therefore, the NOG mouse system is a highly efficient recipient model for the engraftment, proliferation and differentiation of

The transformation of human cells requires additional genetic alterations when compared with their murine counterparts (Hahn et al., 1999; Rangarajan & Weinberg, 2003). This is reflected by the observation that singular expression of several oncogenes induces leukemia in the murine system but fails to transform human CD34+ cells in the NOD/SCID humanized leukemia mouse model. Further evidence of this concept has been demonstrated by gene expression analysis of healthy individuals. Here, oncogenes such as BCR/ABL and AML1/ETO have been frequently detected in individuals completely lacking any sign of disease (Song et al., 2011). As CD34+ cells represent the human hematopoietic stem/progenitor cell population, these cells can be regarded as appropriate target cells used

The following passages provide an overview of recent experiments addressing the leukemogenic activity of several myeloid leukemia associated oncogenes expressed alone or in combination in human hematopoietic CD34+ progenitor cells. In all cases, genes were delivered using gamma-retroviral vectors co-expressing the marker genes eGFP or dNGFR (truncated nerve growth factor receptor), which allowed for the convenient detection of

to study the biological effects of oncogenes in human leukemia development.

**4. The human CD34+ progenitor cell system** 

human cells (Ito et al., 2002).

gene-modified cells by FACS analysis.

mice expressing the truncated protein by transplantation of transduced progenitor cells again revealed a rapid leukemia development (Yan et al., 2004). Consequently, these results led to the investigation and identification of a naturally occurring splice variant of AML1/ETO in t(8;21) patients by sophisticated gene expression analysis of patient material. This splice variant, termed AML1/ETO9A, is almost structurally identical to the truncated form expressed in the leukemic mouse. Expression of this newly discovered splice isoform in mice also induced rapid leukemia development (Yan et al., 2006). In conclusion, these oncogene expression experiments using murine progenitor cells led to the clinical identification of a potent oncogenic AML1/ETO splice variant. Further experiments must be performed to understand whether the AML1/ETO splice isoform is the driving oncogene in human t(8;21) leukemia cells.

The usage of oncogene expressing vectors has also facilitated the understanding of the protein domain structure-function relationship. Biophysical and biochemical experiments have been used to identify highly conserved domains within several oncoproteins, which might represent valuable target structures for molecular intervention. This has been demonstrated with the AML1/ETO fusion protein. Here, a central region, the *nervy* homology region 2 (NHR2), was found to play an essential role in the leukemogenic activity of the fusion protein. Analysis of the crystal structure revealed a tetrameric formation that is essential for the serial colony formation capacity of murine progenitor cells in methylcellulose assays (Liu et al., 2006). By performing additional bioinformatic molecular modeling analyses, five amino acids were identified within the large NHR2 tetramerinterface that are important for oligomer formation. Substitution of these critical residues led to the conversion of tetramers into dimers with the complete loss of the transforming abilities of AML1/ETO. Transplantation of retroviral infected murine bone marrow cells expressing the AML1/ETO protein harboring these five substitutions showed that these amino acids play a critical role in transformation, as the mice failed to develop leukemia. Interestingly, the identified amino acids are clustered in one region at the top of the NHR2 dimer surface, thereby representing a potential target site for molecular intervention (Wichmann et al., 2010). These findings, which reveal the molecular determinants of oncogene activity, are the result of a productive combination of bioinformatics, biochemical and cell-biological analyses. Furthermore, proof of concept of oncogene targeting was demonstrated using a synthetic oligomerization domain. This domain was integrated into the AML1/ETO fusion gene, thereby replacing the NHR2 tetramer domain. Specific inhibitors that disrupt the oligomerization of this synthetic domain within AML1/ETO completely blocked the replating capacity of retrovirally transduced AML1/ETO expressing murine progenitor cells (Kwok et al., 2009).

By expressing leukemia fusion genes in murine cells, one can assess exactly which gene product from a balanced translocation, which often generates two different reciprocal fusion genes, is responsible for driving the leukemic activity. Bursen and colleagues have reported interesting findings regarding the function of the reciprocal fusion proteins, AF4/MLL and MLL/AF4, which are generated by the chromosomal translocation t(4;11) in high-risk infant acute mixed lineage leukemia. They analyzed the leukemia-inducing capacity of these two fusion genes in mice by transplantation of retrovirally transduced Lin-/Sca1+ cells expressing either one or both of the reciprocal fusion genes. They could show that expression of AF4/MLL alone was sufficient to promote leukemia development, while the reciprocal translocation, MLL/AF4, completely lacked leukemogenic potential (Bursen et al., 2010).

mice expressing the truncated protein by transplantation of transduced progenitor cells again revealed a rapid leukemia development (Yan et al., 2004). Consequently, these results led to the investigation and identification of a naturally occurring splice variant of AML1/ETO in t(8;21) patients by sophisticated gene expression analysis of patient material. This splice variant, termed AML1/ETO9A, is almost structurally identical to the truncated form expressed in the leukemic mouse. Expression of this newly discovered splice isoform in mice also induced rapid leukemia development (Yan et al., 2006). In conclusion, these oncogene expression experiments using murine progenitor cells led to the clinical identification of a potent oncogenic AML1/ETO splice variant. Further experiments must be performed to understand whether the AML1/ETO splice isoform is the driving oncogene in

The usage of oncogene expressing vectors has also facilitated the understanding of the protein domain structure-function relationship. Biophysical and biochemical experiments have been used to identify highly conserved domains within several oncoproteins, which might represent valuable target structures for molecular intervention. This has been demonstrated with the AML1/ETO fusion protein. Here, a central region, the *nervy* homology region 2 (NHR2), was found to play an essential role in the leukemogenic activity of the fusion protein. Analysis of the crystal structure revealed a tetrameric formation that is essential for the serial colony formation capacity of murine progenitor cells in methylcellulose assays (Liu et al., 2006). By performing additional bioinformatic molecular modeling analyses, five amino acids were identified within the large NHR2 tetramerinterface that are important for oligomer formation. Substitution of these critical residues led to the conversion of tetramers into dimers with the complete loss of the transforming abilities of AML1/ETO. Transplantation of retroviral infected murine bone marrow cells expressing the AML1/ETO protein harboring these five substitutions showed that these amino acids play a critical role in transformation, as the mice failed to develop leukemia. Interestingly, the identified amino acids are clustered in one region at the top of the NHR2 dimer surface, thereby representing a potential target site for molecular intervention (Wichmann et al., 2010). These findings, which reveal the molecular determinants of oncogene activity, are the result of a productive combination of bioinformatics, biochemical and cell-biological analyses. Furthermore, proof of concept of oncogene targeting was demonstrated using a synthetic oligomerization domain. This domain was integrated into the AML1/ETO fusion gene, thereby replacing the NHR2 tetramer domain. Specific inhibitors that disrupt the oligomerization of this synthetic domain within AML1/ETO completely blocked the replating capacity of retrovirally transduced AML1/ETO expressing

By expressing leukemia fusion genes in murine cells, one can assess exactly which gene product from a balanced translocation, which often generates two different reciprocal fusion genes, is responsible for driving the leukemic activity. Bursen and colleagues have reported interesting findings regarding the function of the reciprocal fusion proteins, AF4/MLL and MLL/AF4, which are generated by the chromosomal translocation t(4;11) in high-risk infant acute mixed lineage leukemia. They analyzed the leukemia-inducing capacity of these two

expressing either one or both of the reciprocal fusion genes. They could show that expression of AF4/MLL alone was sufficient to promote leukemia development, while the reciprocal translocation, MLL/AF4, completely lacked leukemogenic potential (Bursen et al.,

/Sca1+ cells

fusion genes in mice by transplantation of retrovirally transduced Lin-

human t(8;21) leukemia cells.

murine progenitor cells (Kwok et al., 2009).

2010).

Human blood progenitor cells are characterized by the high expression levels of the cell surface marker CD34 and the absence of CD38 expression (CD34+/CD38-). CD34 is a member of the single-pass transmembrane sialomucin protein family, which is expressed in early hematopoietic and vascular-associated tissues; however, little is known about its exact biological function (Furness & McNagny, 2006). There are a variety of ways to obtain hematopoietic CD34+ cells from the human body. The cells can be isolated from the peripheral blood by leukapheresis after stimulation and mobilization of the bone marrow precursors with G-CSF (granulocytic colony stimulating factor). Bone marrow aspiration represents the classical but invasive isolation method, which also allows for the isolation of large numbers of CD34+ cells. A third option involves the usage of placental derived cord blood cells of which the quality/stemness and engraftment potential are superior to that of adult CD34+ stem cells (Hao et al., 1995). The efficient enrichment of human hematopoietic CD34+ cells can be achieved by immunomagnetic cell sorting, which represents a standard and convenient method in laboratory work (Clarke & Davies, 2001). Magnetic labeled CD34 antibodies are used to recover high-purity populations of CD34+ cells by passing cells through magnetic columns. Isolated CD34+ cells can then be used for clinical and experimental purposes. For transgene expression studies, the cells can be manipulated by retroviral transduction immediately after recovery or after a short pre-cultivation in a cytokine cocktail, which usually contains IL-3, IL6 and SCF for cell cycle activation. Like the murine system, transduced human CD34+ cells can be propagated *in vitro* in methylcellulose assays for colony formation as well as in long-term liquid cultures used to assess self-renewal and proliferation/expansion capacities (Figure 1). Furthermore, human CD34+ cells expressing leukemic gene products can be transplanted into severe combined immunodeficient (NOD/SCID) mice (xenotransplants). Additionally, the NOG (NOD/Shiscid/IL-2Rγnull) mice accept heterologous transplanted cells more easily than any other type of immunodeficient nude or NOD/SCID mice. Therefore, the NOG mouse system is a highly efficient recipient model for the engraftment, proliferation and differentiation of human cells (Ito et al., 2002).

The transformation of human cells requires additional genetic alterations when compared with their murine counterparts (Hahn et al., 1999; Rangarajan & Weinberg, 2003). This is reflected by the observation that singular expression of several oncogenes induces leukemia in the murine system but fails to transform human CD34+ cells in the NOD/SCID humanized leukemia mouse model. Further evidence of this concept has been demonstrated by gene expression analysis of healthy individuals. Here, oncogenes such as BCR/ABL and AML1/ETO have been frequently detected in individuals completely lacking any sign of disease (Song et al., 2011). As CD34+ cells represent the human hematopoietic stem/progenitor cell population, these cells can be regarded as appropriate target cells used to study the biological effects of oncogenes in human leukemia development.

The following passages provide an overview of recent experiments addressing the leukemogenic activity of several myeloid leukemia associated oncogenes expressed alone or in combination in human hematopoietic CD34+ progenitor cells. In all cases, genes were delivered using gamma-retroviral vectors co-expressing the marker genes eGFP or dNGFR (truncated nerve growth factor receptor), which allowed for the convenient detection of gene-modified cells by FACS analysis.

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 353

full-blown AML development. Similar results were obtained from analysis of the inv(16) fusion protein CBFB/MYH11. Expression of this fusion gene also caused enhanced proliferation and expansion of CD34+ cells over several months with retention of the engraftment potential in NOD/SCID mice. However, as also observed with AML1/ETO, transplanted mice do not develop leukemia (Wunderlich et al., 2006). Therefore, both oncogenes confer a preleukemic status and subsequent alterations are required for the

To decipher the genetic signature of AML, Abdul-Nabi and colleagues have assessed various AML-associated fusion proteins for their ability to enhance CD34+ cell expansion *ex vivo* and subsequently analyzed their gene expression profiles (Abdul-Nabi et al., 2010). In this study, experiments were also performed with gamma-retroviral vectors expressing the fusion genes AML1/ETO, PML/RAR, MLL/AF9 and NUP98/HOXA9 together with eGFP. To identify key target genes responsible for the expansion of CD34+ cells the authors performed gene array analysis of FACS-sorted eGFP+ cells expressing the indicated oncogenes, which identifies the induced and repressed target genes during the *ex vivo* selection process. One interesting candidate gene that was shown to be specifically upregulated due to both AML1/ETO and PML/RAR expression was the p53-inhibiting molecule MDM2. The authors could further show that activation of the gatekeeper protein p53, by blocking the MDM2-p53 protein-protein interaction with the small molecule compound Nutlin-3, was sufficient to completely block AML1/ETO induced selection and expansion of CD34+ progenitor cells. Therefore, Nutlin-3 is proposed to function as an inhibitor for AML1/ETO positive leukemias by reactivating p53 and consecutively eliminating the leukemic cells via the induction of apoptosis. Two independent groups have shown that retroviral expression of the NUP98/HOXA9 fusion gene, which is found in myelodysplastic syndrome (MDS) and AML patients, resulted in a similar outgrowth of transgene expressing cells with maintenance of self-renewal potential *ex vivo* and increased engraftment capacity in NOD/SCID mice (Chung et al., 2006; Takeda et al., 2006). This outgrowth was accompanied by high levels of HOX gene expression, a typical feature for

Regardless of their remarkable *ex vivo* cell expansion properties, the AML associated gene products described above, PML/RAR, FLT3-ITD, NUP98/HOXA9 and AML1/ETO, were not able to independently induce leukemia in transplanted NOD/SCID mice. Therefore, it is likely that additional genetic alterations are required for full cellular transformation in AML. In this regard it was shown that oncogenic RAS signaling, triggered by N-RasG12D overexpression, does increase AML1/ETO engraftment capacity in NOD/SCID mice but is still not sufficient to overcome the leukemia onset defect in the NOD/SCID mouse model

Interestingly, MLL rearrangements are typically found in aggressive infant or therapy related leukemias (Aplan, 2006). In contrast to AML derived gene products, ectopic expression of either MLL/AF9 or MLL/ENL mixed-lineage leukemia fusion genes, as a single genetic event, results in the development of myeloid or lymphoid leukemias in the NOD/SCID mouse model approximately 100 days after transplantation (Barabe et al., 2007; Wei et al., 2008). Morphological analysis has revealed that AML and B-ALL leukemias are associated with both MLL rearrangements. Remarkably, *ex vivo* expanded cells gave rise to leukemia in NOD/SCID mice, even after 70 days of *in vitro* culture prior to transplantation in sublethally irradiated mice. The reasons for the striking differences between AML and

transition to overt leukemia.

this leukemia entity.

(Chou et al., 2011).

Grignani et al have reported that ectopic expression of PML/RAR in human CD34+ *ex vivo* cultures rapidly induced the cellular differentiation of progenitor cells towards the promyelocytic stage, followed by a block in further terminal differentiation into granulocytes, which resembled typical features of primary human acute promyelocytic leukemia (Grignani et al., 2000). In cytokine deprived liquid cultures, PML/RAR expressing cells survived and continued to expand, whereas control cells stopped proliferation and died by apoptosis. The terminal differentiation block induced by PML/RAR could be overcome by treatment with ATRA, a drug that has dramatically improved the overall survival of APL patients. They further demonstrated that a mutation of the N-CoR repressor binding interface within the PML portion of the fusion protein completely disrupted the observed biological effects, thereby suggesting that N-CoR triggered transcriptional repression is an essential prerequisite for PML/RAR oncogene activity in human hematopoietic progenitor cells.

Among the AML oncogenes, hyperactive tyrosine kinases play a major role in disease development and have gathered much attention since the kinase inhibitor imatinib has been proven to be highly beneficial for BCR/ABL positive CML patients (Druker et al., 1996; Sawyers et al., 2002; Brandts et al., 2007). Chung and colleagues retrovirally expressed a related constitutive active tyrosine kinase, FLT3-ITD, which is frequently found in AML patient samples associated with poor prognosis (Gilliland & Griffin, 2002). Stable expression of FLT3-ITD conferred a strong short-term proliferative signal in human CD34+ cells with enhanced erythropoiesis for two weeks after transduction *ex vivo*. The cells were blocked in terminal differentiation and colony formation was enhanced when compared with control cells. These effects could be partially substituted by expression of a constitutive active STAT5 mutant, a major downstream target of the hyperactive FLT3-ITD kinase. Moreover, FLT3-ITD triggered effects that could be blocked via inhibition with the FLT3-inhibitor tyrphostin (Chung et al., 2005). Despite these significant effects, FLT3-ITD transgeneexpressing CD34+ cells are neither able to expand further in *ex vivo* cultures nor able to induce leukemia in the NOD/SCID mouse transplantation model.

Stable ectopic expression of several leukemogenic fusion genes resulting from chromosomal translocations leads to enhanced *ex vivo* CD34+ cell expansion for up to several months (Abdul-Nabi et al., 2010). This has previously been shown for the t(8;21) associated fusion protein AML1/ETO, which is capable of expanding human CD34+ cells *ex vivo* for several months with a cumulative cell expansion of more than 1015-fold. Retroviral expression of AML1/ETO resulted in a sustained proliferation potential using a cytokine cocktail that included IL3, IL6, SCF, FLT3, GM-CSF, TPO and EPO, all at low concentrations. The cells display the typical differentiation block as observed in myeloid blast cells and grow out from a mixed culture of transduced and non-transduced cells. Even after several weeks in *ex vivo* culture, the cells are capable of colony formation in methylcellulose assays and, more importantly, of engraftment into immunodeficient NOD/SCID mice (Mulloy et al., 2003; Mulloy et al., 2002). Phenotypically, a subpopulation of the cells continues to express CD34, while the majority of the cells express the myeloid specific markers CD13 and CD33, in the absence of erythroid markers. Continuous expansion of the cells completely depends on the CD34+ subgroup. Erythroid differentiation was shown to be blocked by the AML1/ETO fusion protein via direct inhibition of GATA1 transcription factor activity (Choi et al., 2006). However, expression of AML1/ETO does not lead to leukemia development in transplanted immunodeficient NOD/SCID mice, suggesting that additional genetic hits must occur for

Grignani et al have reported that ectopic expression of PML/RAR in human CD34+ *ex vivo* cultures rapidly induced the cellular differentiation of progenitor cells towards the promyelocytic stage, followed by a block in further terminal differentiation into granulocytes, which resembled typical features of primary human acute promyelocytic leukemia (Grignani et al., 2000). In cytokine deprived liquid cultures, PML/RAR expressing cells survived and continued to expand, whereas control cells stopped proliferation and died by apoptosis. The terminal differentiation block induced by PML/RAR could be overcome by treatment with ATRA, a drug that has dramatically improved the overall survival of APL patients. They further demonstrated that a mutation of the N-CoR repressor binding interface within the PML portion of the fusion protein completely disrupted the observed biological effects, thereby suggesting that N-CoR triggered transcriptional repression is an essential prerequisite for PML/RAR oncogene

Among the AML oncogenes, hyperactive tyrosine kinases play a major role in disease development and have gathered much attention since the kinase inhibitor imatinib has been proven to be highly beneficial for BCR/ABL positive CML patients (Druker et al., 1996; Sawyers et al., 2002; Brandts et al., 2007). Chung and colleagues retrovirally expressed a related constitutive active tyrosine kinase, FLT3-ITD, which is frequently found in AML patient samples associated with poor prognosis (Gilliland & Griffin, 2002). Stable expression of FLT3-ITD conferred a strong short-term proliferative signal in human CD34+ cells with enhanced erythropoiesis for two weeks after transduction *ex vivo*. The cells were blocked in terminal differentiation and colony formation was enhanced when compared with control cells. These effects could be partially substituted by expression of a constitutive active STAT5 mutant, a major downstream target of the hyperactive FLT3-ITD kinase. Moreover, FLT3-ITD triggered effects that could be blocked via inhibition with the FLT3-inhibitor tyrphostin (Chung et al., 2005). Despite these significant effects, FLT3-ITD transgeneexpressing CD34+ cells are neither able to expand further in *ex vivo* cultures nor able to

Stable ectopic expression of several leukemogenic fusion genes resulting from chromosomal translocations leads to enhanced *ex vivo* CD34+ cell expansion for up to several months (Abdul-Nabi et al., 2010). This has previously been shown for the t(8;21) associated fusion protein AML1/ETO, which is capable of expanding human CD34+ cells *ex vivo* for several months with a cumulative cell expansion of more than 1015-fold. Retroviral expression of AML1/ETO resulted in a sustained proliferation potential using a cytokine cocktail that included IL3, IL6, SCF, FLT3, GM-CSF, TPO and EPO, all at low concentrations. The cells display the typical differentiation block as observed in myeloid blast cells and grow out from a mixed culture of transduced and non-transduced cells. Even after several weeks in *ex vivo* culture, the cells are capable of colony formation in methylcellulose assays and, more importantly, of engraftment into immunodeficient NOD/SCID mice (Mulloy et al., 2003; Mulloy et al., 2002). Phenotypically, a subpopulation of the cells continues to express CD34, while the majority of the cells express the myeloid specific markers CD13 and CD33, in the absence of erythroid markers. Continuous expansion of the cells completely depends on the CD34+ subgroup. Erythroid differentiation was shown to be blocked by the AML1/ETO fusion protein via direct inhibition of GATA1 transcription factor activity (Choi et al., 2006). However, expression of AML1/ETO does not lead to leukemia development in transplanted immunodeficient NOD/SCID mice, suggesting that additional genetic hits must occur for

activity in human hematopoietic progenitor cells.

induce leukemia in the NOD/SCID mouse transplantation model.

full-blown AML development. Similar results were obtained from analysis of the inv(16) fusion protein CBFB/MYH11. Expression of this fusion gene also caused enhanced proliferation and expansion of CD34+ cells over several months with retention of the engraftment potential in NOD/SCID mice. However, as also observed with AML1/ETO, transplanted mice do not develop leukemia (Wunderlich et al., 2006). Therefore, both oncogenes confer a preleukemic status and subsequent alterations are required for the transition to overt leukemia.

To decipher the genetic signature of AML, Abdul-Nabi and colleagues have assessed various AML-associated fusion proteins for their ability to enhance CD34+ cell expansion *ex vivo* and subsequently analyzed their gene expression profiles (Abdul-Nabi et al., 2010). In this study, experiments were also performed with gamma-retroviral vectors expressing the fusion genes AML1/ETO, PML/RAR, MLL/AF9 and NUP98/HOXA9 together with eGFP. To identify key target genes responsible for the expansion of CD34+ cells the authors performed gene array analysis of FACS-sorted eGFP+ cells expressing the indicated oncogenes, which identifies the induced and repressed target genes during the *ex vivo* selection process. One interesting candidate gene that was shown to be specifically upregulated due to both AML1/ETO and PML/RAR expression was the p53-inhibiting molecule MDM2. The authors could further show that activation of the gatekeeper protein p53, by blocking the MDM2-p53 protein-protein interaction with the small molecule compound Nutlin-3, was sufficient to completely block AML1/ETO induced selection and expansion of CD34+ progenitor cells. Therefore, Nutlin-3 is proposed to function as an inhibitor for AML1/ETO positive leukemias by reactivating p53 and consecutively eliminating the leukemic cells via the induction of apoptosis. Two independent groups have shown that retroviral expression of the NUP98/HOXA9 fusion gene, which is found in myelodysplastic syndrome (MDS) and AML patients, resulted in a similar outgrowth of transgene expressing cells with maintenance of self-renewal potential *ex vivo* and increased engraftment capacity in NOD/SCID mice (Chung et al., 2006; Takeda et al., 2006). This outgrowth was accompanied by high levels of HOX gene expression, a typical feature for this leukemia entity.

Regardless of their remarkable *ex vivo* cell expansion properties, the AML associated gene products described above, PML/RAR, FLT3-ITD, NUP98/HOXA9 and AML1/ETO, were not able to independently induce leukemia in transplanted NOD/SCID mice. Therefore, it is likely that additional genetic alterations are required for full cellular transformation in AML. In this regard it was shown that oncogenic RAS signaling, triggered by N-RasG12D overexpression, does increase AML1/ETO engraftment capacity in NOD/SCID mice but is still not sufficient to overcome the leukemia onset defect in the NOD/SCID mouse model (Chou et al., 2011).

Interestingly, MLL rearrangements are typically found in aggressive infant or therapy related leukemias (Aplan, 2006). In contrast to AML derived gene products, ectopic expression of either MLL/AF9 or MLL/ENL mixed-lineage leukemia fusion genes, as a single genetic event, results in the development of myeloid or lymphoid leukemias in the NOD/SCID mouse model approximately 100 days after transplantation (Barabe et al., 2007; Wei et al., 2008). Morphological analysis has revealed that AML and B-ALL leukemias are associated with both MLL rearrangements. Remarkably, *ex vivo* expanded cells gave rise to leukemia in NOD/SCID mice, even after 70 days of *in vitro* culture prior to transplantation in sublethally irradiated mice. The reasons for the striking differences between AML and

Analysis of Leukemogenic Gene Products in Hematopoietic Progenitor Cells 355

In total, these approaches will lead to a better understanding of the genetic alterations that are required for the onset of cellular transformation. Furthermore, these studies have the potential to propel the development of effective drugs designed to eradicate leukemia cells.

The authors are supported by research grants from the NGFN Cancer Network (grant 01GS0450, TP-10), the Deutsche Krebshilfe (grant 102362, TP-7), the LEOWE OSF (TP-C3) and the LOEWE CGT (startup grant CW). The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und

Abdul-Nabi, A.M.; Yassin, E.R.; Varghese, N.; Deshmukh, H. & Yaseen, N.R. (2010). In vitro

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Bursen, A.; Schwabe, K.; Ruster, B.; Henschler, R.; Ruthardt, M.; Dingermann, T. &

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**6. Acknowledgement** 

Kunst.

**7. References** 

604

141-146

No. 8, pp. e12464

MLL fusion genes in their ability to induce leukemia onset in NOD/SCID mice are completely unknown. A direct comparison of deregulated genes may reveal which additional pathways are activated in MLL leukemic human CD34+ cells.

Recently, a report was published describing a true oncogenic cooperativeness in human CD34+ cell expansion and leukemia induction in NOD/SCID mice (Rizo et al., 2010). The authors convincingly have shown that retroviral co-expression of the CML associated oncogene, BCR/ABL, and the polycomb ring finger oncogene, BMI1, leads to leukemia induction within 4-5 months after transplantation. The isolated human leukemia cells were re-transplantable into secondary recipient mice, which induced leukemia with a shortened latency. *In vitro,* the co-expression of both oncogenes in human CD34+ cells allowed for the establishment of myeloid and lymphoid long-term cultures with self-renewing properties. This report also describes a series of valuable cell culture methods for *in vitro* analysis of CD34+ cell proliferation and self-renewal. Finally, the authors have demonstrated that retroviral introduction of BMI1 into primary leukemia cells from CML chronic-phase patients elevated the proliferative capacity and self-renewal properties of the leukemia cells, while shRNA knockdown of BMI1 in blast crisis CML cells completely blocked their proliferation potential. These results highlight BMI1 as an attractive molecular target for blast crisis CML cells, which is especially important given the rates of BCR/ABL positive leukemic stem cell resistance to kinase inhibitors such as imatinib (Corbin et al., 2011). However, the exact molecular mechanisms of BCR/ABL and BMI1 oncogenic co-operation have to be addressed in further studies.

In conclusion, human CD34+ blood progenitor cells represent a powerful cell biology tool used for the analysis of leukemogenic gene product activity. Methods of isolation and purification of CD34+ cells have significantly improved and are broadly available for researchers. The cells can be propagated *ex vivo* and oncogene activity can be analyzed for a wide range of biological parameters, including differentiation, proliferation, self-renewal, senescence and apoptosis, using standard cell biology methods. CD34+ suspension cells can be cultured *ex vivo* as liquid cultures or on stromal layer cells. With the possibility to transplant these cells into sublethally irradiated NOD/SCID mice this is an ideal model system to study the function of human leukemia-associated oncogenes.

#### **5. Conclusions**

Powerful retroviral expression vectors and standardized enrichment technologies, which are used for the isolation of hematopoietic precursor cells from the murine and human bone marrow compartment, efficiently enabled the accurate modeling of leukemia initiation and progression in the appropriate cell compartment. Currently available expression vectors allow for the simultaneous expression of several genes, including oncogenes, marker genes for detection and selection genes used to isolate modified cells, which represent practical tools for modeling leukemia progression in hematopoietic cells. Among the introduced strategies, the humanized mouse model based on human CD34+ precursor cell transplantation into NOD/SCID mice represents a credible model system to study human leukemia. As described here, the expression of single oncogenes such as BCR/ABL and AML1/ETO, which are primarily found in older patients, does not lead to leukemia development, implying that disease develops as a result of secondary mutations. In contrast, the expression of several MLL fusion proteins, which are typically found in younger patients, was found to be sufficient to trigger leukemia development with high penetrance. In total, these approaches will lead to a better understanding of the genetic alterations that are required for the onset of cellular transformation. Furthermore, these studies have the potential to propel the development of effective drugs designed to eradicate leukemia cells.

#### **6. Acknowledgement**

354 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

MLL fusion genes in their ability to induce leukemia onset in NOD/SCID mice are completely unknown. A direct comparison of deregulated genes may reveal which

Recently, a report was published describing a true oncogenic cooperativeness in human CD34+ cell expansion and leukemia induction in NOD/SCID mice (Rizo et al., 2010). The authors convincingly have shown that retroviral co-expression of the CML associated oncogene, BCR/ABL, and the polycomb ring finger oncogene, BMI1, leads to leukemia induction within 4-5 months after transplantation. The isolated human leukemia cells were re-transplantable into secondary recipient mice, which induced leukemia with a shortened latency. *In vitro,* the co-expression of both oncogenes in human CD34+ cells allowed for the establishment of myeloid and lymphoid long-term cultures with self-renewing properties. This report also describes a series of valuable cell culture methods for *in vitro* analysis of CD34+ cell proliferation and self-renewal. Finally, the authors have demonstrated that retroviral introduction of BMI1 into primary leukemia cells from CML chronic-phase patients elevated the proliferative capacity and self-renewal properties of the leukemia cells, while shRNA knockdown of BMI1 in blast crisis CML cells completely blocked their proliferation potential. These results highlight BMI1 as an attractive molecular target for blast crisis CML cells, which is especially important given the rates of BCR/ABL positive leukemic stem cell resistance to kinase inhibitors such as imatinib (Corbin et al., 2011). However, the exact molecular mechanisms of BCR/ABL and BMI1 oncogenic co-operation

In conclusion, human CD34+ blood progenitor cells represent a powerful cell biology tool used for the analysis of leukemogenic gene product activity. Methods of isolation and purification of CD34+ cells have significantly improved and are broadly available for researchers. The cells can be propagated *ex vivo* and oncogene activity can be analyzed for a wide range of biological parameters, including differentiation, proliferation, self-renewal, senescence and apoptosis, using standard cell biology methods. CD34+ suspension cells can be cultured *ex vivo* as liquid cultures or on stromal layer cells. With the possibility to transplant these cells into sublethally irradiated NOD/SCID mice this is an ideal model

Powerful retroviral expression vectors and standardized enrichment technologies, which are used for the isolation of hematopoietic precursor cells from the murine and human bone marrow compartment, efficiently enabled the accurate modeling of leukemia initiation and progression in the appropriate cell compartment. Currently available expression vectors allow for the simultaneous expression of several genes, including oncogenes, marker genes for detection and selection genes used to isolate modified cells, which represent practical tools for modeling leukemia progression in hematopoietic cells. Among the introduced strategies, the humanized mouse model based on human CD34+ precursor cell transplantation into NOD/SCID mice represents a credible model system to study human leukemia. As described here, the expression of single oncogenes such as BCR/ABL and AML1/ETO, which are primarily found in older patients, does not lead to leukemia development, implying that disease develops as a result of secondary mutations. In contrast, the expression of several MLL fusion proteins, which are typically found in younger patients, was found to be sufficient to trigger leukemia development with high penetrance.

system to study the function of human leukemia-associated oncogenes.

additional pathways are activated in MLL leukemic human CD34+ cells.

have to be addressed in further studies.

**5. Conclusions** 

The authors are supported by research grants from the NGFN Cancer Network (grant 01GS0450, TP-10), the Deutsche Krebshilfe (grant 102362, TP-7), the LEOWE OSF (TP-C3) and the LOEWE CGT (startup grant CW). The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessisches Ministerium für Wissenschaft und Kunst.

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**18** 

 *Norway* 

**Acute Promyelocytic Leukemia: A Model** 

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by a severe bleeding tendency, accumulation of abnormal promyelocytes in the bone marrow and a reciprocal t(15;17) chromosomal translocation that fuses the gene encoding the promyelocytic leukemia protein (PML) to that encoding retinoic acid receptor alpha (RARA) (de Thé & Chen, 2010). During the past 30 years two therapeutic drugs have been introduced into the clinic that have dramatically improved the treatment outcome of this disease (Wang & Chen, 2008). The first of these components was all-trans retinoic acid (ATRA), a vitamin A derivative that significantly increased clinical remission and improved the 5-years disease-free survival rates from below 40% to more than 80% (Huang *et al.*, 1988). The second drug was arsenic trioxide (ATO), a component that was discovered to be remarkably effective in treating APL as a single agent (Sun *et al.*, 1992). Today, most hospitals employ ATRA in combination with chemotherapy as frontline therapy, while ATO is being used for refractory or relapsed patients. Recent clinical studies have also revealed a positive synergistic effect between the two drugs, suggesting that future therapy of newly diagnosed patients may involve a combination of the two reagents (Estey *et al.*, 2006; Hu *et* 

The success of using ATRA and ATO in APL therapy appears to be linked to the ability of these drugs to interact with the fusion oncoprotein PML/RARA, which is produced by the APL-associated t(15;17) translocation, and that causes the disease. ATRA contacts a ligand binding domain present within the RARA moiety of this chimeric protein and promotes differentiation of APL cells along the granulocyte linage (Huang *et al.*, 1988). ATO, on the other hand, has recently been shown to bind one or more cysteine rich motifs within the PML protein (Jeanne *et al.*, 2010; Zhang *et al.*, 2010) and contributes to the cure of APL through a mechanism that involves eradication of leukemic-initiating cells (LICs) (Nasr *et* 

Due to the success of using ATRA and ATO in the clinic, and because of the ability of these drugs to promote clinical remission through a direct contact with PML/RARA, APL has become one of the most attractive model diseases for the development of targeted cancer therapy. The APL cure offers a proof of principle that a cancer can be cured through targeted inactivation of an oncoprotein, and it provides a rationale for the development of novel therapeutic strategies that target fusion oncoproteins produced by chromosomal translocations. In this chapter we will summarize the current knowledge of the biological

**1. Introduction** 

*al.*, 2009; Shen *et al.*, 2004; Wang *et al.*, 2004).

*al*., 2008; Ito *et al.*, 2008; Zheng *et al.*, 2007).

**Disease for Targeted Cancer Therapy** 

Emma Lång and Stig Ove Bøe *Oslo University Hospital, Oslo,* 


### **Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy**

Emma Lång and Stig Ove Bøe *Oslo University Hospital, Oslo, Norway* 

#### **1. Introduction**

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Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by a severe bleeding tendency, accumulation of abnormal promyelocytes in the bone marrow and a reciprocal t(15;17) chromosomal translocation that fuses the gene encoding the promyelocytic leukemia protein (PML) to that encoding retinoic acid receptor alpha (RARA) (de Thé & Chen, 2010). During the past 30 years two therapeutic drugs have been introduced into the clinic that have dramatically improved the treatment outcome of this disease (Wang & Chen, 2008). The first of these components was all-trans retinoic acid (ATRA), a vitamin A derivative that significantly increased clinical remission and improved the 5-years disease-free survival rates from below 40% to more than 80% (Huang *et al.*, 1988). The second drug was arsenic trioxide (ATO), a component that was discovered to be remarkably effective in treating APL as a single agent (Sun *et al.*, 1992). Today, most hospitals employ ATRA in combination with chemotherapy as frontline therapy, while ATO is being used for refractory or relapsed patients. Recent clinical studies have also revealed a positive synergistic effect between the two drugs, suggesting that future therapy of newly diagnosed patients may involve a combination of the two reagents (Estey *et al.*, 2006; Hu *et al.*, 2009; Shen *et al.*, 2004; Wang *et al.*, 2004).

The success of using ATRA and ATO in APL therapy appears to be linked to the ability of these drugs to interact with the fusion oncoprotein PML/RARA, which is produced by the APL-associated t(15;17) translocation, and that causes the disease. ATRA contacts a ligand binding domain present within the RARA moiety of this chimeric protein and promotes differentiation of APL cells along the granulocyte linage (Huang *et al.*, 1988). ATO, on the other hand, has recently been shown to bind one or more cysteine rich motifs within the PML protein (Jeanne *et al.*, 2010; Zhang *et al.*, 2010) and contributes to the cure of APL through a mechanism that involves eradication of leukemic-initiating cells (LICs) (Nasr *et al*., 2008; Ito *et al.*, 2008; Zheng *et al.*, 2007).

Due to the success of using ATRA and ATO in the clinic, and because of the ability of these drugs to promote clinical remission through a direct contact with PML/RARA, APL has become one of the most attractive model diseases for the development of targeted cancer therapy. The APL cure offers a proof of principle that a cancer can be cured through targeted inactivation of an oncoprotein, and it provides a rationale for the development of novel therapeutic strategies that target fusion oncoproteins produced by chromosomal translocations. In this chapter we will summarize the current knowledge of the biological

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 365

and further showed that ATO treatment induced partial differentiation of leukemic cells,

Currently, ATRA in combination with chemotherapy is being employed as frontline therapy for APL, whereas ATO primarily is being used for treatment of cases that are resistant to ATRA or patients suffering from frequent relapses. However, several clinical trials are now assessing the synergistic effect of combining ATRA and ATO with and without chemotherapy. These trials are conducted mainly on the basis of successful studies in animal models, showing a positive effect of ATRA/ATO combinations in APL mice (Jing *et al.*, 2001; Lallemand-Breitenbach *et al.*, 1999). The main conclusion so far from the ongoing clinical studies is that newly diagnosed patients are likely to benefit from ATRA/ATO combination treatment in addition to low-dose chemotherapy (Estey *et al.*, 2006; Hu *et al.*, 2009; Shen *et al.*,

The molecular hallmark of APL is the t(15;17) chromosomal translocation that expresses the fusion oncoprotein PML/RARA. While this genetic aberration is identified in more than 97% of all APL cases, the remaining patients diagnosed with this disease harbor variant translocations that all involve the *RARA* gene in fusion with alternative partners such as the genes encoding promyelocytic leukemia zinc finger (*PLZF*) (Chen *et al.*, 1993), nucleophosmin (*NPM*) (Redner *et al.*, 1996), nuclear matrix associated (*NUMA*) (Wells *et al.*, 1997), or signal transducer and activator of transcription 5b (*STAT5B*) (Arnould *et al.*, 1999). The most compelling evidence that PML/RARA alone can contribute directly to APL development comes from studies in mice showing that expression of this oncoprotein as a transgene leads to development of an APL–like disease. However, these experiments also show that a relatively long latency period is required prior to onset of disease, suggesting the involvement of acquired genetic aberrations in addition to the t(15;17) translocation

The first component of the PML/RARA fusion, the PML protein, is a tumor suppressor (Bernardi *et al.*, 2006; Salomoni & Pandolfi, 2002; Trotman *et al.*, 2006) that functions in multiple cellular processes, including apoptosis (Wang *et al.*, 1998), differentiation (Ito *et al.*, 2008), DNA repair (Bøe *et al.*, 2006; Dellaire *et al.*, 2006a), senescence (Ferbeyre *et al.*, 2000; Pearson *et al.*, 2000), angiogenesis (Bernardi *et al.*, 2006) and virus defence (Everett & Maul, 1994). The human *PML* gene is located on chromosome 15, consists of nine exons and produces several alternatively spliced protein isoforms designated PML I through VII. All of these PML variants contain an identical tripartite (TRIM) motif in their N-terminal region, and a C-terminus that varies due to alternative splicing (Borden, 2002; Fagioli *et al.*, 1992; Jensen *et al.*, 2001; Jul-Larsen *et al.*, 2010; Reymond *et al.*, 2001). The TRIM motif, which comprises a RING finger, two B-boxes and a predicted coiled coil domain, has been shown to be important for PML multimerization, a feature responsible for one of the most striking properties of this protein, namely the ability to generate nuclear structures termed PML

caspase activation and subsequently apoptosis (Soignet *et al.*, 1998).

**3. The mechanism of PML, RARA and PML/RARA** 

**3.1 The role of PML/RARA in APL pathogenesis** 

(Brown *et al.*, 1997; Grisolano *et al.*, 1997).

**3.2 The function of PML** 

**2.3 Present and future APL therapy** 

2004; Wang *et al.*, 2004).

properties of PML, RARA and PML/RARA with particular emphasis on tumorigenesis in APL patients and the molecular mechanisms that underlie the response to ATRA and ATO.

#### **2. APL treatment – a historical perspective**

#### **2.1 The discovery of ATRA-based APL therapy**

APL was first characterized as a distinct clinical entity in 1957 (Hillestad, 1957). Throughout the 1950s and 1960s, this disease had a 100% mortality rate and no effective treatment options. In 1973, chemotherapy by the topoisomerase inhibitor daunorubicin was shown to have some curative effect, yielding a complete remission (CR) rate of 55% (Bernard *et al.*, 1973), and in the early eighties induction therapy based on anthracyclins (daunorubicin, idarubicin among others) and the nucleocide analogue cytosine arabinoside (Ara-C) was found to yield CR rates of up to 80% in newly diagnosed patients (Cunningham *et al.*, 1989; Sanz *et al.*, 1988). However, the patients frequently suffered from one of the inherent drawbacks with induction therapy, namely the release of coagulation factors from dead leukemic cells, causing severe bleedings and increased risk of fatal outcome (Cordonnier *et al.*, 1985; Drapkin *et al.*, 1978; Ruggero *et al.*, 1977). Consequently, most APL patients required intensive platelet and fibrinogen support, and based on the criterion of 5-years disease-free survival (DFS), only 35-45% of the cases were cured (Fenaux *et al.*, 2007). The focus on APL therapy changed in 1978, as it became clear that leukemic cells undergo terminal differentiation upon treatment with differentiating-inducing agents, such as ATRA, Ara-C and 13-cis retinoic acid (Breitman *et al.*, 1981; Degos *et al.*, 1985; Gold *et al.*, 1983; Koeffler *et al.*, 1985; Sachs, 1978). Such differentiation therapy showed an advantage over induction therapy, with respect to incidences of severe bleedings, and led to reduced mortality rates. In 1985, the first attempt to treat APL patients with ATRA was made with promising results, but the percentage of patients with 5-years DFS was still relatively low (less than 50%) (Huang *et al.*, 1987; Huang *et al.*, 1988). Subsequently, optimization trials, combining ATRA with chemotherapy, raised the CR rates up to 90-95% and the 5-years DFS to 86% (Wang & Chen, 2008). In addition, the combination of ATRA and chemotherapy, which currently represents standard frontline APL therapy, helped reducing retinoic acid syndrome (RAS), a potentially fatal side effect caused by induction therapy and manifested in a burst of inflammatory cytokines released from malignant promyelocytes (de Botton *et al.*, 2003; Fenaux *et al.*, 1999; Sanz *et al.*, 1999; Tallman *et al.*, 1997).

#### **2.2 The discovery of ATO-based APL therapy**

Arsenic, in the form of arsenic trioxide (ATO), was first described as an agent that possesses antileukemic properties in the year 1878. In this study, Fowler's solution, a solution of ATO in potassium bicarbonate, was shown to dramatically reduce the number of white blood cells in a patient with chronic myelogenous leukemia (CML) (Cutler & Bradford, 1878). Subsequently, this remedy was used as a primary antileukemic agent until the discovery of radiation therapy in the early 20th century (Forkner & Scott, 1931; Kwong & Todd, 1997). In the 1970s, ATO reappeared as a therapeutic agent for APL as Chinese researchers showed that ailing-1, a mixture of ATO and crude herbal extracts, was effective in the treatment of both *de novo* as well as relapsed cases (Shen *et al.*, 1997; Sun *et al.*, 1992; Zhang *et al.*, 1996). Additional clinical studies showed that ATO, as a single agent, caused complete remission in up to 90% of patients and reduced the relapse rate for high risk patients (Niu *et al.*, 1999; Shen *et al.*, 1997). A research group in the United States confirmed these preliminary studies and further showed that ATO treatment induced partial differentiation of leukemic cells, caspase activation and subsequently apoptosis (Soignet *et al.*, 1998).

#### **2.3 Present and future APL therapy**

364 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

properties of PML, RARA and PML/RARA with particular emphasis on tumorigenesis in APL patients and the molecular mechanisms that underlie the response to ATRA and ATO.

APL was first characterized as a distinct clinical entity in 1957 (Hillestad, 1957). Throughout the 1950s and 1960s, this disease had a 100% mortality rate and no effective treatment options. In 1973, chemotherapy by the topoisomerase inhibitor daunorubicin was shown to have some curative effect, yielding a complete remission (CR) rate of 55% (Bernard *et al.*, 1973), and in the early eighties induction therapy based on anthracyclins (daunorubicin, idarubicin among others) and the nucleocide analogue cytosine arabinoside (Ara-C) was found to yield CR rates of up to 80% in newly diagnosed patients (Cunningham *et al.*, 1989; Sanz *et al.*, 1988). However, the patients frequently suffered from one of the inherent drawbacks with induction therapy, namely the release of coagulation factors from dead leukemic cells, causing severe bleedings and increased risk of fatal outcome (Cordonnier *et al.*, 1985; Drapkin *et al.*, 1978; Ruggero *et al.*, 1977). Consequently, most APL patients required intensive platelet and fibrinogen support, and based on the criterion of 5-years disease-free survival (DFS), only 35-45% of the cases were cured (Fenaux *et al.*, 2007). The focus on APL therapy changed in 1978, as it became clear that leukemic cells undergo terminal differentiation upon treatment with differentiating-inducing agents, such as ATRA, Ara-C and 13-cis retinoic acid (Breitman *et al.*, 1981; Degos *et al.*, 1985; Gold *et al.*, 1983; Koeffler *et al.*, 1985; Sachs, 1978). Such differentiation therapy showed an advantage over induction therapy, with respect to incidences of severe bleedings, and led to reduced mortality rates. In 1985, the first attempt to treat APL patients with ATRA was made with promising results, but the percentage of patients with 5-years DFS was still relatively low (less than 50%) (Huang *et al.*, 1987; Huang *et al.*, 1988). Subsequently, optimization trials, combining ATRA with chemotherapy, raised the CR rates up to 90-95% and the 5-years DFS to 86% (Wang & Chen, 2008). In addition, the combination of ATRA and chemotherapy, which currently represents standard frontline APL therapy, helped reducing retinoic acid syndrome (RAS), a potentially fatal side effect caused by induction therapy and manifested in a burst of inflammatory cytokines released from malignant promyelocytes (de Botton *et* 

**2. APL treatment – a historical perspective 2.1 The discovery of ATRA-based APL therapy** 

*al.*, 2003; Fenaux *et al.*, 1999; Sanz *et al.*, 1999; Tallman *et al.*, 1997).

Arsenic, in the form of arsenic trioxide (ATO), was first described as an agent that possesses antileukemic properties in the year 1878. In this study, Fowler's solution, a solution of ATO in potassium bicarbonate, was shown to dramatically reduce the number of white blood cells in a patient with chronic myelogenous leukemia (CML) (Cutler & Bradford, 1878). Subsequently, this remedy was used as a primary antileukemic agent until the discovery of radiation therapy in the early 20th century (Forkner & Scott, 1931; Kwong & Todd, 1997). In the 1970s, ATO reappeared as a therapeutic agent for APL as Chinese researchers showed that ailing-1, a mixture of ATO and crude herbal extracts, was effective in the treatment of both *de novo* as well as relapsed cases (Shen *et al.*, 1997; Sun *et al.*, 1992; Zhang *et al.*, 1996). Additional clinical studies showed that ATO, as a single agent, caused complete remission in up to 90% of patients and reduced the relapse rate for high risk patients (Niu *et al.*, 1999; Shen *et al.*, 1997). A research group in the United States confirmed these preliminary studies

**2.2 The discovery of ATO-based APL therapy** 

Currently, ATRA in combination with chemotherapy is being employed as frontline therapy for APL, whereas ATO primarily is being used for treatment of cases that are resistant to ATRA or patients suffering from frequent relapses. However, several clinical trials are now assessing the synergistic effect of combining ATRA and ATO with and without chemotherapy. These trials are conducted mainly on the basis of successful studies in animal models, showing a positive effect of ATRA/ATO combinations in APL mice (Jing *et al.*, 2001; Lallemand-Breitenbach *et al.*, 1999). The main conclusion so far from the ongoing clinical studies is that newly diagnosed patients are likely to benefit from ATRA/ATO combination treatment in addition to low-dose chemotherapy (Estey *et al.*, 2006; Hu *et al.*, 2009; Shen *et al.*, 2004; Wang *et al.*, 2004).

#### **3. The mechanism of PML, RARA and PML/RARA**

#### **3.1 The role of PML/RARA in APL pathogenesis**

The molecular hallmark of APL is the t(15;17) chromosomal translocation that expresses the fusion oncoprotein PML/RARA. While this genetic aberration is identified in more than 97% of all APL cases, the remaining patients diagnosed with this disease harbor variant translocations that all involve the *RARA* gene in fusion with alternative partners such as the genes encoding promyelocytic leukemia zinc finger (*PLZF*) (Chen *et al.*, 1993), nucleophosmin (*NPM*) (Redner *et al.*, 1996), nuclear matrix associated (*NUMA*) (Wells *et al.*, 1997), or signal transducer and activator of transcription 5b (*STAT5B*) (Arnould *et al.*, 1999). The most compelling evidence that PML/RARA alone can contribute directly to APL development comes from studies in mice showing that expression of this oncoprotein as a transgene leads to development of an APL–like disease. However, these experiments also show that a relatively long latency period is required prior to onset of disease, suggesting the involvement of acquired genetic aberrations in addition to the t(15;17) translocation (Brown *et al.*, 1997; Grisolano *et al.*, 1997).

#### **3.2 The function of PML**

The first component of the PML/RARA fusion, the PML protein, is a tumor suppressor (Bernardi *et al.*, 2006; Salomoni & Pandolfi, 2002; Trotman *et al.*, 2006) that functions in multiple cellular processes, including apoptosis (Wang *et al.*, 1998), differentiation (Ito *et al.*, 2008), DNA repair (Bøe *et al.*, 2006; Dellaire *et al.*, 2006a), senescence (Ferbeyre *et al.*, 2000; Pearson *et al.*, 2000), angiogenesis (Bernardi *et al.*, 2006) and virus defence (Everett & Maul, 1994). The human *PML* gene is located on chromosome 15, consists of nine exons and produces several alternatively spliced protein isoforms designated PML I through VII. All of these PML variants contain an identical tripartite (TRIM) motif in their N-terminal region, and a C-terminus that varies due to alternative splicing (Borden, 2002; Fagioli *et al.*, 1992; Jensen *et al.*, 2001; Jul-Larsen *et al.*, 2010; Reymond *et al.*, 2001). The TRIM motif, which comprises a RING finger, two B-boxes and a predicted coiled coil domain, has been shown to be important for PML multimerization, a feature responsible for one of the most striking properties of this protein, namely the ability to generate nuclear structures termed PML

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 367

involved in hematopoietic maturation. Consistent with a role in gene repression, PML/RARA has also been shown to recruit the histone methyl transferase SUV39H1 (Carbone *et al.*, 2006), members of the polycomb repressive complex 2 (PRC2) (Villa *et al.*, 2007) and DNA methyltransferases (DNMTs) (Di Croce *et al.*, 2002), proteins that are known to induce a repressive chromatin structure. In addition to increased repressor activity, the PML/RARA fusion also appears to possess a considerable expanded repertoire of target genes compared to the normal RARA protein. This notion is supported by *in vitro* binding studies showing that PML/RARA has a broader and more relaxed DNA binding specificity compared to RARA (Hauksdottir & Privalsky, 2001; Kamashev *et al.*, 2004), and by a genome wide screen revealing a wide range of PML/RARA target genes (Hoemme *et al.*, 2008). The altered DNA binding and transcription repression properties of PML/RARA are partially due to the ability of this chimeric protein to form homodimeres through protein-protein interactions mediated by the TRIM motif of PML (Jansen *et al.*, 1995; Perez *et al.*, 1993). In addition, this chimeric protein has also been shown to form functional complexes with other transcription factors such as RXR and Daxx, a feature that may further contribute to the expanded promoter

PML/RARA is also thought to contribute to malignant transformation and development of APL through inhibition of PML tumor suppressor functions. A dominant negative effect of PML/RARA on this protein is evident by studies demonstrating disruption of nuclear PML bodies into a dispersed microspeckled pattern in cells expressing this oncoprotein (Dyck *et al.*, 1994; Koken *et al.*, 1994; Weis *et al.*, 1994). Interestingly, while disruption of PML NBs by PML/RARA in the nucleus is evident, this oncoprotein readily assembles into MAPPS and CyPNs, the mitotic and cytoplasmic versions of PML NBs, respectively (Jul-Larsen *et al.*, 2009). The disruption of PML NBs in the nucleus may reflect the role of this oncoprotein in

Fig. 1. Structural organization of PML, RARA and PML/RARA. PML contains a RING domain (R), two B boxes (B), a coiled coil (CC) and a variable C-terminus. RARA consists of six regulatory domains (A-F), of which domain C and E harbor the DNA binding domain (DBD) and the ligand binding domain (LBD), respectively. The t (15;17) translocation produces PML/RARA, which retains the N-terminal PML motifs as well as RARA DNA

and ligand binding activity. Arrows indicate protein breakpoints.

binding capacity (Zeisig *et al.*, 2007; Zhu *et al.*, 2005; Zhu *et al.*, 2007).

repression of gene activity.

nuclear bodies (PML NBs) (Lallemand-Breitenbach & de The, 2010). These bodies are highly dynamic and change their morphology and biochemical composition in a cell cycledependent manner. For example, during entry into mitosis, several PML NB resident components, including Daxx, Sp100 and SUMO, are lost concomitant with formation of PML NB aggregates called mitotic assemblies of PML proteins (MAPPs), whereas transition from mitosis to G1-phase coincides with exclusion of PML NBs from the progeny nuclei and complex formation with nucleoporins and microtubule filaments to form cytoplasmic assemblies of PML and nucleoporins (CyPNs) (Chen *et al.*, 2008; Dellaire *et al.*, 2006b; Jul-Larsen *et al.*, 2009). Although, PML NBs have the capacity to recruit a large number of different protein components, PML is the only protein so far that has been shown to be required for their formation. For this reason, it is widely assumed that the ability to assemble these cellular compartments represents an integral part of PML biogenesis. It still remains, however, to clearly define the molecular mechanism involved in PML NB assembly and function.

#### **3.3 The function of RARA**

The second fusion partner, RARA, is a ligand binding transcription factor that contains a DNA binding motif in its central region and a retinoid binding domain at the C-terminus. To generate an active protein complex, this nuclear receptor forms a heterodimer with the RXR family of transcription factors. Upon direct binding to a RA responsive element (RARE) within the regulatory region of a target gene, RARA/RXR complexes promote transcriptional silencing by recruiting co-repressor proteins such as NCOR1, SMRT and histone deacetylase to the promoter-binding complexes. In the presence of physiological concentrations of ligand (i.e. retinoids), a conformational change occurs within the RARA/RXR heterodimer that leads to dissociation of co-repressors and concomitant recruitment of histone acetylases and components of the basic transcription machinery, thus transforming the protein complex from a gene silencer to a gene activator (Bastien & Rochette-Egly, 2004). RARA regulates several genes involved in myeloid progenitor cell differentiation, including c-myc (Bentley & Groudine, 1986; Gowda *et al.*, 1986), C/EBPβ (Duprez *et al.*, 2003), C/EBP (Park *et al.*, 1999) and PU.1 (Mueller *et al.*, 2006), suggesting an important role of this protein in blood cell maturation.

#### **3.4 The function of PML/RARA**

Upon fusion between PML and RARA, the variable C-terminus of the PML protein is lost, whereas the constant N-terminal TRIM motif generally remains intact. In the case of RARA, fusion to PML leads to loss of the first 50 to 60 N-terminal amino acids, a deletion that does not appear to affect the DNA and ligand binding activities of this protein (de Thé *et al.*, 1991). Thus, PML/RARA retains the powerful protein-protein interaction domain of the PML protein, whereas the variable isoform-specific region is replaced by the trans-activating functions of RARA (Fig. 1.).

One of the gained PML/RARA functions that is thought to contribute largely to APL development is the ability of this chimeric protein to form stable transcription repression complexes that are irresponsive to physiological concentrations of retinoids. As a consequence, gene promoters that are targeted by PML/RARA become constitutively repressed, an observation that has led to the general assumption that this oncoprotein causes a block in blood cell differentiation through transcriptional inhibition of key genes

nuclear bodies (PML NBs) (Lallemand-Breitenbach & de The, 2010). These bodies are highly dynamic and change their morphology and biochemical composition in a cell cycledependent manner. For example, during entry into mitosis, several PML NB resident components, including Daxx, Sp100 and SUMO, are lost concomitant with formation of PML NB aggregates called mitotic assemblies of PML proteins (MAPPs), whereas transition from mitosis to G1-phase coincides with exclusion of PML NBs from the progeny nuclei and complex formation with nucleoporins and microtubule filaments to form cytoplasmic assemblies of PML and nucleoporins (CyPNs) (Chen *et al.*, 2008; Dellaire *et al.*, 2006b; Jul-Larsen *et al.*, 2009). Although, PML NBs have the capacity to recruit a large number of different protein components, PML is the only protein so far that has been shown to be required for their formation. For this reason, it is widely assumed that the ability to assemble these cellular compartments represents an integral part of PML biogenesis. It still remains, however, to clearly define the molecular mechanism involved in PML NB assembly

The second fusion partner, RARA, is a ligand binding transcription factor that contains a DNA binding motif in its central region and a retinoid binding domain at the C-terminus. To generate an active protein complex, this nuclear receptor forms a heterodimer with the RXR family of transcription factors. Upon direct binding to a RA responsive element (RARE) within the regulatory region of a target gene, RARA/RXR complexes promote transcriptional silencing by recruiting co-repressor proteins such as NCOR1, SMRT and histone deacetylase to the promoter-binding complexes. In the presence of physiological concentrations of ligand (i.e. retinoids), a conformational change occurs within the RARA/RXR heterodimer that leads to dissociation of co-repressors and concomitant recruitment of histone acetylases and components of the basic transcription machinery, thus transforming the protein complex from a gene silencer to a gene activator (Bastien & Rochette-Egly, 2004). RARA regulates several genes involved in myeloid progenitor cell differentiation, including c-myc (Bentley & Groudine, 1986; Gowda *et al.*, 1986), C/EBPβ (Duprez *et al.*, 2003), C/EBP (Park *et al.*, 1999) and PU.1 (Mueller *et al.*, 2006), suggesting an

Upon fusion between PML and RARA, the variable C-terminus of the PML protein is lost, whereas the constant N-terminal TRIM motif generally remains intact. In the case of RARA, fusion to PML leads to loss of the first 50 to 60 N-terminal amino acids, a deletion that does not appear to affect the DNA and ligand binding activities of this protein (de Thé *et al.*, 1991). Thus, PML/RARA retains the powerful protein-protein interaction domain of the PML protein, whereas the variable isoform-specific region is replaced by the trans-activating

One of the gained PML/RARA functions that is thought to contribute largely to APL development is the ability of this chimeric protein to form stable transcription repression complexes that are irresponsive to physiological concentrations of retinoids. As a consequence, gene promoters that are targeted by PML/RARA become constitutively repressed, an observation that has led to the general assumption that this oncoprotein causes a block in blood cell differentiation through transcriptional inhibition of key genes

and function.

**3.3 The function of RARA** 

important role of this protein in blood cell maturation.

**3.4 The function of PML/RARA** 

functions of RARA (Fig. 1.).

involved in hematopoietic maturation. Consistent with a role in gene repression, PML/RARA has also been shown to recruit the histone methyl transferase SUV39H1 (Carbone *et al.*, 2006), members of the polycomb repressive complex 2 (PRC2) (Villa *et al.*, 2007) and DNA methyltransferases (DNMTs) (Di Croce *et al.*, 2002), proteins that are known to induce a repressive chromatin structure. In addition to increased repressor activity, the PML/RARA fusion also appears to possess a considerable expanded repertoire of target genes compared to the normal RARA protein. This notion is supported by *in vitro* binding studies showing that PML/RARA has a broader and more relaxed DNA binding specificity compared to RARA (Hauksdottir & Privalsky, 2001; Kamashev *et al.*, 2004), and by a genome wide screen revealing a wide range of PML/RARA target genes (Hoemme *et al.*, 2008). The altered DNA binding and transcription repression properties of PML/RARA are partially due to the ability of this chimeric protein to form homodimeres through protein-protein interactions mediated by the TRIM motif of PML (Jansen *et al.*, 1995; Perez *et al.*, 1993). In addition, this chimeric protein has also been shown to form functional complexes with other transcription factors such as RXR and Daxx, a feature that may further contribute to the expanded promoter binding capacity (Zeisig *et al.*, 2007; Zhu *et al.*, 2005; Zhu *et al.*, 2007).

PML/RARA is also thought to contribute to malignant transformation and development of APL through inhibition of PML tumor suppressor functions. A dominant negative effect of PML/RARA on this protein is evident by studies demonstrating disruption of nuclear PML bodies into a dispersed microspeckled pattern in cells expressing this oncoprotein (Dyck *et al.*, 1994; Koken *et al.*, 1994; Weis *et al.*, 1994). Interestingly, while disruption of PML NBs by PML/RARA in the nucleus is evident, this oncoprotein readily assembles into MAPPS and CyPNs, the mitotic and cytoplasmic versions of PML NBs, respectively (Jul-Larsen *et al.*, 2009). The disruption of PML NBs in the nucleus may reflect the role of this oncoprotein in repression of gene activity.

Fig. 1. Structural organization of PML, RARA and PML/RARA. PML contains a RING domain (R), two B boxes (B), a coiled coil (CC) and a variable C-terminus. RARA consists of six regulatory domains (A-F), of which domain C and E harbor the DNA binding domain (DBD) and the ligand binding domain (LBD), respectively. The t (15;17) translocation produces PML/RARA, which retains the N-terminal PML motifs as well as RARA DNA and ligand binding activity. Arrows indicate protein breakpoints.

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 369

*et al.*, 1997). The results from these experiments appear to be in good agreement with studies demonstrating ATO-induced partial differentiation and apoptosis in APL patients or animal models, where the effective serum concentrations of ATO generally ranges from 0.1 to 1.0 µM (Chen *et al.*, 1997; Lallemand-Breitenbach *et al.*, 1999). Interestingly, ATO-mediated differentiation has been shown to become dramatically enhanced in the presence of cyclic adenosine monophosphate (cAMP). The mechanism responsible for this synergistic effect was proposed to be the combined effect of ATO-induced PML/RARA degradation and cAMP-mediated inhibition of cell cycle progression (Guillemin *et al.*, 2002; Zhu *et al.*, 2002). At the molecular level, ATO exerts its therapeutic effect on APL in part by initiating a cascade of biochemical alterations that primarily affect the PML moiety of PML/RARA. Firstly, the presence of arsenic in the cell culture medium has been shown to increase PML and PML/RARA multimerization, an effect that is manifested by decreased solubility of these proteins upon preparation of cell lysates and reduced mobility within PML NBs as determined by analysis of GFP-tagged PML in living cells (Jeanne *et al.*, 2010; Zhang *et al.*, 2010). Concomitant with increased aggregation, PML becomes extensively SUMOylated on at least three different lysine residues. All of the three different SUMO isoforms, including SUMO1, 2 and 3, appear to participate in this reaction, and both mono and poly-SUMOylation events have been reported (Lallemand-Breitenbach *et al.*, 2001; Lallemand-Breitenbach *et al.*, 2008; Muller *et al.*, 1998; Tatham *et al.*, 2008). Subsequent to SUMOylation, a protein called RNF4 binds SUMOylated residues on PML in order to catalyze polyubiquitination, a modification that directs PML and PML/RARA to the proteasome for degradation (Lallemand-Breitenbach *et al.*, 2008; Tatham *et al.*, 2008). Recently, a direct interaction between PML and ATO, that potentially triggers this SUMO-mediated degradation pathway, was mapped to cysteine residues located in the TRIM and B-box

In addition to affecting differentiation of leukemic cells, recent studies have also implicated ATO in clearance of leukemic-initiating cells (LICs), a small population of malignantly transformed cells with stem cell characteristics that frequently are refractory to cancer therapeutic drugs. Consistent with this, PML/RARA expression has been reported to support properties of self-renewal of LICs (Wojiski *et al.*, 2009), and certain characteristics of promyelocytic phenotypes provide the basic properties for the development of APLinitiating LICs (Guibal *et al.*, 2009). Furthermore, a recent study demonstrated LIC clearance in association with ATO-induced PML/RARA degradation by a mechanism that appeared to be uncoupled from the observed cell differentiation (Nasr *et al.*, 2008; Shao *et al.*, 1998). In addition, ATO has been reported to cause increased proliferation of LICs in a chronic myelogenous mouse model, hence sensitizing otherwise therapy-insensitive leukemic cells

The proapoptotic activity of ATO is not specific for APL cells (Akao *et al.*, 1998; Bachleitner-Hofmann *et al.*, 2001; Ishitsuka *et al.*, 1998; Perkins *et al.*, 2000; Rousselot *et al.*, 1999; Wang *et al.*, 1996; Zhang *et al.*, 1998; Zheng *et al.*, 1999), although non-APL tumor cells have been shown to be less sensitive to this drug (Huang *et al.*, 1999). ATO induces apoptosis by downregulation of the antiapoptotic protein Bcl-2, leading to a disturbance in the regulated balance between pro- and antiapoptotic proteins (Akao *et al.*, 1998; Chen *et al.*, 1996; Zhang *et al.*, 1998). In addition, ATO increases radioactive oxygen species (ROS) production in malignant cells. As a consequence, this drug leads to disruption of the mitochondrial membrane potential, followed by cytochrome c release, caspase activation and subsequent

motifs of PML (Jeanne *et al.*, 2010; Zhang *et al.*, 2010).

to Ara-C-based treatment (Ito *et al.*, 2008; Ito *et al.*, 2009).

apoptotic cell death (Jing *et al.*, 1999).

While PML/RARA is constantly expressed in more than 97% of all APL patients, the reciprocal fusion protein RARA/PML, which contains the N-terminus of RARA and variable lengths of the PML C-terminus, is identified in only 70-80% of the cases (Alcalay *et al.*, 1992; Grimwade *et al.*, 1996). Not much is known about the role of this protein in the pathogenesis of APL. However, one study has described a possible link between RARA/PML fusion gene deletions and resistance to ATRA-based therapy (Subramaniyam *et al.*, 2006).

#### **4. The mechanism of ATRA and ATO-mediated APL therapy**

#### **4.1 The mechanism of ATRA-based APL therapy**

Phenotypically, pharmacological concentrations of ATRA lead to effective differentiation of immature APL cells to terminally differentiated granulocytes. From a therapeutic point of view this may be beneficial since the immature malignant cells progress from being highly proliferative and long-lived to arrested and short-lived. In addition, *in vitro* cell culture experiments have shown that ATRA-induced differentiation also coincides with activation of apoptosis (Altucci *et al.*, 2001; Grignani *et al.*, 1998; Martin *et al.*, 1990). The relative contribution of apoptotic cell death versus increased turnover of mature granulocytes to ATRA-induced clearance of tumorigenic cells is not clear. Although ATRA appears to be highly effective in clearing the bulk of proliferative tumor cells, a residual population of cells with detectable t(15;17) translocation almost invariably persist following treatment with this reagent alone, a feature that probably explains the additional need for chemotherapy in order to achieve complete remission (Chen *et al.*, 1991; Chomienne *et al.*, 1990; Huang *et al.*, 1988; Zhu *et al.*, 1995).

At the molecular level, therapeutic doses of ATRA reverse the differentiation block caused by PML/RARA through a direct interaction with the ligand binding site present on the RARA moiety. As for normal RARA, the ligand-receptor interaction induces a change in the PML/RARA protein structural conformation, which leads to release of transcription repressors and subsequent activation of the basal transcription machinery. Coincident with transcription activation, ATRA also induces recruitment of the proteasome to the ligand binding transcription activation domain AF2 of RARA, and subsequent proteasomedependent degradation (Kopf *et al.*, 2000; Zhu *et al.*, 1999). A protein that has been proposed to participate in this pathway is the ubiquitin-activating enzyme E1-like (UBE1L) protein, which itself represents one of the ATRA-induced proteins (Kitareewan *et al.*, 2002). ATRAmediated degradation appears to affect RARA and PML/RARA equally well and may be functionally linked to transcription activation, since mutations in RARA that impairs its DNA binding activity also inhibits ATRA-mediated catabolism (Zhu *et al.*, 1999). The relative contribution of transcriptional activation, differentiation and degradation on therapy remains to be fully elucidated.

#### **4.2 The mechanism of ATO-based APL therapy**

Compared to ATRA, ATO has a more limited ability to induce terminal differentiation of APL cells. *In vitro* studies using cultured cells have revealed a dose-dependent effect of this drug on differentiation and apoptosis (Chen *et al.*, 1997). At high concentrations (0.5-2.0 µM) ATO induced cell death by apoptosis, while at low concentrations (0.1-0.25 µM) this drug caused partial differentiation of APL cells along the granulocyte linage (Cai *et al.*, 2000; Chen

While PML/RARA is constantly expressed in more than 97% of all APL patients, the reciprocal fusion protein RARA/PML, which contains the N-terminus of RARA and variable lengths of the PML C-terminus, is identified in only 70-80% of the cases (Alcalay *et al.*, 1992; Grimwade *et al.*, 1996). Not much is known about the role of this protein in the pathogenesis of APL. However, one study has described a possible link between RARA/PML fusion gene deletions and resistance to ATRA-based therapy (Subramaniyam

Phenotypically, pharmacological concentrations of ATRA lead to effective differentiation of immature APL cells to terminally differentiated granulocytes. From a therapeutic point of view this may be beneficial since the immature malignant cells progress from being highly proliferative and long-lived to arrested and short-lived. In addition, *in vitro* cell culture experiments have shown that ATRA-induced differentiation also coincides with activation of apoptosis (Altucci *et al.*, 2001; Grignani *et al.*, 1998; Martin *et al.*, 1990). The relative contribution of apoptotic cell death versus increased turnover of mature granulocytes to ATRA-induced clearance of tumorigenic cells is not clear. Although ATRA appears to be highly effective in clearing the bulk of proliferative tumor cells, a residual population of cells with detectable t(15;17) translocation almost invariably persist following treatment with this reagent alone, a feature that probably explains the additional need for chemotherapy in order to achieve complete remission (Chen *et al.*, 1991; Chomienne *et al.*,

At the molecular level, therapeutic doses of ATRA reverse the differentiation block caused by PML/RARA through a direct interaction with the ligand binding site present on the RARA moiety. As for normal RARA, the ligand-receptor interaction induces a change in the PML/RARA protein structural conformation, which leads to release of transcription repressors and subsequent activation of the basal transcription machinery. Coincident with transcription activation, ATRA also induces recruitment of the proteasome to the ligand binding transcription activation domain AF2 of RARA, and subsequent proteasomedependent degradation (Kopf *et al.*, 2000; Zhu *et al.*, 1999). A protein that has been proposed to participate in this pathway is the ubiquitin-activating enzyme E1-like (UBE1L) protein, which itself represents one of the ATRA-induced proteins (Kitareewan *et al.*, 2002). ATRAmediated degradation appears to affect RARA and PML/RARA equally well and may be functionally linked to transcription activation, since mutations in RARA that impairs its DNA binding activity also inhibits ATRA-mediated catabolism (Zhu *et al.*, 1999). The relative contribution of transcriptional activation, differentiation and degradation on

Compared to ATRA, ATO has a more limited ability to induce terminal differentiation of APL cells. *In vitro* studies using cultured cells have revealed a dose-dependent effect of this drug on differentiation and apoptosis (Chen *et al.*, 1997). At high concentrations (0.5-2.0 µM) ATO induced cell death by apoptosis, while at low concentrations (0.1-0.25 µM) this drug caused partial differentiation of APL cells along the granulocyte linage (Cai *et al.*, 2000; Chen

**4. The mechanism of ATRA and ATO-mediated APL therapy** 

**4.1 The mechanism of ATRA-based APL therapy** 

1990; Huang *et al.*, 1988; Zhu *et al.*, 1995).

therapy remains to be fully elucidated.

**4.2 The mechanism of ATO-based APL therapy** 

*et al.*, 2006).

*et al.*, 1997). The results from these experiments appear to be in good agreement with studies demonstrating ATO-induced partial differentiation and apoptosis in APL patients or animal models, where the effective serum concentrations of ATO generally ranges from 0.1 to 1.0 µM (Chen *et al.*, 1997; Lallemand-Breitenbach *et al.*, 1999). Interestingly, ATO-mediated differentiation has been shown to become dramatically enhanced in the presence of cyclic adenosine monophosphate (cAMP). The mechanism responsible for this synergistic effect was proposed to be the combined effect of ATO-induced PML/RARA degradation and cAMP-mediated inhibition of cell cycle progression (Guillemin *et al.*, 2002; Zhu *et al.*, 2002). At the molecular level, ATO exerts its therapeutic effect on APL in part by initiating a cascade of biochemical alterations that primarily affect the PML moiety of PML/RARA. Firstly, the presence of arsenic in the cell culture medium has been shown to increase PML and PML/RARA multimerization, an effect that is manifested by decreased solubility of these proteins upon preparation of cell lysates and reduced mobility within PML NBs as determined by analysis of GFP-tagged PML in living cells (Jeanne *et al.*, 2010; Zhang *et al.*, 2010). Concomitant with increased aggregation, PML becomes extensively SUMOylated on at least three different lysine residues. All of the three different SUMO isoforms, including SUMO1, 2 and 3, appear to participate in this reaction, and both mono and poly-SUMOylation events have been reported (Lallemand-Breitenbach *et al.*, 2001; Lallemand-Breitenbach *et al.*, 2008; Muller *et al.*, 1998; Tatham *et al.*, 2008). Subsequent to SUMOylation, a protein called RNF4 binds SUMOylated residues on PML in order to catalyze polyubiquitination, a modification that directs PML and PML/RARA to the proteasome for degradation (Lallemand-Breitenbach *et al.*, 2008; Tatham *et al.*, 2008). Recently, a direct interaction between PML and ATO, that potentially triggers this SUMO-mediated degradation pathway, was mapped to cysteine residues located in the TRIM and B-box motifs of PML (Jeanne *et al.*, 2010; Zhang *et al.*, 2010).

In addition to affecting differentiation of leukemic cells, recent studies have also implicated ATO in clearance of leukemic-initiating cells (LICs), a small population of malignantly transformed cells with stem cell characteristics that frequently are refractory to cancer therapeutic drugs. Consistent with this, PML/RARA expression has been reported to support properties of self-renewal of LICs (Wojiski *et al.*, 2009), and certain characteristics of promyelocytic phenotypes provide the basic properties for the development of APLinitiating LICs (Guibal *et al.*, 2009). Furthermore, a recent study demonstrated LIC clearance in association with ATO-induced PML/RARA degradation by a mechanism that appeared to be uncoupled from the observed cell differentiation (Nasr *et al.*, 2008; Shao *et al.*, 1998). In addition, ATO has been reported to cause increased proliferation of LICs in a chronic myelogenous mouse model, hence sensitizing otherwise therapy-insensitive leukemic cells to Ara-C-based treatment (Ito *et al.*, 2008; Ito *et al.*, 2009).

The proapoptotic activity of ATO is not specific for APL cells (Akao *et al.*, 1998; Bachleitner-Hofmann *et al.*, 2001; Ishitsuka *et al.*, 1998; Perkins *et al.*, 2000; Rousselot *et al.*, 1999; Wang *et al.*, 1996; Zhang *et al.*, 1998; Zheng *et al.*, 1999), although non-APL tumor cells have been shown to be less sensitive to this drug (Huang *et al.*, 1999). ATO induces apoptosis by downregulation of the antiapoptotic protein Bcl-2, leading to a disturbance in the regulated balance between pro- and antiapoptotic proteins (Akao *et al.*, 1998; Chen *et al.*, 1996; Zhang *et al.*, 1998). In addition, ATO increases radioactive oxygen species (ROS) production in malignant cells. As a consequence, this drug leads to disruption of the mitochondrial membrane potential, followed by cytochrome c release, caspase activation and subsequent apoptotic cell death (Jing *et al.*, 1999).

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 371

Fig. 2. Schematic overview of the four main ATRA and ATO-mediated PML/RARA degradation pathways: 1. ATO-induced proteasome-dependent degradation,

4. ATRA/ATO-induced autophagy-mediated degradation.

cleavage defective PML/RARA protein (Uy *et al.*, 2010).

elucidated.

2. ATRA-induced caspase cleavage, 3. ATRA-induced proteasome-dependent degradation,

Two different types of proteases have also been implicated in PML/RARA proteolysis. First, PML/RARA has been shown to be susceptible to a caspase 3-like activity expressed in APL cells and that becomes induced by the presence of ATRA (Nervi *et al.*, 1998). The second protease shown to be involved is neutrophil elastase, a myeloid specific serine protease that is maximally expressed in promyelocytes (Lane & Ley, 2003). The contribution of this protease to APL development is unclear since one study showed enhanced penetrance of PML/RARA in a neutrophil elastase defective mice (Lane & Ley, 2003), while another demonstrated decreased tumorigenesis in a mouse model expressing a neutrophil elastase

PML turnover has also been shown to be regulated by a pathway that involves direct phosphorylation by the casein kinase 2 (CK2) and subsequent ubiquitin-mediated degradation, a mechanism that was proposed to cause decreased PML tumor suppressor activity in lung cancer (Scaglioni *et al.*, 2006). However, the significance of CK2-mediated PML phosphorylation in PML/RARA degradation and APL pathogenesis has not been

#### **4.3 The synergy between ATRA and ATO**

While ATRA and ATO on their own are known to be effective in curing APL, it is also becoming increasingly clear that treatment regiments based on a combination of the two drugs leads to a quicker clinical remission, a more effective clearance of leukemic cells and a significantly longer period of relapse free survival (Estey *et al.*, 2006; Hu *et al.*, 2009; Shen *et al.*, 2004; Wang *et al.*, 2004). This synergistic effect may result due to the ability of both these drugs to cause PML/RARA degradation, a parameter that appears to be critical for the success of APL therapy. In addition, the combined effect of ATO and ATRA may also result due to the ability of the two agents to act on separate targets, both of which are important for disease remission. For example, ATO may be effective in eradicating self-renewable LICs through stimulated PML/RARA degradation, while ATRA represents a more effective differentiating agent, and hence may lead to a more complete clearance of undifferentiated APL cells.

#### **5. Therapy-induced degradation of PML/RARA**

ATRA and ATO-induced therapy of APL may be connected to the ability of these drugs to induce PML/RARA catabolism (Fig. 2.). In agreement with this, reduced PML/RARA expression can be observed in both ATRA and ATO-treated cells, and the two drugs synergize both for their ability to induce oncoprotein degradation as well as for their capacity to promote clinical remission (Hu *et al.*, 2009; Nasr *et al.*, 2008; Shen *et al.*, 2004). An important role of protein degradation for effective APL therapy is also supported by experiments in mice. For example, treatment of an APL mouse model with the proteasome inhibitor bortezomid led to reduced degradation of PML/RARA and concomitant resistance to ATRA and ATO-based therapy (Nasr *et al.*, 2008). In addition, PML/RARA mutated in critical SUMOylation target sites, were found to be more resistant to ATO-mediated degradation compared to unmodified PML/RARA (Lallemand-Breitenbach *et al.*, 2001; Lallemand-Breitenbach *et al.*, 2008).

In addition to proteasome-dependent degradation induced by ATRA and ATO, PML/RARA has also been shown to be amenable for degradation by the lysosomedependent degradation pathway autophagy (Isakson *et al.*, 2010; Klionsky, 2007). This degradation mechanism appears to play a major role both for basal turnover as well as for therapy-induced catabolism of PML/RARA. Indeed, pharmacological inhibitors of autophagy were found to completely prevent ATRA and ATO-stimulated degradation of PML/RARA expressed in the APL cell line NB4 (Isakson *et al.*, 2010). In contrast to proteasome-dependent degradation, autophagy-mediated proteolyses of PML/RARA appears to be independent of a direct interaction between the drugs and the target protein. Instead, ATRA and ATO seem to stimulate autophagy in APL cells primarily through a mechanism that involves the mammalian target of rapamycin (mTOR) and Unc-51-like kinase 1 (ULK1) (Bøe & Simonsen, 2010; Isakson *et al.*, 2010). Furthermore, PML/RARA is highly aggregation prone and therefore a good substrate for this degradation pathway (Isakson *et al.*, 2010; Lallemand-Breitenbach *et al.*, 2001). Aggregates of PML/RARA may form during the process of protein synthesis. In agreement with this, synthesis of PML/RARA has been shown to be associated with endoplasmatic reticulum stress, a feature indicative of aberrant folding during protein synthesis (Khan *et al.*, 2004).

While ATRA and ATO on their own are known to be effective in curing APL, it is also becoming increasingly clear that treatment regiments based on a combination of the two drugs leads to a quicker clinical remission, a more effective clearance of leukemic cells and a significantly longer period of relapse free survival (Estey *et al.*, 2006; Hu *et al.*, 2009; Shen *et al.*, 2004; Wang *et al.*, 2004). This synergistic effect may result due to the ability of both these drugs to cause PML/RARA degradation, a parameter that appears to be critical for the success of APL therapy. In addition, the combined effect of ATO and ATRA may also result due to the ability of the two agents to act on separate targets, both of which are important for disease remission. For example, ATO may be effective in eradicating self-renewable LICs through stimulated PML/RARA degradation, while ATRA represents a more effective differentiating agent, and hence may lead to a more complete clearance of undifferentiated

ATRA and ATO-induced therapy of APL may be connected to the ability of these drugs to induce PML/RARA catabolism (Fig. 2.). In agreement with this, reduced PML/RARA expression can be observed in both ATRA and ATO-treated cells, and the two drugs synergize both for their ability to induce oncoprotein degradation as well as for their capacity to promote clinical remission (Hu *et al.*, 2009; Nasr *et al.*, 2008; Shen *et al.*, 2004). An important role of protein degradation for effective APL therapy is also supported by experiments in mice. For example, treatment of an APL mouse model with the proteasome inhibitor bortezomid led to reduced degradation of PML/RARA and concomitant resistance to ATRA and ATO-based therapy (Nasr *et al.*, 2008). In addition, PML/RARA mutated in critical SUMOylation target sites, were found to be more resistant to ATO-mediated degradation compared to unmodified PML/RARA (Lallemand-Breitenbach *et al.*, 2001;

In addition to proteasome-dependent degradation induced by ATRA and ATO, PML/RARA has also been shown to be amenable for degradation by the lysosomedependent degradation pathway autophagy (Isakson *et al.*, 2010; Klionsky, 2007). This degradation mechanism appears to play a major role both for basal turnover as well as for therapy-induced catabolism of PML/RARA. Indeed, pharmacological inhibitors of autophagy were found to completely prevent ATRA and ATO-stimulated degradation of PML/RARA expressed in the APL cell line NB4 (Isakson *et al.*, 2010). In contrast to proteasome-dependent degradation, autophagy-mediated proteolyses of PML/RARA appears to be independent of a direct interaction between the drugs and the target protein. Instead, ATRA and ATO seem to stimulate autophagy in APL cells primarily through a mechanism that involves the mammalian target of rapamycin (mTOR) and Unc-51-like kinase 1 (ULK1) (Bøe & Simonsen, 2010; Isakson *et al.*, 2010). Furthermore, PML/RARA is highly aggregation prone and therefore a good substrate for this degradation pathway (Isakson *et al.*, 2010; Lallemand-Breitenbach *et al.*, 2001). Aggregates of PML/RARA may form during the process of protein synthesis. In agreement with this, synthesis of PML/RARA has been shown to be associated with endoplasmatic reticulum stress, a feature

indicative of aberrant folding during protein synthesis (Khan *et al.*, 2004).

**4.3 The synergy between ATRA and ATO** 

**5. Therapy-induced degradation of PML/RARA** 

Lallemand-Breitenbach *et al.*, 2008).

APL cells.

Fig. 2. Schematic overview of the four main ATRA and ATO-mediated PML/RARA degradation pathways: 1. ATO-induced proteasome-dependent degradation, 2. ATRA-induced caspase cleavage, 3. ATRA-induced proteasome-dependent degradation, 4. ATRA/ATO-induced autophagy-mediated degradation.

Two different types of proteases have also been implicated in PML/RARA proteolysis. First, PML/RARA has been shown to be susceptible to a caspase 3-like activity expressed in APL cells and that becomes induced by the presence of ATRA (Nervi *et al.*, 1998). The second protease shown to be involved is neutrophil elastase, a myeloid specific serine protease that is maximally expressed in promyelocytes (Lane & Ley, 2003). The contribution of this protease to APL development is unclear since one study showed enhanced penetrance of PML/RARA in a neutrophil elastase defective mice (Lane & Ley, 2003), while another demonstrated decreased tumorigenesis in a mouse model expressing a neutrophil elastase cleavage defective PML/RARA protein (Uy *et al.*, 2010).

PML turnover has also been shown to be regulated by a pathway that involves direct phosphorylation by the casein kinase 2 (CK2) and subsequent ubiquitin-mediated degradation, a mechanism that was proposed to cause decreased PML tumor suppressor activity in lung cancer (Scaglioni *et al.*, 2006). However, the significance of CK2-mediated PML phosphorylation in PML/RARA degradation and APL pathogenesis has not been elucidated.

Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 373

model disease for the development of targeted cancer therapy. For example, the case of APL clearly demonstrates the therapeutic effectiveness of targeting a defined oncoprotein, and since recurrent translocations and expression of fusion oncoproteins similar to that of PML/RARA is a common trait also among other types of cancers (including leukemias and sarcomas), a large number of malignancies, in addition to APL, may benefit from similar targeted therapies. Thus, it will be important to continue identifying therapeutic concepts that contribute to the success of APL therapy and to modulate these concepts for treatment

Since both ATRA and ATO have been shown to exert their therapeutic effects through interactions with specific regions of the PML/RARA oncoprotein, it may be assumed that these drugs will be effective only against APL. However, one should also keep in mind that the ability of ATRA and ATO to mediate cure of APL is regarded as a rather fortuitous discovery and not merely as a result of rational therapeutic design. For this reason, these drugs are likely to have other yet unidentified cellular targets, beside the APL-associated fusion portion, that are important for effective treatment. Evidence for this comes from one of the studies mentioned above showing that both ATRA and ATO-stimulated autophagic degradation of PML/RARA through a mTOR-dependent pathway that does not seem to involve direct interactions between drugs and the oncoprotein (Isakson *et al.*, 2010). In addition, it is also becoming increasingly clear that ATO has the potential to cure a subset of cancers that don't express PML/RARA. For example, induced clearance of LICs has been demonstrated both in PML/RARA positive as well as PML/RARA negative leukemic cells (Ito *et al.*, 2008; Nasr *et al.*, 2008). Furthermore, a phase II clinical study was recently published that showed promising results of using ATO in combination with interferon alpha and zidovudine for treatment of patients with chronic adult T cell leukemia (Kchour *et al.*, 2009), and finally, this drug was found to sensitize glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone (Bornhauser *et al.*, 2007). Thus, it is likely that APL for many years to come will continue to represent an important model disease for targeted and non-targeted effects of ATRA and ATO, while increased understanding of the molecular pathways involved may lead to discoveries of new therapies that are applicable

The work in our laboratory is funded by the Research Council of Norway, The Norwegian

Akao, Y., Mizoguchi, H., Kojima, S., Naoe, T., Ohishi, N. & Yagi, K. (1998). Arsenic induces

regulation of Bcl-2 protein. *British Journal of Haematology* 102, 1055-1060. Alcalay, M., Zangrilli, D., Fagioli, M., Pandolfi, P. P., Mencarelli, A., Lo Coco, F., Biondi, A.,

apoptosis in B-cell leukaemic cell lines in vitro: activation of caspases and down-

Grignani, F. & Pelicci, P. G. (1992). Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia. *Proc Natl Acad Sci U S A* 89, 4840-

Cancer society and South-Eastern Norway Regional Health Authority.

of other cancers.

for other types of cancers.

**8. Acknowledgements** 

**9. References** 

4844.

#### **6. The mechanism of APL therapy resistance**

The second most common translocation associated with APL, the t(11;17) translocation that expresses PLZF/RARA fusion instead of PML/RARA, is generally insensitive to ATRA and ATO-based therapy (Chen *et al.*, 1993; Licht *et al.*, 1995). The poor response of these patients to ATO add support to studies showing that this drug primarily target PML, which is absent in PLZF/RARA. In the case of the poor response to ATRA, on the other hand, the underlying mechanism has been hypothesized to be due to enhanced co-repressor activity conferred by the PLZF moiety of the PLZF-RARA fusion (Grignani *et al.*, 1998; He *et al.*, 1998; Lin *et al.*, 1998). However, the notion that PLZF/RARA is irresponsive to ATRA stimulation has been contradicted in more recent studies demonstrating ATRA-induced gene expression and differentiation also in PLZF/RARA expressing APL cells (Nasr *et al.*, 2008; Petti *et al.*, 2002; Rice *et al.*, 2009). Thus, further work is needed in order to fully understand the mechanism underlying the insensitivity of PLZF/RARA positive APL cells to ATRA.

Resistance to ATRA-mediated therapy is also seen in APL patients that have relapsed following the first clinical remission. Such acquired resistance may be caused by a number of different physiological factors, including increased catabolism, reduced cellular uptake, or increased cytoplasmic sequestration of the therapeutic drugs (Freemantle *et al.*, 2003; Gallagher, 2002). In addition, *in vitro* cell culture experiments, using the APL cell line NB4, have revealed mutations within the PML/RARA gene of subclones with acquired resistance to ATRA. Interestingly, several of these mutations were found clustered at/or near the ligand binding domain of RARA leading to defects in ATRA binding. Since these mutants generally retain their capacity to form complex with RXR and to bind DNA, they have been suggested to act as dominant inhibitors of wild type RARA (Duprez *et al.*, 2000; Kitamura *et al.*, 1997; Nason-Burchenal *et al.*, 1998; Rosenauer *et al.*, 1996; Shao *et al.*, 1997). Mutations in PML/RARA have also been identified in a subset of ATRA-relapsed patients, and these mutations were found to be variably associated with inactivation of ATRA binding (Ding *et al.*, 1998; Gallagher *et al.*, 2006; Imaizumi *et al.*, 1998; Marasca *et al.*, 1999; Takayama *et al.*, 2001; Zhou *et al.*, 2002). Interestingly, one study identified mutations within the intact PML locus of APL patients with ATRA-resistance and poor prognosis (Gurrieri *et al.*, 2004).

Recently, PML/RARA mutations have also been discovered in two APL cases with poor response to ATO (Goto *et al.*, 2011). In both cases, the mutations were located within the second B-box motif of the PML protein. Since the amino acids affected by these mutations were close to a cysteine-rich region, previously proposed to bind ATO (Jeanne *et al.*, 2010), the authors of this paper hypothesized that these mutations may affect interactions between this drug and PML/RARA. Alternatively, the mutated protein may have defects in oligomerization, since the B-box domains are known to function in PML multimerization. Combined, the PML/RARA mutations that have been identified in ATRA and/or ATO–resistant APL cells support the notion that these drugs interact with separate moieties of the fusion protein to induce clinical remission.

#### **7. Perspectives**

During the past 30 years, APL has progressed from a deadly disease to a highly curable malignancy. In addition, the advances that have been made in understanding the pathology and cure of APL at the molecular level have led to the emergence of a highly attractive model disease for the development of targeted cancer therapy. For example, the case of APL clearly demonstrates the therapeutic effectiveness of targeting a defined oncoprotein, and since recurrent translocations and expression of fusion oncoproteins similar to that of PML/RARA is a common trait also among other types of cancers (including leukemias and sarcomas), a large number of malignancies, in addition to APL, may benefit from similar targeted therapies. Thus, it will be important to continue identifying therapeutic concepts that contribute to the success of APL therapy and to modulate these concepts for treatment of other cancers.

Since both ATRA and ATO have been shown to exert their therapeutic effects through interactions with specific regions of the PML/RARA oncoprotein, it may be assumed that these drugs will be effective only against APL. However, one should also keep in mind that the ability of ATRA and ATO to mediate cure of APL is regarded as a rather fortuitous discovery and not merely as a result of rational therapeutic design. For this reason, these drugs are likely to have other yet unidentified cellular targets, beside the APL-associated fusion portion, that are important for effective treatment. Evidence for this comes from one of the studies mentioned above showing that both ATRA and ATO-stimulated autophagic degradation of PML/RARA through a mTOR-dependent pathway that does not seem to involve direct interactions between drugs and the oncoprotein (Isakson *et al.*, 2010). In addition, it is also becoming increasingly clear that ATO has the potential to cure a subset of cancers that don't express PML/RARA. For example, induced clearance of LICs has been demonstrated both in PML/RARA positive as well as PML/RARA negative leukemic cells (Ito *et al.*, 2008; Nasr *et al.*, 2008). Furthermore, a phase II clinical study was recently published that showed promising results of using ATO in combination with interferon alpha and zidovudine for treatment of patients with chronic adult T cell leukemia (Kchour *et al.*, 2009), and finally, this drug was found to sensitize glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone (Bornhauser *et al.*, 2007). Thus, it is likely that APL for many years to come will continue to represent an important model disease for targeted and non-targeted effects of ATRA and ATO, while increased understanding of the molecular pathways involved may lead to discoveries of new therapies that are applicable for other types of cancers.

#### **8. Acknowledgements**

The work in our laboratory is funded by the Research Council of Norway, The Norwegian Cancer society and South-Eastern Norway Regional Health Authority.

#### **9. References**

372 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

The second most common translocation associated with APL, the t(11;17) translocation that expresses PLZF/RARA fusion instead of PML/RARA, is generally insensitive to ATRA and ATO-based therapy (Chen *et al.*, 1993; Licht *et al.*, 1995). The poor response of these patients to ATO add support to studies showing that this drug primarily target PML, which is absent in PLZF/RARA. In the case of the poor response to ATRA, on the other hand, the underlying mechanism has been hypothesized to be due to enhanced co-repressor activity conferred by the PLZF moiety of the PLZF-RARA fusion (Grignani *et al.*, 1998; He *et al.*, 1998; Lin *et al.*, 1998). However, the notion that PLZF/RARA is irresponsive to ATRA stimulation has been contradicted in more recent studies demonstrating ATRA-induced gene expression and differentiation also in PLZF/RARA expressing APL cells (Nasr *et al.*, 2008; Petti *et al.*, 2002; Rice *et al.*, 2009). Thus, further work is needed in order to fully understand the mechanism underlying the insensitivity of PLZF/RARA positive APL cells

Resistance to ATRA-mediated therapy is also seen in APL patients that have relapsed following the first clinical remission. Such acquired resistance may be caused by a number of different physiological factors, including increased catabolism, reduced cellular uptake, or increased cytoplasmic sequestration of the therapeutic drugs (Freemantle *et al.*, 2003; Gallagher, 2002). In addition, *in vitro* cell culture experiments, using the APL cell line NB4, have revealed mutations within the PML/RARA gene of subclones with acquired resistance to ATRA. Interestingly, several of these mutations were found clustered at/or near the ligand binding domain of RARA leading to defects in ATRA binding. Since these mutants generally retain their capacity to form complex with RXR and to bind DNA, they have been suggested to act as dominant inhibitors of wild type RARA (Duprez *et al.*, 2000; Kitamura *et al.*, 1997; Nason-Burchenal *et al.*, 1998; Rosenauer *et al.*, 1996; Shao *et al.*, 1997). Mutations in PML/RARA have also been identified in a subset of ATRA-relapsed patients, and these mutations were found to be variably associated with inactivation of ATRA binding (Ding *et al.*, 1998; Gallagher *et al.*, 2006; Imaizumi *et al.*, 1998; Marasca *et al.*, 1999; Takayama *et al.*, 2001; Zhou *et al.*, 2002). Interestingly, one study identified mutations within the intact PML locus of APL patients with ATRA-resistance and poor prognosis (Gurrieri *et al.*, 2004). Recently, PML/RARA mutations have also been discovered in two APL cases with poor response to ATO (Goto *et al.*, 2011). In both cases, the mutations were located within the second B-box motif of the PML protein. Since the amino acids affected by these mutations were close to a cysteine-rich region, previously proposed to bind ATO (Jeanne *et al.*, 2010), the authors of this paper hypothesized that these mutations may affect interactions between this drug and PML/RARA. Alternatively, the mutated protein may have defects in oligomerization, since the B-box domains are known to function in PML multimerization. Combined, the PML/RARA mutations that have been identified in ATRA and/or ATO–resistant APL cells support the notion that these drugs interact with separate moieties

During the past 30 years, APL has progressed from a deadly disease to a highly curable malignancy. In addition, the advances that have been made in understanding the pathology and cure of APL at the molecular level have led to the emergence of a highly attractive

**6. The mechanism of APL therapy resistance** 

of the fusion protein to induce clinical remission.

**7. Perspectives** 

to ATRA.


Acute Promyelocytic Leukemia: A Model Disease for Targeted Cancer Therapy 375

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**19** 

*România* 

**The Association of the DNA Repair** 

**The Susceptibility and the** 

**Outcome After Therapy** 

*Univ Med & Pharm Tg-Mures* 

**Genes with Acute Myeloid Leukemia:** 

Claudia Bănescu, Carmen Duicu and Minodora Dobreanu

Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a clonal hemopoietic disorder that is frequently associated with genetic instability characterized by a diversity of chromosomal and molecular abnormalities. There are a lot of reports that show that human cancer can be initiated by DNA damage caused by ultraviolet (UV), ionizing radiation, and environmental chemical agents. Many genes encode proteins that function to protect cells against genetic instability through numerous mechanisms, including deoxyribonucleic acid (DNA) repair pathways and protection against oxidative

DNA repair pathways play an important role in maintaining the integrity of the genome, and it is obvious that defects in repair pathways are involved in many different types of

DNA damage repair and cell-cycle checkpoints are the most important defense mechanisms against mutagenic exposures. The most important DNA-repair pathways in human cells are: direct repair, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), Double Strand Break Repair (DSB repair) and translesion DNA synthesis (TLS). Each pathway repairs a different type of lession (D'Andrea, 2010). The NER pathway mainly removes bulky distortions in the shape of the DNA double helix. The BER pathway is responsible for removal of oxidized DNA bases that may arise endogenously or from exogenous agents. The DSB pathway is responsible for repairing double-strand breaks caused by a variety of exposures, including ionizing radiation. There are two distinct and complementary pathways for DSB repair-namely, homologous recombination (HR) and

Recent studies have suggested that DNA damage was related to the pathogenesis of some diseases such as AML. Therefore, some DNA repair genes may be involved in AML

Exposure to carcinogenic and genotoxic compounds causes DNA damage, and the cells have developed multiple DNA repair pathways to protect themselves from different types

susceptibility (Allan, 2004; Kuptsova, 2007; Seedhouse, 2002, 2004; Voso, 2007).

diseases, including leukemia and cancer (Seedhouse, 2002).

nonhomologous end joining (NHEJ).

of DNA damage.

**1. Introduction** 

stress.


### **The Association of the DNA Repair Genes with Acute Myeloid Leukemia: The Susceptibility and the Outcome After Therapy**

Claudia Bănescu, Carmen Duicu and Minodora Dobreanu *Univ Med & Pharm Tg-Mures România* 

#### **1. Introduction**

384 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Zhu, J., Zhou, J., Peres, L., Riaucoux, F., Honore, N., Kogan, S. & de The, H. (2005). A

Zhu, J., Nasr, R., Peres, L. & other authors (2007). RXR is an essential component of the

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*Cell* 7, 143-153.

sumoylation site in PML/RARA is essential for leukemic transformation. *Cancer* 

and cAMP during acute promyelocytic leukemia cell maturation subtends a novel

Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a clonal hemopoietic disorder that is frequently associated with genetic instability characterized by a diversity of chromosomal and molecular abnormalities. There are a lot of reports that show that human cancer can be initiated by DNA damage caused by ultraviolet (UV), ionizing radiation, and environmental chemical agents. Many genes encode proteins that function to protect cells against genetic instability through numerous mechanisms, including deoxyribonucleic acid (DNA) repair pathways and protection against oxidative stress.

DNA repair pathways play an important role in maintaining the integrity of the genome, and it is obvious that defects in repair pathways are involved in many different types of diseases, including leukemia and cancer (Seedhouse, 2002).

DNA damage repair and cell-cycle checkpoints are the most important defense mechanisms against mutagenic exposures. The most important DNA-repair pathways in human cells are: direct repair, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), Double Strand Break Repair (DSB repair) and translesion DNA synthesis (TLS). Each pathway repairs a different type of lession (D'Andrea, 2010). The NER pathway mainly removes bulky distortions in the shape of the DNA double helix. The BER pathway is responsible for removal of oxidized DNA bases that may arise endogenously or from exogenous agents. The DSB pathway is responsible for repairing double-strand breaks caused by a variety of exposures, including ionizing radiation. There are two distinct and complementary pathways for DSB repair-namely, homologous recombination (HR) and nonhomologous end joining (NHEJ).

Recent studies have suggested that DNA damage was related to the pathogenesis of some diseases such as AML. Therefore, some DNA repair genes may be involved in AML susceptibility (Allan, 2004; Kuptsova, 2007; Seedhouse, 2002, 2004; Voso, 2007).

Exposure to carcinogenic and genotoxic compounds causes DNA damage, and the cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage.

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

Reverses damage to restore DNA integrity.

Repair of damaged bases or single-strand

Excision of a variety of helix-distorting DNA

Repair of mispaired

Repair of double strand DNA break

Repair of double strand DNA breaks

Bypass of DNA adducts during DNA

site is removed and finally gap is filled by DNA polymerase.

replication

Table 1. DNA Damage Response Pathways

**2.2 Base Excision Repair** 

DNA breaks

nucleotides

lesions

Direct Repair Mechanism

Base Excision Repair (BER)

Nucleotide Excision Repair

Mismatch Repair

Homologous recombination

Nonhomologous End Joining (NHEJ)

Translesion Synthesis (TLS)

(NER)

(MMR)

(HR)

The Susceptibility and the Outcome After Therapy 387

DNA photolyase, O6-MGMT

OGG1, XRCC1, APE1, PARP,

XPA, XPB, XPC, XPD, XPE, XPF, XPG, RAD23, TFIIH, RPA1, RPA2, RPA3,

MSH2, MSH6, MSH4, PMS1, MLH1, PMS2, MLH3, PMS2L3, PCNA, RPA

RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, RAD52, RAD54, BRCA1, BRCA2, RAD50, NBS1, MRE11

XRCC4, XRCC5, XRCC6, XRCC7, LIG4, XLF,

POLK, POLI, POLZ, REV7, REV1, POLH,

Base excision repair (BER) is the predominant DNA damage repair pathway for the processing of small base lesions, derived from ionizing radiation (Chen et al., 2010), oxidation, hydrolysis, or deamination and alkylation damages. The repair system involves three steps: removal of damaged bases from DNA by DNA glycosylases, then formed abasic

In BER, the removal of a single modified base from one DNA strand is performed by DNA glycosylases, specialized enzymes. Some of these glycosylases show pronounced lesion specificity, others recognize multiple, and structurally different damaged bases. The apurinic/apyrimidinic (AP) site left behind after cleavage of the N-glycosylic bond is hydrolyzed by an AP endonuclease, and the 5'-deoxyribose phosphate is excised by a phosphodiesterase. The resulting single-nucleotide gap is then filled by polymerase β and ligated. Alternatives to this common BER pathway include the excision of a short

PCNA

Kaina et al., 2007; Mitra, 2007

Chaudhry, 2007; Yu et al., 1999; Hazra et al., 2007

Kang et al., 2011; Reardon & Sancar, 2005; Yu et al., 1999

Jiricny, 2006; Martin et al., 2010; Papouli et al.,

Surtees et al., 2004

Li & Heyer, 2008; Sung & Klein,

Burma et al., 2006;

Lehmann, 2006

Shrivastav et al., 2008

2004;

2006;

**Pathway Function Genes Involved References** 

Polymorphisms in DNA repair genes, including those involved in base excision repair (BER), nucleotide excision repair, mismatch repair and double strand break repair have been implicated in carcinogenesis. Common polymorphisms in DNA repair genes may alter protein function and an individual's capacity to repair damaged DNA. Deficits in repair capacity may lead to genetic instability and tumorigenesis.

Studies have noted associations between risk of de novo AML and DNA repair gene polymorphisms (Matullo et al., 2006; Seedhouse et al., 2004). Increased risk of therapyrelated AML was also linked to several gene polymorphisms in base excision repair (BER; XRCC1 Arg399Gln), nucleotide excision repair (NER; XPD Lys751Gln), and DSB repair (RAD51 G135C and XRCC3 Thr241Met) pathways (Allan et al. 2004; Seedhouse et al., 2002, 2004) and may be linked to secondary AML etiology through failure to recognize or excise accumulated DNA lesions.

#### **2. Mechanism of DNA repair**

DNA damage response pathways, some of the genes known to participate in each of these pathways, their modes of action are summarized in Table 1. These categories are not exclusive; there may be functional overlap between repair systems. For example, certain types of base damage can be repaired by base excision repair, nucleotide excision repair or homologous recombinational repair.

DNA repair genes may also have great implications in the therapeutic outcome of certain cancer treatments. Most antileukemic drugs interact with target cell DNA and exert their cytotoxic effects preferentially in replicating cells. In addition to the primary DNA lesions, secondary DNA alterations induced in the course of repair processes also contribute to the cytotoxic effects of DNA-reactive agents (Rajewsky &Müller, 2005).

Although there are several reports on associations between polymorphisms in DNA repair genes and cancer risk (Goode et al., 2002), fewer studies have been conducted to evaluate relationships between DNA repair gene polymorphisms and response to treatment.

#### **2.1 Direct repair mechanism**

This is the simplest repair mechanism compared to other repair system regarding number of molecules involved. It is an enzyme-catalyzed process used to correct the most frequent cause of point mutations in humans.

In direct repair mechanisms, the lesion is removed or reversed by a single step reaction restoring the local sequence to its original state. There are several direct repair enzymes, each having a different substrate. For example, O6- methyl guanine DNA methyltransferase (O6-MT, the product of the MGMT gene) repairs the alkylation damage. MGMT is important in the repair of alkylation damage. The alkyl group from the lesion is transferred to a cysteine residue in the active site of MGMT (Hazra et al., 1997). In 20% of human tumor cell lines the MGMT activity is decreased and the sensivity to alkylating agent is increased (Sancar, 1995), but there are few data which suggest that mutations in the MGMT gene contribute to cancer (Wang L et al, 1997; Yu Z et al, 1999).

The functional status of the O6-MT pathway may be important in patients treated with alkylating agents, for O6-methylation of guanine appears to be an important effect of some members of that class of drugs (Kaina & Christmann, 2002). High levels of O6-MT are often found in AML blasts, which are thereby rendered resistant to certain alkylators (Gerson & Trey, 1988).

Polymorphisms in DNA repair genes, including those involved in base excision repair (BER), nucleotide excision repair, mismatch repair and double strand break repair have been implicated in carcinogenesis. Common polymorphisms in DNA repair genes may alter protein function and an individual's capacity to repair damaged DNA. Deficits in repair

Studies have noted associations between risk of de novo AML and DNA repair gene polymorphisms (Matullo et al., 2006; Seedhouse et al., 2004). Increased risk of therapyrelated AML was also linked to several gene polymorphisms in base excision repair (BER; XRCC1 Arg399Gln), nucleotide excision repair (NER; XPD Lys751Gln), and DSB repair (RAD51 G135C and XRCC3 Thr241Met) pathways (Allan et al. 2004; Seedhouse et al., 2002, 2004) and may be linked to secondary AML etiology through failure to recognize or excise

DNA damage response pathways, some of the genes known to participate in each of these pathways, their modes of action are summarized in Table 1. These categories are not exclusive; there may be functional overlap between repair systems. For example, certain types of base damage can be repaired by base excision repair, nucleotide excision repair or

DNA repair genes may also have great implications in the therapeutic outcome of certain cancer treatments. Most antileukemic drugs interact with target cell DNA and exert their cytotoxic effects preferentially in replicating cells. In addition to the primary DNA lesions, secondary DNA alterations induced in the course of repair processes also contribute to the

Although there are several reports on associations between polymorphisms in DNA repair genes and cancer risk (Goode et al., 2002), fewer studies have been conducted to evaluate

This is the simplest repair mechanism compared to other repair system regarding number of molecules involved. It is an enzyme-catalyzed process used to correct the most frequent

In direct repair mechanisms, the lesion is removed or reversed by a single step reaction restoring the local sequence to its original state. There are several direct repair enzymes, each having a different substrate. For example, O6- methyl guanine DNA methyltransferase (O6-MT, the product of the MGMT gene) repairs the alkylation damage. MGMT is important in the repair of alkylation damage. The alkyl group from the lesion is transferred to a cysteine residue in the active site of MGMT (Hazra et al., 1997). In 20% of human tumor cell lines the MGMT activity is decreased and the sensivity to alkylating agent is increased (Sancar, 1995), but there are few data which suggest that mutations in the MGMT gene

The functional status of the O6-MT pathway may be important in patients treated with alkylating agents, for O6-methylation of guanine appears to be an important effect of some members of that class of drugs (Kaina & Christmann, 2002). High levels of O6-MT are often found in AML blasts, which are thereby rendered resistant to certain alkylators (Gerson &

relationships between DNA repair gene polymorphisms and response to treatment.

cytotoxic effects of DNA-reactive agents (Rajewsky &Müller, 2005).

contribute to cancer (Wang L et al, 1997; Yu Z et al, 1999).

capacity may lead to genetic instability and tumorigenesis.

accumulated DNA lesions.

**2. Mechanism of DNA repair** 

homologous recombinational repair.

**2.1 Direct repair mechanism** 

Trey, 1988).

cause of point mutations in humans.


Table 1. DNA Damage Response Pathways

#### **2.2 Base Excision Repair**

Base excision repair (BER) is the predominant DNA damage repair pathway for the processing of small base lesions, derived from ionizing radiation (Chen et al., 2010), oxidation, hydrolysis, or deamination and alkylation damages. The repair system involves three steps: removal of damaged bases from DNA by DNA glycosylases, then formed abasic site is removed and finally gap is filled by DNA polymerase.

In BER, the removal of a single modified base from one DNA strand is performed by DNA glycosylases, specialized enzymes. Some of these glycosylases show pronounced lesion specificity, others recognize multiple, and structurally different damaged bases. The apurinic/apyrimidinic (AP) site left behind after cleavage of the N-glycosylic bond is hydrolyzed by an AP endonuclease, and the 5'-deoxyribose phosphate is excised by a phosphodiesterase. The resulting single-nucleotide gap is then filled by polymerase β and ligated. Alternatives to this common BER pathway include the excision of a short

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

(Tsukino et al., 2004) cancer development.

(OR=5.36, P<0.001).

(Wilson & Simeonov, 2010).

**APE1** 

**hOGG1** 

The Susceptibility and the Outcome After Therapy 389

The human 8-oxoguanine DNA glycosylase 1 (hOGG1) encoded by the hOGG1 gene can remove 8-hydroxy-2-deoxyguanine (8-OHdG) from damaged DNA as a part of the base excision repair pathway (Kohno et al., 1998). The hOGG1 gene is located on chromosome 3p26.2 (Kohno et al., 1998). Although, several polymorphisms in the hOGG1 gene have been described, the most commonly studied polymorphism is an amino acid change from serine to cysteine at codon 326 (Ser326Cys). Several studies have reported that Ser326Cys polymorphism in OGG1 gene may increase susceptibility to for bladder (Arizono et al., 2008), lung (Park et al., 2004), oesophageal (Xing et al., 2001), gallbladder and gastric

Liddiard et al (2010) provided a study about the importance of 8-oxoguanine in AML including the genotyping of 174 AML patients for the hOGG1 Ser326Cys polymorphism. Using Affymetrix microarrays they showed that the prevalence rate of hOGG1 expression was 33% and correlated strongly with adverse cytogenetics. hOGG1-expressing patients had a worse relapse-free survival and overall survival and an increased risk of relapse at 5-years of follow-up. According to Liddiard et al. (2010) hOGG1 is an important prognostic marker that could be used to sub-stratify AML patients to predict those likely to fail conventional chemotherapies and those likely to benefit from novel therapeutic approaches that modulate DNA repair activity. In a recent study Stanczyk et al. (2011) have demonstrated that Cys/Cys variant of the OGG1 Ser326Cys polymorphism may increase the risk of ALL

The human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the base excision repair pathway, which is the primary mechanism for the repair of DNA damage caused by oxidation and alkylation. The APE1 gene is located on chromosome 14q11.2– q12 and contains five exons. A total of 18 polymorphisms in APE1 have been reported, but the most extensively studied polymorphism is Asp148Glu (Gu et al., 2009). The damaged bases of purine and pyrimidine are recognized and excised by specific DNA glycosylases, leaving abasic sites. Apurinic/apyrimidinic endonuclease then incise the DNA 5' to the abasic sites; further repair proceeds to short-patch (when the gap is only one nucleotide) or long-patch (when the gap is two or more nucleotides) subpathways of base excision repair (Lo et al., 2009). In a hospital-based matched case-control study with 730 lung cancer cases and 730 cancer-free controls Lo et al. (2009) found that the -656T>G variant in the APE1 promoter was associated with a significantly decreased risk for lung cancer. In a study of 320 pediatric patients with acute leukemia, Krajinovic et al. (2002) reported no significant associations between polymorphic APE1 148Glu and XRCC1 194Trp variants and event-free survival. Ji et al. (2020) performed a meta-analysis to explore the association between the APE1 Asn148Glu gene polymorphisms and lung cancer risk. They suggested that the APE1 T1349G (Asp148Glu) polymorphism was not associated with lung cancer risk among Asians or Caucasians. But, the APE1 G allele was an increased risk factor for developing lung cancer among smokers. Pre-clinical and clinical data indicate a role for APE1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs, particularly monofunctional alkylators and antimetabolites

oligonucleotide patch containing the AP site and filling of the gap by polymerase δ or ε. Base alterations caused by a large variety of agents and processes (e.g. spontaneous deamination, ionizing radiation, alkylating agents, DNA replication errors) are processed by BER. A common feature of the DNA lesions recognized by BER glycosylases is that they do not significantly distort the DNA helix (Yu, 2009).

Common single-nucleotide polymorphisms (SNPs) in the 8-oxoguanine glycosylase 1 (OGG1), X-ray repair cross-complementing group1 (XRCC1), and the apyrimidinic endonuclease-endonuclease 1 (APE1) genes in the BER pathway have been studied for their influences in induction of DNA damage (Chen et al., 2010).

#### **XRCC1**

X-ray repair cross-complementing group 1 (XRCC1) is required for repairing single-strand breaks and damaged bases in DNA. The XRCC1 protein interacts with DNA polymerase β, DNA ligase III and polyadenosine diphosphate-ribose polymerases (PARP) involved in excision and recombinational repair pathways (Cardecott, 2003). The XRCC1 has no known enzymatic activity (Thompson et al., 1990) and participates as a scaffold protein in both single-strand break repair and base excision repair activities. The human XRCC1 gene is located on chromosome 19q13.2 which contains 17 exons and encodes a protein of consisting 633 amino acids (Lindahl & Wood, 1999). More than 60 validated single-nucleotide XRCC1 polymorphisms are known. The most studied single nucleotide polymorphisms are Arg194Trp on exon 6, Arg280His on exon 9, and Arg399Gln on exon 10. All these polymorphisms were studied with association to different types of cancer.

The presences of the variant (399Gln and 194Trp) alleles have been shown to be associated with measurable reduced DNA repair capacity and increased risk of several types of cancers (Dufloth et al., 2005; Goode et al., 2002; Hu et al., 2005; Hung et al., 2005). In our study involving 43 patients with acute leukemia and 40 controls, for XRCC1 Arg194Trp and Arg399Gln polymorphism, we observed the relationship of XRCC1 polymorphisms with acute myeloid leukemia (our own unpublished data). Our cases with a codon 194 Trp/Trp homozygous variant as well as the heterozygous Arg/Trp variant had an increased risk of AML with a greater risk in the case of the homozygous codon 194 Trp/Trp allele (OR=2.28, 95% for Trp/Trp). There was a significantly high risk of AML among patients who were carriers of the variant allele 399Gln (OR=2.45, 95% for Gln/Gln and OR=1.90, 95% for Arg/Gln).

A study by Seedhouse et al. (2002) reported that the presence of at least one XRCC1 399Gln allele indicated a protective effect for the allele in controls compared with patients with AML, particularly therapy-related AML (t-AML) (odds ratio OR=0.44; 95% confidence interval CI=0.20~0.93). According to the same study patients who develop AML as a result of therapy for a primary malignancy are more likely to have the wild-type XRCC1 399 arginine allele. El-Din et al. (2010) reported that XRCC1 gene polymorphism is important in the pathogenesis of de novo AML. El-Din et al. (2010) observed that AML patients expressing XRCC1 Arg194Trp polymorphism are at high risk of developing AML; in addition, a significant risk in the development of AML was observed when XRCC1 Arg399Gln polymorphism was present. In a study of 372 patients with acute myeloid leukemia, Kuptsova et al. (2007) reported no significant associations between XRCC1 polymorphisms and treatment outcomes.

#### **hOGG1**

388 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

oligonucleotide patch containing the AP site and filling of the gap by polymerase δ or ε. Base alterations caused by a large variety of agents and processes (e.g. spontaneous deamination, ionizing radiation, alkylating agents, DNA replication errors) are processed by BER. A common feature of the DNA lesions recognized by BER glycosylases is that they do

Common single-nucleotide polymorphisms (SNPs) in the 8-oxoguanine glycosylase 1 (OGG1), X-ray repair cross-complementing group1 (XRCC1), and the apyrimidinic endonuclease-endonuclease 1 (APE1) genes in the BER pathway have been studied for their

X-ray repair cross-complementing group 1 (XRCC1) is required for repairing single-strand breaks and damaged bases in DNA. The XRCC1 protein interacts with DNA polymerase β, DNA ligase III and polyadenosine diphosphate-ribose polymerases (PARP) involved in excision and recombinational repair pathways (Cardecott, 2003). The XRCC1 has no known enzymatic activity (Thompson et al., 1990) and participates as a scaffold protein in both single-strand break repair and base excision repair activities. The human XRCC1 gene is located on chromosome 19q13.2 which contains 17 exons and encodes a protein of consisting 633 amino acids (Lindahl & Wood, 1999). More than 60 validated single-nucleotide XRCC1 polymorphisms are known. The most studied single nucleotide polymorphisms are Arg194Trp on exon 6, Arg280His on exon 9, and Arg399Gln on exon 10. All these

The presences of the variant (399Gln and 194Trp) alleles have been shown to be associated with measurable reduced DNA repair capacity and increased risk of several types of cancers (Dufloth et al., 2005; Goode et al., 2002; Hu et al., 2005; Hung et al., 2005). In our study involving 43 patients with acute leukemia and 40 controls, for XRCC1 Arg194Trp and Arg399Gln polymorphism, we observed the relationship of XRCC1 polymorphisms with acute myeloid leukemia (our own unpublished data). Our cases with a codon 194 Trp/Trp homozygous variant as well as the heterozygous Arg/Trp variant had an increased risk of AML with a greater risk in the case of the homozygous codon 194 Trp/Trp allele (OR=2.28, 95% for Trp/Trp). There was a significantly high risk of AML among patients who were carriers of the variant allele 399Gln (OR=2.45, 95% for Gln/Gln and OR=1.90, 95% for

A study by Seedhouse et al. (2002) reported that the presence of at least one XRCC1 399Gln allele indicated a protective effect for the allele in controls compared with patients with AML, particularly therapy-related AML (t-AML) (odds ratio OR=0.44; 95% confidence interval CI=0.20~0.93). According to the same study patients who develop AML as a result of therapy for a primary malignancy are more likely to have the wild-type XRCC1 399 arginine allele. El-Din et al. (2010) reported that XRCC1 gene polymorphism is important in the pathogenesis of de novo AML. El-Din et al. (2010) observed that AML patients expressing XRCC1 Arg194Trp polymorphism are at high risk of developing AML; in addition, a significant risk in the development of AML was observed when XRCC1 Arg399Gln polymorphism was present. In a study of 372 patients with acute myeloid leukemia, Kuptsova et al. (2007) reported no significant associations between XRCC1

polymorphisms were studied with association to different types of cancer.

not significantly distort the DNA helix (Yu, 2009).

**XRCC1** 

Arg/Gln).

polymorphisms and treatment outcomes.

influences in induction of DNA damage (Chen et al., 2010).

The human 8-oxoguanine DNA glycosylase 1 (hOGG1) encoded by the hOGG1 gene can remove 8-hydroxy-2-deoxyguanine (8-OHdG) from damaged DNA as a part of the base excision repair pathway (Kohno et al., 1998). The hOGG1 gene is located on chromosome 3p26.2 (Kohno et al., 1998). Although, several polymorphisms in the hOGG1 gene have been described, the most commonly studied polymorphism is an amino acid change from serine to cysteine at codon 326 (Ser326Cys). Several studies have reported that Ser326Cys polymorphism in OGG1 gene may increase susceptibility to for bladder (Arizono et al., 2008), lung (Park et al., 2004), oesophageal (Xing et al., 2001), gallbladder and gastric (Tsukino et al., 2004) cancer development.

Liddiard et al (2010) provided a study about the importance of 8-oxoguanine in AML including the genotyping of 174 AML patients for the hOGG1 Ser326Cys polymorphism. Using Affymetrix microarrays they showed that the prevalence rate of hOGG1 expression was 33% and correlated strongly with adverse cytogenetics. hOGG1-expressing patients had a worse relapse-free survival and overall survival and an increased risk of relapse at 5-years of follow-up. According to Liddiard et al. (2010) hOGG1 is an important prognostic marker that could be used to sub-stratify AML patients to predict those likely to fail conventional chemotherapies and those likely to benefit from novel therapeutic approaches that modulate DNA repair activity. In a recent study Stanczyk et al. (2011) have demonstrated that Cys/Cys variant of the OGG1 Ser326Cys polymorphism may increase the risk of ALL (OR=5.36, P<0.001).

#### **APE1**

The human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the base excision repair pathway, which is the primary mechanism for the repair of DNA damage caused by oxidation and alkylation. The APE1 gene is located on chromosome 14q11.2– q12 and contains five exons. A total of 18 polymorphisms in APE1 have been reported, but the most extensively studied polymorphism is Asp148Glu (Gu et al., 2009). The damaged bases of purine and pyrimidine are recognized and excised by specific DNA glycosylases, leaving abasic sites. Apurinic/apyrimidinic endonuclease then incise the DNA 5' to the abasic sites; further repair proceeds to short-patch (when the gap is only one nucleotide) or long-patch (when the gap is two or more nucleotides) subpathways of base excision repair (Lo et al., 2009). In a hospital-based matched case-control study with 730 lung cancer cases and 730 cancer-free controls Lo et al. (2009) found that the -656T>G variant in the APE1 promoter was associated with a significantly decreased risk for lung cancer. In a study of 320 pediatric patients with acute leukemia, Krajinovic et al. (2002) reported no significant associations between polymorphic APE1 148Glu and XRCC1 194Trp variants and event-free survival. Ji et al. (2020) performed a meta-analysis to explore the association between the APE1 Asn148Glu gene polymorphisms and lung cancer risk. They suggested that the APE1 T1349G (Asp148Glu) polymorphism was not associated with lung cancer risk among Asians or Caucasians. But, the APE1 G allele was an increased risk factor for developing lung cancer among smokers. Pre-clinical and clinical data indicate a role for APE1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs, particularly monofunctional alkylators and antimetabolites (Wilson & Simeonov, 2010).

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

(2002), therapy-related AML was not associated with XPD genotypes.

secondary structure such as imperfect palindromes (Bishop et al., 1985).

XPD codon 751 genotypes.

resynthesis and ligation processes.

the refractory and relapsed disease.

**2.4 Mismatch repair** 

The Susceptibility and the Outcome After Therapy 391

evaluated the role of XPD and XRCC1 gene polymorphic variation in response to induction chemotherapy, toxicities and survival in a population of 293 predominantly Caucasian adult patients treated for AML. Kuptsova-Clarkson et al. (2010) had reported that in AML, variation in the XPD gene may be associated with suboptimal DNA repair activity and may thus predispose to therapy-related AML development. In a UK study by Seedhouse et al.

There have been studies of XPD, involved in NER, and survival of patients with AML (Allan et al., 2004). In a study of elderly patients with AML conducted by researchers in the United Kingdom, modestly increased hazard ratio (HR) of 1.30 and 1.19 were found for disease-free and overall survival, respectively, by XPD variant genotypes. However, in a study of pediatric patients with acute myeloid leukemia conducted by the the Children's Oncology Group (Mehta et al., 2006) survival and treatment-related mortality were not associated with

DNA mismatch repair (MMR) plays a critical role in maintaining genomic integrity. MMR is responsible for correction of mismatched basepairs which occurs through processes including misincorporations during DNA replication, formation of heteroduplexes, and

In addition, MMR can also process some types of DNA damage. MMR deficient tumors display widespread alterations in simple repetitive DNA sequences, a phenomenon also called microsatellite instability; MSI (Li, 2008). The repair mechanism is similar to that of excision repair; a patch of nucleotides is removed from one strand, and followed by

There are two types of mismatch repair, long-patch and short-patch which have been found in human cells. In short-patch repair system there are three enzymes possessing nicking activities specific for mismatch repair; T/G specific (Wiebauer & Jiricny, 1989), A/G specific, and all type mismatch nicking enzymes (Yeh et al., 1991). That enzymes have different mode of action, but in either an A/G mismatch or a T/G mismatch, it is usually the guanine that

Long patch can repair all types of mismatches. Long-patch MMR removes a patch of one of the DNA strands from an incision on the to-be-removed strand to 90~170 nucleotides

Defects in the MMR pathway significantly increase the mutation frequency and promote oncogenesis. It has been documented that defects in MMR genes, are the genetic basis for certain types of hereditary and sporadic cancers, including hereditary nonpolyposis colorectal cancer (Mao et al., 2008). There are some studies looking at the incidence of MMR deficiency (microsatellite instability; MSI) in AML (Mao et al., 2008). Genomic instability in AML has led to a search for MSI in AML patients, but the results are quite controversial. While several studies have reported MSI in AML (Das-Gupta et al., 2001; Sheikhha et al., 2002), a study of 132 cases failed to confirm the previous results (Rimsza et al., 2000). According to Rimsza et al. (2000) MMR deficiency was associated with all stages of AML, but the rate of the deficiency was much higher in patients with refractory and relapsed AML than in de novo AML patients, suggesting that the loss of MMR function could contribute to

remains untouched by mismatch specific glycosylases (Wiebauer & Jiricny, 1989).

beyond the mismatch (Fang & Modrich, 1993; Yu et al., 1999).

#### **2.3 Nucleotide Excision Repair**

Nucleotide excision repair is the most studied DNA repair system in humans (de Laat et al., 1999). The NER pathway is responsible for repair of bulky distortions in the shape of the DNA double helix such as chemical adducts, pyrimidine dimers, and cross-links caused by endogenous and environmental lesions (Riedl et al., 2003; van der Wees et al., 2007; Wood, 1989). This pathway may also be important in conferring resistance to chemotherapeutic agents such as platinum-based chemotherapy (Kennedy & D'Andrea, 2006).

NER has five stages: recognition of the bulky damage which distorts the DNA helix; hydrolyzing a phosphodiester bond in the deoxyribose backbone on the 5' side of the lesion; excising the damage; filling in the resultant gap using the information from the complementary strand; closing the nicked DNA to generate intact strand (Yasbin, 2002).

NER can be divided into two subpathways (Transcription coupled NER and Global genomic NER) that differ only in their recognition of helix-distorting DNA damage (Hanawalt, 2002). Global genome repair is a slow process of inspecting the entire genome for damage (Kennedy & D'Andrea AD, 2006). Transcription-coupled repair is more rapid, highly specific and efficient and repairs DNA damage that blocks the progression of RNA polymerase II.

The actual repair mechanism appears to be identical in transcription-coupled and in globalgenome repair. However, the damage recognition in global-genome repair does not involve the RNA polymerase, but is performed by the XPC and HHR23 proteins. Following lesion recognition, however, both repair systems use TFIIH components such as XPB and XPD, as well as the single-strand binding protein RPA and the XPA protein to fully unwind and mark the lesion. The damaged segment of DNA is excised through 5'-incision by the XPF endonuclease and 3'-incision by the XPG endonuclease. The DNA gap is filled by DNA polymerases δ or ε supported by PCNA and sealed by a DNA ligase, presumably DNA ligase I (Kennedy & D'Andrea, 2006; Yu et al., 1999).

#### **XPD**

The XPD gene (xeroderma pigmentosum group D, also known as ERCC2) encodes a DNA helicase involved in nucleotide excision repair pathway. The XPD gene maps to chromosome 19q13.3 and consists of 23 exons (Wang et al., 2008). Its protein is 761 amino acids in length. The XPD protein repairs a wide range of structurally unrelated lesions, such as bulky adducts and thymidine dimers (Braithwaite et al., 1999). The DNA repair process and gene transcription are coupled via activity of the TFIIH complex, a protein complex with functions including transcription, NER, transcription-coupled repair, apoptosis, and cell cycle regulation. XPD protein is involved in maintaining the stability of the TFIIH complex. The XPD gene product has an ATP-dependent DNA helicase activity (Laine et al, 2007).

Because of the biological significance of XPD, the XPD Lys751Gln (2251A>C) polymorphism has been a common subject of studies in different malignant diseases in the last years. Although the XPD 751Gln variant was associated with an increased risk of esophageal cancer and acute lymphoid leukemia (Wang et al., 2008) Allan et al. (2004) investigated XPD Lys751Gln polymorphism in 341 adult British AML patients and observed that the XPD codon 751 polymorphism is an independent prognostic marker for disease-free survival and overall survival in elderly AML patients treated with chemotherapy, and specifically that the glutamine variant was associated with a poorer prognosis relative to the lysine variant. In a pediatric study, Mehta et al. (2006) found no influence of XPD751 genotype on susceptibility to de novo AML in children. In another study, Kuptsova-Clarkson et al. (2010) evaluated the role of XPD and XRCC1 gene polymorphic variation in response to induction chemotherapy, toxicities and survival in a population of 293 predominantly Caucasian adult patients treated for AML. Kuptsova-Clarkson et al. (2010) had reported that in AML, variation in the XPD gene may be associated with suboptimal DNA repair activity and may thus predispose to therapy-related AML development. In a UK study by Seedhouse et al. (2002), therapy-related AML was not associated with XPD genotypes.

There have been studies of XPD, involved in NER, and survival of patients with AML (Allan et al., 2004). In a study of elderly patients with AML conducted by researchers in the United Kingdom, modestly increased hazard ratio (HR) of 1.30 and 1.19 were found for disease-free and overall survival, respectively, by XPD variant genotypes. However, in a study of pediatric patients with acute myeloid leukemia conducted by the the Children's Oncology Group (Mehta et al., 2006) survival and treatment-related mortality were not associated with XPD codon 751 genotypes.

#### **2.4 Mismatch repair**

390 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Nucleotide excision repair is the most studied DNA repair system in humans (de Laat et al., 1999). The NER pathway is responsible for repair of bulky distortions in the shape of the DNA double helix such as chemical adducts, pyrimidine dimers, and cross-links caused by endogenous and environmental lesions (Riedl et al., 2003; van der Wees et al., 2007; Wood, 1989). This pathway may also be important in conferring resistance to chemotherapeutic

NER has five stages: recognition of the bulky damage which distorts the DNA helix; hydrolyzing a phosphodiester bond in the deoxyribose backbone on the 5' side of the lesion; excising the damage; filling in the resultant gap using the information from the complementary strand; closing the nicked DNA to generate intact strand (Yasbin, 2002). NER can be divided into two subpathways (Transcription coupled NER and Global genomic NER) that differ only in their recognition of helix-distorting DNA damage (Hanawalt, 2002). Global genome repair is a slow process of inspecting the entire genome for damage (Kennedy & D'Andrea AD, 2006). Transcription-coupled repair is more rapid, highly specific and efficient and repairs DNA damage that blocks the progression of RNA

The actual repair mechanism appears to be identical in transcription-coupled and in globalgenome repair. However, the damage recognition in global-genome repair does not involve the RNA polymerase, but is performed by the XPC and HHR23 proteins. Following lesion recognition, however, both repair systems use TFIIH components such as XPB and XPD, as well as the single-strand binding protein RPA and the XPA protein to fully unwind and mark the lesion. The damaged segment of DNA is excised through 5'-incision by the XPF endonuclease and 3'-incision by the XPG endonuclease. The DNA gap is filled by DNA polymerases δ or ε supported by PCNA and sealed by a DNA ligase, presumably DNA

The XPD gene (xeroderma pigmentosum group D, also known as ERCC2) encodes a DNA helicase involved in nucleotide excision repair pathway. The XPD gene maps to chromosome 19q13.3 and consists of 23 exons (Wang et al., 2008). Its protein is 761 amino acids in length. The XPD protein repairs a wide range of structurally unrelated lesions, such as bulky adducts and thymidine dimers (Braithwaite et al., 1999). The DNA repair process and gene transcription are coupled via activity of the TFIIH complex, a protein complex with functions including transcription, NER, transcription-coupled repair, apoptosis, and cell cycle regulation. XPD protein is involved in maintaining the stability of the TFIIH complex. The

Because of the biological significance of XPD, the XPD Lys751Gln (2251A>C) polymorphism has been a common subject of studies in different malignant diseases in the last years. Although the XPD 751Gln variant was associated with an increased risk of esophageal cancer and acute lymphoid leukemia (Wang et al., 2008) Allan et al. (2004) investigated XPD Lys751Gln polymorphism in 341 adult British AML patients and observed that the XPD codon 751 polymorphism is an independent prognostic marker for disease-free survival and overall survival in elderly AML patients treated with chemotherapy, and specifically that the glutamine variant was associated with a poorer prognosis relative to the lysine variant. In a pediatric study, Mehta et al. (2006) found no influence of XPD751 genotype on susceptibility to de novo AML in children. In another study, Kuptsova-Clarkson et al. (2010)

XPD gene product has an ATP-dependent DNA helicase activity (Laine et al, 2007).

agents such as platinum-based chemotherapy (Kennedy & D'Andrea, 2006).

ligase I (Kennedy & D'Andrea, 2006; Yu et al., 1999).

**2.3 Nucleotide Excision Repair** 

polymerase II.

**XPD** 

DNA mismatch repair (MMR) plays a critical role in maintaining genomic integrity. MMR is responsible for correction of mismatched basepairs which occurs through processes including misincorporations during DNA replication, formation of heteroduplexes, and secondary structure such as imperfect palindromes (Bishop et al., 1985).

In addition, MMR can also process some types of DNA damage. MMR deficient tumors display widespread alterations in simple repetitive DNA sequences, a phenomenon also called microsatellite instability; MSI (Li, 2008). The repair mechanism is similar to that of excision repair; a patch of nucleotides is removed from one strand, and followed by resynthesis and ligation processes.

There are two types of mismatch repair, long-patch and short-patch which have been found in human cells. In short-patch repair system there are three enzymes possessing nicking activities specific for mismatch repair; T/G specific (Wiebauer & Jiricny, 1989), A/G specific, and all type mismatch nicking enzymes (Yeh et al., 1991). That enzymes have different mode of action, but in either an A/G mismatch or a T/G mismatch, it is usually the guanine that remains untouched by mismatch specific glycosylases (Wiebauer & Jiricny, 1989).

Long patch can repair all types of mismatches. Long-patch MMR removes a patch of one of the DNA strands from an incision on the to-be-removed strand to 90~170 nucleotides beyond the mismatch (Fang & Modrich, 1993; Yu et al., 1999).

Defects in the MMR pathway significantly increase the mutation frequency and promote oncogenesis. It has been documented that defects in MMR genes, are the genetic basis for certain types of hereditary and sporadic cancers, including hereditary nonpolyposis colorectal cancer (Mao et al., 2008). There are some studies looking at the incidence of MMR deficiency (microsatellite instability; MSI) in AML (Mao et al., 2008). Genomic instability in AML has led to a search for MSI in AML patients, but the results are quite controversial. While several studies have reported MSI in AML (Das-Gupta et al., 2001; Sheikhha et al., 2002), a study of 132 cases failed to confirm the previous results (Rimsza et al., 2000). According to Rimsza et al. (2000) MMR deficiency was associated with all stages of AML, but the rate of the deficiency was much higher in patients with refractory and relapsed AML than in de novo AML patients, suggesting that the loss of MMR function could contribute to the refractory and relapsed disease.

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

marker in oral oncology for primary prevention and intervention.

**XRCC5, XRCC6, XRCC7** 

of cancers (Wenshan et al., 2011).

& Jackson, 1999).

risk.

The Susceptibility and the Outcome After Therapy 393

There have been several studies showing that variations of the XRCC4 gene are associated with prostate (Chang et al., 2008), gastric (Chiu et al., 2008), and breast cancer (Fu et al., 2003). Therefore, XRCC4 and the DNA double-strand break repair pathway may serve as a common mechanism of early carcinogenesis. In a recent study, Wu et al. (2008) investigated the association between XRCC4 gene polymorphisms and oral cancer. Their findings suggest that the presence of the A allele of XRCC4 codon 247 was associated with a higher susceptibility to oral cancer, and the A allele of XRCC4 codon 247 may be a useful novel

A key component of the NHEJ pathway is the DNA-dependent Protein Kinase (DNA-PK), which consists of a heterodimeric DNA targeting subunit (i.e., Ku70/Ku80, encoded by XRCC6/XRCC5 genes) and a catalytic subunit DNA-PKcs, encoded by XRCC7 gene (Smith

**XRCC5** (X-ray repair cross-complementing 5) is a gene involved in repair of DNA doublestrand breaks. Abnormal expression of the XRCC5 protein is associated with genomic instability and an increased incidence of cancers. The XRCC5 gene mapped to chromosome 2q35, encodes the 80-kDa subunit of the Ku heterodimer protein, the DNA-binding component of the DNA-dependent protein kinase. The Ku80 is essential for maintaining genomic integrity through its ability to bind DNA double-strand breaks and to facilitate repair by the nonhomologous end-joining pathway in mammalian cells (Taccioli et al., 1994). **XRCC6** (X-ray repair complementing defective repair in Chinese hamster cells 6) is a helicase involved in DNA repair and chromatin remodeling. The XRCC6 gene (also called Ku70) maps to chromosome 22q13.2-q1. Ku70 plays an important role in the DNA doublestrand breaks repair and maintenance of genomic integrity. Genetic variations within human Ku70 have been demonstrated to be associated with increased risk of several types

**XRCC7** (X-ray repair cross complementing group 7) is located on chromosome 8q12, span about 110-180 kb and contains 100 exons. The human XRCC7 gene encodes DNA-PKcs, which is recruited to the site of DSBs by the Ku70/Ku80 heterodimer to form an active DNA-PK complex (Sipley et al., 1995). The expression of Ku70 and XRCC7 is elevated in bladder tumor tissue and head and neck cancer cell lines, respectively (Stronati et al., 2001; Sturgis et al., 1999) and Ku70 may function as a caretaker gene for the development of T-cell lymphomas (Li et al., 1998). XRCC7 encodes DNA-PKcs, which also may have a caretaker role in colon carcinogenesis. Therefore, the variants of the Ku70 and XRCC7 genes could be expected to have an effect on DSB repair, and thus, on carcinogenesis. The Ku70 polymorphism is associated with risk of breast cancer (Fu et al., 2003), and the XRCC7 polymorphism is associated with risk of glioma (Wang et al., 2004). According to Wang et al. (2008) the XRCC7 polymorphism appears to be involved in the etiology of human bladder cancer. This data support the notion that the XRCC7 polymorphism is implicated in cancer

Given the crucial roles of the NHEJ pathway in DNA repair (Gullo et al., 2006), it is possible that the XRCC5, XRCC6 and XRCC7 variants may modulate the risk of cancer, including leukemia. It has been shown that increased NHEJ activity is due to the presence of XRCC5 and XRCC6 protein, which results in genomic instability in myeloid leukemia cells (Gaymes et al., 2002). XRCC5 and XRCC6 may function as a caretaker gene for the development of Tcell lymphomas, while XRCC7 may have a caretaker role in colon carcinogenesis (Wang et

#### **2.5 Double Strand Break repair**

Double strand break repair (DSB) is responsible for the repair of double strand DNA breaks. DNA double strand breaks represent the most important class of DNA damage because, if unrepaired, they can result in a loss of genetic material, chromosome abnormalities and possibly cell death. Moreover, the breaks are prone to nuclease attack with subsequent destruction (Rufer & Morgan, 1992). Double strand breaks (DSBs) can be produced by exogenous agents such as ionizing radiation, some chemotherapeutic drugs, endogenous formed reactive oxygen species. When DNA replication forks encounter DNA single strand breaks or other types of lesion, it might result in formation of DSBs. In addition, DSBs are generated to initiate recombination between homologues chromosomes during meiosis, and also during the immunoglobulin class-switch recombination. Repair of DSBs is more difficult than other type of DNA damage because there is no undamaged template available (Khanna & Jackson, 2001).

Direct DSBs are mainly repaired by non-homologous end joining (Sargent et al., 1997), whereas replication-associated DSBs are repaired by homologous recombination (HR) and related replication repair pathways (Iliakis et al., 2004).

#### **2.5.1 Non-homologous End Joining**

Non homologous end joining (NHEJ) is active in all phases of the cell cycle and is considered to be the most important DSB repair pathway in mammalian cells. The NHEJ pathway is simpler than HR and requires no complementary template. The protein components of NHEJ include the catalytic subunit of DNA protein kinase (DNA-PKCS), the two regulatory subunits of the DNA-PK complex Ku70 and Ku80, DNA ligase IV with its cofactor XRCC4 (the X-ray cross complementing group 4 protein) and the nuclease artemis (Drouet et al., 2005; Khanna & Jackson, 2001). The Ku70/Ku80 (Ku) heterodimer is the first protein to bind to the damaged DNA ends. When bound to the DSB, Ku recruits and activates DNA-PKcs. These proteins play an important role in DNA DSB repair and will act as tumor suppressors. However, either the DNA protein kinase complex, or its three subunits individually, can also act as oncogenes, depending on the compartment of the cell in which they are expressed and on the cell cycle phase (Gullo et al., 2006).

#### **XRCC4**

The X-ray cross-complementing group 4 (XRCC4) gene is one of the specific members of the NHEJ system. Some of the SNPs of XRCC4 have been found to be associated with the susceptibility to different types of cancer. Two single nucleotide polymorphisms (SNPs) of XRCC4, one splicing-site polymorphism (SNP14 rs1805377:A4G) and one intronic polymorphism (SNP1 rs2075685:G4T), have been studied, and the results are conflicting (Allen-Brady et al., 2006; Fu et al., 2003). The protein encoded by XRCC4 consists of 336 amino acid residues distributed among 8 exons, and has a long helical stem domain responsible for multimerization and interaction with DNA ligase IV (Junop et al., 2000). By forming a complex with DNA ligase IV and DNA-dependent protein kinase, XRCC4 functions in the repair of DNA double-strand breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events (Hayden et al, 2007). The NHEJ pathway is required not only for normal development but also for suppression of tumors. Since it is one of the ubiquitous NHEJ components, XRCC4 might be considered as a potential tumor suppressor gene in cancer and leukemia.

There have been several studies showing that variations of the XRCC4 gene are associated with prostate (Chang et al., 2008), gastric (Chiu et al., 2008), and breast cancer (Fu et al., 2003). Therefore, XRCC4 and the DNA double-strand break repair pathway may serve as a common mechanism of early carcinogenesis. In a recent study, Wu et al. (2008) investigated the association between XRCC4 gene polymorphisms and oral cancer. Their findings suggest that the presence of the A allele of XRCC4 codon 247 was associated with a higher susceptibility to oral cancer, and the A allele of XRCC4 codon 247 may be a useful novel marker in oral oncology for primary prevention and intervention.

#### **XRCC5, XRCC6, XRCC7**

392 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Double strand break repair (DSB) is responsible for the repair of double strand DNA breaks. DNA double strand breaks represent the most important class of DNA damage because, if unrepaired, they can result in a loss of genetic material, chromosome abnormalities and possibly cell death. Moreover, the breaks are prone to nuclease attack with subsequent destruction (Rufer & Morgan, 1992). Double strand breaks (DSBs) can be produced by exogenous agents such as ionizing radiation, some chemotherapeutic drugs, endogenous formed reactive oxygen species. When DNA replication forks encounter DNA single strand breaks or other types of lesion, it might result in formation of DSBs. In addition, DSBs are generated to initiate recombination between homologues chromosomes during meiosis, and also during the immunoglobulin class-switch recombination. Repair of DSBs is more difficult than other type of DNA damage because there is no undamaged template available

Direct DSBs are mainly repaired by non-homologous end joining (Sargent et al., 1997), whereas replication-associated DSBs are repaired by homologous recombination (HR) and

Non homologous end joining (NHEJ) is active in all phases of the cell cycle and is considered to be the most important DSB repair pathway in mammalian cells. The NHEJ pathway is simpler than HR and requires no complementary template. The protein components of NHEJ include the catalytic subunit of DNA protein kinase (DNA-PKCS), the two regulatory subunits of the DNA-PK complex Ku70 and Ku80, DNA ligase IV with its cofactor XRCC4 (the X-ray cross complementing group 4 protein) and the nuclease artemis (Drouet et al., 2005; Khanna & Jackson, 2001). The Ku70/Ku80 (Ku) heterodimer is the first protein to bind to the damaged DNA ends. When bound to the DSB, Ku recruits and activates DNA-PKcs. These proteins play an important role in DNA DSB repair and will act as tumor suppressors. However, either the DNA protein kinase complex, or its three subunits individually, can also act as oncogenes, depending on the compartment of the cell

The X-ray cross-complementing group 4 (XRCC4) gene is one of the specific members of the NHEJ system. Some of the SNPs of XRCC4 have been found to be associated with the susceptibility to different types of cancer. Two single nucleotide polymorphisms (SNPs) of XRCC4, one splicing-site polymorphism (SNP14 rs1805377:A4G) and one intronic polymorphism (SNP1 rs2075685:G4T), have been studied, and the results are conflicting (Allen-Brady et al., 2006; Fu et al., 2003). The protein encoded by XRCC4 consists of 336 amino acid residues distributed among 8 exons, and has a long helical stem domain responsible for multimerization and interaction with DNA ligase IV (Junop et al., 2000). By forming a complex with DNA ligase IV and DNA-dependent protein kinase, XRCC4 functions in the repair of DNA double-strand breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events (Hayden et al, 2007). The NHEJ pathway is required not only for normal development but also for suppression of tumors. Since it is one of the ubiquitous NHEJ components, XRCC4 might be considered as a

in which they are expressed and on the cell cycle phase (Gullo et al., 2006).

potential tumor suppressor gene in cancer and leukemia.

**2.5 Double Strand Break repair** 

(Khanna & Jackson, 2001).

**XRCC4** 

**2.5.1 Non-homologous End Joining** 

related replication repair pathways (Iliakis et al., 2004).

A key component of the NHEJ pathway is the DNA-dependent Protein Kinase (DNA-PK), which consists of a heterodimeric DNA targeting subunit (i.e., Ku70/Ku80, encoded by XRCC6/XRCC5 genes) and a catalytic subunit DNA-PKcs, encoded by XRCC7 gene (Smith & Jackson, 1999).

**XRCC5** (X-ray repair cross-complementing 5) is a gene involved in repair of DNA doublestrand breaks. Abnormal expression of the XRCC5 protein is associated with genomic instability and an increased incidence of cancers. The XRCC5 gene mapped to chromosome 2q35, encodes the 80-kDa subunit of the Ku heterodimer protein, the DNA-binding component of the DNA-dependent protein kinase. The Ku80 is essential for maintaining genomic integrity through its ability to bind DNA double-strand breaks and to facilitate repair by the nonhomologous end-joining pathway in mammalian cells (Taccioli et al., 1994). **XRCC6** (X-ray repair complementing defective repair in Chinese hamster cells 6) is a helicase involved in DNA repair and chromatin remodeling. The XRCC6 gene (also called Ku70) maps to chromosome 22q13.2-q1. Ku70 plays an important role in the DNA doublestrand breaks repair and maintenance of genomic integrity. Genetic variations within human Ku70 have been demonstrated to be associated with increased risk of several types of cancers (Wenshan et al., 2011).

**XRCC7** (X-ray repair cross complementing group 7) is located on chromosome 8q12, span about 110-180 kb and contains 100 exons. The human XRCC7 gene encodes DNA-PKcs, which is recruited to the site of DSBs by the Ku70/Ku80 heterodimer to form an active DNA-PK complex (Sipley et al., 1995). The expression of Ku70 and XRCC7 is elevated in bladder tumor tissue and head and neck cancer cell lines, respectively (Stronati et al., 2001; Sturgis et al., 1999) and Ku70 may function as a caretaker gene for the development of T-cell lymphomas (Li et al., 1998). XRCC7 encodes DNA-PKcs, which also may have a caretaker role in colon carcinogenesis. Therefore, the variants of the Ku70 and XRCC7 genes could be expected to have an effect on DSB repair, and thus, on carcinogenesis. The Ku70 polymorphism is associated with risk of breast cancer (Fu et al., 2003), and the XRCC7 polymorphism is associated with risk of glioma (Wang et al., 2004). According to Wang et al. (2008) the XRCC7 polymorphism appears to be involved in the etiology of human bladder cancer. This data support the notion that the XRCC7 polymorphism is implicated in cancer risk.

Given the crucial roles of the NHEJ pathway in DNA repair (Gullo et al., 2006), it is possible that the XRCC5, XRCC6 and XRCC7 variants may modulate the risk of cancer, including leukemia. It has been shown that increased NHEJ activity is due to the presence of XRCC5 and XRCC6 protein, which results in genomic instability in myeloid leukemia cells (Gaymes et al., 2002). XRCC5 and XRCC6 may function as a caretaker gene for the development of Tcell lymphomas, while XRCC7 may have a caretaker role in colon carcinogenesis (Wang et

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

al., 2004) cancers.

nearly 4 fold.

strand transfer reactions.

G135C polymorphism in human carcinogenesis.

**RAD 51** 

The Susceptibility and the Outcome After Therapy 395

increased sensitivity to DNA damaging agents (Griffin, 2002). The human XRCC3 gene is located on chromosome 14q32.3 and consists of 17870 bases. The protein product of XRCC3 gene contains 346 amino acids. According to NCBI SNP database, XRCC3 gene has 111 SNPs. The main polymorphism in this gene involves the change of threonine (Thr) to methionine (Met) at codon 241 in exon 7 (Shen et al., 1998). Little is known about the functional consequences of this variation, although some studies observed a positive relation between the Thr241Met polymorphism and an increased risk for skin (Winsey et al., 2000), bladder (Matullo et al., 2001), breast (Garcia-Closas et al., 2006) and lung (Jacobsen et

A meta-analysis of 48 case–control studies, including 24975 cancer patients and 34209 controls, investigated the associations of the three DNA repair gene XRCC3 polymorphisms (Thr241Met; 4541A4G; 17893A4G) with cancer risk (Han et al., 2006). According to this meta-analysis individuals carrying the XRCC3 Met/Met genotype have a smaller cancer risk compared with the individuals with the Thr/Thr or Thr/Met genotype (OR=1.07; P=0.008; 95% CI=1.02~1.13) (Han et al., 2006). For A4541G, a significantly increased risk was associated with the variant genotypes (G/G+A/G), compared with the wild homozygote A/A genotype (OR=1.09; P=0.004; 95% CI, 1.03~1.15). For A17893G, individuals with the variant genotypes (G/G+A/G) had a significantly decreased cancer risk, compared with individuals with the A/A genotype under a dominant genetic model (OR=0.92; P=0.0004; 95% CI=0.87~0.96). Han et al. (2006) consider that the XRCC3 could not be a major increased risk factor for cancer but it might represent a low-penetrance susceptible gene especially for

In their study Seedhouse et al. (2004) have observed that the presence of variant XRCC3 241Met was associated with an increase in the risk of developing therapy-related AML of more than 8 fold, whereas the increase in risk for the development of de novo AML was

The RAD51 gene plays an important role in homologous recombination and in maintaining the genetic stability of the cell. In HR, RAD51 interacts with and is stabilized by XRCC3, during strand invasion and cross-strand resolution. RAD51 is a central protein in the HR repair pathway binding to DNA and promoting ATP-dependent homologous pairing and

The RAD51 gene is located on chromosome 15q15.1 and consists of 36998 bases. The protein product of RAD51 has 6 domains, one for DNA binding, one for ATPase activity, and the other domains are specific to action of RAD51. According to NCBI SNP database, RAD51 gene has 296 SNPs. The most important polymorphism identified for RAD51 is G135C SNP in 5' untranslated region. The RAD51 G135C polymorphism is associated with RAD51 protein over-expression (Richardson et al., 2004). Regarding the role of RAD51 in the homologous DNA repair mechanism, several studies have examined the relationship between RAD51 G135C polymorphism and risk of certain cancers. However the results from these studies are conflicting. Further studies are needed to establish the role of RAD51

Voso et al. (2007) found an increased frequency of the RAD51 135C allele in AML, mainly in de novo AML, when compared with controls, but not between therapy-related AML (t-AML) and controls. Other reports found increased frequency of the RAD51 135C allele in

cancer of breast, bladder, head and neck, and non-melanoma skin cancer

al., 2009). In a recent study, Wang et al. (2009) have investigated the association between the X-ray repair cross complementing group XRCC5, XRCC6 and XRCC7 polymorphisms and risk of AML in Chinese population. In this case-control study in a southern Chinese population three polymorphisms, XRCC5 2R/1R/0R, XRCC6 -61C>G and XRCC7 6721G>T were investigated. For the XRCC7 6721G>T polymorphism among AML cases and controls no significant association was observed (P=0.68). Significant association was observed (P=0.04) for the XRCC6 -61C > G polymorphism. Their analysis revealed that compared with the XRCC6 -61CC wild type homozygote, the -61CG heterozygotes had a significant 43% decreased risk of AML (adjusted OR=0.57; 95% CI=0.35~0.92) and subjects carrying - 61CG/GG variant genotypes had 45% decrease in risk of AML (adjusted OR=0.55; 95% CI=0.34~0.89). For the XRCC5 2R/1R/0R polymorphism, Wang et al. (2009) found that XRRC5 1R/0R genotype was associated with a 2.60-fold increase in risk of AML (95% CI=1.42~5.92) compared with the 2R/2R genotype. They found a significant association with the polymorphisms of XRCC5 2R/1R/0R, XRCC6 -61C>G and the risk of AML, but there was no evidence for an association between the XRCC7 6721G>T variants and AML.

#### **2.5.2 Homologous recombination**

Homologous recombination (HR) is one of the main pathways for the repair of DNA double strand breaks (DSBs). HR is thought to be particularly important in DNA repair occurring during cellular replication (Rollinson et al., 2007). Although HR is indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes. The repair process is complex and involves many proteins working coordinately. Key players include MRE11, RAD50, NBS1 (MRN), RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, RAD52, RAD54 and BRCA2 (Lieberman, 2008). A number of polymorphic genes involved in this pathway have been studied in AML.

Homologous recombination mechanism starts with degradation of the DNA next to the double-strand break, leaving single-strand ends. Next the single-strand end of the damaged strand invades and binds to its complementary DNA sequence on the homologous duplex. This is associated with the displacement of the cross-complementary strand toward the gap site, where it serves as a template for gap-filling DNA synthesis. The repair sequence is terminated by religation of the repair patches. This mechanism requires the presence of undamaged homologous DNA, and increased activity of this repair pathway has been observed.

RAD51 protein catalyzes homologous recombination through its homologous pairing and strand exchange activities. RAD52 may modulate these activities through its RAD51 interacting region. The ability of RAD52 to induce homologous recombination requires its binding to the p34 subunit of RPA. This RPA binding domain is at amino acids 221–280. Recall that RPA is also involved in NER—this may provide a linkage between these two repair systems (Yu Z et al., 1999).

#### **XRRC3**

The X-ray repair cross-complementing group 3 (XRCC3), codes for a protein participating in homologous recombination repair of DNA double-strand breaks. XRCC3 is a member of the RAD-51-related protein family. RAD-51-like proteins are known to participate in homologous recombination to maintain chromosome stability and repair DNA damage (Brenneman et al., 2000). XRCC3-deficient cells demonstrated genetic instability and increased sensitivity to DNA damaging agents (Griffin, 2002). The human XRCC3 gene is located on chromosome 14q32.3 and consists of 17870 bases. The protein product of XRCC3 gene contains 346 amino acids. According to NCBI SNP database, XRCC3 gene has 111 SNPs. The main polymorphism in this gene involves the change of threonine (Thr) to methionine (Met) at codon 241 in exon 7 (Shen et al., 1998). Little is known about the functional consequences of this variation, although some studies observed a positive relation between the Thr241Met polymorphism and an increased risk for skin (Winsey et al., 2000), bladder (Matullo et al., 2001), breast (Garcia-Closas et al., 2006) and lung (Jacobsen et al., 2004) cancers.

A meta-analysis of 48 case–control studies, including 24975 cancer patients and 34209 controls, investigated the associations of the three DNA repair gene XRCC3 polymorphisms (Thr241Met; 4541A4G; 17893A4G) with cancer risk (Han et al., 2006). According to this meta-analysis individuals carrying the XRCC3 Met/Met genotype have a smaller cancer risk compared with the individuals with the Thr/Thr or Thr/Met genotype (OR=1.07; P=0.008; 95% CI=1.02~1.13) (Han et al., 2006). For A4541G, a significantly increased risk was associated with the variant genotypes (G/G+A/G), compared with the wild homozygote A/A genotype (OR=1.09; P=0.004; 95% CI, 1.03~1.15). For A17893G, individuals with the variant genotypes (G/G+A/G) had a significantly decreased cancer risk, compared with individuals with the A/A genotype under a dominant genetic model (OR=0.92; P=0.0004; 95% CI=0.87~0.96). Han et al. (2006) consider that the XRCC3 could not be a major increased risk factor for cancer but it might represent a low-penetrance susceptible gene especially for cancer of breast, bladder, head and neck, and non-melanoma skin cancer

In their study Seedhouse et al. (2004) have observed that the presence of variant XRCC3 241Met was associated with an increase in the risk of developing therapy-related AML of more than 8 fold, whereas the increase in risk for the development of de novo AML was nearly 4 fold.

#### **RAD 51**

394 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

al., 2009). In a recent study, Wang et al. (2009) have investigated the association between the X-ray repair cross complementing group XRCC5, XRCC6 and XRCC7 polymorphisms and risk of AML in Chinese population. In this case-control study in a southern Chinese population three polymorphisms, XRCC5 2R/1R/0R, XRCC6 -61C>G and XRCC7 6721G>T were investigated. For the XRCC7 6721G>T polymorphism among AML cases and controls no significant association was observed (P=0.68). Significant association was observed (P=0.04) for the XRCC6 -61C > G polymorphism. Their analysis revealed that compared with the XRCC6 -61CC wild type homozygote, the -61CG heterozygotes had a significant 43% decreased risk of AML (adjusted OR=0.57; 95% CI=0.35~0.92) and subjects carrying - 61CG/GG variant genotypes had 45% decrease in risk of AML (adjusted OR=0.55; 95% CI=0.34~0.89). For the XRCC5 2R/1R/0R polymorphism, Wang et al. (2009) found that XRRC5 1R/0R genotype was associated with a 2.60-fold increase in risk of AML (95% CI=1.42~5.92) compared with the 2R/2R genotype. They found a significant association with the polymorphisms of XRCC5 2R/1R/0R, XRCC6 -61C>G and the risk of AML, but there was no evidence for an association between the XRCC7 6721G>T variants and AML.

Homologous recombination (HR) is one of the main pathways for the repair of DNA double strand breaks (DSBs). HR is thought to be particularly important in DNA repair occurring during cellular replication (Rollinson et al., 2007). Although HR is indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes. The repair process is complex and involves many proteins working coordinately. Key players include MRE11, RAD50, NBS1 (MRN), RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, RAD52, RAD54 and BRCA2 (Lieberman, 2008). A number of polymorphic genes

Homologous recombination mechanism starts with degradation of the DNA next to the double-strand break, leaving single-strand ends. Next the single-strand end of the damaged strand invades and binds to its complementary DNA sequence on the homologous duplex. This is associated with the displacement of the cross-complementary strand toward the gap site, where it serves as a template for gap-filling DNA synthesis. The repair sequence is terminated by religation of the repair patches. This mechanism requires the presence of undamaged homologous DNA, and increased activity of this repair pathway has been

RAD51 protein catalyzes homologous recombination through its homologous pairing and strand exchange activities. RAD52 may modulate these activities through its RAD51 interacting region. The ability of RAD52 to induce homologous recombination requires its binding to the p34 subunit of RPA. This RPA binding domain is at amino acids 221–280. Recall that RPA is also involved in NER—this may provide a linkage between these two

The X-ray repair cross-complementing group 3 (XRCC3), codes for a protein participating in homologous recombination repair of DNA double-strand breaks. XRCC3 is a member of the RAD-51-related protein family. RAD-51-like proteins are known to participate in homologous recombination to maintain chromosome stability and repair DNA damage (Brenneman et al., 2000). XRCC3-deficient cells demonstrated genetic instability and

**2.5.2 Homologous recombination** 

repair systems (Yu Z et al., 1999).

observed.

**XRRC3** 

involved in this pathway have been studied in AML.

The RAD51 gene plays an important role in homologous recombination and in maintaining the genetic stability of the cell. In HR, RAD51 interacts with and is stabilized by XRCC3, during strand invasion and cross-strand resolution. RAD51 is a central protein in the HR repair pathway binding to DNA and promoting ATP-dependent homologous pairing and strand transfer reactions.

The RAD51 gene is located on chromosome 15q15.1 and consists of 36998 bases. The protein product of RAD51 has 6 domains, one for DNA binding, one for ATPase activity, and the other domains are specific to action of RAD51. According to NCBI SNP database, RAD51 gene has 296 SNPs. The most important polymorphism identified for RAD51 is G135C SNP in 5' untranslated region. The RAD51 G135C polymorphism is associated with RAD51 protein over-expression (Richardson et al., 2004). Regarding the role of RAD51 in the homologous DNA repair mechanism, several studies have examined the relationship between RAD51 G135C polymorphism and risk of certain cancers. However the results from these studies are conflicting. Further studies are needed to establish the role of RAD51 G135C polymorphism in human carcinogenesis.

Voso et al. (2007) found an increased frequency of the RAD51 135C allele in AML, mainly in de novo AML, when compared with controls, but not between therapy-related AML (t-AML) and controls. Other reports found increased frequency of the RAD51 135C allele in

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

between affected individuals in the same family (Alter, 1993).

disorders with leukemia risk are described below.

AML and certain solid tumours.

pathway causes FA (D'Andrea, 2010).

almost 25%, and FANCE and FANCF for a further 8%.

for DNA damaging chemotherapeutic agents.

esophageal cancers and liver tumors (Alter, 2003; Rosenberg et al, 2008).

The Susceptibility and the Outcome After Therapy 397

Several of these diseases include an inherent predisposition to hematologic malignancies, including AML. The clinical features and molecular characteristics of several of the inherited

**Fanconi anemia** (FA) is a rare disorder with a birth incidence around 3 per million. FA is an autosomal recessive and rarely X-linked syndrome which is characterised by congenital abnormalities, defective haemopoiesis (bone marrow failure) and a high risk of developing

Affected individuals can have mild growth retardation, hypo- or hyperpigmented areas of the skin, skeletal defects including radial limb defects (absent thumb with or without radial aplasia), abnormalities of ribs and hips and scoliosis, cardiac and renal malformations, genital abnormalities (especially undescended testes, testicular agenesis, hypospadias). Other associated anomalies include micropthalmia and developmental delay (Grompe & D'Andrea, 2001). The phenotypic abnormalities are variable and there is marked variability

The Fanconi anemia defect results from biallelic mutation of any one of thirteen known Fanconi anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N). The proteins encoded by these Fanconi anemia genes cooperate in a common DNA repair pathway, referred to as the Fanconi anemia /BRCA pathway. In this pathway, eight of the Fanconi anemia proteins (A, B, C, E, F, G, L, M) are assembled into a core complex that functions as an E3 ubiquitin ligase. This ligase activates in response to DNA damage from a crosslinking drug, adding a 76-amino acid moiety onto two other Fanconi proteins, D2 and I. This monoubiquinated D2/I complex is translocated into chromatin, where it interacts with the downstream Fanconi proteins BRCA2, N, and J. This combination of proteins mediates the DNA repair process. After the repair has occurred, there is another enzyme complex, called USP1, which removes the ubiquitin and inactivates the pathway. Knocking out any of the proteins in this

FANCA, located on chromosome 16q24.3, is the most commonly mutated gene and is altered in 60-65% of FA patients (Steensma, 2007). FANCC and G mutations account for

Approximately one third of patients homozygous for a Fanconi anemia gene mutation will develop a hematologic or solid tumors by the age of 40 years (Kennedy & D'Andrea, 2006). Fanconi anemia patients develop predominately myeloid malignancies (the most common hematologic maliganancy is AML), although numerous other cancers arise, including squamous cell carcinomas of the head and neck or gynecologic system, skin cancers,

Fanconi anemia patients have a systemic DNA repair defect that results in a low tolerance

**Ataxia–telangiectasia** (AT) is a rare autosomal recessive disorder. This human disease is characterized by cerebellar degeneration, immunodeficiency, hypogonadism, growth retardation, genome instability, extreme sensitivity to radiation and predisposition to cancer (Taylor & Byrd, 2005). The disease is caused by homozygous mutations in the gene encoding the ATM protein kinase that plays a critical role in DNA damage detection and regulates DNA double-strand break repair (Mavrou et al, 2008). The ATM gene is located on chromosome 11q23. When ATM is dysfunctional or absent, cells are able to progress

from G1 to S phase and initiate DNA replication in the presence of DNA damage.

t-AML patients compared with controls (Seedhouse et al., 2004), suggesting an effect of RAD51 over-expression during leukemogenesis induced by chemotherapy or radiotherapy.

According to Bhatla et al. (2008) RAD51 gene polymorphism did not influence the outcome of AML therapy in the study of de novo AML patients. On the contrary, Liu et al. (2008) concluded that RAD51 gene polymorphism was significantly related to response to therapy, adverse effects, and prognosis of AML and reported that detection of the RAD51 gene polymorphism genotypes may be useful in selecting individual chemotherapy regimens for patients with AML. Also, Bolufer et al. (2007) reported that the RAD51 gene polymorphism showed significant unfavorable outcome among AML patients.

In their study, Bathla et al. (2008) observed a doubling of risk of AML in children with a RAD51 G135C variant allele and a wild-type XRCC3 Thre241Met genotype. In addition risk of AML was significantly increased in children with at least one variant XRCC3 Thr241Met allele. In antithesis, risk was not significantly elevated in children with variant alleles at both wild-type XRCC3 Thre241Met and RAD51 G135C. Liu et al. (2008) found that XRCC3 gene polymorphism was significantly related to response to therapy and prognosis of AML and reported that detection of the XRCC3 gene polymorphism genotypes may be useful in selecting individual chemotherapy regimens for AML patients.

#### **2.6 Translesion synthesis**

Translesion synthesis is an important mechanism by which cells replicate past DNA damage. The sliding clamp DNA polymerase processivity factors play a central role in this process. The clamps are dimeric in bacteria and trimeric in eukaryotes and archaea, raising the question of whether more than one polymerase can interact with the clamp simultaneously (Lehmann, 2006).

#### **3. Inherited human disease with leukemia susceptibility**

Several studies have demonstrated that the genes involved in DNA repair and maintenance of genome integrity are critically in protecting against mutations that lead to cancer and/or inherited human disease (Table 2).


Table 2. DNA repair defective syndromes

t-AML patients compared with controls (Seedhouse et al., 2004), suggesting an effect of RAD51 over-expression during leukemogenesis induced by chemotherapy or radiotherapy. According to Bhatla et al. (2008) RAD51 gene polymorphism did not influence the outcome of AML therapy in the study of de novo AML patients. On the contrary, Liu et al. (2008) concluded that RAD51 gene polymorphism was significantly related to response to therapy, adverse effects, and prognosis of AML and reported that detection of the RAD51 gene polymorphism genotypes may be useful in selecting individual chemotherapy regimens for patients with AML. Also, Bolufer et al. (2007) reported that the RAD51 gene polymorphism

In their study, Bathla et al. (2008) observed a doubling of risk of AML in children with a RAD51 G135C variant allele and a wild-type XRCC3 Thre241Met genotype. In addition risk of AML was significantly increased in children with at least one variant XRCC3 Thr241Met allele. In antithesis, risk was not significantly elevated in children with variant alleles at both wild-type XRCC3 Thre241Met and RAD51 G135C. Liu et al. (2008) found that XRCC3 gene polymorphism was significantly related to response to therapy and prognosis of AML and reported that detection of the XRCC3 gene polymorphism genotypes may be useful in

Translesion synthesis is an important mechanism by which cells replicate past DNA damage. The sliding clamp DNA polymerase processivity factors play a central role in this process. The clamps are dimeric in bacteria and trimeric in eukaryotes and archaea, raising the question of whether more than one polymerase can interact with the clamp

Several studies have demonstrated that the genes involved in DNA repair and maintenance of genome integrity are critically in protecting against mutations that lead to cancer and/or

FANC-A to FANC-N 16q24.3 Levitus et al., 2004;

NBS1 (Nibrin) 8p21 Digweed et al., 1999;

BLM (RECQL3) 15q26.1 Ellis & German, 1996;

Steensma, 2007;

Varon et al. 2003

Thompson & Schild,

Thompson & Schild,

11q22.3 Savitsky et al., 1995;

2002

2002

showed significant unfavorable outcome among AML patients.

selecting individual chemotherapy regimens for AML patients.

**3. Inherited human disease with leukemia susceptibility** 

Ataxia-telangiectasia

(AT)

Table 2. DNA repair defective syndromes

**Syndrome Gene(s) involved Chromosome References** 

Seckel Syndrome ATR 3q22-24 Casper et al., 2002;

**2.6 Translesion synthesis** 

simultaneously (Lehmann, 2006).

inherited human disease (Table 2).

Fanconi anaemia

syndrome (NBS)

Bloom syndrome

(FA)

(AT)

(BLM)

Nijmegen breakage

Ataxiatelangiectasia Several of these diseases include an inherent predisposition to hematologic malignancies, including AML. The clinical features and molecular characteristics of several of the inherited disorders with leukemia risk are described below.

**Fanconi anemia** (FA) is a rare disorder with a birth incidence around 3 per million. FA is an autosomal recessive and rarely X-linked syndrome which is characterised by congenital abnormalities, defective haemopoiesis (bone marrow failure) and a high risk of developing AML and certain solid tumours.

Affected individuals can have mild growth retardation, hypo- or hyperpigmented areas of the skin, skeletal defects including radial limb defects (absent thumb with or without radial aplasia), abnormalities of ribs and hips and scoliosis, cardiac and renal malformations, genital abnormalities (especially undescended testes, testicular agenesis, hypospadias). Other associated anomalies include micropthalmia and developmental delay (Grompe & D'Andrea, 2001). The phenotypic abnormalities are variable and there is marked variability between affected individuals in the same family (Alter, 1993).

The Fanconi anemia defect results from biallelic mutation of any one of thirteen known Fanconi anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N). The proteins encoded by these Fanconi anemia genes cooperate in a common DNA repair pathway, referred to as the Fanconi anemia /BRCA pathway. In this pathway, eight of the Fanconi anemia proteins (A, B, C, E, F, G, L, M) are assembled into a core complex that functions as an E3 ubiquitin ligase. This ligase activates in response to DNA damage from a crosslinking drug, adding a 76-amino acid moiety onto two other Fanconi proteins, D2 and I. This monoubiquinated D2/I complex is translocated into chromatin, where it interacts with the downstream Fanconi proteins BRCA2, N, and J. This combination of proteins mediates the DNA repair process. After the repair has occurred, there is another enzyme complex, called USP1, which removes the ubiquitin and inactivates the pathway. Knocking out any of the proteins in this pathway causes FA (D'Andrea, 2010).

FANCA, located on chromosome 16q24.3, is the most commonly mutated gene and is altered in 60-65% of FA patients (Steensma, 2007). FANCC and G mutations account for almost 25%, and FANCE and FANCF for a further 8%.

Approximately one third of patients homozygous for a Fanconi anemia gene mutation will develop a hematologic or solid tumors by the age of 40 years (Kennedy & D'Andrea, 2006). Fanconi anemia patients develop predominately myeloid malignancies (the most common hematologic maliganancy is AML), although numerous other cancers arise, including squamous cell carcinomas of the head and neck or gynecologic system, skin cancers, esophageal cancers and liver tumors (Alter, 2003; Rosenberg et al, 2008).

Fanconi anemia patients have a systemic DNA repair defect that results in a low tolerance for DNA damaging chemotherapeutic agents.

**Ataxia–telangiectasia** (AT) is a rare autosomal recessive disorder. This human disease is characterized by cerebellar degeneration, immunodeficiency, hypogonadism, growth retardation, genome instability, extreme sensitivity to radiation and predisposition to cancer (Taylor & Byrd, 2005). The disease is caused by homozygous mutations in the gene encoding the ATM protein kinase that plays a critical role in DNA damage detection and regulates DNA double-strand break repair (Mavrou et al, 2008). The ATM gene is located on chromosome 11q23. When ATM is dysfunctional or absent, cells are able to progress from G1 to S phase and initiate DNA replication in the presence of DNA damage.

The Association of the DNA Repair Genes with Acute Myeloid Leukemia:

the DNA repair mechanisms and risk of AML.

Government under the contract number POSDRU 60782.

No.7 (July 2006), pp. 1306–1310, ISSN: 1538-7755

pp. 3872–3877, ISSN: 1528-0020

pp.9-14, ISSN: 1365-2141.

pp.425-440, ISSN: 1097-0142.

**5. Acknowledgement** 

**6. References** 

6924

8721

The Susceptibility and the Outcome After Therapy 399

to be fully established. There is evidence that some polymorphisms in DNA repair genes play a role in carcinogenesis, notably hOGG1 Ser326Cys, XRCC1 Arg194Trp, XRCC3 Thr241Met, RAD51 G135C and XPD Lys751Gln. Additional studies of these and other DNA repair polymorphisms will provide essential information about the relationships between

This paper is partly supported by the Sectorial operational programme human resources development (SOP HRD), financed from the European social Fund and by the Romanian

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repair of chromosome breaks by homologous recombination. *Mutat Res*, Vol.459,

Approximately one third of AT patients develop cancer, mainly leukemias and lymphomas which develop in childhood and are a common cause of death (Ball & Xiao, 2005; Gumy-Pause et al, 2004). Solid tumors in AT patients are usually adenocarcinoma of the stomach, dysgerminoma, gonadoblastoma and medulloblastoma (Mavrou et al, 2008).

**Bloom's syndrome** (BS) is a rare autosomal recessive syndrome of growth retardation, telangiectasia manifest by facial erythema, immunodeficiency, and skull abnormalities. BS patients also are predisposed to cancer, as they develop mostly leukemias and lymphomas in about half of the patients. This disorder is most commonly found in the Ashkenazi Jewish population resulting from a founder mutation (Ellis et al, 1998). It is characterised by low birth weight, growth deficiency, characteristic facies (long thin face, prominent nose) sunsensitivity, immunodeficiency and infertility in males.

Bloom's syndrome arise through mutations in both copies of the BLM gene, which is located on chromosome 15 at 15q26.1. This gene encodes a member of the RecQ family of DNA helicases (BLM) that is important in maintaining appropriate DNA conformation during chromosomal recombination and repair. Together with topoisomerase III, BLM resolves Holliday junctions during homologous recombination (HR) by a mechanism called double-junction dissolution that is distinct from classical Holliday junction resolution and that prevents erroneous exchange of flanking sequences (Steensma, 2007).

**Nijmegen breakage syndrome** (NBS) is an autosomal recessive disorder that is most commonly found in Eastern Europe. NBS is caused by abnormalities in the NBN gene at 8p21 (Varon et al, 2003), which encodes the protein NBN (NBS1). Complete loss of this polypeptide is lethal. The NBS gene encodes a 95-kDa protein that binds with MRE11 and RAD50 to form a nuclease-containing protein complex that appears to be involved in homologous and nonhomologous recombination. Clinical features include growth retardation, microcephaly, skin findings such as vitiligo and café au lait spots, skeletal defects, immunodeficiency and propensity to infection. Radiation hypersensitivity is a hallmark of the disease, along with a predisposition to cancer, most notably lymphomas (Digweed et al, 1999). The most common cause of death for NBS patients is neoplasia (Steensma, 2007). Although the predominant neoplasm is lymphoma, both lymphoid and myeloid leukemia have been reported (Resnick et al, 2002).

**Seckel syndrome** (SCKL) is a rare autosomal recessive disorder associated with short stature, prenatal and postnatal growth retardation, characteristic craniofacial dysmorphism (bird-headed face including prominent beaked nose, micrognathia and malformed ears), mental deficiency, microcephaly, and skeletal defects (Faivre et al, 2002). Hematological abnormalities, including pancytopenia, myelodysplasia and acute myeloid leukemia, have been reported in some patients with Seckel syndrome (Chanan-Khan et al, 2003; Hayani et al,1994). A gene for Seckel syndrome was mapped on chromosome 3q22.1-q24. The ataxiatelancgiectasia and RAD3-related (ATR) gene is mutated in Seckel syndrome, and encodes an phosphotidylinositol-3-kinase-like kinase which has distinct, but overlapping functions with ATM in co-ordinating the response to DNA damage. ATR is activated by single stranded DNA whilst ATM responds to DNA double strand breaks (Casper et al, 2002; Steensma, 2007).

#### **4. Conclusion**

Genetic variations in genes involved in DNA repair may influence both cancer susceptibility and treatment response. However, in AML, the relevance of these genetic variations remains to be fully established. There is evidence that some polymorphisms in DNA repair genes play a role in carcinogenesis, notably hOGG1 Ser326Cys, XRCC1 Arg194Trp, XRCC3 Thr241Met, RAD51 G135C and XPD Lys751Gln. Additional studies of these and other DNA repair polymorphisms will provide essential information about the relationships between the DNA repair mechanisms and risk of AML.

#### **5. Acknowledgement**

This paper is partly supported by the Sectorial operational programme human resources development (SOP HRD), financed from the European social Fund and by the Romanian Government under the contract number POSDRU 60782.

#### **6. References**

398 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Approximately one third of AT patients develop cancer, mainly leukemias and lymphomas which develop in childhood and are a common cause of death (Ball & Xiao, 2005; Gumy-Pause et al, 2004). Solid tumors in AT patients are usually adenocarcinoma of the stomach,

**Bloom's syndrome** (BS) is a rare autosomal recessive syndrome of growth retardation, telangiectasia manifest by facial erythema, immunodeficiency, and skull abnormalities. BS patients also are predisposed to cancer, as they develop mostly leukemias and lymphomas in about half of the patients. This disorder is most commonly found in the Ashkenazi Jewish population resulting from a founder mutation (Ellis et al, 1998). It is characterised by low birth weight, growth deficiency, characteristic facies (long thin face, prominent nose) sun-

Bloom's syndrome arise through mutations in both copies of the BLM gene, which is located on chromosome 15 at 15q26.1. This gene encodes a member of the RecQ family of DNA helicases (BLM) that is important in maintaining appropriate DNA conformation during chromosomal recombination and repair. Together with topoisomerase III, BLM resolves Holliday junctions during homologous recombination (HR) by a mechanism called double-junction dissolution that is distinct from classical Holliday junction resolution and

**Nijmegen breakage syndrome** (NBS) is an autosomal recessive disorder that is most commonly found in Eastern Europe. NBS is caused by abnormalities in the NBN gene at 8p21 (Varon et al, 2003), which encodes the protein NBN (NBS1). Complete loss of this polypeptide is lethal. The NBS gene encodes a 95-kDa protein that binds with MRE11 and RAD50 to form a nuclease-containing protein complex that appears to be involved in homologous and nonhomologous recombination. Clinical features include growth retardation, microcephaly, skin findings such as vitiligo and café au lait spots, skeletal defects, immunodeficiency and propensity to infection. Radiation hypersensitivity is a hallmark of the disease, along with a predisposition to cancer, most notably lymphomas (Digweed et al, 1999). The most common cause of death for NBS patients is neoplasia (Steensma, 2007). Although the predominant neoplasm is lymphoma, both lymphoid and

**Seckel syndrome** (SCKL) is a rare autosomal recessive disorder associated with short stature, prenatal and postnatal growth retardation, characteristic craniofacial dysmorphism (bird-headed face including prominent beaked nose, micrognathia and malformed ears), mental deficiency, microcephaly, and skeletal defects (Faivre et al, 2002). Hematological abnormalities, including pancytopenia, myelodysplasia and acute myeloid leukemia, have been reported in some patients with Seckel syndrome (Chanan-Khan et al, 2003; Hayani et al,1994). A gene for Seckel syndrome was mapped on chromosome 3q22.1-q24. The ataxiatelancgiectasia and RAD3-related (ATR) gene is mutated in Seckel syndrome, and encodes an phosphotidylinositol-3-kinase-like kinase which has distinct, but overlapping functions with ATM in co-ordinating the response to DNA damage. ATR is activated by single stranded DNA whilst ATM responds to DNA double strand breaks (Casper et al, 2002;

Genetic variations in genes involved in DNA repair may influence both cancer susceptibility and treatment response. However, in AML, the relevance of these genetic variations remains

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**4. Conclusion** 


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**20** 

*Malaysia* 

**Apoptosis and Apoptosis Modulators** 

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults (American Cancer Society, 2010) however overall survival rate remain poor despite

Since the last 50 years, systemic chemotherapy has greatly improved outcome in many types of cancers. The use of continuous infusion Arabinosylcytosine (Ara-C) combined with another agent, usually an anthracycline or anthracenedione, the "3+7" regimen, has been the backbone of induction therapy for AML cases (Yates et al., 1973). An attempt to add other drugs (Preisler et al., 1987) and intensification of the Ara-C dose (Schiller et al., 1992; Weick et al., 1996) to this approach has achieved some degree of success. Currently more work is attempted at improving patient outcome by intensifying the doses of anthracyclines (Lowenberg et al., 2010a) or by adding targeted therapies like gemtuzumabozogamicin

For consolidation therapy, the use of Ara-C with or without other agents has been employed to maintain remission and cure. Allogeneic hematopoietic cell transplantation (HCT) based on initial cytogenetic (Cornelissen et al., 2007; Koreth et al., 2009) and molecular studies (Castaigne et al., 2004) have been proposed as an alternative

Induction therapy aims to produce complete remission (CR) defined as a marrow with less than 5% blast, a neutrophil count greater than 1000/mm3 and a platelet count greater than 100,000/mm3 (Cheson et al., 2003). Majority of younger patients (65-75%) will achieve CR after receiving induction treatment while CR in elderly group is much lower

Patients who do not respond to induction treatment display chemotherapy resistance (Estey et al., 1996). In trials done by the Southwest Oncology Group (SWOG), resistant disease was found in about 33% (patients younger than 56 ) out of 404 patients' enrolled into the studies, 62% for patients in between 56-65 year old, 61% for patients between 66-75 years old and

Resistance is also common at relapse (Estey et al., 1996). Relapse itself could be due to resistance to treatment in a subgroup of leukaemic cells which survived induction therapy

despite CR. Patients usually relapse within two to three years after achieving CR.

**1. Introduction** 

advancement in treatment modality.

(Lowenberg et al., 2010b; Nabhan et al., 2005).

57% for age more than 75 year old (Frederick et al., 2006).

consolidation therapies.

(40-50%).

**in Myeloid Leukemia** 

*University Putra Malaysia* 

Maha Abdullah and Zainina Seman


### **Apoptosis and Apoptosis Modulators in Myeloid Leukemia**

Maha Abdullah and Zainina Seman *University Putra Malaysia Malaysia* 

#### **1. Introduction**

408 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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overview. *Environ Mol Mutagen*, Vol.33, No.1 (February 1999), pp.3–20, ISSN: 0893-

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults (American Cancer Society, 2010) however overall survival rate remain poor despite advancement in treatment modality.

Since the last 50 years, systemic chemotherapy has greatly improved outcome in many types of cancers. The use of continuous infusion Arabinosylcytosine (Ara-C) combined with another agent, usually an anthracycline or anthracenedione, the "3+7" regimen, has been the backbone of induction therapy for AML cases (Yates et al., 1973). An attempt to add other drugs (Preisler et al., 1987) and intensification of the Ara-C dose (Schiller et al., 1992; Weick et al., 1996) to this approach has achieved some degree of success. Currently more work is attempted at improving patient outcome by intensifying the doses of anthracyclines (Lowenberg et al., 2010a) or by adding targeted therapies like gemtuzumabozogamicin (Lowenberg et al., 2010b; Nabhan et al., 2005).

For consolidation therapy, the use of Ara-C with or without other agents has been employed to maintain remission and cure. Allogeneic hematopoietic cell transplantation (HCT) based on initial cytogenetic (Cornelissen et al., 2007; Koreth et al., 2009) and molecular studies (Castaigne et al., 2004) have been proposed as an alternative consolidation therapies.

Induction therapy aims to produce complete remission (CR) defined as a marrow with less than 5% blast, a neutrophil count greater than 1000/mm3 and a platelet count greater than 100,000/mm3 (Cheson et al., 2003). Majority of younger patients (65-75%) will achieve CR after receiving induction treatment while CR in elderly group is much lower (40-50%).

Patients who do not respond to induction treatment display chemotherapy resistance (Estey et al., 1996). In trials done by the Southwest Oncology Group (SWOG), resistant disease was found in about 33% (patients younger than 56 ) out of 404 patients' enrolled into the studies, 62% for patients in between 56-65 year old, 61% for patients between 66-75 years old and 57% for age more than 75 year old (Frederick et al., 2006).

Resistance is also common at relapse (Estey et al., 1996). Relapse itself could be due to resistance to treatment in a subgroup of leukaemic cells which survived induction therapy despite CR. Patients usually relapse within two to three years after achieving CR.

Apoptosis and Apoptosis Modulators in Myeloid Leukemia 411

significant difference in response between P-gp positive and P-gp negative patients (Wen et al., 2006). Nevertheless, an early study revealed by using P-gp reversal modulators, the emergence of drug resistance could be prevented (Futscher et al., 1996). However, a recent randomized phase III trial involving 302 newly diagnosed AML patients, evaluated the effect of P-gp inhibitor valspodar (PSC-833) showed no difference in overall disease survival (Jonathan et al., 2010). Similar result was obtained in another phase III randomized trial involving poor risk AML patients when valspodar was added in the induction therapy

AML is characterized by a high degree of heterogeneity with respect to chromosome abnormalities, gene mutations and expression of multiple genes. The heterogeneous nature of AML has significant clinical impact as there are marked differences in survival following intensive chemotherapy (explained in detail elsewhere in this book). The World Health Organization (WHO) classifies AML by cytogenetics, morphology, immunophenotype and clinical features (Swerdlow et al., 2008). Diagnostic karyotype emerges as the most significant prognostic factor as determined in multivariable analyses that take into account age, type of AML (*de novo* or secondary) and presenting white blood cell count (WBC), and accordingly provides the framework for current risk stratified treatment approaches (Grimwade, 2007). Nevertheless as cytogenetic and molecular genetic aberrations are not mutually exclusive the expression of downstream target genes that encode proteins involved in complex biologic networks are affected (Mrozek et al., 2009) and may alter predictability of standard prognostic markers. Microarray genome-wide gene-expression profiling (GEP) and microRNA-expression profiling assays have revealed AML signatures and may be readily applicable for diagnosis and outcome class prediction in AML (Mrozek et al., 2009). Many of the molecules involved are known mediators of signal transduction

Apoptosis occurs principally via two separate yet interlinked signaling mechanisms: the extrinsic pathway, activated by proapoptotic receptor signals at the cellular surface (members of tumor necrosis factor, TNF, family), and the intrinsic pathway (members of Bcl-2 family), activated by mitochondrial signals from within the cell. These pathways converge through "effector" caspases, which orchestrate the apoptotic program. Nevertheless, each requires different initiation caspases to begin the process. The *extrinsic*  pathway is activated by engagement of death receptors on the cell membrane. The death receptors involved in the extrinsic apoptotic pathway belong to the TNF receptor superfamily that include Fas (CD95 or Apo1), TNFR1 (TNF receptor 1), death receptor 3 (DR3/Wsl-1/APO-3/TRAMP/LARD), death receptor 4 (DR4/TRAIL-R1), death receptor 5 (DR5/TRAIL-R2) and DR6. These receptors are characterized by an intracellular death domain. There are also decoy receptors (i.e. DcR1 and DcR2) that contain no death domain or a truncated death domain and can bind ligand but cannot signal. Therefore, these decoy receptors function as antagonists to inhibit death ligand/death receptor-induced apoptosis. Binding of ligands, such as FasL, tumor necrosis factor-alpha (TNF-alpha) and TNF-related apoptosis-inducing ligand (TRAIL) to their respective membrane receptors Fas, TNF-R and

(Peter et al., 2004)

pathways and apoptosis.

**4. Apoptotic molecules in AML** 

**3. Molecular 'signatures' in AML** 

#### **2. Multi-drug resistance protein as a mechanism of drug resistance**

Development of drug resistance is a major problem in AML therapy. It will eventually occur in most haematological malignancies treated with chemotherapy. Classically, drug resistance is divided into extrinsic and intrinsic (Jean-Pierre et al., 2003). Extrinsic resistance (host factors) refers to the inability of the drug to reach the tumour cell. It occurs when the bioavailability of the oral form varies from patient to patient like poor absorption resulting in low serum levels.

Intrinsic (cellular) resistance is due to properties of the tumour cell. It can be classified as simple resistance, when cells are resistant to only one particular drug, or as multidrug resistance (MDR) when cross resistance is observed among chemotherapeutic drugs with different biochemical targets. Multidrug resistance is more common than simple resistance and it can be due to several mechanisms. The most common pharmacological mechanism involved is due to an active efflux of drugs from the tumour cells or enhanced drug metabolism which prevented the drug from reaching its target in the nucleus.

The most important protein described in MDR cells is P-glycoprotein (P-gp), a transmembrane energy-dependent drug efflux pump, which is most efficient at transporting naturally occurring substances. It is encoded by the MDR1/ABCB1 gene and belongs to a superfamily of ABC (ATP binding cassette) transporters. P-gp expression in AML at initial presentation has been reported to be 20% to 40% (Motoji T et al., 2000). Increase in P-gp expression in leukaemic cells causes reduced intracellular concentration of cytotoxic drugs. There are many drugs used in AML that are transported by P-gp including anthracyclines and anthracenediones like daunorubicin and mitoxantrone, the vinca alkaloids (vincristine and vinblastine) and the epipodophyllotoxins (etoposide and teniposide).

Other ABC transport proteins that have been implicated in MDR include the multi-drug resistance associated proteins (MRP1/ABCC1) and the breast cancer resistance protein (BCRP/ABCG2). All these proteins are not unique to drug resistance cells but expressed in tissue with excretory and secretory functions. However, many studies have found that overexpression of these proteins correlate with poor treatment response (Damiani et al., 2010; Bendarra et al., 2005).

A non-ABC protein, found widely expressed in P-gp negative multidrug resistant cancer cell termed initially as lung resistance related protein (LRP) and now known as major vault protein (MVP) also has been implicated in drug resistance mechanism (Izquierdo et al., 1996; Huh et al., 2006). This protein is involved in bidirectional transportation of a variety of substrates between nucleus and cytoplasm. It is present in many cells and seems to be upregulated in cancer cells and has been found to be an adverse prognostic factor in AML (Styczynski et al., 2007). The expression of P-gp (Leith et al., 1999), MRP and LRP in AML was also found to correlate with advanced age (>60 years) and high white cell count (van delHeuvel et al., 2007). It also correlates with high risk of relapse (Daniela et al., 2007).

There have been extensive trials conducted on AML therapy to circumvent drug resistance like reversion of P-gp, targeted agents against DNA replication and repair, cell cycling and apoptosis.

With the extensive knowledge on P-gp efflux mechanism and its contribution to drug resistance in AML, quinine and cyclosporine were tested to reverse the P-gp action. However, these substances did not significantly improve the response rate in AML (Eric et al., 2003; Solary et al., 1996; Liu et al., 1998; Tallman et al., 1995). Combination of tetrandrine, a potent inhibitor of the MDR-1 efflux pump, with induction therapy also showed no significant difference in response between P-gp positive and P-gp negative patients (Wen et al., 2006). Nevertheless, an early study revealed by using P-gp reversal modulators, the emergence of drug resistance could be prevented (Futscher et al., 1996). However, a recent randomized phase III trial involving 302 newly diagnosed AML patients, evaluated the effect of P-gp inhibitor valspodar (PSC-833) showed no difference in overall disease survival (Jonathan et al., 2010). Similar result was obtained in another phase III randomized trial involving poor risk AML patients when valspodar was added in the induction therapy (Peter et al., 2004)

#### **3. Molecular 'signatures' in AML**

410 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Development of drug resistance is a major problem in AML therapy. It will eventually occur in most haematological malignancies treated with chemotherapy. Classically, drug resistance is divided into extrinsic and intrinsic (Jean-Pierre et al., 2003). Extrinsic resistance (host factors) refers to the inability of the drug to reach the tumour cell. It occurs when the bioavailability of the oral form varies from patient to patient like poor absorption resulting

Intrinsic (cellular) resistance is due to properties of the tumour cell. It can be classified as simple resistance, when cells are resistant to only one particular drug, or as multidrug resistance (MDR) when cross resistance is observed among chemotherapeutic drugs with different biochemical targets. Multidrug resistance is more common than simple resistance and it can be due to several mechanisms. The most common pharmacological mechanism involved is due to an active efflux of drugs from the tumour cells or enhanced drug

The most important protein described in MDR cells is P-glycoprotein (P-gp), a transmembrane energy-dependent drug efflux pump, which is most efficient at transporting naturally occurring substances. It is encoded by the MDR1/ABCB1 gene and belongs to a superfamily of ABC (ATP binding cassette) transporters. P-gp expression in AML at initial presentation has been reported to be 20% to 40% (Motoji T et al., 2000). Increase in P-gp expression in leukaemic cells causes reduced intracellular concentration of cytotoxic drugs. There are many drugs used in AML that are transported by P-gp including anthracyclines and anthracenediones like daunorubicin and mitoxantrone, the vinca alkaloids (vincristine

Other ABC transport proteins that have been implicated in MDR include the multi-drug resistance associated proteins (MRP1/ABCC1) and the breast cancer resistance protein (BCRP/ABCG2). All these proteins are not unique to drug resistance cells but expressed in tissue with excretory and secretory functions. However, many studies have found that overexpression of these proteins correlate with poor treatment response (Damiani et al.,

A non-ABC protein, found widely expressed in P-gp negative multidrug resistant cancer cell termed initially as lung resistance related protein (LRP) and now known as major vault protein (MVP) also has been implicated in drug resistance mechanism (Izquierdo et al., 1996; Huh et al., 2006). This protein is involved in bidirectional transportation of a variety of substrates between nucleus and cytoplasm. It is present in many cells and seems to be upregulated in cancer cells and has been found to be an adverse prognostic factor in AML (Styczynski et al., 2007). The expression of P-gp (Leith et al., 1999), MRP and LRP in AML was also found to correlate with advanced age (>60 years) and high white cell count (van delHeuvel et al., 2007). It also correlates with high risk of relapse (Daniela et al., 2007). There have been extensive trials conducted on AML therapy to circumvent drug resistance like reversion of P-gp, targeted agents against DNA replication and repair, cell cycling and

With the extensive knowledge on P-gp efflux mechanism and its contribution to drug resistance in AML, quinine and cyclosporine were tested to reverse the P-gp action. However, these substances did not significantly improve the response rate in AML (Eric et al., 2003; Solary et al., 1996; Liu et al., 1998; Tallman et al., 1995). Combination of tetrandrine, a potent inhibitor of the MDR-1 efflux pump, with induction therapy also showed no

**2. Multi-drug resistance protein as a mechanism of drug resistance** 

metabolism which prevented the drug from reaching its target in the nucleus.

and vinblastine) and the epipodophyllotoxins (etoposide and teniposide).

in low serum levels.

2010; Bendarra et al., 2005).

apoptosis.

AML is characterized by a high degree of heterogeneity with respect to chromosome abnormalities, gene mutations and expression of multiple genes. The heterogeneous nature of AML has significant clinical impact as there are marked differences in survival following intensive chemotherapy (explained in detail elsewhere in this book). The World Health Organization (WHO) classifies AML by cytogenetics, morphology, immunophenotype and clinical features (Swerdlow et al., 2008). Diagnostic karyotype emerges as the most significant prognostic factor as determined in multivariable analyses that take into account age, type of AML (*de novo* or secondary) and presenting white blood cell count (WBC), and accordingly provides the framework for current risk stratified treatment approaches (Grimwade, 2007). Nevertheless as cytogenetic and molecular genetic aberrations are not mutually exclusive the expression of downstream target genes that encode proteins involved in complex biologic networks are affected (Mrozek et al., 2009) and may alter predictability of standard prognostic markers. Microarray genome-wide gene-expression profiling (GEP) and microRNA-expression profiling assays have revealed AML signatures and may be readily applicable for diagnosis and outcome class prediction in AML (Mrozek et al., 2009). Many of the molecules involved are known mediators of signal transduction pathways and apoptosis.

#### **4. Apoptotic molecules in AML**

Apoptosis occurs principally via two separate yet interlinked signaling mechanisms: the extrinsic pathway, activated by proapoptotic receptor signals at the cellular surface (members of tumor necrosis factor, TNF, family), and the intrinsic pathway (members of Bcl-2 family), activated by mitochondrial signals from within the cell. These pathways converge through "effector" caspases, which orchestrate the apoptotic program. Nevertheless, each requires different initiation caspases to begin the process. The *extrinsic*  pathway is activated by engagement of death receptors on the cell membrane. The death receptors involved in the extrinsic apoptotic pathway belong to the TNF receptor superfamily that include Fas (CD95 or Apo1), TNFR1 (TNF receptor 1), death receptor 3 (DR3/Wsl-1/APO-3/TRAMP/LARD), death receptor 4 (DR4/TRAIL-R1), death receptor 5 (DR5/TRAIL-R2) and DR6. These receptors are characterized by an intracellular death domain. There are also decoy receptors (i.e. DcR1 and DcR2) that contain no death domain or a truncated death domain and can bind ligand but cannot signal. Therefore, these decoy receptors function as antagonists to inhibit death ligand/death receptor-induced apoptosis. Binding of ligands, such as FasL, tumor necrosis factor-alpha (TNF-alpha) and TNF-related apoptosis-inducing ligand (TRAIL) to their respective membrane receptors Fas, TNF-R and

Apoptosis and Apoptosis Modulators in Myeloid Leukemia 413

TRAIL-R1/TRAIL-R2 antibodies appear to be well tolerated *in vivo*. TRAIL/Apo-2L exhibited potent anti-tumor activity and induces little cytotoxic effects in immunodeficient mice xenograft models implanted with several human tumor cell lines (Ashkenazi et al., 1999). However, the *in vivo* half-life of the TRAIL-ligand is very short (<4 minutes) (Kelley et al., 2001). Agonistic TRAIL-R1 and TRAIL-R2 antibodies do not bind to TRAIL decoy receptors, TRAIL-R3 and TRAIL-R4, which are frequently expressed on the membrane of

Fig. 1. Extrinsic and intrinsic pathways in apoptosis (see text for further details).

Antisense therapies involved the use of sequences of single-stranded DNA to complement and bind specific coding regions on mRNA hence forming DNA–mRNA which is then degraded by a ribonuclease, therefore gene expression and translation are prevented. Most widely studied were with XIAP (X-linked inhibitor of apoptosis) and antiapoptotic proteins Bcl-2. Sufficient evidence exists to show that Bcl-2 was overexpressed in AML patients and predictive of worst outcome (Campos et al., 1993; Andreef and Konopleva, 2002). It seemed conceivable that Bcl-2 downregulation might lower the apoptotic threshold of leukemic cells and, through this mechanism, favor response to chemotherapy. Much success has been achieved. A phase I study using oblimersen, an antisense to Bcl-2, added during induction and then consolidation therapy, in elderly AML patients, induced remission in 14/27 patients, of which seven relapsed within 12.6 months (Marcucci et al., 2005); In a multicenter phase II trial, 12/39 relapsed AML patients treated with oblimersen and gentuzamab (anti-

tumor cells.

TRAIL-R induces trimerization of the receptors and recruitment of adaptor proteins such as the Fas-associated death domain (FADD) to the death domain. This then recruits procaspase-8 which then leads to the formation of the oligomeric death-induced signaling complex (DISC). DISC in turn promotes activation of caspase-8 and a cascade of other caspase enzymes that culminates with cell death (reviewed in Elrod and Sun, 2008).

The *intrinsic* pathway is triggered by various extracellular and intracellular stresses, including growth factor deprivation, DNA damage, oncogene induction, hypoxia and cytotoxic drugs. Cellular signals originated by various mechanisms by these different stresses converge on a cellular target represented by mitochondria. Mitochondrial membrane permeability is controlled by pro-apoptotic (Bax, Bak, Bad, Bid, Bim, Bmf, NOXA, PUMA, Bok, Bcl-G, Bfk) and anti-apoptotic (Bcl-2, Bcl-L, Mcl-1, Bcl-w, A1) members of the Bcl-2 family, inducing or preventing heterodimerization of pro-apoptotic members. A series of biochemical events is induced that lead to damage of the outer mitochondrial membrane, with the consequent release of cytochrome c and other pro-apoptotic molecules, such as Smac/DIABLO, from the inner membrane into the cytosol enabling the formation of the apoptosome, a large molecular complex formed by cytochrome c, apoptotic protease activating factor 1 (APAF-1) and caspase-9, and massive activation of caspases. These proteins all play crucial roles for cell survival and the loss of any of these proteins causes major deregulation of survival of some cell types (reviewed in Ashkenazi and Herbst, 2008).

Dysregulation of apoptosis plays an important role in the development of a variety of human pathologies, including cancer and particularly leukemia. The evasion of programmed cell death has been regarded as one of the six essential alterations in cellular physiology that dictate the growth of cancer cells and is a hallmark of virtually all cancers. Moreover, tumors that have alterations in proteins involved in cell death signaling are very frequently resistant to chemotherapy and are difficult to treat with chemotherapeutic agents that primarily act by inducing apoptosis (Testa et al., 2007).

Fas, DR4 and DR5 are generally expressed in both normal and malignant cells. An examination of patients with de novo AML revealed Fas was expressed on eight of nine (89%) patients tested (Tourneur et al., 2004). Another study showed expression of Fas on 62% of 29 AML patients (Min et al., 2004). Fas mutation was observed in 4/28 CML cases and none of the six AML cases tested (Rozenfeld-Granot et al., 2001). DR4 and DR5 mutations detected in cancers including chronic myelogenous leukemia were very low (0– 10.6%) (Liu et al., 2005). On the other hand, DR4 and DR5 receptors were positive in 20 (69%) and 29 (100%) patients, respectively. This study also showed, relapse-free survival was significantly prolonged in patients with CD95-positive AML cells compared with patients with CD95-negative AML cells (73% versus 38% at 3 years; p = 0.047) using univariate analysis (Min et al., 2004). This was however not supported by another study on 99 AML patients where multivariate analysis showed no correlation with overall survival and disease free survival (Brouwer et al., 2001).

Three ligands (TNF-α, FasL and TRAIL) of the TNF-family and their respective four receptors (TNF-R1, Fas, TRAIL-R1 and TRAIL-R2) are potentially important as anti-cancer therapeutics. The demonstration that TNF-α selectively kills tumor cells but not normal cells, set it up for the first molecules to be studied. Unfortunately, marked pro-inflammatory effects precluded its systemic administration (Buzzoni and Butler, 1996). Fas was also excluded as agonistic antibodies triggering Fas activation was highly hepatotoxic causing death in mouse models (Ogasarawa et al., 1993). In contrast, TRAIL and agonistic anti-

TRAIL-R induces trimerization of the receptors and recruitment of adaptor proteins such as the Fas-associated death domain (FADD) to the death domain. This then recruits procaspase-8 which then leads to the formation of the oligomeric death-induced signaling complex (DISC). DISC in turn promotes activation of caspase-8 and a cascade of other

The *intrinsic* pathway is triggered by various extracellular and intracellular stresses, including growth factor deprivation, DNA damage, oncogene induction, hypoxia and cytotoxic drugs. Cellular signals originated by various mechanisms by these different stresses converge on a cellular target represented by mitochondria. Mitochondrial membrane permeability is controlled by pro-apoptotic (Bax, Bak, Bad, Bid, Bim, Bmf, NOXA, PUMA, Bok, Bcl-G, Bfk) and anti-apoptotic (Bcl-2, Bcl-L, Mcl-1, Bcl-w, A1) members of the Bcl-2 family, inducing or preventing heterodimerization of pro-apoptotic members. A series of biochemical events is induced that lead to damage of the outer mitochondrial membrane, with the consequent release of cytochrome c and other pro-apoptotic molecules, such as Smac/DIABLO, from the inner membrane into the cytosol enabling the formation of the apoptosome, a large molecular complex formed by cytochrome c, apoptotic protease activating factor 1 (APAF-1) and caspase-9, and massive activation of caspases. These proteins all play crucial roles for cell survival and the loss of any of these proteins causes major deregulation of survival of some cell types (reviewed in Ashkenazi and Herbst, 2008). Dysregulation of apoptosis plays an important role in the development of a variety of human pathologies, including cancer and particularly leukemia. The evasion of programmed cell death has been regarded as one of the six essential alterations in cellular physiology that dictate the growth of cancer cells and is a hallmark of virtually all cancers. Moreover, tumors that have alterations in proteins involved in cell death signaling are very frequently resistant to chemotherapy and are difficult to treat with chemotherapeutic agents

Fas, DR4 and DR5 are generally expressed in both normal and malignant cells. An examination of patients with de novo AML revealed Fas was expressed on eight of nine (89%) patients tested (Tourneur et al., 2004). Another study showed expression of Fas on 62% of 29 AML patients (Min et al., 2004). Fas mutation was observed in 4/28 CML cases and none of the six AML cases tested (Rozenfeld-Granot et al., 2001). DR4 and DR5 mutations detected in cancers including chronic myelogenous leukemia were very low (0– 10.6%) (Liu et al., 2005). On the other hand, DR4 and DR5 receptors were positive in 20 (69%) and 29 (100%) patients, respectively. This study also showed, relapse-free survival was significantly prolonged in patients with CD95-positive AML cells compared with patients with CD95-negative AML cells (73% versus 38% at 3 years; p = 0.047) using univariate analysis (Min et al., 2004). This was however not supported by another study on 99 AML patients where multivariate analysis showed no correlation with overall survival

Three ligands (TNF-α, FasL and TRAIL) of the TNF-family and their respective four receptors (TNF-R1, Fas, TRAIL-R1 and TRAIL-R2) are potentially important as anti-cancer therapeutics. The demonstration that TNF-α selectively kills tumor cells but not normal cells, set it up for the first molecules to be studied. Unfortunately, marked pro-inflammatory effects precluded its systemic administration (Buzzoni and Butler, 1996). Fas was also excluded as agonistic antibodies triggering Fas activation was highly hepatotoxic causing death in mouse models (Ogasarawa et al., 1993). In contrast, TRAIL and agonistic anti-

caspase enzymes that culminates with cell death (reviewed in Elrod and Sun, 2008).

that primarily act by inducing apoptosis (Testa et al., 2007).

and disease free survival (Brouwer et al., 2001).

TRAIL-R1/TRAIL-R2 antibodies appear to be well tolerated *in vivo*. TRAIL/Apo-2L exhibited potent anti-tumor activity and induces little cytotoxic effects in immunodeficient mice xenograft models implanted with several human tumor cell lines (Ashkenazi et al., 1999). However, the *in vivo* half-life of the TRAIL-ligand is very short (<4 minutes) (Kelley et al., 2001). Agonistic TRAIL-R1 and TRAIL-R2 antibodies do not bind to TRAIL decoy receptors, TRAIL-R3 and TRAIL-R4, which are frequently expressed on the membrane of tumor cells.

Fig. 1. Extrinsic and intrinsic pathways in apoptosis (see text for further details).

Antisense therapies involved the use of sequences of single-stranded DNA to complement and bind specific coding regions on mRNA hence forming DNA–mRNA which is then degraded by a ribonuclease, therefore gene expression and translation are prevented. Most widely studied were with XIAP (X-linked inhibitor of apoptosis) and antiapoptotic proteins Bcl-2. Sufficient evidence exists to show that Bcl-2 was overexpressed in AML patients and predictive of worst outcome (Campos et al., 1993; Andreef and Konopleva, 2002). It seemed conceivable that Bcl-2 downregulation might lower the apoptotic threshold of leukemic cells and, through this mechanism, favor response to chemotherapy. Much success has been achieved. A phase I study using oblimersen, an antisense to Bcl-2, added during induction and then consolidation therapy, in elderly AML patients, induced remission in 14/27 patients, of which seven relapsed within 12.6 months (Marcucci et al., 2005); In a multicenter phase II trial, 12/39 relapsed AML patients treated with oblimersen and gentuzamab (anti-

Apoptosis and Apoptosis Modulators in Myeloid Leukemia 415

expression, through either p53-dependent, or p53-independent mechanisms (Wu et al., 1997; Guan et al., 2001; Sheikh et al., 1998). Etoposide was shown to induce DR5 expression

To complete induction of cell death, chemotherapeutic drugs have to suppress survival mediators in activated signaling pathways. Paclitaxel treatment of transfected MDA MB-435 human breast carcinoma cell line was observed to downregulate phosphorylated Akt (Klos et al., 2003). Nevertheless, chemotherapy induction of cell death is not equal in all cells. Adriamycin produced differential responses in Akt phosphorylation and kinase activity in a panel of breast cancer cell lines. While MCF7, MDA468 and T47D cells showed a dose dependent increase in p-Akt levels; in contrast, SKBR3 and MDA231 cells showed a dosedependent decrease and no or minimal change was detected in MDA361, MDA157 and BT474 cells (Li et al., 2005). The diversity in response may also be predictive of a

Other signaling molecules are activated by chemotherapeutic drugs leading to cell death. Ara-C induced apoptosis in HL-60 cell lines through the activation of p38 (Stadheim et al., 2000). Adriamycin was shown to activate Jnk in a T cell leukemia cell line (Yu et al., 1999). Leukemia cell lines (TF-1 and K562) primed for apoptosis were also revealed to stimulate

Certain cytokines have apoptotic activity. TNF-alpha and IFN-gamma induced the expression of DR5 in a number of cancer cell lines (Meng and El-Deiry, 2000). IFN-gamma had differential effect on induction of death receptors in colon carcinoma cell lines. While it raised the levels of CD95 membrane 6 – 8-fold, it had no effect on the TRAIL-receptors (DR4, DR5, DcR1 and DcR2) (van Geelan et al., 2003). Interferon-alpha was also reported to

In contrast some cytokines exert protective effect from chemotherapeutic drug induced cell death, decreasing the effectiveness of cancer radiotherapy and chemotherapy. Normal hematopoietic cells, like other normal cell types, die by the process of apoptosis when deprived of viability inducing cytokines that include colony stimulating factors (CSFs) and various other cytokines. Induction of apoptosis by cancer chemotherapy such as vincristine, adriamycin, methotrexate and Ara-C was suppressed by IL (interleukin)-6, IL-3, granulocyte-CSF (G-CSF), granulocyte-monocyte CSF (GM-CSF) and IFN-gamma in myeloid leukemia cells (reviewed in Lotem and Sachs, 2002). These cytokines upregulate pro-survival molecules such as Bcl-2 [IL-2, IL-3, stem cell factor (SCF), IFN-gamma], Bcl-xL [IL-3, IL-6, IL-7, IL15, GM-CSF, IFN-gamma and erythropoietin (EPO)] and other apoptosis suppressing genes such as Survivin (Carter et al., 2001), X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis 2 (cIAP2) (Digicylioglu and Lipton, 2001) that are caspase inhibitors and FLICE-like inhibitory protein (FLIP), that may disrupt the ability of cell surface molecules such as Fas to activate apoptosis (Kovalovich et al., 2001) Some myeloid leukemic cells are autonomous and do not require an exogenous source of cytokines for viability (Griffin and Lowenberg,1986), while others do. Thus, it is possible to suppress leukemia not only by cytotoxic agents or by induction of terminal differentiation, but also by decreasing the in vivo supply of apoptosis suppressing cytokines or the response

A characteristic abnormality of leukemia cells is that they are blocked at an early stage of their development. Myeloid leukemic cells however can be induced to differentiate to non dividing mature granulocytes and macrophages by different cytokines, including cytokine

in human acute leukemia cells (Wen et al., 2000).

Jnk and p38 phosphorylation (Tucker et al., 2004)

increase DR5 expression in human hepatoma (Shigeno et al., 2003).

of leukemic cells to these cytokines (reviewed in Sachs, 1996).

heterogeneity in treatment outcome.

CD33) achieved complete remission of which 10/12 survived for more than 6 months (Moore et al., 2004).

XIAP binds and inhibits caspases 3, 7, and 9, mediators of the apoptotic cascade. Downregulation of XIAP using multiple approaches (e.g., antisense, RNAi, knock-out animals and cell lines, immuno-depletion) in vitro and in vivo conditions resulted in increased caspase activation and/or cell death. Antitumor activity was also observed with the use of second generation anti-sense compound, AEG35156, in xenograft models of cancer (Lacasse et al., 2005) Results from clinical trials however, have been variable. While one study on five phase 1 (12–350mg/m2 AEG35156) and eight phase 2 (350 mg/m2 AEG35156) patients showed increased apoptotic cells and increase response (Bing et al., 2011) another study on 27 patients randomized to receive high dose Ara-C and idarubicin with or without AEG35156 (650 mg) found a lower overall response rate in the group which received the anti-XIAP drug (Schimmer et al., 2011).

The analysis of Mcl-1 protein expression in AML showed great heterogeneity, but the levels of the protein do not seem to correlate with response to standard chemotherapy (Kaufmann et al., 1998). Bad and Bcl-xL have been shown to be expressed in normal and leukemic hematopoietic precursor cells. Immature hematopoietic cells do not express Bcl-2 but do express Bcl-xL. CD34 positive cells express Bcl-2, Bcl-xL and Bad. Bcl-2 expression is higher on CD34 positive cells than on AML cells. Phosphorylated Bad was expressed in AML (Andreef et al., 1999).

Potential abnormalities of the various initiator caspases in AML have been explored. Levels of caspase-8, caspase-2 and caspase-3 are heterogeneous in AML. AML with an immature phenotype (i.e., M0 and M1 AML) predominantly express caspase-8L (Mohr et al., 2005).The significance of caspases as prognostic indicators in AML are unclear as current reports are still controversial may be due to the different format of molecules examined (Svingen et al., 2000; Estrov et al., 1998; Holleman et al., 2005).

Expression of pro- and anti-apoptotic molecules continues to be studied in AML to correlate its mutated state, expression, activity or methylated state with treatment outcome (Testa et al., 2007). At present, the prognostic utility of measurements of pro- and antiapoptotic molecules for predicting clinical outcome and response to chemotherapy is uncertain.

#### **5. Drug modulation of signaling, differentiation and apoptotic pathways**

The study of cancer cell biology in predicting treatment outcome cannot stop at the presentation stage as cells continue to be modified by the microenvironment and are ultimately subjected to chemotherapy. While remarkable progress have been achieved in targeted therapies, for most tumors chemo- or radiotherapy is likely to remain in the near future. Both chemo- and radiotherapy are designed to kill cancer cells by damaging nuclear DNA. DNA damage triggers the DNA damage response (DDR) which have three critical goals: (i) halting cell cycle progression and division to prevent transfer of DNA damage to progeny cells; (ii) increasing accessibility of the damage sites to- and engagement of- the DNA damage repair machinery, and (iii) triggering apoptosis to exterminate cells whose damaged DNA cannot successfully be repaired (reviewed in Darzynkiewicz et al., 2009).

Chemotherapeutic drugs such as cisplatin, mitomycin, methotrexate, mitoxantrone, adriamycin, and bleomycin induce Fas expression in human cancer cells, primarily through a p53-dependent mechanism (Muller et al., 1998). Adriamycin, etoposide, Ara-C, cisplatin and camptosar were shown to induce the expression of DR4 and DR5 or only DR5

CD33) achieved complete remission of which 10/12 survived for more than 6 months

XIAP binds and inhibits caspases 3, 7, and 9, mediators of the apoptotic cascade. Downregulation of XIAP using multiple approaches (e.g., antisense, RNAi, knock-out animals and cell lines, immuno-depletion) in vitro and in vivo conditions resulted in increased caspase activation and/or cell death. Antitumor activity was also observed with the use of second generation anti-sense compound, AEG35156, in xenograft models of cancer (Lacasse et al., 2005) Results from clinical trials however, have been variable. While one study on five phase 1 (12–350mg/m2 AEG35156) and eight phase 2 (350 mg/m2 AEG35156) patients showed increased apoptotic cells and increase response (Bing et al., 2011) another study on 27 patients randomized to receive high dose Ara-C and idarubicin with or without AEG35156 (650 mg) found a lower overall response rate in the group which

The analysis of Mcl-1 protein expression in AML showed great heterogeneity, but the levels of the protein do not seem to correlate with response to standard chemotherapy (Kaufmann et al., 1998). Bad and Bcl-xL have been shown to be expressed in normal and leukemic hematopoietic precursor cells. Immature hematopoietic cells do not express Bcl-2 but do express Bcl-xL. CD34 positive cells express Bcl-2, Bcl-xL and Bad. Bcl-2 expression is higher on CD34 positive cells than on AML cells. Phosphorylated Bad was expressed in AML

Potential abnormalities of the various initiator caspases in AML have been explored. Levels of caspase-8, caspase-2 and caspase-3 are heterogeneous in AML. AML with an immature phenotype (i.e., M0 and M1 AML) predominantly express caspase-8L (Mohr et al., 2005).The significance of caspases as prognostic indicators in AML are unclear as current reports are still controversial may be due to the different format of molecules examined (Svingen et al.,

Expression of pro- and anti-apoptotic molecules continues to be studied in AML to correlate its mutated state, expression, activity or methylated state with treatment outcome (Testa et al., 2007). At present, the prognostic utility of measurements of pro- and antiapoptotic molecules for predicting clinical outcome and response to chemotherapy is uncertain.

The study of cancer cell biology in predicting treatment outcome cannot stop at the presentation stage as cells continue to be modified by the microenvironment and are ultimately subjected to chemotherapy. While remarkable progress have been achieved in targeted therapies, for most tumors chemo- or radiotherapy is likely to remain in the near future. Both chemo- and radiotherapy are designed to kill cancer cells by damaging nuclear DNA. DNA damage triggers the DNA damage response (DDR) which have three critical goals: (i) halting cell cycle progression and division to prevent transfer of DNA damage to progeny cells; (ii) increasing accessibility of the damage sites to- and engagement of- the DNA damage repair machinery, and (iii) triggering apoptosis to exterminate cells whose damaged DNA cannot successfully be repaired (reviewed in Darzynkiewicz et al., 2009). Chemotherapeutic drugs such as cisplatin, mitomycin, methotrexate, mitoxantrone, adriamycin, and bleomycin induce Fas expression in human cancer cells, primarily through a p53-dependent mechanism (Muller et al., 1998). Adriamycin, etoposide, Ara-C, cisplatin and camptosar were shown to induce the expression of DR4 and DR5 or only DR5

**5. Drug modulation of signaling, differentiation and apoptotic pathways** 

(Moore et al., 2004).

(Andreef et al., 1999).

received the anti-XIAP drug (Schimmer et al., 2011).

2000; Estrov et al., 1998; Holleman et al., 2005).

expression, through either p53-dependent, or p53-independent mechanisms (Wu et al., 1997; Guan et al., 2001; Sheikh et al., 1998). Etoposide was shown to induce DR5 expression in human acute leukemia cells (Wen et al., 2000).

To complete induction of cell death, chemotherapeutic drugs have to suppress survival mediators in activated signaling pathways. Paclitaxel treatment of transfected MDA MB-435 human breast carcinoma cell line was observed to downregulate phosphorylated Akt (Klos et al., 2003). Nevertheless, chemotherapy induction of cell death is not equal in all cells. Adriamycin produced differential responses in Akt phosphorylation and kinase activity in a panel of breast cancer cell lines. While MCF7, MDA468 and T47D cells showed a dose dependent increase in p-Akt levels; in contrast, SKBR3 and MDA231 cells showed a dosedependent decrease and no or minimal change was detected in MDA361, MDA157 and BT474 cells (Li et al., 2005). The diversity in response may also be predictive of a heterogeneity in treatment outcome.

Other signaling molecules are activated by chemotherapeutic drugs leading to cell death. Ara-C induced apoptosis in HL-60 cell lines through the activation of p38 (Stadheim et al., 2000). Adriamycin was shown to activate Jnk in a T cell leukemia cell line (Yu et al., 1999). Leukemia cell lines (TF-1 and K562) primed for apoptosis were also revealed to stimulate Jnk and p38 phosphorylation (Tucker et al., 2004)

Certain cytokines have apoptotic activity. TNF-alpha and IFN-gamma induced the expression of DR5 in a number of cancer cell lines (Meng and El-Deiry, 2000). IFN-gamma had differential effect on induction of death receptors in colon carcinoma cell lines. While it raised the levels of CD95 membrane 6 – 8-fold, it had no effect on the TRAIL-receptors (DR4, DR5, DcR1 and DcR2) (van Geelan et al., 2003). Interferon-alpha was also reported to increase DR5 expression in human hepatoma (Shigeno et al., 2003).

In contrast some cytokines exert protective effect from chemotherapeutic drug induced cell death, decreasing the effectiveness of cancer radiotherapy and chemotherapy. Normal hematopoietic cells, like other normal cell types, die by the process of apoptosis when deprived of viability inducing cytokines that include colony stimulating factors (CSFs) and various other cytokines. Induction of apoptosis by cancer chemotherapy such as vincristine, adriamycin, methotrexate and Ara-C was suppressed by IL (interleukin)-6, IL-3, granulocyte-CSF (G-CSF), granulocyte-monocyte CSF (GM-CSF) and IFN-gamma in myeloid leukemia cells (reviewed in Lotem and Sachs, 2002). These cytokines upregulate pro-survival molecules such as Bcl-2 [IL-2, IL-3, stem cell factor (SCF), IFN-gamma], Bcl-xL [IL-3, IL-6, IL-7, IL15, GM-CSF, IFN-gamma and erythropoietin (EPO)] and other apoptosis suppressing genes such as Survivin (Carter et al., 2001), X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis 2 (cIAP2) (Digicylioglu and Lipton, 2001) that are caspase inhibitors and FLICE-like inhibitory protein (FLIP), that may disrupt the ability of cell surface molecules such as Fas to activate apoptosis (Kovalovich et al., 2001) Some myeloid leukemic cells are autonomous and do not require an exogenous source of cytokines for viability (Griffin and Lowenberg,1986), while others do. Thus, it is possible to suppress leukemia not only by cytotoxic agents or by induction of terminal differentiation, but also by decreasing the in vivo supply of apoptosis suppressing cytokines or the response of leukemic cells to these cytokines (reviewed in Sachs, 1996).

A characteristic abnormality of leukemia cells is that they are blocked at an early stage of their development. Myeloid leukemic cells however can be induced to differentiate to non dividing mature granulocytes and macrophages by different cytokines, including cytokine

Apoptosis and Apoptosis Modulators in Myeloid Leukemia 417

tumor-host mechanisms as important for effective chemotherapy, there is an immediate

For that purpose, blasts from patients undergoing chemotherapy were collected as a 'natural' and rich source to study response of these cells to the myriad of signals they were subjected to. Even though cells undergo cell death, as white blood cell counts may decline at early stages of chemotherapy, very low percentages of apoptotic cells were detected. Oyan et al. (2009) comparing treated ('3+7', idarubicin + Ara-C ) with untreated AML cells from seven patients, observed upregulation of 113 genes (23 of unknown function) at early time points (2 – 4 hours) and 108 genes at late time points (18 – 24 hours). Among the 113 genes a substantial number (31 genes) were related to the tumor suppressor p53 (Oyan et al., 2009). p53 is implicated to affect a variety of cellular processes, the most undisputed roles of p53 are to induce growth arrest and to induce apoptosis (Bates and Vousden, 1996). p53 is the most commonly mutated gene in a variety of human cancers (Greenblatt et al., 1994). In AML however, mutations of p53 are rare, occurring in approximately 5% to 10% (Fenaux et al., 1992) but in these cases it correlates with worse outcome (Wattel et al., 1994). Wild-type p53 appears to change the balance in expression of apoptosis-inducing versus apoptosis-

In tune with the above, a significant increase in gene expression of the apoptosis facilitators PUMA and Bax and a decrease in the Bcl-2 /Bax ratio as well as Bcl-2 /PUMA were observed for most of the AML samples. The mRNA profile of three other pro-apoptotic mediators Bad , Bak1 and Bim did not change significantly during the first hours, but the level of gene expression varied across patients. Altogether five tumor necrosis factor-related

Induction of ligand to death receptor during chemotherapy was also supported by Devemy et al. (2001) who observed increased TNF transcripts in treated AML cells. We also studied molecular changes in paired AML samples at diagnosis and during chemotherapy ( Ara-C + daunorubicin). We showed increased TNF-alpha was significantly higher in chemo-sensitive patients. Thus, expression of TNF-alpha early during chemotherapy may be a marker to predict good treatment outcome. In chemo-resistant cases, a higher, though not significant,

We observed a significantly higher percentage of chemo-responsive AML patients with blasts cells increased for the expression of IL-6. This was consistent with Devemy et al (2001) who reported that increase of IL-6 transcripts during remission induction therapy of AML patients was accompanied by a fall in blood count and bone marrow cellularity. The role of cytokines in the induction of cell differentiation is well established. Oyan et al (2009) also observed several receptors expressed on monocytic/macrophage lineage cells were upregulated, probably related to chemotherapy induced differentiation of the leukemic cells. Thus, induction of cytokines expression in drug responsive AML patients may be due

Comparing blasts profiles before and during early chemotherapy also revealed upregulation of genes potentially involved in interaction between AML blasts and the host microenvironment. Chemokine receptors CXCR4 and CX3CR1 were upregulated in the late phase after start of chemotherapy indicating intention to home into a microenvironment that favours their growth and survival. This supports the hypothesis that the host response in

receptor genes were modulated 2–4 h after induction therapy (Oyan et al., 2009).

need to investigate these issues in vivo in human cancer (Oyan et al., 2009)

suppressing genes in favor of the former and thus induce apoptosis.

percentage of cases expressed IL-1beta and IL-18 (Maha et al., 2009).

chemotherapy is crucial for persistent remission (Oyan et al., 2009).

to induction of cell differentiation.

independent myeloid leukemic cells that were induced to differentiate with IL-6. Different myeloid leukemic clones however have different blocks and ability to undergo differentiation by cytokines. Our own work on in vitro cultured AML blasts exhibited different degrees of spontaneous apoptosis. Univariate analysis of 13 AML patients revealed blasts with lower levels of cell viability after 72h culture was significantly correlated with a longer disease free survival . Within a smaller number of samples (n=7) we observed blasts with lower levels of cell viability were associated with reduced levels CD34 and higher levels of CD16, indicating an increased level of cell differentiation (Maha et al., 2008). The observations may indicate an abnormal developmental program in leukemic cells which may be reprogrammed epigenetically by appropriate differentiation inducing cytokines. Constitutive expression of transcription factors such as c-myc, c-myb and E2F1 (Gonda and Metcalf, 1984; Blatt et al., 1992; Melamed et al., 1993) as well as others such as the homeobox gene Hox B8 (Hox 2.4) (Blatt et al., 1992) or GATA-1 (Tanaka et al., 2000), disrupted the ability of cells to undergo cytokine induced differentiation (reviewed in Lotem and Sachs, 2002)

Cytokines as a differentiation treatment against leukemia however has been disappointing. Hematopoietic leukemia cell lines of myeloid origin such as K562, U937, HL-60, CS-1, KG-1, MUTZ-3, or ex vivo AML or chronic myeloid leukemia (CML) blasts were modestly permissive to induction of in vitro differentiation by EPO, G-CSF, GM-CSF, IL-4, IL-6, SCF, or synergistic combinations of several cytokines (Leung et al., 2005; Koss et al., 1996; Goliaei et al. 1998; Kamano et al., 1994; Kamijo et al., 1990). A niche for hematopoietic cytokines in differentiation therapy exists in the treatment of congenital neutropenia disorder. The administration of G-CSF to patients has overcome a block of myeloid differentiation leading to a substantial prolongation of their survival (Berliner, 2008).

Clinically, differentiation therapy has been most successful in acute promyelocytic leukemia (APL) using all-trans-retinoic acid (ATRA) as the inducer. This targeted APL cells carrying the chromosomal translocation between chromosomes 15 and 17 [t(15;17)(q22;q21)]. Subsequently, APL therapy was improved with the combination regimen of ATRA with cytotoxic chemotherapy. Currently, complete remission rates of up to 90% to 95% are achievable using ATRA/ATO (arsenic trioxide) and anthracycline-based chemotherapy (Niu et al., 1999; Soignet et al., 2001; Raffoux et al., 2003; Ghavamzadeh et al., 2006; Mathews et al., 2006; Estey et al., 2006; Sanz et al., 2008).

Another targeted treatment with tyrosine kinase inhibitor (TKI) imatinib for the treatment of CMLalso achieved better success. Gefinitib and erlotinib which inhibit the intracellular tyrosine kinase activity of epidermal growth factor receptor (EGFR), induce a differentiation program in myeloid leukemia cells that corresponds to neutrophil maturation (Stegmaier et al., 2005; Boehrer et al., 2008a; Boehrer et al., 2008b).

These results together emphasize further not only the heterogeneity of leukemias but also complexity of host-cancer interaction and its influence on outcome in survival and also during induction of cell death.

#### **6. In vivo drug induced molecular profiles: Potential predictor of drug resistance**

The in vivo molecular changes in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response. As increasing evidence is proposing

independent myeloid leukemic cells that were induced to differentiate with IL-6. Different myeloid leukemic clones however have different blocks and ability to undergo differentiation by cytokines. Our own work on in vitro cultured AML blasts exhibited different degrees of spontaneous apoptosis. Univariate analysis of 13 AML patients revealed blasts with lower levels of cell viability after 72h culture was significantly correlated with a longer disease free survival . Within a smaller number of samples (n=7) we observed blasts with lower levels of cell viability were associated with reduced levels CD34 and higher levels of CD16, indicating an increased level of cell differentiation (Maha et al., 2008). The observations may indicate an abnormal developmental program in leukemic cells which may be reprogrammed epigenetically by appropriate differentiation inducing cytokines. Constitutive expression of transcription factors such as c-myc, c-myb and E2F1 (Gonda and Metcalf, 1984; Blatt et al., 1992; Melamed et al., 1993) as well as others such as the homeobox gene Hox B8 (Hox 2.4) (Blatt et al., 1992) or GATA-1 (Tanaka et al., 2000), disrupted the ability of cells to undergo cytokine induced differentiation

Cytokines as a differentiation treatment against leukemia however has been disappointing. Hematopoietic leukemia cell lines of myeloid origin such as K562, U937, HL-60, CS-1, KG-1, MUTZ-3, or ex vivo AML or chronic myeloid leukemia (CML) blasts were modestly permissive to induction of in vitro differentiation by EPO, G-CSF, GM-CSF, IL-4, IL-6, SCF, or synergistic combinations of several cytokines (Leung et al., 2005; Koss et al., 1996; Goliaei et al. 1998; Kamano et al., 1994; Kamijo et al., 1990). A niche for hematopoietic cytokines in differentiation therapy exists in the treatment of congenital neutropenia disorder. The administration of G-CSF to patients has overcome a block of myeloid differentiation leading

Clinically, differentiation therapy has been most successful in acute promyelocytic leukemia (APL) using all-trans-retinoic acid (ATRA) as the inducer. This targeted APL cells carrying the chromosomal translocation between chromosomes 15 and 17 [t(15;17)(q22;q21)]. Subsequently, APL therapy was improved with the combination regimen of ATRA with cytotoxic chemotherapy. Currently, complete remission rates of up to 90% to 95% are achievable using ATRA/ATO (arsenic trioxide) and anthracycline-based chemotherapy (Niu et al., 1999; Soignet et al., 2001; Raffoux et al., 2003; Ghavamzadeh et al., 2006; Mathews

Another targeted treatment with tyrosine kinase inhibitor (TKI) imatinib for the treatment of CMLalso achieved better success. Gefinitib and erlotinib which inhibit the intracellular tyrosine kinase activity of epidermal growth factor receptor (EGFR), induce a differentiation program in myeloid leukemia cells that corresponds to neutrophil maturation (Stegmaier et

These results together emphasize further not only the heterogeneity of leukemias but also complexity of host-cancer interaction and its influence on outcome in survival and also

The in vivo molecular changes in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response. As increasing evidence is proposing

**6. In vivo drug induced molecular profiles: Potential predictor of drug** 

(reviewed in Lotem and Sachs, 2002)

to a substantial prolongation of their survival (Berliner, 2008).

et al., 2006; Estey et al., 2006; Sanz et al., 2008).

al., 2005; Boehrer et al., 2008a; Boehrer et al., 2008b).

during induction of cell death.

**resistance** 

tumor-host mechanisms as important for effective chemotherapy, there is an immediate need to investigate these issues in vivo in human cancer (Oyan et al., 2009)

For that purpose, blasts from patients undergoing chemotherapy were collected as a 'natural' and rich source to study response of these cells to the myriad of signals they were subjected to. Even though cells undergo cell death, as white blood cell counts may decline at early stages of chemotherapy, very low percentages of apoptotic cells were detected. Oyan et al. (2009) comparing treated ('3+7', idarubicin + Ara-C ) with untreated AML cells from seven patients, observed upregulation of 113 genes (23 of unknown function) at early time points (2 – 4 hours) and 108 genes at late time points (18 – 24 hours). Among the 113 genes a substantial number (31 genes) were related to the tumor suppressor p53 (Oyan et al., 2009). p53 is implicated to affect a variety of cellular processes, the most undisputed roles of p53 are to induce growth arrest and to induce apoptosis (Bates and Vousden, 1996). p53 is the most commonly mutated gene in a variety of human cancers (Greenblatt et al., 1994). In AML however, mutations of p53 are rare, occurring in approximately 5% to 10% (Fenaux et al., 1992) but in these cases it correlates with worse outcome (Wattel et al., 1994). Wild-type p53 appears to change the balance in expression of apoptosis-inducing versus apoptosissuppressing genes in favor of the former and thus induce apoptosis.

In tune with the above, a significant increase in gene expression of the apoptosis facilitators PUMA and Bax and a decrease in the Bcl-2 /Bax ratio as well as Bcl-2 /PUMA were observed for most of the AML samples. The mRNA profile of three other pro-apoptotic mediators Bad , Bak1 and Bim did not change significantly during the first hours, but the level of gene expression varied across patients. Altogether five tumor necrosis factor-related receptor genes were modulated 2–4 h after induction therapy (Oyan et al., 2009).

Induction of ligand to death receptor during chemotherapy was also supported by Devemy et al. (2001) who observed increased TNF transcripts in treated AML cells. We also studied molecular changes in paired AML samples at diagnosis and during chemotherapy ( Ara-C + daunorubicin). We showed increased TNF-alpha was significantly higher in chemo-sensitive patients. Thus, expression of TNF-alpha early during chemotherapy may be a marker to predict good treatment outcome. In chemo-resistant cases, a higher, though not significant, percentage of cases expressed IL-1beta and IL-18 (Maha et al., 2009).

We observed a significantly higher percentage of chemo-responsive AML patients with blasts cells increased for the expression of IL-6. This was consistent with Devemy et al (2001) who reported that increase of IL-6 transcripts during remission induction therapy of AML patients was accompanied by a fall in blood count and bone marrow cellularity. The role of cytokines in the induction of cell differentiation is well established. Oyan et al (2009) also observed several receptors expressed on monocytic/macrophage lineage cells were upregulated, probably related to chemotherapy induced differentiation of the leukemic cells. Thus, induction of cytokines expression in drug responsive AML patients may be due to induction of cell differentiation.

Comparing blasts profiles before and during early chemotherapy also revealed upregulation of genes potentially involved in interaction between AML blasts and the host microenvironment. Chemokine receptors CXCR4 and CX3CR1 were upregulated in the late phase after start of chemotherapy indicating intention to home into a microenvironment that favours their growth and survival. This supports the hypothesis that the host response in chemotherapy is crucial for persistent remission (Oyan et al., 2009).

Apoptosis and Apoptosis Modulators in Myeloid Leukemia 419

profile signaling cell death. This new phenotype corresponded to a sensitive response to chemotherapy. On the other hand, cells may strengthen on pro-survival features which corresponded with resistance to chemotherapy. A few reports, including ours, are lending

Unsurprisingly, chemotherapy-induce phenotype is not confined purely to either a survival or an apoptosis profile but a complex mix of conflicting signals to survive or die in addition to triggers to shut down cell proliferation, induce terminal differentiation or activate inflammatory responses. Thus, further elucidation of these profiles would involve

The immaturity feature in leukemias will undoubtedly be a factor that will further compound the heterogeneity in results obtained. An example is the striking correlations found between lower Bax/Bcl-2 ratio and higher progenitor marker expression, such as CD34, CD117 and CD133 antigens, confirming the link between this apoptotic index and the maturation pathways (Del Principe et al., 2005). Attempts to induce cell death by triggering death receptors has so far achieved mix results with the use of TNF-alpha, Fas ligands and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (i.e., DR4 and DR5). These molecules also selectively kill cancer cells while sparing normal cells (reviewed in Elrod and Sun, 2008). These results indicate a preferential expression of specific death

Selection of lab methods for prognostication depends on the ability to identify lineage, maturation stages, genetic aberrations and activated signal transduction pathways. This feat may include the difficult task of combining surface markers, cytokines (secreted proteins) and phosphorylated proteins (unstable intracellular proteins) in the same tube on the same platform such as flowcytometry. Furthermore many of these proteins such as TNF-a, IL-6, p38 and Jnk have dual function of pro-survival and pro-apoptosis capabilities depending on the stimulating conditions cells are exposed to at that period of time. Precise markers will be required to differentiate these situations. Altogether, all of these add up to an interesting

[1] American Cancer Society. Cancer facts & figures 2010. American Cancer Society Web

[2] Andreeff M, Jiang S, Zhang X, Konopleva M, Estrov Z, Snell VE et al. Expression of Bcl-2

[3] Andreef M, Konopleva M. Mechanisms of drug resistance in AML. Cancer Treat Res

[4] Ashkenazi A, Herbst RS. To kill a tumor cell: the potential of proapoptotic receptor

[5] Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, et al. Safety and

[6] Bates, S., and Vousden, K. H. p53 in signaling checkpoint arrest or apoptosis. Curr. Opin.

[7] Bazzoni F, Butler B. The tumor necrosis factor ligand and receptor families N Engl J Med

related genes in normal and AML progenitors: changes induced by chemotherapy

antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 1999;104:155-

assignment of each of the modulated molecule to its rightful pathway.

support to this hypothesis.

receptors on different tissues.

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We further examined activation of signaling molecules in AML blasts. Chemotherapy increased the percentage of cases showing phosphorylation of the Akt molecules and Forkhead transcription factor (FKHR) but no significant differences were observed between chemo-resistant and chemo-sensitive cases. We however, observed a significantly higher percentage of chemo-resistant cases showing phosphorylation and inactivation of the proapoptotic Bad molecule. A higher percentage of chemo-sensitive cases were phosphorylated for p38, and Jnk (Maha et al., 2009). In summary, we were able to show in chemo-sensitive cases, chemotherapy stimulated IL-6, induced apoptosis by up-regulating TNF-alpha and downregulated phosphorylated Bad. In reverse, in chemo-resistant cases, cells survived by maintaining high levels of phosphorylated Bad maybe through protective role of IL-1b and IL-18 cytokines (Maha et al., 2009).

Most anticancer drugs exert their effects by the induction of apoptosis and/or interfering with cell cycle progression. Often these drugs give rise to specic patterns of cell death and cell cycle arrest that vary according to the drug used and the molecular status of the target cell. Simple in vitro methods may aid in this investigation. Drug cytotoxicity and sensitivity of individual tumor samples was demonstrated by combining cytochrome c and propidium iodide staining of DNA content and detected on flowcytometry. This method elucidated mitochondrial resistance mechanisms which may prove useful in identifying the apoptosissensitive cell cycle phase for a given tumor sample/anticancer drug combination. It offers the opportunity to design personalized drug regimens and to identify new combined treatment modalities (Mohr et al., 2004).

#### **7. Conclusion**

The heterogeneity in AML continues to elude the best methods to characterize them. Genome and proteome-wide analysis has further revealed complexity in the makeup of the leukemic cell. The rapid advancement in targeted therapies implied the urgent need for alternative therapy and the readiness of the community to embrace it. Nevertheless so far, combinatorial medicine still holds out as the best option for successful treatment. If targeted therapies remain the way forward it will eventually bank deeply on the ability to identify molecular signatures in the individual leading to the establishment of personalized medicine.

In the meantime, the mechanisms in leukemogenesis, drug resistance and relapse remain an area of much research. From cell biology to cytogenetics to molecular defects to signaling pathways, all have contributed to a better understanding of cancer biology. New knowledge in epigenetics and microRNA remain to be elucidated.

Current diagnostic and prognostication are based on the assumption that the phenotype of the leukemic cell is static and thus definitive. There is much evidence that suggest otherwise. Activation of oncogenes leads to constitutive expression of signal transduction pathways involved in cell survival and anti-apoptotic activities. These pathways are multiple and made up of a myriad of molecules that are receptive to the environment. The host-cancer microenvironment is a dynamic microcosm of interacting signals and cascading molecules that constantly respond to stimuli in the surrounding to find a balance that maintains survival. In the course of treatment, blast cells are exposed to DNA damaging cytotoxic agents which trigger a gamut of other signaling mediators to exert the opposite effect. It would appear that a struggle ensues in which the strength of the victor determines whether the blast cell would maintain life or be pushed off-balance and replaced with a new

We further examined activation of signaling molecules in AML blasts. Chemotherapy increased the percentage of cases showing phosphorylation of the Akt molecules and Forkhead transcription factor (FKHR) but no significant differences were observed between chemo-resistant and chemo-sensitive cases. We however, observed a significantly higher percentage of chemo-resistant cases showing phosphorylation and inactivation of the proapoptotic Bad molecule. A higher percentage of chemo-sensitive cases were phosphorylated for p38, and Jnk (Maha et al., 2009). In summary, we were able to show in chemo-sensitive cases, chemotherapy stimulated IL-6, induced apoptosis by up-regulating TNF-alpha and downregulated phosphorylated Bad. In reverse, in chemo-resistant cases, cells survived by maintaining high levels of phosphorylated Bad maybe through protective role of IL-1b and

Most anticancer drugs exert their effects by the induction of apoptosis and/or interfering with cell cycle progression. Often these drugs give rise to specic patterns of cell death and cell cycle arrest that vary according to the drug used and the molecular status of the target cell. Simple in vitro methods may aid in this investigation. Drug cytotoxicity and sensitivity of individual tumor samples was demonstrated by combining cytochrome c and propidium iodide staining of DNA content and detected on flowcytometry. This method elucidated mitochondrial resistance mechanisms which may prove useful in identifying the apoptosissensitive cell cycle phase for a given tumor sample/anticancer drug combination. It offers the opportunity to design personalized drug regimens and to identify new combined

The heterogeneity in AML continues to elude the best methods to characterize them. Genome and proteome-wide analysis has further revealed complexity in the makeup of the leukemic cell. The rapid advancement in targeted therapies implied the urgent need for alternative therapy and the readiness of the community to embrace it. Nevertheless so far, combinatorial medicine still holds out as the best option for successful treatment. If targeted therapies remain the way forward it will eventually bank deeply on the ability to identify molecular signatures in the individual leading to the establishment of personalized

In the meantime, the mechanisms in leukemogenesis, drug resistance and relapse remain an area of much research. From cell biology to cytogenetics to molecular defects to signaling pathways, all have contributed to a better understanding of cancer biology. New knowledge

Current diagnostic and prognostication are based on the assumption that the phenotype of the leukemic cell is static and thus definitive. There is much evidence that suggest otherwise. Activation of oncogenes leads to constitutive expression of signal transduction pathways involved in cell survival and anti-apoptotic activities. These pathways are multiple and made up of a myriad of molecules that are receptive to the environment. The host-cancer microenvironment is a dynamic microcosm of interacting signals and cascading molecules that constantly respond to stimuli in the surrounding to find a balance that maintains survival. In the course of treatment, blast cells are exposed to DNA damaging cytotoxic agents which trigger a gamut of other signaling mediators to exert the opposite effect. It would appear that a struggle ensues in which the strength of the victor determines whether the blast cell would maintain life or be pushed off-balance and replaced with a new

IL-18 cytokines (Maha et al., 2009).

treatment modalities (Mohr et al., 2004).

in epigenetics and microRNA remain to be elucidated.

**7. Conclusion** 

medicine.

profile signaling cell death. This new phenotype corresponded to a sensitive response to chemotherapy. On the other hand, cells may strengthen on pro-survival features which corresponded with resistance to chemotherapy. A few reports, including ours, are lending support to this hypothesis.

Unsurprisingly, chemotherapy-induce phenotype is not confined purely to either a survival or an apoptosis profile but a complex mix of conflicting signals to survive or die in addition to triggers to shut down cell proliferation, induce terminal differentiation or activate inflammatory responses. Thus, further elucidation of these profiles would involve assignment of each of the modulated molecule to its rightful pathway.

The immaturity feature in leukemias will undoubtedly be a factor that will further compound the heterogeneity in results obtained. An example is the striking correlations found between lower Bax/Bcl-2 ratio and higher progenitor marker expression, such as CD34, CD117 and CD133 antigens, confirming the link between this apoptotic index and the maturation pathways (Del Principe et al., 2005). Attempts to induce cell death by triggering death receptors has so far achieved mix results with the use of TNF-alpha, Fas ligands and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (i.e., DR4 and DR5). These molecules also selectively kill cancer cells while sparing normal cells (reviewed in Elrod and Sun, 2008). These results indicate a preferential expression of specific death receptors on different tissues.

Selection of lab methods for prognostication depends on the ability to identify lineage, maturation stages, genetic aberrations and activated signal transduction pathways. This feat may include the difficult task of combining surface markers, cytokines (secreted proteins) and phosphorylated proteins (unstable intracellular proteins) in the same tube on the same platform such as flowcytometry. Furthermore many of these proteins such as TNF-a, IL-6, p38 and Jnk have dual function of pro-survival and pro-apoptosis capabilities depending on the stimulating conditions cells are exposed to at that period of time. Precise markers will be required to differentiate these situations. Altogether, all of these add up to an interesting and exciting field of research for the immediate future.

#### **8. References**


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**21** 

**Role of Signaling Pathways in** 

Acute myeloid leukemia (AML) is a cancer wherein dysregulated differentiation, uncontrolled growth and inhibition of apoptosis lead to accumulation of immature myeloid progenitor cells and progression of oncogenic expression (Lowenberg et al., 1999). AML is now seen to be initiated and maintained from a small, self-renewing population of leukemic stem cells (LSCs), which give rise to a progeny of more mature and highly cycling progenitors (colony forming unit-leukemia, CFU-L). CFU-Ls do not self-renew, however they are committed to proliferation and limited differentiation. By doing so, they originate a population of blast cells which constitute the majority of leukemic cells in both the bone marrow and peripheral blood of patients. The exact phenotype of LSCs is still debated, but they are comprised in the CD34+/CD38-/low population (Lane et al., 2009). CD34+/CD38+ leukemic cells were unable to initiate leukemia in immunodeficient mice. It should be noted that only about 50% of AML are able to initiate leukemia in NOD/SCID mice (Testa et al.,

The pathogenesis of leukemia may be explained by two classes of alterations of oncogenic genes as a result of chromosomal aberrations. C*lass I* mutations confers a proliferative and/or survival advantage to the cells. The current list of known leukemogenic class I mutations consists of more than 10 different protein tyrosine kinases (PTK) that undergo constitutive activation either by being fused to different N-terminal partner proteins providing an oligomerization domain, or by activating mutations such as point mutations in their kinase domain or internal tandem repeats (length mutations) in the juxtamembrane domain (Flt3, Kit). Most of these alterations are associated with chronic myeloproliferative disorders such as chronic myeloid leukemia/chronic myelomonocytic leukemia (CML/CMML) or Philadelphia negative myeloproliferative disorders, except activating mutations of Flt3 and Kit which are found almost exclusively in acute leukemia. Flt3 ITD (internal tandem duplication) mutants constitutively activate MAPK, AKT and STAT5, leading to Pim-1 activation and Bcl-xL (B-cell lymphoma) hyperexpression (Minami et al., 2003; Kim et al., 2005). Extracellular c-Kit mutations resulted in c-Kit receptor

**1. Introduction** 

2007)

**2. Leukemogenesis** 

**Acute Myeloid Leukemia** 

Maha Abdullah and Zainina Seman *Immunology Unit, Department of Pathology Faculty of Medicine and Health Sciences* 

*Universiti Putra Malaysia* 

*Malaysia*


### **Role of Signaling Pathways in Acute Myeloid Leukemia**

Maha Abdullah and Zainina Seman

*Immunology Unit, Department of Pathology Faculty of Medicine and Health Sciences Universiti Putra Malaysia Malaysia*

#### **1. Introduction**

428 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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terminal kinase in human leukemia cells: a relevance to apoptosis. Cancer Letter

Acute myeloid leukemia (AML) is a cancer wherein dysregulated differentiation, uncontrolled growth and inhibition of apoptosis lead to accumulation of immature myeloid progenitor cells and progression of oncogenic expression (Lowenberg et al., 1999). AML is now seen to be initiated and maintained from a small, self-renewing population of leukemic stem cells (LSCs), which give rise to a progeny of more mature and highly cycling progenitors (colony forming unit-leukemia, CFU-L). CFU-Ls do not self-renew, however they are committed to proliferation and limited differentiation. By doing so, they originate a population of blast cells which constitute the majority of leukemic cells in both the bone marrow and peripheral blood of patients. The exact phenotype of LSCs is still debated, but they are comprised in the CD34+/CD38-/low population (Lane et al., 2009). CD34+/CD38+ leukemic cells were unable to initiate leukemia in immunodeficient mice. It should be noted that only about 50% of AML are able to initiate leukemia in NOD/SCID mice (Testa et al., 2007)

#### **2. Leukemogenesis**

The pathogenesis of leukemia may be explained by two classes of alterations of oncogenic genes as a result of chromosomal aberrations. C*lass I* mutations confers a proliferative and/or survival advantage to the cells. The current list of known leukemogenic class I mutations consists of more than 10 different protein tyrosine kinases (PTK) that undergo constitutive activation either by being fused to different N-terminal partner proteins providing an oligomerization domain, or by activating mutations such as point mutations in their kinase domain or internal tandem repeats (length mutations) in the juxtamembrane domain (Flt3, Kit). Most of these alterations are associated with chronic myeloproliferative disorders such as chronic myeloid leukemia/chronic myelomonocytic leukemia (CML/CMML) or Philadelphia negative myeloproliferative disorders, except activating mutations of Flt3 and Kit which are found almost exclusively in acute leukemia. Flt3 ITD (internal tandem duplication) mutants constitutively activate MAPK, AKT and STAT5, leading to Pim-1 activation and Bcl-xL (B-cell lymphoma) hyperexpression (Minami et al., 2003; Kim et al., 2005). Extracellular c-Kit mutations resulted in c-Kit receptor

Role of Signaling Pathways in Acute Myeloid Leukemia 431

**aberration** 

HIP1/PDGF R t(5;7)(q33;q11) CMML, atypical

RAB5/PDGF R t(5;17)(q33;p13) CMML, atypical

CEV14/PDGF R t(5;14)(q33;q32) relapse AML NIN1/PDGF R t(1;5)(q23;q33) atypical CML HCMOGT/PDGF R t(5;17)(q33;p11) juvenile CMML TP53BP1/PDGF R t(5;15)(q33;q22) atypical CML

H4/PDGF R t(5;10)(q33;q21) CMML,atypical CML Myomegalin/PDGF R t(1;5)(q23;q33) CMML,atypical CML

TEL/JAK2 t(9;12)(p24;p13) Atypical CML, ALL,

PCM1/JAK2 t(8;9)(p21-22;p23-24) Atypical CML, AML,

**Disease phenotype** 

CML

CML

CML

AML

ALL

myelofibrosis

**PTK involved Fusion gene Chromosomal** 

ABL (9q34) BCR/ABL t(9;22)(q34;q11) CML

(ABL1) TEL/ABL t(9;12)(q34;p13) Atypical CML ARG (1q24) BCR/ARG t(1;22)(q24;q11) Atypical CML (ABL2) TEL/ARG t(1;12)(q24;p13) Atypical CML PDGF R (5q33) TEL/PDGF R t(5;12)(q33;p13) CMML, atypical

PDGF R (4q12) BCR/PDGF R t(4;22)(q12;q11) Atypical CML AK2 (9p24) BCR/JAK2 t(9;22)(p24;q11) CML, atypical CML

TRKC (15q25) TEL/TRKC t(12;15)(p13;q25) AML GFR3 (4p16) TEL/FGFR3 t(4;12)(p16;p13) AML FRK(6q21) TEL/FRK t(6;12)(q21;p13) AML

FLT3 (13q12) ITD (80%), activation loop kinase domain (15%) AML KIT (4q12) JM region, activation loop kinase domain AML JAK2 (9p21) JAK2 V617F mutation 9pLOH PV, ET,

*polycythemia vera; ET: essential thrombocythemia; LOH: loss of heterozygosity* 

FLT3 (13q12) Overexpression MLL alterations ALL/AML

*CML: chronic myeloid leukemia; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; CMML: chronic myelomonocytic leukemia; EMS; ITD: internal tandem duplication; JM: juxtamembrane; PV:* 

Table 1. Deregulated protein tyrosine kinases in myeloid leukemias (taken from Chalandon

Signal transduction is the primary means by which eukaryotic cells respond to external signals from their environment and coordinate complex cellular changes. Extracellular signal is transduced into the cell through ligand-receptor binding, followed by the activation

**B. Gain of function mutations**

**3. Signal Transduction Pathways (STP)** 

**C. Deregulated expression**

and Schwaller, 2005)

**A. Fusion genes**

hyperactivation in response to Kit ligand, with subsequent strong activation of MAPK and PI3K, while codon 816 c-Kit mutations induced constitutive STAT-3 activation and upregulation of Bcl-xL and c-myc (Schnittger et al., 2006)). Other class I alterations are gain of function mutations of the three main RAS isoforms (N-Ras, Ki-Ras, Ha-Ras) which are frequently seen in different myeloid malignancies (Beaupre and Kurzrock, 1999). N-Ras mutations lead to increased activity of the Ras pathway, resulting in increased proliferation and decreased apoptosis (Testa et al., 2007). Overexpression of *class I* mutations is generally sufficient to transform hematopoietic cells to growth-factor independence *in vitro* and to induce a lethal *leukemialike* myeloproliferative disorder in mice (Ilaria, 2004).

(Flt- FMS-Like Tyrosine Kinase 3, STAT- Signal Transducer and Activator of Transcription, MAPK-Mitogen-Activated Protein Kinase)

In contrast to class I mutations, there is a large group of genetic alterations mostly associated with acute leukemia, referred to as *class II* mutations, which impair differentiation of hematopoietic cells and subsequent apoptosis but do not directly provide proliferative and/or survival advantage. Many of them are loss of function mutations (either through fusion formation or point mutations) of transcriptional regulators that are critical for normal hematopoietic development and differentiation. Transcription factor fusion genes include CBF, RAR, MLL, HOX and CBP while loss of function mutations occur in AML1, CEBP/a, PU.1, GATA1 and IKAROS (reviewed in Chalandon and Schwaller, 2005). Via mediators of apoptosis, fusion proteins send anti-apoptotic signals that favor the preferential survival of leukemic cells:PML/RAR-α or CBF/SMMHC through the p53 pathway and AML1/ETO through the Bcl2-related pathway (Klampfer et al., 1996; Britos-bray et al., 1998; Pandolfi, 2001). PML/RARα fusion protein was also shown to exert an anti-apoptotic activity by downmodulating the expression of some death-inducing genes, such as TNF-R1 (Testa et al., 1998) and TRAIL-R1/-R2 (Ricioni et al., 2005). Nucleophosmin acts as a cellular p53 negative regulator to protect hematopoietic cells from stress-induced apoptosis (Lambert and Buckle, 2006). These mutations are usually not sufficient to mimic the human disease in transgenic mice since they do not readily induce a leukemia phenotype. However, after a long latency period, signs of myelodysplasia are often seen with a variable propensity to develop an immature and clonal hematologic disorder closely resembling human AML (reviewed in Chalandon and Schwaller, 2005). Additional mutations, occurring at the level of signal transduction molecules (the receptor tyrosine kinases Flt3 or c-Kit, NRas and Ki-Ras), are required for the generation of disease (reviewed in Testa et al., 2007). This hypothesis is supported by the analysis of unselected blood samples from neonates which showed that about 1% have class II genetic alterations that are detectable by PCR (Greaves et al., 2003). (CBF-core binding factor, RAR –retinoic acid receptor, MLL-mixed lineage leukemia, HOXhomeobox, CBP-CREB binding protein, CEBP/a-CCAAT/enhancer binding protein, PMLpromyelocyte leukemia, SMMHC-smooth muscle myosin heavy chain, TRAIL-tumor necrosis factor–related apoptosis-inducing ligand)

In the same light, being a heterogeneous disease, relapsed AML is unlikely to emanate from one predominant mechanism; instead, there are likely to be multiple biologic factors at play that allow for clinical relapse to occur. These factors likely include multidrug resistance proteins, aberrant signal transduction pathways, survival of leukemia stem cells, microenvironmental interactions, and immune tolerance. Many conditions in the environment select for the development of these target mechanisms, ranging from chemotherapeutic modalities, to signal transduction inhibitors, to upregulation of antileukemic immune responses (reviewed in Lancet and Karp, 2009)

hyperactivation in response to Kit ligand, with subsequent strong activation of MAPK and PI3K, while codon 816 c-Kit mutations induced constitutive STAT-3 activation and upregulation of Bcl-xL and c-myc (Schnittger et al., 2006)). Other class I alterations are gain of function mutations of the three main RAS isoforms (N-Ras, Ki-Ras, Ha-Ras) which are frequently seen in different myeloid malignancies (Beaupre and Kurzrock, 1999). N-Ras mutations lead to increased activity of the Ras pathway, resulting in increased proliferation and decreased apoptosis (Testa et al., 2007). Overexpression of *class I* mutations is generally sufficient to transform hematopoietic cells to growth-factor independence *in vitro* and to

(Flt- FMS-Like Tyrosine Kinase 3, STAT- Signal Transducer and Activator of Transcription,

In contrast to class I mutations, there is a large group of genetic alterations mostly associated with acute leukemia, referred to as *class II* mutations, which impair differentiation of hematopoietic cells and subsequent apoptosis but do not directly provide proliferative and/or survival advantage. Many of them are loss of function mutations (either through fusion formation or point mutations) of transcriptional regulators that are critical for normal hematopoietic development and differentiation. Transcription factor fusion genes include CBF, RAR, MLL, HOX and CBP while loss of function mutations occur in AML1, CEBP/a, PU.1, GATA1 and IKAROS (reviewed in Chalandon and Schwaller, 2005). Via mediators of apoptosis, fusion proteins send anti-apoptotic signals that favor the preferential survival of leukemic cells:PML/RAR-α or CBF/SMMHC through the p53 pathway and AML1/ETO through the Bcl2-related pathway (Klampfer et al., 1996; Britos-bray et al., 1998; Pandolfi, 2001). PML/RARα fusion protein was also shown to exert an anti-apoptotic activity by downmodulating the expression of some death-inducing genes, such as TNF-R1 (Testa et al., 1998) and TRAIL-R1/-R2 (Ricioni et al., 2005). Nucleophosmin acts as a cellular p53 negative regulator to protect hematopoietic cells from stress-induced apoptosis (Lambert and Buckle, 2006). These mutations are usually not sufficient to mimic the human disease in transgenic mice since they do not readily induce a leukemia phenotype. However, after a long latency period, signs of myelodysplasia are often seen with a variable propensity to develop an immature and clonal hematologic disorder closely resembling human AML (reviewed in Chalandon and Schwaller, 2005). Additional mutations, occurring at the level of signal transduction molecules (the receptor tyrosine kinases Flt3 or c-Kit, NRas and Ki-Ras), are required for the generation of disease (reviewed in Testa et al., 2007). This hypothesis is supported by the analysis of unselected blood samples from neonates which showed that about 1% have class II genetic alterations that are detectable by PCR (Greaves et al., 2003). (CBF-core binding factor, RAR –retinoic acid receptor, MLL-mixed lineage leukemia, HOXhomeobox, CBP-CREB binding protein, CEBP/a-CCAAT/enhancer binding protein, PMLpromyelocyte leukemia, SMMHC-smooth muscle myosin heavy chain, TRAIL-tumor

In the same light, being a heterogeneous disease, relapsed AML is unlikely to emanate from one predominant mechanism; instead, there are likely to be multiple biologic factors at play that allow for clinical relapse to occur. These factors likely include multidrug resistance proteins, aberrant signal transduction pathways, survival of leukemia stem cells, microenvironmental interactions, and immune tolerance. Many conditions in the environment select for the development of these target mechanisms, ranging from chemotherapeutic modalities, to signal transduction inhibitors, to upregulation of

induce a lethal *leukemialike* myeloproliferative disorder in mice (Ilaria, 2004).

MAPK-Mitogen-Activated Protein Kinase)

necrosis factor–related apoptosis-inducing ligand)

antileukemic immune responses (reviewed in Lancet and Karp, 2009)


*CML: chronic myeloid leukemia; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; CMML: chronic myelomonocytic leukemia; EMS; ITD: internal tandem duplication; JM: juxtamembrane; PV: polycythemia vera; ET: essential thrombocythemia; LOH: loss of heterozygosity* 

Table 1. Deregulated protein tyrosine kinases in myeloid leukemias (taken from Chalandon and Schwaller, 2005)

#### **3. Signal Transduction Pathways (STP)**

Signal transduction is the primary means by which eukaryotic cells respond to external signals from their environment and coordinate complex cellular changes. Extracellular signal is transduced into the cell through ligand-receptor binding, followed by the activation

Role of Signaling Pathways in Acute Myeloid Leukemia 433

3β (GSK3β). Each of these substrates has a key role in the regulation of cell survival and

The antiapoptotic effects of Akt occur through its phosphorylation of a wide variety of targets. The first antiapoptotic target identified was Bad, a member of the Bcl-2 family. Phosphorylation of Bad at S136 by Akt allows phosphorylated Bad to interact with 14-3-3 proteins, promoting cell survival (Datta et al., 1997; Andreeff et al., 1999). Interaction of Bad with 14-3-3 proteins inhibits the ability of Bad to interact with Bcl-2 and Bcl-xL. This allows Bcl-xL to bind to proapoptotic Bax molecules and prevent the formation of proapoptotic Bax homodimers. However, Bad is also phosphorylated on different sites by members of the

In human cells, Akt phosphorylates and inactivates caspase-9. Overexpression of Akt inhibits cytochrome c-induced activation of caspase-9 (Cardone et al., 1998). Phosphorylation of the Foxo family of transcription factors is also attributed to Akt (Biggs et al 1999); Brunet et al., 1999; Rena et al., 1999; Tang et al., 1999).This phosphorylation results in forkhead transcription factors translocation to the cytoplasm, thus inhibiting transcription of pro-apoptotic genes such as FasL (Brunet et al., 1999). Akt activates transcription of antiapoptotic genes through phosphorylation of IKK and regulation of nuclear factor-kappa B (NF-kB) (Ozes et al., 1999). Akt also promotes cell survival and cell cycle progression by its ability to phosphorylate MDM2 and GSK-3 (Fukumoto et al., 2001; Zhou et al., 2001). Once phosphorylated by Akt, MDM2 translocates to the nucleus and interacts with p300. p300 dissociates from p19ARF, resulting in the degradation of p53 and cell cycle progression. Akt phosphorylates GSK-3, inhibiting its activity. The decreased GSK-3 activity increases stability of catenin and enhances its association with lymphoid enhancer factor/T cell factor (LEF/TCF) (Fukumoto et al., 2001). The catenin–LEF/TCF complex increases transcription of proteins such as cyclin D1 and c-myc, promoting cell cycle progression (Fukumoto et al., 2001). Clearly, Akt can affect both cell cycle progression and apoptosis (reviewed in

MTORC1 is a critical regulator of translation initiation and ribosome biogenesis and plays an evolutionarily conserved role in cell growth control (Wullschleger et al., 2006). The enhanced sensitivity of cancer cells and mouse tumor models exhibiting oncogenic activation of the PI3K-Akt pathway to mTORC1 inhibitors, such as rapamycin, illustrates the importance of mTORC1 activation downstream of Akt (Sabatini, 2006). One of the bestconserved functions of Akt is its role in promoting cell growth (i.e., an increase in cell mass). The predominant mechanism appears to be through activation of mTOR complex 1 (mTORC1 or the mTOR-raptor complex), which is regulated by both nutrients and growth factor signaling. mTORC1 signaling integrates environmental clues (growth factors, hormones, nutrients, stressors) and information from the cell metabolic status. Thus, mTORC1 controls anabolic processes for promoting protein synthesis and cell growth

The JAK/STAT pathway consists of three families of genes: the JAK, or Janus family of tyrosine kinases, the STAT (signal transducers and activators of transcription) family and the CIS/SOCS family, which serves to downregulate the activity of the JAK/STAT pathway (Silvennoinen et al., 1993; Kisseleva et al., 2002; Krebs and Hilton, 2002; Fujitani et al., 1997). The JAK/STAT pathway involves signaling from the cytokine receptor to the nucleus. JAKs are stimulated by activation of a cytokine receptor. Stimulation of JAKs results in STAT

Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT)

proliferation, either directly or through an intermediary.

Raf/MEK/ERK (S112) and PKA (S112, S155) pathways.

Steelman et al., 2004).

(Manning and Cantley, 2007).

of intracellular signaling pathways that involve a series of protein phosphorylation and dephosphorylation, protein-protein interaction, and protein-small molecules interaction (Liu and Zhou, 2004). Cytokines interact with cell-surface receptors initiating signaling cascades that promote cell growth and division, while inhibiting the pathways of apoptotic cell death. The JAK/STAT, Raf/ MEK/ERK and PI3K/Akt signaling pathways are activated by a variety of cytokines that function to potentiate or inhibit hematopoiesis. These include IL (interleukin)-3, IL-7, SCF (stem cell factor), G (granulocyte)-CSF, type I interferons (IFN) and TGF- (transforming growth factor)- beta (Steelman et al., 2004).

The phosphatidylinositol 3-kinase (PI3K)

PI3K /protein kinase B (Akt)/mammalian target of rapamycin (mTOR) (a family of lipid kinases) signaling cascade is crucial to many widely divergent physiological processes which include cell cycle progression, transcription, translation, differentiation, apoptosis, motility, and metabolism (Yuan and Cantley, 2008). The family of PI3K enzymes phosphorylates inositol lipids and comprises three different classes, I, II, and III. Phosphorylated phosphatidylinositol 3,4,5trisphosphate [PtdIns (3,4,5)P3] recruits to the plasma membrane pleckstrin homology (PH) domain-containing proteins, which include phosphoinositide-dependent protein kinase 1 (PDK1) and Akt. The phospholipid products of PI3K activate downstream targets, including PDK, Akt and PKC (Palmer et al., 1995; Toker et al., 1994; Nakanishi et al., 1993).

Class I PI3K is further classified as A [activated by receptor tyrosine kinases (RTKs), Ras, and G-protein coupled receptors (GPCRs)] and B (activated by GPCRs) subtype. Class IA and 1B PI3Ks are heterodimeric enzymes composed of a regulatory and of catalytic subunits (Martelli et al., 2010).

Phosphoinositide-dependent kinase (PDK)

PDK requires the phospholipid product of PI3K for activation. There are believed to be two members of the PDK family – PDK1 and PDK2. Association of Akt with phosphoinositides produces a conformational change allowing Ser473 to be phosphorylated by PDK1 (Scheid et al., 2002).

Protein kinase B (Akt)

Akt is a 57-kDa serine/threonine protein kinase central to cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Activated Akt was originally isolated from cells of the leukemia and lymphoma prone AKR strain of mice (Staal, 1987). It comprises three highly conserved isoforms: Akt1/α, Akt2/β, and Akt3/γ which are functionally different (Staal, 1987; Nicolson and Anderson, 2002; Staal et al., 1988). Once Akt is recruited at the plasma membrane, its activation loop is phosphorylated on Thr308 by PDK1 while the mTOR complex 2 (mTORC2), activated by RTK, phosphorylates Ser473 in the Akt COOH-terminus. Full Akt activation requires both phosphorylation steps. Active Akt migrates to both the cytosol and the nucleus. Nuclear Akt may fulfill important antiapoptotic roles. So far, over 100 Akt substrates have been identified (Manning and Cantley, 2007). Of these, about 40 which mediate the pleiotropic Akt functions have been characterized, including Bad, caspase-9, murine double minute 2 (MDM2), IĸB kinase (IKK) α, proline-rich Akt substrate 40-kDa (PRAS40) 40, the Foxo family of Forkhead box-o transcription factors, apoptosis signal-regulated kinase 1 [ASK1, a negative regulator of proapoptotic c-Jun N-terminal kinase (JNK)], Raf, p27Kip1, p21Cip1, glycogen synthase kinase

of intracellular signaling pathways that involve a series of protein phosphorylation and dephosphorylation, protein-protein interaction, and protein-small molecules interaction (Liu and Zhou, 2004). Cytokines interact with cell-surface receptors initiating signaling cascades that promote cell growth and division, while inhibiting the pathways of apoptotic cell death. The JAK/STAT, Raf/ MEK/ERK and PI3K/Akt signaling pathways are activated by a variety of cytokines that function to potentiate or inhibit hematopoiesis. These include IL (interleukin)-3, IL-7, SCF (stem cell factor), G (granulocyte)-CSF, type I interferons (IFN) and

PI3K /protein kinase B (Akt)/mammalian target of rapamycin (mTOR) (a family of lipid kinases) signaling cascade is crucial to many widely divergent physiological processes which include cell cycle progression, transcription, translation, differentiation, apoptosis, motility, and metabolism (Yuan and Cantley, 2008). The family of PI3K enzymes phosphorylates inositol lipids and comprises three different classes, I, II, and III. Phosphorylated phosphatidylinositol 3,4,5trisphosphate [PtdIns (3,4,5)P3] recruits to the plasma membrane pleckstrin homology (PH) domain-containing proteins, which include phosphoinositide-dependent protein kinase 1 (PDK1) and Akt. The phospholipid products of PI3K activate downstream targets, including PDK, Akt and PKC (Palmer et al., 1995;

Class I PI3K is further classified as A [activated by receptor tyrosine kinases (RTKs), Ras, and G-protein coupled receptors (GPCRs)] and B (activated by GPCRs) subtype. Class IA and 1B PI3Ks are heterodimeric enzymes composed of a regulatory and of catalytic subunits

PDK requires the phospholipid product of PI3K for activation. There are believed to be two members of the PDK family – PDK1 and PDK2. Association of Akt with phosphoinositides produces a conformational change allowing Ser473 to be phosphorylated by PDK1 (Scheid

Akt is a 57-kDa serine/threonine protein kinase central to cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Activated Akt was originally isolated from cells of the leukemia and lymphoma prone AKR strain of mice (Staal, 1987). It comprises three highly conserved isoforms: Akt1/α, Akt2/β, and Akt3/γ which are functionally different (Staal, 1987; Nicolson and Anderson, 2002; Staal et al., 1988). Once Akt is recruited at the plasma membrane, its activation loop is phosphorylated on Thr308 by PDK1 while the mTOR complex 2 (mTORC2), activated by RTK, phosphorylates Ser473 in the Akt COOH-terminus. Full Akt activation requires both phosphorylation steps. Active Akt migrates to both the cytosol and the nucleus. Nuclear Akt may fulfill important antiapoptotic roles. So far, over 100 Akt substrates have been identified (Manning and Cantley, 2007). Of these, about 40 which mediate the pleiotropic Akt functions have been characterized, including Bad, caspase-9, murine double minute 2 (MDM2), IĸB kinase (IKK) α, proline-rich Akt substrate 40-kDa (PRAS40) 40, the Foxo family of Forkhead box-o transcription factors, apoptosis signal-regulated kinase 1 [ASK1, a negative regulator of proapoptotic c-Jun N-terminal kinase (JNK)], Raf, p27Kip1, p21Cip1, glycogen synthase kinase

TGF- (transforming growth factor)- beta (Steelman et al., 2004).

The phosphatidylinositol 3-kinase (PI3K)

Toker et al., 1994; Nakanishi et al., 1993).

Phosphoinositide-dependent kinase (PDK)

(Martelli et al., 2010).

Protein kinase B (Akt)

et al., 2002).

3β (GSK3β). Each of these substrates has a key role in the regulation of cell survival and proliferation, either directly or through an intermediary.

The antiapoptotic effects of Akt occur through its phosphorylation of a wide variety of targets. The first antiapoptotic target identified was Bad, a member of the Bcl-2 family. Phosphorylation of Bad at S136 by Akt allows phosphorylated Bad to interact with 14-3-3 proteins, promoting cell survival (Datta et al., 1997; Andreeff et al., 1999). Interaction of Bad with 14-3-3 proteins inhibits the ability of Bad to interact with Bcl-2 and Bcl-xL. This allows Bcl-xL to bind to proapoptotic Bax molecules and prevent the formation of proapoptotic Bax homodimers. However, Bad is also phosphorylated on different sites by members of the Raf/MEK/ERK (S112) and PKA (S112, S155) pathways.

In human cells, Akt phosphorylates and inactivates caspase-9. Overexpression of Akt inhibits cytochrome c-induced activation of caspase-9 (Cardone et al., 1998). Phosphorylation of the Foxo family of transcription factors is also attributed to Akt (Biggs et al 1999); Brunet et al., 1999; Rena et al., 1999; Tang et al., 1999).This phosphorylation results in forkhead transcription factors translocation to the cytoplasm, thus inhibiting transcription of pro-apoptotic genes such as FasL (Brunet et al., 1999). Akt activates transcription of antiapoptotic genes through phosphorylation of IKK and regulation of nuclear factor-kappa B (NF-kB) (Ozes et al., 1999). Akt also promotes cell survival and cell cycle progression by its ability to phosphorylate MDM2 and GSK-3 (Fukumoto et al., 2001; Zhou et al., 2001). Once phosphorylated by Akt, MDM2 translocates to the nucleus and interacts with p300. p300 dissociates from p19ARF, resulting in the degradation of p53 and cell cycle progression. Akt phosphorylates GSK-3, inhibiting its activity. The decreased GSK-3 activity increases stability of catenin and enhances its association with lymphoid enhancer factor/T cell factor (LEF/TCF) (Fukumoto et al., 2001). The catenin–LEF/TCF complex increases transcription of proteins such as cyclin D1 and c-myc, promoting cell cycle progression (Fukumoto et al., 2001). Clearly, Akt can affect both cell cycle progression and apoptosis (reviewed in Steelman et al., 2004).

MTORC1 is a critical regulator of translation initiation and ribosome biogenesis and plays an evolutionarily conserved role in cell growth control (Wullschleger et al., 2006). The enhanced sensitivity of cancer cells and mouse tumor models exhibiting oncogenic activation of the PI3K-Akt pathway to mTORC1 inhibitors, such as rapamycin, illustrates the importance of mTORC1 activation downstream of Akt (Sabatini, 2006). One of the bestconserved functions of Akt is its role in promoting cell growth (i.e., an increase in cell mass). The predominant mechanism appears to be through activation of mTOR complex 1 (mTORC1 or the mTOR-raptor complex), which is regulated by both nutrients and growth factor signaling. mTORC1 signaling integrates environmental clues (growth factors, hormones, nutrients, stressors) and information from the cell metabolic status. Thus, mTORC1 controls anabolic processes for promoting protein synthesis and cell growth (Manning and Cantley, 2007).

Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT)

The JAK/STAT pathway consists of three families of genes: the JAK, or Janus family of tyrosine kinases, the STAT (signal transducers and activators of transcription) family and the CIS/SOCS family, which serves to downregulate the activity of the JAK/STAT pathway (Silvennoinen et al., 1993; Kisseleva et al., 2002; Krebs and Hilton, 2002; Fujitani et al., 1997). The JAK/STAT pathway involves signaling from the cytokine receptor to the nucleus. JAKs are stimulated by activation of a cytokine receptor. Stimulation of JAKs results in STAT

Role of Signaling Pathways in Acute Myeloid Leukemia 435

Raf-1 is also phosphorylated by Akt which has been associated with inhibition of Raf-1 activity (Wojknowski et al., 1997; Rommel et al., 1999). CAMP-dependent protein kinase (PKA) inhibits Raf-1 (Wu et al., 1993; Schramm et al., 1994; Dumaz et al., 2002). Protein kinase C isoforms (a, b and g) stimulates Raf-1 activity (Sozeri t al., 1992). Raf-1 has been postulated to have important roles in cell cycle progression, activation of the p53 and NF-kB transcription factors and the prevention of apoptosis (reviewed in Steelman et al., 2004) Interactions between the Raf and PI3K/Akt pathways, or crosstalk, is an area of intense research Recently, it was demonstrated that it is more effective to inhibit the growth of Rafand MEK1-transformed hematopoietic cells with inhibitors that target both the

MEK proteins are the primary downstream targets of Raf. The MEK family of genes consists of five genes: MEK1, MEK2, MEK3, MEK4 and MEK5. The structure of MEK consists of an amino-terminal negative regulatory domain and a carboxy-terminal MAP kinase-binding domain, which is necessary for binding and activation of ERKs (Huang et al., 1995; Tanoue et al., 2001; Crews et al., 1992). Deletion of the regulatory MEK1 domain results in constitutive MEK1 and ERK activation. Activated MEK1 could abrogate cytokine dependency of certain hematopoietic cells. Constitutive activity of MEK1 inhibits NF-kB

The main physiological substrates of MEK are the members of the ERK (extracellular signalregulated kinase) or MAPK (mitogen activated protein kinase) family of genes. The ERK family consists of four distinct groups of kinases: ERK, Jun amino terminal kinases (JNK1/2/3), p38MAPK (p38 a/b/g/d) and ERK5. In addition, there are ERK3, ERK4, ERK6, ERK7 and ERK8 kinases, which while related to ERK1 and ERK2 have different modes of activation, and their biochemical roles are not as well characterized. Downstream targets of ERK include the p90Rsk kinase and the CREB, c-Myc and other transcription factors. ERK and p90Rsk can enter the nucleus to phosphorylate transcription factors which can lead to

Cilloni et al. (2007) have presented a comprehensive review on NF-kB. NF-kB proteins are a small group of related and evolutionarily conserved proteins which in mammals consists of five members: Rel (c-Rel), RelA/p65, RelB, p50, and p52 (Ghosh et al., 1998; Hayden et al., 2004).In resting cells, NF-kB proteins arepredominantly cytoplasmic, associating with members of the inhibitory IkB family such as IkBa, IkBb and Ikbe (Ghosh et al., 1998).These interact with NF-kB through multiple ankyrin repeats and as a result inhibit its DNA binding activity. Two NF-kB activation pathways exist; the first is normally triggered in response to infections or exposure to pro-inflammatorycytokines that activate the IkB kinase (IKK) complex leading to phosphorylation-induced IkB degradation, the other pathway leads to selective activation of p52: RelB dimers. This pathway is triggered by certain membersof the tumor necrosis factor (TNF)cytokine family through selective activation of IKKa by the upstream kinase, NF-kappa B-inducing kinase (NIK).In response to many stimuli such as inflammatorycytokines, bacterial lipopolysaccharide, phorbol esters, viral infection or stress, IkB are phosphorylated on two critical serine residues (Senftleben et al., 2001).

Raf/MEK/ERK and PI3K/Akt pathways (Navolanic et al., 2004).

their activation (reviewed in Steelman et al., 2004).

Nuclear factor kappa B (NFkB)

transcription by negatively regulating p38MAPK activity (Carter et al., 2000).

MEK

ERK

transcription factor activity. JAKs are a family of large tyrosine kinases, having molecular weights in the range of 120–140 kDa (1130–1142 aa). Four JAKs (JAK1, JAK2, JAK3 and Tyk2) have been identied in mammals. JAK3 expression is limited to hematopoietic cells (Steelman et al., 2004).

The STAT gene family consists of seven proteins (STAT1,STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6). Upregulation of STAT3 is detected with high frequency inhuman cancer. STAT3 is activated not only by cytokine receptors, such as the receptor for the IL-6 family cytokines, but also growth receptor tyrosine kinases, such as the EGFR family including Her2/Neu, and non-receptor tyrosine kinases such as Src and Abl(Turkson et al., 1998), and is also activated in response to stimulation of G-protein-coupled receptors (GPCR) (Pelletier et al., 2003). Classically, the receptor stimulation by ligand induces STAT3 binding to phosphotyrosine residues of receptors through its SH2 domain and its phosphorylation on a critical tyr705 residue by the receptor itself, or by associated Janus kinase (JAK, Jak1–3, Tyk2) or Src family tyrosine kinases (Yu et al., 2004).

Ras/Raf/MAPK kinase/extracellular signal-regulated kinase pathway (Ras/Raf/MEK/ERK)

The Ras/Raf/MEK/ERK pathway is a central signal transduction pathway, which transmits signals from multiple cell surface receptors to transcription factors in the nucleus (Chang et al., 2003a; Chang et al., 2003b; Chang et al., 2003c). This pathway is frequently referred to as the MAP kinase pathway as MAPK stands for mitogen-activated protein kinase indicating that this pathway can be stimulated by mitogens, cytokines and growth factors. The pathway can be activated by Ras stimulating the membrane translocation of Raf. This pathway also interacts with many different signal transduction pathways including PI3K/Akt and JAK/STAT.

Ras is a small GTP-binding protein, which is the common upstream molecule of several signaling pathways including Raf/MEK/ERK, PI3K/Akt and RalEGF/Ral (Chang et al., 2003a; Chang et al., 2003b; Chang et al., 2003c). There are three different Ras family members: Ha-Ras, Ki-Ras and N-Ras. The Ras proteins show varying abilities to activate the Raf/MEK/ERK and PI3K/Akt cascades, as Ki-Ras has been associated with Raf/MEK/ERK while Ha-Ras is associated with PI3K/Akt activation.

The Raf protein family consists of A-Raf, B-Raf and Raf-1, which are involved in the regulation of proliferation, differentiation and apoptosis induced after cytokine stimulation (Blalock et al., 1999; Mercer et al., 2003; Naumann et al., 1007, Pritchard et al., 1996; Mercer et al., 2002).Raf-1 has many effects on the regulation of apoptosis. Some of these effects occur at the mitochondrial membrane and are independent of MEK and ERK activity. It was observed that overexpression of activated A-Raf abrogates the cytokine dependence of hematopoietic cells. Overexpression of B-Raf in Rat-1 cells results in decreased apoptosis due to inhibition of caspase activity. Raf-1 has important roles in apoptosis as it phosphorylates and inactivates Bad (Wang et al., 1996). Raf-1 phosphorylates and coimmunoprecipitates with Bcl-2, as well as regulates Bag and Bad expression, in BCR/ABLexpressing cells (Salomoni et al., 1998). The ability of Raf proteins to phosphorylate MEK1 varies from B-Raf, Raf-1, A-Raf. The ability of Raf to abrogate cytokine dependency is inversely proportional to their MEK1 activity, with A-Raf, Raf-1, B-Raf (McCubrey et al., 1998; Hoyle et al., 2000). Stimulation of Raf activates MEK1 and ERK resulting in phosphorylation of transcription factors, proliferation, and inhibition of apoptosis (Steelman et al., 2004).

Raf-1 is also phosphorylated by Akt which has been associated with inhibition of Raf-1 activity (Wojknowski et al., 1997; Rommel et al., 1999). CAMP-dependent protein kinase (PKA) inhibits Raf-1 (Wu et al., 1993; Schramm et al., 1994; Dumaz et al., 2002). Protein kinase C isoforms (a, b and g) stimulates Raf-1 activity (Sozeri t al., 1992). Raf-1 has been postulated to have important roles in cell cycle progression, activation of the p53 and NF-kB transcription factors and the prevention of apoptosis (reviewed in Steelman et al., 2004) Interactions between the Raf and PI3K/Akt pathways, or crosstalk, is an area of intense research Recently, it was demonstrated that it is more effective to inhibit the growth of Rafand MEK1-transformed hematopoietic cells with inhibitors that target both the Raf/MEK/ERK and PI3K/Akt pathways (Navolanic et al., 2004).

#### MEK

434 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

transcription factor activity. JAKs are a family of large tyrosine kinases, having molecular weights in the range of 120–140 kDa (1130–1142 aa). Four JAKs (JAK1, JAK2, JAK3 and Tyk2) have been identied in mammals. JAK3 expression is limited to hematopoietic cells

The STAT gene family consists of seven proteins (STAT1,STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6). Upregulation of STAT3 is detected with high frequency inhuman cancer. STAT3 is activated not only by cytokine receptors, such as the receptor for the IL-6 family cytokines, but also growth receptor tyrosine kinases, such as the EGFR family including Her2/Neu, and non-receptor tyrosine kinases such as Src and Abl(Turkson et al., 1998), and is also activated in response to stimulation of G-protein-coupled receptors (GPCR) (Pelletier et al., 2003). Classically, the receptor stimulation by ligand induces STAT3 binding to phosphotyrosine residues of receptors through its SH2 domain and its phosphorylation on a critical tyr705 residue by the receptor itself, or by associated Janus

The Ras/Raf/MEK/ERK pathway is a central signal transduction pathway, which transmits signals from multiple cell surface receptors to transcription factors in the nucleus (Chang et al., 2003a; Chang et al., 2003b; Chang et al., 2003c). This pathway is frequently referred to as the MAP kinase pathway as MAPK stands for mitogen-activated protein kinase indicating that this pathway can be stimulated by mitogens, cytokines and growth factors. The pathway can be activated by Ras stimulating the membrane translocation of Raf. This pathway also interacts with many different signal transduction pathways including

Ras is a small GTP-binding protein, which is the common upstream molecule of several signaling pathways including Raf/MEK/ERK, PI3K/Akt and RalEGF/Ral (Chang et al., 2003a; Chang et al., 2003b; Chang et al., 2003c). There are three different Ras family members: Ha-Ras, Ki-Ras and N-Ras. The Ras proteins show varying abilities to activate the Raf/MEK/ERK and PI3K/Akt cascades, as Ki-Ras has been associated with Raf/MEK/ERK

The Raf protein family consists of A-Raf, B-Raf and Raf-1, which are involved in the regulation of proliferation, differentiation and apoptosis induced after cytokine stimulation (Blalock et al., 1999; Mercer et al., 2003; Naumann et al., 1007, Pritchard et al., 1996; Mercer et al., 2002).Raf-1 has many effects on the regulation of apoptosis. Some of these effects occur at the mitochondrial membrane and are independent of MEK and ERK activity. It was observed that overexpression of activated A-Raf abrogates the cytokine dependence of hematopoietic cells. Overexpression of B-Raf in Rat-1 cells results in decreased apoptosis due to inhibition of caspase activity. Raf-1 has important roles in apoptosis as it phosphorylates and inactivates Bad (Wang et al., 1996). Raf-1 phosphorylates and coimmunoprecipitates with Bcl-2, as well as regulates Bag and Bad expression, in BCR/ABLexpressing cells (Salomoni et al., 1998). The ability of Raf proteins to phosphorylate MEK1 varies from B-Raf, Raf-1, A-Raf. The ability of Raf to abrogate cytokine dependency is inversely proportional to their MEK1 activity, with A-Raf, Raf-1, B-Raf (McCubrey et al., 1998; Hoyle et al., 2000). Stimulation of Raf activates MEK1 and ERK resulting in phosphorylation of transcription factors, proliferation, and inhibition of apoptosis (Steelman

kinase (JAK, Jak1–3, Tyk2) or Src family tyrosine kinases (Yu et al., 2004). Ras/Raf/MAPK kinase/extracellular signal-regulated kinase pathway

(Steelman et al., 2004).

(Ras/Raf/MEK/ERK)

PI3K/Akt and JAK/STAT.

et al., 2004).

while Ha-Ras is associated with PI3K/Akt activation.

MEK proteins are the primary downstream targets of Raf. The MEK family of genes consists of five genes: MEK1, MEK2, MEK3, MEK4 and MEK5. The structure of MEK consists of an amino-terminal negative regulatory domain and a carboxy-terminal MAP kinase-binding domain, which is necessary for binding and activation of ERKs (Huang et al., 1995; Tanoue et al., 2001; Crews et al., 1992). Deletion of the regulatory MEK1 domain results in constitutive MEK1 and ERK activation. Activated MEK1 could abrogate cytokine dependency of certain hematopoietic cells. Constitutive activity of MEK1 inhibits NF-kB transcription by negatively regulating p38MAPK activity (Carter et al., 2000).

#### ERK

The main physiological substrates of MEK are the members of the ERK (extracellular signalregulated kinase) or MAPK (mitogen activated protein kinase) family of genes. The ERK family consists of four distinct groups of kinases: ERK, Jun amino terminal kinases (JNK1/2/3), p38MAPK (p38 a/b/g/d) and ERK5. In addition, there are ERK3, ERK4, ERK6, ERK7 and ERK8 kinases, which while related to ERK1 and ERK2 have different modes of activation, and their biochemical roles are not as well characterized. Downstream targets of ERK include the p90Rsk kinase and the CREB, c-Myc and other transcription factors. ERK and p90Rsk can enter the nucleus to phosphorylate transcription factors which can lead to their activation (reviewed in Steelman et al., 2004).

#### Nuclear factor kappa B (NFkB)

Cilloni et al. (2007) have presented a comprehensive review on NF-kB. NF-kB proteins are a small group of related and evolutionarily conserved proteins which in mammals consists of five members: Rel (c-Rel), RelA/p65, RelB, p50, and p52 (Ghosh et al., 1998; Hayden et al., 2004).In resting cells, NF-kB proteins arepredominantly cytoplasmic, associating with members of the inhibitory IkB family such as IkBa, IkBb and Ikbe (Ghosh et al., 1998).These interact with NF-kB through multiple ankyrin repeats and as a result inhibit its DNA binding activity. Two NF-kB activation pathways exist; the first is normally triggered in response to infections or exposure to pro-inflammatorycytokines that activate the IkB kinase (IKK) complex leading to phosphorylation-induced IkB degradation, the other pathway leads to selective activation of p52: RelB dimers. This pathway is triggered by certain membersof the tumor necrosis factor (TNF)cytokine family through selective activation of IKKa by the upstream kinase, NF-kappa B-inducing kinase (NIK).In response to many stimuli such as inflammatorycytokines, bacterial lipopolysaccharide, phorbol esters, viral infection or stress, IkB are phosphorylated on two critical serine residues (Senftleben et al., 2001).

Role of Signaling Pathways in Acute Myeloid Leukemia 437

The three main signaling pathways are kept in check by naturally occurring inhibitors or tumor suppressor proteins. For example, the JAK/STAT pathway has the SOCS/CIS family of proteins, which serve to limit its effects by a negative feedback pathway. The Raf/MEK/ERK pathway can be negatively regulated by the PI3K/Akt cascade as well as the MKP1 phosphatase, which inactivates phosphorylated ERK. The PI3K/Akt pathway has the PTEN and SHIP phosphatases, which serve to fine-tune its antiapoptotic effects

Genetic events that give rise to leukemic transformation occur through activation of components of receptor tyrosine kinase (RTK) signaling pathways (Liu and Zhou, 2004). These include fusion proteins or gene mutations such as seen with activated TEL-JAK, STAT5A and BCR-ABL. Transforming activity of oncogenic PTK is mediated by parallel activation of several downstream signaling pathways. Final downstream mediators of this complex signaling network are phosphoproteins that translocate to the nucleus and act as transcriptional regulators activating a distinct group of target genes. The oncogenic activity of a given PTK is mediated by several signaling pathways including JAK/STAT,

Oncogenic activity severs dependence of transformed cell on external stimulation for survival. TEL-JAK fusion proteins contain the oligomerization domain of TEL and the tyrosine kinase domains of JAK1, JAK2, JAK3, or TYK2. These efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. STAT5 are constitutively active in TEL-JAK2- and TEL-JAK1-expressing cells (Lacronique et al., 2000). The BCR-ABL oncogene produces an activated tyrosine kinase fusion protein and gain independence from IL-3 for cell growth (Mandanas et al., 1992). Activated forms of Ras, Raf, MEK, PI3K and Akt however, show significant differences in

TEL/JAK2 isoforms, depending on the location of the breakpoints in the JAK2 gene, have been described in acute lymphoblastic leukemia of the B-cell type and atypical CML (Lacronique et al., 1997). Somatically acquired JAK2 mutation (V617F) was detected in 472/944 (50%) of patients with Ph-negative chronic myeloproliferative disorders [including polycythemia vera (PV), idiopathic myelofibrosis (IMF) and essential thrombocytosis (ET)] with predominance in PV (66%) followed by IMF (42%) and ET (26%) (Jones et al., 2005). Recent investigation of novel mutations in JAK2 revealed a higher incidence, ~99% and 55%

Flt-3 mediates its proliferative and antiapoptotic effects through several signaling pathways including the STAT5, Ras/MAPK and PI3K/AKT pathways. Overexpression of Flt-3 was detected in 73% of AML and 78% of ALL patients (Nakao et al., 1996). Flt-3 *length mutations*  (internal tandem duplications (ITD) in the juxtamembrane domain) (Nakao et al., 1996), is observed in more than 20% of adult and more than 10% of pediatric AML patients harbor an Flt-3-ITD (reviewed in Testa et al., 2007). In general, patients with mutant FLT3 show higher cell counts and decreased overall survival. Absence of the wild-type allele in patients with Flt-3-ITD predicted poor prognosis in 82 adult *de novo* AML cases with otherwise normal cytogenetics who received uniform high-dose therapy. Of the the 23 (28%) patients with Flt3-ITD, disease-free survival (DFS) was inferior (P = 0.03), yet overall survival (OS) was not different (P = 0.14) (Whitman et al., 2001). In cytogenetic normal AML patients aged > 60

(reviewed in Steelman et al., 2004).

Ras/MAPK, PI3K/AKT, or NF-kB.

in PV and ET, respectively (Tefferi, 2010).

**4. Aberrant STP and drug resistance in AML** 

the ability to abrogate cytokine dependence (Steelman et al., 2004).

Fig. 1. Signal transduction pathways (refer text for details)

This modification triggers IkB ubiquitination and destruction via the 26Sproteasome degradation machinery. As a consequence, NF-kBis freed to enter the nucleus and regulate transcription of over 150 genes encoding cell adhesion molecules, cytokines, growth factors, components of the immune systems and anti-apoptotic genes such as FLIP (FLICE inhibitory protein), cIAPs (inhibitor of apoptosis), Bcl-2 and Bcl-xL (Aggarwall, 2004). It is also implicated in the regulation of cell proliferation by controlling D-type cyclins (Takebayashi et al., 2003).

Fig. 1. Signal transduction pathways (refer text for details)

cyclins (Takebayashi et al., 2003).

This modification triggers IkB ubiquitination and destruction via the 26Sproteasome degradation machinery. As a consequence, NF-kBis freed to enter the nucleus and regulate transcription of over 150 genes encoding cell adhesion molecules, cytokines, growth factors, components of the immune systems and anti-apoptotic genes such as FLIP (FLICE inhibitory protein), cIAPs (inhibitor of apoptosis), Bcl-2 and Bcl-xL (Aggarwall, 2004). It is also implicated in the regulation of cell proliferation by controlling D-type The three main signaling pathways are kept in check by naturally occurring inhibitors or tumor suppressor proteins. For example, the JAK/STAT pathway has the SOCS/CIS family of proteins, which serve to limit its effects by a negative feedback pathway. The Raf/MEK/ERK pathway can be negatively regulated by the PI3K/Akt cascade as well as the MKP1 phosphatase, which inactivates phosphorylated ERK. The PI3K/Akt pathway has the PTEN and SHIP phosphatases, which serve to fine-tune its antiapoptotic effects (reviewed in Steelman et al., 2004).

#### **4. Aberrant STP and drug resistance in AML**

Genetic events that give rise to leukemic transformation occur through activation of components of receptor tyrosine kinase (RTK) signaling pathways (Liu and Zhou, 2004). These include fusion proteins or gene mutations such as seen with activated TEL-JAK, STAT5A and BCR-ABL. Transforming activity of oncogenic PTK is mediated by parallel activation of several downstream signaling pathways. Final downstream mediators of this complex signaling network are phosphoproteins that translocate to the nucleus and act as transcriptional regulators activating a distinct group of target genes. The oncogenic activity of a given PTK is mediated by several signaling pathways including JAK/STAT, Ras/MAPK, PI3K/AKT, or NF-kB.

Oncogenic activity severs dependence of transformed cell on external stimulation for survival. TEL-JAK fusion proteins contain the oligomerization domain of TEL and the tyrosine kinase domains of JAK1, JAK2, JAK3, or TYK2. These efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. STAT5 are constitutively active in TEL-JAK2- and TEL-JAK1-expressing cells (Lacronique et al., 2000). The BCR-ABL oncogene produces an activated tyrosine kinase fusion protein and gain independence from IL-3 for cell growth (Mandanas et al., 1992). Activated forms of Ras, Raf, MEK, PI3K and Akt however, show significant differences in the ability to abrogate cytokine dependence (Steelman et al., 2004).

TEL/JAK2 isoforms, depending on the location of the breakpoints in the JAK2 gene, have been described in acute lymphoblastic leukemia of the B-cell type and atypical CML (Lacronique et al., 1997). Somatically acquired JAK2 mutation (V617F) was detected in 472/944 (50%) of patients with Ph-negative chronic myeloproliferative disorders [including polycythemia vera (PV), idiopathic myelofibrosis (IMF) and essential thrombocytosis (ET)] with predominance in PV (66%) followed by IMF (42%) and ET (26%) (Jones et al., 2005). Recent investigation of novel mutations in JAK2 revealed a higher incidence, ~99% and 55% in PV and ET, respectively (Tefferi, 2010).

Flt-3 mediates its proliferative and antiapoptotic effects through several signaling pathways including the STAT5, Ras/MAPK and PI3K/AKT pathways. Overexpression of Flt-3 was detected in 73% of AML and 78% of ALL patients (Nakao et al., 1996). Flt-3 *length mutations*  (internal tandem duplications (ITD) in the juxtamembrane domain) (Nakao et al., 1996), is observed in more than 20% of adult and more than 10% of pediatric AML patients harbor an Flt-3-ITD (reviewed in Testa et al., 2007). In general, patients with mutant FLT3 show higher cell counts and decreased overall survival. Absence of the wild-type allele in patients with Flt-3-ITD predicted poor prognosis in 82 adult *de novo* AML cases with otherwise normal cytogenetics who received uniform high-dose therapy. Of the the 23 (28%) patients with Flt3-ITD, disease-free survival (DFS) was inferior (P = 0.03), yet overall survival (OS) was not different (P = 0.14) (Whitman et al., 2001). In cytogenetic normal AML patients aged > 60

Role of Signaling Pathways in Acute Myeloid Leukemia 439

From 50% to 80% of patients with AML display Akt phosphorylated on either Thr308 or Ser473 (or both) (Xu et al., 2003; Min et al., 2003). Univariate analysis of 146 AML patients revealed those with low levels of pAKT had somewhat better CR rates (60% versus 50%; *P=*0.21), longer median CR durations (71 versus 32 weeks; *P=*0.13), and statistically significant longer median survival times (59 versus 30 weeks; *P=*0.02) compared with those with high levels of pAKT. In another study, single analysis of Akt phosphorylated at threonine 308 (Thr308) and serine 473 (Ser473) showed AktThr308(high) patients had significantly shorter overall survival (11 vs 47 months; P=0.01), event-free survival (9 vs 26 months; P=0.005) and relapse-free survival (10 months vs not reached; P=0.02) than Thr308(low) patients. This was not observed for Akt Ser473 (Gallay et al., 2009). Poor prognosis of AML patients with elevated PI3K/Akt/mTOR signaling could be also related to the fact that this pathway controls the expression of the membrane ATP binding cassette (ABC) transporter, multidrug resistance-associated protein 1, associated with a lower survival rate (Tazzari et al., 2007; Schaich et al., 2005). Nevertheless, a more recent report has highlighted that constitutive activation of PI3K/Akt/mTOR signaling could be a favourable prognostic factor in de novo cases of AML. One hypothesis for the lower relapse rate in patients with enhanced PI3K/Akt/mTOR signaling is that it could drive immature leukemic cells (LSCs and CFU-L) into S phase, thus rendering them more susceptible to

The AKT pathway was among the signaling cascades whose simultaneous activation with other pathways, such as PKCα and ERK, was found to confer a poor prognosis in AML (Altman et al., 2011). Eventhough often mutated in human cancer, MMAC1/PTEN gene are

NF-kB has been found to be activated in CD34+/CD38– blast cells derived from patients with *de novo* AML (Guzman et al., 2001; Baumgartner et al., 2002). Leukemic stem cells residing in this population are quiescent or slowly cycling and therefore less sensitive to chemotherapy. They are therefore likely to be responsible for disease relapse and represent the target for future innovative therapies (Bonnett et al.,1997; Lowenberg et al., 1999; Jordan, 2002). Activation of NF-kB in leukemia patients has been well documented though NF-kB activation is not uniform among AML patients. Forty percent of AML patients evaluated presented with increased NF-kB DNA binding activity. These patients are characterized by increased white cell counts at diagnosis and increased blast percentages in the bone marrow suggesting a link between NF-kB and cell proliferation. In particular, cyclin D1, whose expression is regulated by NF-kB. Alternatively, NF-kB action could be due to the induction of genes coding for AML growth factors such as GM-CSF or granulocyte colony-stimulating

The majority of LSCs are quiescent and insensitive to traditional chemotherapeutic drugs. This latter feature explains, at least in part, the difficulties in eradicating this cell population by conventional polychemotherapy. Thus, novel therapeutic strategies for AML eradication should also target LSCs (Misaghian et al., 2009). In AML, aberrant activation of several signal transduction pathways strongly enhances the proliferation and survival of both LSCs and CFU-Ls (McCubrey et al., 2008; Steelman et al., 2008). Therefore, these signaling networks are attractive targets for the development of innovative therapeutic strategies in

infrequent as genetic aberrations in myeloid leukaemia (Aggerholm et al., 2000)

polychemotherapy (Tamburini et al., 2005)

factor (G-CSF) (Cilloni et al., 2007).

AML (Scholl et al., 2008).

years treated on Cancer and Leukemia Group B frontline trials, FLT3-ITD remained associated with shorter disease-free survival (P < .001; hazard ratio 2.10) and overall survival (P < .001; hazard Ratio 1.97) in multivariable analyses (Whitman et al., 2010). Flt3 kinase domain point mutants is mutated in about 35% of AML (Stirewalt et al., 2003). In a study of 481 patients, FLT3 mutation did not have an impact on event-free survival (EFS) in patients with CBF-AML (*P* = .84) and poor-risk AML (*P* = .37). However, while event-free survival was worse in the FLT3-internal tandem duplication (ITD) group (20 weeks vs 41 weeks; P < .00,001) this was not observed for the FLT3-tyrosine kinase domain (TKD) point mutation group (61 weeks vs 41 weeks; P = .15) (Santos et al., 2011).

The profiles of signal transduction that correlated with poor response to chemotherapy showed potentiated STAT5 and STAT3 phosphorylations as well as attenuated STAT1 phosphorylation following cytokine stimulation (Irish et al., 2004)

Ras mutations are frequently observed in certain hematopoietic malignancies including myelodysplastic syndromes, juvenile myelomonocytic leukemia and acute myeloid leukemia (Bartram et al., 1988; Flotho et al., 1999; Stirewalt et al., 2001). lt has been shown to activate both the Raf/MEK/ERK and the PI3K/Akt pathways. Thus, mutations at Ras should theoretically activate both pathways simultaneously. Consequence of this activation may be the increased expression of growth factors that can potentially further activate this cascade by an autocrine loop. Many cytokine and growth factor gene promoters contain binding sites for transcription factors (Ets, Elk, Jun, Fos, CREB) whose activities are often activated by the Raf/MEK/ERK cascade (reviewed in Steelman et al., 2004).

There is increasing evidence that activation of the PI3K/AKT signaling pathway leading to downstream inactivation of Foxo transcription factors, activation of the mammalian target of rapamycin (mTOR), or induction of Skp2 (leading to degradation of the cell cycle inhibitor p27), plays a central role in transformation by several mutated PTK such as BCR/ABL, mutated FLT3 or KIT (Scheijen et al., 2004; Andreu et al., 2005). Emerging evidence suggests that activation of NF-kB involves crosstalk between the PI3K and Ras/MAPK pathways (Gelfanov et al., 2001; Kirchner et al., 2003). Several NF-kB target genes, such as cIAP1/cIAP2, Bcl-xL, or Mcl-1, are well-known inhibitors of apoptosis that may co-mediate the antiapoptotic effect of a constitutively activated PTK (Aichberger et al., 2005).

Expression of transcription factor fusions like AML1/ETO and PML/RARa in leukemic cells leads to induction of several genes associated with WNT signaling (Muller-Tidow et al., 2004). WNT signaling activation was found in a significant fraction of leukemic blasts from patients with AML-M0 (Zheng et al., 2004).

Other causes of PI3K/Akt/mTOR activation in AML may be the result of several factors, including low levels of PP2A, autocrine/paracrine secretion of growth factors such as IGF-1 and VEGF (reviewed in Martelli et al., 2010). Interactions between leukemic cells and bone marrow stromal cells through CXCR4 (a GPCR) which is abundantly expressed on leukemic cell surface where it is up-regulated by hypoxic conditions and its physiological ligand, (Fierro et al., 2009; Fiegl et al., 2009) CXCL12, produced by stromal cells, (Fiegl et al., 2009; Ayala et al., 2009) could result in PI3K/Akt/mTOR activation (Zeng et al., 2009). Furthermore, interactions between β1 integrins on AML cells and stromal fibronectin could lead to pathway activation, (Matsunaga et al., 2003; Matsunaga et al., 2008) possibly through up-regulation of integrin-linked kinase 1 (ILK1) which is involved in Akt phosphorylation on Ser473 in a PI3K-dependent manner in AML cells (Tabe et al., 2007).

PI3K/Akt/ mTOR pathway influences proliferation, survival, and drug resistance of AML cells.

years treated on Cancer and Leukemia Group B frontline trials, FLT3-ITD remained associated with shorter disease-free survival (P < .001; hazard ratio 2.10) and overall survival (P < .001; hazard Ratio 1.97) in multivariable analyses (Whitman et al., 2010). Flt3 kinase domain point mutants is mutated in about 35% of AML (Stirewalt et al., 2003). In a study of 481 patients, FLT3 mutation did not have an impact on event-free survival (EFS) in patients with CBF-AML (*P* = .84) and poor-risk AML (*P* = .37). However, while event-free survival was worse in the FLT3-internal tandem duplication (ITD) group (20 weeks vs 41 weeks; P < .00,001) this was not observed for the FLT3-tyrosine kinase domain (TKD) point

The profiles of signal transduction that correlated with poor response to chemotherapy showed potentiated STAT5 and STAT3 phosphorylations as well as attenuated STAT1

Ras mutations are frequently observed in certain hematopoietic malignancies including myelodysplastic syndromes, juvenile myelomonocytic leukemia and acute myeloid leukemia (Bartram et al., 1988; Flotho et al., 1999; Stirewalt et al., 2001). lt has been shown to activate both the Raf/MEK/ERK and the PI3K/Akt pathways. Thus, mutations at Ras should theoretically activate both pathways simultaneously. Consequence of this activation may be the increased expression of growth factors that can potentially further activate this cascade by an autocrine loop. Many cytokine and growth factor gene promoters contain binding sites for transcription factors (Ets, Elk, Jun, Fos, CREB) whose activities are often

There is increasing evidence that activation of the PI3K/AKT signaling pathway leading to downstream inactivation of Foxo transcription factors, activation of the mammalian target of rapamycin (mTOR), or induction of Skp2 (leading to degradation of the cell cycle inhibitor p27), plays a central role in transformation by several mutated PTK such as BCR/ABL, mutated FLT3 or KIT (Scheijen et al., 2004; Andreu et al., 2005). Emerging evidence suggests that activation of NF-kB involves crosstalk between the PI3K and Ras/MAPK pathways (Gelfanov et al., 2001; Kirchner et al., 2003). Several NF-kB target genes, such as cIAP1/cIAP2, Bcl-xL, or Mcl-1, are well-known inhibitors of apoptosis that may co-mediate

Expression of transcription factor fusions like AML1/ETO and PML/RARa in leukemic cells leads to induction of several genes associated with WNT signaling (Muller-Tidow et al., 2004). WNT signaling activation was found in a significant fraction of leukemic blasts from

Other causes of PI3K/Akt/mTOR activation in AML may be the result of several factors, including low levels of PP2A, autocrine/paracrine secretion of growth factors such as IGF-1 and VEGF (reviewed in Martelli et al., 2010). Interactions between leukemic cells and bone marrow stromal cells through CXCR4 (a GPCR) which is abundantly expressed on leukemic cell surface where it is up-regulated by hypoxic conditions and its physiological ligand, (Fierro et al., 2009; Fiegl et al., 2009) CXCL12, produced by stromal cells, (Fiegl et al., 2009; Ayala et al., 2009) could result in PI3K/Akt/mTOR activation (Zeng et al., 2009). Furthermore, interactions between β1 integrins on AML cells and stromal fibronectin could lead to pathway activation, (Matsunaga et al., 2003; Matsunaga et al., 2008) possibly through up-regulation of integrin-linked kinase 1 (ILK1) which is involved in Akt phosphorylation

PI3K/Akt/ mTOR pathway influences proliferation, survival, and drug resistance of AML

mutation group (61 weeks vs 41 weeks; P = .15) (Santos et al., 2011).

phosphorylation following cytokine stimulation (Irish et al., 2004)

activated by the Raf/MEK/ERK cascade (reviewed in Steelman et al., 2004).

the antiapoptotic effect of a constitutively activated PTK (Aichberger et al., 2005).

on Ser473 in a PI3K-dependent manner in AML cells (Tabe et al., 2007).

patients with AML-M0 (Zheng et al., 2004).

cells.

From 50% to 80% of patients with AML display Akt phosphorylated on either Thr308 or Ser473 (or both) (Xu et al., 2003; Min et al., 2003). Univariate analysis of 146 AML patients revealed those with low levels of pAKT had somewhat better CR rates (60% versus 50%; *P=*0.21), longer median CR durations (71 versus 32 weeks; *P=*0.13), and statistically significant longer median survival times (59 versus 30 weeks; *P=*0.02) compared with those with high levels of pAKT. In another study, single analysis of Akt phosphorylated at threonine 308 (Thr308) and serine 473 (Ser473) showed AktThr308(high) patients had significantly shorter overall survival (11 vs 47 months; P=0.01), event-free survival (9 vs 26 months; P=0.005) and relapse-free survival (10 months vs not reached; P=0.02) than Thr308(low) patients. This was not observed for Akt Ser473 (Gallay et al., 2009). Poor prognosis of AML patients with elevated PI3K/Akt/mTOR signaling could be also related to the fact that this pathway controls the expression of the membrane ATP binding cassette (ABC) transporter, multidrug resistance-associated protein 1, associated with a lower survival rate (Tazzari et al., 2007; Schaich et al., 2005). Nevertheless, a more recent report has highlighted that constitutive activation of PI3K/Akt/mTOR signaling could be a favourable prognostic factor in de novo cases of AML. One hypothesis for the lower relapse rate in patients with enhanced PI3K/Akt/mTOR signaling is that it could drive immature leukemic cells (LSCs and CFU-L) into S phase, thus rendering them more susceptible to polychemotherapy (Tamburini et al., 2005)

The AKT pathway was among the signaling cascades whose simultaneous activation with other pathways, such as PKCα and ERK, was found to confer a poor prognosis in AML (Altman et al., 2011). Eventhough often mutated in human cancer, MMAC1/PTEN gene are infrequent as genetic aberrations in myeloid leukaemia (Aggerholm et al., 2000)

NF-kB has been found to be activated in CD34+/CD38– blast cells derived from patients with *de novo* AML (Guzman et al., 2001; Baumgartner et al., 2002). Leukemic stem cells residing in this population are quiescent or slowly cycling and therefore less sensitive to chemotherapy. They are therefore likely to be responsible for disease relapse and represent the target for future innovative therapies (Bonnett et al.,1997; Lowenberg et al., 1999; Jordan, 2002). Activation of NF-kB in leukemia patients has been well documented though NF-kB activation is not uniform among AML patients. Forty percent of AML patients evaluated presented with increased NF-kB DNA binding activity. These patients are characterized by increased white cell counts at diagnosis and increased blast percentages in the bone marrow suggesting a link between NF-kB and cell proliferation. In particular, cyclin D1, whose expression is regulated by NF-kB. Alternatively, NF-kB action could be due to the induction of genes coding for AML growth factors such as GM-CSF or granulocyte colony-stimulating factor (G-CSF) (Cilloni et al., 2007).

The majority of LSCs are quiescent and insensitive to traditional chemotherapeutic drugs. This latter feature explains, at least in part, the difficulties in eradicating this cell population by conventional polychemotherapy. Thus, novel therapeutic strategies for AML eradication should also target LSCs (Misaghian et al., 2009). In AML, aberrant activation of several signal transduction pathways strongly enhances the proliferation and survival of both LSCs and CFU-Ls (McCubrey et al., 2008; Steelman et al., 2008). Therefore, these signaling networks are attractive targets for the development of innovative therapeutic strategies in AML (Scholl et al., 2008).

Role of Signaling Pathways in Acute Myeloid Leukemia 441

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[6] Altman JK, Sassano A, Platanias LC. Targeting mTOR for the treatment of AML. New

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[9] Bartram CR. Mutations in ras genes in myelocyticleukemias and myelodysplastic

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### **5. Conclusion**

Expression of STP proteins is heterogenous and of prognostic value in AML (Kornblau et al., 2009). These signaling pathways in AML may in the future help rationally select targeted therapies in individual patients (Foran, 2010). While current classification schemes have prognostic relevance they generally do not alter therapeutic recommendations. As knowledge of mutated genes in cancersim proves, our ability to treat patients afflicted with certain diseases will increase substantially. The genetic mutation may affect multiple signal transduction pathways. Targeting multiple pathways may be more efficacious as this approach may suppress or eliminate tumor growth at lower concentrations of the drugs than that required to inhibit growth by targeting a single pathway (Steelmanet al., 2004).

The heterogeneity in AML continues to elude the best methods to characterize them. Genome and proteome-wide analysis has further revealed complexity in the makeup of the leukemic cell. The rapid advancement in targeted therapies implied the urgent need for alternative therapy and the readiness of the community to embrace it. Nevertheless so far, combinatorial medicine still holds out as the best option for successful treatment. If targeted therapies remain the way forward it will eventually bank deeply on the ability to identify molecular signatures in the individual leading to the establishment of personalized medicine.

Novel array technologies enabled the analysis of numerous features at the level of DNA for gene copy number variation, mutations, methylation in addition to mRNA transcription and regulatory microRNA. Emerging technologies to assess protein expression and phosphorylation levels within cells e.g. cytokine and chemokine arrays to assess external forces acting on leukemic cells and phosphoproteins in apoptosis, cell-cycle, and signaltransduction pathways, are highly needed. Protein expression and posttranslational modifications, either alone or in concert with other profiling approaches, could provide independent or complementary information not captured by transcriptional profiles. Protein signature groups, with prognostic information distinct from cytogenetics may reveal underlying similarities indistinguishable by cytogenetics (Kornblau et al., 2009).

Quantitative flow cytometry appears well suited for identifying predictive markers in AML patients because it offers obvious advantages over other techniques (western blot, for example), including rapidness, a much lower number of cells required to perform the assay, and the possibility of identifying different subclones in the leukemic population by coimmunostaining with multiple antibodies to surface antigens (Martelli et al., 2010). The mechanisms in leukemogenesis, drug resistance and relapse remain an area of much research. From cell biology to cytogenetics to molecular defects to signaling pathways, all have contributed to a better understanding of the cancer. New knowledge in epigenetics and microRNA remain to be elucidated.

#### **6. References**


Expression of STP proteins is heterogenous and of prognostic value in AML (Kornblau et al., 2009). These signaling pathways in AML may in the future help rationally select targeted therapies in individual patients (Foran, 2010). While current classification schemes have prognostic relevance they generally do not alter therapeutic recommendations. As knowledge of mutated genes in cancersim proves, our ability to treat patients afflicted with certain diseases will increase substantially. The genetic mutation may affect multiple signal transduction pathways. Targeting multiple pathways may be more efficacious as this approach may suppress or eliminate tumor growth at lower concentrations of the drugs than that required to inhibit growth by targeting a single pathway (Steelmanet al., 2004). The heterogeneity in AML continues to elude the best methods to characterize them. Genome and proteome-wide analysis has further revealed complexity in the makeup of the leukemic cell. The rapid advancement in targeted therapies implied the urgent need for alternative therapy and the readiness of the community to embrace it. Nevertheless so far, combinatorial medicine still holds out as the best option for successful treatment. If targeted therapies remain the way forward it will eventually bank deeply on the ability to identify molecular signatures in the individual leading to the establishment of personalized

Novel array technologies enabled the analysis of numerous features at the level of DNA for gene copy number variation, mutations, methylation in addition to mRNA transcription and regulatory microRNA. Emerging technologies to assess protein expression and phosphorylation levels within cells e.g. cytokine and chemokine arrays to assess external forces acting on leukemic cells and phosphoproteins in apoptosis, cell-cycle, and signaltransduction pathways, are highly needed. Protein expression and posttranslational modifications, either alone or in concert with other profiling approaches, could provide independent or complementary information not captured by transcriptional profiles. Protein signature groups, with prognostic information distinct from cytogenetics may reveal

Quantitative flow cytometry appears well suited for identifying predictive markers in AML patients because it offers obvious advantages over other techniques (western blot, for example), including rapidness, a much lower number of cells required to perform the assay, and the possibility of identifying different subclones in the leukemic population by coimmunostaining with multiple antibodies to surface antigens (Martelli et al., 2010). The mechanisms in leukemogenesis, drug resistance and relapse remain an area of much research. From cell biology to cytogenetics to molecular defects to signaling pathways, all have contributed to a better understanding of the cancer. New knowledge in epigenetics

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**22** 

*1,4Chile, 2.3USA* 

**Epigenetic Changes Associated with** 

Soraya Gutierrez1, Amjad Javed2, Janet Stein3,

*3University of Massachusetts 4Universidad Santo Tomás;* 

Gary Stein3, Sandra Nicovani4, Valentina Fernandez1, Ricardo Alarcon1, Marcela Stuardo1, Milka Martinez1, Marcela Hinojosa1 and Boris Rebolledo-Jaramillo1

**Chromosomal Translocation in Leukemia** 

*1Universidad de Concepcion, Departamento de Bioquimica y Biologia Molecular,* 

Chromosome translocation reflects an abnormality caused by rearrangement of DNA fragments between non-homologous chromosomes. These rearrangements can be visualized by cytogenetic analysis of affected cells. Non-random chromosomal translocations are frequently associated with a variety of cancers, particularly hematological malignancies and childhood sarcomas although recent evidence demonstrates that such translocations are also common in epithelial tumors (Aplan, 2006; Mitelman et al., 2005). Initially considered as random events that get selected, it has become increasingly apparent that chromosomal translocations are influenced by cell type, cell stage and genomic context. Most commonly, these non-random chromosomal translocations are associated with specific hematopoietic cell types. Such chromosomal translocations are commonly used as diagnostic tool and are increasingly utilized to guide therapeutic decisions. Although, the mechanism that causes chromosomal translocations remains largely unknown, it is commonly accepted that they arise from DNA double strand breaks (DSBs) that are misrepaired (Aplan, 2006; Digweed and Sperling, 2004; Betti et al., 2003; Povirk, 2006). For a translocation to occur there are several mechanistic factors required: First there needs to be at least two DSBs in different chromosomes. Second the DSBs must arise close enough both in three-dimensional space and in time. Finally, DNA repair pathways must be available to join the two broken DNA fragments to form the translocation. It is estimated that everyday a normal cell in our body is exposed to approximately 20,000 DNA damaging events (Ames and Shigenaga, 1992). A major source of DNA damage is oxygen free radicals; however there are other endogenous and exogenous sources of DNA damage such as replication, transcription, and genotoxic stress. All of these processes can induce DSBs, but for two DNA fragments to be joined they must necessarily come in close proximity of each other (less than 1.3m) (Chen et al., 1996;

**1. Introduction** 

*2University of Alabama Department of Oral and Maxillofacial Surgery* 


### **Epigenetic Changes Associated with Chromosomal Translocation in Leukemia**

 Soraya Gutierrez1, Amjad Javed2, Janet Stein3, Gary Stein3, Sandra Nicovani4, Valentina Fernandez1, Ricardo Alarcon1, Marcela Stuardo1, Milka Martinez1, Marcela Hinojosa1 and Boris Rebolledo-Jaramillo1 *1Universidad de Concepcion, Departamento de Bioquimica y Biologia Molecular, 2University of Alabama Department of Oral and Maxillofacial Surgery 3University of Massachusetts 4Universidad Santo Tomás; 1,4Chile, 2.3USA* 

#### **1. Introduction**

448 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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973–982.

activated MAP kinase signaling pathway by adenosine 30,50-monophosphate.

Chromosome translocation reflects an abnormality caused by rearrangement of DNA fragments between non-homologous chromosomes. These rearrangements can be visualized by cytogenetic analysis of affected cells. Non-random chromosomal translocations are frequently associated with a variety of cancers, particularly hematological malignancies and childhood sarcomas although recent evidence demonstrates that such translocations are also common in epithelial tumors (Aplan, 2006; Mitelman et al., 2005). Initially considered as random events that get selected, it has become increasingly apparent that chromosomal translocations are influenced by cell type, cell stage and genomic context. Most commonly, these non-random chromosomal translocations are associated with specific hematopoietic cell types. Such chromosomal translocations are commonly used as diagnostic tool and are increasingly utilized to guide therapeutic decisions. Although, the mechanism that causes chromosomal translocations remains largely unknown, it is commonly accepted that they arise from DNA double strand breaks (DSBs) that are misrepaired (Aplan, 2006; Digweed and Sperling, 2004; Betti et al., 2003; Povirk, 2006). For a translocation to occur there are several mechanistic factors required: First there needs to be at least two DSBs in different chromosomes. Second the DSBs must arise close enough both in three-dimensional space and in time. Finally, DNA repair pathways must be available to join the two broken DNA fragments to form the translocation. It is estimated that everyday a normal cell in our body is exposed to approximately 20,000 DNA damaging events (Ames and Shigenaga, 1992). A major source of DNA damage is oxygen free radicals; however there are other endogenous and exogenous sources of DNA damage such as replication, transcription, and genotoxic stress. All of these processes can induce DSBs, but for two DNA fragments to be joined they must necessarily come in close proximity of each other (less than 1.3m) (Chen et al., 1996;

Epigenetic Changes Associated with Chromosomal Translocation in Leukemia 451

Fig. 2. **DNA Double Strand Break Repair Pathways**. Schematic representation of the two main DNA repair pathways: non homologous end joining (NHEJ) and homologous recombination (HR). The key regulatory proteins involved at multiple stages of each

Most of the chromosome translocations that have been analyzed to date show no consistent homologous sequences at the breakpoints regions. However, several structural features like DNase I hypersensitivity, topoisomerase II cleavage sites, DNA fragile sites and matrix or scaffold attachment regions (MARs or SARs) often colocalize with the mapped breakpoints. These observations suggest that chromatin organization may play a role in generation of translocations (Zhang et al., 2002; Strissel et al., 1998; Tanabe et al., 1996; Felix et al., 1995; Stanulla et al., 1997; Zhang and Rowley, 2006). Moreover, chromatin organization also has a prominent role in DNA repair process and therefore may influence both the selection of a DNA repair pathway and the eventual outcome of the DNA repair (Fernandez-Capetillo et al., 2004; Verger and Crossley, 2004; Tsukuda et al., 2005; Murr et al., 2006; Falk et al., 2008; Falk et al., 2010; Misteli and Soutoglou, 2009). Here we will focus on the current knowledge of the role that genomic structural features may have on formation of chromosomal translocations. Particularly we will analyze epigenetic marks associated with chromosome breakpoint regions and unusual DNA conformations among other structural features that

The higher order organization of genomes architecture during interphase of the cell cycle forms chromosomes territories; which are defined as the nuclear space occupied by the DNA of a given chromosome (Cremer and Cremer, 2001; Cremer et al., 2006; Meaburn and Misteli, 2007; Misteli, 2007). Individual chromosomes are organized into open and closed chromatin domains that occupy different spatial compartments. A similar concept of

pathway are indicated.

may alter genomic structural stability.

**2. Higher-order genome organization and translocations** 

Misteli, 2010). These requirements are accommodated in two competing models to explain DSBs proximity in the nucleus: the position first and the breakage first (Figure 1). The position first model suggests that the DNA regions involved in the translocation are in close proximity before DSBs generation. Support for this model comes from the observation that translocation frequencies differ among tissues and is paralleled by tissue-specific organization of the involved chromosomes (Mitelman et al., 2007). For example, the frequency of c-myc translocations to IgH, Ig, or Ig locus in Burkitt's lymphomas correlates with reciprocal distance (Roix et al., 2003). In contrast, the breakage first model proposes that DSBs are produced far apart and then move into close proximity to be repaired. In yeast for example, independent DNA lesions move and colocalize in repair factories (Lisby et al., 2003a; Lisby et al., 2003b). In mammalian cells experimental evidence, ranging from no or limited movement to an extensive movement and clustering of these DSBs breaks, provides support for both models (Kruhlak et al., 2006).

Fig. 1. **Models for Chromosomal Translocation Formation.** In the position first model two DSB that are nearby in the nuclei can be erroneously repair and give rise to a chromosomal translocation. In the breakage first model two DSB that are far apart in the nuclei are brought close together, probably to be repaired, resulting in a translocation.

In eukaryotic cells, DSBs are repaired by two different repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) (Figure 2). HR is a relative error-free repair pathway that required information from a template sequence to repair the damage. This pathway is active during S and G2 phases of the cell cycle when the sister chromatid template is easily available and it can also take place during both mitosis and meiosis. NHEJ is a template independent DSBs repair mechanism. It is an error-prone pathway active throughout the cell cycle and the most commonly used DBSs repair pathway in multicellular eukaryotes. NHEJ is subdivided in classical (C-NHEJ) and alternate (A-NHEJ). The C-NHEJ is responsible for individual intrachromosomal DSBs repair in an homology independent manner, while A-NHEJ works in a micro-homology dependent manner, is more error-prone and seems to be the primary pathway responsible for the generation of chromosomal translocations (Simsek and Jasin, 2010; Boboila et al., 2010).

Misteli, 2010). These requirements are accommodated in two competing models to explain DSBs proximity in the nucleus: the position first and the breakage first (Figure 1). The position first model suggests that the DNA regions involved in the translocation are in close proximity before DSBs generation. Support for this model comes from the observation that translocation frequencies differ among tissues and is paralleled by tissue-specific organization of the involved chromosomes (Mitelman et al., 2007). For example, the frequency of c-myc translocations to IgH, Ig, or Ig locus in Burkitt's lymphomas correlates with reciprocal distance (Roix et al., 2003). In contrast, the breakage first model proposes that DSBs are produced far apart and then move into close proximity to be repaired. In yeast for example, independent DNA lesions move and colocalize in repair factories (Lisby et al., 2003a; Lisby et al., 2003b). In mammalian cells experimental evidence, ranging from no or limited movement to an extensive movement and clustering of these DSBs breaks, provides

Fig. 1. **Models for Chromosomal Translocation Formation.** In the position first model two DSB that are nearby in the nuclei can be erroneously repair and give rise to a chromosomal translocation. In the breakage first model two DSB that are far apart in the nuclei are

In eukaryotic cells, DSBs are repaired by two different repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) (Figure 2). HR is a relative error-free repair pathway that required information from a template sequence to repair the damage. This pathway is active during S and G2 phases of the cell cycle when the sister chromatid template is easily available and it can also take place during both mitosis and meiosis. NHEJ is a template independent DSBs repair mechanism. It is an error-prone pathway active throughout the cell cycle and the most commonly used DBSs repair pathway in multicellular eukaryotes. NHEJ is subdivided in classical (C-NHEJ) and alternate (A-NHEJ). The C-NHEJ is responsible for individual intrachromosomal DSBs repair in an homology independent manner, while A-NHEJ works in a micro-homology dependent manner, is more error-prone and seems to be the primary pathway responsible for the generation of chromosomal translocations (Simsek and Jasin, 2010; Boboila et al.,

brought close together, probably to be repaired, resulting in a translocation.

support for both models (Kruhlak et al., 2006).

2010).

Fig. 2. **DNA Double Strand Break Repair Pathways**. Schematic representation of the two main DNA repair pathways: non homologous end joining (NHEJ) and homologous recombination (HR). The key regulatory proteins involved at multiple stages of each pathway are indicated.

Most of the chromosome translocations that have been analyzed to date show no consistent homologous sequences at the breakpoints regions. However, several structural features like DNase I hypersensitivity, topoisomerase II cleavage sites, DNA fragile sites and matrix or scaffold attachment regions (MARs or SARs) often colocalize with the mapped breakpoints. These observations suggest that chromatin organization may play a role in generation of translocations (Zhang et al., 2002; Strissel et al., 1998; Tanabe et al., 1996; Felix et al., 1995; Stanulla et al., 1997; Zhang and Rowley, 2006). Moreover, chromatin organization also has a prominent role in DNA repair process and therefore may influence both the selection of a DNA repair pathway and the eventual outcome of the DNA repair (Fernandez-Capetillo et al., 2004; Verger and Crossley, 2004; Tsukuda et al., 2005; Murr et al., 2006; Falk et al., 2008; Falk et al., 2010; Misteli and Soutoglou, 2009). Here we will focus on the current knowledge of the role that genomic structural features may have on formation of chromosomal translocations. Particularly we will analyze epigenetic marks associated with chromosome breakpoint regions and unusual DNA conformations among other structural features that may alter genomic structural stability.

#### **2. Higher-order genome organization and translocations**

The higher order organization of genomes architecture during interphase of the cell cycle forms chromosomes territories; which are defined as the nuclear space occupied by the DNA of a given chromosome (Cremer and Cremer, 2001; Cremer et al., 2006; Meaburn and Misteli, 2007; Misteli, 2007). Individual chromosomes are organized into open and closed chromatin domains that occupy different spatial compartments. A similar concept of

Epigenetic Changes Associated with Chromosomal Translocation in Leukemia 453

in lower eukaryote, but the domain that is phosphorylated in response to DSB is present in the C-terminus of other H2A-family members like H2A in S. cerevisiae and H2AZ in D. melanogaster (Downs et al., 2007). Although loss of H2AX does not abrogate DNAdamage checkpoints or repair, it impairs the joining of programmed DNA lesions during immunoglobulin class-switch recombination. These observations suggest that chromatin modifications at a distance are required for bringing together the DNA ends (Petersen et al., 2001; Reina-San-Martin et al., 2003). Moreover, failing to rejoin these programmed DSB in the absence of H2AX result in frequent chromosomal abnormalities (Franco et al., 2006; Ramiro et al., 2006). In addition to phosphorylation, H2A is also modified by acetylation in its N-terminal tail by NuA4, a histone acetyl transferase (HAT). Acetylation seems to be important for the ability of cells to survive after DNA damage (Bird et al.,

The findings that DSB induces a rapid local decrease in the density of the chromatin fiber (Kruhlak et al., 2006) and that nearby nucleosomes are repositioned (Shim et al., 2007) support the idea that ATP-dependent chromatin remodeling factors have an early role in the DNA damage response. Indeed, several reports have demonstrated that the ATP-dependent chromatin remodeling complexes RSC (Remodels the Structure of Chromatin), SWI/SNF (SWItch/Sucrose NonFermenting), INO80 (INOsitol requiring) and SWR (Sick with Rat8 ts) are recruited to DSB, although at different time after the DNA damage. The first complex recruited to the DSB is RSC, which mobilize the nucleosomes near a DSB to new positions. Interestingly, in the absence of RSC the phosphorylation of H2AX is delayed. The other three chromatin remodeling complexes, SWI/SNF, INO80 and SWR, are enriched at sites of DSB at later times suggesting that they are not required for the initial detection or signaling

Additionally, acetylation of conserved residues in the N-terminal tails of H3 and H4 has been found to contribute to both homologous and non-homologous recombination processes. For example, in mammalian cells, the TIP60 and the HAT cofactor TRRAP (transformation/transcription-domain associated protein) are recruited to sites of DSB, where they induce acetylation of H4 and facilitate homologous recombination. Similarly, another HAT, MOF (also known as MYST1) contributes to irradiation-induced acetylation of H4 at lysine 16 (H4K16). Defects on H3 and H4 acetylation have been linked to sensitivity to ionizing radiation and alteration in cell cycle checkpoints (Gupta et al., 2005). Complexes catalyzing the reverse process, i.e. histone deacetylation, have been shown to be enriched at late times at the DSB regions. If the acetylation of histones in the vicinity of DNA damage facilitate the repair process, then it is possible that the role of these histone deacetylase complexes might be to restore the chromatin to its original state once the DNA has been

**4. Histone post-translational modifications in chromosomal rearrangements**  The role of histone modifications on genomic rearrangements has been extensively studied in the V(D)J recombination process. This assembly process depends on a series of sitespecific recombination reactions that are initiated by DSBs produced by RAG1 and RAG2 complex (Bassing et al., 2002). Each rearranging gene segment is flanked by a recombination signal sequence (RSS). The recombinase complex recognizes pair of compatible RSS, introduce DSBs and then channel the reaction products to a DNA repair pathway. Aberrant

of the DSB, but for the subsequent stages of the repair process.

2002).

repaired.

nonrandom nuclear positioning applies to single loci and may be relevant to their translocation potential. For example the BCR and ABL genes, located on chromosomes 9 and 22, whose translocation leads to formation of a fusion protein involved in leukemia, are located in close proximity in normal hematopoietic cells at much higher frequency than would be expected based on a random distribution (Lukasova et al., 1997; Neves et al., 1999; Bartova et al., 2000). The same is true for the human chromosomes 12 and 16, which are frequently translocated in liposarcoma and are found in close proximity in differentiated adipocytes (Kuroda et al., 2004). Similarly, the frequency of c-myc translocations to IgH, Ig or Ig locus in Burkitt's lymphomas correlates with reciprocal nuclear distance. However, it is important to note that clonal oncogenic translocations in tumors are highly selected and therefore cannot be used to unequivocally determine the actual translocation frequency. More unbiased examinations may yet reveal translocations between loci that are not frequently in close proximity. The proximity of two particular loci within the interphase nucleus can be cell-type or tissue-specific. In this context, substantial colocalization of IgH and Ig occurs in activated splenic B cells but not in embryonic stem cells or thymocytes (Wang et al., 2009). Notably, colocalization with IgH is not a characteristic of the entire chromosome 16 on which Ig locus resides. In fact, about 15Mb sequences on either side of Ig do not colocalize; therefore, proximity can be determinant in the context of more narrow areas around specific genes and not with broad chromosome territories.

The internal structure of the chromosomal territories is poorly understood but most probably is formed by a network of looping chromatin fibers. This relatively open structure allows access to gene regulatory factors while simultaneously protect the DNA from the continuous attack of damaging agents. Supporting this view, data from several different loci involved in genomic rearrangements exhibit an altered chromatin conformation in their breakpoint regions. For example, the MLL gene exhibits a strong topoisomerase II cleavage site near exon 12 where genomic breakpoint from therapy related AML (t-AML) patients and infant leukemia patient with MLL translocation have been mapped. Moreover, the same region also exhibit hypersensitivity to DNaseI and is cleaved by S1 and Mung Bean nuclease which specifically recognize and cleave single-strand regions in supercoiled DNA (Strissel et al., 1998; Felix et al., 1995; Stanulla et al., 1997). These features however are not exclusive to MLL gene, as topoisomerase II and DNaseI hypersensitive sites has also been found at the breakpoint regions of AF9, BCL, ABL, *RUNX1*, ETO and CBP among other genes (Greaves, 1996; Zhang et al., 2002; Strissel et al., 1998; Aplan et al., 1996; Relling et al., 1998; Strick et al., 2006; Sperry et al., 1989). However, not all breakpoint regions identified to date for genes involved in translocations colocalize with either topoisomeraseII cleavage sites or DHS, suggesting that other chromatin structural properties maybe involved in determining the location of chromosome breakage.

#### **3. Chromatin organization and DNA repair**

Higher order chromatin structure is not only important for global susceptibility of DNA to damage but may also be relevant for DNA repair. It is well documented that the earliest response to a DSB is the phosphorylation of the histone H2A variant histone H2AX on its C-terminus. Within seconds of DSB formation, the phosphorylated H2AX is present over surrounding regions, spanning thousand to millions of base pairs (Rogakou et al., 1998; Rogakou et al., 1999; Leatherbarrow et al., 2006; Kinner et al., 2008). H2AX is not present

nonrandom nuclear positioning applies to single loci and may be relevant to their translocation potential. For example the BCR and ABL genes, located on chromosomes 9 and 22, whose translocation leads to formation of a fusion protein involved in leukemia, are located in close proximity in normal hematopoietic cells at much higher frequency than would be expected based on a random distribution (Lukasova et al., 1997; Neves et al., 1999; Bartova et al., 2000). The same is true for the human chromosomes 12 and 16, which are frequently translocated in liposarcoma and are found in close proximity in differentiated adipocytes (Kuroda et al., 2004). Similarly, the frequency of c-myc translocations to IgH, Ig or Ig locus in Burkitt's lymphomas correlates with reciprocal nuclear distance. However, it is important to note that clonal oncogenic translocations in tumors are highly selected and therefore cannot be used to unequivocally determine the actual translocation frequency. More unbiased examinations may yet reveal translocations between loci that are not frequently in close proximity. The proximity of two particular loci within the interphase nucleus can be cell-type or tissue-specific. In this context, substantial colocalization of IgH and Ig occurs in activated splenic B cells but not in embryonic stem cells or thymocytes (Wang et al., 2009). Notably, colocalization with IgH is not a characteristic of the entire chromosome 16 on which Ig locus resides. In fact, about 15Mb sequences on either side of Ig do not colocalize; therefore, proximity can be determinant in the context of more narrow

areas around specific genes and not with broad chromosome territories.

location of chromosome breakage.

**3. Chromatin organization and DNA repair** 

The internal structure of the chromosomal territories is poorly understood but most probably is formed by a network of looping chromatin fibers. This relatively open structure allows access to gene regulatory factors while simultaneously protect the DNA from the continuous attack of damaging agents. Supporting this view, data from several different loci involved in genomic rearrangements exhibit an altered chromatin conformation in their breakpoint regions. For example, the MLL gene exhibits a strong topoisomerase II cleavage site near exon 12 where genomic breakpoint from therapy related AML (t-AML) patients and infant leukemia patient with MLL translocation have been mapped. Moreover, the same region also exhibit hypersensitivity to DNaseI and is cleaved by S1 and Mung Bean nuclease which specifically recognize and cleave single-strand regions in supercoiled DNA (Strissel et al., 1998; Felix et al., 1995; Stanulla et al., 1997). These features however are not exclusive to MLL gene, as topoisomerase II and DNaseI hypersensitive sites has also been found at the breakpoint regions of AF9, BCL, ABL, *RUNX1*, ETO and CBP among other genes (Greaves, 1996; Zhang et al., 2002; Strissel et al., 1998; Aplan et al., 1996; Relling et al., 1998; Strick et al., 2006; Sperry et al., 1989). However, not all breakpoint regions identified to date for genes involved in translocations colocalize with either topoisomeraseII cleavage sites or DHS, suggesting that other chromatin structural properties maybe involved in determining the

Higher order chromatin structure is not only important for global susceptibility of DNA to damage but may also be relevant for DNA repair. It is well documented that the earliest response to a DSB is the phosphorylation of the histone H2A variant histone H2AX on its C-terminus. Within seconds of DSB formation, the phosphorylated H2AX is present over surrounding regions, spanning thousand to millions of base pairs (Rogakou et al., 1998; Rogakou et al., 1999; Leatherbarrow et al., 2006; Kinner et al., 2008). H2AX is not present in lower eukaryote, but the domain that is phosphorylated in response to DSB is present in the C-terminus of other H2A-family members like H2A in S. cerevisiae and H2AZ in D. melanogaster (Downs et al., 2007). Although loss of H2AX does not abrogate DNAdamage checkpoints or repair, it impairs the joining of programmed DNA lesions during immunoglobulin class-switch recombination. These observations suggest that chromatin modifications at a distance are required for bringing together the DNA ends (Petersen et al., 2001; Reina-San-Martin et al., 2003). Moreover, failing to rejoin these programmed DSB in the absence of H2AX result in frequent chromosomal abnormalities (Franco et al., 2006; Ramiro et al., 2006). In addition to phosphorylation, H2A is also modified by acetylation in its N-terminal tail by NuA4, a histone acetyl transferase (HAT). Acetylation seems to be important for the ability of cells to survive after DNA damage (Bird et al., 2002).

The findings that DSB induces a rapid local decrease in the density of the chromatin fiber (Kruhlak et al., 2006) and that nearby nucleosomes are repositioned (Shim et al., 2007) support the idea that ATP-dependent chromatin remodeling factors have an early role in the DNA damage response. Indeed, several reports have demonstrated that the ATP-dependent chromatin remodeling complexes RSC (Remodels the Structure of Chromatin), SWI/SNF (SWItch/Sucrose NonFermenting), INO80 (INOsitol requiring) and SWR (Sick with Rat8 ts) are recruited to DSB, although at different time after the DNA damage. The first complex recruited to the DSB is RSC, which mobilize the nucleosomes near a DSB to new positions. Interestingly, in the absence of RSC the phosphorylation of H2AX is delayed. The other three chromatin remodeling complexes, SWI/SNF, INO80 and SWR, are enriched at sites of DSB at later times suggesting that they are not required for the initial detection or signaling of the DSB, but for the subsequent stages of the repair process.

Additionally, acetylation of conserved residues in the N-terminal tails of H3 and H4 has been found to contribute to both homologous and non-homologous recombination processes. For example, in mammalian cells, the TIP60 and the HAT cofactor TRRAP (transformation/transcription-domain associated protein) are recruited to sites of DSB, where they induce acetylation of H4 and facilitate homologous recombination. Similarly, another HAT, MOF (also known as MYST1) contributes to irradiation-induced acetylation of H4 at lysine 16 (H4K16). Defects on H3 and H4 acetylation have been linked to sensitivity to ionizing radiation and alteration in cell cycle checkpoints (Gupta et al., 2005). Complexes catalyzing the reverse process, i.e. histone deacetylation, have been shown to be enriched at late times at the DSB regions. If the acetylation of histones in the vicinity of DNA damage facilitate the repair process, then it is possible that the role of these histone deacetylase complexes might be to restore the chromatin to its original state once the DNA has been repaired.

#### **4. Histone post-translational modifications in chromosomal rearrangements**

The role of histone modifications on genomic rearrangements has been extensively studied in the V(D)J recombination process. This assembly process depends on a series of sitespecific recombination reactions that are initiated by DSBs produced by RAG1 and RAG2 complex (Bassing et al., 2002). Each rearranging gene segment is flanked by a recombination signal sequence (RSS). The recombinase complex recognizes pair of compatible RSS, introduce DSBs and then channel the reaction products to a DNA repair pathway. Aberrant

Epigenetic Changes Associated with Chromosomal Translocation in Leukemia 455

This pattern of highly accessible chromatin structure characterized by H3 and H4 acetylation is also found at the breakpoint regions of other genes involved in translocations like MLL (Khobta et al., 2004), *RUNX1* (Stuardo et al., 2009) and ETO (our unpublished data). Using chromatin immunoprecipitation assays (ChIPs) we analyzed the chromatin structure at intrón 5 of the *RUNX1* gene, where all the translocation points for the (8;21) translocation has been mapped (Figure 3). Our results demonstrate that chromatin organization at intron 5 is completely different in HL-60 hematopoietic cells than in a nonhematopoietic cell (Stuardo et al., 2009). In fact, two distinct features mark the intron 5 in HL-60: a complete lack or significantly reduced levels of histone H1 and an increased

Fig. 3. **Diagrammatic representation of the** *RUNX1* **gene.** Top panel show the exon-intron organization of the gene as well as the two promoters that regulate its expression. Bottom panel show a magnification of intron 5 of the *RUNX1* gene. The three breakpoint clusters (BCR) are indicated as well as the amplification fragments analyzed by ChIP assays

(purple blocks labeled A-U). Dark gray arrows indicate topoisomerase II sites and light gray

The decreased association of histone H1, may indicate an overall enhanced accessibility and hence an increased availability to nucleases or DNA damaging agents. Notably, the region where the DNaseI hypersensitive site has been mapped presents one of the lowest rates of association of histone H1 in myeloid HL-60 cells (Figure 4, region U). Although the complete intron 5 is enriched in acetylated histone H3 (Figure 5), the chromatin organization is not homogeneous throughout the intron, suggesting that particular regions of intron 5 may play a regulatory role in transcription, subnuclear localization or compaction of the *RUNX1* gene. Interestingly, the chromatin organization at intron 5 resembles the chromatin structure adopted by the V(D)J gene segment. During V(D)J recombination, gene segments encoding the variable regions for immunoglobulins and T-cell receptors (TCR) are recombined and assembled in a new configuration. The same recombinase is present in both T and B cells, however recombination of immunoglobulins loci happens only in B cells while TCR loci rearrange only in T cells. Targeting of recombinase activity to specific gene segments is controlled largely by changes in chromatin accessibility in a spatio-temporal manner, and acetylation of histone tails has been shown to be a key event in this process. In fact, acetylation of histone H3 or H4 is elevated in B or T cell type at gene segments that can recombine, and reduced at segments that do not undergo recombination (Maes et al., 2006;

association of hyperacetylated histone H3.

arrow DNase I hypersensitive site.

targeting of RAG proteins can produce chromosomal translocations that are associated with many forms of leukemia or lymphoma. In general, genes segments within recombinationally active loci are mark by the same histone modifications that characterize transcriptionally active genes, i.e. H3 and H4 acetylation as well as H3 trimethylation at lysine 4 (H3K4me3). More recently, it has become evident that the predominant effect of these histone modifications is to recruit the RAG complex to the RSS. In fact, RAG2 through its PHD domain specifically binds to H3K4me3 and mutations that abolish this binding results in greatly impaired V(D)J recombination activity (Liu et al., 2007; Matthews et al., 2007). Additional support for the epigenetic role on V(D)J recombination comes from the observation that in V genes H3 acetylation, although lower than in J genes, exhibit a gradient of enrichment that mirrored the rearrangement frequency. Interestingly, a reciprocal pattern is observed for the repressive modification H3 dimethylation at lysine 9 (H3K9me2) (Espinoza and Feeney, 2005; Espinoza and Feeney, 2007).

H3 trimethylation at lysine 4 (H3K4me3) is also implicated in meiotic recombination. In fact PRDM9, a zinc finger protein that catalyze the trimethylation of H3 at lysine 4, has recently been identified as a major determinant of sequence-specific meiotic recombination (Cheung et al., 2010).

Another histone modification, H3 methylation at lysine 79 (H3K79me), is associated with recombinationally active loci both in yeast and mammalian cell lines (Ng et al., 2003). Moreover, overexpression of DOT1L (a H3K79me specific methyltransferase) together with genotoxic stress and dihydrotestosterone significantly increases formation of chromosomal rearrangement involving the ETS genes, which are a distinguishing feature of prostate cancer (Kumar-Sinha et al., 2008; Lin et al., 2009).

Lin and colleagues (Lin et al., 2009), using prostate cancer as a model to study translocation mechanisms, have shown that after irradiation far more translocations are formed in androgen treated than in control cells. They also demonstrated that the translocation regions, TMPRSS2, ERG and ETV all contain binding sites for the androgen receptor (AR) near their breakpoints and that, after treatment with androgen, there is a rapid recruitment of AR to these sites. Moreover, AR recruitment induces changes in higher-order chromatin structure and epigenetic modifications establishing an open chromatin conformation characteristic of transcribed genes. Another consequence of AR binding was the recruitment of the activation-induced cytidine deaminase (AID), a key factor in somatic hypermutation (SHM) and class switch recombination (CSR) where it contributes to formation of DSB during the process of generating antibody diversity.

Additional data supporting the role of chromatin structure in genomic rearrangements comes from the analysis of the mechanisms involved in formation of t(2;5), a chromosomal translocation associated with anaplastic large cell lymphoma (ALCL). Mathas et al (Mathas et al., 2009) found up-regulation of several genes located near the ALCL translocation breakpoint, regardless of the presence of t(2;5) in the tumor. Moreover, their increased transcriptional activity promotes cell survival and repression of T-cell specific gene expression programs, both characteristics are a hallmark of ALCL (Mathas et al., 2009). Interestingly, cells isolated from ALCL patients lacking t(2;5) were more susceptible to form the (2;5) translocation than control cells. Together these data suggest that deregulation of breakpoint-proximal genes occurs before the formation of translocations and that similar to V(D)J recombination, transcriptional activity and altered chromatin structure predispose cells to chromosomal translocation.

targeting of RAG proteins can produce chromosomal translocations that are associated with many forms of leukemia or lymphoma. In general, genes segments within recombinationally active loci are mark by the same histone modifications that characterize transcriptionally active genes, i.e. H3 and H4 acetylation as well as H3 trimethylation at lysine 4 (H3K4me3). More recently, it has become evident that the predominant effect of these histone modifications is to recruit the RAG complex to the RSS. In fact, RAG2 through its PHD domain specifically binds to H3K4me3 and mutations that abolish this binding results in greatly impaired V(D)J recombination activity (Liu et al., 2007; Matthews et al., 2007). Additional support for the epigenetic role on V(D)J recombination comes from the observation that in V genes H3 acetylation, although lower than in J genes, exhibit a gradient of enrichment that mirrored the rearrangement frequency. Interestingly, a reciprocal pattern is observed for the repressive modification H3 dimethylation at lysine 9

H3 trimethylation at lysine 4 (H3K4me3) is also implicated in meiotic recombination. In fact PRDM9, a zinc finger protein that catalyze the trimethylation of H3 at lysine 4, has recently been identified as a major determinant of sequence-specific meiotic recombination (Cheung

Another histone modification, H3 methylation at lysine 79 (H3K79me), is associated with recombinationally active loci both in yeast and mammalian cell lines (Ng et al., 2003). Moreover, overexpression of DOT1L (a H3K79me specific methyltransferase) together with genotoxic stress and dihydrotestosterone significantly increases formation of chromosomal rearrangement involving the ETS genes, which are a distinguishing feature of prostate

Lin and colleagues (Lin et al., 2009), using prostate cancer as a model to study translocation mechanisms, have shown that after irradiation far more translocations are formed in androgen treated than in control cells. They also demonstrated that the translocation regions, TMPRSS2, ERG and ETV all contain binding sites for the androgen receptor (AR) near their breakpoints and that, after treatment with androgen, there is a rapid recruitment of AR to these sites. Moreover, AR recruitment induces changes in higher-order chromatin structure and epigenetic modifications establishing an open chromatin conformation characteristic of transcribed genes. Another consequence of AR binding was the recruitment of the activation-induced cytidine deaminase (AID), a key factor in somatic hypermutation (SHM) and class switch recombination (CSR) where it contributes to formation of DSB

Additional data supporting the role of chromatin structure in genomic rearrangements comes from the analysis of the mechanisms involved in formation of t(2;5), a chromosomal translocation associated with anaplastic large cell lymphoma (ALCL). Mathas et al (Mathas et al., 2009) found up-regulation of several genes located near the ALCL translocation breakpoint, regardless of the presence of t(2;5) in the tumor. Moreover, their increased transcriptional activity promotes cell survival and repression of T-cell specific gene expression programs, both characteristics are a hallmark of ALCL (Mathas et al., 2009). Interestingly, cells isolated from ALCL patients lacking t(2;5) were more susceptible to form the (2;5) translocation than control cells. Together these data suggest that deregulation of breakpoint-proximal genes occurs before the formation of translocations and that similar to V(D)J recombination, transcriptional activity and altered chromatin structure predispose

(H3K9me2) (Espinoza and Feeney, 2005; Espinoza and Feeney, 2007).

cancer (Kumar-Sinha et al., 2008; Lin et al., 2009).

during the process of generating antibody diversity.

cells to chromosomal translocation.

et al., 2010).

This pattern of highly accessible chromatin structure characterized by H3 and H4 acetylation is also found at the breakpoint regions of other genes involved in translocations like MLL (Khobta et al., 2004), *RUNX1* (Stuardo et al., 2009) and ETO (our unpublished data). Using chromatin immunoprecipitation assays (ChIPs) we analyzed the chromatin structure at intrón 5 of the *RUNX1* gene, where all the translocation points for the (8;21) translocation has been mapped (Figure 3). Our results demonstrate that chromatin organization at intron 5 is completely different in HL-60 hematopoietic cells than in a nonhematopoietic cell (Stuardo et al., 2009). In fact, two distinct features mark the intron 5 in HL-60: a complete lack or significantly reduced levels of histone H1 and an increased association of hyperacetylated histone H3.

Fig. 3. **Diagrammatic representation of the** *RUNX1* **gene.** Top panel show the exon-intron organization of the gene as well as the two promoters that regulate its expression. Bottom panel show a magnification of intron 5 of the *RUNX1* gene. The three breakpoint clusters (BCR) are indicated as well as the amplification fragments analyzed by ChIP assays (purple blocks labeled A-U). Dark gray arrows indicate topoisomerase II sites and light gray arrow DNase I hypersensitive site.

The decreased association of histone H1, may indicate an overall enhanced accessibility and hence an increased availability to nucleases or DNA damaging agents. Notably, the region where the DNaseI hypersensitive site has been mapped presents one of the lowest rates of association of histone H1 in myeloid HL-60 cells (Figure 4, region U). Although the complete intron 5 is enriched in acetylated histone H3 (Figure 5), the chromatin organization is not homogeneous throughout the intron, suggesting that particular regions of intron 5 may play a regulatory role in transcription, subnuclear localization or compaction of the *RUNX1* gene. Interestingly, the chromatin organization at intron 5 resembles the chromatin structure adopted by the V(D)J gene segment. During V(D)J recombination, gene segments encoding the variable regions for immunoglobulins and T-cell receptors (TCR) are recombined and assembled in a new configuration. The same recombinase is present in both T and B cells, however recombination of immunoglobulins loci happens only in B cells while TCR loci rearrange only in T cells. Targeting of recombinase activity to specific gene segments is controlled largely by changes in chromatin accessibility in a spatio-temporal manner, and acetylation of histone tails has been shown to be a key event in this process. In fact, acetylation of histone H3 or H4 is elevated in B or T cell type at gene segments that can recombine, and reduced at segments that do not undergo recombination (Maes et al., 2006;

Epigenetic Changes Associated with Chromosomal Translocation in Leukemia 457

Fig. 6. **Summary of chromatin structure and histone modification in the regions involed in chromosomal translocation.** DSB can occur either in euchromatin or heterochromatin, but they must arise close enough both in time and in space to give rise to a chromosomal translocation. In both cases the regions surrounding the DSB will exhibit an open chromatin conformation either due to its presence in euchromatin or as result of the DNA repair

In addition to the classical B-DNA structure described by Watson and Crick (Watson and Crick, 1953), more than 10 different DNA conformations are documented to date. These alternative DNA conformations include Z-DNA, hairpins/cruciforms, H-DNA (triplexes), slipped DNA and sticky DNA among others (Felsenfeld and RICH, 1957; Wang et al., 1979; Lilley, 1980; Panayotatos and Wells, 1981; Lyamichev et al., 1983; Sen and Gilbert, 1988; Mirkin, 2008). Several studies have shown that these non-canonical DNA structures affect DNA replication and transcription, and contribute to genome instability. For example non-B forming sequences located in c-MYC and BCL-2 genes localize with translocation breaking points. Studies have demonstrated that the H-DNA structure from the human c-MYC gene can induce DSBs in mammalian cells and stimulate genomic instability on mouse chromosomes in transgenic mice (Wang et al., 2008). However, the same sequences are not mutagenic in bacteria, suggesting a requirement for host-specific trans-acting factors to

Palindromic sequences, including palindromic AT-rich repeats (PATRRs), have the potential to form stem-loop structures by intrastrand base pairing within single-stranded DNA. In fact, PATRRs mapped on chromosome 22q11 and other chromosomes, such as 11q23 and 17q11, were found to cause non random chromosomal translocation in sperm cells in the general population (Kato et al., 2006) as well as in cell culture (Inagaki et al., 2009). Polymorphisms within the PATRRs affect the susceptibility to translocation *in vitro*, with longer and more symmetric PATRRs showing a stronger predisposition to translocation events (Kato et al., 2006; Kogo et al., 2007; Inagaki et al., 2009). Therefore, it has been

process.

**5. DNA conformation** 

generate genomic instability.

Maes et al., 2001; McMurry and Krangel, 2000). Moreover, hyperacetylation induced by inhibitors of histone deacetylase complexes (HDAC) rescues recombination defects caused by the elimination of extracellular signals that induce recombination (Durum et al., 1998). These studies suggest that histone hyperacetylation precedes recombination by opening chromatin and promoting access to the recombinase. Thus regardless of the molecular mechanism involved, it seems that an open chromatin conformation is a common requirement for a translocation to take place (Figure 6).

Fig. 4. **Intron 5 of** *RUNX1* **gene exhibit decreased association of histone H1.** Chromatin immunoprecipitation assays were performed with formaldehyde cross-linked chromatin isolated form HL-60 cells. Real-time PCR amplification of ChIP-DNA is shown as fold change over IgG in bar graph for immunoprecipitation with anti H1 antibody. Light gray arrow indicates DNaseI hypersensitive site.

Fig. 5. **Intron 5 of** *RUNX1* **gene is enriched in acetylated histone H3.** Chromatin immunoprecipitation assays were performed with formaldehyde cross-linked chromatin isolated form HL-60 cells. Real-time PCR amplification of ChIP-DNA is shown as fold change over IgG for immunoprecipitation with anti acetylated H3 antibody. Light gray arrow indicates DNaseI hypersensitive site.

Fig. 6. **Summary of chromatin structure and histone modification in the regions involed in chromosomal translocation.** DSB can occur either in euchromatin or heterochromatin, but they must arise close enough both in time and in space to give rise to a chromosomal translocation. In both cases the regions surrounding the DSB will exhibit an open chromatin conformation either due to its presence in euchromatin or as result of the DNA repair process.

#### **5. DNA conformation**

456 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Maes et al., 2001; McMurry and Krangel, 2000). Moreover, hyperacetylation induced by inhibitors of histone deacetylase complexes (HDAC) rescues recombination defects caused by the elimination of extracellular signals that induce recombination (Durum et al., 1998). These studies suggest that histone hyperacetylation precedes recombination by opening chromatin and promoting access to the recombinase. Thus regardless of the molecular mechanism involved, it seems that an open chromatin conformation is a common

Fig. 4. **Intron 5 of** *RUNX1* **gene exhibit decreased association of histone H1.** Chromatin immunoprecipitation assays were performed with formaldehyde cross-linked chromatin isolated form HL-60 cells. Real-time PCR amplification of ChIP-DNA is shown as fold change over IgG in bar graph for immunoprecipitation with anti H1 antibody. Light gray

Fig. 5. **Intron 5 of** *RUNX1* **gene is enriched in acetylated histone H3.** Chromatin immunoprecipitation assays were performed with formaldehyde cross-linked chromatin isolated form HL-60 cells. Real-time PCR amplification of ChIP-DNA is shown as fold change over IgG for immunoprecipitation with anti acetylated H3 antibody. Light gray

requirement for a translocation to take place (Figure 6).

arrow indicates DNaseI hypersensitive site.

arrow indicates DNaseI hypersensitive site.

In addition to the classical B-DNA structure described by Watson and Crick (Watson and Crick, 1953), more than 10 different DNA conformations are documented to date. These alternative DNA conformations include Z-DNA, hairpins/cruciforms, H-DNA (triplexes), slipped DNA and sticky DNA among others (Felsenfeld and RICH, 1957; Wang et al., 1979; Lilley, 1980; Panayotatos and Wells, 1981; Lyamichev et al., 1983; Sen and Gilbert, 1988; Mirkin, 2008). Several studies have shown that these non-canonical DNA structures affect DNA replication and transcription, and contribute to genome instability. For example non-B forming sequences located in c-MYC and BCL-2 genes localize with translocation breaking points. Studies have demonstrated that the H-DNA structure from the human c-MYC gene can induce DSBs in mammalian cells and stimulate genomic instability on mouse chromosomes in transgenic mice (Wang et al., 2008). However, the same sequences are not mutagenic in bacteria, suggesting a requirement for host-specific trans-acting factors to generate genomic instability.

Palindromic sequences, including palindromic AT-rich repeats (PATRRs), have the potential to form stem-loop structures by intrastrand base pairing within single-stranded DNA. In fact, PATRRs mapped on chromosome 22q11 and other chromosomes, such as 11q23 and 17q11, were found to cause non random chromosomal translocation in sperm cells in the general population (Kato et al., 2006) as well as in cell culture (Inagaki et al., 2009). Polymorphisms within the PATRRs affect the susceptibility to translocation *in vitro*, with longer and more symmetric PATRRs showing a stronger predisposition to translocation events (Kato et al., 2006; Kogo et al., 2007; Inagaki et al., 2009). Therefore, it has been

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proposed that the secondary structures adopted by palindromic DNA induce a greater susceptibility to DSBs thus leading to translocations in human (Kurahashi et al., 2000).

There have also been identified chromosomal fragile sites, which are genomic regions especially susceptible to DNA breakage. These fragile sites are non-random specific loci that are stable under normal conditions, but under conditions of partial replication stress can form visible gaps or breaks in metaphase chromosomes (Durkin and Glover, 2007; Richards, 2001). Many different studies have established a connection between DNA fragile sites and the formation of cancer-specific genome rearrangements. However, only recently there has been direct evidence linking breakage at DNA fragile sites to the formation of a cancer specific translocation. Using as model RET/PTC rearrangements; which are commonly found in the papillary thyroid carcinoma (PTC) and in all cases result in the fusion of the tyrosine kinase domain of RET (rearranged in transformation) to the 5' portion of various unrelated genes (Nikiforov, 2008) Gandhi et al (Gandhi et al., 2010) demonstrate that fragile site-inducing chemicals can create DNA breaks within the RET/PTC partner genes and ultimately lead to the formation of RET/PTC rearrangements. Moreover, aphidicolin induced DNA breaks at RET gene were located within intron 11, which is the breakpoint cluster region identified in thyroid cells. Clinical studies have shown that two different rearrangements, RET/PTC1 and RET/PTC3, are more frequent in sporadic and radiationinduced tumors respectively (Fenton et al., 2000; Fugazzola et al., 1995; Nikiforov et al., 1997; Motomura et al., 1998). Interestingly, treatment of cells with aphidicolin (APH), 2 aminopurine (2-AP) and 5-bromodeoxyuridine (BrdU) resulted in the generation of RET/PTC1 but not RET/PTC3 suggesting that sporadic PTC tumors may result from breakage at fragile sites. Although no consensus sequence have been identified in the fragile sites until now, the majority of them can form highly stable non-B DNA structures.

#### **6. Concluding remarks**

Traditionally it has been assumed that translocations arise randomly by stochastic DSB and that enrichment of particular translocations was result of the survival advantage acquired by the cells bearing the translocation. However, more recent results suggest that breaks in the genome occur in a nonrandom fashion and that higher-order chromatin organization maybe, at least in part, responsible for the formation of recurrent translocations. Although, significant progresses have been made in understanding formation of chromosomal translocation, particularly in the area of VDJ recombination, many more questions remain unanswered. For instance, it is still not known what proteins or pathways are involved in formation and maintenance of structure at the breakpoint regions, if these regions have a role in some cellular processes and what signaling pathways or environmental conditions promote chromosomal translocations. The response to these basic research questions may greatly improve diagnostic and therapeutic and may help to develop preventive measures for disorders associated with genomic instability such as chromosomal translocations.

#### **7. Acknowledgments**

This work has been supported by Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT, Grant 1100670) (to SEG);National Institute of Health (Grant R03 TW007170- 01A2) (JLS and SEG).

#### **8. References**

458 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

proposed that the secondary structures adopted by palindromic DNA induce a greater susceptibility to DSBs thus leading to translocations in human (Kurahashi et al., 2000). There have also been identified chromosomal fragile sites, which are genomic regions especially susceptible to DNA breakage. These fragile sites are non-random specific loci that are stable under normal conditions, but under conditions of partial replication stress can form visible gaps or breaks in metaphase chromosomes (Durkin and Glover, 2007; Richards, 2001). Many different studies have established a connection between DNA fragile sites and the formation of cancer-specific genome rearrangements. However, only recently there has been direct evidence linking breakage at DNA fragile sites to the formation of a cancer specific translocation. Using as model RET/PTC rearrangements; which are commonly found in the papillary thyroid carcinoma (PTC) and in all cases result in the fusion of the tyrosine kinase domain of RET (rearranged in transformation) to the 5' portion of various unrelated genes (Nikiforov, 2008) Gandhi et al (Gandhi et al., 2010) demonstrate that fragile site-inducing chemicals can create DNA breaks within the RET/PTC partner genes and ultimately lead to the formation of RET/PTC rearrangements. Moreover, aphidicolin induced DNA breaks at RET gene were located within intron 11, which is the breakpoint cluster region identified in thyroid cells. Clinical studies have shown that two different rearrangements, RET/PTC1 and RET/PTC3, are more frequent in sporadic and radiationinduced tumors respectively (Fenton et al., 2000; Fugazzola et al., 1995; Nikiforov et al., 1997; Motomura et al., 1998). Interestingly, treatment of cells with aphidicolin (APH), 2 aminopurine (2-AP) and 5-bromodeoxyuridine (BrdU) resulted in the generation of RET/PTC1 but not RET/PTC3 suggesting that sporadic PTC tumors may result from breakage at fragile sites. Although no consensus sequence have been identified in the fragile

sites until now, the majority of them can form highly stable non-B DNA structures.

Traditionally it has been assumed that translocations arise randomly by stochastic DSB and that enrichment of particular translocations was result of the survival advantage acquired by the cells bearing the translocation. However, more recent results suggest that breaks in the genome occur in a nonrandom fashion and that higher-order chromatin organization maybe, at least in part, responsible for the formation of recurrent translocations. Although, significant progresses have been made in understanding formation of chromosomal translocation, particularly in the area of VDJ recombination, many more questions remain unanswered. For instance, it is still not known what proteins or pathways are involved in formation and maintenance of structure at the breakpoint regions, if these regions have a role in some cellular processes and what signaling pathways or environmental conditions promote chromosomal translocations. The response to these basic research questions may greatly improve diagnostic and therapeutic and may help to develop preventive measures for disorders associated with genomic instability such as chromosomal translocations.

This work has been supported by Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT, Grant 1100670) (to SEG);National Institute of Health (Grant R03 TW007170-

**6. Concluding remarks** 

**7. Acknowledgments** 

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Eisenreich, T.R., Chen, J., Dickins, R.A., Lowe, S.W., Nussenzweig, A., and Nussenzweig, M.C. (2006). Role of genomic instability and p53 in AID-induced c-

and Nussenzweig, M.C. (2003). H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or

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proximity of translocation-prone gene loci in human lymphomas. Nat Genet *34*,

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**23** 

*Sudan* 

**Myeloid Leukemia: A Molecular Focus** 

The developing world, Africa included, is witnessing an alarming upsurge of cancer incidence. The annual number of new cancer cases is expected to double by 2020 and up to 70% of the 20 million new cases of cancer predicted to occur yearly will be in the developing

One startling disparity, however, between cancer in the developing and developed worlds is that although the overall incidence of cancer in the developing world is half of that observed in the developed world, survival rates in the developing world are often less than one third of site-specific cancers in the developed world (Sener et al., 2005). This emphasizes the duality of the cancer problem in Africa, for being largely a disease of modern life style, occurring against a background of socio-economic disparities and greater burden of

The study of genetic epidemiology of cancers in Africa hence entails the study of peculiar features of gene-environment interaction that may be largely private to Africa. In addition to the state of socio-economic underdevelopment that applies almost to the majority of African states, there is the plethora of extreme environments, wide range of climatic conditions and cultures, but most of all the transition state of the African communities from rural subsistent into urban market oriented life style. Furthermore one has to consider the notorious prevalence of infectious and non infectious diseases that may have a bearing both on the initiation of myeloid leukemia, its prognosis and management, e.g. tuberculosis (Omoti et al., 2009), malaria, other chronic infections, sickle cell disease (Ahmed et al., 2008) and common adaptive traits that could modulate the course of the disease, as well as the role of

One interesting example of a potential trade-off between malignancies and parasitic diseases is CD36 a multiligand receptor associated with a broad array of physiological processes, believed to be under selective pressure from *Plasmodium falciparum,* and deficient or polymorphic in several African populations. The role of CD36 in sickle cell crises and cerebral malaria is debatable. As a receptor for thrombospondin 1, CD36 plays a role in the regulation of angiogenesis, which may be a therapeutic strategy for controlling the dissemination of malignant neoplasm. Moreover, it is commonly expressed on blasts in acute monocytic leukemia, megakaryoblastic leukemia, and erythroleukemia (Ge and

world (Jones et al., 1999; Ferlay et al., 2003; Yach et al., 2004).

**1. Introduction** 

communicable diseases.

oncogenic viruses discussed below.

**on Etiology and Risk Within Africa** 

*Department of Molecular Biology, Institute of Endemic Diseases,* 

Muntaser E. Ibrahim and Emad-Aldin I. Osman

*University of Khartoum, Khartoum,* 

higher-order chromatin fragmentation during the initial stages of apoptosis. Mol Cell Biol *17*, 4070-4079.


### **Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa**

Muntaser E. Ibrahim and Emad-Aldin I. Osman *Department of Molecular Biology, Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan* 

#### **1. Introduction**

464 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

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at atomic resolution. Nature *282*, 680-686.

deoxyribose nucleic acid. Nature *171*, 737-738.

Cell Biol *17*, 4070-4079.

383.

higher-order chromatin fragmentation during the initial stages of apoptosis. Mol

at breakpoint cluster regions may lead to chromosomal translocations found in

cleavage site and a DNase I hypersensitive site colocalize near exon 9 in the MLL

J.L., and Gutierrez, S.E. (2009). Altered chromatin modifications in AML1/RUNX1 breakpoint regions involved in (8;21) translocation. J Cell Physiol *218*, 343-349. Tanabe, S., Bohlander, S.K., Vignon, C.V., Espinosa, R., III, Zhao, N., Strissel, P.L., Zeleznik,

L., and Rowley, J.D. (1996). AF10 is split by MLL and HEAB, a human homolog to a putative Caenorhabditis elegans ATP/GTP-binding protein in an

at a DNA double-strand break site in Saccharomyces cerevisiae. Nature *438*, 379-

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Rowley, J.D. (2002). Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) The developing world, Africa included, is witnessing an alarming upsurge of cancer incidence. The annual number of new cancer cases is expected to double by 2020 and up to 70% of the 20 million new cases of cancer predicted to occur yearly will be in the developing world (Jones et al., 1999; Ferlay et al., 2003; Yach et al., 2004).

One startling disparity, however, between cancer in the developing and developed worlds is that although the overall incidence of cancer in the developing world is half of that observed in the developed world, survival rates in the developing world are often less than one third of site-specific cancers in the developed world (Sener et al., 2005). This emphasizes the duality of the cancer problem in Africa, for being largely a disease of modern life style, occurring against a background of socio-economic disparities and greater burden of communicable diseases.

The study of genetic epidemiology of cancers in Africa hence entails the study of peculiar features of gene-environment interaction that may be largely private to Africa. In addition to the state of socio-economic underdevelopment that applies almost to the majority of African states, there is the plethora of extreme environments, wide range of climatic conditions and cultures, but most of all the transition state of the African communities from rural subsistent into urban market oriented life style. Furthermore one has to consider the notorious prevalence of infectious and non infectious diseases that may have a bearing both on the initiation of myeloid leukemia, its prognosis and management, e.g. tuberculosis (Omoti et al., 2009), malaria, other chronic infections, sickle cell disease (Ahmed et al., 2008) and common adaptive traits that could modulate the course of the disease, as well as the role of oncogenic viruses discussed below.

One interesting example of a potential trade-off between malignancies and parasitic diseases is CD36 a multiligand receptor associated with a broad array of physiological processes, believed to be under selective pressure from *Plasmodium falciparum,* and deficient or polymorphic in several African populations. The role of CD36 in sickle cell crises and cerebral malaria is debatable. As a receptor for thrombospondin 1, CD36 plays a role in the regulation of angiogenesis, which may be a therapeutic strategy for controlling the dissemination of malignant neoplasm. Moreover, it is commonly expressed on blasts in acute monocytic leukemia, megakaryoblastic leukemia, and erythroleukemia (Ge and

Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa 467

fundamentally contrasting life styles and their food cultures are different. The significance of such disparity to all aspects of health and disease is of interest and the study of potential preponderance of these community members to chronic diseases including hematological

The lowest rates of leukemia reported in sub-Saharan Africa probably represent failure of diagnosis or reporting to some extent (Davies, 1973; Fleming, 1993 and Parkin et al., 2005). We should therefore use caution, when drawing conclusions based on the varying prevalence and incidence, as an indication of clustering of cases or an environmental or genetic effect, as this may simply be due to the deficiency of statistics in Africa. The disparity could also be a reflection of the research milieu and capacity of individual countries or research groups, which indeed seem to be the case as most of the current

reports on leukemia emerges from countries with well established science capacities.

Even with the scattered and available data, however, the difference from European and global trends could be observed as well as the evolution of the problem of leukemia. An early report from Uganda found that African children in Uganda showed a great and genuine deficiency of leukemia and an excess of solid lympho-reticular tumors (Davies et

In subsequence, the situation seems to change, a study of pediatric leukemia in Cameroon, showed that Acute Lymphoblastic Leukemia (ALL) comprised 78.6%, while AML 21.4% of all pediatric acute leukemia (Obama et al., 1995). In Egypt, the lymphatic and haemopoietic cancer incidence in 2001 have increased approximately 11-fold compared with the incidence in 1972. Moreover, the incidence of leukemia among infant less than 5 years increased exponentially with a higher incidence among boys (Hosny et al., 2002). In Kenya, leukemia in children below the age of 15 years comprised 37% of leukemia in all ages. Childhood acute leukemia formed 52.3% of all the acute leukemia. AML and ALL occurred, in almost equal proportions 42 % and 46 % respectively (Kasili, 1990). In Ethiopia, a report by Shamebo (1990) showed that the commonest type of leukemia was CML 57.8%, acute leukemia and chronic lymphatic leukemia (CLL) accounted for 21.1% each. Of the acute leukemia, 53.3% were ALL while 46.7% were AML (Shamebo, 1990). A recent study in south Nigeria showed that AML comprises 12.3% and CML 23.9% of all leukemia, with a mean age at diagnosis of 25.6 years and 35.2 years respectively (Nwannadi et al., 2011). In the last 25 years in Sudan CML became the predominant cancer in men, while lymphomas remained the second most common cancer. In women, breast, cervical and ovarian cancer remained the three most common cancers over both time periods, but there was also an increase in the incidence of CML among women (Hamad, 2006), the causes of this high

In Europe AML presents as mainly an adult's disease with a median age at presentation of 64 years, accounting for around 30% of all leukemia in adults, and ~18 000 new patients are diagnosed in Europe each year, representing ~0.6% of all cancers. The annual incidence rate in Europe ranges from two per 100 000/year to four per 100 000/year. In the past decade, the trend in overall incidence of AML has generally been stable or slowly increasing in most European countries, while most cases of CML occur in adults with a median age at presentation around age 60. CML comprises only around 2%–3% of all the leukemia diagnosed in patients <20 years of age but the incidence increases with age slowly until the

malignancies should be investigated.

**2. Incidence and epidemiology** 

incidence of CML are not known.

al., 1965).

Elghetany, 2005). However, CD36 negative AML cells could be found especially in those populations that usually do not express CD36 (like in several African populations). CD36 negative cells appeared less susceptible to trombospondin-1 induced apoptosis, which make leukemic cells less vulnerable to death through this promising therapeutic strategy (Li et al., 2003).

On the other hand, due to the transition state, a number of risk factors believed to represent etiologic determinants of leukemia like use of pesticides, radiation etc. are not yet commonplace in Africa or at least its rural environment and may explain the differential distribution and the focal nature of hematological malignancies in the continent or/and individual countries. The potential impact of transition to a modern life style with the accompanying risks on the emergence and distribution of these diseases is worth our utmost attention.

Cancers in their complex etiology makes an ideal arena for the classical gene versus environment controversy. Those who favor an upper hand for the environment had their views strengthened by results of studies showing that people who migrate from one country to another generally acquire the cancer rates of the new host country, suggesting that environmental or lifestyle factors rather than genetic factors are the key determinants of the international variation in cancer rates. As far as Africa is concerned, African Americans´ disease data, represent a working model to test the role of changing environment and the effect of life style in complex diseases. Interestingly both sides of the argument seem to find support. Chronic myeloid leukemia (CML) patients show worse survival for African American and Hispanics compared to Americans of European origin (Lee et al., 2009). Although the difference in ethnicity data might be argued to reflect socio-economic differences, the current advances in genomics enable the implication of particular genomic regions and genes that explain the ethnic differences in susceptibility to infectious and chronic diseases.

Environmental determinants also falls short of explaining neither the "focality" of cancer types nor the aggressive course of some cancers in Sudan like the breast cancer, a feature that has been claimed to exist across Africa and even among African American women (reviewed by Morris and Mitchell, 2008).

Nutritional factors have also been implicated, adding an extra layer of complexity to the desperately compound picture of cancer etiology. Data on nutrition is greatly deficient in Africa similar to other aspects of genetic epidemiology, although differences in nutritional practices and culture may be key in providing vital clues to the contribution of life style. A study in China (Zhang et al., 2008), for example, suggested that a higher intake of green tea is associated with a reduced risk of adult leukemia. Furthermore, a study by Ross et al. which involved 35.221 older women provided evidence that increased vegetable consumption may decrease the risk of adult leukemia (Ross et al., 2002). Moreover, AML risk was negatively associated with milk intake among women and tea, and positively associated among women with beer, wine and beef (Li et al., 2006). A prospective cohort study by Ma et al. (2009) showed that smoking and total meat intake were risk factors for AML and those who did not drink coffee appeared to have a higher risk of AML.

Africa is still contains one of the last and few enclaves on the planet to harbor communities with distinctive patterns of traditional life styles that once used to characterize human existence. Farmers, pastoralists and hunter-gatherers- like societies do preserve their life style and culture and often coexist in shared terrains. These communities adopt fundamentally contrasting life styles and their food cultures are different. The significance of such disparity to all aspects of health and disease is of interest and the study of potential preponderance of these community members to chronic diseases including hematological malignancies should be investigated.

#### **2. Incidence and epidemiology**

466 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Elghetany, 2005). However, CD36 negative AML cells could be found especially in those populations that usually do not express CD36 (like in several African populations). CD36 negative cells appeared less susceptible to trombospondin-1 induced apoptosis, which make leukemic cells less vulnerable to death through this promising therapeutic strategy (Li et al.,

On the other hand, due to the transition state, a number of risk factors believed to represent etiologic determinants of leukemia like use of pesticides, radiation etc. are not yet commonplace in Africa or at least its rural environment and may explain the differential distribution and the focal nature of hematological malignancies in the continent or/and individual countries. The potential impact of transition to a modern life style with the accompanying risks on the emergence and distribution of these diseases is worth our utmost

Cancers in their complex etiology makes an ideal arena for the classical gene versus environment controversy. Those who favor an upper hand for the environment had their views strengthened by results of studies showing that people who migrate from one country to another generally acquire the cancer rates of the new host country, suggesting that environmental or lifestyle factors rather than genetic factors are the key determinants of the international variation in cancer rates. As far as Africa is concerned, African Americans´ disease data, represent a working model to test the role of changing environment and the effect of life style in complex diseases. Interestingly both sides of the argument seem to find support. Chronic myeloid leukemia (CML) patients show worse survival for African American and Hispanics compared to Americans of European origin (Lee et al., 2009). Although the difference in ethnicity data might be argued to reflect socio-economic differences, the current advances in genomics enable the implication of particular genomic regions and genes that explain the ethnic differences in susceptibility to infectious and

Environmental determinants also falls short of explaining neither the "focality" of cancer types nor the aggressive course of some cancers in Sudan like the breast cancer, a feature that has been claimed to exist across Africa and even among African American women

Nutritional factors have also been implicated, adding an extra layer of complexity to the desperately compound picture of cancer etiology. Data on nutrition is greatly deficient in Africa similar to other aspects of genetic epidemiology, although differences in nutritional practices and culture may be key in providing vital clues to the contribution of life style. A study in China (Zhang et al., 2008), for example, suggested that a higher intake of green tea is associated with a reduced risk of adult leukemia. Furthermore, a study by Ross et al. which involved 35.221 older women provided evidence that increased vegetable consumption may decrease the risk of adult leukemia (Ross et al., 2002). Moreover, AML risk was negatively associated with milk intake among women and tea, and positively associated among women with beer, wine and beef (Li et al., 2006). A prospective cohort study by Ma et al. (2009) showed that smoking and total meat intake were risk factors for

AML and those who did not drink coffee appeared to have a higher risk of AML.

Africa is still contains one of the last and few enclaves on the planet to harbor communities with distinctive patterns of traditional life styles that once used to characterize human existence. Farmers, pastoralists and hunter-gatherers- like societies do preserve their life style and culture and often coexist in shared terrains. These communities adopt

2003).

attention.

chronic diseases.

(reviewed by Morris and Mitchell, 2008).

The lowest rates of leukemia reported in sub-Saharan Africa probably represent failure of diagnosis or reporting to some extent (Davies, 1973; Fleming, 1993 and Parkin et al., 2005). We should therefore use caution, when drawing conclusions based on the varying prevalence and incidence, as an indication of clustering of cases or an environmental or genetic effect, as this may simply be due to the deficiency of statistics in Africa. The disparity could also be a reflection of the research milieu and capacity of individual countries or research groups, which indeed seem to be the case as most of the current reports on leukemia emerges from countries with well established science capacities.

Even with the scattered and available data, however, the difference from European and global trends could be observed as well as the evolution of the problem of leukemia. An early report from Uganda found that African children in Uganda showed a great and genuine deficiency of leukemia and an excess of solid lympho-reticular tumors (Davies et al., 1965).

In subsequence, the situation seems to change, a study of pediatric leukemia in Cameroon, showed that Acute Lymphoblastic Leukemia (ALL) comprised 78.6%, while AML 21.4% of all pediatric acute leukemia (Obama et al., 1995). In Egypt, the lymphatic and haemopoietic cancer incidence in 2001 have increased approximately 11-fold compared with the incidence in 1972. Moreover, the incidence of leukemia among infant less than 5 years increased exponentially with a higher incidence among boys (Hosny et al., 2002). In Kenya, leukemia in children below the age of 15 years comprised 37% of leukemia in all ages. Childhood acute leukemia formed 52.3% of all the acute leukemia. AML and ALL occurred, in almost equal proportions 42 % and 46 % respectively (Kasili, 1990). In Ethiopia, a report by Shamebo (1990) showed that the commonest type of leukemia was CML 57.8%, acute leukemia and chronic lymphatic leukemia (CLL) accounted for 21.1% each. Of the acute leukemia, 53.3% were ALL while 46.7% were AML (Shamebo, 1990). A recent study in south Nigeria showed that AML comprises 12.3% and CML 23.9% of all leukemia, with a mean age at diagnosis of 25.6 years and 35.2 years respectively (Nwannadi et al., 2011). In the last 25 years in Sudan CML became the predominant cancer in men, while lymphomas remained the second most common cancer. In women, breast, cervical and ovarian cancer remained the three most common cancers over both time periods, but there was also an increase in the incidence of CML among women (Hamad, 2006), the causes of this high incidence of CML are not known.

In Europe AML presents as mainly an adult's disease with a median age at presentation of 64 years, accounting for around 30% of all leukemia in adults, and ~18 000 new patients are diagnosed in Europe each year, representing ~0.6% of all cancers. The annual incidence rate in Europe ranges from two per 100 000/year to four per 100 000/year. In the past decade, the trend in overall incidence of AML has generally been stable or slowly increasing in most European countries, while most cases of CML occur in adults with a median age at presentation around age 60. CML comprises only around 2%–3% of all the leukemia diagnosed in patients <20 years of age but the incidence increases with age slowly until the

Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa 469

case of viral oncogenesis that of Epstein Barr Virus (EBV) was established in Africa. EBV is a highly prevalent infection in the adult population in Africa and has been associated with a heterogeneous group of lymphomas, including Burkitt's lymphoma (especially the endemic form in Africa), Hodgkin's disease, NK, and T malignancies with cytotoxic phenotypes, and lymphomas in the immune-compromised patient (congenital immunodeficiency, organ transplantation, AIDS). (Rodriguez-Abreu et al., 2007) but not unequivocally with leukemia. Of the oncogenic retroviruses, the Human T-cell leukemia virus (HTLV) type-1 and type-2 have been identified as being related to the development of rare types of leukemia and lymphoma. HTLV-1 is endemic in certain areas including central Africa, and is associated with the development of adult T-cell leukemia or lymphoma (ATLL), which accounts for about half of the lymphoid malignancies in the endemic areas. The virus is transmitted mainly from mother to child, especially by breastfeeding. Sexual transmission and blood transfusion are minor routes of infection and cell-free blood products are not infectious

The importance of oncogenic viruses stems not only from their transforming oncogenic

 Environmental determinant including infection with high-risk viruses are necessary but not sufficient alone in the development of cancer, as most infections regress without intervention. Thus, genetic host factors and cellular immune responses could be potential modifiers for the risk of developing cancer. In particular, p53 and Rb are considered as the most critical tumor suppressor genes involved in regulating cell division. The polymorphism on p53, which encodes either a Proline or an Arginin amino acid residue at codon 72, has been reported as a possible risk factor for several cancers including breast and

In the last decade the importance of ethnicity, socio-economic and gender differences in relation to disease incidence, diagnosis, and prognosis has been realised. Gender and ethnic differences in these areas should have a focus in health policy in Africa. A study by Lee et al. examined the demographic and clinical features of CML in an ethnically diverse population and found that Hispanic patients present with lower risk profile CML and achieve better treatment responses compared to non-Hispanic patients. The vast majority of their non-Hispanic patients were African American or Asian. This study proposed that biological/genetic factor can contributes to this observed ethnic differences in disease presentation and behavior. Hispanic ethnic group is thought to be the least diverse ethnic group, at the opposite site the African descent is the most diverse ethnic group. African populations are characterized by greater levels of genetic diversity, extensive population substructure, and less linkage disequilibrium (LD) among loci compared to non-African populations (Reich et al., 2001; Campbell and Tishkoff, 2008). Due to the long evolutionary history in Africa there is more genetic diversity within and between populations in the African continent, than between Africans and other peoples in the world (Cavalli-Sforza, 1997). African populations thus vary considerably in their genes. Moreover Africa shows a wide range of environments, climatic, vegetative and zoological. Thus human cancer patters are expected to show a similar degree of diversity, the study of which would contribute to

properties but also from the potential methylating properties of selfish DNA.

(Rodriguez-Abreu et al., 2007).

cervical cancer in Sudan (Eltahir et al, submitted.).

**3.2 Population and ethnic diversity** 

our understanding of their causes.

mid-40s, then more rapidly from about one per 1000 000/year in children <10 years to two per 100 000 in people in the fifth decade to one per 10 000 at age 80. The disease is more common in males. There is no clear evidence of to geographic or ethnic background that predisposes to CML; however, in the United States the incidence is slightly higher in Caucasians than in Blacks or Hispanics (Lee et al., 2009), and it exhibits a male preponderance, in South African Coloured and Black people, and African Americans in comparison to whites but the reason for this is unexplained. (Jacobs et al., 1983). Furthermore, several reports showed an increased incidence of CML during the first 2 decades of life in African subjects (Haddock , 1967; Lowe et al., 1971; Lowenthal et al., 1975), but other reports by Leibowitz et al., (1976) and Jacobs et al., (1983) disagree with this. Jacobs et al. reported that in comparison with Whites and Coloureds, however, the peak incidence for Blacks was lower, lying between the 3rd and 4th decades. The peak incidence for Coloureds was in the 4th and 5th decades, and that for Whites in the 5th and 6th (Jacobs et al., 1983.)

An interesting study in United Arab Emirate (UAE) found that the rate of AML among UAE female nationals was higher than in nationals male and expatriates. The study proposed that chemicals in henna dye, which is used to decorate the body, as well as a lack of sunlight could be behind the increased incidence (Hassan et al., 2009). Henna is applied in many African countries especially Sudan where it is used by vast majority of Sudanese married women.

#### **3. Molecular etiology**

Leukemia in common with other cancers arises from mutations in a single cell, which enable the cell to reproduce excessively, emerging as a dominant clone. A large number of different mutant genes contribute to leukaemogenesis singly or, more often, in combination and many are leukemia sub-type specific (Greaves, 1986). The number of genetic abnormalities is believed to reflect the number of genes that control distinct developmental stages of blood formation and the multiple routes to clonal dominance. These include changes not only in proliferative activity, but in ability to differentiate, in resistance to cell death, in DNA repair activity and in general stability of the genes. Whether any particular mutant genes, or hot spots' for mutations within a gene, are linked to particular DNA damaging agents is a topic of considerable relevance to the molecular epidemiology of cancer in general. For these changes to happen some culprit agents has to come into action namely ionizing radiation, chemicals such as polycylic hydrocarbons and certain drugs, and viruses.

The way how these extrinsic factors affect the cell genetically and epigenetically is the core of the functional research in cancer. Although multiple risk factors have been linked to the development of leukemia, however these known risk factors account for only a small number of observed cases. Few epidemiological studies have explored the relation between lifestyle, dietary factors and the incidence of adult Leukemia, and almost none has addressed the molecular and genetic aspects of these interactions.

#### **3.1 Role of viruses**

For several reasons pertaining to ecology and the human history in Africa, several pathogens have gained access into human genomes through the African gate. This includes major parasites, viral and bacterial diseases. It is not coincidence that the first well proven

mid-40s, then more rapidly from about one per 1000 000/year in children <10 years to two per 100 000 in people in the fifth decade to one per 10 000 at age 80. The disease is more common in males. There is no clear evidence of to geographic or ethnic background that predisposes to CML; however, in the United States the incidence is slightly higher in Caucasians than in Blacks or Hispanics (Lee et al., 2009), and it exhibits a male preponderance, in South African Coloured and Black people, and African Americans in comparison to whites but the reason for this is unexplained. (Jacobs et al., 1983). Furthermore, several reports showed an increased incidence of CML during the first 2 decades of life in African subjects (Haddock , 1967; Lowe et al., 1971; Lowenthal et al., 1975), but other reports by Leibowitz et al., (1976) and Jacobs et al., (1983) disagree with this. Jacobs et al. reported that in comparison with Whites and Coloureds, however, the peak incidence for Blacks was lower, lying between the 3rd and 4th decades. The peak incidence for Coloureds was in the 4th and 5th decades, and that for Whites in the 5th and 6th (Jacobs

An interesting study in United Arab Emirate (UAE) found that the rate of AML among UAE female nationals was higher than in nationals male and expatriates. The study proposed that chemicals in henna dye, which is used to decorate the body, as well as a lack of sunlight could be behind the increased incidence (Hassan et al., 2009). Henna is applied in many African countries especially Sudan where it is used by vast majority of Sudanese married

Leukemia in common with other cancers arises from mutations in a single cell, which enable the cell to reproduce excessively, emerging as a dominant clone. A large number of different mutant genes contribute to leukaemogenesis singly or, more often, in combination and many are leukemia sub-type specific (Greaves, 1986). The number of genetic abnormalities is believed to reflect the number of genes that control distinct developmental stages of blood formation and the multiple routes to clonal dominance. These include changes not only in proliferative activity, but in ability to differentiate, in resistance to cell death, in DNA repair activity and in general stability of the genes. Whether any particular mutant genes, or hot spots' for mutations within a gene, are linked to particular DNA damaging agents is a topic of considerable relevance to the molecular epidemiology of cancer in general. For these changes to happen some culprit agents has to come into action namely ionizing radiation,

The way how these extrinsic factors affect the cell genetically and epigenetically is the core of the functional research in cancer. Although multiple risk factors have been linked to the development of leukemia, however these known risk factors account for only a small number of observed cases. Few epidemiological studies have explored the relation between lifestyle, dietary factors and the incidence of adult Leukemia, and almost none has

For several reasons pertaining to ecology and the human history in Africa, several pathogens have gained access into human genomes through the African gate. This includes major parasites, viral and bacterial diseases. It is not coincidence that the first well proven

chemicals such as polycylic hydrocarbons and certain drugs, and viruses.

addressed the molecular and genetic aspects of these interactions.

et al., 1983.)

women.

**3. Molecular etiology** 

**3.1 Role of viruses** 

case of viral oncogenesis that of Epstein Barr Virus (EBV) was established in Africa. EBV is a highly prevalent infection in the adult population in Africa and has been associated with a heterogeneous group of lymphomas, including Burkitt's lymphoma (especially the endemic form in Africa), Hodgkin's disease, NK, and T malignancies with cytotoxic phenotypes, and lymphomas in the immune-compromised patient (congenital immunodeficiency, organ transplantation, AIDS). (Rodriguez-Abreu et al., 2007) but not unequivocally with leukemia. Of the oncogenic retroviruses, the Human T-cell leukemia virus (HTLV) type-1 and type-2 have been identified as being related to the development of rare types of leukemia and lymphoma. HTLV-1 is endemic in certain areas including central Africa, and is associated with the development of adult T-cell leukemia or lymphoma (ATLL), which accounts for about half of the lymphoid malignancies in the endemic areas. The virus is transmitted mainly from mother to child, especially by breastfeeding. Sexual transmission and blood transfusion are minor routes of infection and cell-free blood products are not infectious (Rodriguez-Abreu et al., 2007).

The importance of oncogenic viruses stems not only from their transforming oncogenic properties but also from the potential methylating properties of selfish DNA.

 Environmental determinant including infection with high-risk viruses are necessary but not sufficient alone in the development of cancer, as most infections regress without intervention. Thus, genetic host factors and cellular immune responses could be potential modifiers for the risk of developing cancer. In particular, p53 and Rb are considered as the most critical tumor suppressor genes involved in regulating cell division. The polymorphism on p53, which encodes either a Proline or an Arginin amino acid residue at codon 72, has been reported as a possible risk factor for several cancers including breast and cervical cancer in Sudan (Eltahir et al, submitted.).

#### **3.2 Population and ethnic diversity**

In the last decade the importance of ethnicity, socio-economic and gender differences in relation to disease incidence, diagnosis, and prognosis has been realised. Gender and ethnic differences in these areas should have a focus in health policy in Africa. A study by Lee et al. examined the demographic and clinical features of CML in an ethnically diverse population and found that Hispanic patients present with lower risk profile CML and achieve better treatment responses compared to non-Hispanic patients. The vast majority of their non-Hispanic patients were African American or Asian. This study proposed that biological/genetic factor can contributes to this observed ethnic differences in disease presentation and behavior. Hispanic ethnic group is thought to be the least diverse ethnic group, at the opposite site the African descent is the most diverse ethnic group. African populations are characterized by greater levels of genetic diversity, extensive population substructure, and less linkage disequilibrium (LD) among loci compared to non-African populations (Reich et al., 2001; Campbell and Tishkoff, 2008). Due to the long evolutionary history in Africa there is more genetic diversity within and between populations in the African continent, than between Africans and other peoples in the world (Cavalli-Sforza, 1997). African populations thus vary considerably in their genes. Moreover Africa shows a wide range of environments, climatic, vegetative and zoological. Thus human cancer patters are expected to show a similar degree of diversity, the study of which would contribute to our understanding of their causes.

Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa 471

Cancers as a group of diseases display the entire range of inheritance modes from the single gene like disorder to the complex inheritance pattern seen in chronic diseases. The paucity of genetic investigation in Africa of cancer susceptibility genes and chromosomal aberrations linked to cancer, makes the picture even more opaque. The few examples discussed above and below demonstrate the great relevance of studying the genetic population structure of African populations and establishing the frequencies of the individual SNPs, Ins/dels and chromosomal abnormalities associated with diseases that may be necessary to define the molecular etiological basis of each cancer. In fact both molecular and genetic abnormalities became an important factor for characterising, treating and risk stratifying of myeloid leukemia. In 2002 the WHO classification of Myeloid Neoplasms showed that AML classification includes specific genetic subcategories; thus, determination of genetic features of the neoplastic cells must be performed if possible. Many recurring genetic abnormalities in the myeloid neoplasms can be identified by advanced molecular and cytogenetic techniques. In the WHO classification, the blast threshold for the diagnosis of AML is reduced from 30% to 20% blasts in the blood or marrow. In addition, patients with the clonal, recurring cytogenetic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22), and t(15;17)(q22;q12) should be considered to have AML regardless of the blast percentage. On the other hand, according to the WHO classification, CML is defined specifically as a myeloproliferative disease that is characterized by the invariable presence of the Philadelphia (Ph) chromosome or the BCR-ABL fusion gene. Although in most cases the diagnosis is easily made from morphologic evaluation of the blood smear, confirmation by genetic studies is essential, particularly in view of the advent of therapy that targets the BCR-ABL fusion protein (Vardiman et al.,

Despite their diagnostic and prognostic values, studies on gene and chromosomal rearrangements associated malignancies is greatly lacking on the African continent, and in spite of the mandatory genotyping of the BCR-ABL as a prerequisite of administering the drug imatinib, in several African countries, there are very few reports on its frequencies. Among the few reports on its association with other leukemias, a multi-country study comprising 181 children with newly diagnosed ALL were tested in laboratories in India, Pakistan, Myanmar, and Sudan, following a common protocol. Across the four countries, the ETV6-RUNX1 (TEL-AML1) fusion gene was present in only 5% of cases. All the positive samples were from children aged 1 to 10 years, in whom the prevalence of this fusion gene, which is associated with good prognosis, was 7.4% (9 out of 121 samples), a much lower rate than reported from Western populations. In the 18 ALL cases tested in Sudan, a notable excess of MLL-AF4 (17%) and BCR-ABL1 (22%) fusion genes was found (Siddiqui et al.,

The significance of studying the frequency of these rearrangements and their relation to pathology, is to establish the level of culpability of the molecular events. A study on Nigerian breast cancer patients suggest that while BRCA1 genomic rearrangement exists, it does not contribute significantly to BRCA1-associated risk in the Nigerian population

Chromosomal translocations in myeloid leukemia yield hybrid RNAs capable of encoding fusion chimeric proteins. The unique amino acid sequences found in these oncogenic fusion proteins represent true tumor-specific antigens that are potentially immunogenic. Although

**3.3 Gene and chromosomal rearrangements** 

2002)

2010).

(Zhang et al., 2011).

The majority of cancers as genetic disease of complex nature involve a multiplicity of genetic loci that cooperates to make the disease happen (the multiple -hit theory). It is expected that such genetic component will be influenced by the genetic background of the population at risk. In one of the few studies on a cancer susceptibility genes background mutational profile in different populations, Africans were found to harbor more mutations in their BRCA2 than populations from the rest of the world (Wagner et al., 1999.) which is expected given their larger effective population size.

Africans also possess a number of genetic adaptations that have evolved in response to diverse climates and diets, as well as exposure to infectious disease (Campbell & Tishkoff, 2008), that diversity may carry great challenge in leukemia presentation and behavior/ prognosis and treatment. Moreover, experimental studies using synthetic peptides identical to the BCR-ABL fusion region in CML patients region have revealed the capability of specific peptides to bind to human leukocyte antigen (HLA) class I molecules (HLA-A2, A3, A11, B8) and class II molecules (HLA-DR1, DR2, DR3, DR4 and DR11). Individuals expressing HLA-A3, B8 or DR4 have a diminished risk for the development of CML in Caucasian populations. A statistically significant increase in the frequency of Cw3 and Cw4 antigens in Caucasians and European CML patients has been reported. Another report in Indian population showed that expression of HLA-Cw6 may result in a protective effect on CML acquisition (Chhaya, 2006). A study in Chinese population indicated that the expression of HLA-A\*30, DRB1\*07 might imply a protective effect on CML acquisition, while B\*81 might be associated with CML susceptibility factors in that population (Miao et al., 2007). These results suggest that the development of CML is apparently associated with HLA phenotypes specific to each population. These data is missed in Africa and we intend to investigate this in Sudanese population.

Common polymorphism like the codon 72 in p53 has been argued to unlikely have major genetic effect since polymorphism in loci with major deleterious nature will not be selected for to reach such frequencies unless it is a balanced polymorphism. (i.e. selected for under the influence of the other allele possessing an favorable adaptive trait). Interestingly It has been proposed that the p53 polymorphism at times when the risk of tumors was not a Human concern, gave a reproductive leverage by increasing reproductive success (Kang et al., 2008). The cost of such trade off will not be visible as long as the conditions that predispose for tumors are absent. In fact the derived allele (arginine), almost reached fixation in some populations.

The p53 codon 72 was studied both in normal Sudanese (Bereir et al., 2002) and in the distribution of the polymorphism in different cancers in Sudan, (not including leukemia). The results show that the different alleles pose different risk ratios in different cancer. The Arg allele which is known to be more resistant to cell death was overrepresented breast carcinoma patients from different linguistic groups as compared to controls with an Odd ratio of 19.44 CI 6.6 – 78.3 *P*<0.0001. In cervical cancer the homozygous Arg genotype was detected in 42.3% (33/78) in cervical cancer patients while the heterozygous arg/pro in 38.5% (30/78) and only 19.2% (15/78) had the pro/pro genotype, with an allele effect of 2.4 (CI 1.12 – 5.33, *P* 0.015). In Burkitt´s lymphoma the opposite seems to be true with a major effect from the Pro allele, where the homozygous Arg accounted for only 6.9%, (OR 0.18 CI 0.02 – 0.89, *P=* 0.018) while the Arg/Pro was 51.7% and pro/pro 41.4% (OR: 0.57, CI 0.23- 1.42, P= 0.1). Possibly indicating the different biological pathways of tumorigenesis (Eltahir et al., submitted).

#### **3.3 Gene and chromosomal rearrangements**

470 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

The majority of cancers as genetic disease of complex nature involve a multiplicity of genetic loci that cooperates to make the disease happen (the multiple -hit theory). It is expected that such genetic component will be influenced by the genetic background of the population at risk. In one of the few studies on a cancer susceptibility genes background mutational profile in different populations, Africans were found to harbor more mutations in their BRCA2 than populations from the rest of the world (Wagner et al., 1999.) which is expected

Africans also possess a number of genetic adaptations that have evolved in response to diverse climates and diets, as well as exposure to infectious disease (Campbell & Tishkoff, 2008), that diversity may carry great challenge in leukemia presentation and behavior/ prognosis and treatment. Moreover, experimental studies using synthetic peptides identical to the BCR-ABL fusion region in CML patients region have revealed the capability of specific peptides to bind to human leukocyte antigen (HLA) class I molecules (HLA-A2, A3, A11, B8) and class II molecules (HLA-DR1, DR2, DR3, DR4 and DR11). Individuals expressing HLA-A3, B8 or DR4 have a diminished risk for the development of CML in Caucasian populations. A statistically significant increase in the frequency of Cw3 and Cw4 antigens in Caucasians and European CML patients has been reported. Another report in Indian population showed that expression of HLA-Cw6 may result in a protective effect on CML acquisition (Chhaya, 2006). A study in Chinese population indicated that the expression of HLA-A\*30, DRB1\*07 might imply a protective effect on CML acquisition, while B\*81 might be associated with CML susceptibility factors in that population (Miao et al., 2007). These results suggest that the development of CML is apparently associated with HLA phenotypes specific to each population. These data is missed in Africa and we intend

Common polymorphism like the codon 72 in p53 has been argued to unlikely have major genetic effect since polymorphism in loci with major deleterious nature will not be selected for to reach such frequencies unless it is a balanced polymorphism. (i.e. selected for under the influence of the other allele possessing an favorable adaptive trait). Interestingly It has been proposed that the p53 polymorphism at times when the risk of tumors was not a Human concern, gave a reproductive leverage by increasing reproductive success (Kang et al., 2008). The cost of such trade off will not be visible as long as the conditions that predispose for tumors are absent. In fact the derived allele (arginine), almost reached

The p53 codon 72 was studied both in normal Sudanese (Bereir et al., 2002) and in the distribution of the polymorphism in different cancers in Sudan, (not including leukemia). The results show that the different alleles pose different risk ratios in different cancer. The Arg allele which is known to be more resistant to cell death was overrepresented breast carcinoma patients from different linguistic groups as compared to controls with an Odd ratio of 19.44 CI 6.6 – 78.3 *P*<0.0001. In cervical cancer the homozygous Arg genotype was detected in 42.3% (33/78) in cervical cancer patients while the heterozygous arg/pro in 38.5% (30/78) and only 19.2% (15/78) had the pro/pro genotype, with an allele effect of 2.4 (CI 1.12 – 5.33, *P* 0.015). In Burkitt´s lymphoma the opposite seems to be true with a major effect from the Pro allele, where the homozygous Arg accounted for only 6.9%, (OR 0.18 CI 0.02 – 0.89, *P=* 0.018) while the Arg/Pro was 51.7% and pro/pro 41.4% (OR: 0.57, CI 0.23- 1.42, P= 0.1). Possibly indicating the different biological pathways of tumorigenesis (Eltahir

given their larger effective population size.

to investigate this in Sudanese population.

fixation in some populations.

et al., submitted).

Cancers as a group of diseases display the entire range of inheritance modes from the single gene like disorder to the complex inheritance pattern seen in chronic diseases. The paucity of genetic investigation in Africa of cancer susceptibility genes and chromosomal aberrations linked to cancer, makes the picture even more opaque. The few examples discussed above and below demonstrate the great relevance of studying the genetic population structure of African populations and establishing the frequencies of the individual SNPs, Ins/dels and chromosomal abnormalities associated with diseases that may be necessary to define the molecular etiological basis of each cancer. In fact both molecular and genetic abnormalities became an important factor for characterising, treating and risk stratifying of myeloid leukemia. In 2002 the WHO classification of Myeloid Neoplasms showed that AML classification includes specific genetic subcategories; thus, determination of genetic features of the neoplastic cells must be performed if possible. Many recurring genetic abnormalities in the myeloid neoplasms can be identified by advanced molecular and cytogenetic techniques. In the WHO classification, the blast threshold for the diagnosis of AML is reduced from 30% to 20% blasts in the blood or marrow. In addition, patients with the clonal, recurring cytogenetic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22), and t(15;17)(q22;q12) should be considered to have AML regardless of the blast percentage. On the other hand, according to the WHO classification, CML is defined specifically as a myeloproliferative disease that is characterized by the invariable presence of the Philadelphia (Ph) chromosome or the BCR-ABL fusion gene. Although in most cases the diagnosis is easily made from morphologic evaluation of the blood smear, confirmation by genetic studies is essential, particularly in view of the advent of therapy that targets the BCR-ABL fusion protein (Vardiman et al., 2002)

Despite their diagnostic and prognostic values, studies on gene and chromosomal rearrangements associated malignancies is greatly lacking on the African continent, and in spite of the mandatory genotyping of the BCR-ABL as a prerequisite of administering the drug imatinib, in several African countries, there are very few reports on its frequencies. Among the few reports on its association with other leukemias, a multi-country study comprising 181 children with newly diagnosed ALL were tested in laboratories in India, Pakistan, Myanmar, and Sudan, following a common protocol. Across the four countries, the ETV6-RUNX1 (TEL-AML1) fusion gene was present in only 5% of cases. All the positive samples were from children aged 1 to 10 years, in whom the prevalence of this fusion gene, which is associated with good prognosis, was 7.4% (9 out of 121 samples), a much lower rate than reported from Western populations. In the 18 ALL cases tested in Sudan, a notable excess of MLL-AF4 (17%) and BCR-ABL1 (22%) fusion genes was found (Siddiqui et al., 2010).

The significance of studying the frequency of these rearrangements and their relation to pathology, is to establish the level of culpability of the molecular events. A study on Nigerian breast cancer patients suggest that while BRCA1 genomic rearrangement exists, it does not contribute significantly to BRCA1-associated risk in the Nigerian population (Zhang et al., 2011).

Chromosomal translocations in myeloid leukemia yield hybrid RNAs capable of encoding fusion chimeric proteins. The unique amino acid sequences found in these oncogenic fusion proteins represent true tumor-specific antigens that are potentially immunogenic. Although

Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa 473

Table 1. The frequencies of T and C alleles of BCR exon 13 SNP in different populations

The study by Gordon et al 2003 suggest that variation among normal individuals may contribute to inter patient heterogeneity in CML. Differences in behaviour of haemopoietic progenitor cells from different normal individuals may be attributable to genetic diversity or other variables. de Haan et al. (2002) concluded that the expression levels of a large number of genes might be responsible for controlling stem cell behaviour. These collections of genes may be analogous to those responsible for the inter-individual diversity in progenitor cell

In CML, the occurrence of additional specific cytogenetic and molecular changes subsequent to the initiation of t(9;22) translocation herald disease progression prior to haematologic and clinical manifestation. These events occur in 50 to 80 percent of patients during the transition from the chronic phase of the disease to the accelerated and blast phases. Minor cytogenetic changes include monosomies of chromosomes 7, 17, and Y; trisomies of chromosomes 17 and 21; and translocation t(3;21)(q26;q22) (Mitelman, 1993). Major changes include trisomy 8, isochromosome i(17q), trisomy 19, and an extra Ph chromosome (double Ph). Trisomy 8 is most common, and isochromosome i(17q) occurs almost exclusively in the myeloid type blast phase (Kantarjian et al., 1987; Derderian et al., 1993; Mitelman, 1993). Molecular abnormalities may correspond to cytogenetic changes. These include abnormalities in p53 (on chromosome 17p13); RB1 (13q14); c-MYC (8q24); p16INK4A (9p21); RAS; and AML–EVI-1, a fusion protein resulting from translocation t(3;21) (q26;q22). Alterations of p53 (deletions, rearrangements, and mutations) occur in 20 to 30 percent of patients with CML in the blast phase (Ahuja et al.,1989) and are associated exclusively with myeloid transformation (Stuppia et al., 1997), whereas abnormalities of RB1 are associated more with lymphoid transformation, although the association is weaker than it was between p53 and myeloid transformation. Mutations of p53 in the progression of CML are associated with an aberrant methylation status of CML cells (Guinn et al., 1997). The introduction of a methyl group causing transcriptional silencing of the calcitonin gene has been found in the transition of chronic-phase CML to blast-phase CML (Malinen et al., 1991). Altered methylation was also described within the M-bcr of cells from patients with chronic-phase CML (Litz et al., 1996). Up to 50 percent of patients with lymphoid transformation have homozygous deletion of p16INK4A (Sill et al., 1995). Alterations of RB1, amplifications of c-

MYC, and mutations of RAS are less frequent (Faderl et al., 1999).

The genetic variation in Africa is poised to constitute major challenge for diagnosis and management. In a world where the diagnosis and prognosis of diseases and particularly cancer is increasingly dependent on molecular approaches such diversity might constitute a

Population T allele C allele African American 0.55 0.45 Sub-Saharan African 0.425 0.575 Asians 0.922 0.078 Chinese 0.91 0.09 Japanese 0.98 0.02 European 0.70 0.30

Data from NCBI/dbSNP/Short Genetic Variations

andethnic groups

behavior

these leukemia-specific fusion proteins have an intracellular location, they might be recognized immunologically by T lymphocytes if peptides derived from the unique sequences are capable of presentation by the major histocompatibility complex (MHC) molecules on Leukemic cells (Bocchia *et al*., 1995**)**. The ability of a series of synthetic peptides corresponding to the junctional sequences of CML-derived BCR-ABL fusion proteins spanning the b3a2 and b2a2 breakpoints to bind to purified class I molecules was studied by Bocchia *et al*. Four peptides derived from b3a2 CML breakpoint bound with high or intermediate affinity to HLA A3, A11, and B8. None of the CML b2a2 junctional peptides showed affinity of this magnitude for the HLA class I molecules tested. Which draw another important conclusion on the significance of the types of BCR-ABL fusion transcripts among populations in relation to vaccine development? The frequencies of the types of the fusion transcript in Ecuadorian population for example consist of 95% b2a2 (Paz-y-Mino et al., 2002) indicating that they may not benefit much from such vaccine.

 Several studies estimated the types of BCR-ABL fusion transcript in CML in different populations. The distribution of transcript type has been studied in European and some other populations (Eisenberg et al., 1988; Lee et al., 1989) with frequencies for b2a2 and b3a2 transcripts being roughly of the order of 40% and 55%, and that for co-expression of b3a2 and b2a2 representing 5% of the cases. A study on an Ecuadorian population, however, registered very different frequencies: 5% for b3a2 and 95% for b2a2 (Paz-y-Mino et al., 2002). In our report in Sudanese patients ( Osman et al., 2010 ), a frequency of 53.5% and 41.9% for b2a2 and b3a2, respectively, was reported, values that are relatively closer to those from a Mexican population (Arana- Trejo et al., 2002). This difference in frequencies may be due to the genetic differences of the populations. Many Controversial reports about the clinical significant of the transcript type in CML were published; however it has a considerable importance in the diagnosis and follow up.

Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the major breakpoint cluster region) has been identified in the eighth position before the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; the corresponding triplets are AAT (T allele) and AAC (C allele), respectively, both coding for asparagine. Therefore, this polymorphism has no implication in the primary structure of BCR and BCR-ABL proteins.

Co-expression of b2a2 and b3a2 transcripts has been linked to two polymorphisms, T to C at exon 13 and A to G at intron 13 (Meissner et al., 1998; Branford et al., 2002). However, in our study by Osman et al. (2010) six PCR products from four patients were sequenced to confirm the products of four b2a2 and two b3a2 and one was found to harbor T to C at exon 13 and expressed only b2a2 transcript which might indicate that this exonic polymorphism is not obligatory for co-expression, as reported by Mondal et al. (2006). Moreover, this polymorphism has no implication on the primary structure of BCR and BCR-ABL proteins. However, since the alteration is located close to the fusion region, it may have a significant influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. This polymorphism could be also a useful marker for the differentiation of normal and rearranged BCR alleles in heterozygotes patients and during follow up of minimal residual disease. The allele frequency for this SNP varied markedly between different world populations, with European attaining intermediate values between African and Asians (Table 1)

The molecular basis of CML is well defined and highly consistent, yet prognosis varies considerably. This could reflect the biological diversity occurring in normal populations.


Data from NCBI/dbSNP/Short Genetic Variations

472 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

these leukemia-specific fusion proteins have an intracellular location, they might be recognized immunologically by T lymphocytes if peptides derived from the unique sequences are capable of presentation by the major histocompatibility complex (MHC) molecules on Leukemic cells (Bocchia *et al*., 1995**)**. The ability of a series of synthetic peptides corresponding to the junctional sequences of CML-derived BCR-ABL fusion proteins spanning the b3a2 and b2a2 breakpoints to bind to purified class I molecules was studied by Bocchia *et al*. Four peptides derived from b3a2 CML breakpoint bound with high or intermediate affinity to HLA A3, A11, and B8. None of the CML b2a2 junctional peptides showed affinity of this magnitude for the HLA class I molecules tested. Which draw another important conclusion on the significance of the types of BCR-ABL fusion transcripts among populations in relation to vaccine development? The frequencies of the types of the fusion transcript in Ecuadorian population for example consist of 95% b2a2 (Paz-y-Mino et al.,

 Several studies estimated the types of BCR-ABL fusion transcript in CML in different populations. The distribution of transcript type has been studied in European and some other populations (Eisenberg et al., 1988; Lee et al., 1989) with frequencies for b2a2 and b3a2 transcripts being roughly of the order of 40% and 55%, and that for co-expression of b3a2 and b2a2 representing 5% of the cases. A study on an Ecuadorian population, however, registered very different frequencies: 5% for b3a2 and 95% for b2a2 (Paz-y-Mino et al., 2002). In our report in Sudanese patients ( Osman et al., 2010 ), a frequency of 53.5% and 41.9% for b2a2 and b3a2, respectively, was reported, values that are relatively closer to those from a Mexican population (Arana- Trejo et al., 2002). This difference in frequencies may be due to the genetic differences of the populations. Many Controversial reports about the clinical significant of the transcript type in CML were published; however it has a considerable

Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the major breakpoint cluster region) has been identified in the eighth position before the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; the corresponding triplets are AAT (T allele) and AAC (C allele), respectively, both coding for asparagine. Therefore, this polymorphism has

Co-expression of b2a2 and b3a2 transcripts has been linked to two polymorphisms, T to C at exon 13 and A to G at intron 13 (Meissner et al., 1998; Branford et al., 2002). However, in our study by Osman et al. (2010) six PCR products from four patients were sequenced to confirm the products of four b2a2 and two b3a2 and one was found to harbor T to C at exon 13 and expressed only b2a2 transcript which might indicate that this exonic polymorphism is not obligatory for co-expression, as reported by Mondal et al. (2006). Moreover, this polymorphism has no implication on the primary structure of BCR and BCR-ABL proteins. However, since the alteration is located close to the fusion region, it may have a significant influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. This polymorphism could be also a useful marker for the differentiation of normal and rearranged BCR alleles in heterozygotes patients and during follow up of minimal residual disease. The allele frequency for this SNP varied markedly between different world populations, with European attaining intermediate values between African

The molecular basis of CML is well defined and highly consistent, yet prognosis varies considerably. This could reflect the biological diversity occurring in normal populations.

2002) indicating that they may not benefit much from such vaccine.

no implication in the primary structure of BCR and BCR-ABL proteins.

importance in the diagnosis and follow up.

and Asians (Table 1)

Table 1. The frequencies of T and C alleles of BCR exon 13 SNP in different populations andethnic groups

The study by Gordon et al 2003 suggest that variation among normal individuals may contribute to inter patient heterogeneity in CML. Differences in behaviour of haemopoietic progenitor cells from different normal individuals may be attributable to genetic diversity or other variables. de Haan et al. (2002) concluded that the expression levels of a large number of genes might be responsible for controlling stem cell behaviour. These collections of genes may be analogous to those responsible for the inter-individual diversity in progenitor cell behavior

In CML, the occurrence of additional specific cytogenetic and molecular changes subsequent to the initiation of t(9;22) translocation herald disease progression prior to haematologic and clinical manifestation. These events occur in 50 to 80 percent of patients during the transition from the chronic phase of the disease to the accelerated and blast phases. Minor cytogenetic changes include monosomies of chromosomes 7, 17, and Y; trisomies of chromosomes 17 and 21; and translocation t(3;21)(q26;q22) (Mitelman, 1993). Major changes include trisomy 8, isochromosome i(17q), trisomy 19, and an extra Ph chromosome (double Ph). Trisomy 8 is most common, and isochromosome i(17q) occurs almost exclusively in the myeloid type blast phase (Kantarjian et al., 1987; Derderian et al., 1993; Mitelman, 1993).

Molecular abnormalities may correspond to cytogenetic changes. These include abnormalities in p53 (on chromosome 17p13); RB1 (13q14); c-MYC (8q24); p16INK4A (9p21); RAS; and AML–EVI-1, a fusion protein resulting from translocation t(3;21) (q26;q22). Alterations of p53 (deletions, rearrangements, and mutations) occur in 20 to 30 percent of patients with CML in the blast phase (Ahuja et al.,1989) and are associated exclusively with myeloid transformation (Stuppia et al., 1997), whereas abnormalities of RB1 are associated more with lymphoid transformation, although the association is weaker than it was between p53 and myeloid transformation. Mutations of p53 in the progression of CML are associated with an aberrant methylation status of CML cells (Guinn et al., 1997). The introduction of a methyl group causing transcriptional silencing of the calcitonin gene has been found in the transition of chronic-phase CML to blast-phase CML (Malinen et al., 1991). Altered methylation was also described within the M-bcr of cells from patients with chronic-phase CML (Litz et al., 1996). Up to 50 percent of patients with lymphoid transformation have homozygous deletion of p16INK4A (Sill et al., 1995). Alterations of RB1, amplifications of c-MYC, and mutations of RAS are less frequent (Faderl et al., 1999).

The genetic variation in Africa is poised to constitute major challenge for diagnosis and management. In a world where the diagnosis and prognosis of diseases and particularly cancer is increasingly dependent on molecular approaches such diversity might constitute a

Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa 475

complete molecular response following administration of imatinib showed significantly (p=0.013) higher in vivo CYP3A activity than patients achieving partial molecular response

In Sudan impact of the distribution of the GSTM1 and GSTT1 genotype leukemic patients was studied in 77 leukemic patients and 107 controls by Tagelsir et al., (Submitted). The results suggest that these genotypes could play a role in the development of leukemia particularly AML. Statistical analysis showed a significant preponderance of null genotype of both genes among pooled cases [*GSTM1* OR 3.45 (95% CI, 1.65 - 7.19); *P* = 0.001] for; *GSTT1* OR 8.57 (95% CI, 3.68 -19.93); *P* < 0.0001]. Double null was also higher in patients compared to controls (*P* = 0.01). When the cases were stratified according to the disease type, AML showed the highest positive predictive value for both loci (*GSTM1 P* < 0.0001, *GSTT1 P* < 0.0001), ALL and CLL showed similar patterns for *GSTT1* (*P* = 0.001) while the Pvalues for *GSTM1* were (0.01) and (0.007) respectively. CML displayed the least positive predictive value for *GSTM1* (0.02), while for *GSTT1* the result was as same as AML (*P* <

When the distribution of the *GSTM1* and *GSTT1* null genotypes were compared between linguistic groups of the control subjects, different percentages were obtained and *GSTT1* null genotypes was statistically different between the Afro-Asiatic and the Nilo-Saharan groups (*P* = 0.01). Difference in frequencies between Africans population is reported; Egyptians 15-29% (Abdel-Rahman, 1996; Hamdy et al., 2003), Tunisians 29% (Hanene et al., 2007) and Zimbabweans 26% (Masimirembwa, et al., 1998). generally speaking the *GSTM1* frequency (10.3%) is close to sub-Saharan Africa range; Nigerians 22% (Zhao et al., 1994) and Zimbabweans 24% (Masimirembwa, et al., 1998). while the previously reported frequency from Sudan is 39% (Tiemersma et al., 2001) . Other African populations frequencies are; Egyptians 44-55% [Abdel-Rahman, 1996 Hamdy et al., 2003), Tunisians 50% (Hanene et al.,

The Tagelsir study suggests an increased risk for leukemia associated with *GSTM1* and *GSTT1* null genotypes and highlights a potential role of genetic make up in leukaemogensis. The most statistically significant association for *GSTT1* observed for both AML and CML may highlight a possible role of GSTT1 enzyme in protection of the myeloid series. Allelic variation in the gene encoding the GST isoform theta (GSTT1) enzymes was found to modulate the rate of benzene metabolism and excretion (Rossi et al., 1999) as well as benzene-induced myelotoxicity (Wan et al., 2002; Chen et al., 2007). AML – which showed the strongest association with both genes– comprises a distinct type of leukemia with different subtypes and is shown to be to somewhat associated with environmental exposure. Epidemiological studies have shown association between AML (M2, M4, and M5) and maternal exposure to marijuana and alcohol, and maternal and paternal exposures to pesticides, (Buckley et al., 1989; Robison et al., 1989; Severson et al., 1993; Shu et al., 1996). In addition to that AML is shown to be associated with exposure to benzene and may arise as therapy related complication after treatment of other cancers (Hoffbrand & Pettit, 2001). Paradoxically, the presence of these genes could not be excluded as a possible risk factor for leukemia as some times these enzymes are involved in bioactivation of some chemicals producing more reactive metabolites that could confer threat to the cell. An example of such risk in other cancer was found in a study on Chinese population which showed that the genotype combination of *GSTM1* and *GSTT1* double positive confers a 4.2-fold higher risk for developing esophageal cancer and a 2.6-fold for esophageal hyperplasia (Lin et al., 1998).

0.0001). Double null, however, showed only association with AML (*P* < 0.0001).

(Green et al., 2010).

2007).

hurdle for future intervention against cancer in general. The anticancer drug imatinib has shown remarkable success in treatment of CML. Though a variety of resistance mechanisms can arise, in the majority of patients resistance coincides with reactivation of the tyrosine kinase activity of the BCR-ABL fusion oncoprotein. This can result from gene amplification and, more importantly, point mutations that disrupt the bind of imatinib to BCR-ABL itself (Nardi et al., 2004). Although there are no indication of resistance so far in Sudan (Alkhatib, 2011), perhaps due to the limited use of the drug, the risk of resistance is proportional to the number of mutations that exist within the kinase domain and even outside the domain which are expected to be higher in Africa given the increased genetic diversity of African populations. The risk of resistance increases with the identification of Novel potential signaling pathways associated with drug resistance (Duy et al., 2011).

The significance of population genetic background extends to diagnostic and prognostic markers that may be applied to populations and to individuals. This is expected to form a trend in the management of diseases of complex inheritance as we learn more of the biological networks in function during diseases and the role of each individual molecule. In a study by Elamin et al., (submitted), aimed at developing biomarkers for breast cancer, Peroxyredoxin V turned to be a potentially useful marker both as a prognostic and treatment marker in Sudanese breast cancer but not among Chinese.

#### **3.4 Drug metabolizing enzymes**

The drug metabolizing enzymes system has been shown to influence the susceptibility, sequel and outcome of cancer treatment. These systems include the Glutathione transferases a family member of genes encoding enzymes involved in the metabolism of many chemicals and shown to be polymorphic with *GSTM1* and *GSTT1* being deleted in proportion of individuals where in the homozygous state results in a phenotypic absence of the corresponding enzyme. These enzymes are considerably important in the detoxification of many environmental compounds and reactive oxygen species, and hence may constitute important cancer predisposition genes.

It includes also the cytochrome P450 enzymes one of the best studied for risk association with cancer (Aqundez, 2004). The P450 shows conflicting and variable degree of association with cancer, possibly reflecting, variation in the role played by these enzymes in carcinogenesis and the genetic background of the population.

 In some populations like those of the Indian subcontinent, the frequencies of homozygous 3/3 genotype and CYP3A5\*3 allele were elevated significantly in the CML group compared to controls (χ²=93.15, df=2, p=0.0001) (Sailaja et al., 2010).

In India also, a statistically significant difference between an AML group and normal control was observed in the case of glutathione-S-transferase M1 null (odds ratio 3.25, 95% confidence interval 1.9-5.58, P<0.001) and N-acetyl transferase 2\*6B (odds ratio 3.04, 95% confidence interval 1.79-5.16, P<0.001) genotypes. Combined deficiency of N-acetyl transferase 2 and glutathione-S-transferase M1 genes produced an odds ratio of 11.91 (95% confidence interval 4.06-34.96, P<0.001). Those with glutathione-S-transferase M1 null genotype and N-acetyl transferase 2\*6B allele are at increased risk of developing AML, and the risk is considerably enhanced in persons with both glutathione-S-transferase M1 and Nacetyl transferase 2 deficiency (Majumdar et al., 2008).

Increased risk of AML has also been reported for combined polymorphisms in detoxification and DNA repair enzymes (Voso et al., 2007), and patients that achieved

hurdle for future intervention against cancer in general. The anticancer drug imatinib has shown remarkable success in treatment of CML. Though a variety of resistance mechanisms can arise, in the majority of patients resistance coincides with reactivation of the tyrosine kinase activity of the BCR-ABL fusion oncoprotein. This can result from gene amplification and, more importantly, point mutations that disrupt the bind of imatinib to BCR-ABL itself (Nardi et al., 2004). Although there are no indication of resistance so far in Sudan (Alkhatib, 2011), perhaps due to the limited use of the drug, the risk of resistance is proportional to the number of mutations that exist within the kinase domain and even outside the domain which are expected to be higher in Africa given the increased genetic diversity of African populations. The risk of resistance increases with the identification of Novel potential

The significance of population genetic background extends to diagnostic and prognostic markers that may be applied to populations and to individuals. This is expected to form a trend in the management of diseases of complex inheritance as we learn more of the biological networks in function during diseases and the role of each individual molecule. In a study by Elamin et al., (submitted), aimed at developing biomarkers for breast cancer, Peroxyredoxin V turned to be a potentially useful marker both as a prognostic and

The drug metabolizing enzymes system has been shown to influence the susceptibility, sequel and outcome of cancer treatment. These systems include the Glutathione transferases a family member of genes encoding enzymes involved in the metabolism of many chemicals and shown to be polymorphic with *GSTM1* and *GSTT1* being deleted in proportion of individuals where in the homozygous state results in a phenotypic absence of the corresponding enzyme. These enzymes are considerably important in the detoxification of many environmental compounds and reactive oxygen species, and hence may constitute

It includes also the cytochrome P450 enzymes one of the best studied for risk association with cancer (Aqundez, 2004). The P450 shows conflicting and variable degree of association with cancer, possibly reflecting, variation in the role played by these enzymes in

 In some populations like those of the Indian subcontinent, the frequencies of homozygous 3/3 genotype and CYP3A5\*3 allele were elevated significantly in the CML group compared

In India also, a statistically significant difference between an AML group and normal control was observed in the case of glutathione-S-transferase M1 null (odds ratio 3.25, 95% confidence interval 1.9-5.58, P<0.001) and N-acetyl transferase 2\*6B (odds ratio 3.04, 95% confidence interval 1.79-5.16, P<0.001) genotypes. Combined deficiency of N-acetyl transferase 2 and glutathione-S-transferase M1 genes produced an odds ratio of 11.91 (95% confidence interval 4.06-34.96, P<0.001). Those with glutathione-S-transferase M1 null genotype and N-acetyl transferase 2\*6B allele are at increased risk of developing AML, and the risk is considerably enhanced in persons with both glutathione-S-transferase M1 and N-

Increased risk of AML has also been reported for combined polymorphisms in detoxification and DNA repair enzymes (Voso et al., 2007), and patients that achieved

signaling pathways associated with drug resistance (Duy et al., 2011).

treatment marker in Sudanese breast cancer but not among Chinese.

carcinogenesis and the genetic background of the population.

to controls (χ²=93.15, df=2, p=0.0001) (Sailaja et al., 2010).

acetyl transferase 2 deficiency (Majumdar et al., 2008).

**3.4 Drug metabolizing enzymes** 

important cancer predisposition genes.

complete molecular response following administration of imatinib showed significantly (p=0.013) higher in vivo CYP3A activity than patients achieving partial molecular response (Green et al., 2010).

In Sudan impact of the distribution of the GSTM1 and GSTT1 genotype leukemic patients was studied in 77 leukemic patients and 107 controls by Tagelsir et al., (Submitted). The results suggest that these genotypes could play a role in the development of leukemia particularly AML. Statistical analysis showed a significant preponderance of null genotype of both genes among pooled cases [*GSTM1* OR 3.45 (95% CI, 1.65 - 7.19); *P* = 0.001] for; *GSTT1* OR 8.57 (95% CI, 3.68 -19.93); *P* < 0.0001]. Double null was also higher in patients compared to controls (*P* = 0.01). When the cases were stratified according to the disease type, AML showed the highest positive predictive value for both loci (*GSTM1 P* < 0.0001, *GSTT1 P* < 0.0001), ALL and CLL showed similar patterns for *GSTT1* (*P* = 0.001) while the Pvalues for *GSTM1* were (0.01) and (0.007) respectively. CML displayed the least positive predictive value for *GSTM1* (0.02), while for *GSTT1* the result was as same as AML (*P* < 0.0001). Double null, however, showed only association with AML (*P* < 0.0001).

When the distribution of the *GSTM1* and *GSTT1* null genotypes were compared between linguistic groups of the control subjects, different percentages were obtained and *GSTT1* null genotypes was statistically different between the Afro-Asiatic and the Nilo-Saharan groups (*P* = 0.01). Difference in frequencies between Africans population is reported; Egyptians 15-29% (Abdel-Rahman, 1996; Hamdy et al., 2003), Tunisians 29% (Hanene et al., 2007) and Zimbabweans 26% (Masimirembwa, et al., 1998). generally speaking the *GSTM1* frequency (10.3%) is close to sub-Saharan Africa range; Nigerians 22% (Zhao et al., 1994) and Zimbabweans 24% (Masimirembwa, et al., 1998). while the previously reported frequency from Sudan is 39% (Tiemersma et al., 2001) . Other African populations frequencies are; Egyptians 44-55% [Abdel-Rahman, 1996 Hamdy et al., 2003), Tunisians 50% (Hanene et al., 2007).

The Tagelsir study suggests an increased risk for leukemia associated with *GSTM1* and *GSTT1* null genotypes and highlights a potential role of genetic make up in leukaemogensis. The most statistically significant association for *GSTT1* observed for both AML and CML may highlight a possible role of GSTT1 enzyme in protection of the myeloid series. Allelic variation in the gene encoding the GST isoform theta (GSTT1) enzymes was found to modulate the rate of benzene metabolism and excretion (Rossi et al., 1999) as well as benzene-induced myelotoxicity (Wan et al., 2002; Chen et al., 2007). AML – which showed the strongest association with both genes– comprises a distinct type of leukemia with different subtypes and is shown to be to somewhat associated with environmental exposure. Epidemiological studies have shown association between AML (M2, M4, and M5) and maternal exposure to marijuana and alcohol, and maternal and paternal exposures to pesticides, (Buckley et al., 1989; Robison et al., 1989; Severson et al., 1993; Shu et al., 1996). In addition to that AML is shown to be associated with exposure to benzene and may arise as therapy related complication after treatment of other cancers (Hoffbrand & Pettit, 2001).

Paradoxically, the presence of these genes could not be excluded as a possible risk factor for leukemia as some times these enzymes are involved in bioactivation of some chemicals producing more reactive metabolites that could confer threat to the cell. An example of such risk in other cancer was found in a study on Chinese population which showed that the genotype combination of *GSTM1* and *GSTT1* double positive confers a 4.2-fold higher risk for developing esophageal cancer and a 2.6-fold for esophageal hyperplasia (Lin et al., 1998).

Myeloid Leukemia: A Molecular Focus on Etiology and Risk Within Africa 477

DNA promoter methylation and histone modification are increasingly recognized as of primary importance in carcinogenesis. The two forms of aberrant methylation, hypomethylation and hypermethylation, are both well documented features of tumor cells (Hanahan and Weinberg, 2000; Jones and Baylin, 2002). The transcriptional silencing of tumor suppressor genes via promoter CpG island hypermethylation constitutes a key tumorigenic process contributing to all the typical hallmarks of a cancer cell that can result from tumor suppressor inactivation. Profiles of tumor suppressor methylation vary according to tumor type (Esteller et al., 2001) and each tumor apparently displays a distinct

Recent studies revealed that specific patterns of DNA methylation characterize AML and help to distinguish AML subtypes. The contribution of this epigenetic dysregulation to leukemogenesis in AML is currently unclear. However, interactions between mutated transcription factors and epigenetic networks have already been shown to be partially responsible for leukemic transformation, for e.g. in acute promyelocytic leukemia (APL). Also, direct mutations in the epigenetic master regulators EZH2 and DNMT3A were recently identified in AML and in diseases leading to secondary leukemia ( Schoofs and

New studies reveal that 20% of individuals with AML harbor somatic mutations in DNMT3A (encoding DNA methyltransferase 3A). Although these leukemia have some gene expression and DNA methylation changes, a direct link between mutant DNMT3A,

The disruption of key protective genes through methylation is not confined to tumor suppression, it extends to other vital genes in protection /susceptibility to cancer like drug metabolizing enzymes, as been reported for the glutathione-S-transferase P1 gene silencing

The burden of myeloid leukemia is expected to rise as part of a global trend of increase in cancer incidence. In Africa the cost of this rise given the compound problems of health systems will be devastating. One pressing need is to challenge the preconception of cancer being a disease of the developed societies and show how this image is changing under the rapid sweep of "globalization. Even now the limited data available on leukemia indicate that the incidence of CML in some African countries may exceed those of the industrialized world. The understanding of cancer complex etiology is a prerequisite for successful

Myeloid leukemia including the chronic subset that behave like a single gene disorder with the predominance of the Ph chromosome, posses complex etiology that includes multiple steps from environmental switches to inactivation of tumor suppression and other guardians of genome integrity and stability and ending with the impact of immune competence. Such complexity renders the handling of each of these potential culprits a daunting task especially in Africa. Without research into the etiology and genetic epidemiology of myeloid leukemia with all possible risk factors considered, including the genetic structure of population at risk, individual genetic effects, role of chronic and concomitant infections, and the possible trade off between infections and malignancies. For

epigenetic changes and pathogenesis remains to be established.

**3.5 Epigenetics** 

'DNA hypermethylation pattern.

Muller-Tidow, 2011).

(Karius et al., 2011)

**4. Future directions** 

management and control efforts.

Fig. 1. The ladder and lever mechanism towards carcinogenesis. As the cancer cell struggles through multiple accumulative genetic events leading to cancer (the ladder), the cell will gain the upper hand in its surrounding tissue environment and eventually metastasize, it encounters various modifications and modulating effects that vary between individuals and communities. The variety of effects and the modifier action is expected to be greater in Africa. The up scaling of the ladder requires a lifting effect from the Environment (lever).

#### **3.5 Epigenetics**

476 Myeloid Leukemia – Basic Mechanisms of Leukemogenesis

Fig. 1. The ladder and lever mechanism towards carcinogenesis. As the cancer cell

be greater in Africa. The up scaling of the ladder requires a lifting effect from the

struggles through multiple accumulative genetic events leading to cancer (the ladder), the cell will gain the upper hand in its surrounding tissue environment and eventually

metastasize, it encounters various modifications and modulating effects that vary between individuals and communities. The variety of effects and the modifier action is expected to

Environment (lever).

DNA promoter methylation and histone modification are increasingly recognized as of primary importance in carcinogenesis. The two forms of aberrant methylation, hypomethylation and hypermethylation, are both well documented features of tumor cells (Hanahan and Weinberg, 2000; Jones and Baylin, 2002). The transcriptional silencing of tumor suppressor genes via promoter CpG island hypermethylation constitutes a key tumorigenic process contributing to all the typical hallmarks of a cancer cell that can result from tumor suppressor inactivation. Profiles of tumor suppressor methylation vary according to tumor type (Esteller et al., 2001) and each tumor apparently displays a distinct 'DNA hypermethylation pattern.

Recent studies revealed that specific patterns of DNA methylation characterize AML and help to distinguish AML subtypes. The contribution of this epigenetic dysregulation to leukemogenesis in AML is currently unclear. However, interactions between mutated transcription factors and epigenetic networks have already been shown to be partially responsible for leukemic transformation, for e.g. in acute promyelocytic leukemia (APL). Also, direct mutations in the epigenetic master regulators EZH2 and DNMT3A were recently identified in AML and in diseases leading to secondary leukemia ( Schoofs and Muller-Tidow, 2011).

New studies reveal that 20% of individuals with AML harbor somatic mutations in DNMT3A (encoding DNA methyltransferase 3A). Although these leukemia have some gene expression and DNA methylation changes, a direct link between mutant DNMT3A, epigenetic changes and pathogenesis remains to be established.

The disruption of key protective genes through methylation is not confined to tumor suppression, it extends to other vital genes in protection /susceptibility to cancer like drug metabolizing enzymes, as been reported for the glutathione-S-transferase P1 gene silencing (Karius et al., 2011)

#### **4. Future directions**

The burden of myeloid leukemia is expected to rise as part of a global trend of increase in cancer incidence. In Africa the cost of this rise given the compound problems of health systems will be devastating. One pressing need is to challenge the preconception of cancer being a disease of the developed societies and show how this image is changing under the rapid sweep of "globalization. Even now the limited data available on leukemia indicate that the incidence of CML in some African countries may exceed those of the industrialized world. The understanding of cancer complex etiology is a prerequisite for successful management and control efforts.

Myeloid leukemia including the chronic subset that behave like a single gene disorder with the predominance of the Ph chromosome, posses complex etiology that includes multiple steps from environmental switches to inactivation of tumor suppression and other guardians of genome integrity and stability and ending with the impact of immune competence. Such complexity renders the handling of each of these potential culprits a daunting task especially in Africa. Without research into the etiology and genetic epidemiology of myeloid leukemia with all possible risk factors considered, including the genetic structure of population at risk, individual genetic effects, role of chronic and concomitant infections, and the possible trade off between infections and malignancies. For

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#### **5. Acknowledgment**

The authors acknowledges financial support from the International Centre of Genetic Engineering and Biotechnology (ICGEB)

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### *Edited by Steffen Koschmieder and Utz Krug*

The current book comprises a series of chapters from experts in the field of myeloid cell biology and myeloid leukemia pathogenesis. It is meant to provide reviews about current knowledge in the area of basic science of acute (AML) and chronic myeloid leukemia (CML) as well as original publications covering specific aspects of these important diseases. Covering the specifics of leukemia biology and pathogenesis by authors from different parts of the World, including America, Europe, Africa, and Asia, this book provides a colorful view on research activities in this field around the globe.

Photo by dzika\_mrowka / iStock

Myeloid Leukemia - Basic Mechanisms of Leukemogenesis

Myeloid Leukemia

Basic Mechanisms of Leukemogenesis

*Edited by Steffen Koschmieder and Utz Krug*