**4.2.4 BMI1**

BMI1 is a polycomb group protein which, together with Ring1 proteins, is part of PRC1 complex that has histone H2A-K119 ubiquitin E3 ligase activity. BMI1 has a role in HOX gene (HOXC13) silencing by H2A ubiquitylation (Cao et al, 2005). BMI1 is also known to be important in the regulation and maintenance of proliferative/self-renewal potential in both normal hematopoietic and leukemic stem cells (Park et al, 2003). Upon knockdown of BMI-1, cells lose their ability to engraft and reconstitute leukemia in mice (Bomken et al., 2010).

#### **4.3 Xenotransplantation model of leukemia**

A key component for understanding the biological mechanisms for tumor heterogeneity is the ability to functionally assess the capacity for limitless proliferative capacity for segregated populations of tumor cells. Unfortunately, for hematologic malignancies, *in vitro* culture assays are not entirely effective as a means of functionally assessing self-renewal capacity. Thus, transplantation assays in which candidate populations are assessed for their ability to establish long-term serial engraftment of recipient animals is the gold standard for assigning limitless proliferative capability, i.e. self-renewal. For murine studies, the availability of syngeneic transplantation models has been responsible in large part for our in depth understanding of the normal murine hematopoietic hierarchy.

Since the 70 s, there is ample evidence supporting the existence of a discrete compartment of slowly cycling leukemic cells that are resistant to standard chemotherapeutic agents. These cell populations were felt to represent the leukemic stem cells and though many observations were consistent with this hypothesis, there was no direct evidence that this was indeed the case. As the first direct evidence for the existence of cancer stem cells, the work of Lapidot et al. (1994) represented a milestone in the history of the leukemic stem cell model. This study identified an infrequent population of leukemic cells capable of recapitulating the human tumor in xenotransplants. A key finding was that the SCID mouse leukemia repopulating cell, SRC, possessed a phenotype that was similar to that of the normal hematopoietic stem cell (CD34+ and CD38−).

#### **4.4 Acute myeloid leukemia**

Acute myeloid leukemia (AML) is a clonal disorder characterized by arrest of differentiation in the myeloid lineage coupled with an accumulation of immature progenitors in the bone marrow, resulting in hematopoietic failure (Pollyea et al., 2011). AML is the most common

Hematopoietic Stem Cell in Acute Myeloid Leukemia Development 273

FLT3, a member of the class III tyrosine kinase receptor family, is expressed in normal hematopoietic progenitors as well as in leukemic blasts, and it plays an important role in cell proliferation, differentiation, and survival. Activation of the FLT3 receptor by the FLT3 ligand leads to receptor dimerization and phosphorylation, and activation of downstream signaling pathways, including the Janus kinase (JAK) 2 signal transducer (JAK2), signal transducer and activator of transcription (STAT) 5, and mitogen-activated protein kinase (MAPK) pathways. Mutations in the FLT3 gene, found in approximately 40% of patients with AML, are believed to promote its autophosphorylation and constitutive activation,

The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. It is a member of the Ig gene superfamily and was first identified as a gene expressed in activated T cells. CD96 was described as a tumor marker AML stem cell (Konopleva & Jordan, 2011). Hosen et al. (2011) showed that AML-LSC can be distinguished from normal HSC by the presence of CD96 expression. This finding suggests that CD96 also may prove to be an excellent target for antibody therapy

Another adhesion molecule, CD44, has been demonstrated to be a key regulator of AML LSCs homing to microenvironmental niches, maintaining a primitive state. CD44 mediates adhesive cell-cell and cell–complex extracellular matrix interactions through binding to its main ligand, hyaluronan, a glycosaminoglycan highly concentrated in the endosteal region. Other ligands include osteopontin, fibronectin, and selectin, all of which are involved in cell trafficking and lodgment. Beyond its adhesion function, CD44 can also transduce multiple intracellular signal transduction pathways when bound to hyaluronan or to specific

Adult hematopoietic stem cells are undifferentiated cells capable of self-renewal and differentiation potential in several cell types that comprise the hematopoietic tissue. These cells have been used in bone marrow transplantation for treatment of hematological malignancies as well as non-hematological diseases. The HSC is the main component in the process of hematopoiesis which, together with the cells that make up the bone marrow stromal environment and other intrinsic and extrinsic factors, orchestrates the entire

However, when this process of cell production is unbalanced, leading to an exacerbated and uncontrolled proliferation of blood progenitor cells, leukemia may develop.The ultimate challenge in coming years will be to understand the stem cell 'programme', particularly the control of self-renewal, in an attempt to develop novel, stem cell-directed therapies. An improved understanding of clonal evolution will be critical if we are to ensure that cancers are not able to evolve mechanisms to evade the new directed therapies. However, reducing the risk of relapse and minimizing long-term side effects should always remain the ultimate

With little doubt, the leukemia stem cell model has had the greatest clinical impact on our understanding and treatment of Philadelphia chromosome positive leukemias. Effective

against LSC because hematopoietic progenitors are regenerated rapidly from HSC.

function-activating monoclonal antibodies (Konopleva & Jordan, 2011).

production of progenitors and terminally differentiated blood cells.

**5. Conclusions** 

goal of understanding the CSCs.

leading to ligand-independent proliferation (Frankfurt et al., 2009).

acute leukemia in adults, affecting roughly three out of 100.000 people. AML patients are predominantly elderly, with a median age at diagnosis of 67 (National Cancer Institute 1975–2007).

In AML, there is wide patient-to-patient heterogeneity in the appearance of the leukemic blasts. Conventionally, AML is classified into seven French–American–British (FAB) subtypes corresponding to the maturation stage of the leukemia (Warner et al., 2004).

The discovery of leukemia-initiating cells in acute myeloid leukemias (AMLs) started with the discovery that the large majority of AML blasts do not proliferate and only a small minority is capable of forming new colonies (Testa, 2011).

A common feature to all AML cases is the arrested aberrant differentiation leading to an accumulation of more than 20% blast cells in the bone marrow (Gilliland & Tallman, 2002). More than 80% of myeloid leukemias are associated with at least one chromosomal rearrangement (Pandolfi, 2001), and over 100 different chromosomal translocations have been cloned (Gilliland & Tallman, 2002). Frequently, these translocations involve genes encoding transcription factors that have been shown to play an important role in hematopoietic lineage development. Thus, alteration of the transcriptional machinery appears to be a common mechanism leading to arrested differentiation (Pandolfi, 2001; Tenen, 2003).

Clinical investigation and experimental animal models suggest that at least two genetic alterations are required for the clinical manifestation of acute leukemia. According to the model proposed by Gilliland & Tallman (2002), cooperation between class I activating mutations and class II mutations that induce termination of differentiation give rise to AML. The class I mutations, such as mutations in the receptor tyrosine kinase genes FLT3 and KIT, RAS family members, and loss of function of neurofibromin 1, confer proliferative and/or survival advantage to hematopoietic progenitors, typically as a consequence of aberrant activation of signal transduction pathways. The class II mutations lead to a halt in differentiation via interference with transcription factors or co-activators (Frankfurt et al., 2009).

While the LSC appears to share many of the cell surface markers previously identified for HSC such as CD34, CD38, HLA-DR, and CD71, there have been several groups who have reported surface markers that are differentially expressed in the two populations. CD90 or Thy-1 is one marker that has been described to be potentially specific of the LSC compartment. Thy-1 is downregulated in normal hematopoiesis as the most primitive stem cells progress toward the progenitor stage. This finding of the lack of expression on LSC might suggest that the primitive stem cell does not contribute to the primary pathological event, or that Thy-1 expression is downregulated as a result of the leukemogenic events (Hope et al., 2003).

The interaction between CXCL12 (stromal cell–derived factor-1 alpha) and its receptor CXCR4 on leukemic progenitor cells contributes to their homing to the bone marrow microenvironment. CXCR4 levels are significantly elevated in leukemic cells from patients with AML and CXCR4 expression is associated with poor outcome (Konopleva & Jordan, 2011). Constitutive activation of the nuclear factor kappa B (NF-kB) pathway in primary human AML stem cells provided evidence that NF-kB plays a significant role in the overall survival of LSCs as well as AML cell types in general. This pathway is strongly implicated as a central target in developing LSC-specific therapies (Konopleva & Jordan, 2011).

FLT3, a member of the class III tyrosine kinase receptor family, is expressed in normal hematopoietic progenitors as well as in leukemic blasts, and it plays an important role in cell proliferation, differentiation, and survival. Activation of the FLT3 receptor by the FLT3 ligand leads to receptor dimerization and phosphorylation, and activation of downstream signaling pathways, including the Janus kinase (JAK) 2 signal transducer (JAK2), signal transducer and activator of transcription (STAT) 5, and mitogen-activated protein kinase (MAPK) pathways. Mutations in the FLT3 gene, found in approximately 40% of patients with AML, are believed to promote its autophosphorylation and constitutive activation, leading to ligand-independent proliferation (Frankfurt et al., 2009).

The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. It is a member of the Ig gene superfamily and was first identified as a gene expressed in activated T cells. CD96 was described as a tumor marker AML stem cell (Konopleva & Jordan, 2011). Hosen et al. (2011) showed that AML-LSC can be distinguished from normal HSC by the presence of CD96 expression. This finding suggests that CD96 also may prove to be an excellent target for antibody therapy against LSC because hematopoietic progenitors are regenerated rapidly from HSC.

Another adhesion molecule, CD44, has been demonstrated to be a key regulator of AML LSCs homing to microenvironmental niches, maintaining a primitive state. CD44 mediates adhesive cell-cell and cell–complex extracellular matrix interactions through binding to its main ligand, hyaluronan, a glycosaminoglycan highly concentrated in the endosteal region. Other ligands include osteopontin, fibronectin, and selectin, all of which are involved in cell trafficking and lodgment. Beyond its adhesion function, CD44 can also transduce multiple intracellular signal transduction pathways when bound to hyaluronan or to specific function-activating monoclonal antibodies (Konopleva & Jordan, 2011).
