**Ikaros in T-Cell Leukemia**

Sinisa Dovat1 and Kimberly J. Payne2

*1Pennsylvania State University College of Medicine 2Loma Linda University United States of America* 

#### **1. Introduction**

96 T-Cell Leukemia

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Although extensive clinical data have established that the loss of Ikaros tumor suppressor activity *via* genetic inactivation is a major contributor to leukemogenesis leading to B-cell ALL, less is known about the role of Ikaros in T-cell leukemia. The T-cell malignancies observed in Ikaros-deficient mice suggest that Ikaros is likely to function as a tumor suppressor in T-cells. In human, multiple studies have identified genetic inactivation of Ikaros (deletion or mutation) to be associated with ~5% of T-ALL cases. Additional studies provide evidence that the functional inactivation of Ikaros due to defects in signal pathways that normally regulate Ikaros activity is likely to be a critical factor in the development of Tcell malignancies. These studies provide a rationale for new chemotherapeutic strategies in the treatment of T-cell leukemia. In this chapter, we summarize the newest advances that shed light on the role of Ikaros in T-cell leukemia.

#### **2. Structure and function of Ikaros**

The *Ikaros* gene, discovered independently by K. Georgopoulos and S. Smale, encodes multiple Ikaros protein isoforms *via* alternate splicing (Lo et al. 1991; Georgopoulos et al. 1992; Hahm et al. 1994). The structure of Ikaros protein reveals several known structural motifs corresponding to distinct functional domains as described below.

#### **2.1 Molecular structure of Ikaros**

#### **2.1.1 DNA-binding domain**

The N-terminal region of the Ikaros protein contains four zinc finger motifs (Fig. 1). Three of these exhibit a typical C2H2 structure with two cysteines and two histidines covalently bound to a zinc atom, while the fourth zinc finger has a CCHC structure. The four N terminal zinc fingers in the Ikaros protein function in DNA binding. Point mutational analysis revealed that zinc fingers #2 and #3 are essential for the DNA-binding function of Ikaros, as well as its localization to pericentromeric heterochromatin (see 2.2.1). Based on DNA-se footprint analysis, it has been suggested that the first zinc finger contributes to DNA binding specificity. The role of the fourth zinc finger in DNA-binding remains unknown, although it has been noted that Ikaros isoforms that lack the fourth zinc finger exhibit a unique expression pattern during hematopoiesis (Payne et al. 2003). Recently, a point mutation in a single allele of the fourth zinc finger has been associated with primary immunodeficiency and pancytopenia in humans (Goldman, et al. In press*.).*

Ikaros in T-Cell Leukemia 99

and/or particular amino acids within the Ikaros protein that are responsible for these functions remain unknown. Further studies are needed to provide more detailed maps of

Ikaros has been shown to regulate transcription by binding DNA at the upstream regulatory elements of its target genes (Georgopoulos et al. 1992; Ernst et al. 1993; Hahm et al. 1994; Molnar and Georgopoulos 1994). Thus, DNA binding is essential for Ikaros function. While Ikaros can directly activate or repress its target genes, subsequent experiments established that Ikaros function as a regulator of gene expression occurs *via* its role in chromatin remodeling. Studies by A. Fisher's group demonstrated that Ikaros is abundantly localized in pericentromeric heterochromatin in cells (Brown et al. 1997). In pericentromeric regions, transcriptionally inactive genes have been shown to selectively associate with Ikaros foci, while transcriptionally active genes do not (Brown et al. 1997). The expression status of several genes that are differentially expressed during T and B cell maturation correlate with their association with Ikaros. Mutations in Ikaros binding sites interferes with the developmentally-regulated shut-down of the 5 gene (Sabbattini et al. 2001), as well as the gene that encodes TdT *(dntt)* (Trinh et al. 2001) during lymphoid differentiation. These data further support a role for Ikaros in gene silencing *via* chromatin remodeling. Experiments by Georgopoulos' group also demonstrated that Ikaros regulates expression of its target genes by recruiting them to pericentromeric heterochromatin, but that recruitment to pericentromeric heterochromatin can lead to activation of the target genes (Koipally et al. 2002). Studies in human T-cells revealed that Ikaros binding to human pericentromeric heterochromatin is regulated in a complex way by the association of different Ikaros isoforms. A model has been proposed whereby Ikaros regulates expression of its target genes by recruiting them to pericentromeric heterochromatin leading to either their activation or repression, and that switching from repression to activation depends on the presence of particular Ikaros isoforms in the Ikaros DNA-binding complex (Ronni et al. 2007; Kim et al. 2009). **Thus, the current hypothesis is that Ikaros binds the upstream region of target genes and aids in their recruitment to pericentromeric heterochromatin, resulting in repression or activation of** 

**2.2.2 Molecular mechanisms of chromatin remodeling and gene regulation by Ikaros** Ikaros associates with histone deacetylase (HDAC)-containing complexes (NuRD and Sin3A and Sin3B) (Koipally et al. 1999). Ikaros directly interacts with the NuRD complex ATPase, Mi-2, and with Sin3A and Sin3B through both its N-terminal and C-terminal regions (Kim et al. 1999; Koipally et al. 1999). The histone deacetylase complex acts as a transcriptional repressor *via* chromatin remodeling. It has been hypothesized that Ikaros recruits histone deacetylase complex to the upstream regulatory elements of its target genes resulting in **chromatin remodeling and repression** of the target genes (Brown et al.

Ikaros has been shown to interact with the CtBP corepressor *in vivo*. This interaction with CtBP is achieved through amino acids at the N-terminal region of Ikaros (Koipally and Georgopoulos 2000). The Ikaros-CtBP complex acts as a transcriptional repressor. This

the functional domains within the Ikaros protein.

**2.2 Ikaros function in regulating gene expression** 

**the target gene (Brown et al. 1997; Liberg et al. 2003).**

1997; Liberg et al. 2003).

**2.2.1 Ikaros regulates gene expression** *via* **chromatin remodeling** 

#### **2.1.2 Dimerization domain**

The C-terminal region of the Ikaros protein contains two zinc finger motifs (Fig. 1). These zinc fingers do not bind DNA, but mediate protein-protein interactions with other Ikaros isoforms, and/or with Ikaros family proteins (Helios, Aiolos, etc.) that share the same motif (Sun et al. 1996). Every described Ikaros isoform contains the two C terminal zinc fingers, thus allowing for the formation of diverse Ikaros dimers with the potential for unique and specific functions. Ikaros binds DNA as a dimer, which underscores the importance of this domain for Ikaros function.

#### **2.1.3 Bipartite activation-repression domain**

All of the Ikaros proteins share a bipartite activation domain, that is adjacent to the two Cterminal zinc fingers (Fig. 1). This domain is responsible for stimulating basal levels of transcription of Ikaros target genes (Sun et al. 1996; Georgopoulos et al. 1997). Within the bipartite activation domain, there are two distinct stretches of amino acids – acidic and hydrophobic. It has been established that acidic amino acids are responsible for transcriptional activation, while the hydrophobic amino acids do not exhibit such an effect (Sun et al. 1996). However, the presence of the hydrophobic amino acids next to the acidic stretch increases their transcriptional activation activity, suggesting that the hydrophobic residues have a functional role in transcriptional regulation (Sun et al. 1996; Georgopoulos et al. 1997).

Fig. 1. Major structural features of the Ikaros protein. The *Ikaros* gene (*Ikzf1)* includes eight coding exons. Exon 1 is untranslated and was not identified in initial reports. Current Genbank sequences do not identify Exon 3B which encodes the 20 amino acid activation domain.

#### **2.1.4 Exon 3B activation domain**

Exon 3B of the *Ikaros* gene encodes a short, 20-amino acid stretch in the N region of the Ikaros protein (Fig. 1). This domain has been described (Hahm et al. 1994; Sun et al. 1999; Payne et al. 2001) and functionally analyzed in human Ikaros (Ronni et al. 2007). This domain has been shown to take part in determining the DNA-binding specificity of the Ikaros protein complex and in regulating the expression of Ikaros target genes (Ronni et al. 2007).

We would like to emphasize that there are functionally significant domains of the Ikaros protein that are as yet unidentified. It is known that Ikaros can act as a transcriptional repressor and that it has a role in chromatin remodeling (see 2.2.2). However, the structures

The C-terminal region of the Ikaros protein contains two zinc finger motifs (Fig. 1). These zinc fingers do not bind DNA, but mediate protein-protein interactions with other Ikaros isoforms, and/or with Ikaros family proteins (Helios, Aiolos, etc.) that share the same motif (Sun et al. 1996). Every described Ikaros isoform contains the two C terminal zinc fingers, thus allowing for the formation of diverse Ikaros dimers with the potential for unique and specific functions. Ikaros binds DNA as a dimer, which underscores the importance of this

All of the Ikaros proteins share a bipartite activation domain, that is adjacent to the two Cterminal zinc fingers (Fig. 1). This domain is responsible for stimulating basal levels of transcription of Ikaros target genes (Sun et al. 1996; Georgopoulos et al. 1997). Within the bipartite activation domain, there are two distinct stretches of amino acids – acidic and hydrophobic. It has been established that acidic amino acids are responsible for transcriptional activation, while the hydrophobic amino acids do not exhibit such an effect (Sun et al. 1996). However, the presence of the hydrophobic amino acids next to the acidic stretch increases their transcriptional activation activity, suggesting that the hydrophobic residues have a functional

Fig. 1. Major structural features of the Ikaros protein. The *Ikaros* gene (*Ikzf1)* includes eight coding exons. Exon 1 is untranslated and was not identified in initial reports. Current Genbank sequences do not identify Exon 3B which encodes the 20 amino acid activation

Exon 3B of the *Ikaros* gene encodes a short, 20-amino acid stretch in the N region of the Ikaros protein (Fig. 1). This domain has been described (Hahm et al. 1994; Sun et al. 1999; Payne et al. 2001) and functionally analyzed in human Ikaros (Ronni et al. 2007). This domain has been shown to take part in determining the DNA-binding specificity of the Ikaros protein complex

We would like to emphasize that there are functionally significant domains of the Ikaros protein that are as yet unidentified. It is known that Ikaros can act as a transcriptional repressor and that it has a role in chromatin remodeling (see 2.2.2). However, the structures

and in regulating the expression of Ikaros target genes (Ronni et al. 2007).

role in transcriptional regulation (Sun et al. 1996; Georgopoulos et al. 1997).

**2.1.2 Dimerization domain** 

domain for Ikaros function.

domain.

**2.1.4 Exon 3B activation domain** 

**2.1.3 Bipartite activation-repression domain** 

and/or particular amino acids within the Ikaros protein that are responsible for these functions remain unknown. Further studies are needed to provide more detailed maps of the functional domains within the Ikaros protein.

#### **2.2 Ikaros function in regulating gene expression 2.2.1 Ikaros regulates gene expression** *via* **chromatin remodeling**

Ikaros has been shown to regulate transcription by binding DNA at the upstream regulatory elements of its target genes (Georgopoulos et al. 1992; Ernst et al. 1993; Hahm et al. 1994; Molnar and Georgopoulos 1994). Thus, DNA binding is essential for Ikaros function. While Ikaros can directly activate or repress its target genes, subsequent experiments established that Ikaros function as a regulator of gene expression occurs *via* its role in chromatin remodeling. Studies by A. Fisher's group demonstrated that Ikaros is abundantly localized in pericentromeric heterochromatin in cells (Brown et al. 1997). In pericentromeric regions, transcriptionally inactive genes have been shown to selectively associate with Ikaros foci, while transcriptionally active genes do not (Brown et al. 1997). The expression status of several genes that are differentially expressed during T and B cell maturation correlate with their association with Ikaros. Mutations in Ikaros binding sites interferes with the developmentally-regulated shut-down of the 5 gene (Sabbattini et al. 2001), as well as the gene that encodes TdT *(dntt)* (Trinh et al. 2001) during lymphoid differentiation. These data further support a role for Ikaros in gene silencing *via* chromatin remodeling. Experiments by Georgopoulos' group also demonstrated that Ikaros regulates expression of its target genes by recruiting them to pericentromeric heterochromatin, but that recruitment to pericentromeric heterochromatin can lead to activation of the target genes (Koipally et al. 2002). Studies in human T-cells revealed that Ikaros binding to human pericentromeric heterochromatin is regulated in a complex way by the association of different Ikaros isoforms. A model has been proposed whereby Ikaros regulates expression of its target genes by recruiting them to pericentromeric heterochromatin leading to either their activation or repression, and that switching from repression to activation depends on the presence of particular Ikaros isoforms in the Ikaros DNA-binding complex (Ronni et al. 2007; Kim et al. 2009). **Thus, the current hypothesis is that Ikaros binds the upstream region of target genes and aids in their recruitment to pericentromeric heterochromatin, resulting in repression or activation of the target gene (Brown et al. 1997; Liberg et al. 2003).**

#### **2.2.2 Molecular mechanisms of chromatin remodeling and gene regulation by Ikaros**

Ikaros associates with histone deacetylase (HDAC)-containing complexes (NuRD and Sin3A and Sin3B) (Koipally et al. 1999). Ikaros directly interacts with the NuRD complex ATPase, Mi-2, and with Sin3A and Sin3B through both its N-terminal and C-terminal regions (Kim et al. 1999; Koipally et al. 1999). The histone deacetylase complex acts as a transcriptional repressor *via* chromatin remodeling. It has been hypothesized that Ikaros recruits histone deacetylase complex to the upstream regulatory elements of its target genes resulting in **chromatin remodeling and repression** of the target genes (Brown et al. 1997; Liberg et al. 2003).

Ikaros has been shown to interact with the CtBP corepressor *in vivo*. This interaction with CtBP is achieved through amino acids at the N-terminal region of Ikaros (Koipally and Georgopoulos 2000). The Ikaros-CtBP complex acts as a transcriptional repressor. This

Ikaros in T-Cell Leukemia 101

Studies by Georgopoulos' group demonstrated that Ikaros binds *in vivo* to a silencer that is located in the first intron of the *Cd4* gene resulting in the suppression of *Cd4* transcription. Further analysis revealed that Ikaros can bind concommitantly with the Mi-2 chromatin remodeler leading to suppression of the Cd4 silencer and upregulation of *Cd4* gene expression. The Ikaros-Mi2 complex aids in recruitment of histone acetyl transferases. Thus Ikaros appeaars to be able to both activate or repress expression of *Cd4 via* chromatin

In mature CD4+ cells, Ikaros was shown to bind *in vivo* to the upstream regulatory element of the *IL-2* gene. Further analysis demonstrated that Ikaros represses expression of the *IL-2* gene in mature CD4+ cells *via* chromatin remodeling. This established Ikaros as a regulator

Studies of Ikaros-deficient mice established Ikaros as a master regulator of T-cell

1. Thymocytes that lack Ikaros expression progress from the double negative (DN) CD4– CD8– to the double positive (DP) CD4+CD8+ stage in the absence of selection (Wang et al. 1996). The proliferative response of thymocytes following TCR ligation is ~9 fold higher in thymocytes from Ikaros null mice, compared to the wild-type. Thus, Ikaros has been identified as a key regulator of the selection checkpoint in T-cell differentiation and a regulator of thymocyte expansion following selection (Winandy

2. Thymocytes from Ikaros-deficient mice downregulate CD8 to become CD4 single positive (SP) thymocytes without positive selection signals that are normally required for differentiation from the DP to the SP stage (Winandy et al. 1999; Urban and Winandy 2004). Ikaros has also been shown to regulate negative selection that occurs during the DP stage (Urban and Winandy 2004). T-cell production in these mice is skewed toward the production of CD4 T-cells. Thus, Ikaros functions as a regulator that controls the TCR selection checkpoint in T-cell development, as well as a regulator of CD4 versus CD8 T-cell fate decisions (Wang et al. 1996; Winandy et al. 1999; Urban and

3. Ikaros-deficient T-cells from all three Ikaros knock-out mice strains possess a lower activation threshold than normal T-cells and enter the cell cycle at an accelerated pace (Avitahl et al. 1999; Winandy et al. 1999). Thus, Ikaros sets the activation threshold in

The Ikaros-defficient mouse described above clearly established that Ikaros has an essential role in T-cell development. Subsequent analysis of heterozygous Ikarosdefficient mice revealed that Ikaros has tumor suppressor activity. Between the second and third month of age, these mice display an aberration in thymic differentiation with an accumulation of triple-positive thymocytes that have intermediate expression levels of CD4, CD8, and the TCR complex (Winandy et al. 1995). At the same time, a polyclonal expansion of mature T lymphocytes occurs in the spleen of these mice. Proliferation

remodeling (Naito et al. 2007).

et al. 1999).

Winandy 2004).

**4. Ikaros and T-cell leukemia** 

of anergy induction in CD4+ T-cells (Thomas et al. 2007).

development and identified several biological functions of Ikaros:

mature T-cells and regulates cell cycle progression.

**4.1 Ikaros-deficient mice develop T-cell leukemia** 

**3.3 T-cell development in Ikaros-defficient mice** 

repression is HDAC-independent, and represents **an additional means by which Ikaros represses transcription** of its target genes (Koipally and Georgopoulos 2000).

Ikaros also associates with Brg-1, a catalytic subunit of the SWI/SNF nucleosome remodeling complex that acts as an activator of gene expression (Kim et al. 1999; O'Neill et al. 2000). It has been hypothesized that Ikaros recruits the SWI/SNF nucleosome remodeling complex to the upstream regions of its target genes (in a similar fashion to the NuRD complex) resulting in **chromatin remodeling and activation** of its target genes. **Thus, Ikaros can act as either an activator or a repressor of its target genes, depending on whether it associates with the NuRD, the CtBP or the SWI/SNF complex.** 

#### **3. Ikaros in T-cell development**

The role of Ikaros in normal T-cell development is demonstrated by evidence that Ikaros regulates the expression of key genes in T-cell differentiation and by the impaired T-cell differentiation observed in Ikaros-defficient mice.

#### **3.1 Ikaros regulates genes critical for the development of T-cells (***TdT***)**

The role of Ikaros in regulating expression of the gene encoding terminal deoxynucleotide transferase (*TdT*) during thymocyte differentiation has been extensively studied (Ernst et al. 1993; Ernst et al. 1996; Trinh et al. 2001; Su et al. 2005). Ikaros has been shown to bind *in vivo* to the D' upstream regulatory element of the *TdT* (*dntt*) gene. This region contains a perfect Ikaros consensus DNA-binding site, and mutational analysis has demonstrated that the presence of this sequence is essential for Ikaros binding to the upstream regulatory element of the *TdT* gene. This same region contains a consensus binding site that is bound *in vivo* by Elf-1, a member of the Ets family of transcription factors. The binding of Elf-1 and Ikaros have been shown to be mutually exclusive. Ikaros binding to the *TdT* upstream regulatory element leads to repositioning of the *TdT* gene to pericentromeric heterochromatin and to repression of *TdT* transcription. Elf-1, in contrast, acts as positive regulator of *TdT* expression. Thus, Ikaros and Elf-1 compete for occupancy of the same upstream control region during thymocyte development and have the opposite effect on *TdT* expression (Trinh et al. 2001). In CD4+CD8+ thymocytes, the upstream region of *TdT* is occupied by Elf-1 leading to expression of the *TdT* gene. During the induction of T-cell differentiation, Ikaros displaces Elf-1 from binding the upstream regulatory element of *TdT*, resulting in the loss of *TdT* expression. The absence of Ikaros results in failure to repress *TdT* expression during thymocyte differentiation leading to impaired T-cell development. The exact molecular mechanisms by which Ikaros displaces Elf-1 during thymocyte differentiation remains to be determined, although there is compelling evidence that the reversible phosphorylation of Ikaros (see 6.1 and 6.2 below) is responsible for this regulation.

#### **3.2 Ikaros regulation of CD4, CD8, and IL-2 expression**

During T-cell development, Ikaros has been demonstrated to bind to the regulatory element of the CD8 gene *in vivo* by chromatin immunoprecipitation (ChIP) assay. Ikaros has been hypothesized to positively regulate expression of the CD8gene during thymocyte differentiation (Harker et al. 2002). This has been supported by evidence that Ikarosdefficient mice have decreased numbers of CD8+ T-cells (see 3.3 below).

repression is HDAC-independent, and represents **an additional means by which Ikaros** 

Ikaros also associates with Brg-1, a catalytic subunit of the SWI/SNF nucleosome remodeling complex that acts as an activator of gene expression (Kim et al. 1999; O'Neill et al. 2000). It has been hypothesized that Ikaros recruits the SWI/SNF nucleosome remodeling complex to the upstream regions of its target genes (in a similar fashion to the NuRD complex) resulting in **chromatin remodeling and activation** of its target genes. **Thus, Ikaros can act as either an activator or a repressor of its target genes, depending on whether it** 

The role of Ikaros in normal T-cell development is demonstrated by evidence that Ikaros regulates the expression of key genes in T-cell differentiation and by the impaired T-cell

The role of Ikaros in regulating expression of the gene encoding terminal deoxynucleotide transferase (*TdT*) during thymocyte differentiation has been extensively studied (Ernst et al. 1993; Ernst et al. 1996; Trinh et al. 2001; Su et al. 2005). Ikaros has been shown to bind *in vivo* to the D' upstream regulatory element of the *TdT* (*dntt*) gene. This region contains a perfect Ikaros consensus DNA-binding site, and mutational analysis has demonstrated that the presence of this sequence is essential for Ikaros binding to the upstream regulatory element of the *TdT* gene. This same region contains a consensus binding site that is bound *in vivo* by Elf-1, a member of the Ets family of transcription factors. The binding of Elf-1 and Ikaros have been shown to be mutually exclusive. Ikaros binding to the *TdT* upstream regulatory element leads to repositioning of the *TdT* gene to pericentromeric heterochromatin and to repression of *TdT* transcription. Elf-1, in contrast, acts as positive regulator of *TdT* expression. Thus, Ikaros and Elf-1 compete for occupancy of the same upstream control region during thymocyte development and have the opposite effect on *TdT* expression (Trinh et al. 2001). In CD4+CD8+ thymocytes, the upstream region of *TdT* is occupied by Elf-1 leading to expression of the *TdT* gene. During the induction of T-cell differentiation, Ikaros displaces Elf-1 from binding the upstream regulatory element of *TdT*, resulting in the loss of *TdT* expression. The absence of Ikaros results in failure to repress *TdT* expression during thymocyte differentiation leading to impaired T-cell development. The exact molecular mechanisms by which Ikaros displaces Elf-1 during thymocyte differentiation remains to be determined, although there is compelling evidence that the reversible phosphorylation of Ikaros (see 6.1 and 6.2 below)

During T-cell development, Ikaros has been demonstrated to bind to the regulatory element of the CD8 gene *in vivo* by chromatin immunoprecipitation (ChIP) assay. Ikaros has been hypothesized to positively regulate expression of the CD8gene during thymocyte differentiation (Harker et al. 2002). This has been supported by evidence that Ikaros-

**represses transcription** of its target genes (Koipally and Georgopoulos 2000).

**3.1 Ikaros regulates genes critical for the development of T-cells (***TdT***)** 

**associates with the NuRD, the CtBP or the SWI/SNF complex.** 

**3. Ikaros in T-cell development** 

is responsible for this regulation.

**3.2 Ikaros regulation of CD4, CD8, and IL-2 expression** 

defficient mice have decreased numbers of CD8+ T-cells (see 3.3 below).

differentiation observed in Ikaros-defficient mice.

Studies by Georgopoulos' group demonstrated that Ikaros binds *in vivo* to a silencer that is located in the first intron of the *Cd4* gene resulting in the suppression of *Cd4* transcription. Further analysis revealed that Ikaros can bind concommitantly with the Mi-2 chromatin remodeler leading to suppression of the Cd4 silencer and upregulation of *Cd4* gene expression. The Ikaros-Mi2 complex aids in recruitment of histone acetyl transferases. Thus Ikaros appeaars to be able to both activate or repress expression of *Cd4 via* chromatin remodeling (Naito et al. 2007).

In mature CD4+ cells, Ikaros was shown to bind *in vivo* to the upstream regulatory element of the *IL-2* gene. Further analysis demonstrated that Ikaros represses expression of the *IL-2* gene in mature CD4+ cells *via* chromatin remodeling. This established Ikaros as a regulator of anergy induction in CD4+ T-cells (Thomas et al. 2007).

#### **3.3 T-cell development in Ikaros-defficient mice**

Studies of Ikaros-deficient mice established Ikaros as a master regulator of T-cell development and identified several biological functions of Ikaros:


#### **4. Ikaros and T-cell leukemia**

#### **4.1 Ikaros-deficient mice develop T-cell leukemia**

The Ikaros-defficient mouse described above clearly established that Ikaros has an essential role in T-cell development. Subsequent analysis of heterozygous Ikarosdefficient mice revealed that Ikaros has tumor suppressor activity. Between the second and third month of age, these mice display an aberration in thymic differentiation with an accumulation of triple-positive thymocytes that have intermediate expression levels of CD4, CD8, and the TCR complex (Winandy et al. 1995). At the same time, a polyclonal expansion of mature T lymphocytes occurs in the spleen of these mice. Proliferation

Ikaros in T-Cell Leukemia 103

have examined human leukemia samples to determine if an alteration of Ikaros' function is associated with the development of hematopoietic malignancies in humans. Increased expression of dominant-negative Ikaros isoforms has been associated with a variety of hematopoietic malignancies in humans. These include childhood ALL (Kuiper et al. 2007; Mullighan et al. 2007; Mullighan et al. 2008; Dovat and Payne 2010; Marcais et al.), adult B cell ALL (Nakase et al. 2000), myelodysplastic syndrome (Crescenzi et al. 2004), AML (Yagi et al. 2002), and adult and juvenile CML (Nakayama et al. 1999). Deletion of an Ikaros allele was detected in over 80% of BCR-ABL1 ALL and the deletion or mutation of Ikaros has been identified as a poor prognostic marker for childhood ALL (Mullighan et al. 2007; Mullighan et al. 2008; Martinelli et al. 2009; Martinelli et al. 2009; Martinelli et al. 2009; Mullighan et al. 2009). These data established Ikaros as a major tumor suppressor in human leukemia. The most compelling data supporting the tumor suppressor activity of Ikaros in human

In T-cell ALL, the initial studies produced somewhat conflicting data. The first report described expression of dominant-negative Ikaros isoforms (using Western blot and RT-PCR) in all 18 pediatric T-cell ALL patients that were studied (Sun et al. 1999), suggesting a strong correlation of the loss of Ikaros function with the development of T-cell ALL. However, subsequent studies on a total of 14 patients with T-cell ALL (both adult and pediatric) did not detect the presence of dominant-negative Ikaros isoforms (using Western blot and RT-PCR) in T-cell ALL (Nakase et al. 2000; Ruiz et al. 2004), although one study detected an association of the expression of a dominant-negative isoform of the Ikaros-

More comprehensive studies that utilized high-resolution CGH-arrays in a total of 81 patients, detected deletion of one copy of Ikaros in 5% of T-cell ALL patients (Kuiper et al. 2007; Maser et al. 2007; Mullighan et al. 2008). The most recent study of 25 cases of human Tcell ALL, that combined Western blot, CGH-array analysis, and sequencing of Ikaros cDNA following RT-PCR, provided a more complete view of the relation of Ikaros and T-cell ALL. This study detected one patient (4%) in which one Ikaros allele had been deleted, while the Ikaros protein produced by the other intact allele exhibited a loss of nuclear localization with an abnormal localization in the cytoplasmic structure (Marcais et al. 2010). This study provided the first definitive functional evidence of the complete loss of Ikaros function and

In summary, studies in human T-cell ALL have demonstrated that genetic inactivation of the *Ikaros* gene by deletion or mutation does occur in human T-cell ALL in at least 5% of cases. Although Ikaros deletion is not as frequent an event in T-cell ALL when compared to B-cell ALL (30%) or BCR-ABL1 ALL (80%), it is a notable cause of T-cell ALL, and needs to be tested in newly diagnosed patients with this disease. The prognostic significance of Ikaros deletion in T-cell ALL remains to be determined. Studies also suggest that functional inactivation of Ikaros plays a significant role in T-cell ALL, although the mechanisms by which Ikaros function is impaired in T-cell ALL is still unknown. Recent findings discussed below provide insights into signal transduction pathways that potentially affect Ikaros

The mechanisms of Ikaros tumor suppressor activity are not well understood. One major obstacle is the paucity of known Ikaros target genes. The best evidence for the mechanisms

hematopoietic malignancies was established for B-cell ALL.

family member – Helios with T-cell ALL (Nakase et al. 2002).

T-cell ALL in humans.

tumor suppressor function in T-cell ALL.

**5. Mechanisms of Ikaros tumor suppressor activity** 

assays established that thymocytes from heterozygous Ikaros-defficient mice have hyperproliferative potential, and that they can proliferate in the absence of TCR stimulation. It is worth noting that these changes precede the malignant transformation of thymocytes described below (Winandy et al. 1995).

After 3 month of age, all of the Ikaros-defficient heterozygous mice develop T-cell leukemia and lymphoma (Winandy et al. 1995). This is manifested by severe generalized lymphadenopathy and splenomegaly, along with an increased number of malignant lymphoblasts in the peripheral blood. Flow cytometry analysis of malignant lymphoblasts established that they are monoclonal in origin, and that malignancy arises in the thymus. The expression analysis of maliganT-cells revealed that the wild type Ikaros copy was lost in these cells, thus they have a loss of Ikaros heterozygosity (Winandy et al. 1995).

An additional Ikaros-defficient biological model provided evidence for the loss of Ikaros tumor suppressor activity in T-cell leukemia. This *Ikaros*-targeted mouse (IKL/L) had the galactosidase (gal) reporter gene inserted in-frame into exon 2 that is present in all known Ikaros isoforms. These mice produce very low levels of Ikaros proteins (Kirstetter et al. 2002; Dumortier et al. 2003; Dumortier et al. 2006). The IKL/L mice exhibit T lineage defects that are identical to those reported in Ikaros null mice including a lowered threshold to activation stimuli and the invariable development of thymic tumors.

These findings strongly suggested that Ikaros acts as a tumor suppressor for T-cell leukemia. They also provide support for the hypothesis that Ikaros regulates normal thymocyte differentiation, and controls the proliferation of thymocytes and mature T-cells in response to TCR signaling.

Further studies directly addressed the question of whether normal Ikaros function is essential and sufficient to induce tumor suppression of T-cell leukemia. The re-introduction of Ikaros *via* retroviral insertion into T leukemia cells that were derived from Ikarosdefficient mice resulted in their cessation of growth (Kathrein et al. 2005). Expression of Ikaros in T-cell leukemia induced T-cell differentiation that was characterized by increased expression of CD4, CD69, CD5, and TCR. These data suggested that Ikaros tumor suppressor activity involves positive regulation of normal thymocyte differentiation, and that a potential mechanism for malignant transformation of Ikaros-defficient thymocytes involves failure of T-cell differentiation.

The induction of T-cell differentiatiom following re-introduction of Ikaros was accompanied by induction of cell cycle arrest at the G0/G1 stage of the cell cycle (Kathrein et al. 2005). The exact mechanism by which Ikaros induces cell cycle arrest remains unknown, although it has been observed that the induction of Ikaros expression in leukemia cells correlates with the increased expression of the cell cycle-dependent kinase inhibitor p27kip1 (Kathrein et al. 2005). One possibility is that Ikaros affects global chromatin remodeling, since restoration of Ikaros activity in T-cell leukemia correlates with a global increase in histone H3 acetylation (Kathrein et al. 2005).

These complementary studies established Ikaros as a *bona fide* tumor suppressor in T-cell leukemia and demonstrate that the lack of Ikaros is the major causative factor of T-cell malignancy in Ikaros-deficient mice.

#### **4.2 Ikaros deficiency in human T-cell leukemia**

Since the discovery of the *Ikaros* gene and the identification of its function as a master regulator of lymphocyte differentiation and a tumor suppressor in mice, a number of studies

assays established that thymocytes from heterozygous Ikaros-defficient mice have hyperproliferative potential, and that they can proliferate in the absence of TCR stimulation. It is worth noting that these changes precede the malignant transformation of

After 3 month of age, all of the Ikaros-defficient heterozygous mice develop T-cell leukemia and lymphoma (Winandy et al. 1995). This is manifested by severe generalized lymphadenopathy and splenomegaly, along with an increased number of malignant lymphoblasts in the peripheral blood. Flow cytometry analysis of malignant lymphoblasts established that they are monoclonal in origin, and that malignancy arises in the thymus. The expression analysis of maliganT-cells revealed that the wild type Ikaros copy was lost in

An additional Ikaros-defficient biological model provided evidence for the loss of Ikaros tumor suppressor activity in T-cell leukemia. This *Ikaros*-targeted mouse (IKL/L) had the galactosidase (gal) reporter gene inserted in-frame into exon 2 that is present in all known Ikaros isoforms. These mice produce very low levels of Ikaros proteins (Kirstetter et al. 2002; Dumortier et al. 2003; Dumortier et al. 2006). The IKL/L mice exhibit T lineage defects that are identical to those reported in Ikaros null mice including a lowered threshold to

These findings strongly suggested that Ikaros acts as a tumor suppressor for T-cell leukemia. They also provide support for the hypothesis that Ikaros regulates normal thymocyte differentiation, and controls the proliferation of thymocytes and mature T-cells in response

Further studies directly addressed the question of whether normal Ikaros function is essential and sufficient to induce tumor suppression of T-cell leukemia. The re-introduction of Ikaros *via* retroviral insertion into T leukemia cells that were derived from Ikarosdefficient mice resulted in their cessation of growth (Kathrein et al. 2005). Expression of Ikaros in T-cell leukemia induced T-cell differentiation that was characterized by increased expression of CD4, CD69, CD5, and TCR. These data suggested that Ikaros tumor suppressor activity involves positive regulation of normal thymocyte differentiation, and that a potential mechanism for malignant transformation of Ikaros-defficient thymocytes

The induction of T-cell differentiatiom following re-introduction of Ikaros was accompanied by induction of cell cycle arrest at the G0/G1 stage of the cell cycle (Kathrein et al. 2005). The exact mechanism by which Ikaros induces cell cycle arrest remains unknown, although it has been observed that the induction of Ikaros expression in leukemia cells correlates with the increased expression of the cell cycle-dependent kinase inhibitor p27kip1 (Kathrein et al. 2005). One possibility is that Ikaros affects global chromatin remodeling, since restoration of Ikaros activity in T-cell leukemia correlates with a global increase in histone H3 acetylation

These complementary studies established Ikaros as a *bona fide* tumor suppressor in T-cell leukemia and demonstrate that the lack of Ikaros is the major causative factor of T-cell

Since the discovery of the *Ikaros* gene and the identification of its function as a master regulator of lymphocyte differentiation and a tumor suppressor in mice, a number of studies

these cells, thus they have a loss of Ikaros heterozygosity (Winandy et al. 1995).

activation stimuli and the invariable development of thymic tumors.

to TCR signaling.

(Kathrein et al. 2005).

involves failure of T-cell differentiation.

malignancy in Ikaros-deficient mice.

**4.2 Ikaros deficiency in human T-cell leukemia** 

thymocytes described below (Winandy et al. 1995).

have examined human leukemia samples to determine if an alteration of Ikaros' function is associated with the development of hematopoietic malignancies in humans. Increased expression of dominant-negative Ikaros isoforms has been associated with a variety of hematopoietic malignancies in humans. These include childhood ALL (Kuiper et al. 2007; Mullighan et al. 2007; Mullighan et al. 2008; Dovat and Payne 2010; Marcais et al.), adult B cell ALL (Nakase et al. 2000), myelodysplastic syndrome (Crescenzi et al. 2004), AML (Yagi et al. 2002), and adult and juvenile CML (Nakayama et al. 1999). Deletion of an Ikaros allele was detected in over 80% of BCR-ABL1 ALL and the deletion or mutation of Ikaros has been identified as a poor prognostic marker for childhood ALL (Mullighan et al. 2007; Mullighan et al. 2008; Martinelli et al. 2009; Martinelli et al. 2009; Martinelli et al. 2009; Mullighan et al. 2009). These data established Ikaros as a major tumor suppressor in human leukemia. The most compelling data supporting the tumor suppressor activity of Ikaros in human hematopoietic malignancies was established for B-cell ALL.

In T-cell ALL, the initial studies produced somewhat conflicting data. The first report described expression of dominant-negative Ikaros isoforms (using Western blot and RT-PCR) in all 18 pediatric T-cell ALL patients that were studied (Sun et al. 1999), suggesting a strong correlation of the loss of Ikaros function with the development of T-cell ALL. However, subsequent studies on a total of 14 patients with T-cell ALL (both adult and pediatric) did not detect the presence of dominant-negative Ikaros isoforms (using Western blot and RT-PCR) in T-cell ALL (Nakase et al. 2000; Ruiz et al. 2004), although one study detected an association of the expression of a dominant-negative isoform of the Ikarosfamily member – Helios with T-cell ALL (Nakase et al. 2002).

More comprehensive studies that utilized high-resolution CGH-arrays in a total of 81 patients, detected deletion of one copy of Ikaros in 5% of T-cell ALL patients (Kuiper et al. 2007; Maser et al. 2007; Mullighan et al. 2008). The most recent study of 25 cases of human Tcell ALL, that combined Western blot, CGH-array analysis, and sequencing of Ikaros cDNA following RT-PCR, provided a more complete view of the relation of Ikaros and T-cell ALL. This study detected one patient (4%) in which one Ikaros allele had been deleted, while the Ikaros protein produced by the other intact allele exhibited a loss of nuclear localization with an abnormal localization in the cytoplasmic structure (Marcais et al. 2010). This study provided the first definitive functional evidence of the complete loss of Ikaros function and T-cell ALL in humans.

In summary, studies in human T-cell ALL have demonstrated that genetic inactivation of the *Ikaros* gene by deletion or mutation does occur in human T-cell ALL in at least 5% of cases. Although Ikaros deletion is not as frequent an event in T-cell ALL when compared to B-cell ALL (30%) or BCR-ABL1 ALL (80%), it is a notable cause of T-cell ALL, and needs to be tested in newly diagnosed patients with this disease. The prognostic significance of Ikaros deletion in T-cell ALL remains to be determined. Studies also suggest that functional inactivation of Ikaros plays a significant role in T-cell ALL, although the mechanisms by which Ikaros function is impaired in T-cell ALL is still unknown. Recent findings discussed below provide insights into signal transduction pathways that potentially affect Ikaros tumor suppressor function in T-cell ALL.

#### **5. Mechanisms of Ikaros tumor suppressor activity**

The mechanisms of Ikaros tumor suppressor activity are not well understood. One major obstacle is the paucity of known Ikaros target genes. The best evidence for the mechanisms

Ikaros in T-Cell Leukemia 105

Ikaros activity would have an oncogenic effect similar to the overexpression of Bcl2 in chronic lymphocytic leukemia (CLL). This would also lead to the development of leukemia

In summary, multiple mechanisms including the loss of Notch pathway inhibition, blocked T-cell differentiation and impaired cell cycle control are likely to play a role in the T-cell leukemogenesis that occurs with the loss of Ikaros activity. However, the specific mechanisms by which Ikaros exerts its tumor suppressor activity remain largely unknown. Identification of additional genes that are regulated by Ikaros will provide more insight on

Despite the fact that Ikaros plays a critical role in regulating T-cell proliferation and differentiation, the level of Ikaros expression remains high throughout the cell cycle and during lymphocyte differentiation. This suggests that Ikaros function is regulated by posttranslational modifications. The role of phosphorylation in regulating Ikaros function has been studied the most extensively. Studies that identified mitosis-specific hyperphopshorylation of Ikaros at an evolutionarily conserved linker sequence, provided evidence that the cell cycle-specific phosphorylation of Ikaros regulates its DNA-binding ability and nuclear localization during mitosis (Dovat et al. 2002). This provided the first

Subsequent studies demonstrated that Ikaros is a direct substrate of Casein Kinase II (CK2) and that CK2-mediated phosphorylation regulates multiple functions of Ikaros in normal

Experiments performed by Georgopoulos' group identified several amino acids located in the C-terminal region of the Ikaros protein that are directly phoshorylated by CK2. Mutational analysis of phosphoacceptor sites revealed that CK2-mediated phosphorylation

Studies by Dovat's group identified additional phosphorylation sites in the N-terminal region of the Ikaros protein that are phosphorylated by CK2 (Gurel et al. 2008). Experiments with Ikaros phosphomimetic mutants (that mimic constitutive phosphorylation) and phosphoresistant mutants (that mimic constitutive dephosphorylation) revealed that CK2 mediated phosphorylation of two amino acids located in the N-terminal region of Ikaros controls two essential functions of Ikaros: 1) Ikaros' DNA-binding activity and 2) Ikaros' subcellular localization to pericentromeric heterochromatin (Gurel et al. 2008). Increased phosphorylation of Ikaros by CK2 results in severely decreased DNA-binding affinity and the loss of pericentromeric localization, thus **CK2-mediated phosphorylation leads to** 

**6.2 CK2-mediated phosphorylation controls Ikaros function in T-cell differentiation**  The significance of CK2-mediated phosphorylation for normal T-cell development was underscored by the discovery that Ikaraos in CD4+CD8+ thymocytes is phosphorylated at multiple sites by CK2 (Gurel et al. 2008). As described above, in thymocytes, Ikaros binds to the upstream regulatory region of its target gene, *TdT,* leading to repression of *TdT*  transcription. Phosphorylation of Ikaros in CD4+CD8+ thymocytes by CK2 decreases its

regulates Ikaros' ability to control cell cycle progression during the G1/S transition.

that is more highly resistance to chemotherapy.

**6. Regulation of Ikaros' function in T-cell leukemia** 

evidence that phosphorylation can regulate Ikaros function in cells.

**inactivation of Ikaros function** (Gurel et al. 2008).

**6.1 Phosphorylation of Ikaros by CK2 kinase inactivates the Ikaros protein** 

this important process.

and leukemia cells:

of tumor suppression by Ikaros in T-cell ALL comes from studies that identified the role of Ikaros in the Notch pathway.

#### **5.1 Ikaros-mediated repression of the downstream effectors of the Notch pathway (Deltex and Hes1)**

The Notch pathway is essential for normal T-cell differentiation. However, activation of the Notch 1 gene has been found in over 50% of T-ALLs (Weng et al. 2004) and T-ALL leukemia cells often express the Notch target genes Hes-1 and pT (Chiaramonte et al. 2005). The intracelular domain of NotchIC forms a complex with the Notch transcription factor RBP-Jk/CSL and the cofactor Mastermind. This complex activates expression of Notch pathway target genes. Studies by S. Chen's group demonstrated that the Notch pathway is activated in the T-cell leukemia that develops in Ikaros-defficient mice (Dumortier et al. 2006). Additional analysis showed that Ikaros directly downregulates a Notch target gene, Hes-1. Ikaros directly competes with CSL for binding to the upstream regulator element of Hes-1 (Kleinmann et al. 2008). Since CSL acts as a stimulator of transcription, and Ikaros represses transcription of Hes-1, Ikaros counteracts the pro-oncogenic Notch signaling in T-cell ALL. Repression of Hes-1 by Ikaros likely involves chromatin remodeling since Ikaros binding to the upstream regulatory region of Hes-1 leads to a decrease in histone H3 acetylation (Kathrein et al. 2008), and Ikaros-defficient mice have reduced trimethylation of histone H3 at the K27 residue (Kleinmann et al. 2008).

A link between Ikaros deciency and Notch activation in T-cell ALL had been suggested by Beverly and Capobianco (Beverly and Capobianco 2003). They found synergism between Notch activation and the inactivation of Ikaros in T-cell leukemogenesis. Sequence analysis of the consensus binding sequence for CSL and Ikaros revealed remarkable similarities and the authors hypothesized that Ikaros may interfere with CSL binding and Notch signaling (Beverly and Capobianco 2003).

Additional experiments showed that Ikaros represses another target gene of the Notch signaling pathway – Deltex1 (Kathrein et al. 2008). Similarly to the regulation of Hes-1, Ikaros competes with CSL for binding to the upstream regulatory region of Deltex1 and represses expression of Deltex1 by chromatin remodeling. Ikaros binding to the upstream region of Deltex1 also results in decreased histone H3 acetylation (Kathrein et al. 2008).

These data strongly suggest that one of the mechanisms by which Ikaros suppresses leukemogenesis in T-cells involves inhibition of the Notch signal transduction pathway.

#### **5.2 Additional mechanisms of Ikaros tumor suppressor activity in T-cell ALL**

One possible explanation for why the lack of Ikaros function leads to leukemia is the fact that Ikaros regulates expression of several genes that are essential for normal T-cell development (section 3 above). Often, malignant transformation is characterized by a failure (or arrest) of normal differentiation. Thus, the absence of Ikaros activity and its subsequent impact on T-cell differentiation is likely to be an important step toward the development of leukemia.

It has been demonstrated that Ikaros can negatively regulate cell cycle progression at the G1/S transition (Gomez-del Arco et al. 2004), thus the absence of Ikaros would impair the G1/S check point in the regulation of the cell cycle.

Several reports suggested that Ikaros downregulates *Bcl-xL* expression (Yagi et al. 2002; Ezzat et al. 2006; Kano et al. 2008). Thus, Ikaros might regulate apoptosis, and the lack of

of tumor suppression by Ikaros in T-cell ALL comes from studies that identified the role of

The Notch pathway is essential for normal T-cell differentiation. However, activation of the Notch 1 gene has been found in over 50% of T-ALLs (Weng et al. 2004) and T-ALL leukemia cells often express the Notch target genes Hes-1 and pT (Chiaramonte et al. 2005). The intracelular domain of NotchIC forms a complex with the Notch transcription factor RBP-Jk/CSL and the cofactor Mastermind. This complex activates expression of Notch pathway target genes. Studies by S. Chen's group demonstrated that the Notch pathway is activated in the T-cell leukemia that develops in Ikaros-defficient mice (Dumortier et al. 2006). Additional analysis showed that Ikaros directly downregulates a Notch target gene, Hes-1. Ikaros directly competes with CSL for binding to the upstream regulator element of Hes-1 (Kleinmann et al. 2008). Since CSL acts as a stimulator of transcription, and Ikaros represses transcription of Hes-1, Ikaros counteracts the pro-oncogenic Notch signaling in T-cell ALL. Repression of Hes-1 by Ikaros likely involves chromatin remodeling since Ikaros binding to the upstream regulatory region of Hes-1 leads to a decrease in histone H3 acetylation (Kathrein et al. 2008), and Ikaros-defficient mice have reduced trimethylation of histone H3

A link between Ikaros deciency and Notch activation in T-cell ALL had been suggested by Beverly and Capobianco (Beverly and Capobianco 2003). They found synergism between Notch activation and the inactivation of Ikaros in T-cell leukemogenesis. Sequence analysis of the consensus binding sequence for CSL and Ikaros revealed remarkable similarities and the authors hypothesized that Ikaros may interfere with CSL binding and Notch signaling

Additional experiments showed that Ikaros represses another target gene of the Notch signaling pathway – Deltex1 (Kathrein et al. 2008). Similarly to the regulation of Hes-1, Ikaros competes with CSL for binding to the upstream regulatory region of Deltex1 and represses expression of Deltex1 by chromatin remodeling. Ikaros binding to the upstream region of Deltex1 also results in decreased histone H3 acetylation (Kathrein et al. 2008). These data strongly suggest that one of the mechanisms by which Ikaros suppresses leukemogenesis in T-cells involves inhibition of the Notch signal transduction pathway.

One possible explanation for why the lack of Ikaros function leads to leukemia is the fact that Ikaros regulates expression of several genes that are essential for normal T-cell development (section 3 above). Often, malignant transformation is characterized by a failure (or arrest) of normal differentiation. Thus, the absence of Ikaros activity and its subsequent impact on T-cell differentiation is likely to be an important step toward the

It has been demonstrated that Ikaros can negatively regulate cell cycle progression at the G1/S transition (Gomez-del Arco et al. 2004), thus the absence of Ikaros would impair the

Several reports suggested that Ikaros downregulates *Bcl-xL* expression (Yagi et al. 2002; Ezzat et al. 2006; Kano et al. 2008). Thus, Ikaros might regulate apoptosis, and the lack of

**5.2 Additional mechanisms of Ikaros tumor suppressor activity in T-cell ALL** 

**5.1 Ikaros-mediated repression of the downstream effectors of the Notch pathway** 

Ikaros in the Notch pathway.

at the K27 residue (Kleinmann et al. 2008).

(Beverly and Capobianco 2003).

development of leukemia.

G1/S check point in the regulation of the cell cycle.

**(Deltex and Hes1)**

Ikaros activity would have an oncogenic effect similar to the overexpression of Bcl2 in chronic lymphocytic leukemia (CLL). This would also lead to the development of leukemia that is more highly resistance to chemotherapy.

In summary, multiple mechanisms including the loss of Notch pathway inhibition, blocked T-cell differentiation and impaired cell cycle control are likely to play a role in the T-cell leukemogenesis that occurs with the loss of Ikaros activity. However, the specific mechanisms by which Ikaros exerts its tumor suppressor activity remain largely unknown. Identification of additional genes that are regulated by Ikaros will provide more insight on this important process.

### **6. Regulation of Ikaros' function in T-cell leukemia**

#### **6.1 Phosphorylation of Ikaros by CK2 kinase inactivates the Ikaros protein**

Despite the fact that Ikaros plays a critical role in regulating T-cell proliferation and differentiation, the level of Ikaros expression remains high throughout the cell cycle and during lymphocyte differentiation. This suggests that Ikaros function is regulated by posttranslational modifications. The role of phosphorylation in regulating Ikaros function has been studied the most extensively. Studies that identified mitosis-specific hyperphopshorylation of Ikaros at an evolutionarily conserved linker sequence, provided evidence that the cell cycle-specific phosphorylation of Ikaros regulates its DNA-binding ability and nuclear localization during mitosis (Dovat et al. 2002). This provided the first evidence that phosphorylation can regulate Ikaros function in cells.

Subsequent studies demonstrated that Ikaros is a direct substrate of Casein Kinase II (CK2) and that CK2-mediated phosphorylation regulates multiple functions of Ikaros in normal and leukemia cells:

Experiments performed by Georgopoulos' group identified several amino acids located in the C-terminal region of the Ikaros protein that are directly phoshorylated by CK2. Mutational analysis of phosphoacceptor sites revealed that CK2-mediated phosphorylation regulates Ikaros' ability to control cell cycle progression during the G1/S transition.

Studies by Dovat's group identified additional phosphorylation sites in the N-terminal region of the Ikaros protein that are phosphorylated by CK2 (Gurel et al. 2008). Experiments with Ikaros phosphomimetic mutants (that mimic constitutive phosphorylation) and phosphoresistant mutants (that mimic constitutive dephosphorylation) revealed that CK2 mediated phosphorylation of two amino acids located in the N-terminal region of Ikaros controls two essential functions of Ikaros: 1) Ikaros' DNA-binding activity and 2) Ikaros' subcellular localization to pericentromeric heterochromatin (Gurel et al. 2008). Increased phosphorylation of Ikaros by CK2 results in severely decreased DNA-binding affinity and the loss of pericentromeric localization, thus **CK2-mediated phosphorylation leads to inactivation of Ikaros function** (Gurel et al. 2008).

#### **6.2 CK2-mediated phosphorylation controls Ikaros function in T-cell differentiation**

The significance of CK2-mediated phosphorylation for normal T-cell development was underscored by the discovery that Ikaraos in CD4+CD8+ thymocytes is phosphorylated at multiple sites by CK2 (Gurel et al. 2008). As described above, in thymocytes, Ikaros binds to the upstream regulatory region of its target gene, *TdT,* leading to repression of *TdT*  transcription. Phosphorylation of Ikaros in CD4+CD8+ thymocytes by CK2 decreases its

Ikaros in T-Cell Leukemia 107

role in T-cell differentiation, ability to regulate cell cycle progression, DNA-binding affinity, chromatin remodeling capability (by controlling its subcellular localization to pericentromeric heterochromatin), regulation of gene expression, and protein stability. This suggests that the pro-oncogenic activity of CK2 involves inactivation of the *Ikaros* gene, while the tumor suppressor activity of PP1 is mediated by preserving the tumor suppressor function of Ikaros. These data also strongly suggest that in leukemia cells that have increased activity of CK2, but no deletion/mutation in the *Ikaros* gene, the Ikaros protein that is present is unlikely to be a functionally active Ikaros protein with tumor suppressor activity. Instead, recent discoveries suggest that the functional inactivation of Ikaros by CK2-mediated hyperphopshorylation might be one important mechanism leading to T-cell leukemia. This provides new insight into the role of Ikaros, CK2, and PP1 in T-cell leukemia, and identifies CK2 kinase as a potential target for novel chemotherapy for T-cell ALL.

Fig. 2. CK2 kinase-mediated hyperphosphorylation results in functional inactivation of Ikaros and T-cell ALL. Hyperphosphorylation of Ikaros by CK2 kinase leads to the loss of Ikaros' ability to bind DNA, control cell cycle progression, regulate chromatin remodelling, regulate gene expression and to increased degradation of the Ikaros protein. The loss of

Ikaros tumor suppressor function leads to the development of T-cell ALL.

DNA-binding affinity toward the upstream regulatory element of *TdT*, which results in occupancy of this region by the transcription factor Elf-1 and expression of *TdT*. During induction of thymocyte differentiation, Ikaros undergoes dephosphorylation at amino acids #13 and #294 (Gurel et al. 2008). Differentiation-specific dephosphorylation of Ikaros results in its increased DNA-binding affinity toward the upstream regulatory element of *TdT*, displacement of Elf-1, and repression of *TdT* transcription. These data demonstrate that the regulation of Ikaros phosphorylation by CK2 plays an important role in T-cell differentiation and suggest that increased activity of CK2 kinase in thymocytes would lead to impaired Tcell development due to interference with normal Ikaros function.

#### **6.3 CK2-mediated phosphorylation leads to ubiquitination and degradation of the Ikaros protein**

Further functional analysis of Ikaros phosphorylation revealed that CK2-mediated phosphorylation occurs at PEST sequences in the Ikaros protein. PEST sequences are comprised of a region that is rich in Proline (P), Gluamate (E), Serine (S) and Threonine (T). It has been demonstrated that phosophorylation of the serines or threonines located within the PEST sequence typically results in increased degradation of the respective protein. Ikaros contains two PEST sequences that are phosphorylated *in vivo* by CK2 (Popescu et al. 2009). Phosphorylation at PEST sequences leads to decreased half-life and increased degradation of Ikaros, resulting in a low level of Ikaros protein in cells. Hyperphoshorylated Ikaros undergoes polyubiquitination, which leads to its degradation *via* the ubiquitin/ proteasome pathway (Popescu et al. 2009).

#### **6.4 Dephosphorylation of Ikaros by PP1 opposes CK2-mediated phosphorylation**

The discovery that Ikaros is a direct substrate for Protein Phosphatase 1 (PP1) (Popescu et al. 2009), a well-known tumor suppressor protein, provided additional evidence that phosphorylation plays an extremely important role in the regulation of Ikaros activity in normal hematopoiesis and in leukemia. Ikaros interacts with PP1 directly by binding *via* a PP1 consensus recognition motif that is located near the C-terminal zinc fingers. Dephosphorylation of Ikaros by PP1 is essential for preservation of Ikaros DNA-binding activity and subcelular localization to pericentromeric heterochromatin (Popescu et al. 2009). Mutated Ikaros protein that is unable to interact with PP1 undergoes accelerated degradation *via* the ubiquitin pathway. This results in a 5- to 10-fold decrease in the level of Ikaros protein in cells, when compared to wild type Ikaros (Popescu et al. 2009). Thus, PP1 mediated dephosphorylation of Ikaros counteracts the effect of CK2-mediated phosphorylation of Ikaros. This provides evidence that two major signal transduction pathways – one involving CK2 and the other PP1 – converge on the Ikaros protein (Fig. 2).

These pathways exert opposite effects on Ikaros function and thus regulate T-cell differentiation and proliferation.

Both CK2 and PP1 proteins are known to play a critical role in malignant transformation. Increased expression of CK2 during T-cell differentiation results in the development of Tcell leukemia in mice (Seldin and Leder 1995). Furthermore, CK2 activity is elevated in many other types of human malignancies (Phan-Dinh-Tuy et al. 1985; Pinna 1997; Roig et al. 1999; Kim et al. 2007). The activity of CK2 and PP1 is closely tied to the ability of cells to proliferate, and thus the balance of their function is essential to prevent malignant transformation. Both CK2 and PP1 exerts strong effects on multiple functions of Ikaros – its

DNA-binding affinity toward the upstream regulatory element of *TdT*, which results in occupancy of this region by the transcription factor Elf-1 and expression of *TdT*. During induction of thymocyte differentiation, Ikaros undergoes dephosphorylation at amino acids #13 and #294 (Gurel et al. 2008). Differentiation-specific dephosphorylation of Ikaros results in its increased DNA-binding affinity toward the upstream regulatory element of *TdT*, displacement of Elf-1, and repression of *TdT* transcription. These data demonstrate that the regulation of Ikaros phosphorylation by CK2 plays an important role in T-cell differentiation and suggest that increased activity of CK2 kinase in thymocytes would lead to impaired T-

**6.3 CK2-mediated phosphorylation leads to ubiquitination and degradation of the** 

**6.4 Dephosphorylation of Ikaros by PP1 opposes CK2-mediated phosphorylation**  The discovery that Ikaros is a direct substrate for Protein Phosphatase 1 (PP1) (Popescu et al. 2009), a well-known tumor suppressor protein, provided additional evidence that phosphorylation plays an extremely important role in the regulation of Ikaros activity in normal hematopoiesis and in leukemia. Ikaros interacts with PP1 directly by binding *via* a PP1 consensus recognition motif that is located near the C-terminal zinc fingers. Dephosphorylation of Ikaros by PP1 is essential for preservation of Ikaros DNA-binding activity and subcelular localization to pericentromeric heterochromatin (Popescu et al. 2009). Mutated Ikaros protein that is unable to interact with PP1 undergoes accelerated degradation *via* the ubiquitin pathway. This results in a 5- to 10-fold decrease in the level of Ikaros protein in cells, when compared to wild type Ikaros (Popescu et al. 2009). Thus, PP1 mediated dephosphorylation of Ikaros counteracts the effect of CK2-mediated phosphorylation of Ikaros. This provides evidence that two major signal transduction pathways – one involving CK2 and the other PP1 – converge on the Ikaros protein (Fig. 2). These pathways exert opposite effects on Ikaros function and thus regulate T-cell

Both CK2 and PP1 proteins are known to play a critical role in malignant transformation. Increased expression of CK2 during T-cell differentiation results in the development of Tcell leukemia in mice (Seldin and Leder 1995). Furthermore, CK2 activity is elevated in many other types of human malignancies (Phan-Dinh-Tuy et al. 1985; Pinna 1997; Roig et al. 1999; Kim et al. 2007). The activity of CK2 and PP1 is closely tied to the ability of cells to proliferate, and thus the balance of their function is essential to prevent malignant transformation. Both CK2 and PP1 exerts strong effects on multiple functions of Ikaros – its

Further functional analysis of Ikaros phosphorylation revealed that CK2-mediated phosphorylation occurs at PEST sequences in the Ikaros protein. PEST sequences are comprised of a region that is rich in Proline (P), Gluamate (E), Serine (S) and Threonine (T). It has been demonstrated that phosophorylation of the serines or threonines located within the PEST sequence typically results in increased degradation of the respective protein. Ikaros contains two PEST sequences that are phosphorylated *in vivo* by CK2 (Popescu et al. 2009). Phosphorylation at PEST sequences leads to decreased half-life and increased degradation of Ikaros, resulting in a low level of Ikaros protein in cells. Hyperphoshorylated Ikaros undergoes polyubiquitination, which leads to its degradation *via* the ubiquitin/

cell development due to interference with normal Ikaros function.

proteasome pathway (Popescu et al. 2009).

differentiation and proliferation.

**Ikaros protein** 

role in T-cell differentiation, ability to regulate cell cycle progression, DNA-binding affinity, chromatin remodeling capability (by controlling its subcellular localization to pericentromeric heterochromatin), regulation of gene expression, and protein stability. This suggests that the pro-oncogenic activity of CK2 involves inactivation of the *Ikaros* gene, while the tumor suppressor activity of PP1 is mediated by preserving the tumor suppressor function of Ikaros. These data also strongly suggest that in leukemia cells that have increased activity of CK2, but no deletion/mutation in the *Ikaros* gene, the Ikaros protein that is present is unlikely to be a functionally active Ikaros protein with tumor suppressor activity. Instead, recent discoveries suggest that the functional inactivation of Ikaros by CK2-mediated hyperphopshorylation might be one important mechanism leading to T-cell leukemia. This provides new insight into the role of Ikaros, CK2, and PP1 in T-cell leukemia, and identifies CK2 kinase as a potential target for novel chemotherapy for T-cell ALL.

Fig. 2. CK2 kinase-mediated hyperphosphorylation results in functional inactivation of Ikaros and T-cell ALL. Hyperphosphorylation of Ikaros by CK2 kinase leads to the loss of Ikaros' ability to bind DNA, control cell cycle progression, regulate chromatin remodelling, regulate gene expression and to increased degradation of the Ikaros protein. The loss of Ikaros tumor suppressor function leads to the development of T-cell ALL.

Ikaros in T-Cell Leukemia 109

This work was supported in part by an R01 HL095120 grant, a St. Baldrick's Foundation Career Development Award, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (SD). This work was supported by the Department of Pathology and Human Anatomy and the Center for Health Dispartities and Molecular Medicine, Loma Linda University School of Medicine (to KJP) and by a Grant for Research and School Partnerships from Loma Linda University

Avitahl, N., S. Winandy, C. Friedrich, B. Jones, Y. Ge and K. Georgopoulos (1999). Ikaros

Beverly, L. J. and A. J. Capobianco (2003). Perturbation of Ikaros isoform selection by MLV

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Chiaramonte, R., A. Basile, E. Tassi, E. Calzavara, V. Cecchinato, V. Rossi, A. Biondi and P.

Crescenzi, B., R. La Starza, S. Romoli, D. Beacci, C. Matteucci, G. Barba, A. Aventin, P.

Dovat, S., T. Ronni, D. Russell, R. Ferrini, B. S. Cobb and S. T. Smale (2002). A common

Dumortier, A., R. Jeannet, P. Kirstetter, E. Kleinmann, M. Sellars, N. R. dos Santos, C.

Dumortier, A., P. Kirstetter, P. Kastner and S. Chan (2003). Ikaros regulates neutrophil

Ernst, P., K. Hahm and S. T. Smale (1993). Both LyF-1 and an Ets protein interact with a

Ernst, P., K. Hahm, L. Trinh, J. N. Davis, M. F. Roussel, C. W. Turck and S. T. Smale (1996).

deletions in 5q- associated malignancies, Haematologica 89(3):281-285. Dovat, S. and K. J. Payne (2010). Tumor suppression in T-cell leukemia--the role of Ikaros,

sets thresholds for T-cell activation and regulates chromosome propagation,

integration is a cooperative event in Notch(IC)-induced T-cell leukemogenesis,

Association of transcriptionally silent genes with Ikaros complexes at centromeric

Comi (2005). A wide role for NOTCH1 signaling in acute leukemia, Cancer Lett

Marynen, S. Ciolli, C. Nozzoli, M. F. Martelli and C. Mecucci (2004). Submicroscopic

mechanism for mitotic inactivation of C2H2 zinc finger DNA-binding domains,

Thibault, J. Barths, J. Ghysdael, J. A. Punt, P. Kastner and S. Chan (2006). Notch activation is an early and critical event during T-Cell leukemogenesis in Ikaros-

critical promoter element in the murine terminal transferase gene, Mol Cell Biol

A potential role for Elf-1 in terminal transferase gene regulation, Mol Cell Biol

**8. Acknowledgements** 

(to KJP).

**9. References** 

Immunity 10(3): 333-343.

Cancer Cell 3(6): 551-564.

Leuk Res 34(4): 416-417.

Genes Dev 16(23): 2985-2990.

deficient mice, Mol Cell Biol 26(1): 209-220.

differentiation, Blood 101(6): 2219-2226.

219(1): 113-120.

13(5): 2982-2992.

16(11): 6121-6131.

heterochromatin, Cell 91(6): 845-854.

#### **7. Conclusion: The role of Ikaros in T-cell leukemia – overall hypothesis**

Numerous clinical and experimental studies established the absence of Ikaros activity as a causative and/or contributory factor to the development of T-cell ALL. While genetic inactivation of Ikaros is evident in about 5% of T-cell ALL, recent evidence suggests that the functional inactivation of Ikaros by CK2 kinase-mediated phosphorylation is an important factor for the development of T-cell ALL. **With regard to Ikaros activity, we propose that there are three types of T-cell leukemia (Fig. 3):** 


The prognostic and therapeutic significance of these three types of T-cell leukemia will be the subject of intense investigation in the future.

Fig. 3. Three types of T-cell leukemia with regards to Ikaros activity. Genetic and functional inactivation of Ikaros, in addition to changes in previously identified pathways, lead to Tcell leukemogenesis.

Numerous clinical and experimental studies established the absence of Ikaros activity as a causative and/or contributory factor to the development of T-cell ALL. While genetic inactivation of Ikaros is evident in about 5% of T-cell ALL, recent evidence suggests that the functional inactivation of Ikaros by CK2 kinase-mediated phosphorylation is an important factor for the development of T-cell ALL. **With regard to Ikaros activity, we propose that** 

4. T-cell leukemia with the presence of genetic inactivation (deletion or mutation) of at

5. T-cell leukemia with functional inactivation of the Ikaros protein in leukemia cells due

6. T-cell leukemia with intact Ikaros function, but with defects in other genes and proteins

The prognostic and therapeutic significance of these three types of T-cell leukemia will be

Fig. 3. Three types of T-cell leukemia with regards to Ikaros activity. Genetic and functional inactivation of Ikaros, in addition to changes in previously identified pathways, lead to T-

least one Ikaros allele – responsible for ~5% of T-cell leukemia.

that regulate T-cell proliferation and differentiation.

**7. Conclusion: The role of Ikaros in T-cell leukemia – overall hypothesis**

**there are three types of T-cell leukemia (Fig. 3):** 

the subject of intense investigation in the future.

to overexpression of CK2 kinase.

cell leukemogenesis.

#### **8. Acknowledgements**

This work was supported in part by an R01 HL095120 grant, a St. Baldrick's Foundation Career Development Award, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (SD). This work was supported by the Department of Pathology and Human Anatomy and the Center for Health Dispartities and Molecular Medicine, Loma Linda University School of Medicine (to KJP) and by a Grant for Research and School Partnerships from Loma Linda University (to KJP).

#### **9. References**


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**7** 

*Austria* 

**p16INK4A – Connecting Cell Cycle Control to Cell** 

To expand without limitation cancerous cells have to acquire the ability to proliferate without external or internal restrictions. Whereas almost all normal cells within a body will not proliferate without external mitogenic stimuli cancer cells gain defects in growth factor signaling control that mimic external stimulation and thus allow them to divide. However, this unrestricted proliferation also activates an internal barrier to transformation, i.e. it induces the expression of so called cell cycle brakes that stop unrestricted growth at the very basal level of the cell cycle. These cell cycle inhibitors prevent increased cycling by controlling the activity of cyclin/CDK holoenzyms which are the central promoters of cell cycle progression. These cell cycle inhibitors or "brakes" are divided into two families, the INK4 inhibitors and the Cip/Kip inhibitor family. In this chapter we will mainly focus on

the INK4 inhibitor protein p16INK4A which is encoded by the INK4A gene locus.

than "just inhibiting cyclin-dependent kinases".

Inactivation of the INK4A gene locus occurs frequently in primary tumor cells of T-cell acute lymphoblastic leukemia (T-ALL), suggesting a critical role of this locus in disease development. Its deletion predicts relapse in childhood acute lymphoblastic leukemia (ALL). Interestingly, this gene locus also represents a main barrier to the generation of induced progenitor stem (iPS) cells and evidence has been provided that downregulation of p16INK4A is associated with enhanced self-renewal and proliferative capacity of hematopoietic stem cells. This suggests that the inactivation of this tumor suppressor in immature pre-cancerous cells might allow them to overcome replicative senescence. Importantly, p16INK4A exerts its effects not only on cell cycle control but changes cell death sensitivity of T-ALL cells by affecting glucocorticoid sensitivity, death receptor-mediatedand intrinsic programmed cell death pathways that are controlled at the level of BCL2 proteins. These effects of p16INK4A on the cell death machinery in ALL cells support the notion that loss of the INK4A gene locus has a two-sided effect during the development of ALL, on one hand it allows unrestricted proliferation of ALL progenitor cells and on the other hand this deletion rescues leukemic cells from cell death. Thus, the effects of p16INK4A in hematopoietic cells and during leukemia development seem to be much more

**1. Introduction** 

**Death Regulation in Human Leukemia** 

Petra Obexer1,3, Judith Hagenbuchner1,3

*1Department of Pediatrics IV and 2Department of Pediatrics II and 3Tyrolean Cancer Research Institute Medical University Innsbruck* 

Markus Holzner3 and Michael J. Ausserlechner2,3

Yagi, T., S. Hibi, M. Takanashi, G. Kano, Y. Tabata, T. Imamura, T. Inaba, A. Morimoto, S. Todo and S. Imashuku (2002). High frequency of Ikaros isoform 6 expression in acute myelomonocytic and monocytic leukemias: implications for upregulation of the antiapoptotic protein Bcl-XL in leukemogenesis, Blood 99(4): 1350-1355.

## **p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia**

Petra Obexer1,3, Judith Hagenbuchner1,3

Markus Holzner3 and Michael J. Ausserlechner2,3 *1Department of Pediatrics IV and 2Department of Pediatrics II and 3Tyrolean Cancer Research Institute Medical University Innsbruck Austria* 

#### **1. Introduction**

114 T-Cell Leukemia

Yagi, T., S. Hibi, M. Takanashi, G. Kano, Y. Tabata, T. Imamura, T. Inaba, A. Morimoto, S.

1350-1355.

Todo and S. Imashuku (2002). High frequency of Ikaros isoform 6 expression in acute myelomonocytic and monocytic leukemias: implications for upregulation of the antiapoptotic protein Bcl-XL in leukemogenesis, Blood 99(4):

> To expand without limitation cancerous cells have to acquire the ability to proliferate without external or internal restrictions. Whereas almost all normal cells within a body will not proliferate without external mitogenic stimuli cancer cells gain defects in growth factor signaling control that mimic external stimulation and thus allow them to divide. However, this unrestricted proliferation also activates an internal barrier to transformation, i.e. it induces the expression of so called cell cycle brakes that stop unrestricted growth at the very basal level of the cell cycle. These cell cycle inhibitors prevent increased cycling by controlling the activity of cyclin/CDK holoenzyms which are the central promoters of cell cycle progression. These cell cycle inhibitors or "brakes" are divided into two families, the INK4 inhibitors and the Cip/Kip inhibitor family. In this chapter we will mainly focus on the INK4 inhibitor protein p16INK4A which is encoded by the INK4A gene locus.

> Inactivation of the INK4A gene locus occurs frequently in primary tumor cells of T-cell acute lymphoblastic leukemia (T-ALL), suggesting a critical role of this locus in disease development. Its deletion predicts relapse in childhood acute lymphoblastic leukemia (ALL). Interestingly, this gene locus also represents a main barrier to the generation of induced progenitor stem (iPS) cells and evidence has been provided that downregulation of p16INK4A is associated with enhanced self-renewal and proliferative capacity of hematopoietic stem cells. This suggests that the inactivation of this tumor suppressor in immature pre-cancerous cells might allow them to overcome replicative senescence. Importantly, p16INK4A exerts its effects not only on cell cycle control but changes cell death sensitivity of T-ALL cells by affecting glucocorticoid sensitivity, death receptor-mediatedand intrinsic programmed cell death pathways that are controlled at the level of BCL2 proteins. These effects of p16INK4A on the cell death machinery in ALL cells support the notion that loss of the INK4A gene locus has a two-sided effect during the development of ALL, on one hand it allows unrestricted proliferation of ALL progenitor cells and on the other hand this deletion rescues leukemic cells from cell death. Thus, the effects of p16INK4A in hematopoietic cells and during leukemia development seem to be much more than "just inhibiting cyclin-dependent kinases".

p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 117

lack functional pRB, either by mutation or due to viral proteins that target and inactivate pRB, usually show high levels of p16INK4A (Aagaard et al, 1995). In such cells CDK4 and CDK6 do not interact with D-type cyclins but form stable, long half-life, binary complexes with p16INK4A (Parry et al, 1995) demonstrating that the cell cycle inhibitory effect of

Fig. 1. Dynamic accumulation and assembly of cyclin/CDK complexes in different phases of the cell cycle. Schematic presentation of the cell cycle with emphasis on the G1/S checkpoint

However nature has found an elegant way of redundancy to compensate for the loss of p16INK4A function in pRB-deficient tumors by an alternative protein. Adjacent to the INK4A gene locus on chromosome 9p21 an additional promoter region exists that produces a transcript which includes exon 2 and exon 3 of p16INK4A but has an alternate exon 1 (exon1β) (Figure 2). Since the exon 2 is translated in an alternative reading frame (ARF) the resulting protein is completely unrelated to the gene product of INK4A although it shares parts of the mRNA sequence (Quelle et al, 1995). p16INK4A and ARF have no similarities in amino acid composition and are two completely different proteins with distinct functions – but both act as efficient tumor suppressors (Figure 2). This is demonstrated by the fact that mice that are deficient for either ARF or p16INK4A have increased susceptibility to spontaneous or carcinogen-induced cancers (Sharpless et al, 2001; Kamijo et al, 1997). p16INK4A-deficient mice show a much less spontaneous tumor rate than ARF null mice. p16INK4A ablation leads to spontaneous sarcomas and lymphomas within 17 months (Sharpless et al, 2001), whereas the onset of spontaneous sarcomas, carcinomas and lymphomas in ARF-null mice is already observed at the age of 9 months (Kamijo et al, 1997). However, it is difficult to compare results from ARF null mice with the situation in human cells since human (p14ARF) and mouse p19ARF share only about 50% sequence homology. In addition there are apparent regulatory and functional differences between human and mouse ARF. Whereas in senescent mouse fibroblasts p19ARF accumulates and is critical for senescence-induced growth arrest, the human p14ARF seems not involved and is dispensable for this process (Sharpless et al, 2004). In human cells, the senescence process is

p16INK4A depends on the functionality of the tumor suppressor pRB.

regulation by CDK inhibitors, detailed explanation in the text.

#### **2. Stop or go: A general overview on cell cycle entry**

Proliferating eukaryotic cells pass through a complex cell division cycle that is divided into four phases: the gap-phase before DNA replication (G1-phase), the phase of DNA synthesis (S-phase), the gap after DNA synthesis (G2-phase) and mitosis (M-phase). Whereas the length of DNA-synthesis, G2 and M-phase are relatively constant within different cell types, the greatest variation is observed in the duration of G1. Highly proliferative cells pass through this phase within a few hours; differentiated cells, on the other hand, may stay in this cell cycle stage for months, years, or even life time. This situation of final differentiation is called G0. The G0/G1 cell cycle state is usually considered as the time window, in which a cell decides whether to proliferate or to arrest.

Progression through the cell cycle is governed by a family of serine-threonine kinases called cyclin-dependent kinases (CDKs) that associate with different cyclins in distinct phases of the cell cycle (Murray and Hunt 1993; Sherr and Roberts 2004). Whereas the cellular concentration of CDK proteins does not vary significantly during cell cycle, the levels of cyclins undergo dramatic changes during the different phases. They reach characteristic peak levels in specific cell cycle phases and are degraded in other phases.

When quiescent cells re-enter the cell cycle, D-type and E-type cyclins are synthesized sequentially during the G1 interval; both types are rate-limiting for S-phase entry (Quelle et al, 1993). Cyclin Ds assemble with CDK4 (Matsushime et al, 1992) and CDK6 (Meyerson and Harlow 1994) and these complexes are activated by phosphorylation via the so called cyclin activating kinase (Murray and Hunt 1993). Critical substrates of G1 cyclin/CDK complexes are the retinoblastoma gene product pRB and its family members p107 and p130, which are transcriptional repressors that are bound to transcription factors essential for S-phase entry (Cobrinik 2005; Grana et al, 1998). Upon phosphorylation on distinct sites by both, cyclin D/CDK4/6 and cyclin E/CDK2 complexes pRB and its family members lose their function as transcriptional repressors thereby activating the transcription of genes essential for Sphase progression. The activity of CDK4, CDK6 and CDK2 is regulated by mitogenic hormones and by binding of CDK inhibitors (CDKI) of the INK4A family, like p16INK4A, and of the Cip/Kip family, i.e. p21Cip1, p27Kip1 and p57Kip2 (Sherr and Roberts 1999; Ekholm and Reed 2000) (see Figure 1).

#### **3. The strange case of INK4A: One gene locus that codes for two unrelated tumor suppressors**

The INK4A gene locus on chromosome 9p21 codes for the two functionally fully unrelated tumor suppressor genes p16INK4A and ARF (known as p14ARF in human and p19ARF in mouse). As outlined above, p16INK4A/CDKN2A was originally identified as a cyclindependent kinase inhibitor that, like its family members p15INK4B, p18INK4C and p19INK4D, binds to the kinase subunits CDK4 and CDK6 of D-type cyclins. Cyclin D1, D2 and D3 govern the decision of cell cycle entry from quiescent G0 state. In contrast to other cyclins the activity of cyclin Ds/CDK4 and CDK6 holoenzymes is controlled by various survival signaling pathways e.g. by Ras/Raf signaling, by the PI3K/PKB pathway or by the Wnt signaling pathway which are frequently perturbed in cancer cells thereby leading to aberrant cell cycle entry and cell proliferation. p16INK4A and other inhibitors of the INK4 family (INK4 means "inhibitor of kinase 4") thereby represent a main barrier to increased proliferation of cells with defects in growth factor signaling control. Human tumor cells that

Proliferating eukaryotic cells pass through a complex cell division cycle that is divided into four phases: the gap-phase before DNA replication (G1-phase), the phase of DNA synthesis (S-phase), the gap after DNA synthesis (G2-phase) and mitosis (M-phase). Whereas the length of DNA-synthesis, G2 and M-phase are relatively constant within different cell types, the greatest variation is observed in the duration of G1. Highly proliferative cells pass through this phase within a few hours; differentiated cells, on the other hand, may stay in this cell cycle stage for months, years, or even life time. This situation of final differentiation is called G0. The G0/G1 cell cycle state is usually considered as the time window, in which a

Progression through the cell cycle is governed by a family of serine-threonine kinases called cyclin-dependent kinases (CDKs) that associate with different cyclins in distinct phases of the cell cycle (Murray and Hunt 1993; Sherr and Roberts 2004). Whereas the cellular concentration of CDK proteins does not vary significantly during cell cycle, the levels of cyclins undergo dramatic changes during the different phases. They reach characteristic

When quiescent cells re-enter the cell cycle, D-type and E-type cyclins are synthesized sequentially during the G1 interval; both types are rate-limiting for S-phase entry (Quelle et al, 1993). Cyclin Ds assemble with CDK4 (Matsushime et al, 1992) and CDK6 (Meyerson and Harlow 1994) and these complexes are activated by phosphorylation via the so called cyclin activating kinase (Murray and Hunt 1993). Critical substrates of G1 cyclin/CDK complexes are the retinoblastoma gene product pRB and its family members p107 and p130, which are transcriptional repressors that are bound to transcription factors essential for S-phase entry (Cobrinik 2005; Grana et al, 1998). Upon phosphorylation on distinct sites by both, cyclin D/CDK4/6 and cyclin E/CDK2 complexes pRB and its family members lose their function as transcriptional repressors thereby activating the transcription of genes essential for Sphase progression. The activity of CDK4, CDK6 and CDK2 is regulated by mitogenic hormones and by binding of CDK inhibitors (CDKI) of the INK4A family, like p16INK4A, and of the Cip/Kip family, i.e. p21Cip1, p27Kip1 and p57Kip2 (Sherr and Roberts 1999;

**3. The strange case of INK4A: One gene locus that codes for two unrelated** 

The INK4A gene locus on chromosome 9p21 codes for the two functionally fully unrelated tumor suppressor genes p16INK4A and ARF (known as p14ARF in human and p19ARF in mouse). As outlined above, p16INK4A/CDKN2A was originally identified as a cyclindependent kinase inhibitor that, like its family members p15INK4B, p18INK4C and p19INK4D, binds to the kinase subunits CDK4 and CDK6 of D-type cyclins. Cyclin D1, D2 and D3 govern the decision of cell cycle entry from quiescent G0 state. In contrast to other cyclins the activity of cyclin Ds/CDK4 and CDK6 holoenzymes is controlled by various survival signaling pathways e.g. by Ras/Raf signaling, by the PI3K/PKB pathway or by the Wnt signaling pathway which are frequently perturbed in cancer cells thereby leading to aberrant cell cycle entry and cell proliferation. p16INK4A and other inhibitors of the INK4 family (INK4 means "inhibitor of kinase 4") thereby represent a main barrier to increased proliferation of cells with defects in growth factor signaling control. Human tumor cells that

peak levels in specific cell cycle phases and are degraded in other phases.

**2. Stop or go: A general overview on cell cycle entry** 

cell decides whether to proliferate or to arrest.

Ekholm and Reed 2000) (see Figure 1).

**tumor suppressors** 

lack functional pRB, either by mutation or due to viral proteins that target and inactivate pRB, usually show high levels of p16INK4A (Aagaard et al, 1995). In such cells CDK4 and CDK6 do not interact with D-type cyclins but form stable, long half-life, binary complexes with p16INK4A (Parry et al, 1995) demonstrating that the cell cycle inhibitory effect of p16INK4A depends on the functionality of the tumor suppressor pRB.

Fig. 1. Dynamic accumulation and assembly of cyclin/CDK complexes in different phases of the cell cycle. Schematic presentation of the cell cycle with emphasis on the G1/S checkpoint regulation by CDK inhibitors, detailed explanation in the text.

However nature has found an elegant way of redundancy to compensate for the loss of p16INK4A function in pRB-deficient tumors by an alternative protein. Adjacent to the INK4A gene locus on chromosome 9p21 an additional promoter region exists that produces a transcript which includes exon 2 and exon 3 of p16INK4A but has an alternate exon 1 (exon1β) (Figure 2). Since the exon 2 is translated in an alternative reading frame (ARF) the resulting protein is completely unrelated to the gene product of INK4A although it shares parts of the mRNA sequence (Quelle et al, 1995). p16INK4A and ARF have no similarities in amino acid composition and are two completely different proteins with distinct functions – but both act as efficient tumor suppressors (Figure 2). This is demonstrated by the fact that mice that are deficient for either ARF or p16INK4A have increased susceptibility to spontaneous or carcinogen-induced cancers (Sharpless et al, 2001; Kamijo et al, 1997). p16INK4A-deficient mice show a much less spontaneous tumor rate than ARF null mice. p16INK4A ablation leads to spontaneous sarcomas and lymphomas within 17 months (Sharpless et al, 2001), whereas the onset of spontaneous sarcomas, carcinomas and lymphomas in ARF-null mice is already observed at the age of 9 months (Kamijo et al, 1997). However, it is difficult to compare results from ARF null mice with the situation in human cells since human (p14ARF) and mouse p19ARF share only about 50% sequence homology. In addition there are apparent regulatory and functional differences between human and mouse ARF. Whereas in senescent mouse fibroblasts p19ARF accumulates and is critical for senescence-induced growth arrest, the human p14ARF seems not involved and is dispensable for this process (Sharpless et al, 2004). In human cells, the senescence process is

p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 119

silencing, retains the ability to be re-activated during differentiation processes (Li et al, 2009). It is well established that the rate of induced pluripotent stem cells generated from somatic cells significantly drops with the age of the organism they are obtained from. In humans also the cellular levels of p16INK4A increase with age. Back to back several different groups showed in 2009 that the INK4A gene locus critically impairs successful reprogramming to pluripotent stem cells and that it represents a main barrier to iPS cell programming (Utikal et al, 2009; Marion et al, 2009; Banito et al, 2009; Li et al, 2009). Also in these papers the differences of p16INK4A and ARF between murine and human cells become evident: In mouse cells, the ARF-p53 pathway has more impact on preventing the generation of pluripotent stem cells from somatic cells, whereas p16INK4A seems to play a minor role in the mouse. In human fibroblasts, knockdown of ARF does not affect at all the generation of iPS cells whereas knockdown of p16INK4A significantly improved reprogramming efficiency. This suggests that depending on the species, either p16INK4A or ARF represent a barrier to "back-differentiation" of normal somatic cells and prevent the induction of "stemness" in cells that have differentiated into a certain lineage. This makes this gene locus so important for the medical application of induced progenitor cells to e.g. replace damaged tissue of a patient, but also underlines that p16INK4A and/or ARF may be critical for maintaining tissue architecture and function in complex organisms by preventing uncontrolled expansion or "development" of somatic cells with stem cell-like abilities.

**5. Regulation of INK4A in hematopoietic stem cells and their progenitors** 

As discussed before the INK4A gene locus represents a main barrier to the generation of iPS cells. In hematopoietic stem cells and many other stem cell types e.g. neuronal stem cells (Molofsky et al, 2006), the INK4A gene locus is not active. In particular the downregulation and silencing of p16INK4A seems to be essential for the enhanced self-renewal and proliferative capacity of human hematopoietic stem cells (Janzen et al, 2006). At the transcriptional level the INK4A expression is modulated by three main regulators, beta lymphoma Mo-MLV insertion region (BMI1), ETS1 and inhibitor of DNA binding 1 (Id1) whereas age-related induction of p16INK4A and ARF in human cells is mainly related to the balance between ETS1 and Id1 proteins (Ohtani et al, 2001). The reversible silencing of this gene locus in hematopoietic stem cells can be ascribed to the activity of the BMI1 protein. BMI1 belongs to the polycomb group genes, which are transcriptional repressors that control gene expression patterns during differentiation and development (Simon and Kingston 2009). The polycomb group genes fall into two subgroups that are either part of polycomb repression complex 1 (PRC1) or polycomb-repression complex 2 (PRC2). PRC2 is the so called "initiation complex" that functions as a histone-methyltransferase which specifically methylates histone H3 on lysine 27 causing gene silencing. As outlined above methylation of histone H3 on lysine 27 (H3K27me3) and on lysine 4 (H3K4me3) are hallmarks of "bivalent" chromatin that is silenced but retains the ability to be reactivated upon cell differentiation processes. BMI1 is part of PRC1 which is the so called "maintenance complex" that in a second step recognizes trimethylated H3K27. BMI1 directly associates with the INK4A locus and it was demonstrated that repression of the INK4A gene locus depends on the continuous presence of the PRC2 complex (Bracken et al, 2007). Several lines of evidence suggest that BMI1 is critical for maintaining "stemness" at least in human hematopoietic stem cells (Figure 2). In cord blood hematopoietic cells BMI1 expression is highest expressed in the hematopoietic stem cell population and gradually

mainly controlled by the unrelated twin p16INK4A and the relevance of p14ARF seems also limited for other processes such as prevention of "stemness" in normal human fibroblasts by the expression of specific transcription factors (see below).

Human or murine ARF proteins do not contain any recognizable structural motifs and probably need to interact with other proteins to form functional complexes. The first discovered and best-defined function of ARF is the induction of p53 via inhibition of the p53-degrading E3-ubiquitine ligase MDM2 (mouse) or HDM2 (human) (Sherr 2006). In situations of increased cell cycle progression, e.g. when oncogenic signaling stimulates cell cycle entry or loss of pRB function, ARF is transcriptionally induced via E2F1/DP1 and binds to and inhibits MDM2 or HDM2, respectively. This leads to accumulation of p53 and induction of p53-induced response genes, such as p21Cip1 that interferes with cell cycle progression, or the proapoptotic BCL-2 proteins BBC3/PUMA and PMAIP1/Noxa that induce programmed cell death (Villunger et al, 2003). More recently other functions of ARF have been discovered: in response to oncogenic stress ARF enters the nucleolus and retards rRNA transcription thereby inhibiting ribosome biosynthesis (Itahana et al, 2003) and ARF influences the ATM/ATR kinases during the DNA-damage response in a p53-independent manner (Rocha et al, 2005). Moreover, it was shown that independent of p53 ARF antagonizes the activity of two other critical factors for cell cycle entry, namely E2F and Myc (Sherr 2006). The detailed investigation of how p16INK4A and ARF control cell survival and proliferation in distinct cell types will remain a highly interesting field of research in the next years, in particular the physiologic role of these two unrelated twins in nontransformed somatic cells.

#### **4. The role of the INK4A locus for the generation of induced pluripotent stem cells (iPS)**

Tissue repair and permanent replacement of damaged or aged cells are essential for the life of complex organisms and usually depend on a distinct, unspecialized stem cell population with almost unlimited proliferative capacity. With age, the capacity of these stem cells to proliferate and generate progenitors declines, which may also contribute for many agerelated symptoms. The reprogramming of normal, differentiated cells by the three transcription factors Oct4, Klf4, and Sox2 has opened a completely new field of research and raised the hope to regenerate almost all cell types and tissues within the human body by generating stem cells from somatic cells of a patient. However the process that is initiated by these three transcription factors works at low efficiency and remains poorly understood. In embryonal stem cells and in induced progenitor stem (iPS) cells the INK4A gene locus is completely silenced and neither p16INK4A nor ARF are expressed. The lack of INK4A proteins seems to be a hallmark of different kinds of stem cells. Interestingly, this epigenetic silencing is not due to enhanced DNA-methylation of the INK4A promoters but results from so called "bivalent chromatin" which is present at the INK4A gene locus (Ohm et al, 2007). "Bivalent" means that repressive methylation marks (H3K27me3) and activating methylation marks (H3K4me3) are present on the same histone molecule, leading to a chromatin state that silences gene expression but can be reversed during differentiation of a cell. Such domains are characteristics of embryonal stem cells and are frequently associated with binding sites for Oct4 and Sox2. Although such binding sites are absent in the INK4A gene locus the presence of these bivalent domains suggests that the INK4A gene locus adopts a silent configuration in stem cells, but, in contrast to DNA-methylation-induced

mainly controlled by the unrelated twin p16INK4A and the relevance of p14ARF seems also limited for other processes such as prevention of "stemness" in normal human fibroblasts by

Human or murine ARF proteins do not contain any recognizable structural motifs and probably need to interact with other proteins to form functional complexes. The first discovered and best-defined function of ARF is the induction of p53 via inhibition of the p53-degrading E3-ubiquitine ligase MDM2 (mouse) or HDM2 (human) (Sherr 2006). In situations of increased cell cycle progression, e.g. when oncogenic signaling stimulates cell cycle entry or loss of pRB function, ARF is transcriptionally induced via E2F1/DP1 and binds to and inhibits MDM2 or HDM2, respectively. This leads to accumulation of p53 and induction of p53-induced response genes, such as p21Cip1 that interferes with cell cycle progression, or the proapoptotic BCL-2 proteins BBC3/PUMA and PMAIP1/Noxa that induce programmed cell death (Villunger et al, 2003). More recently other functions of ARF have been discovered: in response to oncogenic stress ARF enters the nucleolus and retards rRNA transcription thereby inhibiting ribosome biosynthesis (Itahana et al, 2003) and ARF influences the ATM/ATR kinases during the DNA-damage response in a p53-independent manner (Rocha et al, 2005). Moreover, it was shown that independent of p53 ARF antagonizes the activity of two other critical factors for cell cycle entry, namely E2F and Myc (Sherr 2006). The detailed investigation of how p16INK4A and ARF control cell survival and proliferation in distinct cell types will remain a highly interesting field of research in the next years, in particular the physiologic role of these two unrelated twins in non-

**4. The role of the INK4A locus for the generation of induced pluripotent stem** 

Tissue repair and permanent replacement of damaged or aged cells are essential for the life of complex organisms and usually depend on a distinct, unspecialized stem cell population with almost unlimited proliferative capacity. With age, the capacity of these stem cells to proliferate and generate progenitors declines, which may also contribute for many agerelated symptoms. The reprogramming of normal, differentiated cells by the three transcription factors Oct4, Klf4, and Sox2 has opened a completely new field of research and raised the hope to regenerate almost all cell types and tissues within the human body by generating stem cells from somatic cells of a patient. However the process that is initiated by these three transcription factors works at low efficiency and remains poorly understood. In embryonal stem cells and in induced progenitor stem (iPS) cells the INK4A gene locus is completely silenced and neither p16INK4A nor ARF are expressed. The lack of INK4A proteins seems to be a hallmark of different kinds of stem cells. Interestingly, this epigenetic silencing is not due to enhanced DNA-methylation of the INK4A promoters but results from so called "bivalent chromatin" which is present at the INK4A gene locus (Ohm et al, 2007). "Bivalent" means that repressive methylation marks (H3K27me3) and activating methylation marks (H3K4me3) are present on the same histone molecule, leading to a chromatin state that silences gene expression but can be reversed during differentiation of a cell. Such domains are characteristics of embryonal stem cells and are frequently associated with binding sites for Oct4 and Sox2. Although such binding sites are absent in the INK4A gene locus the presence of these bivalent domains suggests that the INK4A gene locus adopts a silent configuration in stem cells, but, in contrast to DNA-methylation-induced

the expression of specific transcription factors (see below).

transformed somatic cells.

**cells (iPS)** 

silencing, retains the ability to be re-activated during differentiation processes (Li et al, 2009). It is well established that the rate of induced pluripotent stem cells generated from somatic cells significantly drops with the age of the organism they are obtained from. In humans also the cellular levels of p16INK4A increase with age. Back to back several different groups showed in 2009 that the INK4A gene locus critically impairs successful reprogramming to pluripotent stem cells and that it represents a main barrier to iPS cell programming (Utikal et al, 2009; Marion et al, 2009; Banito et al, 2009; Li et al, 2009). Also in these papers the differences of p16INK4A and ARF between murine and human cells become evident: In mouse cells, the ARF-p53 pathway has more impact on preventing the generation of pluripotent stem cells from somatic cells, whereas p16INK4A seems to play a minor role in the mouse. In human fibroblasts, knockdown of ARF does not affect at all the generation of iPS cells whereas knockdown of p16INK4A significantly improved reprogramming efficiency. This suggests that depending on the species, either p16INK4A or ARF represent a barrier to "back-differentiation" of normal somatic cells and prevent the induction of "stemness" in cells that have differentiated into a certain lineage. This makes this gene locus so important for the medical application of induced progenitor cells to e.g. replace damaged tissue of a patient, but also underlines that p16INK4A and/or ARF may be critical for maintaining tissue architecture and function in complex organisms by preventing uncontrolled expansion or "development" of somatic cells with stem cell-like abilities.

#### **5. Regulation of INK4A in hematopoietic stem cells and their progenitors**

As discussed before the INK4A gene locus represents a main barrier to the generation of iPS cells. In hematopoietic stem cells and many other stem cell types e.g. neuronal stem cells (Molofsky et al, 2006), the INK4A gene locus is not active. In particular the downregulation and silencing of p16INK4A seems to be essential for the enhanced self-renewal and proliferative capacity of human hematopoietic stem cells (Janzen et al, 2006). At the transcriptional level the INK4A expression is modulated by three main regulators, beta lymphoma Mo-MLV insertion region (BMI1), ETS1 and inhibitor of DNA binding 1 (Id1) whereas age-related induction of p16INK4A and ARF in human cells is mainly related to the balance between ETS1 and Id1 proteins (Ohtani et al, 2001). The reversible silencing of this gene locus in hematopoietic stem cells can be ascribed to the activity of the BMI1 protein. BMI1 belongs to the polycomb group genes, which are transcriptional repressors that control gene expression patterns during differentiation and development (Simon and Kingston 2009). The polycomb group genes fall into two subgroups that are either part of polycomb repression complex 1 (PRC1) or polycomb-repression complex 2 (PRC2). PRC2 is the so called "initiation complex" that functions as a histone-methyltransferase which specifically methylates histone H3 on lysine 27 causing gene silencing. As outlined above methylation of histone H3 on lysine 27 (H3K27me3) and on lysine 4 (H3K4me3) are hallmarks of "bivalent" chromatin that is silenced but retains the ability to be reactivated upon cell differentiation processes. BMI1 is part of PRC1 which is the so called "maintenance complex" that in a second step recognizes trimethylated H3K27. BMI1 directly associates with the INK4A locus and it was demonstrated that repression of the INK4A gene locus depends on the continuous presence of the PRC2 complex (Bracken et al, 2007). Several lines of evidence suggest that BMI1 is critical for maintaining "stemness" at least in human hematopoietic stem cells (Figure 2). In cord blood hematopoietic cells BMI1 expression is highest expressed in the hematopoietic stem cell population and gradually

p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 121

give rise to leukemia. Due to the unusual structure of this gene locus, mutations often affect both, p16INK4A and p14ARF gene products, thereby deleting the gatekeepers of two essential check points. This may explain why mutations of the INK4A gene locus are observed in almost all human cancers. However, the effects of the INK4A gene products are also cell lineage specific, which might explain why some malignancies, for example T-ALL, show very high frequencies of homozygous deletion of the INK4A gene locus. Deletions frequently affect exon 2, thereby destroying both, p16INK4A and p14ARF, but there are also patients with alterations in either exon 1α or exon 1β which only affect one of the tumor suppressors (Cayuela et al, 1996; Cayuela et al, 1997; Hebert et al, 1994; Quelle et al, 1995). In addition methylation of the p16INK4A promoter was reported in T-ALL patients, leading to permanent silencing of the INK4A gene (Gardie et al, 1998; Drexler 1998). From a prognostic point of view inactivation of the INK4A locus seems also highly important for human lymphoblastic leukemia. Loss of INK4A predicts relaps in children with acute lymphoblastic leukemia suggesting a critical role of this locus in disease development and also highlighting the need for additional therapies to treat this subgroup of T-ALL patients (Kees et al, 1997); (Okuda et al, 1995). Several attempts have been undertaken to better understand, why inactivation of the INK4A gene locus is critical in particular for the development of hematopoietic malignancies. One oncogene that is thought to initiate T-cell leukemia is the TAL1/SCL oncogene. TAL1/SCL expressing T-ALL patients have a high incidence (up to 90%) of deletion of exon 2 in the INK4A gene locus. Although TAL1/SCL overexpression induces leukemia in transgenic mice, leukemia by this oncogene is characterized by a long latency suggesting that additional genetic events are required (Condorelli et al, 1996). To elucidate the contribution of the INK4A gene locus to leukemogenesis Shank-Calvo and colleagues (Shank-Calvo et al, 2006) mated TAL1 transgenic mice with single knockout mice for either p16INK4A or p19ARF. Of note, each of these mice developed T-cell leukemia rapidly, indicating that loss of either p16INK4A or

The fact that the INK4A genes are inactivated at high incidence in hematopoietic malignancies might be ascribed to the specific roles of these two proteins in slowing down the proliferation of hematopoietic progenitor cells, as discussed above. INK4A-/- mice possess increased thymus size and cellularity suggesting involvement of p16INK4A in the control of thymocyte proliferation. These animals exhibit increased numbers of CD4 and CD8 T lymphocytes in thymus and spleen (Bianchi et al, 2006) which also reflects increased proliferative potential. By using somatic, tissue specific ablation of p16INK4A in the T- or the B-lymphoid progenitor cells it was recently demonstrated that in the T-cell lineage loss of p16INK4A attenuated age-dependent thymic involution or increased production of naive T-cells. In the B-cell lineage p16INK4A inactivation significantly accelerated lymphoid tumorigenesis. Interestingly, the animals mainly suffered from tumors that manifested in the central nervous system but still expressed CD45 leukocyte-common antigen. These tumor cells were negative for a neuromeningeal marker, proving that they are not brain tumors and expressed B-lymphocyte markers demonstrating their B-cell origin (Liu et al, 2011). In this paper the authors argued therefore that in the T-cell linage p16INK4A merely regulates cell senescence in the mouse, whereas in the B-cell lineage loss of p16INK4A contributes to lymphoid cancer. These results are contradictory to the high prevalence of p16INK4A loss in human T-cell leukemia progenitor cells and may be ascribed to the already above discussed differences of the role of p16INK4A and ARF between mice and men. Senescence, cell cycle arrest and cell death are three possible physiologic fates of a cell

p19ARF accelerates TAL1-induced leukemia in mice.

decreases when these cells maturate into more differentiated progenitor cells. Overexpression of BMI1 enhances the self-renewal of hematopoietic stem cells, increases the engraftment potential and results in stem cell maintenance. Knockdown of BMI1 in cord blood CD34+ and in acute myeloid leukemia (AML) CD34+ cells reduces progenitorforming capacity, stem cell marker expression and long-term culture-initiating cell frequencies significantly suggesting that loss of BMI impairs the maintenance of stem cells and progenitor cells. In parallel, because of the gene-silencing effect of the BMI1 containing PRC1 complex on the INK4A gene locus, loss of BMI1 in C34+ cord blood and AML cells causes the induction of p14ARF and p16INK4A, significantly increased apoptosis and the production of cellular reactive oxygen species (Rizo et al, 2009). Lack of BMI1 in hematopoietic cells from BMI1 knockout mice also resulted in an increased expression of p16INK4A and p19ARF. The fact that the deletion of INK4A/ARF in the BMI1-/ background partly restored the self-renewal capacity of hematopoietic stem cells demonstrates the importance of silencing of the INK4A gene locus by BMI1 for the maintenance of hematopoietic stem cells (Oguro et al, 2006).

Fig. 2. The INK4A gene locus generates the two unrelated tumor suppressor proteins p16INK4A and ARF by alternative promoter usage and splicing, which are subjected to a complex regulation in hematopoietic stem cells and progenitor cells. Stress and senescence activate p16INK4A via Ets1/2 transcription factors and accelerated cell cycle entry triggers ARF expression. Id1 heterodimerizes and blocks Ets1/2 in young unstressed cells or stem cells. In stem cells BMI1 as part of the polycomb complex PRC2 causes epigenetic gene silencing of the entire locus by bivalent histone methylation (see text).

#### **6. Is p16INK4A critical for the development of hematologic malignancies?**

The almost unlimited replicative capacity of stem cells and efficient generation of progenitors may be a double edged sword for a multicellular organism. On one hand this proliferative capacity allows efficient repair, regeneration and plasticity of tissues, on the other hand it increases the risk of acquiring genetic defects in this stem cell population that may result in hyperproliferative diseases, among them malignant transformation and cancer. Considering the fact that the INK4A gene locus codes for two tumor suppressors with completely different functions which either critically control the pRB or the p53 gene network, it becomes clear that genetic abnormalities of this gene locus may have a dramatic impact on progression of a damaged hematopoietic stem cell into precancerous cells that

decreases when these cells maturate into more differentiated progenitor cells. Overexpression of BMI1 enhances the self-renewal of hematopoietic stem cells, increases the engraftment potential and results in stem cell maintenance. Knockdown of BMI1 in cord blood CD34+ and in acute myeloid leukemia (AML) CD34+ cells reduces progenitorforming capacity, stem cell marker expression and long-term culture-initiating cell frequencies significantly suggesting that loss of BMI impairs the maintenance of stem cells and progenitor cells. In parallel, because of the gene-silencing effect of the BMI1 containing PRC1 complex on the INK4A gene locus, loss of BMI1 in C34+ cord blood and AML cells causes the induction of p14ARF and p16INK4A, significantly increased apoptosis and the production of cellular reactive oxygen species (Rizo et al, 2009). Lack of BMI1 in hematopoietic cells from BMI1 knockout mice also resulted in an increased expression of p16INK4A and p19ARF. The fact that the deletion of INK4A/ARF in the BMI1-/ background partly restored the self-renewal capacity of hematopoietic stem cells demonstrates the importance of silencing of the INK4A gene locus by BMI1 for the

Fig. 2. The INK4A gene locus generates the two unrelated tumor suppressor proteins p16INK4A and ARF by alternative promoter usage and splicing, which are subjected to a complex regulation in hematopoietic stem cells and progenitor cells. Stress and senescence activate p16INK4A via Ets1/2 transcription factors and accelerated cell cycle entry triggers ARF expression. Id1 heterodimerizes and blocks Ets1/2 in young unstressed cells or stem cells. In stem cells BMI1 as part of the polycomb complex PRC2 causes epigenetic gene

**6. Is p16INK4A critical for the development of hematologic malignancies?** 

The almost unlimited replicative capacity of stem cells and efficient generation of progenitors may be a double edged sword for a multicellular organism. On one hand this proliferative capacity allows efficient repair, regeneration and plasticity of tissues, on the other hand it increases the risk of acquiring genetic defects in this stem cell population that may result in hyperproliferative diseases, among them malignant transformation and cancer. Considering the fact that the INK4A gene locus codes for two tumor suppressors with completely different functions which either critically control the pRB or the p53 gene network, it becomes clear that genetic abnormalities of this gene locus may have a dramatic impact on progression of a damaged hematopoietic stem cell into precancerous cells that

silencing of the entire locus by bivalent histone methylation (see text).

maintenance of hematopoietic stem cells (Oguro et al, 2006).

give rise to leukemia. Due to the unusual structure of this gene locus, mutations often affect both, p16INK4A and p14ARF gene products, thereby deleting the gatekeepers of two essential check points. This may explain why mutations of the INK4A gene locus are observed in almost all human cancers. However, the effects of the INK4A gene products are also cell lineage specific, which might explain why some malignancies, for example T-ALL, show very high frequencies of homozygous deletion of the INK4A gene locus. Deletions frequently affect exon 2, thereby destroying both, p16INK4A and p14ARF, but there are also patients with alterations in either exon 1α or exon 1β which only affect one of the tumor suppressors (Cayuela et al, 1996; Cayuela et al, 1997; Hebert et al, 1994; Quelle et al, 1995). In addition methylation of the p16INK4A promoter was reported in T-ALL patients, leading to permanent silencing of the INK4A gene (Gardie et al, 1998; Drexler 1998). From a prognostic point of view inactivation of the INK4A locus seems also highly important for human lymphoblastic leukemia. Loss of INK4A predicts relaps in children with acute lymphoblastic leukemia suggesting a critical role of this locus in disease development and also highlighting the need for additional therapies to treat this subgroup of T-ALL patients (Kees et al, 1997); (Okuda et al, 1995). Several attempts have been undertaken to better understand, why inactivation of the INK4A gene locus is critical in particular for the development of hematopoietic malignancies. One oncogene that is thought to initiate T-cell leukemia is the TAL1/SCL oncogene. TAL1/SCL expressing T-ALL patients have a high incidence (up to 90%) of deletion of exon 2 in the INK4A gene locus. Although TAL1/SCL overexpression induces leukemia in transgenic mice, leukemia by this oncogene is characterized by a long latency suggesting that additional genetic events are required (Condorelli et al, 1996). To elucidate the contribution of the INK4A gene locus to leukemogenesis Shank-Calvo and colleagues (Shank-Calvo et al, 2006) mated TAL1 transgenic mice with single knockout mice for either p16INK4A or p19ARF. Of note, each of these mice developed T-cell leukemia rapidly, indicating that loss of either p16INK4A or p19ARF accelerates TAL1-induced leukemia in mice.

The fact that the INK4A genes are inactivated at high incidence in hematopoietic malignancies might be ascribed to the specific roles of these two proteins in slowing down the proliferation of hematopoietic progenitor cells, as discussed above. INK4A-/- mice possess increased thymus size and cellularity suggesting involvement of p16INK4A in the control of thymocyte proliferation. These animals exhibit increased numbers of CD4 and CD8 T lymphocytes in thymus and spleen (Bianchi et al, 2006) which also reflects increased proliferative potential. By using somatic, tissue specific ablation of p16INK4A in the T- or the B-lymphoid progenitor cells it was recently demonstrated that in the T-cell lineage loss of p16INK4A attenuated age-dependent thymic involution or increased production of naive T-cells. In the B-cell lineage p16INK4A inactivation significantly accelerated lymphoid tumorigenesis. Interestingly, the animals mainly suffered from tumors that manifested in the central nervous system but still expressed CD45 leukocyte-common antigen. These tumor cells were negative for a neuromeningeal marker, proving that they are not brain tumors and expressed B-lymphocyte markers demonstrating their B-cell origin (Liu et al, 2011). In this paper the authors argued therefore that in the T-cell linage p16INK4A merely regulates cell senescence in the mouse, whereas in the B-cell lineage loss of p16INK4A contributes to lymphoid cancer. These results are contradictory to the high prevalence of p16INK4A loss in human T-cell leukemia progenitor cells and may be ascribed to the already above discussed differences of the role of p16INK4A and ARF between mice and men. Senescence, cell cycle arrest and cell death are three possible physiologic fates of a cell

p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 123

based on different tetracycline-activated transactivators and repressors (Ausserlechner et al, 2006). In Molt4 T-ALL cells, for example, p16INK4A causes increased sensitivity to UVirradiation, which is associated with induction of the pro-apoptotic BH3-only protein

In principle programmed cell death can be initiated by a number of different signals originating either from outside of the cell (extrinsic pathway) or from intrinsic signals (Strasser 2005). Soluble or cell bound death ligands such as FASLG/FAS ligand or TRAIL bind to their cognate receptors, thereby inducing the formation of the so called deathinducing signaling complex (DISC) which contains the adaptor molecule FADD and procaspase-8. The autocatalytic cleavage and activation of procaspase-8 triggers the downstream caspase cascade. Mitochondria are central decision makers of apoptosis that integrate death signals originating from DNA-damage, growth factor withdrawal, glucocorticoid-treatment and anoikis. These stimuli trigger cell death either by directly regulating cell survival/cell death genes, or by deregulating cellular networks, which leads to apoptosis. Pro- and anti-apoptotic BCL2 proteins, referred to as the "BCL2-rheostat", are involved in this cell death decision either as direct targets or as sensors for cellular stress. BCL2-proteins can be divided into three groups. The prosurvival multi-domain BCL2 proteins BCL2, BCL2L2/Bcl-w, BCL2L1/Bcl-xL, BCL2A1/A1 and MCL1 share four BCL2 homology (BH) domains, whereas multi-domain proapoptotic proteins BAX and BAK1/Bak are characterized by three BH-domains (Strasser 2005). The third group is the family of BH3-only proteins which contain only the BH3-domain and heterodimerize with prosurvival BCL2-proteins. The two models that have been proposed for apoptosis

Fig. 3. Expression of p16INK4A induces growth arrest in the G1 phase of the cell cycle and shifts the balance of pro- and anti-apoptotic BCL2 proteins towards the edge of cell death

induction by BH3-only proteins suggest that either strong BH3-only proteins such as BCL2L11/Bim, truncated Bid or BBC3/Puma directly activate BAX or BAK1 ("direct activator/de-repressor model") (Kim et al, 2006) or that these BH3-only proteins neutralize the pro-survival function of anti-apoptotic BCL2-proteins ("displacement model") (Labi et

BBC3/Puma (Obexer et al, 2009a).

(detailed explanations in the text).

that may be triggered by the two tumor suppressors of the INK4A gene locus. In the next chapter we will discuss how p16INK4A affects programmed cell death.

#### **7. p16INK4A regulates programmed cell death and death sensitivity in leukemia cells**

When a cell is hit by a genotoxic insult it will either try to repair the genetic damage or, if not possible, undergo programmed cell death. Otherwise the mutation will be inherited to the daughter cells, which may give rise to precancerous cells that slowly proceed into malignancy. Slowing down cell cycle progression upon genotoxic stress may therefore help cells to get time for efficient damage repair and thereby prevent programmed cell death. On the other hand cell cycle arrest in the presence of oncogenic signaling may constitute a death signal per se since apoptosis may be the only way for a precancerous cell to respond to this "conflicting signaling" situation. INK4A gene products are not expressed in hematopoietic stem cells and therefore may not play an essential role for the quiescence state that every stem cell has to enter after division to preserve its replicatory potential. Instead, INK4A proteins accumulate in the progenitor cell population and seem to limit their proliferation. As a consequence of p16INK4A deficiency rapid movement through the cell cycle may sensitize leukemia cells to genotoxic stress, which has been shown in different types of cancer cells. In p16INK4A-deficient MEFs and U2OS osteosarkoma cells the lack of p16INK4A sensitizes these cells to cell death induction by UV irradiation, whereas p16INK4A-proficient cells are largely resistant. The authors also observed that UV-induced apoptosis in p16INK4A-deficient cells coincided with decreased levels of pro-survival BCL2 and increased levels of pro-apoptotic BAX proteins (Al Mohanna et al, 2004). Moreover, in p16INK4A-deficient mice increased numbers of CD4 and CD8 T-cells are found in thymus and spleen and these increased cell numbers correlated with reduced T-cell apoptosis in the thymus rather than increased proliferation rates (Bianchi et al, 2006). The increased rates of DNA-synthesis in p16INK4A-deficient cells may expose an "archilles heel" to DNAdamage-induced cell death, but there are also data that highlight that p16INK4A reconstitution sensitizes T-ALL cells to certain forms of apoptosis. This suggests that p16INK4A may exert also additional effects beyond cell cycle inhibition, such as the regulation of proteins critical for cell death initiation and cell death decision in mammalian cells. We demonstrated already ten years ago that tetracycline-regulated p16INK4A reconstitution in p16INK4A-deficient CCRF-CEM cells, a human T-ALL cell line, sensitizes these leukemia cells to physiologic levels of cortisol, a glucocorticoid that also plays a significant role during T-cell selection and T-cell maturation in the thymus. In this early study we were able to show that p16INK4A significantly induces the expression of endogenous glucocorticoid receptor thereby markedly lowering the threshold for glucocorticoid-induced apoptosis (Ausserlechner et al, 2001). In this paper we discussed that loss of p16INK4A in precancerous hematopoietic cells may render them resistant to glucocorticoid-induced cell death by physiologic levels of cortisol. Since the strikingly increased sensitivity was difficult to explain by increases in receptor levels alone we sought for additional mechanisms further downstream of the glucocorticoid receptor that may contribute to p16INK4A-regulated death sensitivity. Indeed, also in T-ALL cells activation of p16INK4A affects the balance of death inducers and death protectors at the level of mitochondria, but also activates death ligands. For studying p16INK4A effects in different

T-ALL cell lines we applied a tightly controlled tetracycline-regulated expression system,

that may be triggered by the two tumor suppressors of the INK4A gene locus. In the next

When a cell is hit by a genotoxic insult it will either try to repair the genetic damage or, if not possible, undergo programmed cell death. Otherwise the mutation will be inherited to the daughter cells, which may give rise to precancerous cells that slowly proceed into malignancy. Slowing down cell cycle progression upon genotoxic stress may therefore help cells to get time for efficient damage repair and thereby prevent programmed cell death. On the other hand cell cycle arrest in the presence of oncogenic signaling may constitute a death signal per se since apoptosis may be the only way for a precancerous cell to respond to this "conflicting signaling" situation. INK4A gene products are not expressed in hematopoietic stem cells and therefore may not play an essential role for the quiescence state that every stem cell has to enter after division to preserve its replicatory potential. Instead, INK4A proteins accumulate in the progenitor cell population and seem to limit their proliferation. As a consequence of p16INK4A deficiency rapid movement through the cell cycle may sensitize leukemia cells to genotoxic stress, which has been shown in different types of cancer cells. In p16INK4A-deficient MEFs and U2OS osteosarkoma cells the lack of p16INK4A sensitizes these cells to cell death induction by UV irradiation, whereas p16INK4A-proficient cells are largely resistant. The authors also observed that UV-induced apoptosis in p16INK4A-deficient cells coincided with decreased levels of pro-survival BCL2 and increased levels of pro-apoptotic BAX proteins (Al Mohanna et al, 2004). Moreover, in p16INK4A-deficient mice increased numbers of CD4 and CD8 T-cells are found in thymus and spleen and these increased cell numbers correlated with reduced T-cell apoptosis in the thymus rather than increased proliferation rates (Bianchi et al, 2006). The increased rates of DNA-synthesis in p16INK4A-deficient cells may expose an "archilles heel" to DNAdamage-induced cell death, but there are also data that highlight that p16INK4A reconstitution sensitizes T-ALL cells to certain forms of apoptosis. This suggests that p16INK4A may exert also additional effects beyond cell cycle inhibition, such as the regulation of proteins critical for cell death initiation and cell death decision in mammalian cells. We demonstrated already ten years ago that tetracycline-regulated p16INK4A reconstitution in p16INK4A-deficient CCRF-CEM cells, a human T-ALL cell line, sensitizes these leukemia cells to physiologic levels of cortisol, a glucocorticoid that also plays a significant role during T-cell selection and T-cell maturation in the thymus. In this early study we were able to show that p16INK4A significantly induces the expression of endogenous glucocorticoid receptor thereby markedly lowering the threshold for glucocorticoid-induced apoptosis (Ausserlechner et al, 2001). In this paper we discussed that loss of p16INK4A in precancerous hematopoietic cells may render them resistant to glucocorticoid-induced cell death by physiologic levels of cortisol. Since the strikingly increased sensitivity was difficult to explain by increases in receptor levels alone we sought for additional mechanisms further downstream of the glucocorticoid receptor that may contribute to p16INK4A-regulated death sensitivity. Indeed, also in T-ALL cells activation of p16INK4A affects the balance of death inducers and death protectors at the level of mitochondria, but also activates death ligands. For studying p16INK4A effects in different T-ALL cell lines we applied a tightly controlled tetracycline-regulated expression system,

**7. p16INK4A regulates programmed cell death and death sensitivity in** 

chapter we will discuss how p16INK4A affects programmed cell death.

**leukemia cells** 

based on different tetracycline-activated transactivators and repressors (Ausserlechner et al, 2006). In Molt4 T-ALL cells, for example, p16INK4A causes increased sensitivity to UVirradiation, which is associated with induction of the pro-apoptotic BH3-only protein BBC3/Puma (Obexer et al, 2009a).

In principle programmed cell death can be initiated by a number of different signals originating either from outside of the cell (extrinsic pathway) or from intrinsic signals (Strasser 2005). Soluble or cell bound death ligands such as FASLG/FAS ligand or TRAIL bind to their cognate receptors, thereby inducing the formation of the so called deathinducing signaling complex (DISC) which contains the adaptor molecule FADD and procaspase-8. The autocatalytic cleavage and activation of procaspase-8 triggers the downstream caspase cascade. Mitochondria are central decision makers of apoptosis that integrate death signals originating from DNA-damage, growth factor withdrawal, glucocorticoid-treatment and anoikis. These stimuli trigger cell death either by directly regulating cell survival/cell death genes, or by deregulating cellular networks, which leads to apoptosis. Pro- and anti-apoptotic BCL2 proteins, referred to as the "BCL2-rheostat", are involved in this cell death decision either as direct targets or as sensors for cellular stress. BCL2-proteins can be divided into three groups. The prosurvival multi-domain BCL2 proteins BCL2, BCL2L2/Bcl-w, BCL2L1/Bcl-xL, BCL2A1/A1 and MCL1 share four BCL2 homology (BH) domains, whereas multi-domain proapoptotic proteins BAX and BAK1/Bak are characterized by three BH-domains (Strasser 2005). The third group is the family of BH3-only proteins which contain only the BH3-domain and heterodimerize with prosurvival BCL2-proteins. The two models that have been proposed for apoptosis

Fig. 3. Expression of p16INK4A induces growth arrest in the G1 phase of the cell cycle and shifts the balance of pro- and anti-apoptotic BCL2 proteins towards the edge of cell death (detailed explanations in the text).

induction by BH3-only proteins suggest that either strong BH3-only proteins such as BCL2L11/Bim, truncated Bid or BBC3/Puma directly activate BAX or BAK1 ("direct activator/de-repressor model") (Kim et al, 2006) or that these BH3-only proteins neutralize the pro-survival function of anti-apoptotic BCL2-proteins ("displacement model") (Labi et

p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 125

Fig. 4. p16INK4A expression differentially affects the sensitivity of T-ALL cells to various death-inducing agents. T-ALL cells expressing p16INK4A in a tetracycline-regulated manner were cultured for 24 hours in the presence of doxycycline (200 ng/ml) to switch on p16INK4A expression and were then treated with the therapeutic agents doxorubicin (8 µM), etoposide (8 µM), vincristine (8 µM), dexamethasone (10 nM) and bortezomib (10 nM) for additional 24 hours. To activate death receptor signaling an anti-Fas-receptor antibody (clone CH11, 0.1 µg/ml) was applied for 4 hours. Cells were resuspended in hypotonic propidium-iodide containing buffer according Nicoletti et al (Nicoletti et al, 1991) and the percentage of apoptotic cells was assessed in a Beckman-Coulter FC500 flow cytometer.

However, additional levels of apoptosis modulation by p16INK4A in T-ALL cells exist. One protein that is highly expressed in many cancers e.g. in up to 65% of acute lymphoblastic Bcell leukemia is the anti-apoptotic protein BIRC5/Survivin (Troeger et al, 2007). Survivin belongs to the family of Inhibitor of Apoptosis Proteins (IAPs) that are characterized by so called BIR domains that allow them to interact with caspases and other molecules involved in cell death signaling. Survivin has also additional functions since it acts as a chromosomal passenger protein and blocks apoptosis induction at the level of mitochondria (Obexer et al, 2009b). Recently, it was shown that mice overexpressing Survivin in hematopoietic stem cells show a high incidence of hematologic tumors. This pro-oncogenic effect of Survivin was not due to increased proliferative potential but to increased death resistance of

Interestingly, the loss of p16INK4A during leukemogenesis apparently contributes to high levels of Survivin in T-ALL cells: in leukemia cells engineered to express p16INK4A the Survivin steady state expression levels are completely repressed upon p16INK4A induction (Figure 5A). To directly assess the relevance of Survivin-repression for changes in apoptosis sensitivity we retrovirally transduced human Survivin into T-ALL cells with conditional p16INK4A expression. As shown in Figure 5A this ectopic Survivin compensates for the loss of the endogenous protein during p16INK4A expression. Interestingly, ectopic Survivin prevented FAS-induced death sensitization (Figure 5B), but did not affect the increased sensitivity of p16INK4A-expressing T-ALL cells to glucocorticoid-induced cell death (Figure 5C). These results suggest that in T-ALL cells Survivin interferes with death-receptor-

Shown is the mean of three independent experiments.

hematologic cells (Small et al, 2010).

al, 2006; Willis et al, 2007). Upon cell death decision BAX or BAK1 oligomerize in the mitochondrial outer membrane which causes cytochrome *c* release from mitochondria and activation of the further downstream apoptosome complex.

Interestingly, the apoptosis-regulatory effect of conditional reconstitution of p16INK4A in T-ALL leukemia cells depends on the therapeutic agent that is applied to the cells: p16INK4A markedly protects against programmed cell death induced by doxorubicin, etoposide and vinblastine (Figure 4), but in parallel sensitizes these cells to FAS- and GC-induced apoptosis (Figure 4 and (Obexer et al, 2009a)). Since p16INK4A induces an almost complete cell cycle arrest in these cells and uncouples growth from cell cycle arrest (Ausserlechner et al, 2005) we believe that this protective effect is merely a consequence of accumulation of cells in the G1 phase of the cell cycle. DNA-damaging agents such as doxorubicin or etoposide and tubulin-destabilzing compounds such as vinblastine exert their effects mainly on proliferating cells in S-phase or mitosis, respectively. Slowly proliferating cells or cells that are arrested in the G1-phase of the cell cycle may be therefore more resistant (Bacher et al, 2006). However, conditional p16INK4A also protects T-ALL cells against bortezomibinduced cell death (Figure 4). Although, the proteasome-inhibitor bortezomib/VelcadeTM may perturb the correct degradation of cell cycle regulators such as cyclins, it has been shown that bortezomib is highly effective on slowly proliferating cancer cells such as chronic myeloid leukemia. This suggests that p16INK4A has a direct or indirect affect on cell death regulators at the level of death receptors and/or BCL2-proteins. When analyzing the expression of death receptors and their ligands it became evident that p16INK4A induces FAS-ligand mRNA expression in these cells, but does not change the FAS-receptor expression. Since CCRF-CEM cells are FAS-sensitive, this might cause an autocrine death signal that lowers death resistance in general since also the FAS downstream effectors Caspase-8 and Bid showed accelerated cleavage in p16INK4A expressing cells upon FASinduced apoptosis. However, by introducing a dominant negative FADD mutant that blocks death receptor signaling we provided evidence that increased death sensitivity in p16INK4A expressing T-ALL cells can be ascribed to distinct changes in the composition of pro- and anti-apoptotic BCL2-proteins at the level of mitochondria (Obexer et al, 2009a). Whereas the potent pro-apoptotic protein BBC3/Puma accumulated in p16INK4A expressing CCRF-CEM and Molt4 cells, the pro-survival proteins BCL2 and MCL1 were downregulated after 24 hours and completely lost after 48 hours of p16INK4A expression. Although p16INK4A-expressing T-ALL cells did not undergo programmed cell death spontaneously within 48 hours, these significant changes in the balance of pro- and antiapoptotic BCL2-proteins reduce the capacity of T-ALL cells to cope with additional apoptotic signals such as binding of death-ligands or glucocorticoid-induced cell death (Figure 3). We demonstrated the relevance of Puma, BCL2 and MCL1 by retroviral overexpression and knock down, but did not further investigate other forms of cell death (Obexer et al, 2009a). Concerning the observation that p16INK4A expression renders T-ALL cells less sensitive to bortezomib, we found that p16INK4A reconstitution induced rapid loss of the BH3-only protein PMAIP1/Noxa in both CCRF-CEM T-ALL and Molt4 T-ALL cells (Obexer et al, 2009a). Noxa is a "weak" BH3-only protein that does not neutralize all pro-survival BCL2-proteins, but preferentially binds to MCL1, BCL2A1/A1 (Willis et al, 2007) and, as recently shown by our group also to BCL2L1/Bcl-xL. Noxa acts as a sensitizer that critically regulates the sensitivity to e.g. proteasome-inhibition-induced cell death (Hagenbuchner et al, 2010). The rapid loss of Noxa upon p16INK4A reconstitution might therefore explain, why p16INK4A-expressing T-ALL cells show reduced sensitivity to bortezomib-induced apoptosis.

al, 2006; Willis et al, 2007). Upon cell death decision BAX or BAK1 oligomerize in the mitochondrial outer membrane which causes cytochrome *c* release from mitochondria and

Interestingly, the apoptosis-regulatory effect of conditional reconstitution of p16INK4A in T-ALL leukemia cells depends on the therapeutic agent that is applied to the cells: p16INK4A markedly protects against programmed cell death induced by doxorubicin, etoposide and vinblastine (Figure 4), but in parallel sensitizes these cells to FAS- and GC-induced apoptosis (Figure 4 and (Obexer et al, 2009a)). Since p16INK4A induces an almost complete cell cycle arrest in these cells and uncouples growth from cell cycle arrest (Ausserlechner et al, 2005) we believe that this protective effect is merely a consequence of accumulation of cells in the G1 phase of the cell cycle. DNA-damaging agents such as doxorubicin or etoposide and tubulin-destabilzing compounds such as vinblastine exert their effects mainly on proliferating cells in S-phase or mitosis, respectively. Slowly proliferating cells or cells that are arrested in the G1-phase of the cell cycle may be therefore more resistant (Bacher et al, 2006). However, conditional p16INK4A also protects T-ALL cells against bortezomibinduced cell death (Figure 4). Although, the proteasome-inhibitor bortezomib/VelcadeTM may perturb the correct degradation of cell cycle regulators such as cyclins, it has been shown that bortezomib is highly effective on slowly proliferating cancer cells such as chronic myeloid leukemia. This suggests that p16INK4A has a direct or indirect affect on cell death regulators at the level of death receptors and/or BCL2-proteins. When analyzing the expression of death receptors and their ligands it became evident that p16INK4A induces FAS-ligand mRNA expression in these cells, but does not change the FAS-receptor expression. Since CCRF-CEM cells are FAS-sensitive, this might cause an autocrine death signal that lowers death resistance in general since also the FAS downstream effectors Caspase-8 and Bid showed accelerated cleavage in p16INK4A expressing cells upon FASinduced apoptosis. However, by introducing a dominant negative FADD mutant that blocks death receptor signaling we provided evidence that increased death sensitivity in p16INK4A expressing T-ALL cells can be ascribed to distinct changes in the composition of pro- and anti-apoptotic BCL2-proteins at the level of mitochondria (Obexer et al, 2009a). Whereas the potent pro-apoptotic protein BBC3/Puma accumulated in p16INK4A expressing CCRF-CEM and Molt4 cells, the pro-survival proteins BCL2 and MCL1 were downregulated after 24 hours and completely lost after 48 hours of p16INK4A expression. Although p16INK4A-expressing T-ALL cells did not undergo programmed cell death spontaneously within 48 hours, these significant changes in the balance of pro- and antiapoptotic BCL2-proteins reduce the capacity of T-ALL cells to cope with additional apoptotic signals such as binding of death-ligands or glucocorticoid-induced cell death (Figure 3). We demonstrated the relevance of Puma, BCL2 and MCL1 by retroviral overexpression and knock down, but did not further investigate other forms of cell death (Obexer et al, 2009a). Concerning the observation that p16INK4A expression renders T-ALL cells less sensitive to bortezomib, we found that p16INK4A reconstitution induced rapid loss of the BH3-only protein PMAIP1/Noxa in both CCRF-CEM T-ALL and Molt4 T-ALL cells (Obexer et al, 2009a). Noxa is a "weak" BH3-only protein that does not neutralize all pro-survival BCL2-proteins, but preferentially binds to MCL1, BCL2A1/A1 (Willis et al, 2007) and, as recently shown by our group also to BCL2L1/Bcl-xL. Noxa acts as a sensitizer that critically regulates the sensitivity to e.g. proteasome-inhibition-induced cell death (Hagenbuchner et al, 2010). The rapid loss of Noxa upon p16INK4A reconstitution might therefore explain, why p16INK4A-expressing T-ALL cells show reduced sensitivity to

activation of the further downstream apoptosome complex.

bortezomib-induced apoptosis.

Fig. 4. p16INK4A expression differentially affects the sensitivity of T-ALL cells to various death-inducing agents. T-ALL cells expressing p16INK4A in a tetracycline-regulated manner were cultured for 24 hours in the presence of doxycycline (200 ng/ml) to switch on p16INK4A expression and were then treated with the therapeutic agents doxorubicin (8 µM), etoposide (8 µM), vincristine (8 µM), dexamethasone (10 nM) and bortezomib (10 nM) for additional 24 hours. To activate death receptor signaling an anti-Fas-receptor antibody (clone CH11, 0.1 µg/ml) was applied for 4 hours. Cells were resuspended in hypotonic propidium-iodide containing buffer according Nicoletti et al (Nicoletti et al, 1991) and the percentage of apoptotic cells was assessed in a Beckman-Coulter FC500 flow cytometer. Shown is the mean of three independent experiments.

However, additional levels of apoptosis modulation by p16INK4A in T-ALL cells exist. One protein that is highly expressed in many cancers e.g. in up to 65% of acute lymphoblastic Bcell leukemia is the anti-apoptotic protein BIRC5/Survivin (Troeger et al, 2007). Survivin belongs to the family of Inhibitor of Apoptosis Proteins (IAPs) that are characterized by so called BIR domains that allow them to interact with caspases and other molecules involved in cell death signaling. Survivin has also additional functions since it acts as a chromosomal passenger protein and blocks apoptosis induction at the level of mitochondria (Obexer et al, 2009b). Recently, it was shown that mice overexpressing Survivin in hematopoietic stem cells show a high incidence of hematologic tumors. This pro-oncogenic effect of Survivin was not due to increased proliferative potential but to increased death resistance of hematologic cells (Small et al, 2010).

Interestingly, the loss of p16INK4A during leukemogenesis apparently contributes to high levels of Survivin in T-ALL cells: in leukemia cells engineered to express p16INK4A the Survivin steady state expression levels are completely repressed upon p16INK4A induction (Figure 5A). To directly assess the relevance of Survivin-repression for changes in apoptosis sensitivity we retrovirally transduced human Survivin into T-ALL cells with conditional p16INK4A expression. As shown in Figure 5A this ectopic Survivin compensates for the loss of the endogenous protein during p16INK4A expression. Interestingly, ectopic Survivin prevented FAS-induced death sensitization (Figure 5B), but did not affect the increased sensitivity of p16INK4A-expressing T-ALL cells to glucocorticoid-induced cell death (Figure 5C). These results suggest that in T-ALL cells Survivin interferes with death-receptor-

p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 127

cell cycle regulation (and de-regulation) in normal and malignant cells. Despite all this effort and progress, continuously new aspects of p16INK4A and ARF are discovered, which highlight their diverse functions beyond the inhibition of the cell division cycle. In this chapter we reviewed current findings on how these INK4A-encoded genes are regulated in hematopoietic stem cells and that these proteins also represent a barrier to the artificial generation of pluripotent stem cells from normal differentiated tissue. In addition, both proteins also contribute to death sensitivity: ARF by activating p53 via a well defined pathway, p16INK4A by directly or indirectly affecting the expression and activity of critical death regulators such as BCL2 proteins, death ligands or pro-survival proteins such as Survivin. Under normal, physiologic conditions these significant changes in death sensitivity may determine a deadly barrier for cells that try to return to a less differentiated state and also for precancerous cells that lose proliferative control. These novel findings implicate that INK4A proteins are not "only cell cycle brakes" but serve as gatekeepers that

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**9. References**

induced apoptosis triggered by FAS-ligand. Therefore, the effects of p16INK4A in leukemia cells by far extend the generally described effect as an inhibitor of cell cycle progression.

Fig. 5. High levels of p16INK4A in human CCRF-CEM T-ALL cells repress the IAP family member Survivin, which is critical for increased FAS-induced cell death, but not for p16INK4A-induced sensitization to glucocorticoid-induced apoptosis. A) CEM/Ctr cells stably express the reverse tet-transactivator rtTA, CEM/p16 cells are derivatives that contain the rtTA and express human p16INK4A under the control of a tetracycline responsive CMV promoter (Ausserlechner et al, 2001). In presence of 200 ng/ml doxycycline, p16INK4A expression is induced, which causes complete loss of Survivin within 48 hour. Retrovirally transduced Survivin compensates for the loss of the endogenous protein in CEM/p16-Survivin cells. Re-expression of p16INK4A accelerates death-receptor-induced (anti-FAS anibody, 0.1 mg/ml, for four hours) and glucocorticoidinduced apoptosis (10 nM dexamethasone, 24 hours) as shown in B und C (Obexer et al, 2009a). Ectopic expression of Survivin did not change the sensitivity to dexamethasone (C), but prevents increased sensitivity to Fas-induced apoptosis (B). The amount of apoptotic cells was assessed by flow cytometric analysis of propidium-iodide stained nuclei. Each bar represents the mean of three independent experiments.

#### **8. Conclusions**

The INK4A genes were discovered in the mid-90s of the last century and intensive studies on their function and regulation have contributed significantly to our current knowledge on cell cycle regulation (and de-regulation) in normal and malignant cells. Despite all this effort and progress, continuously new aspects of p16INK4A and ARF are discovered, which highlight their diverse functions beyond the inhibition of the cell division cycle. In this chapter we reviewed current findings on how these INK4A-encoded genes are regulated in hematopoietic stem cells and that these proteins also represent a barrier to the artificial generation of pluripotent stem cells from normal differentiated tissue. In addition, both proteins also contribute to death sensitivity: ARF by activating p53 via a well defined pathway, p16INK4A by directly or indirectly affecting the expression and activity of critical death regulators such as BCL2 proteins, death ligands or pro-survival proteins such as Survivin. Under normal, physiologic conditions these significant changes in death sensitivity may determine a deadly barrier for cells that try to return to a less differentiated state and also for precancerous cells that lose proliferative control. These novel findings implicate that INK4A proteins are not "only cell cycle brakes" but serve as gatekeepers that keep the doors closed for those cells that want some piece of "stemness".

#### **9. References**

126 T-Cell Leukemia

induced apoptosis triggered by FAS-ligand. Therefore, the effects of p16INK4A in leukemia cells by far extend the generally described effect as an inhibitor of cell cycle progression.

Fig. 5. High levels of p16INK4A in human CCRF-CEM T-ALL cells repress the IAP family member Survivin, which is critical for increased FAS-induced cell death, but not for p16INK4A-induced sensitization to glucocorticoid-induced apoptosis. A) CEM/Ctr cells stably express the reverse tet-transactivator rtTA, CEM/p16 cells are derivatives that contain the rtTA and express human p16INK4A under the control of a tetracycline responsive CMV promoter (Ausserlechner et al, 2001). In presence of 200 ng/ml doxycycline, p16INK4A expression is induced, which causes complete loss of Survivin within 48 hour. Retrovirally transduced Survivin compensates for the loss of the

endogenous protein in CEM/p16-Survivin cells. Re-expression of p16INK4A accelerates death-receptor-induced (anti-FAS anibody, 0.1 mg/ml, for four hours) and glucocorticoidinduced apoptosis (10 nM dexamethasone, 24 hours) as shown in B und C (Obexer et al, 2009a). Ectopic expression of Survivin did not change the sensitivity to dexamethasone (C), but prevents increased sensitivity to Fas-induced apoptosis (B). The amount of apoptotic cells was assessed by flow cytometric analysis of propidium-iodide stained nuclei. Each bar

The INK4A genes were discovered in the mid-90s of the last century and intensive studies on their function and regulation have contributed significantly to our current knowledge on

represents the mean of three independent experiments.

**8. Conclusions** 


p16INK4A – Connecting Cell Cycle Control to Cell Death Regulation in Human Leukemia 129

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**8** 

 *Japan* 

**Accumulation of Specific Epigenetic** 

*1Okayama University, Okayama, 2Imamura Bun-in Hospital, Kagoshima* 

**Abnormalities During Development and** 

**Progression of T Cell Leukemia/Lymphoma** 

Takashi Oka1, Hiaki Sato1, Mamoru Ouchida1, Atae Utsunomiya2, Daisuke Ennishi1, Mitsune Tanimoto1 and Tadashi Yoshino1

The genetic abnormalities found in various types of leukemia and lymphoma do not provide a complete picture of the molecular mechanism(s) responsible for hematopoietic malignancies. Aberrant changes in epigenetics, including systems controlling DNA methylation, histone modifications, chromatin remodeling and miRNAs, are additional mechanisms that contribute to the malignant phenotype. DNA methylation is one of the basic mechanisms that controls the development and differentiation, and maintains the normal physiological status, in mammalian cells. DNA methylation is also involved in the regulation of imprinted gene expression and X-chromosome inactivation, and in the finetuning of tissue specific differentiation and development from stem cells. However, aberrant promoter hypermethylation of CpG islands leads to epigenetic silencing of multiple genes, including tumor suppressor genes, and has been recognized as an important mechanism involved in carcinogenesis. Furthermore, multiple genes have been shown to be methylated simultaneously (a condition termed the CpG island methylator phenotype: CIMP) in various types of human malignancies. This mechanism is a fundamental process involved in the development of many tumors. A comprehensive knowledge of the methylation profile of a given tumor may provide important information for risk assessment, diagnosis, monitoring,

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignant disease of CD4 positive T lymphocytes caused by infection with human T-lymphotropic virus type I (HTLV-1). HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years. Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical event(s) involved in the progression have not been characterized in details. The pathogenesis of HTLV-1 has been investigated intensively in terms of the viral regulatory proteins HTLV-1 Tax and Rex, which are supposed to play key roles in the HTLV-1 leukemogenesis/lymphomagenesis, as well as the HTLV-1 basic leucine zipper factor (HBZ). The mechanism(s) underlying the progression of ATLL have been reported from various genetic aspects, including specific chromosome abnormalities and changes in

**1. Introduction** 

and treatments.


### **Accumulation of Specific Epigenetic Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma**

Takashi Oka1, Hiaki Sato1, Mamoru Ouchida1, Atae Utsunomiya2, Daisuke Ennishi1, Mitsune Tanimoto1 and Tadashi Yoshino1 *1Okayama University, Okayama, 2Imamura Bun-in Hospital, Kagoshima Japan* 

#### **1. Introduction**

130 T-Cell Leukemia

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(2004). The differential impact of p16(INK4a) or p19(ARF) deficiency on cell growth

The genetic abnormalities found in various types of leukemia and lymphoma do not provide a complete picture of the molecular mechanism(s) responsible for hematopoietic malignancies. Aberrant changes in epigenetics, including systems controlling DNA methylation, histone modifications, chromatin remodeling and miRNAs, are additional mechanisms that contribute to the malignant phenotype. DNA methylation is one of the basic mechanisms that controls the development and differentiation, and maintains the normal physiological status, in mammalian cells. DNA methylation is also involved in the regulation of imprinted gene expression and X-chromosome inactivation, and in the finetuning of tissue specific differentiation and development from stem cells. However, aberrant promoter hypermethylation of CpG islands leads to epigenetic silencing of multiple genes, including tumor suppressor genes, and has been recognized as an important mechanism involved in carcinogenesis. Furthermore, multiple genes have been shown to be methylated simultaneously (a condition termed the CpG island methylator phenotype: CIMP) in various types of human malignancies. This mechanism is a fundamental process involved in the development of many tumors. A comprehensive knowledge of the methylation profile of a given tumor may provide important information for risk assessment, diagnosis, monitoring, and treatments.

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignant disease of CD4 positive T lymphocytes caused by infection with human T-lymphotropic virus type I (HTLV-1). HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years. Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical event(s) involved in the progression have not been characterized in details. The pathogenesis of HTLV-1 has been investigated intensively in terms of the viral regulatory proteins HTLV-1 Tax and Rex, which are supposed to play key roles in the HTLV-1 leukemogenesis/lymphomagenesis, as well as the HTLV-1 basic leucine zipper factor (HBZ). The mechanism(s) underlying the progression of ATLL have been reported from various genetic aspects, including specific chromosome abnormalities and changes in

Accumulation of Specific Epigenetic

fashion throughout cell division.

left by DNMT1 (Jones & Liang, 2009).

are known to carry out methylation and maintenance.

**2.1 DNA methylation** 

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 133

In the case of eukaryotes, especially in vertebrates, 5-methylcytosine is the predominant modified base in DNA. The 5'-methylation of cytosine residues is a physical modification, and does not inhibit its pairing with guanine nucleotide. In mammals, cytosine methylation is essentially confined to the sequence 5'-CpG-3' (Razin & Riggs, 1980). In certain areas of the genome of mammals, especially in regulatory regions of genes like promoters and enhancers, a high concentration of these CpG dinucleotides is found, and these are referred to as "CpG islands" (CGIs). The methyl-residue is exposed to the major groove of double stranded DNA, and the modification of cytosine in regulatory regions results in the alteration (inhibition or activation) of the interactions between DNA and DNA binding proteins. The methylation of cytosine is catalyzed by DNA methyltransferase enzymes, which can transfer the methyl residue supplied by S-adenosylmethionine (SAM) to cytosine on DNA. In mammals, three DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B,

DNA methyltransferase I (DNMT1) is known as a "maintenance methyltransferase". This enzyme has been shown to have a 10-fold preference for hemi-methylated sites as a substrate. This enzyme can transfer a methyl residue specifically to a newly synthesized strand after semi-conservative replication of methylated DNA, and copies the methylation status of the parental DNA during the division of somatic cells. UHRF1 (also known as NP95) preferentially binds to hemi-methylated sites, and it has been suggested that DNMT1 might be recruited to DNA replication foci by UHRF1 (Sharif et al., 2007; Bostick et al., 2007). Mammalian cells use DNMT1 primarily to maintain the DNA methylation profile in a stable

*De novo* methylation of unmodified DNA is required to form methylation patterns in response to embryogenesis, cell differentiation and extracellular signals. DNMT3A and DNMT3B are known as "*de novo* methyltransferases" which are used to methylate previously unmethylated DNA during development and differentiation. These DNMTs function to discontinuously change the methylation profile for specific compartments of the genome in a tissue-specific manner in vertebrates. In mammals, during the early stage of embryonic development and the early development of primordial germ cells, DNA methylation is erased, followed by introduction of DNA methylation by *de novo* methyltransferases at different sites (Reik, 2007). The *de novo* methylation is carried by DNMT3A and DNMT3B during the early stage of embryonic development, and DNMT3A and its cofactor, DNMT3-like (DBMT3L), are active during germ cell development. Recently, a new model was proposed, in which DNMT3A and DNMT3B, compartmentalized to CpG islands, complete the methylation process and correct errors

In general, the DNA methylation profile is associated with gene repression, and CpG island methylation is involved in the regulation of imprinted gene expression and X-chromosome inactivation, in addition to the fine-tuning of the specific differentiation of cells and their development from stem cells (Csankovszki et al., 2001; Jones & Takai, 2001; Kaneda et al., 2004; Meissner et al., 2008). Aberrant methylation of DNA is also known to be associated with many diseases including malignant tumors, imprinting disorders, and neuronal diseases. Aberrant promoter hypermethylation of tumor suppressor genes is a prevalent phenomenon in human cancers, as well as malignant leukemia/lymphoma, and inhibits the expression of these genes, leading to tumorigenesis in these cells. Recently, it has been

the characteristic HTLV-1 Tax and Rex protein expression pattern, although the detailed mechanism(s) triggering the onset and progression of ATLL remains to be elucidated.

In this chapter, the current state of knowledge about the epigenetic abnormalities that occur during the development and progression of T cell leukemia/lymphoma, especially during adult T- cell leukemia/lymphoma (ATLL), will be reviewed, as will the basic mechanism of epigenetic regulation of gene expression and various clinical aspects of T cell leukemia/lymphoma. In addition, the relevance of this knowledge to leukemia/lymphoma risk assessment, prevention and early detection will be discussed.

#### **2. Epigenetic regulation on gene expression**

The term "epigenetics" was coined by Conrad H. Waddinton in the 1940s, fusing the word "genetics" with "epigenesis". The classical definition proposed by Waddinton involves the heritability of a phenotype, passed on through either mitosis or meiosis. Recently, epigenetics has been proposed as "a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence" (Berger et al., 2009). The pigenetic regulation of gene expression falls mainly into two categories, DNA methylation and histone modification (Figure 1).

Fig. 1. (A) DNAmethylation of CpG islands in the 5' transcriptional regulatory region occurs gene silencing. (B) Histone acetylation and deacetylation regulate gene expression. (C) DNA methylation recruits methyl-CpG binding proteins such as MeCP2, MBD1, MBD2 and MBD4, followed by association with co-repressors such as HDAC complexes, resulting in gene silencing. (D) Histone modification and gene expression state. Ac, acetylation; Me, methylation; H3, histone 3; K, lysine.

#### **2.1 DNA methylation**

132 T-Cell Leukemia

the characteristic HTLV-1 Tax and Rex protein expression pattern, although the detailed

In this chapter, the current state of knowledge about the epigenetic abnormalities that occur during the development and progression of T cell leukemia/lymphoma, especially during adult T- cell leukemia/lymphoma (ATLL), will be reviewed, as will the basic mechanism of epigenetic regulation of gene expression and various clinical aspects of T cell leukemia/lymphoma. In addition, the relevance of this knowledge to leukemia/lymphoma

The term "epigenetics" was coined by Conrad H. Waddinton in the 1940s, fusing the word "genetics" with "epigenesis". The classical definition proposed by Waddinton involves the heritability of a phenotype, passed on through either mitosis or meiosis. Recently, epigenetics has been proposed as "a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence" (Berger et al., 2009). The pigenetic regulation of gene expression falls mainly into two categories, DNA methylation and

Fig. 1. (A) DNAmethylation of CpG islands in the 5' transcriptional regulatory region occurs gene silencing. (B) Histone acetylation and deacetylation regulate gene expression. (C) DNA methylation recruits methyl-CpG binding proteins such as MeCP2, MBD1, MBD2 and MBD4, followed by association with co-repressors such as HDAC complexes, resulting in gene silencing. (D) Histone modification and gene expression state. Ac, acetylation; Me,

mechanism(s) triggering the onset and progression of ATLL remains to be elucidated.

risk assessment, prevention and early detection will be discussed.

**2. Epigenetic regulation on gene expression** 

histone modification (Figure 1).

methylation; H3, histone 3; K, lysine.

In the case of eukaryotes, especially in vertebrates, 5-methylcytosine is the predominant modified base in DNA. The 5'-methylation of cytosine residues is a physical modification, and does not inhibit its pairing with guanine nucleotide. In mammals, cytosine methylation is essentially confined to the sequence 5'-CpG-3' (Razin & Riggs, 1980). In certain areas of the genome of mammals, especially in regulatory regions of genes like promoters and enhancers, a high concentration of these CpG dinucleotides is found, and these are referred to as "CpG islands" (CGIs). The methyl-residue is exposed to the major groove of double stranded DNA, and the modification of cytosine in regulatory regions results in the alteration (inhibition or activation) of the interactions between DNA and DNA binding proteins. The methylation of cytosine is catalyzed by DNA methyltransferase enzymes, which can transfer the methyl residue supplied by S-adenosylmethionine (SAM) to cytosine on DNA. In mammals, three DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B, are known to carry out methylation and maintenance.

DNA methyltransferase I (DNMT1) is known as a "maintenance methyltransferase". This enzyme has been shown to have a 10-fold preference for hemi-methylated sites as a substrate. This enzyme can transfer a methyl residue specifically to a newly synthesized strand after semi-conservative replication of methylated DNA, and copies the methylation status of the parental DNA during the division of somatic cells. UHRF1 (also known as NP95) preferentially binds to hemi-methylated sites, and it has been suggested that DNMT1 might be recruited to DNA replication foci by UHRF1 (Sharif et al., 2007; Bostick et al., 2007). Mammalian cells use DNMT1 primarily to maintain the DNA methylation profile in a stable fashion throughout cell division.

*De novo* methylation of unmodified DNA is required to form methylation patterns in response to embryogenesis, cell differentiation and extracellular signals. DNMT3A and DNMT3B are known as "*de novo* methyltransferases" which are used to methylate previously unmethylated DNA during development and differentiation. These DNMTs function to discontinuously change the methylation profile for specific compartments of the genome in a tissue-specific manner in vertebrates. In mammals, during the early stage of embryonic development and the early development of primordial germ cells, DNA methylation is erased, followed by introduction of DNA methylation by *de novo* methyltransferases at different sites (Reik, 2007). The *de novo* methylation is carried by DNMT3A and DNMT3B during the early stage of embryonic development, and DNMT3A and its cofactor, DNMT3-like (DBMT3L), are active during germ cell development. Recently, a new model was proposed, in which DNMT3A and DNMT3B, compartmentalized to CpG islands, complete the methylation process and correct errors left by DNMT1 (Jones & Liang, 2009).

In general, the DNA methylation profile is associated with gene repression, and CpG island methylation is involved in the regulation of imprinted gene expression and X-chromosome inactivation, in addition to the fine-tuning of the specific differentiation of cells and their development from stem cells (Csankovszki et al., 2001; Jones & Takai, 2001; Kaneda et al., 2004; Meissner et al., 2008). Aberrant methylation of DNA is also known to be associated with many diseases including malignant tumors, imprinting disorders, and neuronal diseases. Aberrant promoter hypermethylation of tumor suppressor genes is a prevalent phenomenon in human cancers, as well as malignant leukemia/lymphoma, and inhibits the expression of these genes, leading to tumorigenesis in these cells. Recently, it has been

Accumulation of Specific Epigenetic

Cernilogar & Orlando, 2005; Cunliffe, 2003).

(Kouzarides, 2007).

**2.3 Transcriptional regulation of genes** 

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 135

islands and histone hyperacetylation. The transcribed body of an active gene is characterized by trimethylation of histone 3 at lysine 36 (H3K36me3), while transcriptionally repressed genes exhibit the trimethylation of histone 3 at lysine 27 (H3K27me3). Permanently silenced genes are characterized by trimethylation at lysine 9 (H3K9me3), with histone hypoacetylation and hypermethylation of CpG islands on their promoters. For these histone methylations, polycomb group (PcG) and Trithorax group (Trx) proteins work on alternative systems of epigenetic memory to regulate gene expression and chromatin structure via modification of histone tails in a heritable manner (Bantignies & Cavalli, 2006;

The multiprotein polycomb complexes are important mediators of transcriptional repression. The PRC2 (Polycomb repressive complex) is responsible for adding methyl groups to H3K27 (Kirmizis et al., 2004). The catalytic component of the PRC2 complex is EZH2, a histone methyltransferase. The cofactors SUZ12 and EED induce EZH2 activity and interact with nucleosomes. The H3K27-methylated histones recruit the PRC1 complex, and PC2, a component of the PRC1 complex, binds to H3K27-methylated histones and blocks gene activation by interfering with the movement of nucleosomes. H3K27-methylated histones also recruit the PRC2 complex to nucleosomes of the nascent DNA strand during DNA replication to continue gene silencing (Hansen et al., 2008). The mutation and overexpression of EZH2 has been reported in malignant cells, especially in diffuse large B-cell lymphomas (Morin et al., 2010). Histone demethylation is mediated by the Jumonji domain (JMJD) enzymes, which remove tri-, di- or monomethyl modifications. H3K27me3 is similarly removed by the JMJD3 and UTX proteins. Alterations of UTX have been found in a variety of tumors (van Haaften et al., 2009). However, the mechanism by which loss of the

Trithorax (Trx) group molecules, such as the MLL/ALL family of genes are methyltransferases for H3K4, and positively regulate the expression of target genes, including multiple HOX genes. MLL is a frequent target for recurrent translocations in acute leukemias that may be characterized as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or mixed lineage leukemia (MLL). More than 50 different MLL fusion partners have been identified so far. Leukemogenic MLL translocations encode MLL fusion proteins that have lost H3K4 methyltransferase activity, and loss of H3K4 methyltransferase activity is strongly associated with disorders of hematopoietic progenitor cells. LSD1 (lysine-specific demethylase-1) removes di- and monomethyl modifications from H3K4

In addition to epigenetic regulation by DNA methylation and histone modifications, the other major regulation system is concerned with transcriptional regulation as a result of remodeling of the chromatin structure. Chromatin is actively remodeled by the SWI/SNF family protein complexes, referred to as chromatin remodeling complexes, which have DNA helicase activity (DNA-dependent ATPase activity) to alter the histone-DNA contacts. Chromatin remodeling complexes carry out transient unwrapping of the DNA end from histone octamers, forming a DNA loop, and moving nucleosomes to different translational positions (sliding). These chromatin remodeling complexes are mainly thought to exert activities that precede transcriptional activation of genes. Among the ATPase subunit group (SMARCA1-6) of chromatin remodeling complexes, SMARCA2/BRM and

H3K27 methylation system leads to cancer remains poorly characterized.

reported that aberrant promoter hypermethylation, referred to as the CpG island methylator phenotype (CIMP), is associated with specific clinical conditions in colorectal cancer, brain tumors, and malignant leukemia/lymphoma, as we will describe later in detail. On the other hand, it is also known that genome-wide hypomethylation is commonly observed in human tumors, and global loss of DNA methylation leads to widespread tumorigenesis as a result of chromosomal instability (Holm et al., 2005).

#### **2.2 Histone modification**

Large eukaryotic genomes in the nucleus are tightly packed, forming the fundamental repeating units referred to as nucleosomes. The nucleosome core particle consists of approximately 147 base pairs of DNA wrapped in left-handed superhelical turns around a histone octamer consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. The N-terminal tail domains of histones comprise 25~30% of the mass of individual histones, and pass through a channel formed by the minor grooves of two DNA strands, and protrude from the surface of the chromatin. The tails of histones are subject to many posttranslational modifications, including methylation of arginines, methylation, acetylation, ubiquitination, ADP-ribosylation, and sumolation of lysines, and phosphorylation of serine and threonine residues. These modifications on the tail domains are considered to be a histone language that is read by other proteins. This language is referred to as the "histone code" (Strahl & Allis, 2000), and also as the "epigenetic code" with regard to histone modification and DNA methylation.

#### **2.2.1 Histone acetylation**

Histone acetylation that occurs at multiple lysine residues of histone 3 (H3) and histone 4 (H4) is associated with active transcription, commonly observed in euchromatin, and is usually carried out by a variety of histone acetyltransferase complexes (HATs) such as p300, CBP and MOZ, which are known as fusion genes in acute myeloid leukemia. Histone acetylation results in a change in the net charge of nucleosomes, which can lead to the decrease of inter- or intranucleosomal DNA-histone interactions. On the other hand, deacetylation of histones occurs as a result of interactions with histone deacetylase complexes (HDACs), and is associated with transcriptional repression. Histone deacetylase complexes, HDAC1 and HDAC2, contain the SIN3 complex and MiNuRD (nucleosome remodeling and deacetylase) complex, and these complexes interact with methylated DNA on gene promoters through methylated DNA binding proteins, MeCP2 and MBD2/MBD3, respectively. SIRT1 is an NAD(+)-dependent histone deacetylase, and is a stress-response and chromatin-silencing factor, which is involved in various nuclear events such as transcription, DNA replication, and DNA repair (Abdelmohsen et al., 2007). The PML-RARA fusion protein induces a block on hematopoietic differentiation and acute promyelocytic leukemia by inactivating target genes via its ability to recruit HDAC3, MBD1 and DNA methyltransferases (Villa et al., 2006). The AML protein, a partner of fusion proteins detected in acute myeloid leukemia, interacts with p300, CBP, MOZ, PML, SIN3A and HDAC.

#### **2.2.2 Histone methylation**

Promoter regions in actively transcribed genes are marked by the presence of a trimethyl mark on histone 3 lysine 4 (H3K4me3), in addition to hypomethylated promoter CpG

reported that aberrant promoter hypermethylation, referred to as the CpG island methylator phenotype (CIMP), is associated with specific clinical conditions in colorectal cancer, brain tumors, and malignant leukemia/lymphoma, as we will describe later in detail. On the other hand, it is also known that genome-wide hypomethylation is commonly observed in human tumors, and global loss of DNA methylation leads to widespread tumorigenesis as a

Large eukaryotic genomes in the nucleus are tightly packed, forming the fundamental repeating units referred to as nucleosomes. The nucleosome core particle consists of approximately 147 base pairs of DNA wrapped in left-handed superhelical turns around a histone octamer consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. The N-terminal tail domains of histones comprise 25~30% of the mass of individual histones, and pass through a channel formed by the minor grooves of two DNA strands, and protrude from the surface of the chromatin. The tails of histones are subject to many posttranslational modifications, including methylation of arginines, methylation, acetylation, ubiquitination, ADP-ribosylation, and sumolation of lysines, and phosphorylation of serine and threonine residues. These modifications on the tail domains are considered to be a histone language that is read by other proteins. This language is referred to as the "histone code" (Strahl & Allis, 2000), and also as the "epigenetic code"

Histone acetylation that occurs at multiple lysine residues of histone 3 (H3) and histone 4 (H4) is associated with active transcription, commonly observed in euchromatin, and is usually carried out by a variety of histone acetyltransferase complexes (HATs) such as p300, CBP and MOZ, which are known as fusion genes in acute myeloid leukemia. Histone acetylation results in a change in the net charge of nucleosomes, which can lead to the decrease of inter- or intranucleosomal DNA-histone interactions. On the other hand, deacetylation of histones occurs as a result of interactions with histone deacetylase complexes (HDACs), and is associated with transcriptional repression. Histone deacetylase complexes, HDAC1 and HDAC2, contain the SIN3 complex and MiNuRD (nucleosome remodeling and deacetylase) complex, and these complexes interact with methylated DNA on gene promoters through methylated DNA binding proteins, MeCP2 and MBD2/MBD3, respectively. SIRT1 is an NAD(+)-dependent histone deacetylase, and is a stress-response and chromatin-silencing factor, which is involved in various nuclear events such as transcription, DNA replication, and DNA repair (Abdelmohsen et al., 2007). The PML-RARA fusion protein induces a block on hematopoietic differentiation and acute promyelocytic leukemia by inactivating target genes via its ability to recruit HDAC3, MBD1 and DNA methyltransferases (Villa et al., 2006). The AML protein, a partner of fusion proteins detected in acute myeloid leukemia, interacts with p300, CBP, MOZ, PML, SIN3A

Promoter regions in actively transcribed genes are marked by the presence of a trimethyl mark on histone 3 lysine 4 (H3K4me3), in addition to hypomethylated promoter CpG

result of chromosomal instability (Holm et al., 2005).

with regard to histone modification and DNA methylation.

**2.2 Histone modification** 

**2.2.1 Histone acetylation** 

and HDAC.

**2.2.2 Histone methylation** 

islands and histone hyperacetylation. The transcribed body of an active gene is characterized by trimethylation of histone 3 at lysine 36 (H3K36me3), while transcriptionally repressed genes exhibit the trimethylation of histone 3 at lysine 27 (H3K27me3). Permanently silenced genes are characterized by trimethylation at lysine 9 (H3K9me3), with histone hypoacetylation and hypermethylation of CpG islands on their promoters. For these histone methylations, polycomb group (PcG) and Trithorax group (Trx) proteins work on alternative systems of epigenetic memory to regulate gene expression and chromatin structure via modification of histone tails in a heritable manner (Bantignies & Cavalli, 2006; Cernilogar & Orlando, 2005; Cunliffe, 2003).

The multiprotein polycomb complexes are important mediators of transcriptional repression. The PRC2 (Polycomb repressive complex) is responsible for adding methyl groups to H3K27 (Kirmizis et al., 2004). The catalytic component of the PRC2 complex is EZH2, a histone methyltransferase. The cofactors SUZ12 and EED induce EZH2 activity and interact with nucleosomes. The H3K27-methylated histones recruit the PRC1 complex, and PC2, a component of the PRC1 complex, binds to H3K27-methylated histones and blocks gene activation by interfering with the movement of nucleosomes. H3K27-methylated histones also recruit the PRC2 complex to nucleosomes of the nascent DNA strand during DNA replication to continue gene silencing (Hansen et al., 2008). The mutation and overexpression of EZH2 has been reported in malignant cells, especially in diffuse large B-cell lymphomas (Morin et al., 2010). Histone demethylation is mediated by the Jumonji domain (JMJD) enzymes, which remove tri-, di- or monomethyl modifications. H3K27me3 is similarly removed by the JMJD3 and UTX proteins. Alterations of UTX have been found in a variety of tumors (van Haaften et al., 2009). However, the mechanism by which loss of the H3K27 methylation system leads to cancer remains poorly characterized.

Trithorax (Trx) group molecules, such as the MLL/ALL family of genes are methyltransferases for H3K4, and positively regulate the expression of target genes, including multiple HOX genes. MLL is a frequent target for recurrent translocations in acute leukemias that may be characterized as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or mixed lineage leukemia (MLL). More than 50 different MLL fusion partners have been identified so far. Leukemogenic MLL translocations encode MLL fusion proteins that have lost H3K4 methyltransferase activity, and loss of H3K4 methyltransferase activity is strongly associated with disorders of hematopoietic progenitor cells. LSD1 (lysine-specific demethylase-1) removes di- and monomethyl modifications from H3K4 (Kouzarides, 2007).

#### **2.3 Transcriptional regulation of genes**

In addition to epigenetic regulation by DNA methylation and histone modifications, the other major regulation system is concerned with transcriptional regulation as a result of remodeling of the chromatin structure. Chromatin is actively remodeled by the SWI/SNF family protein complexes, referred to as chromatin remodeling complexes, which have DNA helicase activity (DNA-dependent ATPase activity) to alter the histone-DNA contacts. Chromatin remodeling complexes carry out transient unwrapping of the DNA end from histone octamers, forming a DNA loop, and moving nucleosomes to different translational positions (sliding). These chromatin remodeling complexes are mainly thought to exert activities that precede transcriptional activation of genes. Among the ATPase subunit group (SMARCA1-6) of chromatin remodeling complexes, SMARCA2/BRM and

Accumulation of Specific Epigenetic

lymphoma (Tomita et al., 2007).

underlying immunodeficiency (Armitage et al., 1998).

this entity is also described in other sections in this issue.

**3.2.1 Initial assessment and staging of T-cell lymphomas** 

assessment of T-cell lymphoma patients (Kako et al., 2007).

**3.2 Management of T-cell lymphomas** 

marker of the disease burden.

**3.3.1 Initial chemotherapy** 

**3.3 Treatment** 

(Suzuki et al., 2000).

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 137

definite histopathological pattern. In particular, cutaneous PTCL has specific histological features, and this lymphoma is defined as a distinct subtype; cutaneous T-cell lymphoma, in the WHO classification. Therefore, PTCL-NOS is often diagnosed by demonstration of a Tcell lineage. The relatively high proportion of patients with PTCL-NOS described in some series of T-cell lymphomas might thus reflect inadequate classification into other T-cell lymphoma subtypes. The clinical behavior of PTCL-NOS is not specific, but it generally has an aggressive clinical course similar to aggressive B-cell lymphoma, but the outcome of PTCL-NOS is poorer than that of aggressive B-cell lymphomas, such as diffuse large B-cell

The clinicopathological features of ALCL depend on the presence of anaplastic large cell lymphoma kinase (ALK). ALK-positive ALCL typicallys arise in 20-30-year-old patients, and mainly in males (Suzuki et al., 2000). The presentation can be both nodal and extranodal, involving the skin, bones, soft tissues, lungs, and liver. On the other hand, ALKnegative ALCL occurs primarily in elderly patients, and its presentation is usually nodal. The prognosis of ALCL is clearly divided into two groups by ALK expression, with the ALK-positive ALCL patients having a better prognosis than the ALK-negative patients

AITL occurs in elderly patients, who are often initially described as having an atypical reactive process with generalized lymphoadenopathy, skin rash, hepatosplenomegaly, fever and hypergammaglobulinemia. The prognosis of AITL is poor and comparable to that of PTCL-NOS, and many patients will die of infectious complications that may be the result of

Other uncommon T-cell lymphomas include enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia/lymphoma (ATLL), hepatosplenic T-cell lymphoma (10), and subcutaneous panniculitis-like T-cell lymphoma. EATL is associated with gluten-sensitive enteropathy and has a fatal clinical course. ATLL is caused by infection with HTLV-1, and

In the process of diagnosing T-cell lymphoma, the assessment of viral infection should be done as early as possible. The histological features and immunophenotype of ATLL are not specific among other T-cell lymphomas, and the detection of HTLV-1 is the only clue to the diagnosis of ATLL. The detection of EBV infection in the serum and lymphoma tissue is also important in T-cell lymphoma patients. In NK-cell lymphoma, the detection of EBV in tissues is an important diagnostic tool. When EBV is detectable in lymphoma or nonlymphoma cells, quantification of EBV DNA by quantitative PCR is a useful surrogate

Radiological procedures including CT and MRI are critical methods used in the staging of Tcell lymphomas. In addition, [18F]-fluorodeoxyglucose (FDG) has recently been reported to be avid in T-cell lymphoma patients, and PET/CT might be a useful procedure for the initial

In the past several decades, conventional anthracycline-based chemotherapy has been the mainstay for the treatment of lymphoma, including T-cell lymphoma. The large

SMARCA4/BRG1 interact with chromatin-modifying enzymes, such as HDAC1, HDAC2, SIN3, and poly (ADP-ribose) polymerase (PARP) 1, and methyl-CpG binding protein MeCP2 (Calvin et al., 2010; Harikrishnan et al., 2005; Sif et al., 2001). In many tumor cells, alterations of the SMARCA2, 4, and 6 genes have been reported (Gunduz et al., 2005; Wong et al., 2000; Yano et al., 2004).

Chromatin remodeling and epigenetic regulation are involved in the intricate control of gene expression. The methyl-CpG binding protein, MeCP2, is involved in histone methyltransferase activity (Fuks et al., 2003) and in regulating DNA methyltransferase DNMT1 (Kimura & Shiota, 2003). Methylated CpG islands in the 5' transcriptional regulatory region recruit methyl-CpG binding proteins such as MeCP2, MBD1, MBD2 and MBD4, followed by association with co-repressors such as HDAC complexes, histone methyltransferases, and chromatin remodeling complexes, thus resulting in the formation of a repressive chromatin structure that leads to gene silencing. This may provide "epigenetic memory" by helping progeny cells to "remember" their cellular identity (Bird, 2002). The epigenetic landscape of the whole genome is different in malignant cells compared to that in normal cells. Epigenetic processes have been implicated in the development of various malignancies, including leukemia/lymphoma, in which the repression or silencing of tumor suppressor genes is remarkably common (Costello et al., 2000; Esteller, 2005; Esteller et al., 2001; Herman & Baylin, 2003; Miremadi et al., 2007).

#### **3. Clinical characteristics of T-cell lymphoma**

T-cell lymphoma is distinct clinicopathological entity classified by the WHO. T-cell lymphoma is a neoplasm with geographical variations in frequency, and the pathogenesis and clinical behavior, including the prognosis, are different from other lymphomas, such as B-cell lymphoma and Hodgkin's lymphoma. In this section, we mainly discuss the clinical features and management of T-cell lymphoma.

#### **3.1 Clinical features of T-cell lymphoma**

#### **3.1.1 Epidemiology**

The incidence of T-cell lymphoma demonstrates interesting geographical variations; in North America and Europe, about 5-10% of lymphomas are T-cell lymphomas (Anderson et al., 2002). However, in Asia, T-cell and natural killer (NK)-cell lymphomas account for 15- 25% of all lymphomas (Au et al., 2005). The higher prevalence of T-cell lymphoma in Asia is reported to be influenced by endemic virus infections, such as human T-cell lymphotropic virus type-I (HTLV-1) and Epstein-Barr virus (EBV). The establishment of management recommendations by Asian oncologists in collaboration with international experts is urgently needed.

#### **3.1.2 Clinical behavior of T-cell lymphoma**

The WHO's classification includes 15 different T-cell lymphomas. Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL) account for 70-80% of T-cell lymphomas (Armitage et al., 1998). The other subtypes of T-cell lymphoma are rare entities.

PTCL-NOS is a heterogeneous subtype that cannot be defined as another specific T-cell lymphoma. Both nodal and extranodal sites can be involved in this lymphoma. The nodal type can be well characterized histologically, but the extranodal type often does not show a definite histopathological pattern. In particular, cutaneous PTCL has specific histological features, and this lymphoma is defined as a distinct subtype; cutaneous T-cell lymphoma, in the WHO classification. Therefore, PTCL-NOS is often diagnosed by demonstration of a Tcell lineage. The relatively high proportion of patients with PTCL-NOS described in some series of T-cell lymphomas might thus reflect inadequate classification into other T-cell lymphoma subtypes. The clinical behavior of PTCL-NOS is not specific, but it generally has an aggressive clinical course similar to aggressive B-cell lymphoma, but the outcome of PTCL-NOS is poorer than that of aggressive B-cell lymphomas, such as diffuse large B-cell lymphoma (Tomita et al., 2007).

The clinicopathological features of ALCL depend on the presence of anaplastic large cell lymphoma kinase (ALK). ALK-positive ALCL typicallys arise in 20-30-year-old patients, and mainly in males (Suzuki et al., 2000). The presentation can be both nodal and extranodal, involving the skin, bones, soft tissues, lungs, and liver. On the other hand, ALKnegative ALCL occurs primarily in elderly patients, and its presentation is usually nodal. The prognosis of ALCL is clearly divided into two groups by ALK expression, with the ALK-positive ALCL patients having a better prognosis than the ALK-negative patients (Suzuki et al., 2000).

AITL occurs in elderly patients, who are often initially described as having an atypical reactive process with generalized lymphoadenopathy, skin rash, hepatosplenomegaly, fever and hypergammaglobulinemia. The prognosis of AITL is poor and comparable to that of PTCL-NOS, and many patients will die of infectious complications that may be the result of underlying immunodeficiency (Armitage et al., 1998).

Other uncommon T-cell lymphomas include enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia/lymphoma (ATLL), hepatosplenic T-cell lymphoma (10), and subcutaneous panniculitis-like T-cell lymphoma. EATL is associated with gluten-sensitive enteropathy and has a fatal clinical course. ATLL is caused by infection with HTLV-1, and this entity is also described in other sections in this issue.

#### **3.2 Management of T-cell lymphomas**

#### **3.2.1 Initial assessment and staging of T-cell lymphomas**

In the process of diagnosing T-cell lymphoma, the assessment of viral infection should be done as early as possible. The histological features and immunophenotype of ATLL are not specific among other T-cell lymphomas, and the detection of HTLV-1 is the only clue to the diagnosis of ATLL. The detection of EBV infection in the serum and lymphoma tissue is also important in T-cell lymphoma patients. In NK-cell lymphoma, the detection of EBV in tissues is an important diagnostic tool. When EBV is detectable in lymphoma or nonlymphoma cells, quantification of EBV DNA by quantitative PCR is a useful surrogate marker of the disease burden.

Radiological procedures including CT and MRI are critical methods used in the staging of Tcell lymphomas. In addition, [18F]-fluorodeoxyglucose (FDG) has recently been reported to be avid in T-cell lymphoma patients, and PET/CT might be a useful procedure for the initial assessment of T-cell lymphoma patients (Kako et al., 2007).

#### **3.3 Treatment**

136 T-Cell Leukemia

SMARCA4/BRG1 interact with chromatin-modifying enzymes, such as HDAC1, HDAC2, SIN3, and poly (ADP-ribose) polymerase (PARP) 1, and methyl-CpG binding protein MeCP2 (Calvin et al., 2010; Harikrishnan et al., 2005; Sif et al., 2001). In many tumor cells, alterations of the SMARCA2, 4, and 6 genes have been reported (Gunduz et al., 2005; Wong

Chromatin remodeling and epigenetic regulation are involved in the intricate control of gene expression. The methyl-CpG binding protein, MeCP2, is involved in histone methyltransferase activity (Fuks et al., 2003) and in regulating DNA methyltransferase DNMT1 (Kimura & Shiota, 2003). Methylated CpG islands in the 5' transcriptional regulatory region recruit methyl-CpG binding proteins such as MeCP2, MBD1, MBD2 and MBD4, followed by association with co-repressors such as HDAC complexes, histone methyltransferases, and chromatin remodeling complexes, thus resulting in the formation of a repressive chromatin structure that leads to gene silencing. This may provide "epigenetic memory" by helping progeny cells to "remember" their cellular identity (Bird, 2002). The epigenetic landscape of the whole genome is different in malignant cells compared to that in normal cells. Epigenetic processes have been implicated in the development of various malignancies, including leukemia/lymphoma, in which the repression or silencing of tumor suppressor genes is remarkably common (Costello et al., 2000; Esteller, 2005; Esteller et al.,

T-cell lymphoma is distinct clinicopathological entity classified by the WHO. T-cell lymphoma is a neoplasm with geographical variations in frequency, and the pathogenesis and clinical behavior, including the prognosis, are different from other lymphomas, such as B-cell lymphoma and Hodgkin's lymphoma. In this section, we mainly discuss the clinical

The incidence of T-cell lymphoma demonstrates interesting geographical variations; in North America and Europe, about 5-10% of lymphomas are T-cell lymphomas (Anderson et al., 2002). However, in Asia, T-cell and natural killer (NK)-cell lymphomas account for 15- 25% of all lymphomas (Au et al., 2005). The higher prevalence of T-cell lymphoma in Asia is reported to be influenced by endemic virus infections, such as human T-cell lymphotropic virus type-I (HTLV-1) and Epstein-Barr virus (EBV). The establishment of management recommendations by Asian oncologists in collaboration with international experts is

The WHO's classification includes 15 different T-cell lymphomas. Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL) account for 70-80% of T-cell lymphomas

PTCL-NOS is a heterogeneous subtype that cannot be defined as another specific T-cell lymphoma. Both nodal and extranodal sites can be involved in this lymphoma. The nodal type can be well characterized histologically, but the extranodal type often does not show a

(Armitage et al., 1998). The other subtypes of T-cell lymphoma are rare entities.

et al., 2000; Yano et al., 2004).

2001; Herman & Baylin, 2003; Miremadi et al., 2007).

**3. Clinical characteristics of T-cell lymphoma** 

features and management of T-cell lymphoma.

**3.1 Clinical features of T-cell lymphoma** 

**3.1.2 Clinical behavior of T-cell lymphoma** 

**3.1.1 Epidemiology** 

urgently needed.

#### **3.3.1 Initial chemotherapy**

In the past several decades, conventional anthracycline-based chemotherapy has been the mainstay for the treatment of lymphoma, including T-cell lymphoma. The large

Accumulation of Specific Epigenetic

**3.3.3 Novel therapeutic agents** 

treatment options.

the EFS was only 7 months (O'Connor, 2010).

disease, were often observed (Gallamini et al., 2007).

**leukemia-lymphoma -The clinical aspects-4.1 Epidemiology, etiology, and leukemogenesis** 

international study of romideptin in PTCL patients is ongoing.

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 139

(EFS) rates were 57% and 53%, respectively, in almost non-complete response (CR) patients (Le Gouill et al., 2008). However the 1-year TRM was 32% in patients treated using a myeloablative conditioning regimen, indicating that further prospective trials, including

Several new agents, including molecular targeting drugs, have been studied. Gemcitabine has been investigated in several combination chemotherapy regimens. When gemcitabine was combined with etoposide and CHOP for the treatment of 26 patients with T-cell lymphoma, favorable results were demonstrated, including an overall response rate of 77%, with 62% achieving a CR. However, 54% of patients experienced severe neutropenia, and

Alemtuzumab is a humanized monoclonal antibody against CD52, which is expressed on both T cells and B cells. In 24 patients with T-cell lymphomas, alemtuzumab plus CHOP treatment resulted in a CR in 71% of patients, and a 1-year OS of 70%, and 2-year OS of 53%. However, severe infective complications, such as invasive aspergillosis and cytomegalovirus

Romidepin was the first histone deacetylase inhibitor (HDACi) to show efficacy in patients with PTCL or cutaneous T-cell lymphoma (CTCL). In a report of four patients treated in a phase 1 study, one patient with PTCL-NOS had a CR, and prompted a subsequent phase 2 study to assess its efficacy in patients with CTCL (Sandor et al., 2002). These two trials resulted in the FDA approval of the agent for patients with CTCL. Romideptin was also studied in patients with PTCL in a multicenter study; leading to an overall response rate of 33%, with a CR rate of 11% (Piekarz et al., 2009). On the basis of these results, a confirmatory

In conclusion, T-cell lymphoma is a distinct subtype of lymphoma, based on its unique epidemiology and clinical behavior. However, the optimal treatment strategy is undefined, and a prognostic model remains unclear due to the rarity of this entity. PTCL, the most common T-cell lymphoma, has a poor prognosis when patients are treated with conventional chemotherapy, and a large scale study is needed to establish more effective chemotherapy regimens, including HSCT. Novel targeted agents have been and are currently being examined for efficacy against the disease and to decrease the toxicity for the patients, and an improved understanding of the biology of PTCLs may give rise to new

**4. Leukemogenesis/lymphomagenesis and the progression of adult T-cell** 

Adult T-cell leukemia-lymphoma (ATLL) is a mature T-cell malignancy, caused by human T-cell leukemia virus type-I (HTLV-1)(Poiesz *et al*., 1980), and is characterized by lymphadenopathy, hepatosplenomegaly, skin lesions, the appearance of abnormal lymphocytes with convoluted or lobulated nuclei in the peripheral blood (PB) and specific geographic distributions (Uchiyama *et al*., 1977). ATLL cells are often resistant to conventional chemotherapeutic agents associated with the expression of P-glycoprotein (Kuwazuru *et al*., 1990) or functional lung resistance-related protein (Ohno *et al*., 2001), and

reduced induction stem cell transplantation, will be necessary.

international group trial established that cyclophosphamide, doxorubicin, vincristine, and predonisone (CHOP) was equally effective and less toxic than intensive second and third generation chemotherapy for aggressive lymphoma (Fisher et al., 1993). CHOP or CHOPtype chemotherapy is now considered to be the standard treatment for peripheral T-cell lymphomas, including PTCL-NOS, AITL and ALCL. However, the results of treatment with a CHOP-like regimen for T-cell lymphoma is poor, with 5-year overall survival (OS) of 10- 45% (Armitage et al., 1998; López-Guillermo et al., 1998). Due to the low incidence of T-cell lymphoma, the optimum treatment regimen for T-cell lymphoma has not been studied prospectively in randomized controlled trials, and no effective regimen other than CHOP has been established. Although ALK-positive ALCL patients have a good prognosis even when treated using the CHOP regimen (Suzuki et al., 2000), other PTCL patients will need more efficacious regimens.

Recently, modern dose-intense regimens have been investigated for aggressive lymphoma. A cyclophosphamide, doxorubicin, vincristine, dexamethasone (hyper CVAD) regimen was reported to be effective against Burkitt's lymphoma or mantle cell lymphoma, and a study of the hyper CVAD regimen for T-cell lymphoma patients showed a 3-year OS that was similar to that obtained using CHOP (49% and 43%)(Escalón et al., 2005). A French group showed that the cyclophosphamide, doxorubicin, vincristine, bleomycin, and prednisone (ACVBP) regimen was associated with a significant better 5-year OS than CHOP (46% vs 38%) in a randomized trial of patients with various types of aggressive lymphomas (Tilly et al., 2003). However, T-cell lymphoma patients accounted for only 15% of the cases evaluated in this study. Randomized trials will be necessary for more accurate assessment of the efficacy of this regimen for T-cell lymphoma.

#### **3.3.2 Hematopoietic stem cell transplantation**

Because of the generally poor outcome obtained with initial conventional chemotherapy, high-dose chemotherapy with autologous stem cell transplantation (ASCT) has been considered as a part of initial treatment for T-cell lymphoma. Numerous studies have shown favorable outcomes with low treatment-related mortality (TRM) (median OS was 50-70 months), particularly in advanced stage patients (Rodriguezet al., 2003 & 2007; Feyler et al., 2007). One study excluding ALK-positive ALCL patients, who are known to have a good prognosis with chemotherapy alone, showed that the median OS was 54 months, which was similar to the results of chemotherapy alone (Mounier et al., 2004). The trial conducted by the EBMT including 146 AILT patients reported that the median OS was 59 months, with low TRM (7%), indicating that ASCT should be considered as a useful treatment strategy for AITL patients (Kyriakou et al., 2008). Although most studies were retrospective and included ALK-positive ALCL patients, the favorable results and low toxicity indicated that ASCT is a promising strategy for PTCL patients. To clarify the patients who would benefit most from ASCT, further investigations in a prospective randomized setting are warranted.

Allogeneic HSCT is considered as a salvage treatment for relapsed or refractory patients. Corradini et al conducted a Phase 2 study of 17 relapsed or refractory patients, and showed that there was a good outcome, with 64% and 80% 1-year disease-free survival (DFS) and OS respectively. Of interest, several patients responded to donor lymphocyte infusion, suggesting that there was a graft-versus lymphoma effect (Corradini et al., 2004). Another author reported their retrospective experience with seventy-seven PTCL patients who received an allogeneic HSCT. This study showed that the 5-year OS and event-free survival (EFS) rates were 57% and 53%, respectively, in almost non-complete response (CR) patients (Le Gouill et al., 2008). However the 1-year TRM was 32% in patients treated using a myeloablative conditioning regimen, indicating that further prospective trials, including reduced induction stem cell transplantation, will be necessary.

#### **3.3.3 Novel therapeutic agents**

138 T-Cell Leukemia

international group trial established that cyclophosphamide, doxorubicin, vincristine, and predonisone (CHOP) was equally effective and less toxic than intensive second and third generation chemotherapy for aggressive lymphoma (Fisher et al., 1993). CHOP or CHOPtype chemotherapy is now considered to be the standard treatment for peripheral T-cell lymphomas, including PTCL-NOS, AITL and ALCL. However, the results of treatment with a CHOP-like regimen for T-cell lymphoma is poor, with 5-year overall survival (OS) of 10- 45% (Armitage et al., 1998; López-Guillermo et al., 1998). Due to the low incidence of T-cell lymphoma, the optimum treatment regimen for T-cell lymphoma has not been studied prospectively in randomized controlled trials, and no effective regimen other than CHOP has been established. Although ALK-positive ALCL patients have a good prognosis even when treated using the CHOP regimen (Suzuki et al., 2000), other PTCL patients will need

Recently, modern dose-intense regimens have been investigated for aggressive lymphoma. A cyclophosphamide, doxorubicin, vincristine, dexamethasone (hyper CVAD) regimen was reported to be effective against Burkitt's lymphoma or mantle cell lymphoma, and a study of the hyper CVAD regimen for T-cell lymphoma patients showed a 3-year OS that was similar to that obtained using CHOP (49% and 43%)(Escalón et al., 2005). A French group showed that the cyclophosphamide, doxorubicin, vincristine, bleomycin, and prednisone (ACVBP) regimen was associated with a significant better 5-year OS than CHOP (46% vs 38%) in a randomized trial of patients with various types of aggressive lymphomas (Tilly et al., 2003). However, T-cell lymphoma patients accounted for only 15% of the cases evaluated in this study. Randomized trials will be necessary for more accurate assessment of the

Because of the generally poor outcome obtained with initial conventional chemotherapy, high-dose chemotherapy with autologous stem cell transplantation (ASCT) has been considered as a part of initial treatment for T-cell lymphoma. Numerous studies have shown favorable outcomes with low treatment-related mortality (TRM) (median OS was 50-70 months), particularly in advanced stage patients (Rodriguezet al., 2003 & 2007; Feyler et al., 2007). One study excluding ALK-positive ALCL patients, who are known to have a good prognosis with chemotherapy alone, showed that the median OS was 54 months, which was similar to the results of chemotherapy alone (Mounier et al., 2004). The trial conducted by the EBMT including 146 AILT patients reported that the median OS was 59 months, with low TRM (7%), indicating that ASCT should be considered as a useful treatment strategy for AITL patients (Kyriakou et al., 2008). Although most studies were retrospective and included ALK-positive ALCL patients, the favorable results and low toxicity indicated that ASCT is a promising strategy for PTCL patients. To clarify the patients who would benefit most from ASCT, further investigations in a prospective

Allogeneic HSCT is considered as a salvage treatment for relapsed or refractory patients. Corradini et al conducted a Phase 2 study of 17 relapsed or refractory patients, and showed that there was a good outcome, with 64% and 80% 1-year disease-free survival (DFS) and OS respectively. Of interest, several patients responded to donor lymphocyte infusion, suggesting that there was a graft-versus lymphoma effect (Corradini et al., 2004). Another author reported their retrospective experience with seventy-seven PTCL patients who received an allogeneic HSCT. This study showed that the 5-year OS and event-free survival

more efficacious regimens.

efficacy of this regimen for T-cell lymphoma.

randomized setting are warranted.

**3.3.2 Hematopoietic stem cell transplantation** 

Several new agents, including molecular targeting drugs, have been studied. Gemcitabine has been investigated in several combination chemotherapy regimens. When gemcitabine was combined with etoposide and CHOP for the treatment of 26 patients with T-cell lymphoma, favorable results were demonstrated, including an overall response rate of 77%, with 62% achieving a CR. However, 54% of patients experienced severe neutropenia, and the EFS was only 7 months (O'Connor, 2010).

Alemtuzumab is a humanized monoclonal antibody against CD52, which is expressed on both T cells and B cells. In 24 patients with T-cell lymphomas, alemtuzumab plus CHOP treatment resulted in a CR in 71% of patients, and a 1-year OS of 70%, and 2-year OS of 53%. However, severe infective complications, such as invasive aspergillosis and cytomegalovirus disease, were often observed (Gallamini et al., 2007).

Romidepin was the first histone deacetylase inhibitor (HDACi) to show efficacy in patients with PTCL or cutaneous T-cell lymphoma (CTCL). In a report of four patients treated in a phase 1 study, one patient with PTCL-NOS had a CR, and prompted a subsequent phase 2 study to assess its efficacy in patients with CTCL (Sandor et al., 2002). These two trials resulted in the FDA approval of the agent for patients with CTCL. Romideptin was also studied in patients with PTCL in a multicenter study; leading to an overall response rate of 33%, with a CR rate of 11% (Piekarz et al., 2009). On the basis of these results, a confirmatory international study of romideptin in PTCL patients is ongoing.

In conclusion, T-cell lymphoma is a distinct subtype of lymphoma, based on its unique epidemiology and clinical behavior. However, the optimal treatment strategy is undefined, and a prognostic model remains unclear due to the rarity of this entity. PTCL, the most common T-cell lymphoma, has a poor prognosis when patients are treated with conventional chemotherapy, and a large scale study is needed to establish more effective chemotherapy regimens, including HSCT. Novel targeted agents have been and are currently being examined for efficacy against the disease and to decrease the toxicity for the patients, and an improved understanding of the biology of PTCLs may give rise to new treatment options.

#### **4. Leukemogenesis/lymphomagenesis and the progression of adult T-cell leukemia-lymphoma -The clinical aspects-**

#### **4.1 Epidemiology, etiology, and leukemogenesis**

Adult T-cell leukemia-lymphoma (ATLL) is a mature T-cell malignancy, caused by human T-cell leukemia virus type-I (HTLV-1)(Poiesz *et al*., 1980), and is characterized by lymphadenopathy, hepatosplenomegaly, skin lesions, the appearance of abnormal lymphocytes with convoluted or lobulated nuclei in the peripheral blood (PB) and specific geographic distributions (Uchiyama *et al*., 1977). ATLL cells are often resistant to conventional chemotherapeutic agents associated with the expression of P-glycoprotein (Kuwazuru *et al*., 1990) or functional lung resistance-related protein (Ohno *et al*., 2001), and

Accumulation of Specific Epigenetic

the hypercalcemia associated with ATLL.

**4.3 Hematological and laboratory features** 

and organ infiltration of ATLL cells (Dewan *et al*., 2008).

**4.4 Diagnosis and classification** 

**4.2 Clinical features** 

(Shimoyama *et al*., 1991).

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 141

The signs or symptoms frequently seen at the onset of ATLL include lymph node swelling, hepatosplenomegaly and skin lesions. ATLL patients also often suffer from abdominal symptoms such as abdominal pain or refractory diarrhea due to infiltration of ATLL cells into the gastrointestinal (GI) tract (Utsunomiya *et al*., 1988), and headache or disturbance of consciousness due to infiltration of ATLL cells into the central nervous systems (CNS). In addition, cough or dyspnea due to pleural effusion or lung infiltration of ATLL cells, abdominal distension due to lymph node swelling in the abdominal cavity, hepatosplenomegaly and/or ascites often distress ATLL patients. General fatigue, muscle weakness, constipation, and disturbance of consciousness are also seen, and are caused by

Opportunistic infections are common in ATLL patients due to impairment of cellular immunity. In particular, fungal (cutaneous, pulmonary, oral, esophageal and meningeal) and protozoal (*Pneumocystis carinii, Strongyloides stercoralis*) infections are often seen at diagnosis, mainly in the acute or chronic, rather than the lymphoma type, of ATLL

Leukocytes often increase from moderate to marked levels in leukemic type ATLL, while anemia and thrombocytopenia are rarely seen or mild, if they occur at all. Increases in the serum level of lactate dehydrogenase (LDH), serum calcium and soluble interleukin-2 receptor (sIL-2R) are frequently observed. Neutrophilia and/or eosinophilia are also observed due to the increased level of cytokines produced by the ATLL cells. Eosinophilia is a poor prognostic factor in ATLL patients (Utsunomiya *et al*., 2007). Hypercalcemia occurs more frequently in patients with aggressive ATLL, not only at the onset but also at relapse or upon transformation from an indolent to aggressive form. The mechanism underlying hypercalcemia is thought to be associated with the expression of parathyroid hormone-related peptides (PTHrP) (Watanabe *et a*l., 1990) or tumor necrosis factor-β (TNF-β) (Ishibashi *et al*., 1991). In addition, over expression of receptor activator of nuclear factor-κB ligand (RANKL) on ATLL cells was found to correlate with hypercalcemia in ATLL patients (Nosaka *et al*., 2002). The tumor suppressor lung cancer 1 (TSLC1) gene was initially identified as a novel cell surface marker for ATLL. Afterward the expression of TSLC1 was found to be associated with tumor growth

ATLL is diagnosed as peripheral T-cell leukemia or lymphoma by cytology and the surface phenotype of tumor cells, and/or pathology combined with immunohistochemical findings. Positivity for anti-HTLV-1 antibodies in the sera is mandatory for a diagnosis of ATLL. Most ATLL cells have a CD4+CD8- surface phenotype, and other unusual phenotypes such as CD4+CD8+, CD4-CD8+, CD4-CD8- are seen in about 20% of ATLL patients. The patients with these unusual phenotypes have a poorer prognosis than the patients with the typical phenotype (Kamihira, *et al*., 1992). ATLL cells also express CD25, CCR4 and FoxP3. Histologically, the lymph nodes are occupied by diffuse proliferation of lymphoma cells with resultant destruction of the lymph node structure. Extranodal lesions such as those in the GI tract, skin or lungs should be diagnosed by histological examination. In addition to the presence of HTLV-1 antibodies in the sera, the detection of monoclonal integration of HTLV-1 proviral DNA in leukemia cells or tumor cells is necessary for a definite diagnosis of ATLL.

ATLL patients often present with opportunistic infections (Shimoyama *et al*., 1991). At present, the therapeutic outcomes of patients with acute or lymphoma type ATLL are still very poor.

It is estimated that over one million peoples infected by HTLV-1 live in Japan (Yamaguchi *et al*., 2002) and that 15-20 million peoples are infected worldwide (Proietti *et al*., 2005). Only a small percentage of HTLV-1 carriers develop ATLL at a median age of 67 in Japan, whose median age is older than those in other countries. The cumulative risk of ATLL development in HTLV-1 carriers from 30 to 79 years of age was estimated to be 2.1% for females and 6.6% for males (Arisawa *et al*., 2000). Recently, the Joint Study on Predisposing Factors on ATLL Development (JSPFAD) Group performed a large scale cohort study between 2002 and 2008 for HTLV-1 carriers in order to clarify the risk factors for the development of ATLL. During this period, 14 cases out of 1,218 HTLV-1 carriers developed ATLL. This study revealed 4 major risk factors for the development of ATLL in HTLV-1 carriers using a multivariate analysis, i.e., high HTLV-1 proviral loads (in other words, an increase in HTLV-1 infected cells) in the PB, advanced age (over 40 years of age), the existence of a family history of ATLL, and detecting HTLV-1 antibody positivity during treatment for other diseases (Iwanaga *et al*., 2010). Familial ATLL cases were reported by several researchers (Miyamoto *et al*., 1985; Ratner *et al*., 1990; Wilks *et al*., 1993). Surprisingly, we experienced a family with accumulated familial ATLL, in which six of seven siblings (excluding one who died during World War II) developed acute type ATLL between 1978 and 1989 (Nomura *et al*., 2006).

In HTLV-1 leukemogenesis, the HTLV-1 viral protein Tax activates nuclear factor-κB (NFκB), represses p53, and is associated with various other protein-protein interactions (Yoshida, 2001). In particular, Tax plays an important role in the early phase of HTLV-1 leukemogenesis by immortalization of HTLV-1 infected T cells. On the other hand, cells expressing Tax are eradicated by the normal immune surveillance system by Tax- specific cytotoxic T lymphocytes (CTL). The accumulation of gene impairment finally results in leukemogenesis/lymphomagenesis of ATLL in HTLV-1 infected cells that escape from the CTL. However, ATLL cells frequently lack Tax expression or carry deletions in the Tax gene. Therefore, the Tax gene has been suggested to be non-essential for the proliferation of ATLL cells. On the other hand, HTLV-1 basic leucine zipper factor gene (HBZ) is expressed on the ATLL cells in all ATLL patients, and supports the proliferation of ATLL cells (Satou *et al*., 2006). HBZ is now considered to be vital for the leukemogenesis and progression of ATLL. Interestingly, there is a distinct mechanism of flower cell formation in ATLL cells which is a characteristic feature of the acute type ATLL demonstrated by Fukuda *et al* (2005). The

multilobulated nuclear formation in ATLL cells is induced by overactivation of phosphatidylinositol 3-kinase signaling cascades resulting from disruption of phosphatidylinositol-3,4,5-triphosphate inositol phosphatases such as the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP). Moreover this aberrantly activated signaling pathway is suggested to have an essential role in the development of ATLL in patients.

Recently, it has been reported that ATLL cells are derived from regulatory T (Treg) cells or helper T cell type 2 (Th2) cells both of which express CD4 and CD25 on their cell surface. Because ATLL cells express CC chemokine receptor 4 (CCR4) which is expressed on both Treg and Th2 cells, and forkhead/winged helix transcription factor (FoxP3) which is expressed on Treg cells in most ATLL patients, ATLL cells are now thought to be mainly of Treg cell origin (Karube *et al*., 2004).

#### **4.2 Clinical features**

140 T-Cell Leukemia

ATLL patients often present with opportunistic infections (Shimoyama *et al*., 1991). At present, the therapeutic outcomes of patients with acute or lymphoma type ATLL are still

It is estimated that over one million peoples infected by HTLV-1 live in Japan (Yamaguchi *et al*., 2002) and that 15-20 million peoples are infected worldwide (Proietti *et al*., 2005). Only a small percentage of HTLV-1 carriers develop ATLL at a median age of 67 in Japan, whose median age is older than those in other countries. The cumulative risk of ATLL development in HTLV-1 carriers from 30 to 79 years of age was estimated to be 2.1% for females and 6.6% for males (Arisawa *et al*., 2000). Recently, the Joint Study on Predisposing Factors on ATLL Development (JSPFAD) Group performed a large scale cohort study between 2002 and 2008 for HTLV-1 carriers in order to clarify the risk factors for the development of ATLL. During this period, 14 cases out of 1,218 HTLV-1 carriers developed ATLL. This study revealed 4 major risk factors for the development of ATLL in HTLV-1 carriers using a multivariate analysis, i.e., high HTLV-1 proviral loads (in other words, an increase in HTLV-1 infected cells) in the PB, advanced age (over 40 years of age), the existence of a family history of ATLL, and detecting HTLV-1 antibody positivity during treatment for other diseases (Iwanaga *et al*., 2010). Familial ATLL cases were reported by several researchers (Miyamoto *et al*., 1985; Ratner *et al*., 1990; Wilks *et al*., 1993). Surprisingly, we experienced a family with accumulated familial ATLL, in which six of seven siblings (excluding one who died during World War II) developed acute type ATLL between 1978

In HTLV-1 leukemogenesis, the HTLV-1 viral protein Tax activates nuclear factor-κB (NFκB), represses p53, and is associated with various other protein-protein interactions (Yoshida, 2001). In particular, Tax plays an important role in the early phase of HTLV-1 leukemogenesis by immortalization of HTLV-1 infected T cells. On the other hand, cells expressing Tax are eradicated by the normal immune surveillance system by Tax- specific cytotoxic T lymphocytes (CTL). The accumulation of gene impairment finally results in leukemogenesis/lymphomagenesis of ATLL in HTLV-1 infected cells that escape from the CTL. However, ATLL cells frequently lack Tax expression or carry deletions in the Tax gene. Therefore, the Tax gene has been suggested to be non-essential for the proliferation of ATLL cells. On the other hand, HTLV-1 basic leucine zipper factor gene (HBZ) is expressed on the ATLL cells in all ATLL patients, and supports the proliferation of ATLL cells (Satou *et al*., 2006). HBZ is now considered to be vital for the leukemogenesis and progression of ATLL. Interestingly, there is a distinct mechanism of flower cell formation in ATLL cells which is a characteristic feature of the acute type ATLL demonstrated by Fukuda *et al* (2005). The multilobulated nuclear formation in ATLL cells is induced by overactivation of phosphatidylinositol 3-kinase signaling cascades resulting from disruption of phosphatidylinositol-3,4,5-triphosphate inositol phosphatases such as the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP). Moreover this aberrantly activated signaling pathway is suggested to have an essential role in the development of ATLL in patients. Recently, it has been reported that ATLL cells are derived from regulatory T (Treg) cells or helper T cell type 2 (Th2) cells both of which express CD4 and CD25 on their cell surface. Because ATLL cells express CC chemokine receptor 4 (CCR4) which is expressed on both Treg and Th2 cells, and forkhead/winged helix transcription factor (FoxP3) which is expressed on Treg cells in most ATLL patients, ATLL cells are now thought to be mainly of

very poor.

and 1989 (Nomura *et al*., 2006).

Treg cell origin (Karube *et al*., 2004).

The signs or symptoms frequently seen at the onset of ATLL include lymph node swelling, hepatosplenomegaly and skin lesions. ATLL patients also often suffer from abdominal symptoms such as abdominal pain or refractory diarrhea due to infiltration of ATLL cells into the gastrointestinal (GI) tract (Utsunomiya *et al*., 1988), and headache or disturbance of consciousness due to infiltration of ATLL cells into the central nervous systems (CNS). In addition, cough or dyspnea due to pleural effusion or lung infiltration of ATLL cells, abdominal distension due to lymph node swelling in the abdominal cavity, hepatosplenomegaly and/or ascites often distress ATLL patients. General fatigue, muscle weakness, constipation, and disturbance of consciousness are also seen, and are caused by the hypercalcemia associated with ATLL.

Opportunistic infections are common in ATLL patients due to impairment of cellular immunity. In particular, fungal (cutaneous, pulmonary, oral, esophageal and meningeal) and protozoal (*Pneumocystis carinii, Strongyloides stercoralis*) infections are often seen at diagnosis, mainly in the acute or chronic, rather than the lymphoma type, of ATLL (Shimoyama *et al*., 1991).

#### **4.3 Hematological and laboratory features**

Leukocytes often increase from moderate to marked levels in leukemic type ATLL, while anemia and thrombocytopenia are rarely seen or mild, if they occur at all. Increases in the serum level of lactate dehydrogenase (LDH), serum calcium and soluble interleukin-2 receptor (sIL-2R) are frequently observed. Neutrophilia and/or eosinophilia are also observed due to the increased level of cytokines produced by the ATLL cells. Eosinophilia is a poor prognostic factor in ATLL patients (Utsunomiya *et al*., 2007). Hypercalcemia occurs more frequently in patients with aggressive ATLL, not only at the onset but also at relapse or upon transformation from an indolent to aggressive form. The mechanism underlying hypercalcemia is thought to be associated with the expression of parathyroid hormone-related peptides (PTHrP) (Watanabe *et a*l., 1990) or tumor necrosis factor-β (TNF-β) (Ishibashi *et al*., 1991). In addition, over expression of receptor activator of nuclear factor-κB ligand (RANKL) on ATLL cells was found to correlate with hypercalcemia in ATLL patients (Nosaka *et al*., 2002). The tumor suppressor lung cancer 1 (TSLC1) gene was initially identified as a novel cell surface marker for ATLL. Afterward the expression of TSLC1 was found to be associated with tumor growth and organ infiltration of ATLL cells (Dewan *et al*., 2008).

#### **4.4 Diagnosis and classification**

ATLL is diagnosed as peripheral T-cell leukemia or lymphoma by cytology and the surface phenotype of tumor cells, and/or pathology combined with immunohistochemical findings. Positivity for anti-HTLV-1 antibodies in the sera is mandatory for a diagnosis of ATLL. Most ATLL cells have a CD4+CD8- surface phenotype, and other unusual phenotypes such as CD4+CD8+, CD4-CD8+, CD4-CD8- are seen in about 20% of ATLL patients. The patients with these unusual phenotypes have a poorer prognosis than the patients with the typical phenotype (Kamihira, *et al*., 1992). ATLL cells also express CD25, CCR4 and FoxP3. Histologically, the lymph nodes are occupied by diffuse proliferation of lymphoma cells with resultant destruction of the lymph node structure. Extranodal lesions such as those in the GI tract, skin or lungs should be diagnosed by histological examination. In addition to the presence of HTLV-1 antibodies in the sera, the detection of monoclonal integration of HTLV-1 proviral DNA in leukemia cells or tumor cells is necessary for a definite diagnosis of ATLL.

Accumulation of Specific Epigenetic

**4.6 Spontaneous regression** 

new immunological therapy for ATLL patients.

suffering from symptomatic skin lesions.

**4.7 Therapy** 

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 143

marked skin manifestations suddenly occur in previously indolent ATLL, often accompanied by marked leukocytosis, an increase in the serum LDH, sIL-2R and/or hypercalcemia. In particular, the sIL-2R level has been considered to be an indicator of disease progression and prognosis (Kamihira *et al*., 1994). Multi-step aberrant CpG island hyper-methylation was detected in ATLL patients, which was associated with the progression and transformation (crisis) of ATLL (Sato *et al*., 2010). Clonal evolution of ATLL

Few ATLL patients show spontaneous regression of tumors (Shimamoto *et al*., 1993). We experienced two chronic type ATLL patients, both of whom had a poor prognostic factor (increased serum LDH), who obtained a complete remission (CR) without any therapeutic intervention. In one patient, the systemic lymphadenopathy and ATLL cells in the PB disappeared, and the serum LDH level was normalized after surgical excision of an inguinal lymph node. However, he suffered from bone pain due to multiple bone lesions infiltrated by ATLL cells about 10 months after the CR. In another patient, the leukocytes and abnormal lymphocytes in the PB, and the serum LDH level gradually decreased to normal range. The ATLL cells in her PB had disappeared completely about 6 years after the diagnosis of ATLL without any therapy. She is now in an HTLV-1 carrier state, and has been free from ATLL for about 7 years after the complete disappearance of the ATLL cells in her PB. Although the mechanisms of spontaneous regression of ATLL have not been elucidated, it is suggested that the cytotoxic activity of peripheral mononuclear cells or the apoptosis of ATLL cells are associated with this phenomenon (Jinnohara *et al*., 1997; Matsushita *et al*., 1999). Clarification of this interesting phenomenon might be useful for the development of

Treatment for patients with ATLL differs according to the clinical subtypes. It therefore is very important to make an accurate diagnosis of the clinical subtype of ATLL in order to ensure that the appropriate therapy is selected. In patients with indolent ATLL including those with the smoldering type or the chronic type without any unfavorable prognostic factors, watchful waiting is the standard of care in Japan except when the patients are

Generally, intensive combination chemotherapy for aggressive ATLL has been performed immediately after the diagnosis because the prognoses of aggressive ATLL are poorer than those of other non-Hodgkin's lymphomas (NHL) free from HTLV-1 infection (Shimoyama *et al*., 1988). The results of chemotherapy in studies performed by the Japan Clinical Oncology Group-Lymphoma Study Group (JCOG-LSG) from the 1980's to early 1990's were unsatisfactory for ATLL. The CR rate was 17-42%, and the median OS time was 5-13 months, and the OS rate at 3 years was only 13-24% (Uozumi, 2010). Recently, Tsukasaki *et al* (2007) reported the results of a randomized phase III trial for aggressive ATLL. They revealed that the CR rate was higher in the patients treated with sequential combination chemotherapy consisting of VCAP (vincristine, cyclophosphamide, doxorubicin, and prednisone), AMP (doxorubicin, ranimustine, and prednisone), and VECP (vindesine, etpoposide, carboplatin, and prednisone) (mLSG15) than in those treated with biweekly CHOP (vincristine, cyclophosphamide, doxorubicin, and prednisone: bi-CHOP) (40% vs

cells often occurs at the time of acute transformation in ATLL patients.

After the diagnosis of ATLL, subclassification of ATLL should be performed to determine the optimal therapeutic regimen. ATLL is divided into four clinical subtypes; the acute, lymphoma, chronic and smoldering types, according to the percentage of ATLL cells in the PB, the involvement of the CNS, bone, peritoneum, pleura and GI tract, and whether there are increases in the serum LDH and calcium (Table 1) (Shimoyama *et al*, 1991). An increase in the serum LDH and blood urea nitrogen, and a decrease in the serum albumin level are poor prognostic factors in patients with chronic type ATLL, so patients who have at least one of these poor prognostic factors have been considered to belong to the unfavorable subgroup (Shimoyama, 1994). The acute, lymphoma and chronic types with at least one of poor prognostic factors are considered to be aggressive ATLL, while chronic type, without any poor prognostic factors, and the smoldering types are called indolent ATLL.


Table 1. Diagnostic criteria for clinical subtype of ATLL

N: normal upper limit, CNS: central nervous system, GI tract: gastrointestinal tract.

\* : No essential qualification except terms required for other subtype(s).

\*2: No essential qualification if other terms are fulfilled, but histology-proven malignant lesion(s) is

required in case abnormal T-lymphocytes are less than 5% in peripheral blood.

\*3: Accompanied by T-lymphocytosis (3.5×109/l or more).

\*4: In case abnormal T-lymphocytes are less than 5% in peripheral blood, histology-proven tumor lesion is required.

A specific subtype of ATLL whose main lesions are limited to the skin, and does not have marked leukemic cells (<5%), a serum LDH level without exceeding 1.5-fold the normal upper limit, and a serum calcium level in the normal range was proposed as cutaneous type ATLL. The percentage of abnormal T-lymphocytes in the PB of such patients is less than 5% (Amano *et al*., 2008).

#### **4.5 Progression/acute transformation**

Indolent ATLL often progresses into acute type ATLL during the long period of the natural course of the disease. The rapid growth of lymph nodes, hepatosplenomegaly, and/or marked skin manifestations suddenly occur in previously indolent ATLL, often accompanied by marked leukocytosis, an increase in the serum LDH, sIL-2R and/or hypercalcemia. In particular, the sIL-2R level has been considered to be an indicator of disease progression and prognosis (Kamihira *et al*., 1994). Multi-step aberrant CpG island hyper-methylation was detected in ATLL patients, which was associated with the progression and transformation (crisis) of ATLL (Sato *et al*., 2010). Clonal evolution of ATLL cells often occurs at the time of acute transformation in ATLL patients.

#### **4.6 Spontaneous regression**

142 T-Cell Leukemia

After the diagnosis of ATLL, subclassification of ATLL should be performed to determine the optimal therapeutic regimen. ATLL is divided into four clinical subtypes; the acute, lymphoma, chronic and smoldering types, according to the percentage of ATLL cells in the PB, the involvement of the CNS, bone, peritoneum, pleura and GI tract, and whether there are increases in the serum LDH and calcium (Table 1) (Shimoyama *et al*, 1991). An increase in the serum LDH and blood urea nitrogen, and a decrease in the serum albumin level are poor prognostic factors in patients with chronic type ATLL, so patients who have at least one of these poor prognostic factors have been considered to belong to the unfavorable subgroup (Shimoyama, 1994). The acute, lymphoma and chronic types with at least one of poor prognostic factors are considered to be aggressive ATLL, while chronic type, without

any poor prognostic factors, and the smoldering types are called indolent ATLL.

Table 1. Diagnostic criteria for clinical subtype of ATLL

\*3: Accompanied by T-lymphocytosis (3.5×109/l or more).

**4.5 Progression/acute transformation** 

is required.

(Amano *et al*., 2008).

\* : No essential qualification except terms required for other subtype(s).

N: normal upper limit, CNS: central nervous system, GI tract: gastrointestinal tract.

required in case abnormal T-lymphocytes are less than 5% in peripheral blood.

\*2: No essential qualification if other terms are fulfilled, but histology-proven malignant lesion(s) is

\*4: In case abnormal T-lymphocytes are less than 5% in peripheral blood, histology-proven tumor lesion

A specific subtype of ATLL whose main lesions are limited to the skin, and does not have marked leukemic cells (<5%), a serum LDH level without exceeding 1.5-fold the normal upper limit, and a serum calcium level in the normal range was proposed as cutaneous type ATLL. The percentage of abnormal T-lymphocytes in the PB of such patients is less than 5%

Indolent ATLL often progresses into acute type ATLL during the long period of the natural course of the disease. The rapid growth of lymph nodes, hepatosplenomegaly, and/or Few ATLL patients show spontaneous regression of tumors (Shimamoto *et al*., 1993). We experienced two chronic type ATLL patients, both of whom had a poor prognostic factor (increased serum LDH), who obtained a complete remission (CR) without any therapeutic intervention. In one patient, the systemic lymphadenopathy and ATLL cells in the PB disappeared, and the serum LDH level was normalized after surgical excision of an inguinal lymph node. However, he suffered from bone pain due to multiple bone lesions infiltrated by ATLL cells about 10 months after the CR. In another patient, the leukocytes and abnormal lymphocytes in the PB, and the serum LDH level gradually decreased to normal range. The ATLL cells in her PB had disappeared completely about 6 years after the diagnosis of ATLL without any therapy. She is now in an HTLV-1 carrier state, and has been free from ATLL for about 7 years after the complete disappearance of the ATLL cells in her PB. Although the mechanisms of spontaneous regression of ATLL have not been elucidated, it is suggested that the cytotoxic activity of peripheral mononuclear cells or the apoptosis of ATLL cells are associated with this phenomenon (Jinnohara *et al*., 1997; Matsushita *et al*., 1999). Clarification of this interesting phenomenon might be useful for the development of new immunological therapy for ATLL patients.

#### **4.7 Therapy**

Treatment for patients with ATLL differs according to the clinical subtypes. It therefore is very important to make an accurate diagnosis of the clinical subtype of ATLL in order to ensure that the appropriate therapy is selected. In patients with indolent ATLL including those with the smoldering type or the chronic type without any unfavorable prognostic factors, watchful waiting is the standard of care in Japan except when the patients are suffering from symptomatic skin lesions.

Generally, intensive combination chemotherapy for aggressive ATLL has been performed immediately after the diagnosis because the prognoses of aggressive ATLL are poorer than those of other non-Hodgkin's lymphomas (NHL) free from HTLV-1 infection (Shimoyama *et al*., 1988). The results of chemotherapy in studies performed by the Japan Clinical Oncology Group-Lymphoma Study Group (JCOG-LSG) from the 1980's to early 1990's were unsatisfactory for ATLL. The CR rate was 17-42%, and the median OS time was 5-13 months, and the OS rate at 3 years was only 13-24% (Uozumi, 2010). Recently, Tsukasaki *et al* (2007) reported the results of a randomized phase III trial for aggressive ATLL. They revealed that the CR rate was higher in the patients treated with sequential combination chemotherapy consisting of VCAP (vincristine, cyclophosphamide, doxorubicin, and prednisone), AMP (doxorubicin, ranimustine, and prednisone), and VECP (vindesine, etpoposide, carboplatin, and prednisone) (mLSG15) than in those treated with biweekly CHOP (vincristine, cyclophosphamide, doxorubicin, and prednisone: bi-CHOP) (40% vs

Accumulation of Specific Epigenetic

**5.2 Epigenetic abnormalities in leukemia and lymphoma** 

might be a crucial target for cancer risk assessment and chemoprevention.

**phosphatase (SHP1) in hemetopoietic cell malignancies** 

**5.3 Frequent gene silencing of hematopoietic cell-specific protein tyrosine** 

Genome-wide studies of gene expression on a genomic scale using cDNA microarrays make it easy to measure the transcription levels of almost every gene at once. Various types of leukemia/lymphoma have been analyzed using cDNA microarrays to investigate the molecular basis of leukemogenesis/ lymphomagenesis. From the cDNA microarray analyses of gene expression pattern of the human NK/T cell line (NK-YS), followed by comprehensive and systematic tissue microarrays, RT-PCR and Western blotting analysis, it has been demonstrated that strongly decreased expression of hematopoietic cell specific protein-tyrosine-phosphatase *SHP1* mRNA was present in malignant cells (Oka et al., 2001). A further analysis using standard immunohistochemistry and tissue microarrays, which utilized 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphomas and more than 95% of malignant leukemia/lymphoma patient specimens of DLBCL, follicular lymphoma (FL), Hodgkin's lymphoma (HL) (Hodgkin's disease (HD)), mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), ATLL and plasmacytoma were negative for SHP1 protein expression. The promoter region of the *SHP1* gene has been revealed to be highly methylated in patient samples of adult T cell leukemia (methylation frequency: 90%), natural killer (NK)/T cell

et al. 2010).

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 145

pressures. Pathogenic factors may be considered such an environmental pressure (Arens & Schoenberger; 2010). Consequently, cellular differentiation and adaptation might be considered as an epigenetic phenomenon. Many of the recent epigenetic investigations have focused on DNA methylation, histone modifications and chromatin remodeling. Non-coding RNAs, such miRNAs, also play important roles in epigenetic pathways (Thai

Lymphoma and leukemia, as well as other cancers, have been thought to be predominantly induced by acquired genetic changes such as mutations, deletions, and amplifications of genes and chromosome translocations. However, it is now becoming clear that microenvironment-mediated epigenetic alterations also play important roles. Although many genetic changes have been reported, it is difficult to discriminate cause from consequence. It is also unclear whether genetic or epigenetic changes occur first. Recent data suggest that cancer has a fundamentally common basis that is grounded in a polyclonal epigenetic disruption of stem/progenitor cells, mediated by 'tumor-progenitor genes'. Furthermore, tumor cell heterogeneity is due, in part, to epigenetic variation in progenitor cells, and epigenetic plasticity, together with genetic lesions, drives tumor progression (Feinburg et al, 2006). The epigenetic disruption of key genes is supposed to occur at the earliest stage of cancer development. Some of the most convincing evidence for epigenetic disruption of progenitor cells derives from the ubiquitous nature of genome-wide hypomethylation, which is present in almost of all malignant tumors. In addition, genesilencing induced by hypermethylation of genes involved in DNA repair (MGMT, hMLH1), cell cycle progression (p16INK4a, p15INK4b, p14ARF), signal transducing molecules (SHP1), apoptosis (DAPK) and cell adhesion (CHD1, HCAD) (Flanagan, 2007) is also common. Therefore, non-neoplastic, but epigenetically disrupted, stem/progenitor cells

25%, respectively). Furthermore, the OS rate at 3 years was higher in the mLSG15 arm than in the bi-CHOP arm (24% vs 13%, respectively) (Tsukasaki *et al*., 2007).

On the other hand, Bazarbachi *et al* (2010) reported that excellent results were obtained using combination therapy with zidovudine (AZT) and interferon-α (IFN) for ATLL patients. The OS rate at 5 years was 46% for their 75 patients who received first-line antiviral therapy. In particular, the OS rate at 5 years for patients with the chronic and smoldering types of ATLL was 100%. However, the results for aggressive type ATLL obtained using AZT/IFN therapy were inferior to those obtained during the JCOG-LSG study (JCOG9303, JCOG9801)(Yamada *et al*., 2001; Tsukasaki *et al*., 2007). Nevertheless, as the results of chemotherapy for aggressive ATLL are unsatisfactory, new strategies using approaches other than conventional chemotherapy are needed for ATLL to improve the survival of the patients.

We previously reported that allogeneic hematopoietic stem cell transplantation (allo-HSCT) was useful for aggressive ATLL (Utsunomiya *et al*., 2001). Following our report, many other researchers reported the possibility of long-term survival in ATLL patients who received allo-HSCT using conventional or reduced intensity conditioning (Fukushima *et al*., 2005; Okamura *et al*., 2005; Shiratori *et al*., 2008; Hishizawa *et al*., 2010). A graft-versus-Tax (Gv-Tax) response in ATLL patients after allo-HSCT was demonstrated by Harashima *et al* (2004). The Gv-Tax response, which has been suggested to induce a graft versus-ATLL (Gv-ATLL) effect may bring about the eradication of not only ATLL cells but also of HTLV-1 infected cells in general (Okamura *et al*., 2005; Yonekura *et al*., 2008).

New agents, especially an anti-CCR4 antibody (KW-0761) are promising for ATLL therapy. Recently, promising results for relapsed ATLL patients who had been treated by intravenous administration of KW-0761 indicated that the overall response rate was 31% in a phase I study (Yamamoto *et al*., 2010) and 50% in a phase II study (Ishida *et al*., 2010). Other novel agents, such as lenalidomide (a thalidomide analogue) and bortezomib, which inhibits proteasome and thereby inhibits activation of NF-κB, are now being evaluated in clinical trials for relapsed ATLL in Japan. In addition, immunotherapy using dendric cells stimulated by Tax peptides is now being prepared for ATLL patients who had previously obtained remission by chemotherapy.

In conclusion, ATLL presents diverse features, and the mechanisms of leukemogenesis induced by HTLV-1 development and the progression of ATLL have not been well elucidated. Clarification of these mechanisms will therefore give ATLL patients a chance to obtain a cure. Furthermore, our final goals are not only to cure ATLL patients, but also to completely eradicate HTLV-1 by preventing HTLV-1 infection or by eradicating infections once they are established.

#### **5. Epigenetics of leukemia and lymphoma**

#### **5.1 Modulation of the expression profile in the immune system through epigenetic mechanism**

Epigenetic mechanisms control the development and differentiation, and maintain the normal physiological status in mammalian cells, and epigenetic events link a subjects' genotype to their phenotype. Epigenetic regulatory mechanisms are a central system to control the differentiation and function of the immune system and to ensure an appropriate gene expression profile in immune cells (Natoli G, 2010). This mechanism changes the gene expression profile, permitting cells to adapt to multiple environmental pressures. Pathogenic factors may be considered such an environmental pressure (Arens & Schoenberger; 2010). Consequently, cellular differentiation and adaptation might be considered as an epigenetic phenomenon. Many of the recent epigenetic investigations have focused on DNA methylation, histone modifications and chromatin remodeling. Non-coding RNAs, such miRNAs, also play important roles in epigenetic pathways (Thai et al. 2010).

#### **5.2 Epigenetic abnormalities in leukemia and lymphoma**

144 T-Cell Leukemia

25%, respectively). Furthermore, the OS rate at 3 years was higher in the mLSG15 arm than

On the other hand, Bazarbachi *et al* (2010) reported that excellent results were obtained using combination therapy with zidovudine (AZT) and interferon-α (IFN) for ATLL patients. The OS rate at 5 years was 46% for their 75 patients who received first-line antiviral therapy. In particular, the OS rate at 5 years for patients with the chronic and smoldering types of ATLL was 100%. However, the results for aggressive type ATLL obtained using AZT/IFN therapy were inferior to those obtained during the JCOG-LSG study (JCOG9303, JCOG9801)(Yamada *et al*., 2001; Tsukasaki *et al*., 2007). Nevertheless, as the results of chemotherapy for aggressive ATLL are unsatisfactory, new strategies using approaches other than conventional chemotherapy are needed for ATLL to improve the

We previously reported that allogeneic hematopoietic stem cell transplantation (allo-HSCT) was useful for aggressive ATLL (Utsunomiya *et al*., 2001). Following our report, many other researchers reported the possibility of long-term survival in ATLL patients who received allo-HSCT using conventional or reduced intensity conditioning (Fukushima *et al*., 2005; Okamura *et al*., 2005; Shiratori *et al*., 2008; Hishizawa *et al*., 2010). A graft-versus-Tax (Gv-Tax) response in ATLL patients after allo-HSCT was demonstrated by Harashima *et al* (2004). The Gv-Tax response, which has been suggested to induce a graft versus-ATLL (Gv-ATLL) effect may bring about the eradication of not only ATLL cells but also of HTLV-1

New agents, especially an anti-CCR4 antibody (KW-0761) are promising for ATLL therapy. Recently, promising results for relapsed ATLL patients who had been treated by intravenous administration of KW-0761 indicated that the overall response rate was 31% in a phase I study (Yamamoto *et al*., 2010) and 50% in a phase II study (Ishida *et al*., 2010). Other novel agents, such as lenalidomide (a thalidomide analogue) and bortezomib, which inhibits proteasome and thereby inhibits activation of NF-κB, are now being evaluated in clinical trials for relapsed ATLL in Japan. In addition, immunotherapy using dendric cells stimulated by Tax peptides is now being prepared for ATLL patients who had previously

In conclusion, ATLL presents diverse features, and the mechanisms of leukemogenesis induced by HTLV-1 development and the progression of ATLL have not been well elucidated. Clarification of these mechanisms will therefore give ATLL patients a chance to obtain a cure. Furthermore, our final goals are not only to cure ATLL patients, but also to completely eradicate HTLV-1 by preventing HTLV-1 infection or by eradicating infections

**5.1 Modulation of the expression profile in the immune system through epigenetic** 

Epigenetic mechanisms control the development and differentiation, and maintain the normal physiological status in mammalian cells, and epigenetic events link a subjects' genotype to their phenotype. Epigenetic regulatory mechanisms are a central system to control the differentiation and function of the immune system and to ensure an appropriate gene expression profile in immune cells (Natoli G, 2010). This mechanism changes the gene expression profile, permitting cells to adapt to multiple environmental

in the bi-CHOP arm (24% vs 13%, respectively) (Tsukasaki *et al*., 2007).

infected cells in general (Okamura *et al*., 2005; Yonekura *et al*., 2008).

survival of the patients.

obtained remission by chemotherapy.

**5. Epigenetics of leukemia and lymphoma** 

once they are established.

**mechanism** 

Lymphoma and leukemia, as well as other cancers, have been thought to be predominantly induced by acquired genetic changes such as mutations, deletions, and amplifications of genes and chromosome translocations. However, it is now becoming clear that microenvironment-mediated epigenetic alterations also play important roles. Although many genetic changes have been reported, it is difficult to discriminate cause from consequence. It is also unclear whether genetic or epigenetic changes occur first. Recent data suggest that cancer has a fundamentally common basis that is grounded in a polyclonal epigenetic disruption of stem/progenitor cells, mediated by 'tumor-progenitor genes'. Furthermore, tumor cell heterogeneity is due, in part, to epigenetic variation in progenitor cells, and epigenetic plasticity, together with genetic lesions, drives tumor progression (Feinburg et al, 2006). The epigenetic disruption of key genes is supposed to occur at the earliest stage of cancer development. Some of the most convincing evidence for epigenetic disruption of progenitor cells derives from the ubiquitous nature of genome-wide hypomethylation, which is present in almost of all malignant tumors. In addition, genesilencing induced by hypermethylation of genes involved in DNA repair (MGMT, hMLH1), cell cycle progression (p16INK4a, p15INK4b, p14ARF), signal transducing molecules (SHP1), apoptosis (DAPK) and cell adhesion (CHD1, HCAD) (Flanagan, 2007) is also common. Therefore, non-neoplastic, but epigenetically disrupted, stem/progenitor cells might be a crucial target for cancer risk assessment and chemoprevention.

#### **5.3 Frequent gene silencing of hematopoietic cell-specific protein tyrosine phosphatase (SHP1) in hemetopoietic cell malignancies**

Genome-wide studies of gene expression on a genomic scale using cDNA microarrays make it easy to measure the transcription levels of almost every gene at once. Various types of leukemia/lymphoma have been analyzed using cDNA microarrays to investigate the molecular basis of leukemogenesis/ lymphomagenesis. From the cDNA microarray analyses of gene expression pattern of the human NK/T cell line (NK-YS), followed by comprehensive and systematic tissue microarrays, RT-PCR and Western blotting analysis, it has been demonstrated that strongly decreased expression of hematopoietic cell specific protein-tyrosine-phosphatase *SHP1* mRNA was present in malignant cells (Oka et al., 2001). A further analysis using standard immunohistochemistry and tissue microarrays, which utilized 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphomas and more than 95% of malignant leukemia/lymphoma patient specimens of DLBCL, follicular lymphoma (FL), Hodgkin's lymphoma (HL) (Hodgkin's disease (HD)), mantle cell lymphoma (MCL), peripheral T-cell lymphoma (PTCL), ATLL and plasmacytoma were negative for SHP1 protein expression. The promoter region of the *SHP1* gene has been revealed to be highly methylated in patient samples of adult T cell leukemia (methylation frequency: 90%), natural killer (NK)/T cell

Accumulation of Specific Epigenetic

malignancies associated with infectious agents.

**6.2 Epigenetic changes induced by virus infection** 

normal tissues is also common in non-virus-related cancers (Esteller, 2006)

critical early events in all malignant tumors (Flanagan, 2007).

**progression of ATLL** 

**6.3 Accumulation of epigenetic abnormalities during the development and** 

ATLL is an aggressive malignant disease of CD4-positive T lymphocytes caused by infection with HTLV-1 (Poiesz et al., 1980; Hinuma et al., 1981). HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40–60 years (Tajima et al., 1990). Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical events in its progression have not been well characterized. The pathogenesis of HTLV-1 has been intensively investigated in terms of the viral regulatory proteins HTLV-1 Tax and Rex, which are supposed to play key roles in the HTLV-1 leukemogenesis/lymphomagenesis, as well as the HTLV-1 basic leucine zipper factor

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 147

are also associated with human cancers. The molecular mechanisms by which these infectious agents contribute to the carcinogenesis and lymphomagenesis are not always clear. However, some of the evidence discussed below suggests an important role for epigenetic changes and aberrant DNA methylation in the onset and progression of

More than 20% of cancers have been causally linked to human pathogens (Zur Hausen, 2009). Why virus infection is sometimes controlled, and on the other occasions leads to the progression to malignant tumors is still mystery. However, recent evidence suggests that epigenetic changes induced by infection play a causative role. Oncogenic viruses have been revealed to increase DNA methylation activity and decrease histone acetylation activity (Flanagan, 2007). The latent membrane protein 1 (LMP-1), one of the virus proteins of EBV, has been shown to be an oncoprotein with transforming activity. LMP-1 activates DNMT1, DNMT3a and DNMT3b to initiate epigenetic alterations, followed by hypermethylation and gene silencing of the *E-cadherin* gene (Tsai et al., 2002). Human epithelial cells expressing LMP-1 have been shown to have higher invasive activity, in accordance with reduced expression of the *E-cadherin* gene (Kim et al., 2000). Integration-defective HIV-I was shown to increase DNMT1 expression, followed by increased methylation of CpG islands in the promoter region of the *p16INK4A* and *IFN-gamma* genes to induce gene silencing (Fang et al., 2001; Mikovits et al., 1998). Overall increases in DNA methyltransferase activity in malignant cells compared with

The ability to alter histone modifications and chromatin structure is also common to many oncogenic viruses, including EBV, HPV, adenoviruses and HTLV-1. EBV nuclear antigens EBNA2 and EBNA 3c alter histone acetylation by interacting with p300/CBP, PCAF histone acetyltransferase (HAT) complexes or with histone deacetylase (HDAC), respectively (Wang et al., 2000; Knight et al., 2003). The HPV E6 oncoprotein binds and inhibits the histone acetyltransferase activity of the p300/CBP complex (Patel et al., 1999). The HTLV-1 Tax protein also interacts with the p300/CBP complex to mediate transcriptional repression (Kwok et al., 1996). Disruption or alteration of p300/CBP histone acetyltransferase activity is common to many oncogenic viruses, suggesting that it may be one of the critical early events in virus-induced tumorigenesis. Further evidence of the early involvement of p300/CBP in various non-viral cancers has also been observed, suggesting that abrogation or perturbation of the histone acetyltransferase activity of p300/CBP may be one of the

lymphoma (91%), diffuse large B-cell lymphoma (93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%). The methylation frequency was significantly higher in high grade-MALT lymphoma cases (100%) than in low grade-MALT lymphoma cases (70%), correlating well with the frequency of the lack of SHP1 protein in high grade- (80%) and low grade-MALT lymphoma (54%) (Oka et al., 2002; Koyama et al., 2003). This suggests that the *SHP1* gene silencing with aberrant CpG methylation is related to the progression of lymphoma, in addition to the malignant transformation. Furthermore, the promoter methylation of the *SHP1* gene was clearly correlated with the clinical stage, such as complete remission or relapse. Loss of heterozygosity with microsatellite markers near the *SHP1* gene was shown in 79% of informative ALL cases. These findings indicate that the *SHP1* gene is a relevant novel biomarker of a wide range of hematopoietic malignancies. Additionally, these results suggest that loss of *SHP1* gene expression plays an important role in multistep lymphomagenesis/leukemogenesis.

SHP1 negatively regulates the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway (Chim et al., 2004a; Chim et al., 2004b). SHP1 in myeloma showed hypermethylation, with constitutive STAT3 phosphorylation. Demethylating reagent-treated myeloma samples showed restored SHP1 expression in accordance with down-regulation of phosphorylated STAT3 (Chim et al., 2004a). SHP1 methylation thus leading to the induction of epigenetic activation of the Jak/STAT pathway might play a key role in the pathogenesis of myeloma. Similarly, frequent methylation of SHP1 was observed in mantle cell and follicular lymphomas (Oka et al., 2001 & 2002; Chim et al., 2004c) and also in acute myeloid leukemia (Oka et al., 2001; Chim et al., 2004b). The hypermethylation of SHP1 led to the activation of the Jak/STAT signaling pathway, along with the upregulation of cyclin D1 and *BCL2,* and could be the basis for the lymomagenesis of follicular lymphoma (Koyama et al., 2003; Chim et al., 2004c).

#### **6. Epigenetic alterations induced by infectious agents**

#### **6.1 Oncogenic infectious agents**

Infectious agents, including viruses, bacteria and parasites, have been reported to be associated with various human malignancies (Oka et al., 2011). These include Epstein-Barr virus (EBV), human T lymphotropic virus type-I (HTLV-1), human T lymphotropic virus type-II (HTLV-2), hepatitis viruses (hepatitis B virus (HBV) and hepatitis C virus (HCV)), human papilloma virus (HPV), polyoma viruses (JC virus, BK virus, SV40) and Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 (KSHV/HHV-8). EBV is associated with Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL), NK/T lymphoma, nasopharyngeal carcinoma and Hodgkin's disease (Lindstrom et al., 2002; Kwong et al., 2002; Bravender, 2010). HTLV-1 is associated with adult T-cell leukemia/lymphoma (ATLL) (Poiesz et al. 1980; Hinuma et al., 1981; Yoshida et al., 1982), HTLV-2 with hairy cell leukemia (Feuer et al., 2005; Kaplan, 1993; Hielle, 1991), HHV-8 with Kaposi's sarcoma and primary effusion lymphomas (Zhang et al., 2010; Du, 2007), HBV and HCV with hepatocellular carcinoma (HCC) (Miroux et al., 2010; Alavian et al., 2010), HPV with cervical carcinoma (Tota et al., 2010; Grce et al., 2010) and JCV with brain and colon cancer (Parkin, 2006; Selgrad et al.,2009). The bacterium *Helicobacter pylori*, a major contributor to gastric cancer and MALT lymphoma, and parasitic infections such as particular *Schistosoma hematobium*, a major cause of bladder cancer in Egypt, and liver flukes (Zur Hausen, 2009) are also associated with human cancers. The molecular mechanisms by which these infectious agents contribute to the carcinogenesis and lymphomagenesis are not always clear. However, some of the evidence discussed below suggests an important role for epigenetic changes and aberrant DNA methylation in the onset and progression of malignancies associated with infectious agents.

#### **6.2 Epigenetic changes induced by virus infection**

146 T-Cell Leukemia

lymphoma (91%), diffuse large B-cell lymphoma (93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%). The methylation frequency was significantly higher in high grade-MALT lymphoma cases (100%) than in low grade-MALT lymphoma cases (70%), correlating well with the frequency of the lack of SHP1 protein in high grade- (80%) and low grade-MALT lymphoma (54%) (Oka et al., 2002; Koyama et al., 2003). This suggests that the *SHP1* gene silencing with aberrant CpG methylation is related to the progression of lymphoma, in addition to the malignant transformation. Furthermore, the promoter methylation of the *SHP1* gene was clearly correlated with the clinical stage, such as complete remission or relapse. Loss of heterozygosity with microsatellite markers near the *SHP1* gene was shown in 79% of informative ALL cases. These findings indicate that the *SHP1* gene is a relevant novel biomarker of a wide range of hematopoietic malignancies. Additionally, these results suggest that loss of *SHP1* gene expression plays an important role in multistep

SHP1 negatively regulates the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway (Chim et al., 2004a; Chim et al., 2004b). SHP1 in myeloma showed hypermethylation, with constitutive STAT3 phosphorylation. Demethylating reagent-treated myeloma samples showed restored SHP1 expression in accordance with down-regulation of phosphorylated STAT3 (Chim et al., 2004a). SHP1 methylation thus leading to the induction of epigenetic activation of the Jak/STAT pathway might play a key role in the pathogenesis of myeloma. Similarly, frequent methylation of SHP1 was observed in mantle cell and follicular lymphomas (Oka et al., 2001 & 2002; Chim et al., 2004c) and also in acute myeloid leukemia (Oka et al., 2001; Chim et al., 2004b). The hypermethylation of SHP1 led to the activation of the Jak/STAT signaling pathway, along with the upregulation of cyclin D1 and *BCL2,* and could be the basis for the lymomagenesis of follicular lymphoma

Infectious agents, including viruses, bacteria and parasites, have been reported to be associated with various human malignancies (Oka et al., 2011). These include Epstein-Barr virus (EBV), human T lymphotropic virus type-I (HTLV-1), human T lymphotropic virus type-II (HTLV-2), hepatitis viruses (hepatitis B virus (HBV) and hepatitis C virus (HCV)), human papilloma virus (HPV), polyoma viruses (JC virus, BK virus, SV40) and Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 (KSHV/HHV-8). EBV is associated with Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL), NK/T lymphoma, nasopharyngeal carcinoma and Hodgkin's disease (Lindstrom et al., 2002; Kwong et al., 2002; Bravender, 2010). HTLV-1 is associated with adult T-cell leukemia/lymphoma (ATLL) (Poiesz et al. 1980; Hinuma et al., 1981; Yoshida et al., 1982), HTLV-2 with hairy cell leukemia (Feuer et al., 2005; Kaplan, 1993; Hielle, 1991), HHV-8 with Kaposi's sarcoma and primary effusion lymphomas (Zhang et al., 2010; Du, 2007), HBV and HCV with hepatocellular carcinoma (HCC) (Miroux et al., 2010; Alavian et al., 2010), HPV with cervical carcinoma (Tota et al., 2010; Grce et al., 2010) and JCV with brain and colon cancer (Parkin, 2006; Selgrad et al.,2009). The bacterium *Helicobacter pylori*, a major contributor to gastric cancer and MALT lymphoma, and parasitic infections such as particular *Schistosoma hematobium*, a major cause of bladder cancer in Egypt, and liver flukes (Zur Hausen, 2009)

lymphomagenesis/leukemogenesis.

(Koyama et al., 2003; Chim et al., 2004c).

**6.1 Oncogenic infectious agents** 

**6. Epigenetic alterations induced by infectious agents** 

More than 20% of cancers have been causally linked to human pathogens (Zur Hausen, 2009). Why virus infection is sometimes controlled, and on the other occasions leads to the progression to malignant tumors is still mystery. However, recent evidence suggests that epigenetic changes induced by infection play a causative role. Oncogenic viruses have been revealed to increase DNA methylation activity and decrease histone acetylation activity (Flanagan, 2007). The latent membrane protein 1 (LMP-1), one of the virus proteins of EBV, has been shown to be an oncoprotein with transforming activity. LMP-1 activates DNMT1, DNMT3a and DNMT3b to initiate epigenetic alterations, followed by hypermethylation and gene silencing of the *E-cadherin* gene (Tsai et al., 2002). Human epithelial cells expressing LMP-1 have been shown to have higher invasive activity, in accordance with reduced expression of the *E-cadherin* gene (Kim et al., 2000). Integration-defective HIV-I was shown to increase DNMT1 expression, followed by increased methylation of CpG islands in the promoter region of the *p16INK4A* and *IFN-gamma* genes to induce gene silencing (Fang et al., 2001; Mikovits et al., 1998). Overall increases in DNA methyltransferase activity in malignant cells compared with normal tissues is also common in non-virus-related cancers (Esteller, 2006)

The ability to alter histone modifications and chromatin structure is also common to many oncogenic viruses, including EBV, HPV, adenoviruses and HTLV-1. EBV nuclear antigens EBNA2 and EBNA 3c alter histone acetylation by interacting with p300/CBP, PCAF histone acetyltransferase (HAT) complexes or with histone deacetylase (HDAC), respectively (Wang et al., 2000; Knight et al., 2003). The HPV E6 oncoprotein binds and inhibits the histone acetyltransferase activity of the p300/CBP complex (Patel et al., 1999). The HTLV-1 Tax protein also interacts with the p300/CBP complex to mediate transcriptional repression (Kwok et al., 1996). Disruption or alteration of p300/CBP histone acetyltransferase activity is common to many oncogenic viruses, suggesting that it may be one of the critical early events in virus-induced tumorigenesis. Further evidence of the early involvement of p300/CBP in various non-viral cancers has also been observed, suggesting that abrogation or perturbation of the histone acetyltransferase activity of p300/CBP may be one of the critical early events in all malignant tumors (Flanagan, 2007).

#### **6.3 Accumulation of epigenetic abnormalities during the development and progression of ATLL**

ATLL is an aggressive malignant disease of CD4-positive T lymphocytes caused by infection with HTLV-1 (Poiesz et al., 1980; Hinuma et al., 1981). HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40–60 years (Tajima et al., 1990). Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical events in its progression have not been well characterized. The pathogenesis of HTLV-1 has been intensively investigated in terms of the viral regulatory proteins HTLV-1 Tax and Rex, which are supposed to play key roles in the HTLV-1 leukemogenesis/lymphomagenesis, as well as the HTLV-1 basic leucine zipper factor

Accumulation of Specific Epigenetic

induced lymphomagenesis and leukemogenesis.

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 149

It will be of interest to determine whether there is a direct link between HTLV-1 induction of DNMTs causing CIMP and hypermethylation of specific target genes, and how or what kind of viruses induce deregulation of the epigenetic machinery. Such discoveries may provide new insights into the understanding of the molecular mechanisms responsible for virus-

The HTLV-1 Tax protein has been demonstrated to activate the nuclear factor-κB (NF-κB) and Akt pathways as major cellular pro-survival pathways (Yoshida, 2001). However, Tax transcripts are detected in only about 40% of transformed ATLL cells and are sometimes mutated. On the other hand, it has been demonstrated that the Hbz transcript is ubiquitously expressed in all ATLL cells, and possesses a pro-proliferative function in cells (Satou et al., 2006). It has therefore been proposed that Tax initiates transformation, while HBZ is required to maintain the transformed phenotype late in ATLL when Tax expression is extinguished (Matsuoka & Jeang, 2011). During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. The loss of Tax expression *in vivo* could facilitate the escape of HTLV-1 infected cells from CTL-surveillance to induce disease progression. In the Bovine Leukemia Virus (BLV)-induced ovine (sheep) leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion (Merimi et at., 2007). They showed that there was a correlation between the complete suppression of provirus expression and tumor onset, providing experimental evidence that virus and Tax silencing are critical, if not mandatory, for the progression to overt malignancy. This suggests that epigenetic and/or genetic changes in the host genome induced by HTLV-1 infection are

crucial for the onset and progression, independent of virus genome expression.

maintained by daughter cells though epigenetic machinery.

**6.4 Possible link to host-pathogen interaction** 

This raises questions about whether it might be possible to maintain the leukemic phenotype, on for cells to progress to ATLL without Tax expression. One possibility is that the genetic changes are associated with multipolar mitosis and aneuploidy. Aberrant centrosome replication is linked to oncogenesis, disregulating the intact spindle assembly checkpoint, accurate centrosome cycle and proper cytokinesis (Chi & Jeang, 2007). A second possibility is that there is aberrant expression of miRNAs (microRNAs) in ATLL leukemic cells, which occur independent of Tax expression. Yeung et al. reported that the tumor suppressor protein, TP53INP1, in HTLV-1 infected/transformed cells was targeted for repression by upregulated expression of miR-93 and miR-130b (Yeung et al., 2008). Pichler et al also reported that TP53INP1 was targeted in HTLV-1 infected/transformed cells by miR-21, -24, 146a and -155 (Pichler et al., 2008). Bellon et al described that ATLL cells show increased expression of miR155 (Bellon et al., 2009). These aberrant expression levels of onco-miR may disregulate downstream gene expression. A third possibility is that aberrant gene expression induced by epigenetic abnormalities, including aberrant DNA methylation, abnormal changes in histone modifications and dysregulation of chromatin remodeling, are

Experimental interspecies-transmission of BLV to sheep shows the shorter latency period preceding disease onset: leukemia occurs usually 1-4 years after infection in contrast to 4-10 years in cows. In addition, the incidence of virus-induced leukemia is much higher: almost all infected sheep will succumb within normal life time compared to only about 5% in cattle, suggesting that it is related to the lack of natural transmission of BLV to sheep (Florins et al.,

(HBZ) (Matsuoka et al., 2003, 2007; Gaudray et al.2002). The mechanism responsible for the progression of ATLL have been investigated from various genetic aspects, including specific chromosome abnormalities (Okamoto et al., 1989; Oka et al.1992, 2006; Ariyama et al.1999; Fujimoto et al., 1999), changes in the characteristic HTLV-1 Tax, Rex and HBZ protein expression patterns (Oka et al., 1992; Selgrad et al., 2009) and aberrant expression of the *SHP1* (Oka et al., 2002, 2006), *P53* (Yamato et al., 1993; Tawara et al., 2006), *DRS* (Shimakage et al. 2007), and *ASY/Nogo* (Shimakage et al. 2006) genes, although the detailed mechanisms triggering the onset and progression of ATLL remains to be elucidated. Frequent epigenetic aberration of DNA hypermethylation associated with *SHP1* gene silencing has been identified in a wide range of hematopoietic malignancies (Oka et al., 2001, 2002; Koyama et al., 2003). Recently, the number of genes methylated CpG islands, including the *SHP1*, *P15*, *P16*, *P73*, *HCAD*, *DAPK*, and *MGMT* genes, has been reported to increase with disease progression, and aberrant hypermethylation in specific genes has been detected even in HTLV-1 carriers, and correlated with eventual progression to ATLL (Sato et al., 2010). CIMP was observed most frequently in the lymphoma type ATLL, and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes, and the increased incidence of CIMP were shown to be unfavorable prognostic factors for ATLL (Sato et al., 2010) and correlated with a shorter overall survival as calculated by a Kaplan-Meyer analysis. These findings strongly suggest that the multi-step accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression and prognosis of ATLL, and that CpG methylation and CIMP may provide new diagnostic and prognostic biomarkers for patients with this disease (Figure 2).

Fig. 2. Natural course from infection of human T lymphotropic virus type-I (HTLV-1) to onset and progression of adult T-cell leukemia/lymphoma (ATLL). Accumulation of genetic and epigenetic changes in host and virus genome during long latent period induce onset of ATLL.

(HBZ) (Matsuoka et al., 2003, 2007; Gaudray et al.2002). The mechanism responsible for the progression of ATLL have been investigated from various genetic aspects, including specific chromosome abnormalities (Okamoto et al., 1989; Oka et al.1992, 2006; Ariyama et al.1999; Fujimoto et al., 1999), changes in the characteristic HTLV-1 Tax, Rex and HBZ protein expression patterns (Oka et al., 1992; Selgrad et al., 2009) and aberrant expression of the *SHP1* (Oka et al., 2002, 2006), *P53* (Yamato et al., 1993; Tawara et al., 2006), *DRS* (Shimakage et al. 2007), and *ASY/Nogo* (Shimakage et al. 2006) genes, although the detailed mechanisms triggering the onset and progression of ATLL remains to be elucidated. Frequent epigenetic aberration of DNA hypermethylation associated with *SHP1* gene silencing has been identified in a wide range of hematopoietic malignancies (Oka et al., 2001, 2002; Koyama et al., 2003). Recently, the number of genes methylated CpG islands, including the *SHP1*, *P15*, *P16*, *P73*, *HCAD*, *DAPK*, and *MGMT* genes, has been reported to increase with disease progression, and aberrant hypermethylation in specific genes has been detected even in HTLV-1 carriers, and correlated with eventual progression to ATLL (Sato et al., 2010). CIMP was observed most frequently in the lymphoma type ATLL, and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes, and the increased incidence of CIMP were shown to be unfavorable prognostic factors for ATLL (Sato et al., 2010) and correlated with a shorter overall survival as calculated by a Kaplan-Meyer analysis. These findings strongly suggest that the multi-step accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression and prognosis of ATLL, and that CpG methylation and CIMP may provide new diagnostic

and prognostic biomarkers for patients with this disease (Figure 2).

Fig. 2. Natural course from infection of human T lymphotropic virus type-I (HTLV-1) to onset and progression of adult T-cell leukemia/lymphoma (ATLL). Accumulation of genetic and epigenetic changes in host and virus genome during long latent period induce onset of

ATLL.

It will be of interest to determine whether there is a direct link between HTLV-1 induction of DNMTs causing CIMP and hypermethylation of specific target genes, and how or what kind of viruses induce deregulation of the epigenetic machinery. Such discoveries may provide new insights into the understanding of the molecular mechanisms responsible for virusinduced lymphomagenesis and leukemogenesis.

The HTLV-1 Tax protein has been demonstrated to activate the nuclear factor-κB (NF-κB) and Akt pathways as major cellular pro-survival pathways (Yoshida, 2001). However, Tax transcripts are detected in only about 40% of transformed ATLL cells and are sometimes mutated. On the other hand, it has been demonstrated that the Hbz transcript is ubiquitously expressed in all ATLL cells, and possesses a pro-proliferative function in cells (Satou et al., 2006). It has therefore been proposed that Tax initiates transformation, while HBZ is required to maintain the transformed phenotype late in ATLL when Tax expression is extinguished (Matsuoka & Jeang, 2011). During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. The loss of Tax expression *in vivo* could facilitate the escape of HTLV-1 infected cells from CTL-surveillance to induce disease progression. In the Bovine Leukemia Virus (BLV)-induced ovine (sheep) leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion (Merimi et at., 2007). They showed that there was a correlation between the complete suppression of provirus expression and tumor onset, providing experimental evidence that virus and Tax silencing are critical, if not mandatory, for the progression to overt malignancy. This suggests that epigenetic and/or genetic changes in the host genome induced by HTLV-1 infection are crucial for the onset and progression, independent of virus genome expression.

This raises questions about whether it might be possible to maintain the leukemic phenotype, on for cells to progress to ATLL without Tax expression. One possibility is that the genetic changes are associated with multipolar mitosis and aneuploidy. Aberrant centrosome replication is linked to oncogenesis, disregulating the intact spindle assembly checkpoint, accurate centrosome cycle and proper cytokinesis (Chi & Jeang, 2007). A second possibility is that there is aberrant expression of miRNAs (microRNAs) in ATLL leukemic cells, which occur independent of Tax expression. Yeung et al. reported that the tumor suppressor protein, TP53INP1, in HTLV-1 infected/transformed cells was targeted for repression by upregulated expression of miR-93 and miR-130b (Yeung et al., 2008). Pichler et al also reported that TP53INP1 was targeted in HTLV-1 infected/transformed cells by miR-21, -24, 146a and -155 (Pichler et al., 2008). Bellon et al described that ATLL cells show increased expression of miR155 (Bellon et al., 2009). These aberrant expression levels of onco-miR may disregulate downstream gene expression. A third possibility is that aberrant gene expression induced by epigenetic abnormalities, including aberrant DNA methylation, abnormal changes in histone modifications and dysregulation of chromatin remodeling, are maintained by daughter cells though epigenetic machinery.

#### **6.4 Possible link to host-pathogen interaction**

Experimental interspecies-transmission of BLV to sheep shows the shorter latency period preceding disease onset: leukemia occurs usually 1-4 years after infection in contrast to 4-10 years in cows. In addition, the incidence of virus-induced leukemia is much higher: almost all infected sheep will succumb within normal life time compared to only about 5% in cattle, suggesting that it is related to the lack of natural transmission of BLV to sheep (Florins et al.,

Accumulation of Specific Epigenetic

responses (Peart et al., 2003).

cell leukemia/lymphoma.

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 151

the responsive subset of T cell lymphomas has its origin in an as-yet unknown chromosomal rearrangement that recruits the class I HDACs to the promoter of a gene, and T cell lymphoma is therefore distinctly susceptible to different therapeutic interventions that affect HDACs (Piekarz et al., 2009b). In particular, Vorinostat (suberoylanilide hydroxamic acid, SAHA), which is a hydroxamic acid derivative that inhibits both class I and II HDACs, showed a good response for the treatment of relapsed and refractory cutaneous T-cell lymphoma (CTCL) (O'Connor et al., 2006; Mann et al., 2007; Duvic et al., 2007; Olsen et al., 2007; Garcia-Manero et al., 2008). Romidepsin (depsipeptide, FR901228, FK228, NSC 630176) is generally classified as a broad-spectrum inhibitor, as it inhibits class II enzymes. Romidepin was the first HDACi reported to show efficacy as monotherapy (complete or partial response) in patients with PTCL and CTCL (Piekarz et al., 2001). Favorable responses have been confirmed in CLL (Byrd et al., 2005; Dai et al., 2008; Inoue et al., 2009), CTCL (Piekarz et al., 2009b; Bates et al., 2010; Whittaker et al., 2010), and in additional PTCL patients (Bates et al., 2010; Piekarz et al., 2011). Panobinostat (LBH589) induces clinical responses in patients with refractory CTCL (Ellis et al., 2008). Peart et al. described that the specific attributes of each individual HDACi could be clarified, and that "matching" an individual HDACi to particular tumors or genetic profiles might help improve the clinical

The two main analogs of DNMT inhibitors, such as DAC and 5-AC, are incorporated into DNA to trap and target DNMTs for degradation. The subsequent absence of these enzymes during DNA synthesis causes hypomethylation, and finally, reactivation of silenced gene expression in the daughter cells. The activated gene expression has effects on multiple pathways, contributing to a clinical response (Yoo et al., 2006). However, caution should be exercised, because the hypomethylation resulting from treatment these drugs can also likely activate oncogenes that are generally known to be silenced (e.g., *COX2, EGFR*, etc) (Toyota et al., 2005). Recent data show that hypomethylation by treatment with a single DAC is insufficient for the induction of gene expression (Si et al., 2010). Therefore, combination therapies using DNA demethylating agents with HDACi are well established. Indeed, HDACi enhance the activation of aberrantly methylated tumor suppressor gene promoters in tumor cells by DNA demethylating agents (Cameron et al., 1999; Steiner et al., 2005). These results suggest that potentiation of DAC-mediated gene induction by HDACi may be more complex than mere additive activities. However, the previous trials have mostly involved patients with AML and MDS (Silverman et al., 2009), not including those with T

Approximately 30–40% cases of PTCL-NOS express CCR4+, and CCR4 expression is an unfavorable prognostic factor (Ohshima et al., 2004; Ishida et al., 2004). Additionally, PTCL originating from a CCR4+ Treg cell often shows a tendency to be ''PTCL-NOS with genomic alterations'' (Ishida et al., 2011). Tumor cells from most ATLL patients are characterized by the Treg phenotype(CD4+CD25+CCR4+FOXP3+)(Yoshie et al., 2002; Karube et al., 2008). Consequently, anti-CCR4 mAbs (KW-0761) have been developed, and have shown notable

Interestingly, a recent investigation showed that the CCR4 expression on human CD4+ T cells is regulated by histone H3 acetylation and methylation (Singh et al., 2010). In ATLL, it was noted that the indolent type is associated with a worse survival (mean survival time: 4.1 years) (Takasaki et al., 2010), and the proliferation of HTLV-1 infected cells seems to determine the viral burden during the carrier state (Matsuoka et al., 2011). These reports

anti-tumor effects (Yamamoto et al., 2010; Ishii et al., 2010).

2008). In nature it is often observed that interspecies transmission of viruses results in a high incidence of disease in the new host. Genetic analyses of several human and simian T-cell leukemia virus type-I (HTLV-1/STLV-1) strains of African and Asian origin suggest recent interspecies transfer between species within primate genera, including humans. The phylogenetic analyses suggest that at least three independent human-simian exchanges have occurred during the evolution of these retroviruses (Dekaban et al., 1996). The incidence of ATLL within normal lifetime is about 5%, suggesting that HTLV-1 is in the process to establish a new relationship to human as a natural host. Elucidation of symbiotic evolution mechanisms may provides new insights to find out the strategy to reduce the virulence of HTLV-1 and suppress the onset of diseases.

#### **7. Epigenetic therapy for leukemia/ lymphoma**

Abnormalities of the epigenetic machinery have been associated with a broad range of diseases, including hematologic disorders and malignant leukemia/lymphoma. The malignancies have specific epigenetic profiles related to their histological type, and show many common phenotypes such as self-sufficiency of growth signals, resistance to antiproliferative or pro-apoptotic signals, and so on. As previously reported, epigenetic markers can be used for various clinical applications, including for determing the risk of the onset and progression, for early detection, prediction of prognosis, and for predicting treatment outcomes and evaluating the response to treatment.

Moreover, there are already several systems with high sensitivity for detecting epigenetic profiles, such as the methylation-specific polymerase chain reaction (MSP) assay, which have been developed using leukemia/lymphoma samples (Oka et al., 2002; Sato et al., 2010). The epigenetic modifications are characterized by reversible reactions. On the basis of this point, inhibitors to reverse these modifications as therapeutic interventions have been developed and exploited, and good results have been reported for various malignant leukemias/lymphomas.

It is important to determine why T cell leukemia/lymphoma shows a worse prognosis than other disease, and to use this information to design a effective treatment. It is noteworthy that epigenetic therapy is now regarded as an innovative approach to the treatment of T cell leukemia/lymphoma (Piekarz et al., 2009a). In fact, treatment of tumor cells with epigenetic drugs can induce a range of antitumor effects, including apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune responses, and angiogenesis (Bolden et al., 2006). The current drugs targeted for epigenetic mechanisms are categorized as either histone deacetylase (HDAC) inhibitors (HDACi) such as vorinostat, romidepsin and DNA methyltransferase (DNMT) inhibitors, such as 5-aza-2'-deoxycytidine (DAC) or 5 azacytidine (5-AC).

HDACi have diverse structures, and include sodium butyrate, vorinostat, MS-275, TSA, and FK228 (Prince et al., 2009). However, regardless of their structures, similarities have been observed with regard to their efficacy, and their timing- and dose-dependence, although some profiles on gene expression induced by HDACi seem to be agent-specific (Gray et al., 2004; Peart et al., 2005). Several HDACi have also been reported to predominantly improve the patient prognosis (Prince et al., 2009). However, the mechanism responsible for the marked efficacy of HDACi in T cell lymphoma is not yet understood, nor is there an understanding of the differences among the various HDACi. Piekarz et al. speculated that

2008). In nature it is often observed that interspecies transmission of viruses results in a high incidence of disease in the new host. Genetic analyses of several human and simian T-cell leukemia virus type-I (HTLV-1/STLV-1) strains of African and Asian origin suggest recent interspecies transfer between species within primate genera, including humans. The phylogenetic analyses suggest that at least three independent human-simian exchanges have occurred during the evolution of these retroviruses (Dekaban et al., 1996). The incidence of ATLL within normal lifetime is about 5%, suggesting that HTLV-1 is in the process to establish a new relationship to human as a natural host. Elucidation of symbiotic evolution mechanisms may provides new insights to find out the strategy to reduce the

Abnormalities of the epigenetic machinery have been associated with a broad range of diseases, including hematologic disorders and malignant leukemia/lymphoma. The malignancies have specific epigenetic profiles related to their histological type, and show many common phenotypes such as self-sufficiency of growth signals, resistance to antiproliferative or pro-apoptotic signals, and so on. As previously reported, epigenetic markers can be used for various clinical applications, including for determing the risk of the onset and progression, for early detection, prediction of prognosis, and for predicting treatment

Moreover, there are already several systems with high sensitivity for detecting epigenetic profiles, such as the methylation-specific polymerase chain reaction (MSP) assay, which have been developed using leukemia/lymphoma samples (Oka et al., 2002; Sato et al., 2010). The epigenetic modifications are characterized by reversible reactions. On the basis of this point, inhibitors to reverse these modifications as therapeutic interventions have been developed and exploited, and good results have been reported for various malignant

It is important to determine why T cell leukemia/lymphoma shows a worse prognosis than other disease, and to use this information to design a effective treatment. It is noteworthy that epigenetic therapy is now regarded as an innovative approach to the treatment of T cell leukemia/lymphoma (Piekarz et al., 2009a). In fact, treatment of tumor cells with epigenetic drugs can induce a range of antitumor effects, including apoptosis, cell cycle arrest, differentiation and senescence, modulation of immune responses, and angiogenesis (Bolden et al., 2006). The current drugs targeted for epigenetic mechanisms are categorized as either histone deacetylase (HDAC) inhibitors (HDACi) such as vorinostat, romidepsin and DNA methyltransferase (DNMT) inhibitors, such as 5-aza-2'-deoxycytidine (DAC) or 5-

HDACi have diverse structures, and include sodium butyrate, vorinostat, MS-275, TSA, and FK228 (Prince et al., 2009). However, regardless of their structures, similarities have been observed with regard to their efficacy, and their timing- and dose-dependence, although some profiles on gene expression induced by HDACi seem to be agent-specific (Gray et al., 2004; Peart et al., 2005). Several HDACi have also been reported to predominantly improve the patient prognosis (Prince et al., 2009). However, the mechanism responsible for the marked efficacy of HDACi in T cell lymphoma is not yet understood, nor is there an understanding of the differences among the various HDACi. Piekarz et al. speculated that

virulence of HTLV-1 and suppress the onset of diseases.

**7. Epigenetic therapy for leukemia/ lymphoma** 

outcomes and evaluating the response to treatment.

leukemias/lymphomas.

azacytidine (5-AC).

the responsive subset of T cell lymphomas has its origin in an as-yet unknown chromosomal rearrangement that recruits the class I HDACs to the promoter of a gene, and T cell lymphoma is therefore distinctly susceptible to different therapeutic interventions that affect HDACs (Piekarz et al., 2009b). In particular, Vorinostat (suberoylanilide hydroxamic acid, SAHA), which is a hydroxamic acid derivative that inhibits both class I and II HDACs, showed a good response for the treatment of relapsed and refractory cutaneous T-cell lymphoma (CTCL) (O'Connor et al., 2006; Mann et al., 2007; Duvic et al., 2007; Olsen et al., 2007; Garcia-Manero et al., 2008). Romidepsin (depsipeptide, FR901228, FK228, NSC 630176) is generally classified as a broad-spectrum inhibitor, as it inhibits class II enzymes. Romidepin was the first HDACi reported to show efficacy as monotherapy (complete or partial response) in patients with PTCL and CTCL (Piekarz et al., 2001). Favorable responses have been confirmed in CLL (Byrd et al., 2005; Dai et al., 2008; Inoue et al., 2009), CTCL (Piekarz et al., 2009b; Bates et al., 2010; Whittaker et al., 2010), and in additional PTCL patients (Bates et al., 2010; Piekarz et al., 2011). Panobinostat (LBH589) induces clinical responses in patients with refractory CTCL (Ellis et al., 2008). Peart et al. described that the specific attributes of each individual HDACi could be clarified, and that "matching" an individual HDACi to particular tumors or genetic profiles might help improve the clinical responses (Peart et al., 2003).

The two main analogs of DNMT inhibitors, such as DAC and 5-AC, are incorporated into DNA to trap and target DNMTs for degradation. The subsequent absence of these enzymes during DNA synthesis causes hypomethylation, and finally, reactivation of silenced gene expression in the daughter cells. The activated gene expression has effects on multiple pathways, contributing to a clinical response (Yoo et al., 2006). However, caution should be exercised, because the hypomethylation resulting from treatment these drugs can also likely activate oncogenes that are generally known to be silenced (e.g., *COX2, EGFR*, etc) (Toyota et al., 2005). Recent data show that hypomethylation by treatment with a single DAC is insufficient for the induction of gene expression (Si et al., 2010). Therefore, combination therapies using DNA demethylating agents with HDACi are well established. Indeed, HDACi enhance the activation of aberrantly methylated tumor suppressor gene promoters in tumor cells by DNA demethylating agents (Cameron et al., 1999; Steiner et al., 2005). These results suggest that potentiation of DAC-mediated gene induction by HDACi may be more complex than mere additive activities. However, the previous trials have mostly involved patients with AML and MDS (Silverman et al., 2009), not including those with T cell leukemia/lymphoma.

Approximately 30–40% cases of PTCL-NOS express CCR4+, and CCR4 expression is an unfavorable prognostic factor (Ohshima et al., 2004; Ishida et al., 2004). Additionally, PTCL originating from a CCR4+ Treg cell often shows a tendency to be ''PTCL-NOS with genomic alterations'' (Ishida et al., 2011). Tumor cells from most ATLL patients are characterized by the Treg phenotype(CD4+CD25+CCR4+FOXP3+)(Yoshie et al., 2002; Karube et al., 2008). Consequently, anti-CCR4 mAbs (KW-0761) have been developed, and have shown notable anti-tumor effects (Yamamoto et al., 2010; Ishii et al., 2010).

Interestingly, a recent investigation showed that the CCR4 expression on human CD4+ T cells is regulated by histone H3 acetylation and methylation (Singh et al., 2010). In ATLL, it was noted that the indolent type is associated with a worse survival (mean survival time: 4.1 years) (Takasaki et al., 2010), and the proliferation of HTLV-1 infected cells seems to determine the viral burden during the carrier state (Matsuoka et al., 2011). These reports

Accumulation of Specific Epigenetic

**9. Acknowledgements** 

79, 428-437.

**10. References**

Abnormalities During Development and Progression of T Cell Leukemia/Lymphoma 153

dynamic nature of microbe-host interactions and the human epigenome itself with regard to

The authors would like to acknowledge to John Wiley & Sons Ltd for kindly giving us a permission to reproduce Table I, which has been originally published in *Br J Haematol* (1991)

Abdelmohsen, K., Pullmann, R.Jr., Lal, A., Kim, H.H., Galban, S., Yang, X., Blethrow, J.D.,

Alavian, S.M., Behnava, B., Tabatabaei, S.V. (2010). Comparative efficacy and overall safety

systematic review and meta-analysis. *Eur J Clin Pharmacol 66*, 1071-1079. Amano, M., Kurokawa, M., Ogata, K., Itoh, H., Kataoka, H., and Setoyama, M. (2008). New

locations. *Non-Hodgkin's Lymphoma Classification Project* 9, 717-720.

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the various diseases. Such findings will greatly assist in improving human health.

suggest that early detection and treatment are essential for preventing transformation, or for decreasing the tumor burden in patients with the disease. Tax expression is regulated by the SUV39H1 histone methyltransferase (Kamoi et al., 2006) and HDAC1 (Ego et al., 2002), which negatively regulate the viral gene expression. These findings indicate that the presence of epigenetic abnormalities, including those that occur as a result of Tax regulation, play crucial roles in the pathogenesis of ATLL. A previous report showed that a histone deacetylase inhibitor, valproate, reduced the HTLV-1 proviral load in HAM/TSP through induction of tax gene expression and subsequent activation of CTLs (Lezin et al., 2007). However, it is important to note that the downstream effectors affected by these epigenetic agents have not been elucidated, although their primary enzymatic targets are known. In addition, it is necessary to confirm the optimal dosing schedule, potency, pharmacology, and longterm toxicity for each cell type.

Recent reports have evaluated additional combinations of HDACi with other agents, such as anthracyclines, in patients with AML and MDS (Zxu et al., 2010) and AMG 655 (anti-TRAIL receptor 2 antibody) in patients with various B cell lymphomas (National Cancer Institute (NCI), USA; http://www.cancer.gov). It appears that combination therapy using epigenetic agents with another therapy, such as immunotherapy, will make it possible to create an effective treatment strategy for intractable T cell leukemia/lymphoma. Additional larger studies of epigenetic therapy in subjects with intractable T cell leukemia/lymphoma are warranted.

#### **8. Conclusions and perspective**

Increased activity of DNA methyltransferases and decreases in p300/CBP-mediated histone acetylation are common in both virus-induced and non-viral malignancies, which suggests that epigenetic therapy would be effective for a wide range of malignancies. Aberrant DNA methylation has been shown to be the most consistent molecular changes present in many neoplasms. Hypermethylation of specific target genes, which can be detected at various stages and in different types of lymphomas and leukemias, can be detected with high sensitivity and accuracy. In the near future, we hope to be able to identify the specific signature of the methylation profile and biomarkers of hypermethylated genes for each specific type and stage of malignancy. Moreover, some epigenetic markers might be present prior to the development of lymphoma and leukemia. Thus, epigenetic markers may crucial for identifying the risk of leukemia/lymphoma development and also indicate the possibility of cancer prevention for such high-risk patients. Epigenetic changes, in contrast to genetic changes, can be easily reversed by the use of therapeutic interventions at various stages. The hypermethylated genes found in various cancers, in addition to leukemia/lymphoma, seem to be particularly sensitive to reactivation by demethylating reagents and HDACi. Therefore, restoration of multiple gene functions at the same time may be possible by therapeutic targeting of DNA methylation and histone acetylation. This could have profound implications for the diagnosis and treatment of malignancies.

The newer technologies that enable the global analyses of the epigenome are developing with remarkable speed, and include methods such as ChIP-on-chip (Chromatin ImmunoPrecipitation with microarray) and ChIP-sequencing, with deep sequencing by next generation sequencers for mapping global methylation and chromatin modifications, which will provide information about the landscape of infection-induced alterations, and about the dynamic nature of microbe-host interactions and the human epigenome itself with regard to the various diseases. Such findings will greatly assist in improving human health.

#### **9. Acknowledgements**

The authors would like to acknowledge to John Wiley & Sons Ltd for kindly giving us a permission to reproduce Table I, which has been originally published in *Br J Haematol* (1991) 79, 428-437.

#### **10. References**

152 T-Cell Leukemia

suggest that early detection and treatment are essential for preventing transformation, or for decreasing the tumor burden in patients with the disease. Tax expression is regulated by the SUV39H1 histone methyltransferase (Kamoi et al., 2006) and HDAC1 (Ego et al., 2002), which negatively regulate the viral gene expression. These findings indicate that the presence of epigenetic abnormalities, including those that occur as a result of Tax regulation, play crucial roles in the pathogenesis of ATLL. A previous report showed that a histone deacetylase inhibitor, valproate, reduced the HTLV-1 proviral load in HAM/TSP through induction of tax gene expression and subsequent activation of CTLs (Lezin et al., 2007). However, it is important to note that the downstream effectors affected by these epigenetic agents have not been elucidated, although their primary enzymatic targets are known. In addition, it is necessary to confirm the optimal dosing schedule, potency, pharmacology,

Recent reports have evaluated additional combinations of HDACi with other agents, such as anthracyclines, in patients with AML and MDS (Zxu et al., 2010) and AMG 655 (anti-TRAIL receptor 2 antibody) in patients with various B cell lymphomas (National Cancer Institute (NCI), USA; http://www.cancer.gov). It appears that combination therapy using epigenetic agents with another therapy, such as immunotherapy, will make it possible to create an effective treatment strategy for intractable T cell leukemia/lymphoma. Additional larger studies of epigenetic therapy in subjects with intractable T cell

Increased activity of DNA methyltransferases and decreases in p300/CBP-mediated histone acetylation are common in both virus-induced and non-viral malignancies, which suggests that epigenetic therapy would be effective for a wide range of malignancies. Aberrant DNA methylation has been shown to be the most consistent molecular changes present in many neoplasms. Hypermethylation of specific target genes, which can be detected at various stages and in different types of lymphomas and leukemias, can be detected with high sensitivity and accuracy. In the near future, we hope to be able to identify the specific signature of the methylation profile and biomarkers of hypermethylated genes for each specific type and stage of malignancy. Moreover, some epigenetic markers might be present prior to the development of lymphoma and leukemia. Thus, epigenetic markers may crucial for identifying the risk of leukemia/lymphoma development and also indicate the possibility of cancer prevention for such high-risk patients. Epigenetic changes, in contrast to genetic changes, can be easily reversed by the use of therapeutic interventions at various stages. The hypermethylated genes found in various cancers, in addition to leukemia/lymphoma, seem to be particularly sensitive to reactivation by demethylating reagents and HDACi. Therefore, restoration of multiple gene functions at the same time may be possible by therapeutic targeting of DNA methylation and histone acetylation. This could have

profound implications for the diagnosis and treatment of malignancies.

The newer technologies that enable the global analyses of the epigenome are developing with remarkable speed, and include methods such as ChIP-on-chip (Chromatin ImmunoPrecipitation with microarray) and ChIP-sequencing, with deep sequencing by next generation sequencers for mapping global methylation and chromatin modifications, which will provide information about the landscape of infection-induced alterations, and about the

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**9** 

*Japan* 

Mariko Tomita

*University of the Ryukyus* 

*Department Pathology and Oncology Graduate School of Medical Science* 

**Roles of MicroRNA in T-Cell Leukemia** 

Previously, cancer researchers have been focused on the genes that code proteins. They have considered those effects on tumorigenesis. However, discoveries of microRNAs (miRNAs) have shed a light on the role of non-protein-coding RNAs in tumorigenesis. The first small non-coding RNA, named *lin-4* in *Caenorhabditis elegans* (*C. elegans*) was described by Lee et al in 1993 (Lee et al., 1993). *Lin-4* codes miRNA that regulates the timing of *C. elegans* larval development by translational repression (Ambros, 2000). Since then, many miRNAs in different organisms such as plants, *C. elegans*, Drosophila, and mammals including humans have been discovered substantially. Up to now, the human genome is predicted to encode as

miRNAs belong to a class of regulatory genes that are single-stranded 19-25 nucleotides non-cording RNAs and are generated from endogenous hairpin-shaped transcripts (Kim, 2005). miRNA genes are located either within the introns or exons of protein-coding genes (70%) or in intergenic regions (30%). More than 50% of mammalian miRNAs are located within the intronic regions of protein-coding genes. Most of the intronic or exonic miRNAs are transcribed in parallel with their host genes, indicating that these miRNAs use their host genes transcriptional machinalies. On the other hand, miRNAs produced from intergenic

The first step of miRNA synthesis is the transcription of primary miRNA (pri-miRNA) from *miRNA* genes (Fig.1). pri-miRNAs are transcribed in a RNA polymerase II (Pol II) dependent manner as several hundreds or thousands of nucleotides long polyadenylated RNAs. In the nucleus, the pri-miRNA is processed to a precursor miRNA (pre-miRNA) of 60-100 nucleotides in length with a stem-loop structure by the nuclear protein Drosha that belongs to class II RNase III. Drosha interacts with its cofactor DGCR8 (the DiGeorge syndrome critical region gene 8 protein). Then, the pre-miRNA is exported from the nucleus to the cytoplasm by the Exportin 5/Ran-GTP complex. In the cytoplasm, the pre-miRNA is cleaved by class III RNase III, Dicer, which is a 200-kDa protein and miRNA is produced. Primary function of miRNAs in the cytoplasm is the negative regulation of gene expression by binding to complementary target sequences in the 3' untranslated region (UTR) of mRNA. Binding of a miRNA to the target mRNA typically leads to translational repression or degradation of mRNA, which means that miRNAs repress the expression of the target genes. In mammals, miRNAs guide the RNA induced silencing complex (RISC) to

regions are transcribed separately from internal promoters (Rodriguez et al., 2004).

**1. Introduction** 

many as 1,000 miRNAs.

Hotta, T., and Shimoyama, M. (2001). A new G-CSF-supported combination chemotherapy, LSG15, for adult T-cell leukemia-lymphoma: Japan Clinical Oncology Group Study 9303. *Br J Haematol* 113, 375-382.


### **Roles of MicroRNA in T-Cell Leukemia**

#### Mariko Tomita

*Department Pathology and Oncology Graduate School of Medical Science University of the Ryukyus Japan* 

#### **1. Introduction**

168 T-Cell Leukemia

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from cell lines of human adult T-cell leukemia and its implication in the disease.

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sarcoma-associated herpesvirus (human herpesvirus type 8)-associated extracavitary lymphoma: Report of a case in an HIV-positive patient with simultaneous kaposi

2009 American Society of Hematology annual meeting highlights*. J Hematol Oncol*,

Previously, cancer researchers have been focused on the genes that code proteins. They have considered those effects on tumorigenesis. However, discoveries of microRNAs (miRNAs) have shed a light on the role of non-protein-coding RNAs in tumorigenesis. The first small non-coding RNA, named *lin-4* in *Caenorhabditis elegans* (*C. elegans*) was described by Lee et al in 1993 (Lee et al., 1993). *Lin-4* codes miRNA that regulates the timing of *C. elegans* larval development by translational repression (Ambros, 2000). Since then, many miRNAs in different organisms such as plants, *C. elegans*, Drosophila, and mammals including humans have been discovered substantially. Up to now, the human genome is predicted to encode as many as 1,000 miRNAs.

miRNAs belong to a class of regulatory genes that are single-stranded 19-25 nucleotides non-cording RNAs and are generated from endogenous hairpin-shaped transcripts (Kim, 2005). miRNA genes are located either within the introns or exons of protein-coding genes (70%) or in intergenic regions (30%). More than 50% of mammalian miRNAs are located within the intronic regions of protein-coding genes. Most of the intronic or exonic miRNAs are transcribed in parallel with their host genes, indicating that these miRNAs use their host genes transcriptional machinalies. On the other hand, miRNAs produced from intergenic regions are transcribed separately from internal promoters (Rodriguez et al., 2004).

The first step of miRNA synthesis is the transcription of primary miRNA (pri-miRNA) from *miRNA* genes (Fig.1). pri-miRNAs are transcribed in a RNA polymerase II (Pol II) dependent manner as several hundreds or thousands of nucleotides long polyadenylated RNAs. In the nucleus, the pri-miRNA is processed to a precursor miRNA (pre-miRNA) of 60-100 nucleotides in length with a stem-loop structure by the nuclear protein Drosha that belongs to class II RNase III. Drosha interacts with its cofactor DGCR8 (the DiGeorge syndrome critical region gene 8 protein). Then, the pre-miRNA is exported from the nucleus to the cytoplasm by the Exportin 5/Ran-GTP complex. In the cytoplasm, the pre-miRNA is cleaved by class III RNase III, Dicer, which is a 200-kDa protein and miRNA is produced. Primary function of miRNAs in the cytoplasm is the negative regulation of gene expression by binding to complementary target sequences in the 3' untranslated region (UTR) of mRNA. Binding of a miRNA to the target mRNA typically leads to translational repression or degradation of mRNA, which means that miRNAs repress the expression of the target genes. In mammals, miRNAs guide the RNA induced silencing complex (RISC) to

Roles of MicroRNA in T-Cell Leukemia 171

stress responses (Bartel, 2004). Moreover, one target gene usually contains binding sites for multiple miRNAs, allowing miRNAs to form more complex regulatory networks of gene

Over the recent years, many miRNAs have been implicated in many human cancers. The first evidence for the importance of miRNAs in human cancer was the discovery of the loss of miR-15a/miR-16-1 cluster in chronic lymphocytic leukemia (CLL) (Calin et al., 2002). Since then, aberrant numerous miRNAs expression has been shown to be involved in the

Many miRNAs have been shown to function as oncogenes in human cancers. Among them, miR-155, which is encoded by nucleotides 241–262 of B-cell integration cluster (*BIC),* is the first miRNAs linked with cancer (Metzler et al., 2004). After the discovery, many research groups have shown that *miR-155* is highly expressed in various human cancers, including childrend's Burkitt's lymphoma (Metzler et al., 2004), Hodgkin's lymphoma, CLL (Kluiver et al., 2005), primary mediastinal non-Hodgkin's lymphoma (Calin et al., 2005), acute myelogenous leukemia (Garzon et al., 2008), lung cancer, breast cancer (Volinia et al., 2006), and pancreatic cancer (Greither et al., 2010). *miR-155* transgenic mice develop acute lymphoblastic leukemia (ALL) and high-grade lymphoma (Costinean et al.,

On the other hand, many miRNA encoding genes have been shown as tumor suppressor genes. As mentioned above, miR-15a/miR-16-1 cluster was the first to establish the link between miRNAs and cancer. This study showed loss of miR-15a/miR-16-1 cluster, which are located at chromosome 13q14, a region deleted in more than 65% of B cell CLL (Calin et al., 2002). These miRNAs induce apoptosis through the negative regulation of the antiapoptotic gene *Bcl2* (Cimmino et al., 2005). Indeed, down-regulation of miR-15a/miR-16-1 has been associated with the pathogenesis of CLL (Calin et al., 2008). These data support the

In this review, we introduce the accumulating evidences for the central roles that miRNAs play in hematological malignancy, in particular focusing on their role in T-cell leukemia and

Acute lymphoblastic leukemia (ALL) is the most common neoplasm in children, while it is relatively rare in adults. Although ALL originates from either B or T-cell progenitors, most cases are of B-cell ALL (B-ALL) (Pui & Evans, 2006). The less common type, T-cell ALL (T-ALL) is induced by the transformation of T-cell progenitors, and is diagnosed in 10-15% of children and 25% of adults with ALL (Copelan & McGuire, 1995). Molecular mechanisms of leukemogenesis of T-ALL have been investigated intensively. Recent studies revealed that 50-70% of T-ALLs have gain-of-function mutations in *Notch1*, a gene that is essential for Tcell development (Ferrando, 2009). miRNAs expression profiles in T- and B-ALL are highly associated with the lineage from which the leukemia derived (Lu et al., 2005). Some miRNAs can be discriminative of T-ALL versus B-ALL (Fulci et al., 2009). Although

2009). These results support the idea that miR-155 plays a role as oncogene *in vivo*.

idea that miR-15a/miR-16-1 plays a role as tumor-suppressor genes.

**3. microRNA in T-cell leukemia and lymphoma** 

**3.1 Acute lymphoblastic T-cell leukemia/lymphoma (T-ALL)** 

expression (Lewis et al., 2003).

development of human cancers.

lymphoma.

**2. microRNA in cancer** 

Fig. 1. miRNA biogenesis

complementary target sites of their specific target mRNAs, where endonucleolytically active Ago protein cleaves the mRNAs (Martinez et al., 2002). In contrast, other miRNAs predominantly bind to partially complementary target sites. Such imperfect binding between miRNAs and target mRNAs leads to repression of translation and/or deadenylation, followed by destabilization of the target mRNAs (Pillai et al., 2007). miRNAs are estimated to regulate more than 30% of mRNAs. Therefore, miRNAs have many roles in biological processes, such as development, differentiation, cell proliferation, apoptosis, and stress responses (Bartel, 2004). Moreover, one target gene usually contains binding sites for multiple miRNAs, allowing miRNAs to form more complex regulatory networks of gene expression (Lewis et al., 2003).

#### **2. microRNA in cancer**

170 T-Cell Leukemia

complementary target sites of their specific target mRNAs, where endonucleolytically active Ago protein cleaves the mRNAs (Martinez et al., 2002). In contrast, other miRNAs predominantly bind to partially complementary target sites. Such imperfect binding between miRNAs and target mRNAs leads to repression of translation and/or deadenylation, followed by destabilization of the target mRNAs (Pillai et al., 2007). miRNAs are estimated to regulate more than 30% of mRNAs. Therefore, miRNAs have many roles in biological processes, such as development, differentiation, cell proliferation, apoptosis, and

Fig. 1. miRNA biogenesis

Over the recent years, many miRNAs have been implicated in many human cancers. The first evidence for the importance of miRNAs in human cancer was the discovery of the loss of miR-15a/miR-16-1 cluster in chronic lymphocytic leukemia (CLL) (Calin et al., 2002). Since then, aberrant numerous miRNAs expression has been shown to be involved in the development of human cancers.

Many miRNAs have been shown to function as oncogenes in human cancers. Among them, miR-155, which is encoded by nucleotides 241–262 of B-cell integration cluster (*BIC),* is the first miRNAs linked with cancer (Metzler et al., 2004). After the discovery, many research groups have shown that *miR-155* is highly expressed in various human cancers, including childrend's Burkitt's lymphoma (Metzler et al., 2004), Hodgkin's lymphoma, CLL (Kluiver et al., 2005), primary mediastinal non-Hodgkin's lymphoma (Calin et al., 2005), acute myelogenous leukemia (Garzon et al., 2008), lung cancer, breast cancer (Volinia et al., 2006), and pancreatic cancer (Greither et al., 2010). *miR-155* transgenic mice develop acute lymphoblastic leukemia (ALL) and high-grade lymphoma (Costinean et al., 2009). These results support the idea that miR-155 plays a role as oncogene *in vivo*.

On the other hand, many miRNA encoding genes have been shown as tumor suppressor genes. As mentioned above, miR-15a/miR-16-1 cluster was the first to establish the link between miRNAs and cancer. This study showed loss of miR-15a/miR-16-1 cluster, which are located at chromosome 13q14, a region deleted in more than 65% of B cell CLL (Calin et al., 2002). These miRNAs induce apoptosis through the negative regulation of the antiapoptotic gene *Bcl2* (Cimmino et al., 2005). Indeed, down-regulation of miR-15a/miR-16-1 has been associated with the pathogenesis of CLL (Calin et al., 2008). These data support the idea that miR-15a/miR-16-1 plays a role as tumor-suppressor genes.

In this review, we introduce the accumulating evidences for the central roles that miRNAs play in hematological malignancy, in particular focusing on their role in T-cell leukemia and lymphoma.

#### **3. microRNA in T-cell leukemia and lymphoma**

#### **3.1 Acute lymphoblastic T-cell leukemia/lymphoma (T-ALL)**

Acute lymphoblastic leukemia (ALL) is the most common neoplasm in children, while it is relatively rare in adults. Although ALL originates from either B or T-cell progenitors, most cases are of B-cell ALL (B-ALL) (Pui & Evans, 2006). The less common type, T-cell ALL (T-ALL) is induced by the transformation of T-cell progenitors, and is diagnosed in 10-15% of children and 25% of adults with ALL (Copelan & McGuire, 1995). Molecular mechanisms of leukemogenesis of T-ALL have been investigated intensively. Recent studies revealed that 50-70% of T-ALLs have gain-of-function mutations in *Notch1*, a gene that is essential for Tcell development (Ferrando, 2009). miRNAs expression profiles in T- and B-ALL are highly associated with the lineage from which the leukemia derived (Lu et al., 2005). Some miRNAs can be discriminative of T-ALL versus B-ALL (Fulci et al., 2009). Although

Roles of MicroRNA in T-Cell Leukemia 173

ATLL is an aggressive lymphoproliferative disorder that occurs in individuals infected with human T-cell leukemia virus type 1 (HTLV-1) (Matsuoka & Jeang, 2007). HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years (Tajima, 1990). More than 20 million people are infected with HTLV-1 worldwide. ATLL occurs mainly in regions where HTLV-1 is endemic, mainly southern Japan, West Africa, and the Caribbean basin. ATLL is classified into four clinical subtypes termed acute, lymphoma, smoldering, and chronic. The prognosis of ATLL patients remains poor with a median survival time of 13 months in aggressive cases (Yamada et al., 2001). HTLV-1 encodes a protein Tax in its genome. The malignant growth and survival of HTLV-1-infected T-cells can be attributed to Tax, that is a modulator of many transcription factors and associations with molecules of signal transduction pathways that alter expression of host-cell genes involved in proliferation, apoptosis, and genetic stability (Marriott & Semmes, 2005; Boxus et al., 2008). Recent studies have shown the interactions between HTLV-1 and the miRNA regulatory

miRNA expression profiling studies in HTLV-1-infected T-cell lines and ATLL patients samples have been performed by some groups (Pichler et al., 2008; Yeung et al., 2008; Bellon et al., 2009). Pichler et al. demonstrated that 4 miRNAs (miR-21, miR-24, miR-146a, and miR-155) are upregulated and miR-223 is downregulated in HTLV-1-transfected cells by realtime RT-PCR to analyze selected sets of miRNAs that already been implicated in oncogenic transformation (Pichler et al., 2008). Bellon et al. idenentified aberrant expression of hematopoietic-specific miRNAs included miR-150, miR-155, miR-223, miR-142-3p, and miR142-5p (upregulated) and miR-181a, miR-132, miR-125a, and miR-146b (downregulated) in ATLL cells versus control peripheral blood mononuclear cells (PBMC) and CD4+ T-cells, and HTLV-1-infected cells lines *in vitro* and uncultured *ex vivo* ATLL cells (Bellon et al., 2009). These results were confirmed by real-time RT-PCR in additional ATLL cases and infected cell lines. They also demonstrated that treatment of HTLV-1-infected cell lines with an NF-B inhibitor (pathenolide) or JNK inhibitor (JNK II) resulted in reduced levels of the

Moreover, Yeung et al. demonstrated that 6 miRNAs (miR-9, miR-17-3p, miR-20b, miR-93, miR-130b, and miR-18a) are upregulated and 9 mi-RNAs (miR-1, miR-144, miR-126, miR-130a, miR-199a\*, miR-338, miR-432, miR-335, and miR-337) are downregulated both in HTLV-1-transformed cell lines and primary ATLL cells (Yeung et al., 2008). To distinguish miRNAs that are responding to proliferative stimuli, they also examined PBMC exposed to phorbol-12-myristate 13-acetate (PMA) compared to untreated PBMC. By these comparisons, they identified 3 miRNAs (miR-93, miR-130b, and miR-18a) that were upregulated in ATLL cells, HTLV-1-infected cell lines and PMA-treated cells, an additional miRNA, miR-335, was downregulated in all three cell types. Then they focused on miR-93 and miR-130b, those were confirmed increased expression in the ATLL samples by real-time RT-PCR. These miRNAs served to regulate tumor protein 53-induced nuclear protein 1 (TP53INP1), that is a cellular tumor suppressor protein whose activity governs cellular survival and proliferation (Yeung et al., 2008). Pichler et al. have shown that TP53INP1 is a potential mRNA target of miR-21, miR-24, miR-146a, and miR-155 (Pichler et al., 2008) .TP53INP1 is induced by the p53 response triggered by various stress treatments such as gamma irradiation, UV irradiation, and oxidative stress. Accumulation of TP53INP1 results in a block in the cell cycle at G1 (Tomasini et al., 2003) and triggers apoptosis through increased phosphorylation of p53 on Ser46 (Okamura et al., 2001) and upregulation of

**3.2 Adult T-cell leukemia/lymphoma (ATLL)** 

miR-155 precursor (Bellon et al., 2009).

network.

miRNAs that associated with leukemogenesis of B-ALL have been well documented (Lawrie, 2008), a few studies have been demonstrated association between particular miRNA and pathogenesis of T-ALL.

Recently, Mavrakis et al. have revealed association between miR-19 and leukemogenesis in T-ALL (Mavrakis et al., 2010). miR-19 was identified within the miR-17-92 cluster. The cluster is located at human chromosome 13q31 in a genomic region that is often amplified in many human cancers (Lu et al., 2005; Nagel et al., 2009). This cluster is also implicated in human hematopoietic malignancies (He et al., 2005; Mendell, 2008; Xiao et al., 2008). Xiao et al. have shown that miR-17-92 cluster is highly expressed in hematopoietic tumors and promotes lymphomagenesis *in vivo* (Xiao et al., 2008). Indeed, retroviral expression of miR-17-92 cluster genes accelerates c-Myc-induced B-cell lymphoma (Mu et al., 2009). The miR-17-92 cluster encodes 15 miRNAs including miR-19 with overlapping functions in development (Ventura et al., 2008). More recently, Mavrakis et al. demonstrated that miR-19 expresses at levels seen in other human tumors, enhances lymphocyte survival and is sufficient to cooperate with Notch1 in T-ALL *in vivo*. They found a 5-17 fold increase in miR-19 expression in T-ALL, and less for other miRNAs in the miR-17-92 cluster. miR-19 has a distinct ability to enhance lymphocyte survival *in vitro*. miR-19 target genes were identified by a large-scale short hairpin RNA screening, including multiple negative regulators in PI3K pathway such as PTEN, Bim, AMP-activated kinase (Prkaa1), and PP2A (Mavrakis & Wendel, 2010). The expression of these genes is regulated by miR-19 in lymphocytes, indicating that miR-19 produces a coordinate clampdown on multiple negative regulators of PI3K-related survival signals (Mavrakis et al., 2010).

Bhatia et al. recently reported downregulation of the expression of miR-196b in the human T-cell leukemia cell line, and T-ALL patients samples (Bhatia et al., 2011). Same group has shown that miR-196b has the capacity to downregulation the overamplified *c-myc* gene, recognized as a common pathogenomic feature leading to many cancers including B-ALL (Bhatia et al., 2010). In addition, they have demonstrated that miR-196b downregulation several *c-myc* effector genes like human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase enzyme responsible for unlimited proliferative potential of cancerous cells, Bcl-2, the anti-apoptotic protein involved in inhibition of cellular apoptosis, and apoptosis antagonizing transcription factor (AATF). Indeed, restoration of miR-196b in EB-3 cells derived from a Burkitt lymphoma leads to significant downregulation of *c-myc* and its effector genes and qualifies for tumor suppressor function in B-ALL (Bhatia et al., 2010). On the contrary, miR-196b loses its ability to down regulate *c-myc* gene in T-ALL as a consequence of mutations in its target binding region in 3'UTR of *c-myc* gene (Bhatia et al., 2011). Although miR-196b is implicated to have different functions in T-ALL from in B-ALL, the role of miR-196b on leukemogenesis of T-ALL is still unclear. Another group's recent study has shown that miR-196a and miR-196b as regulators of the oncogenic ETS transcription factor *ERG* (Coskun et al., 2011). *ERG* has been known as playing important physiological and oncogenic roles in hematopoiesis (Baldus et al., 2006). It is also a prognostic factor in a subset of adult patients with T-ALL (Baldus et al., 2006; Loughran et al., 2008). They found that miR-196a and miR-196b expression was associated with an immature immunophenotype (CD34 positive) in T-ALL patients (Coskun et al., 2011). These findings indicate miR-196a and miR-196b as *ERG* regulators and implicate a potential role for these miRNAs in T-ALL.

miRNAs that associated with leukemogenesis of B-ALL have been well documented (Lawrie, 2008), a few studies have been demonstrated association between particular

Recently, Mavrakis et al. have revealed association between miR-19 and leukemogenesis in T-ALL (Mavrakis et al., 2010). miR-19 was identified within the miR-17-92 cluster. The cluster is located at human chromosome 13q31 in a genomic region that is often amplified in many human cancers (Lu et al., 2005; Nagel et al., 2009). This cluster is also implicated in human hematopoietic malignancies (He et al., 2005; Mendell, 2008; Xiao et al., 2008). Xiao et al. have shown that miR-17-92 cluster is highly expressed in hematopoietic tumors and promotes lymphomagenesis *in vivo* (Xiao et al., 2008). Indeed, retroviral expression of miR-17-92 cluster genes accelerates c-Myc-induced B-cell lymphoma (Mu et al., 2009). The miR-17-92 cluster encodes 15 miRNAs including miR-19 with overlapping functions in development (Ventura et al., 2008). More recently, Mavrakis et al. demonstrated that miR-19 expresses at levels seen in other human tumors, enhances lymphocyte survival and is sufficient to cooperate with Notch1 in T-ALL *in vivo*. They found a 5-17 fold increase in miR-19 expression in T-ALL, and less for other miRNAs in the miR-17-92 cluster. miR-19 has a distinct ability to enhance lymphocyte survival *in vitro*. miR-19 target genes were identified by a large-scale short hairpin RNA screening, including multiple negative regulators in PI3K pathway such as PTEN, Bim, AMP-activated kinase (Prkaa1), and PP2A (Mavrakis & Wendel, 2010). The expression of these genes is regulated by miR-19 in lymphocytes, indicating that miR-19 produces a coordinate clampdown on multiple negative regulators of

Bhatia et al. recently reported downregulation of the expression of miR-196b in the human T-cell leukemia cell line, and T-ALL patients samples (Bhatia et al., 2011). Same group has shown that miR-196b has the capacity to downregulation the overamplified *c-myc* gene, recognized as a common pathogenomic feature leading to many cancers including B-ALL (Bhatia et al., 2010). In addition, they have demonstrated that miR-196b downregulation several *c-myc* effector genes like human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase enzyme responsible for unlimited proliferative potential of cancerous cells, Bcl-2, the anti-apoptotic protein involved in inhibition of cellular apoptosis, and apoptosis antagonizing transcription factor (AATF). Indeed, restoration of miR-196b in EB-3 cells derived from a Burkitt lymphoma leads to significant downregulation of *c-myc* and its effector genes and qualifies for tumor suppressor function in B-ALL (Bhatia et al., 2010). On the contrary, miR-196b loses its ability to down regulate *c-myc* gene in T-ALL as a consequence of mutations in its target binding region in 3'UTR of *c-myc* gene (Bhatia et al., 2011). Although miR-196b is implicated to have different functions in T-ALL from in B-ALL, the role of miR-196b on leukemogenesis of T-ALL is still unclear. Another group's recent study has shown that miR-196a and miR-196b as regulators of the oncogenic ETS transcription factor *ERG* (Coskun et al., 2011). *ERG* has been known as playing important physiological and oncogenic roles in hematopoiesis (Baldus et al., 2006). It is also a prognostic factor in a subset of adult patients with T-ALL (Baldus et al., 2006; Loughran et al., 2008). They found that miR-196a and miR-196b expression was associated with an immature immunophenotype (CD34 positive) in T-ALL patients (Coskun et al., 2011). These findings indicate miR-196a and miR-196b as *ERG* regulators and implicate a potential role

miRNA and pathogenesis of T-ALL.

PI3K-related survival signals (Mavrakis et al., 2010).

for these miRNAs in T-ALL.

#### **3.2 Adult T-cell leukemia/lymphoma (ATLL)**

ATLL is an aggressive lymphoproliferative disorder that occurs in individuals infected with human T-cell leukemia virus type 1 (HTLV-1) (Matsuoka & Jeang, 2007). HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years (Tajima, 1990). More than 20 million people are infected with HTLV-1 worldwide. ATLL occurs mainly in regions where HTLV-1 is endemic, mainly southern Japan, West Africa, and the Caribbean basin. ATLL is classified into four clinical subtypes termed acute, lymphoma, smoldering, and chronic. The prognosis of ATLL patients remains poor with a median survival time of 13 months in aggressive cases (Yamada et al., 2001). HTLV-1 encodes a protein Tax in its genome. The malignant growth and survival of HTLV-1-infected T-cells can be attributed to Tax, that is a modulator of many transcription factors and associations with molecules of signal transduction pathways that alter expression of host-cell genes involved in proliferation, apoptosis, and genetic stability (Marriott & Semmes, 2005; Boxus et al., 2008). Recent studies have shown the interactions between HTLV-1 and the miRNA regulatory network.

miRNA expression profiling studies in HTLV-1-infected T-cell lines and ATLL patients samples have been performed by some groups (Pichler et al., 2008; Yeung et al., 2008; Bellon et al., 2009). Pichler et al. demonstrated that 4 miRNAs (miR-21, miR-24, miR-146a, and miR-155) are upregulated and miR-223 is downregulated in HTLV-1-transfected cells by realtime RT-PCR to analyze selected sets of miRNAs that already been implicated in oncogenic transformation (Pichler et al., 2008). Bellon et al. idenentified aberrant expression of hematopoietic-specific miRNAs included miR-150, miR-155, miR-223, miR-142-3p, and miR142-5p (upregulated) and miR-181a, miR-132, miR-125a, and miR-146b (downregulated) in ATLL cells versus control peripheral blood mononuclear cells (PBMC) and CD4+ T-cells, and HTLV-1-infected cells lines *in vitro* and uncultured *ex vivo* ATLL cells (Bellon et al., 2009). These results were confirmed by real-time RT-PCR in additional ATLL cases and infected cell lines. They also demonstrated that treatment of HTLV-1-infected cell lines with an NF-B inhibitor (pathenolide) or JNK inhibitor (JNK II) resulted in reduced levels of the miR-155 precursor (Bellon et al., 2009).

Moreover, Yeung et al. demonstrated that 6 miRNAs (miR-9, miR-17-3p, miR-20b, miR-93, miR-130b, and miR-18a) are upregulated and 9 mi-RNAs (miR-1, miR-144, miR-126, miR-130a, miR-199a\*, miR-338, miR-432, miR-335, and miR-337) are downregulated both in HTLV-1-transformed cell lines and primary ATLL cells (Yeung et al., 2008). To distinguish miRNAs that are responding to proliferative stimuli, they also examined PBMC exposed to phorbol-12-myristate 13-acetate (PMA) compared to untreated PBMC. By these comparisons, they identified 3 miRNAs (miR-93, miR-130b, and miR-18a) that were upregulated in ATLL cells, HTLV-1-infected cell lines and PMA-treated cells, an additional miRNA, miR-335, was downregulated in all three cell types. Then they focused on miR-93 and miR-130b, those were confirmed increased expression in the ATLL samples by real-time RT-PCR. These miRNAs served to regulate tumor protein 53-induced nuclear protein 1 (TP53INP1), that is a cellular tumor suppressor protein whose activity governs cellular survival and proliferation (Yeung et al., 2008). Pichler et al. have shown that TP53INP1 is a potential mRNA target of miR-21, miR-24, miR-146a, and miR-155 (Pichler et al., 2008) .TP53INP1 is induced by the p53 response triggered by various stress treatments such as gamma irradiation, UV irradiation, and oxidative stress. Accumulation of TP53INP1 results in a block in the cell cycle at G1 (Tomasini et al., 2003) and triggers apoptosis through increased phosphorylation of p53 on Ser46 (Okamura et al., 2001) and upregulation of

Roles of MicroRNA in T-Cell Leukemia 175

Although several miRNA profiling experiments accumulate miRNA subsets up or downregulated in HTLV-1-infected T-cell lines and ATLL patient's samples, data of miRNA expression profiles are not consistent in these reports. This inconsistency could be due to methodological differences in the techniques employed to prepare RNA from the cells

Among the miRNA expression profilings, miR-146a was found to be activated by Tax in an NF-B-dependent manner (Pichler et al., 2008). Two potential NF-B binding sites were identified in the miR-146a promoter. They found that the proximal NF-B binding site on the *miR-146a* gene is responsible for transcriptional activation by Tax (Pichler et al., 2008). Recently, our group demonstrated that *miR-146a* gene expression is activated by Tax in NF- B-dependent manner (Tomita et al., 2011). We found that the miR-146a promoter was highly bound by NF-B complexes in HTLV-1-infected cells, while treatment with the NF- B inhibitor Bay11-7082 reduced binding and interfered with expression of the miR-146a (Tomita et al., 2011). In contrast to Picheler's results, we observed binding between NF-B protein and distal NF-B binding site, not proximal one (Fig. 2). Moreover, we observed that miR-146a plays an important role in the growth of HTLV-1-infected T-cells (Tomita et al., 2011). Treatment of HTLV-1-infected cell lines with an anti-miR-146a inhibitor interfered with their growth and increased the expression levels of TRAF6, a predicted target for miR-146a. On the other hand, a growth-enhancing effect was observed in HTLV-1-infected cell line forced to overexpress miR-146a. These results suggest that miR-146a might be a good

Recently, Sasaki et al. demonstrated that ATLL cells showed a decreased level of miR-101 and miR-128a expression compared with the cells from HTLV-1 carriers (Sasaki et al., 2011). Moreover, there was a clear inverse correlation between *Enhancer of zeste homolog 2* (EZH2) expression and miR-101 expression or *EZH2* expression and miR-128a expression, suggesting that increased EZH2 is caused by the decrease in these miRNAs expression (Sasaki et al., 2011). EZH2 is a critical component of polycomb repressive complex 2 (PRC2), which mediates epigenetic gene silencing through trimethylation of H3K27 (Cao et al., 2002; Czermin et al., 2002). ATLL patients with high *EZH2* expression showed shorter survival than patients with low *EZH2* expression (Sasaki et al., 2011), indicating that increased EZH2

Sézary syndrome is a rare aggressive form of primary cutaneous T-cell lymphoma characterized by erythroderma, generalized lymphadenopathy, and the presence of neoplastic cerebriform nucleated CD4+ T-cells (Sézary cells) in peripheral blood. Patients with Sézary syndrome have a high leukemic burden and a poor prognostic outcome, with an estimated 5-year survival of only 24% (Willemze et al., 2005). Mycosis fungoides, the most common cutaneous T-cell lymphoma, is a malignancy of mature, skin-homing T-cells. Sézary syndrome is often considered to represent a leukemic phase of mycosis fungoides. Recently, Ballabio et al. performed miRNA profile of CD4+ T-cells from Sézary syndrome patients. They identified 114 miRNAs specifically expressed in Sézary syndrome (Ballabio et al., 2010). They demonstrated that levels of 4 microRNAs (miR-150, miR-191, miR-15a, and miR-16) correctly predicted diagnosis of Sézary syndrome with 100% accuracy, whereas miR-223 and miR-17-5p were 96% accurate. Further analysis revealed that levels of miR-223 distinguished Sézary syndrome samples from healthy controls and patients with mycosis

and/or to hybridize probes to microarray.

therapeutic target in ATLL.

plays a role in the process of ATLL progression.

**3.3 Sézary syndrome and mycosis fungoides** 

selected p53-responsive genes such as p53AIP1 (Okamura et al., 2001), p21 and Bax (Tomasini et al., 2001). These results suggest that miRNAs enhance cell growth and suppress apoptosis through targeting TP53INP1.

Fig. 2. Schematic representation of the effects of Tax on miR-146a expression (Tomita et al., 2011)

selected p53-responsive genes such as p53AIP1 (Okamura et al., 2001), p21 and Bax (Tomasini et al., 2001). These results suggest that miRNAs enhance cell growth and suppress

Fig. 2. Schematic representation of the effects of Tax on miR-146a expression (Tomita et

apoptosis through targeting TP53INP1.

al., 2011)

Although several miRNA profiling experiments accumulate miRNA subsets up or downregulated in HTLV-1-infected T-cell lines and ATLL patient's samples, data of miRNA expression profiles are not consistent in these reports. This inconsistency could be due to methodological differences in the techniques employed to prepare RNA from the cells and/or to hybridize probes to microarray.

Among the miRNA expression profilings, miR-146a was found to be activated by Tax in an NF-B-dependent manner (Pichler et al., 2008). Two potential NF-B binding sites were identified in the miR-146a promoter. They found that the proximal NF-B binding site on the *miR-146a* gene is responsible for transcriptional activation by Tax (Pichler et al., 2008). Recently, our group demonstrated that *miR-146a* gene expression is activated by Tax in NF- B-dependent manner (Tomita et al., 2011). We found that the miR-146a promoter was highly bound by NF-B complexes in HTLV-1-infected cells, while treatment with the NF- B inhibitor Bay11-7082 reduced binding and interfered with expression of the miR-146a (Tomita et al., 2011). In contrast to Picheler's results, we observed binding between NF-B protein and distal NF-B binding site, not proximal one (Fig. 2). Moreover, we observed that miR-146a plays an important role in the growth of HTLV-1-infected T-cells (Tomita et al., 2011). Treatment of HTLV-1-infected cell lines with an anti-miR-146a inhibitor interfered with their growth and increased the expression levels of TRAF6, a predicted target for miR-146a. On the other hand, a growth-enhancing effect was observed in HTLV-1-infected cell line forced to overexpress miR-146a. These results suggest that miR-146a might be a good therapeutic target in ATLL.

Recently, Sasaki et al. demonstrated that ATLL cells showed a decreased level of miR-101 and miR-128a expression compared with the cells from HTLV-1 carriers (Sasaki et al., 2011). Moreover, there was a clear inverse correlation between *Enhancer of zeste homolog 2* (EZH2) expression and miR-101 expression or *EZH2* expression and miR-128a expression, suggesting that increased EZH2 is caused by the decrease in these miRNAs expression (Sasaki et al., 2011). EZH2 is a critical component of polycomb repressive complex 2 (PRC2), which mediates epigenetic gene silencing through trimethylation of H3K27 (Cao et al., 2002; Czermin et al., 2002). ATLL patients with high *EZH2* expression showed shorter survival than patients with low *EZH2* expression (Sasaki et al., 2011), indicating that increased EZH2 plays a role in the process of ATLL progression.

#### **3.3 Sézary syndrome and mycosis fungoides**

Sézary syndrome is a rare aggressive form of primary cutaneous T-cell lymphoma characterized by erythroderma, generalized lymphadenopathy, and the presence of neoplastic cerebriform nucleated CD4+ T-cells (Sézary cells) in peripheral blood. Patients with Sézary syndrome have a high leukemic burden and a poor prognostic outcome, with an estimated 5-year survival of only 24% (Willemze et al., 2005). Mycosis fungoides, the most common cutaneous T-cell lymphoma, is a malignancy of mature, skin-homing T-cells. Sézary syndrome is often considered to represent a leukemic phase of mycosis fungoides. Recently, Ballabio et al. performed miRNA profile of CD4+ T-cells from Sézary syndrome patients. They identified 114 miRNAs specifically expressed in Sézary syndrome (Ballabio et al., 2010). They demonstrated that levels of 4 microRNAs (miR-150, miR-191, miR-15a, and miR-16) correctly predicted diagnosis of Sézary syndrome with 100% accuracy, whereas miR-223 and miR-17-5p were 96% accurate. Further analysis revealed that levels of miR-223 distinguished Sézary syndrome samples from healthy controls and patients with mycosis

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WK., Thiel, E. & Baldus, CD. (2011). The role of microRNA-196a and microRNA-

fungoides in more than 90% of samples (Ballabio et al., 2010). miR-342 expression in Sézary syndrome was negatively regulated by miR-199a\* expression. Transfection with either miR-342 or miR-199a\* inhibitor resulted in a significant increase in levels of apoptosis of SeAx cells, suggesting that downregulation of miR-342 plays an important role in the pathogenesis of Sézary syndrome by inhibiting apoptosis (Ballabio et al., 2010). These data indicate that miRNAs are important in the pathogenesis of Sézary syndrome and provide possibilities for the diagnosis and treatment of this disease.

#### **3.4 Anaplastic large-cell lymphoma**

Anaplastic large cell lymphoma (ALCL) with anaplastic lymphoma kinase (ALK)-positive is a T-cell lymphoma consisting of lymphoid cells that are usually large with abundant cytoplasm and pleomorphic, often horseshoe-shaped nuclei, with *ALK* gene rearrangements. It tends to occur in children or young adults. The most commonly involved extranodal sites are skin, bone, soft tissues, lung and liver. The 5-year survival of ALCL with ALK-positive patients is about 70%, in contrast to ALCL with ALK-negative, which shows poor prognosis (Swerdlow et al., 2008). Recently, Merkel et al. demonstrated that five members of the miR-17–92 cluster were expressed more highly in ALCL with ALK-positive, whereas miR-155 was expressed more than 10-fold higher in ALCL with ALK-negative. Moreover, miR-101 was downregulated in all ALCL model systems, but forced expression of miR-101 attenuated cell proliferation only in ALK-positive and not in ALK-negative cell lines, suggesting different modes of ALK-dependent regulation of its target proteins (Merkel et al., 2010). For future therapeutical and diagnostic application, it will be interesting to study the physiological implications and prognostic value of the identified miRNA profiles.

#### **4. Conclusion**

Molecular targeting therapy based on miRNAs hold great promise for the development of more effective and less toxic personalized treatment strategies against cancer. Approach of targeting therapy needs deeper knowledge of the molecular changes that associate with development and progression of the diseases. The research on miRNAs is rapidly progressing from *in vitro* to *in vivo* and this becomes a powerful tool for molecular targeting therapy for human cancers. Although there is emerging evidence that miRNAs are involved in the pathogenesis of many cancers, including B-cell lymphomas, there are very little published data on the involvement of miRNAs in human T-cell leukemias/lymphomas that were discussed in this review. It is necessary to accumulate more molecular data that indicate association between miRNAs and T-cell leukemia/lymphoma.

#### **5. Acknowledgment**

I appreciate Dr. Shuji Tomita for valuable discussions and critical reading.

#### **6. References**

Ambros, V. (2000). Control of developmental timing in Caenorhabditis elegans. Current Opinion in Genetics & Development 10(4): 428-433.

Baldus, CD., Burmeister, T., Martus, P., Schwartz, S., Gokbuget, N., Bloomfield, CD., Hoelzer, D., Thiel, E. & Hofmann, WK. (2006). High expression of the ETS

fungoides in more than 90% of samples (Ballabio et al., 2010). miR-342 expression in Sézary syndrome was negatively regulated by miR-199a\* expression. Transfection with either miR-342 or miR-199a\* inhibitor resulted in a significant increase in levels of apoptosis of SeAx cells, suggesting that downregulation of miR-342 plays an important role in the pathogenesis of Sézary syndrome by inhibiting apoptosis (Ballabio et al., 2010). These data indicate that miRNAs are important in the pathogenesis of Sézary syndrome and provide

Anaplastic large cell lymphoma (ALCL) with anaplastic lymphoma kinase (ALK)-positive is a T-cell lymphoma consisting of lymphoid cells that are usually large with abundant cytoplasm and pleomorphic, often horseshoe-shaped nuclei, with *ALK* gene rearrangements. It tends to occur in children or young adults. The most commonly involved extranodal sites are skin, bone, soft tissues, lung and liver. The 5-year survival of ALCL with ALK-positive patients is about 70%, in contrast to ALCL with ALK-negative, which shows poor prognosis (Swerdlow et al., 2008). Recently, Merkel et al. demonstrated that five members of the miR-17–92 cluster were expressed more highly in ALCL with ALK-positive, whereas miR-155 was expressed more than 10-fold higher in ALCL with ALK-negative. Moreover, miR-101 was downregulated in all ALCL model systems, but forced expression of miR-101 attenuated cell proliferation only in ALK-positive and not in ALK-negative cell lines, suggesting different modes of ALK-dependent regulation of its target proteins (Merkel et al., 2010). For future therapeutical and diagnostic application, it will be interesting to study the

physiological implications and prognostic value of the identified miRNA profiles.

indicate association between miRNAs and T-cell leukemia/lymphoma.

I appreciate Dr. Shuji Tomita for valuable discussions and critical reading.

Opinion in Genetics & Development 10(4): 428-433.

Molecular targeting therapy based on miRNAs hold great promise for the development of more effective and less toxic personalized treatment strategies against cancer. Approach of targeting therapy needs deeper knowledge of the molecular changes that associate with development and progression of the diseases. The research on miRNAs is rapidly progressing from *in vitro* to *in vivo* and this becomes a powerful tool for molecular targeting therapy for human cancers. Although there is emerging evidence that miRNAs are involved in the pathogenesis of many cancers, including B-cell lymphomas, there are very little published data on the involvement of miRNAs in human T-cell leukemias/lymphomas that were discussed in this review. It is necessary to accumulate more molecular data that

Ambros, V. (2000). Control of developmental timing in Caenorhabditis elegans. Current

Baldus, CD., Burmeister, T., Martus, P., Schwartz, S., Gokbuget, N., Bloomfield, CD.,

Hoelzer, D., Thiel, E. & Hofmann, WK. (2006). High expression of the ETS

possibilities for the diagnosis and treatment of this disease.

**3.4 Anaplastic large-cell lymphoma** 

**4. Conclusion** 

**5. Acknowledgment** 

**6. References** 

transcription factor ERG predicts adverse outcome in acute T-lymphoblastic leukemia in adults. Journal of Clinical Oncology 24(29): 4714-4720.


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**10** 

**Mechanisms of Humoral Hypercalcemia of** 

Hypercalcemia is one of the most common paraneoplastic syndromes. The incidence of hypercalcemia is 50-90% in adult T-cell leukemia/lymphoma (ATLL), 27-35% in lung cancer, 25-30% in breast cancer, 7-30% in multiple myeloma, and less than 10% in other types of cancer patients (Mundy & Martin, 1982; Roodman, 1997). Patients with severe hypercalcemia (>12 mg/dL; > 6.0 mM) usually develop neuromuscular, gastrointestinal and renal symptoms including lethargy, depression, anorexia, nausea, vomiting, polyuria and polydipsia. Patients with serum calcium concentrations >15 mg/dL (7.6 mM) can develop renal failure or cardiovascular abnormalities with arrhythmias and coma (Mundy & Martin, 1982). Depending on the sources of the stimulating factors, hypercalcemia in cancer can be divided into 3 types: (1) humoral hypercalcemia of malignancy (HHM) in which humoral factors secreted by tumor cells directly or indirectly affect cells in the target organs including bone, kidney and intestine that regulate calcium homeostasis; (2) local osteolytic hypercalcemia in which factors secreted by either primary or metastatic tumor cells locally in the bone microenvironment stimulate osteoclastic bone resorption; and (3) primary hyperparathyroidism that coexists with the malignancy (Stewart, 2005). This review will focus on HHM, although some types of cancers may induce both HHM and local osteolytic

hypercalcemia, since several factors can function both systemically and locally.

Humoral hypercalcemia of malignancy (HHM) is characterized by (1) circulating humoral factors derived from cancer cells; (2) uncoupling of bone formation and bone resorption; (3) increased renal calcium reabsorption even though there is hypercalciuria caused by increased Ca2+ in the glomerular filtrate. In contrast to HHM in primary hyperparathyroidism, bone formation and resorption are both increased resulting in fibrous

HHM is a common complication of certain lymphoma/leukemias; squamous cell carcinomas (e.g., of the lung or other organs); renal and breast carcinomas, and occasionally other tumors (Stewart, 2002). Factors secreted by the cancer cells (see Table 1 below) stimulate osteoclastic bone resorption (directly or indirectly through osteoblasts) by increasing the activity and/or survival of osteoclast precursors or mature osteoclasts. Most

**2. Overview of humoral hypercalcemia of malignancy** 

osteodystrophy in patients with longstanding disease.

**1. Introduction** 

**Malignancy in Leukemia/Lymphoma** 

Katherine N. Weibaecher and Thomas J. Rosol *Ohio State University and Washington University (KNW)* 

Sherry T. Shu, Wessel P. Dirksen,

*United States of America* 

trimethylation of lysine 27 on histone H3 in adult T-cell leukemia/lymphoma as a target for epigenetic therapy. Haematologica 96(5): 712-719.


## **Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma**

 Sherry T. Shu, Wessel P. Dirksen, Katherine N. Weibaecher and Thomas J. Rosol *Ohio State University and Washington University (KNW) United States of America* 

#### **1. Introduction**

180 T-Cell Leukemia

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Chimenti, S., Diaz-Perez, JL., Duncan, LM., Grange, F., Harris, NL., Kempf, W., Kerl, H., Kurrer, M., Knobler, R., Pimpinelli, N., Sander, C., Santucci, M., Sterry, W., Vermeer, MH., Wechsler, J., Whittaker, S. & Meijer, CJ. (2005). WHO-EORTC classification for cutaneous lymphomas. Blood 105(10): 3768-3785.Xiao, C., Srinivasan, L., Calado, DP., Patterson, HC., Zhang, B., Wang, J., Henderson, JM., Kutok, JL. & Rajewsky, K. (2008). Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nature Immunology

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Jeang, KT. (2008). Roles for microRNAs, miR-93 and miR-130b, and tumor protein 53-induced nuclear protein 1 tumor suppressor in cell growth dysregulation by Hypercalcemia is one of the most common paraneoplastic syndromes. The incidence of hypercalcemia is 50-90% in adult T-cell leukemia/lymphoma (ATLL), 27-35% in lung cancer, 25-30% in breast cancer, 7-30% in multiple myeloma, and less than 10% in other types of cancer patients (Mundy & Martin, 1982; Roodman, 1997). Patients with severe hypercalcemia (>12 mg/dL; > 6.0 mM) usually develop neuromuscular, gastrointestinal and renal symptoms including lethargy, depression, anorexia, nausea, vomiting, polyuria and polydipsia. Patients with serum calcium concentrations >15 mg/dL (7.6 mM) can develop renal failure or cardiovascular abnormalities with arrhythmias and coma (Mundy & Martin, 1982). Depending on the sources of the stimulating factors, hypercalcemia in cancer can be divided into 3 types: (1) humoral hypercalcemia of malignancy (HHM) in which humoral factors secreted by tumor cells directly or indirectly affect cells in the target organs including bone, kidney and intestine that regulate calcium homeostasis; (2) local osteolytic hypercalcemia in which factors secreted by either primary or metastatic tumor cells locally in the bone microenvironment stimulate osteoclastic bone resorption; and (3) primary hyperparathyroidism that coexists with the malignancy (Stewart, 2005). This review will focus on HHM, although some types of cancers may induce both HHM and local osteolytic hypercalcemia, since several factors can function both systemically and locally.

#### **2. Overview of humoral hypercalcemia of malignancy**

Humoral hypercalcemia of malignancy (HHM) is characterized by (1) circulating humoral factors derived from cancer cells; (2) uncoupling of bone formation and bone resorption; (3) increased renal calcium reabsorption even though there is hypercalciuria caused by increased Ca2+ in the glomerular filtrate. In contrast to HHM in primary hyperparathyroidism, bone formation and resorption are both increased resulting in fibrous osteodystrophy in patients with longstanding disease.

HHM is a common complication of certain lymphoma/leukemias; squamous cell carcinomas (e.g., of the lung or other organs); renal and breast carcinomas, and occasionally other tumors (Stewart, 2002). Factors secreted by the cancer cells (see Table 1 below) stimulate osteoclastic bone resorption (directly or indirectly through osteoblasts) by increasing the activity and/or survival of osteoclast precursors or mature osteoclasts. Most

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 183

regulated by NF-κB, TGF-β, and/or Ras-MAPK signaling (Nadella et al., 2007; Richard et al., 2005). In adult T-cell leukemia/lymphoma, the promoter of PTHrP can be activated by

The functions of PTHrP depend on the activation of different signal transduction pathways, including cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and protein lipase C/protein kinase C (PLC/PKC) cascades. In addition, PTHrP fragments have different functions that depend on the tissue-specific expression of distinct receptors. Activation of the cAMP-PKA pathway is required for PTHrP to induce bone resorption through the PTH1R, which recognizes both PTH and PTHrP (Greenfield et al., 1995). Besides the catabolic effect of PTHrP on bone, intermittent administration or pulsatile secretion of the N-terminal fragment of PTH (1-34) and PTHrP (1-36) by a neuroendocrine tumor, such as islet cell carcinoma, can induce bone anabolic effects (Takeuchi et al., 2002). When osteoblastic cells were transiently exposed to the C-terminus of PTHrP (107-139), anabolic effects were induced by the upregulation of vascular endothelial growth factor

**3.2 Receptor activator of nuclear kappa-B ligand (RANKL) and osteoprotegerin (OPG)**  RANKL belongs to the tumor necrosis factor (TNF) family and represents one of the most common mediators for inducing osteoclastic bone resorption. Mature osteoblasts produce two forms of RANKL, a membrane-bound and secreted form. Both are important for osteoclast stimulation (Leibbrandt & Penninger, 2008). The expression of RANKL in osteoblasts and stromal cells is activated by the RUNX2 transcriptional factor under the regulation of PTHrP, 1,25-dihydroxyvitamin D3, and prostaglandins (Lipton et al., 2009). RANKL binds and activates its receptor, RANK, which is expressed on the surface of osteoclasts. The binding of RANKL to RANK activates at least three major signaling pathways, NFAT, p38 and JNK, resulting in up-regulation of genes that are required for induction of osteoclast fusion, differentiation, activation and survival (Leibbrandt & Penninger, 2008). The secreted form of RANKL is important for the recruitment of osteoclast precursors and osteoclastogenesis. RANKL is an essential activator for normal bone

remodeling. RANKL knockout mice have severe osteopetrosis (Lomaga et al., 1999).

activity (Horwood et al., 1998).

OPG, or osteoclastogenesis inhibitory factor (OCIF), is a secreted member of the tumor necrosis receptor superfamily that is expressed by osteoblasts and functions as a RANKL 'decoy receptor' (Simonet et al., 1997). OPG binds to RANKL and blocks its interaction with RANK on osteoclast precursors (Lacey et al., 1998); therefore, it is a potent inhibitor of osteoclast formation and bone resorption. OPG knockout mice have early-onset osteopenia (Bucay et al., 1998; Mizuno et al., 1998), whereas OPG overexpressing mice develop osteopetrosis that results from the failure to form osteoclasts (Simonet et al., 1997). PTH and PTHrP suppress OPG expression by downregulating the promoter of OPG through activation of the cAMP/PKA pathway (Yang et al., 2002). PTHrP, on the other hand, stimulates RANKL expression to induce bone resorption. The ratio between RANKL and OPG levels in osteoblasts is a key factor in the regulation of osteoclast

In ATLL, the expression of RANKL in tumor cells correlated with hypercalcemia in patients. RANKL on the surface of leukemia cells induced osteoclastogenesis through direct contact with precursor cells (Nosaka et al., 2002). The direct activation of osteoclasts by tumor cells may play a key role in the decoupling of bone formation and bone resorption in HMM.

the binding of HTLV-1 oncoprotein, Tax (Ejima et al., 1993; Watanabe et al., 1990).

receptor 2 (VEGFR2) through PKC/ERK activation (de Gortazar et al., 2006).

cancer-derived hypercalcemic factors stimulate osteoclastic bone resorption indirectly by inducing osteoclast-stimulating factors from osteoblasts or bone stromal cells. Under normal physiological conditions, increased serum calcium concentration can be compensated for by decreasing the intestinal calcium absorption, increasing renal calcium excretion, decreasing PTH secretion from the parathyroid glands, and decreasing bone resorption. However, secretion of the cancer-related factor, parathyroid hormone-related protein (PTHrP), also increases renal calcium reabsorption in the kidney through the activation of parathyroid hormone receptor 1 (PTH1R), which facilitates the development of HHM.


Table 1. Factors associated with HHM that cause bone formation (anabolic action) or resorption (catabolic action) in bone.

#### **3. Factors involved in the pathogenesis of HHM**

#### **3.1 Parathyroid hormone-related protein (PTHrP)**

PTHrP was first cloned by Suva et al. (Suva et al., 1987) and purified by Broadus et al. (Broadus et al., 1988). PTHrP is widely expressed in normal tissues and functions as an endocrine, autocrine, paracrine and intracrine hormone. PTHrP is a polyhormone that results from alternative mRNA splicing and post-translational proteolytic processing. During development, PTHrP is essential for the growth and regulation of endochondral bone (Karaplis et al., 1994; Wysolmerski et al., 1998), epithelial-mesenchymal interactions in mammary gland (Wysolmerski et al., 1998), and has important functions in many other tissues. PTHrP knockout mice die soon after birth due to asphyxia caused by developmental abnormalities of bones in the thorax (Karaplis et al., 1994). PTHrP has been shown to be the principal factor in most cases of cancer-induced HHM. The expression of PTHrP is up-

cancer-derived hypercalcemic factors stimulate osteoclastic bone resorption indirectly by inducing osteoclast-stimulating factors from osteoblasts or bone stromal cells. Under normal physiological conditions, increased serum calcium concentration can be compensated for by decreasing the intestinal calcium absorption, increasing renal calcium excretion, decreasing PTH secretion from the parathyroid glands, and decreasing bone resorption. However, secretion of the cancer-related factor, parathyroid hormone-related protein (PTHrP), also increases renal calcium reabsorption in the kidney through the activation of parathyroid

**Factors Origin Target cells/molecule Function in bone**  PTHrP Cancer cells Osteoblast Catabolic and

OPG Osteoblast RANKL Inhibit RANKL MIP-1 Cancer cells Osteoblast Catabolic

TNF-α, IL-1, IL-6, IL-17 T-cells Osteoclast Catabolic

Table 1. Factors associated with HHM that cause bone formation (anabolic action) or

PTHrP was first cloned by Suva et al. (Suva et al., 1987) and purified by Broadus et al. (Broadus et al., 1988). PTHrP is widely expressed in normal tissues and functions as an endocrine, autocrine, paracrine and intracrine hormone. PTHrP is a polyhormone that results from alternative mRNA splicing and post-translational proteolytic processing. During development, PTHrP is essential for the growth and regulation of endochondral bone (Karaplis et al., 1994; Wysolmerski et al., 1998), epithelial-mesenchymal interactions in mammary gland (Wysolmerski et al., 1998), and has important functions in many other tissues. PTHrP knockout mice die soon after birth due to asphyxia caused by developmental abnormalities of bones in the thorax (Karaplis et al., 1994). PTHrP has been shown to be the principal factor in most cases of cancer-induced HHM. The expression of PTHrP is up-

T-cells Osteoclast

cancer cells Osteoclast Catabolic

Intestines, kidney, osteoclast, osteoblast, parathyroid chief cells

Calcium-sensing receptor (CaR) on bone and cancer cells and parathyroid chief cells

anabolic

Increases calcium in blood

Inhibit osteoclast formation and/or function

Anabolic (bone cells) Catabolic (cancer cells) Decreases PTH secretion

hormone receptor 1 (PTH1R), which facilitates the development of HHM.

Kidney, cancer cells, tumorassociated macrophages

> Bone Kidney Intestinal tract

**3. Factors involved in the pathogenesis of HHM** 

**3.1 Parathyroid hormone-related protein (PTHrP)** 

RANKL Osteoblast,

Calcitriol

OPG, IL-3, IL-4, IL-10, IL-13, IFN-β, IFN-γ, GM-CSF, and sFRPs

resorption (catabolic action) in bone.

Calcium (Ca+2)

regulated by NF-κB, TGF-β, and/or Ras-MAPK signaling (Nadella et al., 2007; Richard et al., 2005). In adult T-cell leukemia/lymphoma, the promoter of PTHrP can be activated by the binding of HTLV-1 oncoprotein, Tax (Ejima et al., 1993; Watanabe et al., 1990).

The functions of PTHrP depend on the activation of different signal transduction pathways, including cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and protein lipase C/protein kinase C (PLC/PKC) cascades. In addition, PTHrP fragments have different functions that depend on the tissue-specific expression of distinct receptors. Activation of the cAMP-PKA pathway is required for PTHrP to induce bone resorption through the PTH1R, which recognizes both PTH and PTHrP (Greenfield et al., 1995).

Besides the catabolic effect of PTHrP on bone, intermittent administration or pulsatile secretion of the N-terminal fragment of PTH (1-34) and PTHrP (1-36) by a neuroendocrine tumor, such as islet cell carcinoma, can induce bone anabolic effects (Takeuchi et al., 2002). When osteoblastic cells were transiently exposed to the C-terminus of PTHrP (107-139), anabolic effects were induced by the upregulation of vascular endothelial growth factor receptor 2 (VEGFR2) through PKC/ERK activation (de Gortazar et al., 2006).

#### **3.2 Receptor activator of nuclear kappa-B ligand (RANKL) and osteoprotegerin (OPG)**

RANKL belongs to the tumor necrosis factor (TNF) family and represents one of the most common mediators for inducing osteoclastic bone resorption. Mature osteoblasts produce two forms of RANKL, a membrane-bound and secreted form. Both are important for osteoclast stimulation (Leibbrandt & Penninger, 2008). The expression of RANKL in osteoblasts and stromal cells is activated by the RUNX2 transcriptional factor under the regulation of PTHrP, 1,25-dihydroxyvitamin D3, and prostaglandins (Lipton et al., 2009). RANKL binds and activates its receptor, RANK, which is expressed on the surface of osteoclasts. The binding of RANKL to RANK activates at least three major signaling pathways, NFAT, p38 and JNK, resulting in up-regulation of genes that are required for induction of osteoclast fusion, differentiation, activation and survival (Leibbrandt & Penninger, 2008). The secreted form of RANKL is important for the recruitment of osteoclast precursors and osteoclastogenesis. RANKL is an essential activator for normal bone remodeling. RANKL knockout mice have severe osteopetrosis (Lomaga et al., 1999).

OPG, or osteoclastogenesis inhibitory factor (OCIF), is a secreted member of the tumor necrosis receptor superfamily that is expressed by osteoblasts and functions as a RANKL 'decoy receptor' (Simonet et al., 1997). OPG binds to RANKL and blocks its interaction with RANK on osteoclast precursors (Lacey et al., 1998); therefore, it is a potent inhibitor of osteoclast formation and bone resorption. OPG knockout mice have early-onset osteopenia (Bucay et al., 1998; Mizuno et al., 1998), whereas OPG overexpressing mice develop osteopetrosis that results from the failure to form osteoclasts (Simonet et al., 1997). PTH and PTHrP suppress OPG expression by downregulating the promoter of OPG through activation of the cAMP/PKA pathway (Yang et al., 2002). PTHrP, on the other hand, stimulates RANKL expression to induce bone resorption. The ratio between RANKL and OPG levels in osteoblasts is a key factor in the regulation of osteoclast activity (Horwood et al., 1998).

In ATLL, the expression of RANKL in tumor cells correlated with hypercalcemia in patients. RANKL on the surface of leukemia cells induced osteoclastogenesis through direct contact with precursor cells (Nosaka et al., 2002). The direct activation of osteoclasts by tumor cells may play a key role in the decoupling of bone formation and bone resorption in HMM.

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 185

concentration has been reported in acute nonlymphocytic leukemia patients with HHM

Interferon (IFN)-γ disrupts JAK/STAT signaling in osteoblasts to induce the expression of OPG and Wnt, causing decreased cathepsin K and tartrate-resistant acid phosphatase (TRAP) in mature osteoclasts (Gallo et al., 2008; Gillespie, 2007). Transgenic mice with the HTLV-1 viral oncoprotein, Tax, and knockout of IFN-γ developed more severe osteolytic bone lesions and increased osteoclast activity. Administration of IFN-γ to mice transplanted with Tax-positive tumors inhibited tumor growth and decreased hypercalcemia, suggesting

Cytokines have been shown to play a more important role in the pathogenesis of HHM in patients with lymphoma and leukemia compared to patients with carcinoma. However, further investigation is needed to clarify the additive and synergistic roles of cytokines in causing hypercalcemia because multiple cytokines are often produced by the tumor cells.

MIP-1α is a pro-inflammatory chemokine expressed by many different cell types including macrophages, dendritic cells and lymphocytes. It normally functions as a chemoattractant for T-cells, macrophages, and other proinflammatory cells at the site of inflammation. It also regulates the transendothelial migration of NK cells, monocytes and dendritic cells (Maurer & von, 2004). Therefore, it plays an important role in autoimmune and inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, asthma, and organ transplant rejection (Maurer & von, 2004). MIP-1α binds to several chemokine (c-c motif) G-protein-coupled receptors including CCR1, CCR5 and CCR9, which are expressed in lymphocytes and monocytes/macrophages. It activates several signaling pathways, including PI3K, PLC, PKC, MAP kinase and JAK/STAT pathways (Tsubaki et al., 2007). MIP-1α can potently inhibit the binding of HIV to CCR5 on macrophages (Baba

Both CCR1 and CCR5 are expressed in bone marrow stromal cells. MIP-1α is chemotactic for osteoclasts and osteoclast precursors (Fuller et al., 1995). MIP-1α was first identified as a putative osteoclastogenic factor in a human myeloma cDNA expression library derived from marrow samples of myeloma patients (Zlotnik & Yoshie, 2000). Subsequently, CCR1 and CCR5 expression was demonstrated in human and murine multiple myeloma cell lines (Menu et al., 2006). Furthermore, MIP-1α enhances osteoclast formation induced by IL-6, PTHrP and RANKL in multiple myeloma (Han et al., 2001) and increases adhesion of myeloma cells to bone marrow cells through its binding to both CCR1 and CCR5 receptors (Oba et al., 2005). *In vivo,* mice bearing Chinese hamster ovary cells that overexpress MIP-1α develop more severe osteolytic lesions after intramuscular

The mechanisms by which MIP-1α induces osteoclastic bone resorption are controversial. One study demonstrated that a RANKL-dependent pathway was responsible for activation of osteoclasts (Tsubaki et al., 2007). MIP-1α enhances RANKL expression in mouse bone marrow stromal cells and osteoblasts through the MAPK and PI3K/Akt signaling pathways. On the other hand, RANK-Fc did not block the activation of osteoclasts by MIP-1α, indicating that MIP-1α used a RANKL-independent pathway to increase bone resorption (Han et al., 2001). Therefore, it has been suggested that MIP-1α is a RANKL-independent osteoclastogenic factor that acts directly on osteoclast precursors (Choi et al., 2000). In any case, it is apparent that MIP-1α is a significant osteoclast activator. The role of MIP-1 in

a protective role for IFN-γ in HHM development (Xu and Hurchla et al., 2009).

**3.4 Macrophage inflammatory protein-1 alpha (MIP-1α)** 

et al., 1999; Maurer & von, 2004).

inoculation (Oyajobi et al., 2003).

(Kounami et al., 2004).

Therefore, RANKL from either osteoblasts or tumor cells is an important mediator of bone resorption in many forms of HHM.

#### **3.3 Cytokines**

The increased secretion of inflammatory cytokines from cancer cells, such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, IL-8, M-CSF, CCL2 and CXCL12, is one of the key mechanisms by which NF-κB is constitutively activated in tumor cells (Lu & Stark, 2004). The proinflammatory cytokine, TNF-α, is a critical factor in the pathogenesis of many inflammatory and non-inflammatory diseases which are characterized by increased osteoclastic bone resorption including rheumatoid arthritis (Chu et al., 1991), osteoporosis (Horowitz, 1993), osteomyelitis (Meghji et al., 1998) and aseptic loosening (an osteolysis syndrome caused by macrophages in joint replacements) (Merkel et al., 1999). TNF-α mediates lipopolysaccharide-stimulated osteoclastogenesis through the p55 receptor expressed on bone marrow macrophages and activation of NF-κB (Abu-Amer et al., 1997; Abu-Amer et al., 1998). Whether osteoclastogenesis induced by TNF-α depends on RANKL or not remains controversial. Some studies have shown that without exogenous RANKL, TNF-α is sufficient to induce osteoclast differentiation (Kudo et al., 2002), and the increase of osteoclastogenesis was inhibited by OPG (Hounoki et al., 2008). However, Boyle et al. have shown that administration of TNF-α to RANK-deficient mice failed to induce osteoclastogenesis and restore hypocalcemia, suggesting that TNF-α cannot substitute for RANKL (Li et al., 2000). Teitelbaum et al. have reported that TNF-α alone was not able to induce osteoclastogenesis in murine osteoclast precursors; rather, RANKL was required for TNF-α to stimulate osteoclast precursors to form osteoclasts (Lam et al., 2000). Regardless, TNF-α has shown its potential to be a therapeutic target for bone resorption. Increased TNFα levels have been found in patients with advanced chronic lymphocytic leukemia (CLL) and acute nonlymphocytic leukemia with HHM (Ferrajoli et al., 2002).

Increased plasma IL-6 has been found in cancer patients with HHM, including patients with multiple myeloma, squamous cell carcinoma of the liver, acute nonlymphocytic leukemia, and adult T-cell leukemia/lymphoma (Asanuma et al., 2002; Kounami et al., 2004; Roodman, 1997). IL-6 increases osteoclast recruitment by binding to its receptor on osteoblasts and inducing the signal of transducer and activator of transcription (STAT)- 1/3 and mitogen-activated protein kinase (MAPK) signaling pathways (Sims et al., 2004). It also enhances the effects of PTH and PTHrP and mediates the effects of inflammatory cytokines, such as IL-1 and TNF-α, on osteoclast formation (Roodman, 2001). IL-6 does not directly increase RANKL expression; therefore, its osteoclastogenic effect is RANKLindependent (Hofbauer et al., 2000). However, there is no correlation between serum IL-6 and HHM in cancer patients, suggesting that IL-6 functions additively or synergistically with other factors and may be a redundant factor for development of HHM (Vanderschueren et al., 1994).

Granulocyte macrophage-colony stimulating factor (GM-CSF) also plays a role in osteoclastogenesis and it has two distinct effects on osteoclast activity depending upon the presence of RANKL. GM-CSF increases the proliferation of osteoclast progenitors when RANKL is absent. On the other hand, it induces osteoclast progenitors to differentiate into dendritic cells when RANKL is present (Gillespie, 2007).

Macrophage colony-stimulating factor (M-CSF) is essential for osteoclast differentiation and proliferation of osteoclast progenitor cells (Tanaka et al., 1993). Increased serum M-CSF

Therefore, RANKL from either osteoblasts or tumor cells is an important mediator of bone

The increased secretion of inflammatory cytokines from cancer cells, such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, IL-8, M-CSF, CCL2 and CXCL12, is one of the key mechanisms by which NF-κB is constitutively activated in tumor cells (Lu & Stark, 2004). The proinflammatory cytokine, TNF-α, is a critical factor in the pathogenesis of many inflammatory and non-inflammatory diseases which are characterized by increased osteoclastic bone resorption including rheumatoid arthritis (Chu et al., 1991), osteoporosis (Horowitz, 1993), osteomyelitis (Meghji et al., 1998) and aseptic loosening (an osteolysis syndrome caused by macrophages in joint replacements) (Merkel et al., 1999). TNF-α mediates lipopolysaccharide-stimulated osteoclastogenesis through the p55 receptor expressed on bone marrow macrophages and activation of NF-κB (Abu-Amer et al., 1997; Abu-Amer et al., 1998). Whether osteoclastogenesis induced by TNF-α depends on RANKL or not remains controversial. Some studies have shown that without exogenous RANKL, TNF-α is sufficient to induce osteoclast differentiation (Kudo et al., 2002), and the increase of osteoclastogenesis was inhibited by OPG (Hounoki et al., 2008). However, Boyle et al. have shown that administration of TNF-α to RANK-deficient mice failed to induce osteoclastogenesis and restore hypocalcemia, suggesting that TNF-α cannot substitute for RANKL (Li et al., 2000). Teitelbaum et al. have reported that TNF-α alone was not able to induce osteoclastogenesis in murine osteoclast precursors; rather, RANKL was required for TNF-α to stimulate osteoclast precursors to form osteoclasts (Lam et al., 2000). Regardless, TNF-α has shown its potential to be a therapeutic target for bone resorption. Increased TNFα levels have been found in patients with advanced chronic lymphocytic leukemia (CLL)

and acute nonlymphocytic leukemia with HHM (Ferrajoli et al., 2002).

Increased plasma IL-6 has been found in cancer patients with HHM, including patients with multiple myeloma, squamous cell carcinoma of the liver, acute nonlymphocytic leukemia, and adult T-cell leukemia/lymphoma (Asanuma et al., 2002; Kounami et al., 2004; Roodman, 1997). IL-6 increases osteoclast recruitment by binding to its receptor on osteoblasts and inducing the signal of transducer and activator of transcription (STAT)- 1/3 and mitogen-activated protein kinase (MAPK) signaling pathways (Sims et al., 2004). It also enhances the effects of PTH and PTHrP and mediates the effects of inflammatory cytokines, such as IL-1 and TNF-α, on osteoclast formation (Roodman, 2001). IL-6 does not directly increase RANKL expression; therefore, its osteoclastogenic effect is RANKLindependent (Hofbauer et al., 2000). However, there is no correlation between serum IL-6 and HHM in cancer patients, suggesting that IL-6 functions additively or synergistically with other factors and may be a redundant factor for development of HHM

Granulocyte macrophage-colony stimulating factor (GM-CSF) also plays a role in osteoclastogenesis and it has two distinct effects on osteoclast activity depending upon the presence of RANKL. GM-CSF increases the proliferation of osteoclast progenitors when RANKL is absent. On the other hand, it induces osteoclast progenitors to differentiate into

Macrophage colony-stimulating factor (M-CSF) is essential for osteoclast differentiation and proliferation of osteoclast progenitor cells (Tanaka et al., 1993). Increased serum M-CSF

resorption in many forms of HHM.

(Vanderschueren et al., 1994).

dendritic cells when RANKL is present (Gillespie, 2007).

**3.3 Cytokines** 

concentration has been reported in acute nonlymphocytic leukemia patients with HHM (Kounami et al., 2004).

Interferon (IFN)-γ disrupts JAK/STAT signaling in osteoblasts to induce the expression of OPG and Wnt, causing decreased cathepsin K and tartrate-resistant acid phosphatase (TRAP) in mature osteoclasts (Gallo et al., 2008; Gillespie, 2007). Transgenic mice with the HTLV-1 viral oncoprotein, Tax, and knockout of IFN-γ developed more severe osteolytic bone lesions and increased osteoclast activity. Administration of IFN-γ to mice transplanted with Tax-positive tumors inhibited tumor growth and decreased hypercalcemia, suggesting a protective role for IFN-γ in HHM development (Xu and Hurchla et al., 2009).

Cytokines have been shown to play a more important role in the pathogenesis of HHM in patients with lymphoma and leukemia compared to patients with carcinoma. However, further investigation is needed to clarify the additive and synergistic roles of cytokines in causing hypercalcemia because multiple cytokines are often produced by the tumor cells.

#### **3.4 Macrophage inflammatory protein-1 alpha (MIP-1α)**

MIP-1α is a pro-inflammatory chemokine expressed by many different cell types including macrophages, dendritic cells and lymphocytes. It normally functions as a chemoattractant for T-cells, macrophages, and other proinflammatory cells at the site of inflammation. It also regulates the transendothelial migration of NK cells, monocytes and dendritic cells (Maurer & von, 2004). Therefore, it plays an important role in autoimmune and inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, asthma, and organ transplant rejection (Maurer & von, 2004). MIP-1α binds to several chemokine (c-c motif) G-protein-coupled receptors including CCR1, CCR5 and CCR9, which are expressed in lymphocytes and monocytes/macrophages. It activates several signaling pathways, including PI3K, PLC, PKC, MAP kinase and JAK/STAT pathways (Tsubaki et al., 2007). MIP-1α can potently inhibit the binding of HIV to CCR5 on macrophages (Baba et al., 1999; Maurer & von, 2004).

Both CCR1 and CCR5 are expressed in bone marrow stromal cells. MIP-1α is chemotactic for osteoclasts and osteoclast precursors (Fuller et al., 1995). MIP-1α was first identified as a putative osteoclastogenic factor in a human myeloma cDNA expression library derived from marrow samples of myeloma patients (Zlotnik & Yoshie, 2000). Subsequently, CCR1 and CCR5 expression was demonstrated in human and murine multiple myeloma cell lines (Menu et al., 2006). Furthermore, MIP-1α enhances osteoclast formation induced by IL-6, PTHrP and RANKL in multiple myeloma (Han et al., 2001) and increases adhesion of myeloma cells to bone marrow cells through its binding to both CCR1 and CCR5 receptors (Oba et al., 2005). *In vivo,* mice bearing Chinese hamster ovary cells that overexpress MIP-1α develop more severe osteolytic lesions after intramuscular inoculation (Oyajobi et al., 2003).

The mechanisms by which MIP-1α induces osteoclastic bone resorption are controversial. One study demonstrated that a RANKL-dependent pathway was responsible for activation of osteoclasts (Tsubaki et al., 2007). MIP-1α enhances RANKL expression in mouse bone marrow stromal cells and osteoblasts through the MAPK and PI3K/Akt signaling pathways. On the other hand, RANK-Fc did not block the activation of osteoclasts by MIP-1α, indicating that MIP-1α used a RANKL-independent pathway to increase bone resorption (Han et al., 2001). Therefore, it has been suggested that MIP-1α is a RANKL-independent osteoclastogenic factor that acts directly on osteoclast precursors (Choi et al., 2000). In any case, it is apparent that MIP-1α is a significant osteoclast activator. The role of MIP-1 in

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 187

which secrete a soluble form of RANKL. TNF-α is also expressed by activated T-cells to act in concert with RANKL. IL-1, IL-6 and IL-17 secreted from T-cells increase RANKL expression in osteoblasts. This mechanism is important for rheumatoid arthritis where TH17 cells have been shown to be an immunomodulator of osteoclastic bone resorption (Sato et al., 2006). In addition, T-cells produce IL-7 which increase bone resorption in a RANKLindependent mechanism (Weitzmann & Pacifici, 2005). T-cells also play a major role in postmenopausal osteoporosis induced by decreased estrogen levels and decreased

In contrast, T-cells can also exert an antiresorptive effect directly by secreting OPG, IL-3, IL-4, IL-10, IL-13, IFN-β, IFN-γ, GM-CSF, and secreted frizzled-related proteins (sFRPs) to inhibit osteoclastogenesis (Quinn & Gillespie, 2005). In addition, T-cells can inhibit osteoclast formation and activity indirectly by expressing GM-CSF (induced by the upregulation of IL-18 in bone microenvironment), IFN-γ (by IL-12) and OPG (by leptin) (Horwood et al., 2001). It will be important to understand the effects of T-cells on osteoblast and stromal cell function, as well as signaling in the immune response to further understand

50-90% of ATLL patients develop HHM and osteolytic lesions in the long bones and calvaria (Olivo et al., 2008). Bone resorption may act as a 'vicious cycle' for ATLL growth in bone, since factors released by resorbing bone increased the growth of ATLL and HTLV-1-infected T-cells *in vitro* (Shu et al., 2010). The mechanisms by which HTLV-1 induces HHM are not completely known. We and others have demonstrated that ATLL primary cell lines (T-cell lines derived from leukemic ATLL patients) and *in vitro* HTLV-1 transformed T-cell lines express and secrete PTHrP, particularly transcripts from the P3 promoter (Nadella et al., 2007; Richard et al., 2005; Shu et al., 2010) (Figure 1). The expression of PTHrP was upregulated by both oncoviral protein Tax-dependent and -independent pathways. Tax cooperates with Ets to activate the P3 promoter of PTHrP, while constitutive activation of NF-κB in ATLL cells contributes to expression of the PTHrP P2 promoter (Nadella et al., 2007; Richard et al., 2005). Despite the essential role of PTHrP in HHM in carcinomas (such as lung, breast and prostate cancers), the correlation between the plasma PTHrP and HHM in ATLL patients has been controversial. It has been concluded that PTHrP is not the sole factor that induces HHM in ATLL, but it likely plays an important cooperative or synergistic

In ATLL, there were increased plasma MIP-1α concentrations in mice with human ATLL cells and HHM (Shu et al., 2007). In a human clinical study, plasma MIP-1α concentrations had a strong correlation with HHM in ATLL patients (Okada et al., 2004). MIP-1α expression in ATLL cells is induced by Tax and increased plasma calcium concentrations may further up-regulate MIP-1α expression through the calmodulin-dependent protein kinase kinase (CaM-KK) cascade (Matsumoto et al., 2008; Sharma & May, 1999). Treatment with a neutralizing MIP-1α antibody decreased osteoclast formation induced by ATLL cells

The increase in RANKL expression observed in the ATL leukemic cells has been shown to correlate with the occurrence of HHM in ATLL patients (Nosaka et al., 2002). Although the levels of RANKL expression were not high in HTLV-1-infected cell lines *in vitro* (Shu et al.,

transforming growth factor (TGF)-β expression in bone cells (Gillespie, 2007).

the interactions between the immune system and bone biology.

**4. HHM in leukemia/lymphoma in humans and animals** 

**4.1 Adult T-cell leukemia lymphoma (ATLL)** 

role with other humoral factors (Figure 1).

*in vitro* (Okada et al., 2004).

HHM is highlighted by the fact that HTLV-1 infected T-cells express and secrete MIP-1α (Shu et al., 2007; Shu et al., 2010), and the increased serum levels of MIP-1α correlated well with the development of HHM in HTLV-1-infected patients (Okada et al., 2004).

#### **3.5 1α,25-Dihydroxyvitamin D (Calcitriol)**

Vitamin D is an essential hormone for calcium homeostasis. Its active form, 1α,25 dihydroxyvitamin D3 or calcitriol, is synthesized by hydroxylation of vitamin D in the kidney. The renal 25-hydroxyvitamin D 1-α hydroxylase is the rate limiting enzyme for production of calcitriol. Calcitriol increases calcium absorption from the intestinal tract, increases osteoclastic bone resorption, and decreases PTH gene expression in the parathyroid glands (Guise et al., 2005). Calcitriol is an uncommon primary cause of HHM in patients with leukemia or lymphoma even though serum calcitriol concentrations may be increased in up to 50% of patients (Seymour & Gagel, 1993). Some lymphomas or tumorassociated macrophages express 25-hydroxyvitamin D 1-α hydroxylase, which is responsible for the increased production of calcitriol (Hewison et al., 2003).

#### **3.6 Cell membrane calcium-sensing receptor (CaR)**

CaR is expressed on multiple cell types including the chief cells of the parathyroid gland where it regulates PTH expression and secretion. Under normal conditions, CaR, a G protein-coupled receptor, senses extracellular calcium concentrations and activates downstream signaling pathways, such as the PLC/inositol trisphosphate (IP3) and ERK1/2 pathways, in a tissue-specific manner (Saidak et al., 2009). In bone, CaR is expressed in osteoblasts, osteoclasts, stromal cells, monocytes-macrophages, and chondrocytes. CaR promotes osteoblast proliferation, differentiation and mineralization (Sharan et al., 2008). It also mediates osteoclast differentiation and apoptosis. Therefore, CaR in bone cells promotes the bone formation phase of bone remodeling (Yamaguchi, 2008). CaR is also expressed in some cancer cells, such as breast and prostate cancers (Liao et al., 2006). However, in these cells, increased extracellular calcium leads to increased PTHrP expression (Chattopadhyay, 2006). Increased PTHrP induces osteoclastic bone resorption and renal calcium reabsorption, resulting in HHM. The positive feedback loop formed by PTHrP, calcium and CaR is a unique phenomena in HHM. In addition to the specific activation of PTHrP expression, gain-of-function mutations in CaR have been demonstrated in breast cancer. Lorch *et al*. have found single nucleotide polymorphisms in CaR in human lung squamous cell carcinoma (Lorch et al., 2011). Functional evaluation of a nonconservative amino acid substitution (R990G) in CaR induced HHM in patients with lung squamous cell carcinoma. Dysregulation of PTHrP expression and HHM caused by CaR signaling has been demonstrated in breast and prostate cancer (Saidak et al., 2009). However, the role of CaR in HHM induced by lymphoma/leukemia remains to be determined.

#### **3.7 Role of T cells in calcium homeostasis**

The skeletal and immune systems share many regulatory molecules and systems. An interdisciplinary research area called "Osteoimmunology" has been developed recently to understand the interplay between these two systems. Cytokines, receptors, signaling molecules and transcriptional factors, and their signaling pathways are comprehensively reviewed by Takayanagi (Takayanagi, 2007). T-cells have both pro- and antiresorptive effects on osteoclasts. The proresorptive effect is present in osteoclast-stimulating T-cells, which secrete a soluble form of RANKL. TNF-α is also expressed by activated T-cells to act in concert with RANKL. IL-1, IL-6 and IL-17 secreted from T-cells increase RANKL expression in osteoblasts. This mechanism is important for rheumatoid arthritis where TH17 cells have been shown to be an immunomodulator of osteoclastic bone resorption (Sato et al., 2006). In addition, T-cells produce IL-7 which increase bone resorption in a RANKLindependent mechanism (Weitzmann & Pacifici, 2005). T-cells also play a major role in postmenopausal osteoporosis induced by decreased estrogen levels and decreased transforming growth factor (TGF)-β expression in bone cells (Gillespie, 2007).

In contrast, T-cells can also exert an antiresorptive effect directly by secreting OPG, IL-3, IL-4, IL-10, IL-13, IFN-β, IFN-γ, GM-CSF, and secreted frizzled-related proteins (sFRPs) to inhibit osteoclastogenesis (Quinn & Gillespie, 2005). In addition, T-cells can inhibit osteoclast formation and activity indirectly by expressing GM-CSF (induced by the upregulation of IL-18 in bone microenvironment), IFN-γ (by IL-12) and OPG (by leptin) (Horwood et al., 2001). It will be important to understand the effects of T-cells on osteoblast and stromal cell function, as well as signaling in the immune response to further understand the interactions between the immune system and bone biology.

#### **4. HHM in leukemia/lymphoma in humans and animals**

#### **4.1 Adult T-cell leukemia lymphoma (ATLL)**

186 T-Cell Leukemia

HHM is highlighted by the fact that HTLV-1 infected T-cells express and secrete MIP-1α (Shu et al., 2007; Shu et al., 2010), and the increased serum levels of MIP-1α correlated well

Vitamin D is an essential hormone for calcium homeostasis. Its active form, 1α,25 dihydroxyvitamin D3 or calcitriol, is synthesized by hydroxylation of vitamin D in the kidney. The renal 25-hydroxyvitamin D 1-α hydroxylase is the rate limiting enzyme for production of calcitriol. Calcitriol increases calcium absorption from the intestinal tract, increases osteoclastic bone resorption, and decreases PTH gene expression in the parathyroid glands (Guise et al., 2005). Calcitriol is an uncommon primary cause of HHM in patients with leukemia or lymphoma even though serum calcitriol concentrations may be increased in up to 50% of patients (Seymour & Gagel, 1993). Some lymphomas or tumorassociated macrophages express 25-hydroxyvitamin D 1-α hydroxylase, which is

CaR is expressed on multiple cell types including the chief cells of the parathyroid gland where it regulates PTH expression and secretion. Under normal conditions, CaR, a G protein-coupled receptor, senses extracellular calcium concentrations and activates downstream signaling pathways, such as the PLC/inositol trisphosphate (IP3) and ERK1/2 pathways, in a tissue-specific manner (Saidak et al., 2009). In bone, CaR is expressed in osteoblasts, osteoclasts, stromal cells, monocytes-macrophages, and chondrocytes. CaR promotes osteoblast proliferation, differentiation and mineralization (Sharan et al., 2008). It also mediates osteoclast differentiation and apoptosis. Therefore, CaR in bone cells promotes the bone formation phase of bone remodeling (Yamaguchi, 2008). CaR is also expressed in some cancer cells, such as breast and prostate cancers (Liao et al., 2006). However, in these cells, increased extracellular calcium leads to increased PTHrP expression (Chattopadhyay, 2006). Increased PTHrP induces osteoclastic bone resorption and renal calcium reabsorption, resulting in HHM. The positive feedback loop formed by PTHrP, calcium and CaR is a unique phenomena in HHM. In addition to the specific activation of PTHrP expression, gain-of-function mutations in CaR have been demonstrated in breast cancer. Lorch *et al*. have found single nucleotide polymorphisms in CaR in human lung squamous cell carcinoma (Lorch et al., 2011). Functional evaluation of a nonconservative amino acid substitution (R990G) in CaR induced HHM in patients with lung squamous cell carcinoma. Dysregulation of PTHrP expression and HHM caused by CaR signaling has been demonstrated in breast and prostate cancer (Saidak et al., 2009). However, the role of CaR in

The skeletal and immune systems share many regulatory molecules and systems. An interdisciplinary research area called "Osteoimmunology" has been developed recently to understand the interplay between these two systems. Cytokines, receptors, signaling molecules and transcriptional factors, and their signaling pathways are comprehensively reviewed by Takayanagi (Takayanagi, 2007). T-cells have both pro- and antiresorptive effects on osteoclasts. The proresorptive effect is present in osteoclast-stimulating T-cells,

with the development of HHM in HTLV-1-infected patients (Okada et al., 2004).

responsible for the increased production of calcitriol (Hewison et al., 2003).

HHM induced by lymphoma/leukemia remains to be determined.

**3.7 Role of T cells in calcium homeostasis** 

**3.6 Cell membrane calcium-sensing receptor (CaR)** 

**3.5 1α,25-Dihydroxyvitamin D (Calcitriol)** 

50-90% of ATLL patients develop HHM and osteolytic lesions in the long bones and calvaria (Olivo et al., 2008). Bone resorption may act as a 'vicious cycle' for ATLL growth in bone, since factors released by resorbing bone increased the growth of ATLL and HTLV-1-infected T-cells *in vitro* (Shu et al., 2010). The mechanisms by which HTLV-1 induces HHM are not completely known. We and others have demonstrated that ATLL primary cell lines (T-cell lines derived from leukemic ATLL patients) and *in vitro* HTLV-1 transformed T-cell lines express and secrete PTHrP, particularly transcripts from the P3 promoter (Nadella et al., 2007; Richard et al., 2005; Shu et al., 2010) (Figure 1). The expression of PTHrP was upregulated by both oncoviral protein Tax-dependent and -independent pathways. Tax cooperates with Ets to activate the P3 promoter of PTHrP, while constitutive activation of NF-κB in ATLL cells contributes to expression of the PTHrP P2 promoter (Nadella et al., 2007; Richard et al., 2005). Despite the essential role of PTHrP in HHM in carcinomas (such as lung, breast and prostate cancers), the correlation between the plasma PTHrP and HHM in ATLL patients has been controversial. It has been concluded that PTHrP is not the sole factor that induces HHM in ATLL, but it likely plays an important cooperative or synergistic role with other humoral factors (Figure 1).

In ATLL, there were increased plasma MIP-1α concentrations in mice with human ATLL cells and HHM (Shu et al., 2007). In a human clinical study, plasma MIP-1α concentrations had a strong correlation with HHM in ATLL patients (Okada et al., 2004). MIP-1α expression in ATLL cells is induced by Tax and increased plasma calcium concentrations may further up-regulate MIP-1α expression through the calmodulin-dependent protein kinase kinase (CaM-KK) cascade (Matsumoto et al., 2008; Sharma & May, 1999). Treatment with a neutralizing MIP-1α antibody decreased osteoclast formation induced by ATLL cells *in vitro* (Okada et al., 2004).

The increase in RANKL expression observed in the ATL leukemic cells has been shown to correlate with the occurrence of HHM in ATLL patients (Nosaka et al., 2002). Although the levels of RANKL expression were not high in HTLV-1-infected cell lines *in vitro* (Shu et al.,

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 189

Hypercalcemia in de novo ANLL patients can be caused by either local osteolytic hypercalcemia or HHM. Elevated circulating concentrations of several humoral factors, including PTHrP, TNF-α, IL-6, and M-CSF, in de novo ANLL patients with HHM have been reported supporting a role for HHM in ANLL (Kounami et al., 2004). Generalized osteoporosis with normal renal function was observed in these patients, indicating that the

**Cancer Type Incidence of HHM Humoral factor(s)** 

lymphoma Rare PTHrP, IL-6, 1,25-

ATLL 50-90% PTHrP, MIP-1α, RANKL, IL-1,

leukemia Rare PTHrP, IL-6, TNF-α, M-CSF

cell lymphoma Rare 1,25-dihydroxyvitamin D

Has been reported Unknown

40% in mediastinal lymphoma; 10-20% in multicentric lymphoma

Feline lymphoma Has been reported PTHrP

lymphoma Has been reported Unknown

animals. Other undefined factors or cytokines may also be involved.

Table 2. Incidence and humoral factors of HHM in leukemia/lymphoma in humans and

Increased PTHrP, IL-6, and 1,25-dihydroxyvitamin D concentrations have been reported in the serum from patients with diffuse large B cell lymphoma that developed hypercalcemia (Amezyane et al., 2008; Chang et al., 2008). Diffuse osteolytic lesions and nephrocalcinosis

Hypercalcemia has been reported in patients with primary cutaneous B-cell lymphoma (Habra et al., 2007; Narimatsu et al., 2003). Increased serum 1,25-dihydroxyvitamin D and undetectable PTH and PTHrP levels were found in one patient. The pathogenesis of

HHM occurs in 10-40% of dogs with T-cell lymphoma (Fournel-Fleury et al., 2002). Increased circulating PTHrP and 1,25-dihydroxyvitamin D were found in dogs with lymphoma and HHM, but the serum concentrations did not always correlate with

TNF-β, IL-6

dihydroxyvitamin D

PTHrP, 1,25-dihydroxyvitamin D

**4.2 Other leukemias/lymphomas that develop HHM (Table 2) 4.2.1 De novo acute nonlymphocytic leukemia (ANLL)** 

increased calcium was mainly from bone.

Diffuse large B cell

Acute nonlymphocytic

Primary cutaneous B-

Feline leukemia virus

leukemia/lymphoma

**4.2.2 Diffuse large B-cell lymphoma** 

**4.2.3 Primary cutaneous B-cell lymphoma** 

hypercalcemia in these patients has not been determined.

**4.2.4 HHM in animals with leukemia/lymphoma** 

also occurred in these patients.

Avain malignant

Canine T-cell lymphoma

associated

2010), leukemic cells isolated from ATLL patients did have up-regulation of RANKL expression (Nosaka et al., 2002). In addition, HTLV-1 infected leukocytes *in vitro* were able to convert 25-dihydroxyvitamin D3 to its active form, 1α,25-dihydroxyvitamin D3 or calcitriol (Fetchick et al., 1986). High levels of calcitriol have been found in ATLL patients with hypercalcemia (Johnston & Hammond, 1992; Seymour & Gagel, 1993)

Fig. 1. HHM in ATLL is caused by increased osteoclastic bone resorption. Cancer cells secrete bone regulatory factors, including PTHrP, MIP-1, calcitriol, and RANKL to increase osteoclast activity. The resorbing bone secretes bone-derived growth factors that increase ATLL cell growth. In osteoblasts, the RANKL/OPG expression ratio increases when cells are co-cultured with T-cell leukemia lines. Antibodies against HTLV-1 Gp46 can inhibit OPG and contributes to the pathogenesis of hypercalcemia.

In addition to the direct effects of factors secreted from ATLL cells on osteoclasts, we recently found decreased OPG expression and secretion in osteoblasts that were co-cultured with leukemic T-cell lines (Shu et al., 2010). This suggests that the regulation of gene expression in osteoblasts by leukemic T-cells plays an indirect role in HHM in ATLL. Finally, an endogenous antibody recognizing the HTLV-1 viral envelope protein Gp46-197 can occur in ATLL patients, which correlated with disease progression. The amino acid sequence of Gp46-197 is homologous with the C-terminus of OPG. Rabbits immunized with the Gp46-197 peptide developed hypercalcemia and died. Sprague-Dawley rats injected with Gp46-197 peptide developed decreased bone mineral density and hypercalcemia. Administration of recombinant human OPG restored femoral bone growth. These data suggest that HTLV-1 Gp46 contributes to the pathogenesis of hypercalcemia due to cross-reactive antibodies in the patients that antagonize the action of OPG (Sagara et al., 2009).

Other factors proposed to be humoral factors for HHM in ATLL include TNF-β (lymphotoxin) (Ishibashi et al., 1992), IL-6 (Chiba et al., 2009), and IL-1 (Wano et al., 1987).

#### **4.2 Other leukemias/lymphomas that develop HHM (Table 2) 4.2.1 De novo acute nonlymphocytic leukemia (ANLL)**

188 T-Cell Leukemia

2010), leukemic cells isolated from ATLL patients did have up-regulation of RANKL expression (Nosaka et al., 2002). In addition, HTLV-1 infected leukocytes *in vitro* were able to convert 25-dihydroxyvitamin D3 to its active form, 1α,25-dihydroxyvitamin D3 or calcitriol (Fetchick et al., 1986). High levels of calcitriol have been found in ATLL patients with

Fig. 1. HHM in ATLL is caused by increased osteoclastic bone resorption. Cancer cells secrete bone regulatory factors, including PTHrP, MIP-1, calcitriol, and RANKL to increase osteoclast activity. The resorbing bone secretes bone-derived growth factors that increase ATLL cell growth. In osteoblasts, the RANKL/OPG expression ratio increases when cells are co-cultured with T-cell leukemia lines. Antibodies against HTLV-1 Gp46 can inhibit

In addition to the direct effects of factors secreted from ATLL cells on osteoclasts, we recently found decreased OPG expression and secretion in osteoblasts that were co-cultured with leukemic T-cell lines (Shu et al., 2010). This suggests that the regulation of gene

Finally, an endogenous antibody recognizing the HTLV-1 viral envelope protein Gp46-197 can occur in ATLL patients, which correlated with disease progression. The amino acid sequence of Gp46-197 is homologous with the C-terminus of OPG. Rabbits immunized with the Gp46-197 peptide developed hypercalcemia and died. Sprague-Dawley rats injected with Gp46-197 peptide developed decreased bone mineral density and hypercalcemia. Administration of recombinant human OPG restored femoral bone growth. These data suggest that HTLV-1 Gp46 contributes to the pathogenesis of hypercalcemia due to cross-reactive antibodies in the patients that antagonize the action

Other factors proposed to be humoral factors for HHM in ATLL include TNF-β (lymphotoxin) (Ishibashi et al., 1992), IL-6 (Chiba et al., 2009), and IL-1 (Wano et al., 1987).

expression in osteoblasts by leukemic T-cells plays an indirect role in HHM in ATLL.

OPG and contributes to the pathogenesis of hypercalcemia.

of OPG (Sagara et al., 2009).

hypercalcemia (Johnston & Hammond, 1992; Seymour & Gagel, 1993)

Hypercalcemia in de novo ANLL patients can be caused by either local osteolytic hypercalcemia or HHM. Elevated circulating concentrations of several humoral factors, including PTHrP, TNF-α, IL-6, and M-CSF, in de novo ANLL patients with HHM have been reported supporting a role for HHM in ANLL (Kounami et al., 2004). Generalized osteoporosis with normal renal function was observed in these patients, indicating that the increased calcium was mainly from bone.


Table 2. Incidence and humoral factors of HHM in leukemia/lymphoma in humans and animals. Other undefined factors or cytokines may also be involved.

#### **4.2.2 Diffuse large B-cell lymphoma**

Increased PTHrP, IL-6, and 1,25-dihydroxyvitamin D concentrations have been reported in the serum from patients with diffuse large B cell lymphoma that developed hypercalcemia (Amezyane et al., 2008; Chang et al., 2008). Diffuse osteolytic lesions and nephrocalcinosis also occurred in these patients.

#### **4.2.3 Primary cutaneous B-cell lymphoma**

Hypercalcemia has been reported in patients with primary cutaneous B-cell lymphoma (Habra et al., 2007; Narimatsu et al., 2003). Increased serum 1,25-dihydroxyvitamin D and undetectable PTH and PTHrP levels were found in one patient. The pathogenesis of hypercalcemia in these patients has not been determined.

#### **4.2.4 HHM in animals with leukemia/lymphoma**

HHM occurs in 10-40% of dogs with T-cell lymphoma (Fournel-Fleury et al., 2002). Increased circulating PTHrP and 1,25-dihydroxyvitamin D were found in dogs with lymphoma and HHM, but the serum concentrations did not always correlate with

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 191

a potent (but transient) inhibitor of osteoclastic bone resorption. The mechanisms of action have been shown to involve several signaling pathways including cAMP/PKA, PKC, and pyk2/src activity. It has also been shown that calcitonin up-regulates renal 1α-hydroxylase, an important enzyme for the synthesis of calcitriol, through the binding of C/EBPβ and brahma-related gene 1 (BRG1), an ATPase in the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, on the 1α-hydroxylase promoter (Zhong et al., 2009). Because of its potent inhibitory effect on bone resorption, calcitonin has clinical applications in the treatment of Paget's disease, osteoporosis, and hypercalcemia. However, due to its short duration of action, calcitonin may not be suitable for treatment of chronic hypercalcemia. Salmon calcitonin is used clinically due to its greater potency compared to

Corticosteroids have been used for the treatment of hypercalcemia induced by multiple myeloma and certain types of lymphoma. Corticosteroids act by decreasing calcium absorption in gastrointestinal tract, decreasing bone resorption, and increasing renal calcium excretion (Unal et al., 2008; Yarbro et al., 2003). Corticosteroids generally are not effective in

Bortezomib is a selective proteasome inhibitor that decreases the ubiquination of IκB, the inhibitor for NF-κB, thereby stabilizing IκB and inhibiting NF-κB. NF-κB is necessary for osteoclast function and constitutively activated NF-κB plays an important role in ATLL cells. Bortezomib has been shown to inhibit tumor growth in models of ATLL (Mitra-Kaushik et al., 2004b; Nasr et al., 2005; Satou et al., 2004; Shu et al., 2007; Tan & Waldmann, 2002). Shu et al. reported that bortezomib not only decreased tumor burden in mice bearing ATLL cells, but also decreased the severity of HHM (Shu et al., 2007). Although the decrease in serum calcium concentrations was mainly due to the decreased tumor burden, several studies have shown that bortezomib inhibited osteoclastogenesis by decreasing p38, AP-1 and NF-κB activity in osteoclasts (von Metzler et al., 2007) and increased bone formation by decreasing the expression of Dkk-1, a potent osteoblast inhibitor (Drake & Rajkumar, 2009; Heider et al., 2009; Pennisi et al., 2009; Qiang et al., 2009; Terpos et al., 2006). Bortezomib is now a standard of care for treatment of multiple myeloma patients. Several clinical trials involving bortezomib are ongoing for the treatment of prostate cancer, nonsmall cell lung

**5.5 Humanized anti-parathyroid hormone-related protein antibody and small molecule** 

Although bisphophonates have been widely used for treatment of HHM to inhibit osteoclastic bone resorption, they do not function on the kidney to decrease renal calcium reabsorption. To eliminate the actions of PTHrP in bone and kidney, an anti-PTHrP antibody has been developed and tested (Sato et al., 2003). Anti-PTHrP antibody prevented hypercalcemia and skeletal metastasis, but not visceral metastasis, induced by PTHrPproducing cancer cells in mice (Guise & Mundy, 1996; Sato et al., 2003). Although it has been difficult to identify small molecule antagonists for type B G-protein-coupled receptors, including the PTH1R, two compounds have been recently developed that antagonize PTH1R. SW106 was discovered by Bristol-Myers-Squibb Company by screening compounds

human calcitonin (Zaidi et al., 2002).

patients with HHM induced by solid tumors.

cancer, acute myelogenous leukemia, and other cancers.

**antagonists of the PTH/PTHrP receptor (PTH1R)** 

**5.3 Corticosteroids** 

**5.4 Bortezomib (PS-341)** 

hypercalcemia. Therefore, it was speculated that additional cytokines are involved in the pathogenesis of HHM in dogs with lymphoma (Mellanby et al., 2006; Rosol et al., 1992). In a xenograft mouse model of canine lymphoma, there was increased expression of TNF-α in the tumor *in vivo* (Nadella et al., 2008). Bone histomorphometry indicated that increased osteoclastic bone resorption was the major cause of HHM in these mice. Increased circulating PTHrP has also been reported in cats with lymphoma and HHM (Bolliger et al., 2002). Cats may also develop HHM due to unknown humoral factors induced by feline leukemia virus infection (Engelman et al., 1985). Hypercalcemia has also been reported in an Amazon parrot with lymphoma (de Wit et al., 2003).

#### **5. Therapy of HHM in leukemia/lymphoma**

For urgent care, saline hydration is the first step to correct hypercalcemia by diluting the serum calcium concentration and increasing the clearance of calcium by the kidneys. Treatment of the underlying leukemia/lymphoma, including chemotherapy and radiation therapy, is necessary. Several drugs have been used for long-term management of hypercalcemia associated with HHM.

#### **5.1 Bisphosphonates**

Bisphosphonates are structural analogs of pyrophosphoric acid and have been widely used to treat cancer patients with hypercalcemia, heritable skeletal disorders in children, and postmenopausal and glucocorticoid-induced osteoporosis patients with significant bone loss (Fleisch, 1997). Intravenous aminobisphosphonates are a standard of care for the treatment of HHM. The newest generation of bisphosphonates, nitrogen-containing aminobisphosphonates, inhibits farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) synthetases in the mevalonic acid metabolic pathway resulting in decreased prenylation of low-molecularweight G proteins including Ras. Functional Ras signaling is necessary for osteoclast activity and survival (Fleisch, 1998). It also has been suggested that bisphosphonates exert a cytotoxic effect on HTLV-1-infected T-cells. However, high doses of bisphosphonates were needed for cytotoxic effects *in vitro* and *in vivo* (Gao et al, 2005; Ishikawa et al., 2007; Shu et al., 2007; Hirbe et al., 2009). The clinical relevance of a direct effect of bisphosphonates on ATLL cells needs to be further evaluated. Bisphosphonates have a high affinity for bone mineral and bind to hydroxyapatite crystals *in vivo.* Therefore, bisphosphonates are rapidly depleted from the circulation and extracellular fluid and become highly concentrated in bone. Bisphosphonate therapy may have complications including hypocalcemia, osteonecrosis of the jaw, low bone turnover with pathologic fractures, and increased incidence of atrial fibrillation (Drake et al., 2008). In addition to the effects on osteoclasts, a new bisphosphonate, YM527/ONO-5920, decreased MIP-1α expression and secretion through inhibition of the transient increase of phosphorylated ERK1/2 and Akt in mouse myeloma cells after lipopolysaccharide (LPS) stimulation (Drake & Rajkumar, 2009). Since MIP-1α has been shown to play an important role in cancer cell growth and osteolysis in multiple myeloma, bisphosphonates may be useful for inhibiting the growth of myeloma cells and to prevent osteolysis by decreasing MIP-1α expression. This may also apply to ATLL. Commonly used bisphosphonates include pamidronate and zoledronic acid.

#### **5.2 Calcitonin**

Calcitonin is a 32-amino acid peptide secreted by thyroid C-cells. It inhibits calcium absorption in the intestine and reabsorption of calcium and phosphate in renal tubules. It is a potent (but transient) inhibitor of osteoclastic bone resorption. The mechanisms of action have been shown to involve several signaling pathways including cAMP/PKA, PKC, and pyk2/src activity. It has also been shown that calcitonin up-regulates renal 1α-hydroxylase, an important enzyme for the synthesis of calcitriol, through the binding of C/EBPβ and brahma-related gene 1 (BRG1), an ATPase in the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, on the 1α-hydroxylase promoter (Zhong et al., 2009). Because of its potent inhibitory effect on bone resorption, calcitonin has clinical applications in the treatment of Paget's disease, osteoporosis, and hypercalcemia. However, due to its short duration of action, calcitonin may not be suitable for treatment of chronic hypercalcemia. Salmon calcitonin is used clinically due to its greater potency compared to human calcitonin (Zaidi et al., 2002).

#### **5.3 Corticosteroids**

190 T-Cell Leukemia

hypercalcemia. Therefore, it was speculated that additional cytokines are involved in the pathogenesis of HHM in dogs with lymphoma (Mellanby et al., 2006; Rosol et al., 1992). In a xenograft mouse model of canine lymphoma, there was increased expression of TNF-α in the tumor *in vivo* (Nadella et al., 2008). Bone histomorphometry indicated that increased osteoclastic bone resorption was the major cause of HHM in these mice. Increased circulating PTHrP has also been reported in cats with lymphoma and HHM (Bolliger et al., 2002). Cats may also develop HHM due to unknown humoral factors induced by feline leukemia virus infection (Engelman et al., 1985). Hypercalcemia has also been reported in

For urgent care, saline hydration is the first step to correct hypercalcemia by diluting the serum calcium concentration and increasing the clearance of calcium by the kidneys. Treatment of the underlying leukemia/lymphoma, including chemotherapy and radiation therapy, is necessary. Several drugs have been used for long-term management of

Bisphosphonates are structural analogs of pyrophosphoric acid and have been widely used to treat cancer patients with hypercalcemia, heritable skeletal disorders in children, and postmenopausal and glucocorticoid-induced osteoporosis patients with significant bone loss (Fleisch, 1997). Intravenous aminobisphosphonates are a standard of care for the treatment of HHM. The newest generation of bisphosphonates, nitrogen-containing aminobisphosphonates, inhibits farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) synthetases in the mevalonic acid metabolic pathway resulting in decreased prenylation of low-molecularweight G proteins including Ras. Functional Ras signaling is necessary for osteoclast activity and survival (Fleisch, 1998). It also has been suggested that bisphosphonates exert a cytotoxic effect on HTLV-1-infected T-cells. However, high doses of bisphosphonates were needed for cytotoxic effects *in vitro* and *in vivo* (Gao et al, 2005; Ishikawa et al., 2007; Shu et al., 2007; Hirbe et al., 2009). The clinical relevance of a direct effect of bisphosphonates on ATLL cells needs to be further evaluated. Bisphosphonates have a high affinity for bone mineral and bind to hydroxyapatite crystals *in vivo.* Therefore, bisphosphonates are rapidly depleted from the circulation and extracellular fluid and become highly concentrated in bone. Bisphosphonate therapy may have complications including hypocalcemia, osteonecrosis of the jaw, low bone turnover with pathologic fractures, and increased incidence of atrial fibrillation (Drake et al., 2008). In addition to the effects on osteoclasts, a new bisphosphonate, YM527/ONO-5920, decreased MIP-1α expression and secretion through inhibition of the transient increase of phosphorylated ERK1/2 and Akt in mouse myeloma cells after lipopolysaccharide (LPS) stimulation (Drake & Rajkumar, 2009). Since MIP-1α has been shown to play an important role in cancer cell growth and osteolysis in multiple myeloma, bisphosphonates may be useful for inhibiting the growth of myeloma cells and to prevent osteolysis by decreasing MIP-1α expression. This may also apply to ATLL. Commonly used bisphosphonates include

Calcitonin is a 32-amino acid peptide secreted by thyroid C-cells. It inhibits calcium absorption in the intestine and reabsorption of calcium and phosphate in renal tubules. It is

an Amazon parrot with lymphoma (de Wit et al., 2003).

**5. Therapy of HHM in leukemia/lymphoma** 

hypercalcemia associated with HHM.

pamidronate and zoledronic acid.

**5.2 Calcitonin** 

**5.1 Bisphosphonates** 

Corticosteroids have been used for the treatment of hypercalcemia induced by multiple myeloma and certain types of lymphoma. Corticosteroids act by decreasing calcium absorption in gastrointestinal tract, decreasing bone resorption, and increasing renal calcium excretion (Unal et al., 2008; Yarbro et al., 2003). Corticosteroids generally are not effective in patients with HHM induced by solid tumors.

#### **5.4 Bortezomib (PS-341)**

Bortezomib is a selective proteasome inhibitor that decreases the ubiquination of IκB, the inhibitor for NF-κB, thereby stabilizing IκB and inhibiting NF-κB. NF-κB is necessary for osteoclast function and constitutively activated NF-κB plays an important role in ATLL cells. Bortezomib has been shown to inhibit tumor growth in models of ATLL (Mitra-Kaushik et al., 2004b; Nasr et al., 2005; Satou et al., 2004; Shu et al., 2007; Tan & Waldmann, 2002). Shu et al. reported that bortezomib not only decreased tumor burden in mice bearing ATLL cells, but also decreased the severity of HHM (Shu et al., 2007). Although the decrease in serum calcium concentrations was mainly due to the decreased tumor burden, several studies have shown that bortezomib inhibited osteoclastogenesis by decreasing p38, AP-1 and NF-κB activity in osteoclasts (von Metzler et al., 2007) and increased bone formation by decreasing the expression of Dkk-1, a potent osteoblast inhibitor (Drake & Rajkumar, 2009; Heider et al., 2009; Pennisi et al., 2009; Qiang et al., 2009; Terpos et al., 2006). Bortezomib is now a standard of care for treatment of multiple myeloma patients. Several clinical trials involving bortezomib are ongoing for the treatment of prostate cancer, nonsmall cell lung cancer, acute myelogenous leukemia, and other cancers.

#### **5.5 Humanized anti-parathyroid hormone-related protein antibody and small molecule antagonists of the PTH/PTHrP receptor (PTH1R)**

Although bisphophonates have been widely used for treatment of HHM to inhibit osteoclastic bone resorption, they do not function on the kidney to decrease renal calcium reabsorption. To eliminate the actions of PTHrP in bone and kidney, an anti-PTHrP antibody has been developed and tested (Sato et al., 2003). Anti-PTHrP antibody prevented hypercalcemia and skeletal metastasis, but not visceral metastasis, induced by PTHrPproducing cancer cells in mice (Guise & Mundy, 1996; Sato et al., 2003). Although it has been difficult to identify small molecule antagonists for type B G-protein-coupled receptors, including the PTH1R, two compounds have been recently developed that antagonize PTH1R. SW106 was discovered by Bristol-Myers-Squibb Company by screening compounds

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 193

mice (Feuer et al., 1993). Richard et al. characterized the tumorigenesis and HHM of this cell line in SCID/beige mice (Richard et al., 2001). SCID/beige mice developed severe hypercalcemia one month after intraperitoneal injection of RV-ATL cells. The mice had bone loss due to increased osteoclastic bone resorption. Shu et al. introduced the luciferase gene into the RV-ATL cells by lentiviral infection and developed a bioluminescent model of HHM in ATLL for preclinical studies (Shu et al., 2007). It was found that both zoledronic acid, a nitrogen-containing bisphosphonate, and PS-341, a selective proteasome inhibitor, decreased tumor burden and HHM in mice. No complication of using the combination of

PS-341 and zoledronic acid was observed in this preclinical study (Shu et al., 2007).

The laboratory of Dr. Takashi Uchiyama developed a mouse xenograft model by injecting cells from the lymph node of a lymphoma-type ATLL patient intraperitoneally into SCID mice (Imada et al., 1996). The mice developed tumors and hypercalcemia within three weeks. There was a marked increase in serum C-terminal PTHrP concentrations with decreased bone formation rates reported in the mice (Takaori-Kondo et al., 1998). However, no significant increase in bone resorption measured by bone histomorphometry was

Recently, a NOD/SCID mouse model using human ATLL MET-1 cells has been developed (Phillips et al., 2000). The mice developed leukemia and HHM after intraperitoneal injection. Marked infiltration of tumor cells in multiple organs including spleen, lungs, liver, lymph nodes was observed. Increased plasma PTHrP concentrations in the mice and expression of PTHrP and RANKL in MET-1 cells were reported (Parrula et al., 2009). It is not known whether the HHM in this model was caused by an increase in bone resorption or

Transgenic mice have been used to study the pathogenesis and role of the HTLV-1 viral oncoprotein, Tax. Several Tax transgenic mice have been developed using different promoters. A transgenic mouse model overexpressing Tax under the regulation of the HTLV-1 long terminal repeat (LTR) was generated (Ruddle et al., 1993). These mice developed neurofibromas and adrenal medullary tumors, but did not develop leukemia or lymphoma. Unexpectedly, a significant increase in bone remodeling with a net increase in bone volume was observed. This was surprising because the authors also demonstrated that osteoclasts were increased in number, size and degree of multinucleation, which would have been expected to lead to a net decrease in bone volume. It was not reported whether

The laboratory of Dr. Lee Ratner developed a tissue-specific Tax overexpressing mouse model (Grossman et al., 1995). Tax expression in the mice was under the regulation of the human granzyme B promoter, which limited Tax expression primarily to activated CD4+ and CD8+ Tcells and NK cells. The mice developed mild hypercalcemia and multifocal osteolytic bone lesions, especially in the tail, with increased osteoclastic bone resorption (Gao et al., 2005). The

**6.1.2 Uchiyama human xenograft mouse model of ATLL** 

**6.1.3 Human MET-1 xenograft mouse model of ATLL** 

**6.1.4 HTLV-1 LTR-Tax transgenic model** 

**6.1.5 Human granzyme B-Tax transgenic mice** 

other mechanisms.

HHM developed in the mice.

observed in the mice, which is in contrast to ATLL patients with HHM.

that inhibited targets downstream of the PTHR1 (Carter et al., 2007). The benzoxazepinone non-peptide inhibits the cAMP response induced by a PTH1R agonist. The pharmacological behavior of SW106 is not yet completely understood. A similar compound, 1,3,4 benzotriazepine was identified and serves as a base molecule to develop non-peptide PTH1R antagonist derivatives (McDonald et al., 2007). Using a radioligand binding assay that measures cAMP production, N-1 anilino-substituted compounds were identified that have up to a 1000-fold more potency at inhibiting PTH1R compared to the original compound. Efforts are ongoing to determine the effects of these compounds on bone metastasis, hypercalcemia and hyperparathyroidism.

#### **5.6 RANKL inhibitors**

Ever since RANKL and OPG were discovered to be major regulators of osteoclastic bone resorption, RANK-Fc, Fc-OPG, antibodies targeting RANKL or inhibitors imitating OPG function have been developed for the treatment of osteolytic bone diseases (Schwarz & Ritchlin, 2007). RANK-Fc was generated by combining the carboxyl-terminus of RANK with the Fc portion of human IgG1. It has been shown to decrease tumor burden in two multiple myeloma mouse models, inhibit prostate cancer bone metastasis, and decrease the incidence of lung metastasis and bone lysis in a osteosarcoma mouse model (Lamoureux et al., 2008; Sordillo & Pearse, 2003; Zhang et al., 2003). Fc-OPG was generated by combining the Fc portion of the immunoglobulin heavy chain to the amino-terminus of OPG. The inhibitory effect of Fc-OPG on bone resorption is similar to pamidronate (Bekker et al., 2001; Body et al., 2003). However, Fc-OPG and RANK-Fc have been replaced by denosumab (Amgen), which is a fully human monoclonal antibody developed using the xenomouse technology that specifically inhibits primate RANKL (Green, 1999). Denosumab does not bind to murine RANKL, human TRAIL, or other TNF family proteins and has a longer half-life than Fc-OPG (Kostenuik et al., 2009). Denosumab has been tested in patients with varying diseases/conditions, including osteoporosis, treatment-induced bone loss, bone metastases, multiple myeloma and rheumatoid arthritis, and is now a standard of care for patients with solid tumor bone metastases (Schwarz & Ritchlin, 2007). Denosumab can significantly delay or prevent skeleton-related events (SREs), including hypercalcemia, in patients with bone metastasis (Castellano et al., 2011). Due to its longer half life, higher specificity and lower toxicity, the therapeutic potential of denosumab is superior to that of Fc-OPG and RANK-Fc (Schwarz & Ritchlin, 2007).

#### **6. Development of** *in vivo* **models of HHM in leukemia/lymphoma**

#### **6.1 Mouse models of HHM and ATLL**

Animal models of ATLL are divided into infectious models, which are useful to study viral infection, viral transmission and the immune response, and pathogenesis models, which are useful for preclinical therapy studies. Pathogenesis models, including tumor xenografts and transgenic mice that develop tumors, are useful to study the development and treatment of HHM in ATLL. Unfortunately, there are few animal models of ATLL that develop HHM. The following animal models are currently available for studying HHM in ATLL.

#### **6.1.1 Human RV-ATL xenograft mouse model of ATLL**

The RV-ATL model was first developed by the laboratory of Dr. Irvin Chen by injecting RV-ATL cells, derived from an ATLL patient, into severe combined immunodeficient (SCID)

that inhibited targets downstream of the PTHR1 (Carter et al., 2007). The benzoxazepinone non-peptide inhibits the cAMP response induced by a PTH1R agonist. The pharmacological behavior of SW106 is not yet completely understood. A similar compound, 1,3,4 benzotriazepine was identified and serves as a base molecule to develop non-peptide PTH1R antagonist derivatives (McDonald et al., 2007). Using a radioligand binding assay that measures cAMP production, N-1 anilino-substituted compounds were identified that have up to a 1000-fold more potency at inhibiting PTH1R compared to the original compound. Efforts are ongoing to determine the effects of these compounds on bone

Ever since RANKL and OPG were discovered to be major regulators of osteoclastic bone resorption, RANK-Fc, Fc-OPG, antibodies targeting RANKL or inhibitors imitating OPG function have been developed for the treatment of osteolytic bone diseases (Schwarz & Ritchlin, 2007). RANK-Fc was generated by combining the carboxyl-terminus of RANK with the Fc portion of human IgG1. It has been shown to decrease tumor burden in two multiple myeloma mouse models, inhibit prostate cancer bone metastasis, and decrease the incidence of lung metastasis and bone lysis in a osteosarcoma mouse model (Lamoureux et al., 2008; Sordillo & Pearse, 2003; Zhang et al., 2003). Fc-OPG was generated by combining the Fc portion of the immunoglobulin heavy chain to the amino-terminus of OPG. The inhibitory effect of Fc-OPG on bone resorption is similar to pamidronate (Bekker et al., 2001; Body et al., 2003). However, Fc-OPG and RANK-Fc have been replaced by denosumab (Amgen), which is a fully human monoclonal antibody developed using the xenomouse technology that specifically inhibits primate RANKL (Green, 1999). Denosumab does not bind to murine RANKL, human TRAIL, or other TNF family proteins and has a longer half-life than Fc-OPG (Kostenuik et al., 2009). Denosumab has been tested in patients with varying diseases/conditions, including osteoporosis, treatment-induced bone loss, bone metastases, multiple myeloma and rheumatoid arthritis, and is now a standard of care for patients with solid tumor bone metastases (Schwarz & Ritchlin, 2007). Denosumab can significantly delay or prevent skeleton-related events (SREs), including hypercalcemia, in patients with bone metastasis (Castellano et al., 2011). Due to its longer half life, higher specificity and lower toxicity, the therapeutic potential of denosumab is superior to that of Fc-OPG and RANK-Fc

**6. Development of** *in vivo* **models of HHM in leukemia/lymphoma** 

The following animal models are currently available for studying HHM in ATLL.

**6.1.1 Human RV-ATL xenograft mouse model of ATLL** 

Animal models of ATLL are divided into infectious models, which are useful to study viral infection, viral transmission and the immune response, and pathogenesis models, which are useful for preclinical therapy studies. Pathogenesis models, including tumor xenografts and transgenic mice that develop tumors, are useful to study the development and treatment of HHM in ATLL. Unfortunately, there are few animal models of ATLL that develop HHM.

The RV-ATL model was first developed by the laboratory of Dr. Irvin Chen by injecting RV-ATL cells, derived from an ATLL patient, into severe combined immunodeficient (SCID)

metastasis, hypercalcemia and hyperparathyroidism.

**5.6 RANKL inhibitors** 

(Schwarz & Ritchlin, 2007).

**6.1 Mouse models of HHM and ATLL** 

mice (Feuer et al., 1993). Richard et al. characterized the tumorigenesis and HHM of this cell line in SCID/beige mice (Richard et al., 2001). SCID/beige mice developed severe hypercalcemia one month after intraperitoneal injection of RV-ATL cells. The mice had bone loss due to increased osteoclastic bone resorption. Shu et al. introduced the luciferase gene into the RV-ATL cells by lentiviral infection and developed a bioluminescent model of HHM in ATLL for preclinical studies (Shu et al., 2007). It was found that both zoledronic acid, a nitrogen-containing bisphosphonate, and PS-341, a selective proteasome inhibitor, decreased tumor burden and HHM in mice. No complication of using the combination of PS-341 and zoledronic acid was observed in this preclinical study (Shu et al., 2007).

#### **6.1.2 Uchiyama human xenograft mouse model of ATLL**

The laboratory of Dr. Takashi Uchiyama developed a mouse xenograft model by injecting cells from the lymph node of a lymphoma-type ATLL patient intraperitoneally into SCID mice (Imada et al., 1996). The mice developed tumors and hypercalcemia within three weeks. There was a marked increase in serum C-terminal PTHrP concentrations with decreased bone formation rates reported in the mice (Takaori-Kondo et al., 1998). However, no significant increase in bone resorption measured by bone histomorphometry was observed in the mice, which is in contrast to ATLL patients with HHM.

#### **6.1.3 Human MET-1 xenograft mouse model of ATLL**

Recently, a NOD/SCID mouse model using human ATLL MET-1 cells has been developed (Phillips et al., 2000). The mice developed leukemia and HHM after intraperitoneal injection. Marked infiltration of tumor cells in multiple organs including spleen, lungs, liver, lymph nodes was observed. Increased plasma PTHrP concentrations in the mice and expression of PTHrP and RANKL in MET-1 cells were reported (Parrula et al., 2009). It is not known whether the HHM in this model was caused by an increase in bone resorption or other mechanisms.

#### **6.1.4 HTLV-1 LTR-Tax transgenic model**

Transgenic mice have been used to study the pathogenesis and role of the HTLV-1 viral oncoprotein, Tax. Several Tax transgenic mice have been developed using different promoters. A transgenic mouse model overexpressing Tax under the regulation of the HTLV-1 long terminal repeat (LTR) was generated (Ruddle et al., 1993). These mice developed neurofibromas and adrenal medullary tumors, but did not develop leukemia or lymphoma. Unexpectedly, a significant increase in bone remodeling with a net increase in bone volume was observed. This was surprising because the authors also demonstrated that osteoclasts were increased in number, size and degree of multinucleation, which would have been expected to lead to a net decrease in bone volume. It was not reported whether HHM developed in the mice.

#### **6.1.5 Human granzyme B-Tax transgenic mice**

The laboratory of Dr. Lee Ratner developed a tissue-specific Tax overexpressing mouse model (Grossman et al., 1995). Tax expression in the mice was under the regulation of the human granzyme B promoter, which limited Tax expression primarily to activated CD4+ and CD8+ Tcells and NK cells. The mice developed mild hypercalcemia and multifocal osteolytic bone lesions, especially in the tail, with increased osteoclastic bone resorption (Gao et al., 2005). The

Mechanisms of Humoral Hypercalcemia of Malignancy in Leukemia/Lymphoma 195

Abu-Amer, Y.; Ross, F. P.; McHugh, K. P.; Livolsi, A.; Peyron, J. F. & Teitelbaum, S. L. (1998).

Amezyane, T.; Lecoules, S.; Bordier, L.; Blade, J. S.; Desrame, J.; Bechade, D.; Coutant, G. &

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Bolliger, A. P.; Graham, P. A.; Richard, V.; Rosol, T. J.; Nachreiner, R. F. & Refsal, K. R.

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mice have increased serum IL-6 concentrations, which is a potent osteoclast activator. When the mice were crossed to mice that overexpressed OPG, they were protected from the development of osteolytic lesions and soft tissue tumors, indicating that increased bone resorption in Tax transgenic mice was induced, at least in part, through a RANKL-dependent pathway. The mouse model also has been used for preclinical studies and zoledronic acid not only prevented the osteolytic bone lesions but also decreased tumor burden. After crossing the mice with IFN-γ knockout mice, the resulting Tax+IFN-γ-/- mice had accelerated tumor formation, dissemination, and death, when compared with Tax+IFN-γ+/- or Tax+IFN-γ+/+ mice (Mitra-Kaushik et al., 2004a). The mice also develop increased osteolytic bone lesions, increased osteoclast formation and more severe hypercalcemia compared to Tax+IFN-γ+/+ mice (Xu et al., 2009). These data indicate that IFN-γ may contribute to the host defense systems that prevent HTLV-1-induced malignancy, bone metastasis and HHM.

#### **6.2 Mouse model of canine lymphoma and HHM**

A bioluminescent NOD/SCID mouse model of canine T-cell lymphoma and HHM has been developed (Nadella et al., 2008). The mice developed multicentric lymphoma in the mesenteric lymph nodes after intraperitoneal injection of tumor cells. Moderate to marked splenomegaly and enlarged thymuses were observed. There was increased osteoclastic bone resorption in trabecular bone in mice with lymphoma. HHM developed 6-8 weeks after injection of tumor cells. The increase in plasma PTHrP concentrations likely played a central role in HHM in the mice. The cause of canine T-cell lymphoma is unknown and retroviruses have not been identified as a cause of lymphoma in dogs (in contrast to humans and cats).

#### **7. Conclusion**

HHM is a life-threatening complication in certain patients with lymphoma or leukemia. As outlined in this review, progress has been made in elucidating the mechanisms by which humoral factors from neoplastic lymphocytes induce HHM, including increased osteoclastic bone resorption and renal calcium reabsorption. However, further efforts are needed to fully understand the pathogenesis of HHM, including the endocrine or paracrine role of interactions between tumor-associated and host-produced cytokines. PTHrP plays a major endocrine and paracrine role in HHM, but the effects of other factors secreted from tumor cells or host cells cannot be neglected. For example, the expression of RANKL in ATLL cells suggests that ATLL cells may function directly as inducers of osteoclastic bone resorption. Additional effective treatments are needed for this paraneoplastic syndrome. Small molecules or humanized antibodies targeting essential factors or their receptors may be an attractive future therapeutic strategy for treatment of HHM.

#### **8. Acknowledgements**

This work was supported by the National Cancer Institute (P01 CA100730) and the National Center for Research Resources (T32 RR07073). We thank Tim Vojt for the illustrations.

#### **9. References**

Abu-Amer, Y.; Ross, F. P.; Edwards, J. & Teitelbaum, S. L. (1997). Lipopolysaccharidestimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor. J.Clin.Invest, 100, No.6, pp. 1557-1565, 0021-9738

mice have increased serum IL-6 concentrations, which is a potent osteoclast activator. When the mice were crossed to mice that overexpressed OPG, they were protected from the development of osteolytic lesions and soft tissue tumors, indicating that increased bone resorption in Tax transgenic mice was induced, at least in part, through a RANKL-dependent pathway. The mouse model also has been used for preclinical studies and zoledronic acid not only prevented the osteolytic bone lesions but also decreased tumor burden. After crossing the mice with IFN-γ knockout mice, the resulting Tax+IFN-γ-/- mice had accelerated tumor formation, dissemination, and death, when compared with Tax+IFN-γ+/- or Tax+IFN-γ+/+ mice (Mitra-Kaushik et al., 2004a). The mice also develop increased osteolytic bone lesions, increased osteoclast formation and more severe hypercalcemia compared to Tax+IFN-γ+/+ mice (Xu et al., 2009). These data indicate that IFN-γ may contribute to the host defense systems

A bioluminescent NOD/SCID mouse model of canine T-cell lymphoma and HHM has been developed (Nadella et al., 2008). The mice developed multicentric lymphoma in the mesenteric lymph nodes after intraperitoneal injection of tumor cells. Moderate to marked splenomegaly and enlarged thymuses were observed. There was increased osteoclastic bone resorption in trabecular bone in mice with lymphoma. HHM developed 6-8 weeks after injection of tumor cells. The increase in plasma PTHrP concentrations likely played a central role in HHM in the mice. The cause of canine T-cell lymphoma is unknown and retroviruses have not been identified as a cause of lymphoma in dogs (in contrast to humans and cats).

HHM is a life-threatening complication in certain patients with lymphoma or leukemia. As outlined in this review, progress has been made in elucidating the mechanisms by which humoral factors from neoplastic lymphocytes induce HHM, including increased osteoclastic bone resorption and renal calcium reabsorption. However, further efforts are needed to fully understand the pathogenesis of HHM, including the endocrine or paracrine role of interactions between tumor-associated and host-produced cytokines. PTHrP plays a major endocrine and paracrine role in HHM, but the effects of other factors secreted from tumor cells or host cells cannot be neglected. For example, the expression of RANKL in ATLL cells suggests that ATLL cells may function directly as inducers of osteoclastic bone resorption. Additional effective treatments are needed for this paraneoplastic syndrome. Small molecules or humanized antibodies targeting essential factors or their receptors may be an

This work was supported by the National Cancer Institute (P01 CA100730) and the National Center for Research Resources (T32 RR07073). We thank Tim Vojt for the illustrations.

Abu-Amer, Y.; Ross, F. P.; Edwards, J. & Teitelbaum, S. L. (1997). Lipopolysaccharide-

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stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55

that prevent HTLV-1-induced malignancy, bone metastasis and HHM.

**6.2 Mouse model of canine lymphoma and HHM** 

attractive future therapeutic strategy for treatment of HHM.

**7. Conclusion** 

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A.; Katodritou, E.; Verrou, E.; Vervessou, E. C.; Dimopoulos, M. A. & Croucher, P. I. (2006). Bortezomib reduces serum dickkopf-1 and receptor activator of nuclear factor-kappaB ligand concentrations and normalises indices of bone remodelling in patients with relapsed multiple myeloma. Br.J.Haematol., 135, No.5, pp. 688-692,

Fujiwara, K.; Matsuoka, H. & Nishida, S. (2007). Macrophage inflammatory protein-1alpha (MIP-1alpha) enhances a receptor activator of nuclear factor kappaB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways. Mol.Cell Biochem., 304, No.1-2, pp. 53-60,


**11** 

*Japan* 

**The Role of T-Cell Leukemia Translocation-**

Shigeru Kotake, Toru Yago, Manabu Kawamoto and Yuki Nanke

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-κB ligand (RANKL), TNF-α, IL-6, IL-17, and IFN-γ. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibits human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported. In the current review paper, we

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption by expressing cytokines such as RANKL, TNF, IL-6, IL-17, and IFN (Horwood et al. 1999; Kawai et al. 2006; Kobayashi et al. 2000; Kotake et al.1996; 1999; 2001; 2005; Takayanagi et al. 2000; Yago et al. 2007, Yago et al. 2009). In addition to these cytokines, however, other factors expressed in synovial tissues may play a role in resorbing bone. To identify novel peptides or proteins expressed in synovial tissues of patients with RA that regulate human osteoclastogenesis, we purified proteins from synovial tissues of patients with RA, using gel filtration chromatography, reverse-aspect HPLC, and mass spectrometry. We finally demonstrated that a peptide derived from the extra-cellular domain of T-cell leukemia translocation-associated gene (TCTA) protein inhibits both RANKL-induced human osteoclastogenesis and pit formation of mature human osteoclasts (Kotake et al.

In 1995, Aplan et al. cloned and characterized a novel gene at the site of a t(1;3)(p34;p21) translocation breakpoint in T-cell acute lymphoblastic leukemia, designating this gene as TCTA (Aplan et al. 1995). TCTA is also reported as T-cell leukemia translocation-altered gene. TCTA mRNA is expressed ubiquitously in normal tissues, with the highest levels of expression in the kidney. TCTA has been conserved throughout evolution in organisms ranging from *Drosophila* to humans. A short open reading frame encodes a protein of 103 amino acid residues, Mr 11,300, without strong homology to any previously reported proteins. Of note, genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene

summarized papers on TCTA protein and our recent findings.

**1. Introduction** 

2009b, 2009c).

**Associated Gene (TCTA) Protein in** 

 *Institute of Rheumatology, Tokyo Women's Medical University* 

**Human Osteoclastogenesis** 


### **The Role of T-Cell Leukemia Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis**

Shigeru Kotake, Toru Yago, Manabu Kawamoto and Yuki Nanke  *Institute of Rheumatology, Tokyo Women's Medical University Japan* 

#### **1. Introduction**

206 T-Cell Leukemia

Zhong, Y.; Armbrecht, H. J. & Christakos, S. (2009). Calcitonin, a regulator of the 25-

Zlotnik, A. & Yoshie, O. (2000). Chemokines: a new classification system and their role in

immunity. Immunity., 12, No.2, pp. 121-127, 1074-7613

11069, 0021-9258

hydroxyvitamin D3 1alpha-hydroxylase gene. J.Biol.Chem., 284, No.17, pp. 11059-

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-κB ligand (RANKL), TNF-α, IL-6, IL-17, and IFN-γ. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibits human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported. In the current review paper, we summarized papers on TCTA protein and our recent findings.

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption by expressing cytokines such as RANKL, TNF, IL-6, IL-17, and IFN (Horwood et al. 1999; Kawai et al. 2006; Kobayashi et al. 2000; Kotake et al.1996; 1999; 2001; 2005; Takayanagi et al. 2000; Yago et al. 2007, Yago et al. 2009). In addition to these cytokines, however, other factors expressed in synovial tissues may play a role in resorbing bone. To identify novel peptides or proteins expressed in synovial tissues of patients with RA that regulate human osteoclastogenesis, we purified proteins from synovial tissues of patients with RA, using gel filtration chromatography, reverse-aspect HPLC, and mass spectrometry. We finally demonstrated that a peptide derived from the extra-cellular domain of T-cell leukemia translocation-associated gene (TCTA) protein inhibits both RANKL-induced human osteoclastogenesis and pit formation of mature human osteoclasts (Kotake et al. 2009b, 2009c).

In 1995, Aplan et al. cloned and characterized a novel gene at the site of a t(1;3)(p34;p21) translocation breakpoint in T-cell acute lymphoblastic leukemia, designating this gene as TCTA (Aplan et al. 1995). TCTA is also reported as T-cell leukemia translocation-altered gene. TCTA mRNA is expressed ubiquitously in normal tissues, with the highest levels of expression in the kidney. TCTA has been conserved throughout evolution in organisms ranging from *Drosophila* to humans. A short open reading frame encodes a protein of 103 amino acid residues, Mr 11,300, without strong homology to any previously reported proteins. Of note, genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene

The Role of T-Cell Leukemia

**2.2 Origin of osteoclasts** 

inhibits bone resorption by human osteoclasts.

(ODF)' expressed on osteoblasts (Suda et al. 1995).

is essential to retain both the structure and strength of normal bone.

Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis 209

structure, necessary for bone resorption, and in motile osteoclasts, podosomes are organized into lamellipodia (Latin lamella, a thin leaf; Greek pous, foot), the structure responsible for cell movement. Thus, the presence of actin rings and lamellipodia is mutually exclusive (Sarrazin et al. 2004). In 2004, Sarrazin et al. showed, using mature human osteoclasts extracted from the femurs and tibias of human fetuses, that osteoclasts have two subtypes of EP receptors, prostaglandin E2 (PGE2), EP3 and EP4, that mediate different actions of PGE2 on these cells; activation of EP4 receptors inhibits actin ring formation and activation of EP3 receptors increases the number of lamellipodia (Sarrazin et al. 2004). Thus, PGE2 directly

The cooperation of osteoclasts and osteoblasts is critical to maintain skeletal integrity in normal bones. After bone resorption by osteoclasts on normal bone tissues, osteoblasts subsequently rebuild bone in the lacunae resorbed by osteoclasts; this mechanism is called 'bone remodeling'. When the activity or number of osteoclasts is elevated compared with osteoblasts, the bone becomes fragile, that is, 'osteoporotic'. In addition, bone remodeling is disrupted in all bone diseases associated with changes in bone mass. Thus, bone remodeling

The origin of osteoclasts was unclear until the late 1980s. In 1988, Takahashi et al. established a co-culture system using mouse spleen cells and osteoblasts to induce osteoclastogenesis *in vitro*, demonstrating that the origin of osteoclasts is hematopoietic cells and that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts (Takahashi et al. 1988b). The precursor of osteoclasts was then revealed to be colony-forming unit–macrophage (CFU–M) or CFU– granulocyte/macrophage (CFU–GM) in bone marrow or spleen in mice. In 1990, Udagawa et al. demonstrated that osteoclasts are formed from murine macrophages (Udagawa et al. 1990). From these findings, Suda et al. hypothesized that bone marrow hemopoietic cells differentiate into osteoclasts through the stimulation of 'osteoclast-differentiation factor

Finally, ODF, now termed RANKL, which induces osteoclastogenesis from monocytes or macrophages, was independently cloned by three groups in 1997 (Fig. 2) (Udagawa et al. 2002). RANKL is a member of the TNF superfamily of cytokines. The protein constructs a trimeric complex to bind its receptor, receptor activator NF-κB (RANK)(Lam et al. 2001). A decoy receptor is also cloned, which is designated as 'osteoprotegerin (OPG)' (Udagawa et al. 2002). In 2000–2001, we and other groups showed that T cells expressing RANKL induce osteoclastogenesis (Kong et al. 1999; Horwood et al. 1999; Kotake et al. 2001); in particular, we demonstrated osteoclastogenesis using human cells (Kotake et al. 2001), whereas others used murine cells (Kong et al. 1999, Horwood et al. 1999). In addition, in 2009, we reported that, in human osteoclastogenesis induced by RANKL, T-cell leukemia translocation-

associated gene (TCTA) protein is required for cellular fusion (Kotake et al. 2009b).

Culture systems were developed to form osteoclasts *in vitro* in 1981–1988. In 1981, Testa et al. first succeeded in forming osteoclast-like multinucleated cells from feline marrow cells in long-term Dexter cultures (Testa et al. 1981). In 1984, using this feline marrow culture system, Ibbotson et al. showed that the formation of osteoclast-like cells is greatly stimulated

**2.3 Development of culture systems to form osteoclasts** *in vitro*

(Aplan et al. 1995). On the other hand, in 2005, it has been reported that TCTA interacts with SMA- and MAD- related protein 4 (SMAD4) in a proteome-scale map of the human proteinprotein interaction network (Rual et al. 2005); however, the function of TCTA has not been clarified.

### **2. Osteoclast**

#### **2.1 Structure and function of osteoclasts**

Osteoclasts are unique multinucleated cells whose specialized function is to resorb calcified tissues (Fig. 1) (Kotake et al. 2005). On the surface of bone, osteoclasts develop a specialized adhesion structure, the 'podosome', which subsequently undergoes reorganization into sealing zones (Luxenburg et al. 2007). These ring-like adhesion structures, i.e., actin rings, seal osteoclasts to the surface of bone. In the sealed resorption lacuna, localized acidification is driven by carbonic anhydrase II and vacuolar H(+)-ATPase in osteoclasts; carbonic anhydrase II produces protons and vacuolar H(+)-ATPase transfers them into the lacuna. In acidified lacuna, cathepsin-K and matrix metalloproteinase-9 (MMP-9) are released from osteoclasts to degrade calcified tissues (Fuller et al. 2007).

c-Fms, M-CSF receptor; CTR, calcitonin receptor; MMP, matrix metalloproteinase; OSCAR, osteoclastassociated receptor; RANK, receptor activator of NFκB; TRAF, TNF receptor-associated factor; TRAP, tartrate-resistant acid phosphatase.

Fig. 1. Schematic structure of osteoclast

Osteoclasts express unique cell adhesion structures called podosomes, which contain actin filaments. Podosomes are organized differently depending on the activity of the osteoclast; in bone-resorbing osteoclasts, podosomes form the actin ring, representing a gasket-like structure, necessary for bone resorption, and in motile osteoclasts, podosomes are organized into lamellipodia (Latin lamella, a thin leaf; Greek pous, foot), the structure responsible for cell movement. Thus, the presence of actin rings and lamellipodia is mutually exclusive (Sarrazin et al. 2004). In 2004, Sarrazin et al. showed, using mature human osteoclasts extracted from the femurs and tibias of human fetuses, that osteoclasts have two subtypes of EP receptors, prostaglandin E2 (PGE2), EP3 and EP4, that mediate different actions of PGE2 on these cells; activation of EP4 receptors inhibits actin ring formation and activation of EP3 receptors increases the number of lamellipodia (Sarrazin et al. 2004). Thus, PGE2 directly inhibits bone resorption by human osteoclasts.

The cooperation of osteoclasts and osteoblasts is critical to maintain skeletal integrity in normal bones. After bone resorption by osteoclasts on normal bone tissues, osteoblasts subsequently rebuild bone in the lacunae resorbed by osteoclasts; this mechanism is called 'bone remodeling'. When the activity or number of osteoclasts is elevated compared with osteoblasts, the bone becomes fragile, that is, 'osteoporotic'. In addition, bone remodeling is disrupted in all bone diseases associated with changes in bone mass. Thus, bone remodeling is essential to retain both the structure and strength of normal bone.

#### **2.2 Origin of osteoclasts**

208 T-Cell Leukemia

(Aplan et al. 1995). On the other hand, in 2005, it has been reported that TCTA interacts with SMA- and MAD- related protein 4 (SMAD4) in a proteome-scale map of the human proteinprotein interaction network (Rual et al. 2005); however, the function of TCTA has not been

Osteoclasts are unique multinucleated cells whose specialized function is to resorb calcified tissues (Fig. 1) (Kotake et al. 2005). On the surface of bone, osteoclasts develop a specialized adhesion structure, the 'podosome', which subsequently undergoes reorganization into sealing zones (Luxenburg et al. 2007). These ring-like adhesion structures, i.e., actin rings, seal osteoclasts to the surface of bone. In the sealed resorption lacuna, localized acidification is driven by carbonic anhydrase II and vacuolar H(+)-ATPase in osteoclasts; carbonic anhydrase II produces protons and vacuolar H(+)-ATPase transfers them into the lacuna. In acidified lacuna, cathepsin-K and matrix metalloproteinase-9 (MMP-9) are released from

c-Fms, M-CSF receptor; CTR, calcitonin receptor; MMP, matrix metalloproteinase; OSCAR, osteoclastassociated receptor; RANK, receptor activator of NFκB; TRAF, TNF receptor-associated factor; TRAP,

Osteoclasts express unique cell adhesion structures called podosomes, which contain actin filaments. Podosomes are organized differently depending on the activity of the osteoclast; in bone-resorbing osteoclasts, podosomes form the actin ring, representing a gasket-like

clarified.

**2. Osteoclast** 

**2.1 Structure and function of osteoclasts** 

tartrate-resistant acid phosphatase.

Fig. 1. Schematic structure of osteoclast

osteoclasts to degrade calcified tissues (Fuller et al. 2007).

The origin of osteoclasts was unclear until the late 1980s. In 1988, Takahashi et al. established a co-culture system using mouse spleen cells and osteoblasts to induce osteoclastogenesis *in vitro*, demonstrating that the origin of osteoclasts is hematopoietic cells and that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts (Takahashi et al. 1988b). The precursor of osteoclasts was then revealed to be colony-forming unit–macrophage (CFU–M) or CFU– granulocyte/macrophage (CFU–GM) in bone marrow or spleen in mice. In 1990, Udagawa et al. demonstrated that osteoclasts are formed from murine macrophages (Udagawa et al. 1990). From these findings, Suda et al. hypothesized that bone marrow hemopoietic cells differentiate into osteoclasts through the stimulation of 'osteoclast-differentiation factor (ODF)' expressed on osteoblasts (Suda et al. 1995).

Finally, ODF, now termed RANKL, which induces osteoclastogenesis from monocytes or macrophages, was independently cloned by three groups in 1997 (Fig. 2) (Udagawa et al. 2002). RANKL is a member of the TNF superfamily of cytokines. The protein constructs a trimeric complex to bind its receptor, receptor activator NF-κB (RANK)(Lam et al. 2001). A decoy receptor is also cloned, which is designated as 'osteoprotegerin (OPG)' (Udagawa et al. 2002). In 2000–2001, we and other groups showed that T cells expressing RANKL induce osteoclastogenesis (Kong et al. 1999; Horwood et al. 1999; Kotake et al. 2001); in particular, we demonstrated osteoclastogenesis using human cells (Kotake et al. 2001), whereas others used murine cells (Kong et al. 1999, Horwood et al. 1999). In addition, in 2009, we reported that, in human osteoclastogenesis induced by RANKL, T-cell leukemia translocationassociated gene (TCTA) protein is required for cellular fusion (Kotake et al. 2009b).

#### **2.3 Development of culture systems to form osteoclasts** *in vitro*

Culture systems were developed to form osteoclasts *in vitro* in 1981–1988. In 1981, Testa et al. first succeeded in forming osteoclast-like multinucleated cells from feline marrow cells in long-term Dexter cultures (Testa et al. 1981). In 1984, using this feline marrow culture system, Ibbotson et al. showed that the formation of osteoclast-like cells is greatly stimulated

The Role of T-Cell Leukemia

especially in patients with RA.

an *in vivo* study.

**immunology and cell biology** 

Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis 211

**2.5 Geranylgeranylacetone Inhibits the formation and function of human osteoclasts**  The anti-ulcer drug geranylgeranylacetone (GGA), known as teprenon, is frequently used with nonstroidal anti-inflammatory drugs (NSAIDs) in Japan. In 2005, we demonstrated that GGA inhibits the formation and function of human osteoclasts and prevents bone loss in tail-suspended rats and ovariectomized rats (Nanke et al. 2005). Vitamin K is also used to protect against osteoporosis. It has been reported that the inhibitory effect of vitamin K2 (menatetrenone) on bone resorption may be related to its side chain. GGA has almost the

We hypothesized that GGA also has an inhibitory effect on osteoclastogenesis both *in vitro* and *in vivo*. GGA in pharmacological concentrations directly inhibited osteoclastogenesis from human monocytes induced by soluble RANKL. In addition, GGA induced the degradation of actin rings in mature osteoclasts, which was reversed by adding geranylgeranylpyrophosphatase. Moreover, GGA increased the bone mineral density of the total femur, proximal metaphysis, and diaphysis of femur in ovariectomized rats. GGA also prevented bone loss induced by hindlimb unloading in tail-suspended rats. These results indicate that GGA prevents bone loss by maintaining a positive balance of bone turnover through suppression of both the formation and activity of osteoclasts. In addition, in 2009, we also reported that GGA induces cell death in fibroblast-like synoviocytes from patients with RA (Nanke et al. 2009). Thus, GGA could be used to prevent and improve osteoporosis,

**3. 'Human osteoclastology' - Difference between human and mouse in** 

In basic science, murine cells are usually used, because these cells and experimental tools for them can be easily obtained. In addition, it is possible to create transgenic and knockout murine models. Indeed, biological science has made marked progress using murine cells and disease models. On the other hand, there are many disadvantages in studies using human cells, e.g., it is difficult to obtain human cells. In addition, it is impossible to perform

However, to investigate the pathogenesis of RA, it is critical to investigate osteoclastogenesis using human cells. In bone cell biology, some cytokines show different functions between humans and mice. For example, M-CSF induces colony formation in mouse cells. In human cells, however, M-CSF usually induces the differentiation of progenitor cells of monocytes rather than colony formation, although Motoyoshi et al. initially reported that M-CSF induces colony formation in human macrophages in 1982 (Motoyoshi et al. 1982). On the other hand, some cytokines show different expressions between humans and mice; Kanamaru et al. reported that the expression of membrane-bound RANKL is limited in human T cells compared with mouse T cells (Kanamaru 2004). Moreover, the mouse CD4+CD25+ regulatory subset can be isolated from all CD25+ T cells regardless of their level of CD25 expression; however, when similar criteria are followed to isolate these cells from human blood, CD25+ cells (high and low together) do not exhibit an anergic phenotype or significant suppressive function. In 2001, Baecher-Allan et al. demonstrated that, in humans, CD4+CD25**high** exhibit all the properties of regulatory T cells (Baecher-Allan et al. 2001). We also measured the percentages of CD4+CD25**high** T cells as regulatory T cells in patients with Behcet's disease (Nanke et al. 2008). In addition, Amadi-Obi et al. reported that a major immunological difference between humans and mice is the presence of CD4+T cells

same chemical structure as the side chain of menatetenone.

OPG, osteoprotegerin; RANK, receptor activator of NFκB, RANKL, RANK ligand.

Fig. 2. Differentiation and activation of osteoclasts. A RANK-RANKL system induces both osteoclastogenesis from monocytes and the activation of mature osteoclasts.

by osteotropic hormones, such as 1,25(OH)2D3, PTH, and PGE2 (Ibbotson et al. 1984). In 1987, MacDonald et al. reported the formation of multinucleated cells that respond to osteotropic hormones in long-term human bone marrow cultures (MacDonald et al. 1987). In 1988, Takahashi et al. and in 1989, Hattersley et al. used marrow cells of mice to examine osteoclast-like cell formation from their progenitor cells (Takahashi et al. 1988a, Hattersley et al. 1989). Moreover, in 1988, Takahashi et al. established an innovative co-culture system using mouse spleen cells and osteoblasts to induce osteoclastogenesis *in vitro* (Takahashi et al. 1988b). Thus, since 1981, studies using osteoclastogenesis *in vitro* have been developed, and PGE2 was shown to up-regulate murine osteoclastogenesis using the marrow culture system *in vitro*.

#### **2.4 Role of osteoclasts in the pathogenesis of RA**

Osteoclasts also play an important role in the pathogenesis of rheumatoid arthritis (RA). Since 1984, it has been reported that in bone destruction of RA, many activated osteoclasts are detected on the surface of eroded bone in the interface with synovial tissues (Shimizu et al. 1985). In addition, we have demonstrated that osteoclasts are detected in synovial tissues as well as eroded bone from patients with RA (Kotake et al. 1996). We have also reported that the number of precursor cells of osteoclasts increases in bone marrow adjacent to joints with arthritis (Kotake et al. 1992). Moreover, the amount of cytokines that induces osteoclastogenesis, such as IL-1, TNF-α and IL-6, is elevated in synovial tissues of patients with RA, while the amount of cytokines that induces osteoclastogenesis, such as IL-4 and IL-10, is decreased (Kotake et al. 1992; Kotake et al. 1996b; Kotake et al. 1997). Thus, patients with RA are likely to suffer from joint destruction as well as systemic osteoporosis, in which the number of osteoclasts increases, suggesting that osteoclasts play a critical role in the pathogenesis of RA.

OPG, osteoprotegerin; RANK, receptor activator of NFκB, RANKL, RANK ligand.

osteoclastogenesis from monocytes and the activation of mature osteoclasts.

**2.4 Role of osteoclasts in the pathogenesis of RA** 

system *in vitro*.

pathogenesis of RA.

Fig. 2. Differentiation and activation of osteoclasts. A RANK-RANKL system induces both

by osteotropic hormones, such as 1,25(OH)2D3, PTH, and PGE2 (Ibbotson et al. 1984). In 1987, MacDonald et al. reported the formation of multinucleated cells that respond to osteotropic hormones in long-term human bone marrow cultures (MacDonald et al. 1987). In 1988, Takahashi et al. and in 1989, Hattersley et al. used marrow cells of mice to examine osteoclast-like cell formation from their progenitor cells (Takahashi et al. 1988a, Hattersley et al. 1989). Moreover, in 1988, Takahashi et al. established an innovative co-culture system using mouse spleen cells and osteoblasts to induce osteoclastogenesis *in vitro* (Takahashi et al. 1988b). Thus, since 1981, studies using osteoclastogenesis *in vitro* have been developed, and PGE2 was shown to up-regulate murine osteoclastogenesis using the marrow culture

Osteoclasts also play an important role in the pathogenesis of rheumatoid arthritis (RA). Since 1984, it has been reported that in bone destruction of RA, many activated osteoclasts are detected on the surface of eroded bone in the interface with synovial tissues (Shimizu et al. 1985). In addition, we have demonstrated that osteoclasts are detected in synovial tissues as well as eroded bone from patients with RA (Kotake et al. 1996). We have also reported that the number of precursor cells of osteoclasts increases in bone marrow adjacent to joints with arthritis (Kotake et al. 1992). Moreover, the amount of cytokines that induces osteoclastogenesis, such as IL-1, TNF-α and IL-6, is elevated in synovial tissues of patients with RA, while the amount of cytokines that induces osteoclastogenesis, such as IL-4 and IL-10, is decreased (Kotake et al. 1992; Kotake et al. 1996b; Kotake et al. 1997). Thus, patients with RA are likely to suffer from joint destruction as well as systemic osteoporosis, in which the number of osteoclasts increases, suggesting that osteoclasts play a critical role in the

#### **2.5 Geranylgeranylacetone Inhibits the formation and function of human osteoclasts**

The anti-ulcer drug geranylgeranylacetone (GGA), known as teprenon, is frequently used with nonstroidal anti-inflammatory drugs (NSAIDs) in Japan. In 2005, we demonstrated that GGA inhibits the formation and function of human osteoclasts and prevents bone loss in tail-suspended rats and ovariectomized rats (Nanke et al. 2005). Vitamin K is also used to protect against osteoporosis. It has been reported that the inhibitory effect of vitamin K2 (menatetrenone) on bone resorption may be related to its side chain. GGA has almost the same chemical structure as the side chain of menatetenone.

We hypothesized that GGA also has an inhibitory effect on osteoclastogenesis both *in vitro* and *in vivo*. GGA in pharmacological concentrations directly inhibited osteoclastogenesis from human monocytes induced by soluble RANKL. In addition, GGA induced the degradation of actin rings in mature osteoclasts, which was reversed by adding geranylgeranylpyrophosphatase. Moreover, GGA increased the bone mineral density of the total femur, proximal metaphysis, and diaphysis of femur in ovariectomized rats. GGA also prevented bone loss induced by hindlimb unloading in tail-suspended rats. These results indicate that GGA prevents bone loss by maintaining a positive balance of bone turnover through suppression of both the formation and activity of osteoclasts. In addition, in 2009, we also reported that GGA induces cell death in fibroblast-like synoviocytes from patients with RA (Nanke et al. 2009). Thus, GGA could be used to prevent and improve osteoporosis, especially in patients with RA.

#### **3. 'Human osteoclastology' - Difference between human and mouse in immunology and cell biology**

In basic science, murine cells are usually used, because these cells and experimental tools for them can be easily obtained. In addition, it is possible to create transgenic and knockout murine models. Indeed, biological science has made marked progress using murine cells and disease models. On the other hand, there are many disadvantages in studies using human cells, e.g., it is difficult to obtain human cells. In addition, it is impossible to perform an *in vivo* study.

However, to investigate the pathogenesis of RA, it is critical to investigate osteoclastogenesis using human cells. In bone cell biology, some cytokines show different functions between humans and mice. For example, M-CSF induces colony formation in mouse cells. In human cells, however, M-CSF usually induces the differentiation of progenitor cells of monocytes rather than colony formation, although Motoyoshi et al. initially reported that M-CSF induces colony formation in human macrophages in 1982 (Motoyoshi et al. 1982). On the other hand, some cytokines show different expressions between humans and mice; Kanamaru et al. reported that the expression of membrane-bound RANKL is limited in human T cells compared with mouse T cells (Kanamaru 2004). Moreover, the mouse CD4+CD25+ regulatory subset can be isolated from all CD25+ T cells regardless of their level of CD25 expression; however, when similar criteria are followed to isolate these cells from human blood, CD25+ cells (high and low together) do not exhibit an anergic phenotype or significant suppressive function. In 2001, Baecher-Allan et al. demonstrated that, in humans, CD4+CD25**high** exhibit all the properties of regulatory T cells (Baecher-Allan et al. 2001). We also measured the percentages of CD4+CD25**high** T cells as regulatory T cells in patients with Behcet's disease (Nanke et al. 2008). In addition, Amadi-Obi et al. reported that a major immunological difference between humans and mice is the presence of CD4+T cells

The Role of T-Cell Leukemia

CDW52 antigens.

We would like to present our findings as follows.

Thirty fractions were obtained by the reverse-aspect HPLC.

**5.2 Purification of proteins from synovial tissues of patients with RA** 

Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis 213

to identify novel peptides or proteins expressed in synovial tissues of patients with RA that regulate human osteoclastogenesis since 1996. We purified proteins from synovial tissues of patients with RA, using gel filtration chromatography, reverse-aspect HPLC, and mass spectrometry. We finally demonstrated that a peptide derived from the extracellular domain of TCTA protein inhibited both RANKL-induced human osteoclastogenesis and pit formation of mature human osteoclasts (Kotake et al. 2009b).

Sixty-five grams of synovial tissues were obtained from 5 patients with RA at total knee replacement. The crude extract was obtained by homogenization of the synovial tissues. NH3Ac buffer was added to the freeze-dried crude extract. The supernatant was then applied to the column equilibrated with NH3Ac buffer and divided into two fractions, low molecular weight (MW) and high MW by first gel filtration chromatography. The low MW fraction was further applied to the column equilibrated with PBS by the second gel filtration chromatography. Proteins were eluted and protein concentration (A280) was monitored by UV absorption. Each fraction was added to the culture for human osteoclastogenesis to evaluate the activity on the osteoclastogenesis. Two fractions showed the inhibitory activity of osteoclastogenesis. These fractions were then subjected to ion-exchange chromatography. The "flow through" from the column showed the inhibitory activity on the osteoclastogenesis. The "flow through" was then applied to second gel filtration chromatography. Ten fractions from gel filtration chromatography showed the inhibitory activity on osteoclastogenesis. Thus, these fractions were applied to reverse-aspect HPLC.

After each fraction obtained by reverse-aspect HPLC was concentrated, amino acid sequences were determined using a protein sequencer. Amino acid sequences (3-5 mers) were determined in 8 fractions. In addition, we tried to determine the sequence of each fraction by mass spectrometry; however, the sequences were not determined, although the total molecular weight of each peptide was speculated to be less than 1000 Da. Thus, we synthesized 8 peptides according to the sequences determined using the protein sequencer

We finally revealed that the amino acid sequence of **Glycine-Glutamine-Asparagine (Gly-Gln-Asn; GQN)** alone in 8 synthesized peptides showed inhibitory activity in human osteoclastogenesis; **GQN** dose-dependently inhibited human osteoclastogenesis from

When searching for proteins that include **GQN** using FASTA search, we found only 2 proteins in human proteins, CDW52 antigen (CAMPATH-1 antigen) and TCTA. In the FASTA search, it was possible to identify 3 mer peptides, but we did not find any other human proteins. Using "PeptideCutter", by which it is possible to predict the site cleaved by enzymes in peptides, we synthesized 4 peptides from a sequence of CDW52 antigen and one peptide from a sequence of TCTA protein, which included **GQN**. A peptide from TCTA protein showed inhibitory activity on human osteoclastogenesis, but not peptides from

and evaluated the effect of each peptide on human osteoclastogenesis in vitro.

**5.3 A small peptide including GQN inhibits human osteoclastogenesis** 

peripheral monocytes by RANKL (IC50: around 30 M)(Kotake et al. 2009b).

producing IL-17, Th17 cells, in the peripheral blood of healthy humans, but not mice (Amadi-Obi et al. 2007). Recently, several studies have also demonstrated the existence of substantial numbers of human CD4+ T cells that are able to produce both IL-17 and IFN-γ; the term "Th17/Th1" cells has been proposed (Annunziato et al. 2009).

In addition, two groups have reported the effects of PGE2 on human osteoclastogenesis from monocytes alone stimulated by RANKL in the absence of osteoblasts, contrary to many reports on the stimulatory effects of PGE2 on murine cells. In 1999, Itonaga et al. reported that PGE2 inhibits osteoclast formation induced by RANKL in human peripheral blood mononuclear cell cultures (Itonaga et al. 1999). In 2005, Take et al. demonstrated that, unlike mouse macrophage cultures, PGE2 strongly inhibits RANKL-induced osteoclast formation in human CD14+ cell cultures (Take et al. 2005). In addition, they showed that human osteoclast progenitors produce a soluble unidentified factor(s) in response to PGE2 that strongly inhibits RANKL-induced osteoclast formation not only in human CD14+ cell cultures but also in mouse macrophage cultures. They tried to identify the soluble factors, and concluded that CD14+ cells produce an inhibitor(s) that does not correspond to known inhibitory factors, such as GM-CSF, IFN-γ, and IL-4. Thus, these reports demonstrated the possibility that PGE2 differently plays a direct role in osteoclastogenesis from monocytes alone in the absence of osteoblastic cells between humans and mice.

Thus, in the study of human diseases, it is essential to investigate human osteoclastogenesis using human cells; the differences in species used in studies are critical to discuss the function of cytokines. We suggest that the term 'human osteoclastology' be used to describe studies on human osteoclastogenesis (Kotake 2009a, Kotake 2010).

#### **4. T-cell leukemia translocation-associated gene (TCTA)**

In 1995, Aplan et al. cloned and characterized a novel gene at the site of a t(1;3)(p34;p21) translocation breakpoint in T-cell acute lymphoblastic leukemia, designating this gene as TCTA (Aplan et al. 1995). TCTA mRNA is expressed ubiquitously in normal tissues, with the highest levels of expression in the kidney. TCTA has been conserved throughout evolution in organisms ranging from *Drosophila* to humans. A short open reading frame encodes a protein of 103 amino acid residues, Mr 11,300, without strong homology to any previously reported proteins. Of note, genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene (Aplan et al. 1995). On the other hand, in 2005, it has been reported that TCTA interacts with SMA- and MAD- related protein 4 (SMAD4) in a proteome-scale map of the human protein-protein interaction network (Supplementary Table S2, line 6175) (Rual et al. 2005); however, the function of TCTA has not been clarified.

#### **5. Role of TCTA protein in human osteoclastogenesis**

#### **5.1 Hypothesis**

Synovial tissues of patients with RA include factors regulating bone resorption by expressing cytokines such as RANKL, TNF, IL-6, IL-17, and IFN as described in Introduction. In addition to these cytokines, however, other factors expressed in synovial tissues may play a role in resorbing bone. We hypothesized that a novel factor in synovial tissues of patients with RA regulates osteoclastogenesis. To test this hypothesis, we tried

producing IL-17, Th17 cells, in the peripheral blood of healthy humans, but not mice (Amadi-Obi et al. 2007). Recently, several studies have also demonstrated the existence of substantial numbers of human CD4+ T cells that are able to produce both IL-17 and IFN-γ;

In addition, two groups have reported the effects of PGE2 on human osteoclastogenesis from monocytes alone stimulated by RANKL in the absence of osteoblasts, contrary to many reports on the stimulatory effects of PGE2 on murine cells. In 1999, Itonaga et al. reported that PGE2 inhibits osteoclast formation induced by RANKL in human peripheral blood mononuclear cell cultures (Itonaga et al. 1999). In 2005, Take et al. demonstrated that, unlike mouse macrophage cultures, PGE2 strongly inhibits RANKL-induced osteoclast formation in human CD14+ cell cultures (Take et al. 2005). In addition, they showed that human osteoclast progenitors produce a soluble unidentified factor(s) in response to PGE2 that strongly inhibits RANKL-induced osteoclast formation not only in human CD14+ cell cultures but also in mouse macrophage cultures. They tried to identify the soluble factors, and concluded that CD14+ cells produce an inhibitor(s) that does not correspond to known inhibitory factors, such as GM-CSF, IFN-γ, and IL-4. Thus, these reports demonstrated the possibility that PGE2 differently plays a direct role in osteoclastogenesis from monocytes

Thus, in the study of human diseases, it is essential to investigate human osteoclastogenesis using human cells; the differences in species used in studies are critical to discuss the function of cytokines. We suggest that the term 'human osteoclastology' be used to describe

In 1995, Aplan et al. cloned and characterized a novel gene at the site of a t(1;3)(p34;p21) translocation breakpoint in T-cell acute lymphoblastic leukemia, designating this gene as TCTA (Aplan et al. 1995). TCTA mRNA is expressed ubiquitously in normal tissues, with the highest levels of expression in the kidney. TCTA has been conserved throughout evolution in organisms ranging from *Drosophila* to humans. A short open reading frame encodes a protein of 103 amino acid residues, Mr 11,300, without strong homology to any previously reported proteins. Of note, genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene (Aplan et al. 1995). On the other hand, in 2005, it has been reported that TCTA interacts with SMA- and MAD- related protein 4 (SMAD4) in a proteome-scale map of the human protein-protein interaction network (Supplementary Table S2, line 6175) (Rual et al. 2005); however, the function of TCTA has not been

Synovial tissues of patients with RA include factors regulating bone resorption by expressing cytokines such as RANKL, TNF, IL-6, IL-17, and IFN as described in Introduction. In addition to these cytokines, however, other factors expressed in synovial tissues may play a role in resorbing bone. We hypothesized that a novel factor in synovial tissues of patients with RA regulates osteoclastogenesis. To test this hypothesis, we tried

the term "Th17/Th1" cells has been proposed (Annunziato et al. 2009).

alone in the absence of osteoblastic cells between humans and mice.

studies on human osteoclastogenesis (Kotake 2009a, Kotake 2010).

**4. T-cell leukemia translocation-associated gene (TCTA)** 

**5. Role of TCTA protein in human osteoclastogenesis** 

clarified.

**5.1 Hypothesis** 

to identify novel peptides or proteins expressed in synovial tissues of patients with RA that regulate human osteoclastogenesis since 1996. We purified proteins from synovial tissues of patients with RA, using gel filtration chromatography, reverse-aspect HPLC, and mass spectrometry. We finally demonstrated that a peptide derived from the extracellular domain of TCTA protein inhibited both RANKL-induced human osteoclastogenesis and pit formation of mature human osteoclasts (Kotake et al. 2009b). We would like to present our findings as follows.

#### **5.2 Purification of proteins from synovial tissues of patients with RA**

Sixty-five grams of synovial tissues were obtained from 5 patients with RA at total knee replacement. The crude extract was obtained by homogenization of the synovial tissues. NH3Ac buffer was added to the freeze-dried crude extract. The supernatant was then applied to the column equilibrated with NH3Ac buffer and divided into two fractions, low molecular weight (MW) and high MW by first gel filtration chromatography. The low MW fraction was further applied to the column equilibrated with PBS by the second gel filtration chromatography. Proteins were eluted and protein concentration (A280) was monitored by UV absorption. Each fraction was added to the culture for human osteoclastogenesis to evaluate the activity on the osteoclastogenesis. Two fractions showed the inhibitory activity of osteoclastogenesis. These fractions were then subjected to ion-exchange chromatography. The "flow through" from the column showed the inhibitory activity on the osteoclastogenesis. The "flow through" was then applied to second gel filtration chromatography. Ten fractions from gel filtration chromatography showed the inhibitory activity on osteoclastogenesis. Thus, these fractions were applied to reverse-aspect HPLC. Thirty fractions were obtained by the reverse-aspect HPLC.

After each fraction obtained by reverse-aspect HPLC was concentrated, amino acid sequences were determined using a protein sequencer. Amino acid sequences (3-5 mers) were determined in 8 fractions. In addition, we tried to determine the sequence of each fraction by mass spectrometry; however, the sequences were not determined, although the total molecular weight of each peptide was speculated to be less than 1000 Da. Thus, we synthesized 8 peptides according to the sequences determined using the protein sequencer and evaluated the effect of each peptide on human osteoclastogenesis in vitro.

#### **5.3 A small peptide including GQN inhibits human osteoclastogenesis**

We finally revealed that the amino acid sequence of **Glycine-Glutamine-Asparagine (Gly-Gln-Asn; GQN)** alone in 8 synthesized peptides showed inhibitory activity in human osteoclastogenesis; **GQN** dose-dependently inhibited human osteoclastogenesis from peripheral monocytes by RANKL (IC50: around 30 M)(Kotake et al. 2009b).

When searching for proteins that include **GQN** using FASTA search, we found only 2 proteins in human proteins, CDW52 antigen (CAMPATH-1 antigen) and TCTA. In the FASTA search, it was possible to identify 3 mer peptides, but we did not find any other human proteins. Using "PeptideCutter", by which it is possible to predict the site cleaved by enzymes in peptides, we synthesized 4 peptides from a sequence of CDW52 antigen and one peptide from a sequence of TCTA protein, which included **GQN**. A peptide from TCTA protein showed inhibitory activity on human osteoclastogenesis, but not peptides from CDW52 antigens.

The Role of T-Cell Leukemia

**days, but not 24 h** 

not 24 h (Kotake et al. 2009b).

**human osteoclasts** 

(Kotake et al. 2009b).

CSF (Kotake et al. 2009b).

**macrophages** 

scrambled peptide as a control (Kotake et al. 2009b).

**monocytes and fusion of mature osteoclasts** 

Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis 215

osteoclasts. Peptide A appeared to weakly inhibit the expression of protein of NFATc1 in osteoclasts in immunohistological staining for NFATc1. Peptide A (0.8 – 3.2 M) also weakly, but not significantly, inhibited the expression of mRNA of NFATc1 in osteoclasts. In addition, peptide A did not inhibit the expression of mRNA of cathepsin K using the

**5.6 Peptide A inhibits pit formation of mature human osteoclasts in the culture of 3** 

We then examined the effect of peptide A on pit formation of mature human osteoclasts on OsteologicR. Mature osteoclasts formed from monocytes in culture with RANKL and M-CSF for 14 days were removed from plates. The mature osteoclasts then cultured on OsteologicR discs with peptide A or a scrambled peptide in the presence of RANKL and M-CSF for 24 h or 3 days. Peptide A as well as a scrambled peptide did not inhibit pit formation for 24 h. However, peptide A significantly inhibited pit formation in the culture of 3 days, although a scrambled peptide did not inhibit pit formation at all. Thus, peptide A showed inhibitory activity on mature human osteoclasts in the culture of 3 days, but

**5.7 Peptide A suppresses the formation of large osteoclasts in the culture of mature** 

We then investigated the effects of peptide A on mature osteoclasts cultured with sRANKL and M-CSF for 3 days. In the control well with sRANKL alone in the absence of peptide A, larger osteoclasts were formed than osteoclasts cultured with sRANKL and peptide A. Huge osteoclasts were detected in the control well alone, but not in the well cultured with peptide A. Osteoclasts in the well cultured with peptide A showed usual size. The number of huge osteoclasts in the control well cultured without peptide A was significantly higher than in the well cultured with peptide A. Thus, we hypothesized that TCTA plays a role in the fusion of monocytes/macrophages, preosteoclasts and osteoclasts (Kotake et al. 2009b).

**5.8 Polyclonal antibodies against TCTA inhibit both human osteoclastogenesis from** 

We constructed indirect competitive enzyme-linked immunosorbent assay (ELISA) using 2 polyclonal antibodies against TCTA protein. Standard curves showed specificity and high affinity of the antibodies against TCTA protein. To investigate our hypothesis further, we cultured monocytes with M-CSF and sRANKL in the presence of the polyclonal antibody, #1 or #2, both of which significantly inhibited human osteoclastogenesis, reducing the size of osteoclasts. In addition, polyclonal antibody #2 inhibited the fusion of mature osteoclasts

**5.9 TCTA protein is immunohistologically detected on human osteoclasts and** 

Using antibody #1, TCTA protein was immunohistologically detected on human osteoclasts induced by sRANKL and M-CSF. TCTA protein was detected in the central area of cells or in the peripheral area of cells. TCTA protein stained in the peripheral area of cells was observed in both a pre-osteoclast with 2 nuclei and an osteoclast with 4 nuclei, using fluorescent microscopy. TCTA protein also detected human macrophages cultured with M-

**5.4 Peptide A from TCTA strongly inhibits human, but not mouse, osteoclastogenesis**  We then synthesized another 2 peptides, **GQN**GSTPDGSTHFPSWEMAANEPLKTHRE and **GQN**GSTPDGSTHFPSWEMAAN, using 'PeptideCutter', shown as "peptide A" (MW 3182.4) and "peptide A2", respectively, from a sequence of TCTA gene protein (Fig. 3). Osteoclastogenesis was more potently inhibited by these peptides than by **GQN** or **GQN**GST. Peptide A most strongly inhibited osteoclastogenesis; IC50 level was around 1.6 M. Osteoclastogenesis induced by soluble RANKL (sRANKL) was not inhibited by adding 1.6 M of a scrambled peptide, SPFTGTKGSWNETAHPDHGNEERQAPMSL (MW, 3182.4), randomly designed from the sequence of peptide A. We finally synthesized 3 more peptides from a sequence of TCTA protein, also using "PeptideCutter": "peptide B": **GQN**GSTPDGSTHF, "peptide C": PGLG**GQN**GSTPDGSTHF, and "peptide D": GFYGNTVTGLYHRPGLG**GQN**GSTPDGSTHFPSWEMAANEPLKTHRE, which is the whole of the human extra-cellular domain (Fig. 3). Sequences of both peptide B and peptide C are included in a human/mouse identical sequence of TCTA protein (Fig. 3).


Structure of TCTA showing both intra-cellular (white bar) and extra-cellular (meshed bar) domains. Gray bar shows the human/mouse identical sequence in the extra-cellular domain.

Fig. 3. Structure of TCTA and sequences of peptides

Both peptide B and peptide C showed a weak inhibitory effect on human osteoclastogenesis from human peripheral monocytes. Peptide D did not show inhibitory activity. On the other hand, we also examined the effects of these peptides on mouse osteoclastogenesis; however, in contrast to our expectation, peptides B and C, which are included in the mouse sequence, did not inhibit ddY mouse osteoclastogenesis from mouse bone marrow cells stimulated by sRANKL and M-CSF. Peptides A and D did not show the inhibitory effects on mouse cells as we expected. Peptide A did not show cytotoxicity on human monocytes or cell proliferation on human monocytes (Kotake et al. 2009b).

#### **5.5 Peptide A failed to inhibit the expression of protein and mRNA of NFATc1, and mRNA of cathepsin K**

We then examined the effect of peptide A on the expression of both protein and mRNA of NFATc1, a key regulator of osteoclastogenesis under the signals of RANK-RANKL, in

**5.4 Peptide A from TCTA strongly inhibits human, but not mouse, osteoclastogenesis**  We then synthesized another 2 peptides, **GQN**GSTPDGSTHFPSWEMAANEPLKTHRE and **GQN**GSTPDGSTHFPSWEMAAN, using 'PeptideCutter', shown as "peptide A" (MW 3182.4) and "peptide A2", respectively, from a sequence of TCTA gene protein (Fig. 3). Osteoclastogenesis was more potently inhibited by these peptides than by **GQN** or **GQN**GST. Peptide A most strongly inhibited osteoclastogenesis; IC50 level was around 1.6 M. Osteoclastogenesis induced by soluble RANKL (sRANKL) was not inhibited by adding 1.6 M of a scrambled peptide, SPFTGTKGSWNETAHPDHGNEERQAPMSL (MW, 3182.4), randomly designed from the sequence of peptide A. We finally synthesized 3 more peptides from a sequence of TCTA protein, also using "PeptideCutter": "peptide B": **GQN**GSTPDGSTHF, "peptide C": PGLG**GQN**GSTPDGSTHF, and "peptide D": GFYGNTVTGLYHRPGLG**GQN**GSTPDGSTHFPSWEMAANEPLKTHRE, which is the whole of the human extra-cellular domain (Fig. 3). Sequences of both peptide B and peptide

C are included in a human/mouse identical sequence of TCTA protein (Fig. 3).

Structure of TCTA showing both intra-cellular (white bar) and extra-cellular (meshed bar) domains.

Both peptide B and peptide C showed a weak inhibitory effect on human osteoclastogenesis from human peripheral monocytes. Peptide D did not show inhibitory activity. On the other hand, we also examined the effects of these peptides on mouse osteoclastogenesis; however, in contrast to our expectation, peptides B and C, which are included in the mouse sequence, did not inhibit ddY mouse osteoclastogenesis from mouse bone marrow cells stimulated by sRANKL and M-CSF. Peptides A and D did not show the inhibitory effects on mouse cells as we expected. Peptide A did not show cytotoxicity on human monocytes or cell

**5.5 Peptide A failed to inhibit the expression of protein and mRNA of NFATc1, and** 

We then examined the effect of peptide A on the expression of both protein and mRNA of NFATc1, a key regulator of osteoclastogenesis under the signals of RANK-RANKL, in

Gray bar shows the human/mouse identical sequence in the extra-cellular domain.

Fig. 3. Structure of TCTA and sequences of peptides

proliferation on human monocytes (Kotake et al. 2009b).

**mRNA of cathepsin K** 

osteoclasts. Peptide A appeared to weakly inhibit the expression of protein of NFATc1 in osteoclasts in immunohistological staining for NFATc1. Peptide A (0.8 – 3.2 M) also weakly, but not significantly, inhibited the expression of mRNA of NFATc1 in osteoclasts. In addition, peptide A did not inhibit the expression of mRNA of cathepsin K using the scrambled peptide as a control (Kotake et al. 2009b).

#### **5.6 Peptide A inhibits pit formation of mature human osteoclasts in the culture of 3 days, but not 24 h**

We then examined the effect of peptide A on pit formation of mature human osteoclasts on OsteologicR. Mature osteoclasts formed from monocytes in culture with RANKL and M-CSF for 14 days were removed from plates. The mature osteoclasts then cultured on OsteologicR discs with peptide A or a scrambled peptide in the presence of RANKL and M-CSF for 24 h or 3 days. Peptide A as well as a scrambled peptide did not inhibit pit formation for 24 h. However, peptide A significantly inhibited pit formation in the culture of 3 days, although a scrambled peptide did not inhibit pit formation at all. Thus, peptide A showed inhibitory activity on mature human osteoclasts in the culture of 3 days, but not 24 h (Kotake et al. 2009b).

#### **5.7 Peptide A suppresses the formation of large osteoclasts in the culture of mature human osteoclasts**

We then investigated the effects of peptide A on mature osteoclasts cultured with sRANKL and M-CSF for 3 days. In the control well with sRANKL alone in the absence of peptide A, larger osteoclasts were formed than osteoclasts cultured with sRANKL and peptide A. Huge osteoclasts were detected in the control well alone, but not in the well cultured with peptide A. Osteoclasts in the well cultured with peptide A showed usual size. The number of huge osteoclasts in the control well cultured without peptide A was significantly higher than in the well cultured with peptide A. Thus, we hypothesized that TCTA plays a role in the fusion of monocytes/macrophages, preosteoclasts and osteoclasts (Kotake et al. 2009b).

#### **5.8 Polyclonal antibodies against TCTA inhibit both human osteoclastogenesis from monocytes and fusion of mature osteoclasts**

We constructed indirect competitive enzyme-linked immunosorbent assay (ELISA) using 2 polyclonal antibodies against TCTA protein. Standard curves showed specificity and high affinity of the antibodies against TCTA protein. To investigate our hypothesis further, we cultured monocytes with M-CSF and sRANKL in the presence of the polyclonal antibody, #1 or #2, both of which significantly inhibited human osteoclastogenesis, reducing the size of osteoclasts. In addition, polyclonal antibody #2 inhibited the fusion of mature osteoclasts (Kotake et al. 2009b).

#### **5.9 TCTA protein is immunohistologically detected on human osteoclasts and macrophages**

Using antibody #1, TCTA protein was immunohistologically detected on human osteoclasts induced by sRANKL and M-CSF. TCTA protein was detected in the central area of cells or in the peripheral area of cells. TCTA protein stained in the peripheral area of cells was observed in both a pre-osteoclast with 2 nuclei and an osteoclast with 4 nuclei, using fluorescent microscopy. TCTA protein also detected human macrophages cultured with M-CSF (Kotake et al. 2009b).

The Role of T-Cell Leukemia

as mentioned in section 3 in this article.

a putative counterpart of TCTA protein.

http://sosui.proteome.bio.tuat.ac.jp)

TCTA protein. (Structure of TCTA is derived from SOSUI;

Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis 217

important than the other molecules for fusion, such as, dendritic cell-specific transmembrane protein (DC-STAMP) (Kukita et al. 2004; Yagi et al. 2005), CD9 (Yi et al. 2006), CD47 (Lundberg et al. 2007; Yago et al. 2006), macrophage fusion protein (MFR)( Lundberg et al. 2007; Saginario et al. 1998; Yago et al. 2006), E-cadherin (Mbalaviele et al. 1995; Vignery 2000; Vignery 2005), meltrin- (ADAM12) (Abe et al. 1999), or CD44 (Suzuki et al. 2002). These findings also underline the difference of differentiation of osteoclasts between human and mice, supporting the importance of the term, 'Human osteoclastology',

TCTA protein plays an important role in cellular fusion in human osteoclastogenesis from monocytes and mature osteoclasts. Both peptide A and antibodies block the interaction between TCTA protein and

Fig. 4. Possible mechanism of human osteoclastogenesis by peptide A and antibodies against

In conclusion, we demonstrated that peptide A and polyclonal antibody against TCTA protein inhibited not only human osteoclastogenesis from monocytes but also the further maturation of mature human osteoclasts *in vitro* (Kotake et al. 2009b). Our findings suggest that TCTA protein plays an important role in cellular fusion in human osteoclastogenesis from monocytes and mature osteoclasts. Thus, peptide A may show the same inhibitory function *in vivo*, offering an effective therapeutic approach for inhibiting bone resorption.

#### **5.10 Other findings on Peptide A**

Peptide A as well as a scrambled peptide failed to disrupt the structure of actin rings of mature osteoclasts in the culture of 24h and 3 days. The amount of TCTA mRNA was significantly lower in human osteoclasts induced by sRANKL and M-CSF than in human macrophages cultured with M-CSF alone. Peptide A or the scrambled peptide, did not reduce the amount of TCTA mRNA in osteoclasts. TCTA mRNA was detected in human osteoclasts, monocytes, fibroblast-like synoviocytes, T cells, and PBMC by RT-PCR. TCTA protein was immunohistologically detected in cultured fibroblast-like synoviocytes using polyclonal anti-TCTA antibodies #1. TCTA protein was also immunohistologically detected in synovial tissues using polyclonal anti-TCTA antibodies #1. TCTA protein-positive cells were detected in synovial lining cells, but not in lymphoid folliculi with many lymphocytes. TCTA protein was significantly detected in human monocytes by flow cytometry using polyclonal anti-TCTA antibody #1 (Kotake et al. 2009b).

#### **6. A novel hypothesis**

We demonstrated that a novel peptide derived from the amino acid sequence of the extracellular domain of TCTA protein inhibited not only RANKL-induced human osteoclastogenesis from monocytes but also fusion of activated mature human osteoclasts (Kotake et al. 2009b). In the culture of mature osteoclasts, peptide A suppressed the formation of large mature osteoclasts after 3 days in the presence of sRANKL. In addition, although peptide A inhibited human osteoclastogenesis from monocytes with sRANKL, peptide A did not show significant inhibition of both protein and mRNA of NFATc1. The inhibitory effects of peptide A were not different among early, middle, and late phases of culture. In addition, the structure of actin rings of mature osteoclasts was not disrupted by peptide A. Thus, taken together, our findings suggest that the extracelluler domain of TCTA may play a role in the fusion of osteoclast precursors and mature osteoclasts, and that peptide A may block the interaction of TCTA and a putative counterpart of TCTA (Fig. 4). Supporting our putative mechanism, Saginario et al. reported that, because the soluble extracellular domain of macrophage fusion receptor (MFR) prevents the fusion of macrophages *in vitro*, MFR belongs to the fusion machinery of macrophages (Sarginario et al. 1998).

Several studies have reported that peptides inhibit osteoclastogenesis (Choi et al, 2001; Ikeda et al. 2004; Jimi et al. 2004). Jimi et al. reported that, using a cell-permeable peptide inhibitor of the I-B-kinase complex, the peptide inhibits RANKL-stimulated NF-B activation and osteoclastogenesis both *in vitro* and *in vivo*. They also showed that this peptide significantly reduces the severity of collagen-induced arthritis in mice by reducing levels of TNFand IL-1, abrogating joint swelling and reducing the destruction of bone and cartilage (Jimi et al. 2004). They used 20 M of peptides to inhibit RANKLinduced mice osteoclastogenesis, whereas our peptide A inhibited RANKL-induced human osteoclastogenesis at 1.6 M of IC50. Thus, our peptide more effectively inhibits osteoclastogenesis, although the different efficacy of the peptides may be derived from the different species used: humans and mice.

The reasons why peptides B and C, included in the mouse sequence, did not inhibit the osteoclastogenesis from mouse bone marrow cells remain unclear. We used only 3 peptides, peptide B, peptide C, and mouse "peptide A", in the culture of mouse cells. The other peptides, including GQN, may inhibit mouse osteoclastogenesis. On the other hand, even if TCTA protein plays a role in fusion as discussed above, TCTA protein in mice may be less important than the other molecules for fusion, such as, dendritic cell-specific transmembrane protein (DC-STAMP) (Kukita et al. 2004; Yagi et al. 2005), CD9 (Yi et al. 2006), CD47 (Lundberg et al. 2007; Yago et al. 2006), macrophage fusion protein (MFR)( Lundberg et al. 2007; Saginario et al. 1998; Yago et al. 2006), E-cadherin (Mbalaviele et al. 1995; Vignery 2000; Vignery 2005), meltrin- (ADAM12) (Abe et al. 1999), or CD44 (Suzuki et al. 2002). These findings also underline the difference of differentiation of osteoclasts between human and mice, supporting the importance of the term, 'Human osteoclastology', as mentioned in section 3 in this article.

216 T-Cell Leukemia

Peptide A as well as a scrambled peptide failed to disrupt the structure of actin rings of mature osteoclasts in the culture of 24h and 3 days. The amount of TCTA mRNA was significantly lower in human osteoclasts induced by sRANKL and M-CSF than in human macrophages cultured with M-CSF alone. Peptide A or the scrambled peptide, did not reduce the amount of TCTA mRNA in osteoclasts. TCTA mRNA was detected in human osteoclasts, monocytes, fibroblast-like synoviocytes, T cells, and PBMC by RT-PCR. TCTA protein was immunohistologically detected in cultured fibroblast-like synoviocytes using polyclonal anti-TCTA antibodies #1. TCTA protein was also immunohistologically detected in synovial tissues using polyclonal anti-TCTA antibodies #1. TCTA protein-positive cells were detected in synovial lining cells, but not in lymphoid folliculi with many lymphocytes. TCTA protein was significantly detected in human monocytes by flow cytometry using

We demonstrated that a novel peptide derived from the amino acid sequence of the extracellular domain of TCTA protein inhibited not only RANKL-induced human osteoclastogenesis from monocytes but also fusion of activated mature human osteoclasts (Kotake et al. 2009b). In the culture of mature osteoclasts, peptide A suppressed the formation of large mature osteoclasts after 3 days in the presence of sRANKL. In addition, although peptide A inhibited human osteoclastogenesis from monocytes with sRANKL, peptide A did not show significant inhibition of both protein and mRNA of NFATc1. The inhibitory effects of peptide A were not different among early, middle, and late phases of culture. In addition, the structure of actin rings of mature osteoclasts was not disrupted by peptide A. Thus, taken together, our findings suggest that the extracelluler domain of TCTA may play a role in the fusion of osteoclast precursors and mature osteoclasts, and that peptide A may block the interaction of TCTA and a putative counterpart of TCTA (Fig. 4). Supporting our putative mechanism, Saginario et al. reported that, because the soluble extracellular domain of macrophage fusion receptor (MFR) prevents the fusion of macrophages *in vitro*, MFR belongs

Several studies have reported that peptides inhibit osteoclastogenesis (Choi et al, 2001; Ikeda et al. 2004; Jimi et al. 2004). Jimi et al. reported that, using a cell-permeable peptide inhibitor of the I-B-kinase complex, the peptide inhibits RANKL-stimulated NF-B activation and osteoclastogenesis both *in vitro* and *in vivo*. They also showed that this peptide significantly reduces the severity of collagen-induced arthritis in mice by reducing levels of TNFand IL-1, abrogating joint swelling and reducing the destruction of bone and cartilage (Jimi et al. 2004). They used 20 M of peptides to inhibit RANKLinduced mice osteoclastogenesis, whereas our peptide A inhibited RANKL-induced human osteoclastogenesis at 1.6 M of IC50. Thus, our peptide more effectively inhibits osteoclastogenesis, although the different efficacy of the peptides may be derived from

The reasons why peptides B and C, included in the mouse sequence, did not inhibit the osteoclastogenesis from mouse bone marrow cells remain unclear. We used only 3 peptides, peptide B, peptide C, and mouse "peptide A", in the culture of mouse cells. The other peptides, including GQN, may inhibit mouse osteoclastogenesis. On the other hand, even if TCTA protein plays a role in fusion as discussed above, TCTA protein in mice may be less

**5.10 Other findings on Peptide A** 

**6. A novel hypothesis** 

polyclonal anti-TCTA antibody #1 (Kotake et al. 2009b).

to the fusion machinery of macrophages (Sarginario et al. 1998).

the different species used: humans and mice.

TCTA protein plays an important role in cellular fusion in human osteoclastogenesis from monocytes and mature osteoclasts. Both peptide A and antibodies block the interaction between TCTA protein and a putative counterpart of TCTA protein.

Fig. 4. Possible mechanism of human osteoclastogenesis by peptide A and antibodies against TCTA protein. (Structure of TCTA is derived from SOSUI; http://sosui.proteome.bio.tuat.ac.jp)

In conclusion, we demonstrated that peptide A and polyclonal antibody against TCTA protein inhibited not only human osteoclastogenesis from monocytes but also the further maturation of mature human osteoclasts *in vitro* (Kotake et al. 2009b). Our findings suggest that TCTA protein plays an important role in cellular fusion in human osteoclastogenesis from monocytes and mature osteoclasts. Thus, peptide A may show the same inhibitory function *in vivo*, offering an effective therapeutic approach for inhibiting bone resorption.

The Role of T-Cell Leukemia

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Translocation-Associated Gene (TCTA) Protein in Human Osteoclastogenesis 219

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osteoclastogenesis. *J Clin Invest* Vol.103:1345–1352.

R.B., Valverde, P., Dibart, S., Li, Y.P., Miranda, L.A., Ernst, C.W., Izumi, Y., & Taubman, M.A. (2006). B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. *Am J Pathol* Vol.169:987-998. Kobayashi, K., Takahashi, N., Jimi, E., Udagawa, N., Takami, M., Kotake, S., Nakagawa, N.,

Kinosaki, M., Yamaguchi, K., Shima, N., Yasuda, H., Morinaga, T., Higashio, K., Martin, T.J., & Suda, T. (2000). Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK

R., McCabe, S., Wong, T., Campagnuolo, G., Moran, E., Bogoch, E.R., Van, G., Nguyen, L.T., Ohashi, P.S., Lacey, D.L., Fish, E., Boyle, W.J., & Penninger, J.M. (1999). Activated T cells regulate bone loss and joint destruction in adjuvant

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Gourley, M.F., Klippel, J.H., & Wilder, R.L. (1997). In vivo gene expression of type 1 and type 2 cytokines in synovial tissues from patients in early stages of rheumatoid, reactive, and undifferentiated arthritis. *Proc Assoc Am Physicians*, Vol.109:286–301. Kotake, S., Schumacher, H.R. Jr., Arayssi, T.K., Gerard, H.C., Branigan, P.J., Hudson, A.P.,

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**12** 

*Japan* 

**Retrovirus Infection and Retinoid** 

*1Department of Hematology, National Hospital Organization* 

*2Department of Hematology, Kinki University School of Medicine* 

*Osaka Minami Medical Center* 

Yasuhiro Maeda1, Masaya Kawauchi1,2, Chikara Hirase2,

Terufumi Yamaguchi2, Jun-ichi Miyatake1,2 and Itaru Matsumura2

Human T cell leukemia virus type I (HTLV-I) is a human retrovirus that is an etiologic agent of adult T cell leukemia/lymphoma (ATL/ATLL) ( Hinuma et al., 1981, Uchiyama et al., 1977). Adult ATL/ATLL is an aggressive lymphoid neoplasm associated with human T-cell leukemia virus type 1 (HTLV-1) (Hinuma et al, 1982). ATL, the first human disease found to be associated with retroviral infection, usually occurs in native individuals from HTLV-1 endemic regions, i.e. southern Japan, the Caribbean, intertropical Africa, and Brazil (Kaplan et al., 1993, Gessain 1996). The HTLV-1 provirus is clonally integrated in CD4+, CD25+ activated T lymphocytes, which are leukemic cells characteristic of ATL. The exact mechanism of HTLV-1-induced tumorigenesis has not been fully elucidated, although HTLV-1 infection appears to represent the first event in a multi-step oncogenic process. (Franchini 1995). Diversity in the clinical features of ATL has been noted and four clinical subtypes of ATL have been defined: the acute form, the chronic form, the smoldering form, and the ATL lymphoma type (Shimoyama 1991). The acute and lymphoma types of ATL have a poor prognosis with a median survival of about six months (Shimoyama 1991). This extremely bad outcome is mainly due to an intrinsic resistance of the leukemic cells to conventional or even high doses of chemotherapy and to a severe immuno-suppression (Hermine et al., 1998, Bozarbachi & Hermine, 2001) reported, but a high toxicity and transplant-related mortality were observed in immuno-compromised patients (Borg et al., 1996, Ljungman et al., 1994, Sobue et al., 1987, Rio et al., 1980). A more effective therapy is therefore needed. Vitamin A and its analogs (retinoid) influence the growth and differentiation of normal and malignant cells, and have been shown to possess anticarcinogenic and antitumor activities in vitro and in vivo (Lotan 1980, Smith et al., 1992). Retinoic acid (RA) influences the clonal growth of normal human myeloid cells and induces the differentiation of both HL-60 cells (classified as a celll from a myeloblastic leukemia) and fresh human acute promyelocytic leukemia cells into normal granulocytes (Tobler et al., 1986, Breitman et al., 1980, Koeffler 1983). It has been reported that RA inhibits the growth of some tumor cells (Lotan 1979, Marth et al., 1986, Jetten et al., 1998)). Tax is a specific gene of ATL that immortalizes human T-cells (Tanaka et al., 1990). Tax, a 40 kD protein, is a transcription trans-activator of HTLV-1 that interacts with cellular transcriptional factors to activate HTLV-1 gene expression and HTLV-1 transformation of human T lymphocytes

**1. Introduction** 


### **Retrovirus Infection and Retinoid**

Yasuhiro Maeda1, Masaya Kawauchi1,2, Chikara Hirase2, Terufumi Yamaguchi2, Jun-ichi Miyatake1,2 and Itaru Matsumura2 *1Department of Hematology, National Hospital Organization Osaka Minami Medical Center 2Department of Hematology, Kinki University School of Medicine Japan* 

#### **1. Introduction**

222 T-Cell Leukemia

Udagawa, N., Kotake, S., Kamatani, N., Takahashi, N., & Suda, T. (2002). The molecular mechanism of osteoclastogenesis in rheumatoid arthritis. *Arthritis Res* Vol.4:281–289. Yagi, M., Miyamoto, T., Sawatani, Y., Iwamoto, K., Hosogane, N., Fujita, N., Morita, K.,

Yago, T., Nanke, Y., Kawamoto, M., Furuya, T., Kobashigawa, T., Kamatani, N., Kotake, S.

Yago, T., Nanke, Y., Kawamoto, M., Furuya, T., Kobashigawa, T., Kamatani, N., Kotake, S.

giant cells. *J Exp Med* Vol.202:345-351.

*Int J Exp Pathol* Vol.81:291-304.

Vol.202:337-340.

*Arthritis Rheum* Vol.50 (supple):1394, S354 (abst).

activity. *Biochem Biophys Res Commun* Vol.347:178-184.

Ninomiya, K., Suzuki, T., Miyamoto, K., Oike, Y., Takeya, M., Toyama, Y., & Suda, T. (2005). DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body

(2006). Antibodies against CD47 expressed on monocytes in rheumatoid arthritis patients inhibits human osteoclastogenesis by blocking fusion of monocytes.

(2007). IL-23 induces human osteoclastogenesis via IL-17 in vitro, and anti-IL-23 antibody attenuates collagen-induced arthritis in rats. *Arthritis Res Ther* Vol.9:R96. Yago, T., Nanke, Y., Ichikawa, N., Kobashigawa, T., Mogi, M., Kamatani, N., & Kotake, S.

(2009). IL-17 induces osteoclastogenesis from human monocytes alone in the absence of osteoblasts, which is potently inhibited by anti-TNF-alpha antibody: a novel mechanism of osteoclastogenesis by IL-17. *J Cell Biochem* Vol.108:947–955. Yi, T., Kim, H.J., Cho, J.Y., Woo, K.M., Ryoo, H.M., Kim, G.S., & Baek, J.H. (2006).

Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK

Vignery A. (2000). Osteoclasts and giant cells: macrophage-macrophage fusion mechanism.

Vignery, A. (2005). Macrophage fusion: the making of osteoclasts and giant cells. *J Exp Med*

Human T cell leukemia virus type I (HTLV-I) is a human retrovirus that is an etiologic agent of adult T cell leukemia/lymphoma (ATL/ATLL) ( Hinuma et al., 1981, Uchiyama et al., 1977). Adult ATL/ATLL is an aggressive lymphoid neoplasm associated with human T-cell leukemia virus type 1 (HTLV-1) (Hinuma et al, 1982). ATL, the first human disease found to be associated with retroviral infection, usually occurs in native individuals from HTLV-1 endemic regions, i.e. southern Japan, the Caribbean, intertropical Africa, and Brazil (Kaplan et al., 1993, Gessain 1996). The HTLV-1 provirus is clonally integrated in CD4+, CD25+ activated T lymphocytes, which are leukemic cells characteristic of ATL. The exact mechanism of HTLV-1-induced tumorigenesis has not been fully elucidated, although HTLV-1 infection appears to represent the first event in a multi-step oncogenic process. (Franchini 1995). Diversity in the clinical features of ATL has been noted and four clinical subtypes of ATL have been defined: the acute form, the chronic form, the smoldering form, and the ATL lymphoma type (Shimoyama 1991). The acute and lymphoma types of ATL have a poor prognosis with a median survival of about six months (Shimoyama 1991). This extremely bad outcome is mainly due to an intrinsic resistance of the leukemic cells to conventional or even high doses of chemotherapy and to a severe immuno-suppression (Hermine et al., 1998, Bozarbachi & Hermine, 2001) reported, but a high toxicity and transplant-related mortality were observed in immuno-compromised patients (Borg et al., 1996, Ljungman et al., 1994, Sobue et al., 1987, Rio et al., 1980). A more effective therapy is therefore needed. Vitamin A and its analogs (retinoid) influence the growth and differentiation of normal and malignant cells, and have been shown to possess anticarcinogenic and antitumor activities in vitro and in vivo (Lotan 1980, Smith et al., 1992). Retinoic acid (RA) influences the clonal growth of normal human myeloid cells and induces the differentiation of both HL-60 cells (classified as a celll from a myeloblastic leukemia) and fresh human acute promyelocytic leukemia cells into normal granulocytes (Tobler et al., 1986, Breitman et al., 1980, Koeffler 1983). It has been reported that RA inhibits the growth of some tumor cells (Lotan 1979, Marth et al., 1986, Jetten et al., 1998)). Tax is a specific gene of ATL that immortalizes human T-cells (Tanaka et al., 1990). Tax, a 40 kD protein, is a transcription trans-activator of HTLV-1 that interacts with cellular transcriptional factors to activate HTLV-1 gene expression and HTLV-1 transformation of human T lymphocytes

Retrovirus Infection and Retinoid 225

(Maeda et al., 1996). Thus, there is a possibility that specific target cells of RA may be ATL

We next investigated NFtranscription activity by CAT assay with pCD12-CAT. Spontaneous enhancement of CAT activity for NFwas detected. CAT activity determined with percent conversion was decreased after treatment with ATRA (% conversion: 60.8% to 21.0%). These results suggested that growth inhibition and CD25 down-regulation by ATRA occurred via the NFB signaling pathway (Nawata et al., 2001).Further, we demonstrated typical apoptosis on PBMCs obtained from ATL patients after treatment with ATRA for 48 hrs (Maeda et al., 1996). CAT-measured NFB activity was also significantly decreased on these PBMCs after treatment with ATRA for 24 hrs (Nawata et al., 2001). It has been reported that NF is activated by Tax protein, which induces the degradation of I-, which molecule is known to contribute to constitutive activation of NFin ATL cells for cytokine gene, receptor gene and cell proliferation. We carried out a CAT assay for NF using pCD12-CAT on ATL-2 cells in the presence or absence of ATRA (Nawata et al., 2001). Enhanced CAT activity determined with percent conversion was detected on ATL-2 cells (% conversion: 60.8%). It has been reported that Tax-mediated increases in NFnuclear translocation result from direct interaction of Tax and MEKK1, leading to enhanced Ikkphosphorylation of IkB (Yim et al., 1998, Mori et al., 1992). Furthermore, Arima et al. reported that Tax is capable of inducing nuclear expression of all four Fspecies (p50, p55, p75 and p85) in primary ATL cells of acute type patients (Arima et al., 1999), and inhibition of apoptosis has been reported to be essential for activation of NF Our results possible indicate that the enhanced CAT activity for NFmay reveal that NFB protects against apoptosis. After treatment with ATRA, NFB activity decreased significantly (% conversion: 21.0%) on ATL-2 cells. Furthermore, we also transfected the *tax* gene in the expression vector (pCMV-Tax-neo) into the HTLV-I negative T cell line Jurkat (Nawata et al., 2001), and examined the effects of ATRA on cell growth. Interestingly, ATRA inhibited the growth of these transient transformants, but had no effect on the growth of control cells transformed with neomycin-resistance gene alone (Nawata et al., 2001). Taken together, these results indicate that the difference in the sensitivity to ATRA may be dependent on the expression of Tax. However, Mori et al. have reported that NFconstitutively activates in primary ATL cells as well as HTLV- Ⅰpositive T cell line TL-Om1 independent of Tax protein (Mori et al., 1992). In summary, we have shown that ATRA could inhibit growth of the ATL cells and induce their apoptosis with suppressed NF-B transcriptional activity. These results suggest that the target molecule of ATRA may be Tax or some molecule in the Tax-NF-B signaling pathway, and that the existence of Tax would thus enhance the sensitivity to ATRA. Further study will be needed to determine whether ATRA exert its effects directly, or via some intermediary factor. Plans to administer ATRA to ATL patients in a clinical setting were currently

cells in peripheral blood.

**2.1 Inhibition of NF-B transcription activity** 

undertaken in our laboratory (Maeda et al., 2000, 2004, 2008).

In ATL, ADF that is homologous to thioredoxin (TRX) (Tagaya et al., 1989) have been reported to be not only a CD25 inducer, but also an active reducing molecule for active oxygen species. It was reported that the activity of thioredoxin reductase (TRX-R) from

**3. Effects of thiol compounds** 

(Tanaka et al., 1990, Feuer & Chen 1992). Tax activates HTLV-1 gene expression by increasing the binding of the cyclic AMP-responsive element-binding protein/activating transcription factor (CREB/ATF) proteins and the coactivator CBP (CREB binding protein) to the three 21-bp repeats in the long terminal repeat of HTLV-1 (Zhao & Giam 1991, Kwoak et al, 1996), and also activates immediate early genes (c-fos, c-jun, egr-1, and egr-2), a receptor gene (IL-2Rα), and cytokine genes (IL-2, IL-6, TGF-β, GM-CSF) (Tanaka et al., 1990, Feuer & Chen 1992). Furthermore, tax interacts with the ankyrin motifs in I-κB and NF-κB p105 and dissociates from or interferes with the complex I-κB/NF-κB, which is involved in the transcriptional activation of NF-κB in the cytoplasm (Hirai et al., 1994). It has also been shown that NF-κB was transported into nuclei and activated to induce the expression of cytokine and receptor genes (Feuer & Chen 1992, Baeuerle 1991). Inhibition of NF-kB activity is related for induction of apoptosis, and thus the Rel/NF-κB family plays important roles in the proliferation and differentiation of various cells in vitro. Already, Mori et al. have reported that NF-κB is constitutively activated in primary ATL cells as well as in the HTLV-1-positive T-cell line TL-Om1 independent of Tax protein (Mori et al., 1999). Furthermore, we have suggested that the target molecule of all-*trans* retinoic acid (ATRA) may be tax or some molecule in the tax- NF-κB signal pathway (Nawata et al., 2001). At the present time, the mechanism of ATRA's effect in ATL cells is not clear. In this article, we showed effects of ATRA in the aspect of 1) growth inhibition and CD25 down-regulation, 2) inhibition of NF-�B transcription, 3) effects of thiol compound, 4) effects for skin involvement, 5) mechanism of ATRA action, 6) clinical application, 7) effects for HIV infection.

#### **2. Growth inhibition and down-regulation IL-2RCD25 by ATRA**

We initially assessed the effect of ATRA to HTLV-I positive T cell lines, HUT102 and ATL-2 cells. When those cells were treated with ATRA, cell proliferation was decreased significantly (Miyatake & Maeda, 1997). To assess the effect of ATRA to the cell surface antigen, we observed the expression of IL-2RCD25 by flow cytometry. Incubation of those HTLV-I positive T-cell lines for 48hrs with 10-5 M ATRA for 48 h also resulted in downregulation of CD25 expression (Miyatake & Maeda, 1997). Two peaks were apparent on FACS analysis of those cells, treated with ATRA, suggesting the existence of sensitive and resistant clones to ATRA. HTLVI negative cell lines, Jurkat and MOLT-4, were incubated with ATRA for 48hrs and assayed for cell proliferation. However, no growth inhibition was observed on both T cell lines (Miyatake & Maeda, 1997). The mechanism responsible for the difference in sensitivity of HUT102 cell clones to RA with regard to down-regulation of CD25 is not clear. However, this difference may be attributable to: (i) Differences in the expression of retinoic acid receptors (RARs) (Petkovich et al., 1987, Giguere et al., 1987), or retinoid X receptors (RXRs) (Heyman et al., 1992, Zhang et al., 1992). These receptors expression may be associated with the sensitivity to RA. (ii) Differences in the expression of cytosolic retinoic acid binding proteins (CRABPs), which binds RA before its transfer to the nucleus and acts as an intracellular antagonist of RA action (Maden et al., 1981, Eller et al., 1992, Siegenthaler et al., 1992, Wei et al., 1989). The extent of CRABP expression would be expected to correlate with RA resistance. And (iii) differences in the expression of antioxidant including ATL-derived factor (ADF). Indeed, our study showed that incubation with ATRA for 48hrs resulted in inhibition of growth for PBMCs and in induction of apoptosis from some patients with ATL, but not for PBMCs from normal individuals

(Tanaka et al., 1990, Feuer & Chen 1992). Tax activates HTLV-1 gene expression by increasing the binding of the cyclic AMP-responsive element-binding protein/activating transcription factor (CREB/ATF) proteins and the coactivator CBP (CREB binding protein) to the three 21-bp repeats in the long terminal repeat of HTLV-1 (Zhao & Giam 1991, Kwoak et al, 1996), and also activates immediate early genes (c-fos, c-jun, egr-1, and egr-2), a receptor gene (IL-2Rα), and cytokine genes (IL-2, IL-6, TGF-β, GM-CSF) (Tanaka et al., 1990, Feuer & Chen 1992). Furthermore, tax interacts with the ankyrin motifs in I-κB and NF-κB p105 and dissociates from or interferes with the complex I-κB/NF-κB, which is involved in the transcriptional activation of NF-κB in the cytoplasm (Hirai et al., 1994). It has also been shown that NF-κB was transported into nuclei and activated to induce the expression of cytokine and receptor genes (Feuer & Chen 1992, Baeuerle 1991). Inhibition of NF-kB activity is related for induction of apoptosis, and thus the Rel/NF-κB family plays important roles in the proliferation and differentiation of various cells in vitro. Already, Mori et al. have reported that NF-κB is constitutively activated in primary ATL cells as well as in the HTLV-1-positive T-cell line TL-Om1 independent of Tax protein (Mori et al., 1999). Furthermore, we have suggested that the target molecule of all-*trans* retinoic acid (ATRA) may be tax or some molecule in the tax- NF-κB signal pathway (Nawata et al., 2001). At the present time, the mechanism of ATRA's effect in ATL cells is not clear. In this article, we showed effects of ATRA in the aspect of 1) growth inhibition and CD25 down-regulation, 2) inhibition of NF-�B transcription, 3) effects of thiol compound, 4) effects for skin involvement, 5) mechanism of ATRA action, 6) clinical application, 7) effects for HIV

**2. Growth inhibition and down-regulation IL-2RCD25 by ATRA** 

We initially assessed the effect of ATRA to HTLV-I positive T cell lines, HUT102 and ATL-2 cells. When those cells were treated with ATRA, cell proliferation was decreased significantly (Miyatake & Maeda, 1997). To assess the effect of ATRA to the cell surface antigen, we observed the expression of IL-2RCD25 by flow cytometry. Incubation of those HTLV-I positive T-cell lines for 48hrs with 10-5 M ATRA for 48 h also resulted in downregulation of CD25 expression (Miyatake & Maeda, 1997). Two peaks were apparent on FACS analysis of those cells, treated with ATRA, suggesting the existence of sensitive and resistant clones to ATRA. HTLVI negative cell lines, Jurkat and MOLT-4, were incubated with ATRA for 48hrs and assayed for cell proliferation. However, no growth inhibition was observed on both T cell lines (Miyatake & Maeda, 1997). The mechanism responsible for the difference in sensitivity of HUT102 cell clones to RA with regard to down-regulation of CD25 is not clear. However, this difference may be attributable to: (i) Differences in the expression of retinoic acid receptors (RARs) (Petkovich et al., 1987, Giguere et al., 1987), or retinoid X receptors (RXRs) (Heyman et al., 1992, Zhang et al., 1992). These receptors expression may be associated with the sensitivity to RA. (ii) Differences in the expression of cytosolic retinoic acid binding proteins (CRABPs), which binds RA before its transfer to the nucleus and acts as an intracellular antagonist of RA action (Maden et al., 1981, Eller et al., 1992, Siegenthaler et al., 1992, Wei et al., 1989). The extent of CRABP expression would be expected to correlate with RA resistance. And (iii) differences in the expression of antioxidant including ATL-derived factor (ADF). Indeed, our study showed that incubation with ATRA for 48hrs resulted in inhibition of growth for PBMCs and in induction of apoptosis from some patients with ATL, but not for PBMCs from normal individuals

infection.

(Maeda et al., 1996). Thus, there is a possibility that specific target cells of RA may be ATL cells in peripheral blood.

#### **2.1 Inhibition of NF-B transcription activity**

We next investigated NFtranscription activity by CAT assay with pCD12-CAT. Spontaneous enhancement of CAT activity for NFwas detected. CAT activity determined with percent conversion was decreased after treatment with ATRA (% conversion: 60.8% to 21.0%). These results suggested that growth inhibition and CD25 down-regulation by ATRA occurred via the NFB signaling pathway (Nawata et al., 2001).Further, we demonstrated typical apoptosis on PBMCs obtained from ATL patients after treatment with ATRA for 48 hrs (Maeda et al., 1996). CAT-measured NFB activity was also significantly decreased on these PBMCs after treatment with ATRA for 24 hrs (Nawata et al., 2001). It has been reported that NF is activated by Tax protein, which induces the degradation of I-, which molecule is known to contribute to constitutive activation of NFin ATL cells for cytokine gene, receptor gene and cell proliferation. We carried out a CAT assay for NF using pCD12-CAT on ATL-2 cells in the presence or absence of ATRA (Nawata et al., 2001). Enhanced CAT activity determined with percent conversion was detected on ATL-2 cells (% conversion: 60.8%). It has been reported that Tax-mediated increases in NFnuclear translocation result from direct interaction of Tax and MEKK1, leading to enhanced Ikkphosphorylation of IkB (Yim et al., 1998, Mori et al., 1992). Furthermore, Arima et al. reported that Tax is capable of inducing nuclear expression of all four Fspecies (p50, p55, p75 and p85) in primary ATL cells of acute type patients (Arima et al., 1999), and inhibition of apoptosis has been reported to be essential for activation of NF Our results possible indicate that the enhanced CAT activity for NFmay reveal that NFB protects against apoptosis. After treatment with ATRA, NFB activity decreased significantly (% conversion: 21.0%) on ATL-2 cells. Furthermore, we also transfected the *tax* gene in the expression vector (pCMV-Tax-neo) into the HTLV-I negative T cell line Jurkat (Nawata et al., 2001), and examined the effects of ATRA on cell growth. Interestingly, ATRA inhibited the growth of these transient transformants, but had no effect on the growth of control cells transformed with neomycin-resistance gene alone (Nawata et al., 2001). Taken together, these results indicate that the difference in the sensitivity to ATRA may be dependent on the expression of Tax. However, Mori et al. have reported that NFconstitutively activates in primary ATL cells as well as HTLV- Ⅰpositive T cell line TL-Om1 independent of Tax protein (Mori et al., 1992). In summary, we have shown that ATRA could inhibit growth of the ATL cells and induce their apoptosis with suppressed NF-B transcriptional activity. These results suggest that the target molecule of ATRA may be Tax or some molecule in the Tax-NF-B signaling pathway, and that the existence of Tax would thus enhance the sensitivity to ATRA. Further study will be needed to determine whether ATRA exert its effects directly, or via some intermediary factor. Plans to administer ATRA to ATL patients in a clinical setting were currently undertaken in our laboratory (Maeda et al., 2000, 2004, 2008).

#### **3. Effects of thiol compounds**

In ATL, ADF that is homologous to thioredoxin (TRX) (Tagaya et al., 1989) have been reported to be not only a CD25 inducer, but also an active reducing molecule for active oxygen species. It was reported that the activity of thioredoxin reductase (TRX-R) from

Retrovirus Infection and Retinoid 227

skin eruption with chronic ATL. After detection of proviral DNA in the skin by Southern blot analysis, ATRA (60 mg/day) was administered. The skin biopsy exhibited dense lymphoid infiltrates with atypical cytological features in the dermis. The infiltrate was composed mainly of medium to large cells with irregular nuclei. Neoplastic cells showed mild epidermotropism. There was a clinical and histological improvement after ATRA therapy was given for 4 weeks. Furthermore, proviral DNA for HTLV-I by Southern blot analysis in skin became to be negative after treatment with ATRA. These results indicated that ATRA may be a useful agent for skin involvement of ATL. Adverse effects were seen in 6 of 8 patients, these effects were temporally and generally mild (3 cases of headache, 2 cases of dry skin, 1 case of skin pigmentation). This confirms that as it has been reported ATRA only shows toxicities in a few cases. We had 2 cases that did not respond to ATRA, indicative of ATRA resistant cases. Differences between good responders and resistant cases should be investigated, including the mechanism of ATRA action for skin involvement.

At the present time, the mechanism of ATRA's effect in ATL cells is not clear. We observed two critical points; 1) whether ATRA suppresses HTLV-1 replication, and 2) whether ATRA decreases RT activity via a direct reaction. To confirm the anti-retroviral effect of ATRA, detection of HTLV-1 proviral DNA load using real time PCR was carried out in five HTLV-1-positive T-cell lines treated with VP-16, AZT, and ATRA for 48 and 72 hours. HTLV-1 proviral DNA load was only decreased by VP-16 in MT-2. HTLV-1 proviral DNA load was significantly suppressed by AZT in the HTLV-1-positive T-cell lines (ATL-2 and MT-2 at 48 hours, and ATL-2, MT-2, MT-4 and ED40515 at 72 hours) (Yamaguchi et al., 2005). Furthermore, HTLV-1 proviral DNA load was also significantly decreased by ATRA in HTLV-1-positive T-cell lines (all five HTLV-1-positive T-cell lines at 48 hours, and ATL-2, HUT102, MT-4 and ED40515 at 72 hours). These results suggested that ATRA might act as a RT inhibitor (Yamaguchi et al., 2005). Moreover, HTLV-1 tax mRNA load was significantly suppressed by ATRA (HUT102 and MT-2 at 48hours). As ATRA reduced HTLV-1 proviral DNA load, we observed whether it degrades the RT that participates in the cycle of retroviral replication (Yamaguchi et al., 2005). HTLV-1-positive T-cell lines (1×105/ml: total 20ml) were cultured with 10-5 M ATRA, 64 μM AZT or control reagent. Using the RT detection assay, we measured the RT activity of cell lysates. It was observed that ATRA significantly suppressed the activity in HTLV-1-positive T-cell lines (MT-4 and ED40515 at 48 hours, and HUT102, ED40515, MT-2 and MT-4 at 72 hours). In summary, we found that ATRA reduce HTLV-1 proviral DNA at mRNA level and RT activity of HTLV-1. These results suggest that the mechanism of ATRA's action may be dichotomized into inhibition of NF-κB transcriptional activity related to HTLV-1 and inhibition of RT (Yamaguchi et al., 2005). In another aspect on ATRA mechanism, we focused on the role of retinoids in inducing cellular senescence during the treatment of ATL (Maeda et al., 2011). Cellular senescence was detected by staining for senescence-associated -galactosidase (SA -Gal). SA Gal-positive cells were observed during the spontaneous culture without retinoids (ATRA or Am-80) in HTLV-I (+) T-cell lines (HUT102, MT-2, MT-4, ED40515, and ATL-2), but not in HTLV-I (-) T-cell lines (Jurkat and MOLT-4). On treatment with ATRA or Am-80, the number of SA -Gal-positive cells significantly increased in the HTLV-I (+) T-cell lines, but not in the HTLV-I (-) ones. P16INK4a expression was enhanced in all the HTLV-I (+) T-cell lines, but not in the HTLV-I (-) T-cell lines. A telomeric repeat amplification protocol (TRAP)

**5. Mechanism of ATRA action for ATL cells** 

melanoma tissue was inhibited remarkably by 13-*cis* RA (Shallreuter & Wood 1990). Cellular redox status modulates various aspects of cellular function when oxidative stress occurs. The balance of oxidative/anti-oxidative influences may play an important role in the modulation of cellular function. It has been reported that L-cysteine and L-cystine act as a buffer of the redox potential of the environment in cells or serum (Bannai 1984, Miura et al., 1992). To study the effects of exogenous thiol compounds on the sensitivity to retinoid in a HTLV-I (+) T cell line, ATL-2 cells (Maeda et al., 1985) were cultured with thiol compounds (10-5 M L-cystine, 10-4 M GSH and 1 g/ml TRX), following addition of ATRA or 13-*cis* RA. Significant growth inhibition was seen in ATL-2 cells when 10-5 M RA was added. Unexpectedly, similar growth inhibition of ATL-2 cells was shown with each thiol compound added to ATL-2 cells (Miyatake et al., 1998, 2000). These unexpected results may be explained by differences in uptake time into the cells between RA and thiol compounds. Next, we preincubated ATL-2 cells with each thiol compound (1 g/ml recombinant ADF, 1 g/ml TRX, 10-5 M L-cystine and 10-4 M GSH) for 24 hrs, and 10-5 M ATRA or 13-*cis* was added to ATL-2 cells in thiol-depleted medium. The reduction rate was decreased significantly by preincubation with the thiol compounds. Especially, preincubation of ATL-2 with L-cyctine or GSH resulted in complete restoration of growth despite the inhibitory effects of RA, this phenomenon suggested that it helped to increase the redox potential of the intracellular environment. Intracellular L-cystine is converted to L-cysteine, which is an active thiol compound that is utilized for GSH synthesis (Bannai 1984) and depletion of Lcystine results in a reduction of intracellular GSH content. These processes are antagonized by antioxidants such as cysteine and GSH (Miura et al., 1992). However, no restoration of growth was obtained in thiol-untreated ATL-2 cells. These reports suggested that Lcystine/GSH and ADF/TRX systems cooperate to support the adjustment of intracellular redox states against several oxidants and, thereby, promote the growth and viability of lymphocytes. Our results suggest that the imbalance of intracellular redox potential in HTLV-I (+) T cell lines may be associated strongly with the sensitivity to RA and exogenous thiol compounds may prepare the intracellular environment to become resistant to RA. In other words, cystine/GSH and ADF/TRX redox systems may act against RA, an antioxidant.

#### **4. Effects of skin involvement**

ATL is characterized by infiltration of various tissues by circulating ATL cells. Especially, skin lesions occur in 50% of ATL patients. We observed the effects of ATRA on skin involvement in ATL patients. Eight patients with ATL (2 cases acute type, 5 chronic type and 1 smoldering type) were selected (Maeda et al., 2004). Cutaneous lesions included erythematous plaques, papules, nodules, erythroderma, and tumors. Patients were scheduled to receive oral ATRA 45mg/m2 daily. During treatment with ATRA, there was no chemotherapy or glucocorticoid therapy administered. Patients were monitored for safety and anti-tumor effect by regular physical examination and laboratory studies including complete and differential blood count and standard chemistry performed at the baseline and repeated at weeks 1, 2, 3 and 4. Skin biopsy was carried out before and after treatment with ATRA Complete response required all skin eruptions coming macroscopically negative. ATRA was effective for skin involvement in 6 patients (Maeda et al., 2004). A typical case is shown below; Case: A 42-year-old Japanese woman was referred to our hospital because of

melanoma tissue was inhibited remarkably by 13-*cis* RA (Shallreuter & Wood 1990). Cellular redox status modulates various aspects of cellular function when oxidative stress occurs. The balance of oxidative/anti-oxidative influences may play an important role in the modulation of cellular function. It has been reported that L-cysteine and L-cystine act as a buffer of the redox potential of the environment in cells or serum (Bannai 1984, Miura et al., 1992). To study the effects of exogenous thiol compounds on the sensitivity to retinoid in a HTLV-I (+) T cell line, ATL-2 cells (Maeda et al., 1985) were cultured with thiol compounds (10-5 M L-cystine, 10-4 M GSH and 1 g/ml TRX), following addition of ATRA or 13-*cis* RA. Significant growth inhibition was seen in ATL-2 cells when 10-5 M RA was added. Unexpectedly, similar growth inhibition of ATL-2 cells was shown with each thiol compound added to ATL-2 cells (Miyatake et al., 1998, 2000). These unexpected results may be explained by differences in uptake time into the cells between RA and thiol compounds. Next, we preincubated ATL-2 cells with each thiol compound (1 g/ml recombinant ADF, 1 g/ml TRX, 10-5 M L-cystine and 10-4 M GSH) for 24 hrs, and 10-5 M ATRA or 13-*cis* was added to ATL-2 cells in thiol-depleted medium. The reduction rate was decreased significantly by preincubation with the thiol compounds. Especially, preincubation of ATL-2 with L-cyctine or GSH resulted in complete restoration of growth despite the inhibitory effects of RA, this phenomenon suggested that it helped to increase the redox potential of the intracellular environment. Intracellular L-cystine is converted to L-cysteine, which is an active thiol compound that is utilized for GSH synthesis (Bannai 1984) and depletion of Lcystine results in a reduction of intracellular GSH content. These processes are antagonized by antioxidants such as cysteine and GSH (Miura et al., 1992). However, no restoration of growth was obtained in thiol-untreated ATL-2 cells. These reports suggested that Lcystine/GSH and ADF/TRX systems cooperate to support the adjustment of intracellular redox states against several oxidants and, thereby, promote the growth and viability of lymphocytes. Our results suggest that the imbalance of intracellular redox potential in HTLV-I (+) T cell lines may be associated strongly with the sensitivity to RA and exogenous thiol compounds may prepare the intracellular environment to become resistant to RA. In other words, cystine/GSH and ADF/TRX redox systems may act against RA, an

ATL is characterized by infiltration of various tissues by circulating ATL cells. Especially, skin lesions occur in 50% of ATL patients. We observed the effects of ATRA on skin involvement in ATL patients. Eight patients with ATL (2 cases acute type, 5 chronic type and 1 smoldering type) were selected (Maeda et al., 2004). Cutaneous lesions included erythematous plaques, papules, nodules, erythroderma, and tumors. Patients were scheduled to receive oral ATRA 45mg/m2 daily. During treatment with ATRA, there was no chemotherapy or glucocorticoid therapy administered. Patients were monitored for safety and anti-tumor effect by regular physical examination and laboratory studies including complete and differential blood count and standard chemistry performed at the baseline and repeated at weeks 1, 2, 3 and 4. Skin biopsy was carried out before and after treatment with ATRA Complete response required all skin eruptions coming macroscopically negative. ATRA was effective for skin involvement in 6 patients (Maeda et al., 2004). A typical case is shown below; Case: A 42-year-old Japanese woman was referred to our hospital because of

antioxidant.

**4. Effects of skin involvement** 

skin eruption with chronic ATL. After detection of proviral DNA in the skin by Southern blot analysis, ATRA (60 mg/day) was administered. The skin biopsy exhibited dense lymphoid infiltrates with atypical cytological features in the dermis. The infiltrate was composed mainly of medium to large cells with irregular nuclei. Neoplastic cells showed mild epidermotropism. There was a clinical and histological improvement after ATRA therapy was given for 4 weeks. Furthermore, proviral DNA for HTLV-I by Southern blot analysis in skin became to be negative after treatment with ATRA. These results indicated that ATRA may be a useful agent for skin involvement of ATL. Adverse effects were seen in 6 of 8 patients, these effects were temporally and generally mild (3 cases of headache, 2 cases of dry skin, 1 case of skin pigmentation). This confirms that as it has been reported ATRA only shows toxicities in a few cases. We had 2 cases that did not respond to ATRA, indicative of ATRA resistant cases. Differences between good responders and resistant cases should be investigated, including the mechanism of ATRA action for skin involvement.

#### **5. Mechanism of ATRA action for ATL cells**

At the present time, the mechanism of ATRA's effect in ATL cells is not clear. We observed two critical points; 1) whether ATRA suppresses HTLV-1 replication, and 2) whether ATRA decreases RT activity via a direct reaction. To confirm the anti-retroviral effect of ATRA, detection of HTLV-1 proviral DNA load using real time PCR was carried out in five HTLV-1-positive T-cell lines treated with VP-16, AZT, and ATRA for 48 and 72 hours. HTLV-1 proviral DNA load was only decreased by VP-16 in MT-2. HTLV-1 proviral DNA load was significantly suppressed by AZT in the HTLV-1-positive T-cell lines (ATL-2 and MT-2 at 48 hours, and ATL-2, MT-2, MT-4 and ED40515 at 72 hours) (Yamaguchi et al., 2005). Furthermore, HTLV-1 proviral DNA load was also significantly decreased by ATRA in HTLV-1-positive T-cell lines (all five HTLV-1-positive T-cell lines at 48 hours, and ATL-2, HUT102, MT-4 and ED40515 at 72 hours). These results suggested that ATRA might act as a RT inhibitor (Yamaguchi et al., 2005). Moreover, HTLV-1 tax mRNA load was significantly suppressed by ATRA (HUT102 and MT-2 at 48hours). As ATRA reduced HTLV-1 proviral DNA load, we observed whether it degrades the RT that participates in the cycle of retroviral replication (Yamaguchi et al., 2005). HTLV-1-positive T-cell lines (1×105/ml: total 20ml) were cultured with 10-5 M ATRA, 64 μM AZT or control reagent. Using the RT detection assay, we measured the RT activity of cell lysates. It was observed that ATRA significantly suppressed the activity in HTLV-1-positive T-cell lines (MT-4 and ED40515 at 48 hours, and HUT102, ED40515, MT-2 and MT-4 at 72 hours). In summary, we found that ATRA reduce HTLV-1 proviral DNA at mRNA level and RT activity of HTLV-1. These results suggest that the mechanism of ATRA's action may be dichotomized into inhibition of NF-κB transcriptional activity related to HTLV-1 and inhibition of RT (Yamaguchi et al., 2005). In another aspect on ATRA mechanism, we focused on the role of retinoids in inducing cellular senescence during the treatment of ATL (Maeda et al., 2011). Cellular senescence was detected by staining for senescence-associated -galactosidase (SA -Gal). SA Gal-positive cells were observed during the spontaneous culture without retinoids (ATRA or Am-80) in HTLV-I (+) T-cell lines (HUT102, MT-2, MT-4, ED40515, and ATL-2), but not in HTLV-I (-) T-cell lines (Jurkat and MOLT-4). On treatment with ATRA or Am-80, the number of SA -Gal-positive cells significantly increased in the HTLV-I (+) T-cell lines, but not in the HTLV-I (-) ones. P16INK4a expression was enhanced in all the HTLV-I (+) T-cell lines, but not in the HTLV-I (-) T-cell lines. A telomeric repeat amplification protocol (TRAP)

Retrovirus Infection and Retinoid 229

model means multi-target therapy, and indicated that if one pathway is blocked by some factors, the other one will be available. Furthermore, we should recognize the differences between the clinical outcome and experimental results *in vitro*. We examined the differences in several clinical parameters (LDH, AL-P, sIL-2R, and age) between cases of NC and PR. However, no significant difference was observed (data not shown). Other intrinsic factors (i.e., retinoic acid receptor (RAR)-� expression, cellular retinoic acid binding protein (CRABP expression etc.) need to be investigated carefully. We previously established a myeloid cell line with retinoid resistance. The cells expressed multi drug resistance 1 (MDR-1) mRNA and p-glycoprotein cell surface protein, we assessed whether verapamil and ATRA would induce the differentiation of the cells, however, they did not. An increased expression of cellular retinoic acid-binding protein (CRABP)- was also detected on the cells compared with that of HL-60. These results suggest that high level of expression of CRABP-may contribute to be the mechanism of ATRA resistance (Sumimoto et al., 2000). Further, serum concentration of ATRA would be an important factor, especially trough level should be measured in each case. In the present study, the common adverse effects of ATRA were temporal and generally mild (5 patients had headaches, 2 had liver dysfunction, 2 had hyperlipidemia, and 1 had anorexia). Moreover, the adverse effects ranged between CTC grade 1 and 3. As mentioned above, ATRA may be useful in treating some ATL patients and may also be used in combination with other chemical agents. When ATRA used with conventional chemotherapy, we suggested that dose of anti-neoplastic agents could be reduced significantly. Further, the nonmyeloablative chemotherapy will be able to reduce the opportunities of severe infection and hemorrhagic disorder in the clinical course. In conclusion, we firmly believe that treatment with ATRA can provide some benefits to

Finally, we concluded that the mechanism of ATRA's action may be dichotomized into the inhibition of NF-κB's transcriptional activity related to HTLV-1 and inhibition of RT (Yamaguchi et al., 2005). It was reported that vitamin A supplementation reduced HIVassociated disease and slowed the progression toward AIDS (Fawzi et al., 2002). Maciaszek et al. reported that ATRA repressed HIV-1 long terminal repeat-directed expression in THP-1 monocytes (Maciaszek et al., 1998). Furthermore, Hanley et al. reported that a synthetic pan-retinoic acid receptor antagonist, BMS-204 493, activated replication of HIV-1 in a dosedependent manner (Hanley et al., 2004). This phenomenon suggested that ATRA-induced transactivation of cellular gene expression is required for the viral replication (Recio et al., 2000). On the other hand, it was reported that RA stimulates transcription of HIV in human neuronal cells. The HIV-1 proviral DNA load in 8E5 cells (HIV positive T-cell line) was significantly reduced by ATRA as well as AZT. Furthermore, ATRA affected viral replication in the three HIV patients. Further, HIV proviral DNA load on treatment with AZT, 10-5 M ATRA or 10-7 M ATRA. Interestingly, ATRA could reduce viral replication not only in the 8E5 cell line but in the primary lymphocytes from HIV patients. Regarding ATRA and HIV infection, there are several interesting reports (Calvo et al., 1997, Kudva et al., 2004). Briefly, four patients were diagnosed with HIV infection and APL at the same time. The use of HAART was not reported in three of these cases. All three patients with APL and HIV infection treated with ATRA achieved a complete remission (Calvo et al., 1997, Kudva et al., 2004). Furthermore, the CD4+ cell count decreased during therapy, but

clinicians and ATL patients.

**7. Effects of HIV infection** 

assay revealed that telomerase activity was not inhibited in retinoid-treated HTLV-I (+) Tcell lines; this indicated premature senescence (data not shown). We observed cellular senescence in HTLV-I (+) T-cell lines and in fresh primary cells obtained from patients with acute ATL. The grade of cellular senescence was greater for the HUT102, MT-2, MT-4, and ATL-2 cells than the ED40515 cells, which do not express *Tax* mRNA because of a nonsense mutation. This is an additional report pointing to *Tax* as an oncogene, and oncogene induced senescence (OIS) was possibly induced. These cells cannot re-enter the cell cycle or undergo tumorigenesis once senescence is triggered. OIS is caused by the accumulation of DNA damage. This DNA damage is, in turn, caused by oncogene-driven accumulation of reactive oxygen species (ROS) (Maeda et al., 2011). Chemotherapy using antineoplastic agents that decrease OIS and reduce cellular senescence may rejuvenate these cells and finally induce chemotherapy resistance. In conclusion, retinoids may be a reasonable agent for ATL with facilitating cellular senescence (Maeda et al., 2011).

#### **6. Clinical application**

We confirmed the clinical effects of ATRA in 20 ATL patients (Maeda et al., 2008). The median age was 56 years (range, 35–73). In total, 7 men and 13 women were enrolled in the study. Of these, 7 patients presented with the acute type; 3, lymphoma; 4, chronic; and 6, smoldering. The performance status (PS) of the patients ranged between 0 and 2, and 10 patients (50%) had skin involvement and 7 (35%), liver dysfunction. The treatment efficacy was as follows: CR, 0% of the patients; PR, 40%; NC, 45%; and PD, 15%. In the 7 acute patients, a PR was achieved in 2 (28.5%); NC, 2 (28.5%); and a PD, 3 (42.8%). In all the 3 lymphoma-type patients, a PR (100%) was achieved. In the 4 chronic-type patients, a PR was achieved in 1 (25%) and NC was observed in the remaining 3 (75%). Among the 6 smoldering-type patients, a PR was achieved in 2 (33.3 %) and NC was observed in 4 (66.6%). Adverse effects were noted in 10 of the 20 patients (50%). These effects were generally mild (headache in 5 patients; liver dysfunction, 2; hyperlipidemia, 2; and anorexia, 1). No hematological toxicity was observed. Considering the results described above, we indicated that ATRA has a therapeutic effect on ATL and should be the first choice for treating ATL. However, in fact, the present study showed no CR, which is not consistent with the results obtained in previous *in vitro* studies (Miyatake & Maeda 1997, Nawata et al., 2001). Interestingly, in the analysis among subtypes, ATL of the lymphoma-type showed a better PR rate than ATL of the acute-type (Maeda et al., 2008). In conclusion, the causes leading to a favorable response for ATRA treatment remain unknown. However, our clinical trial of ATRA for skin involvement demonstrated that ATRA was effective in the treatment of skin involvement in 6 of 8 patients (74%) (Maeda et al., 2004). Taken together, these results show that ATRA may have potential in the treatment of tumor formation with ATL cells than intravascular ATL cells. The present study showed that some patients are sensitive to ATRA while some are resistant. To elucidate the mechanism of resistance to ATRA, we focused on the intracellular redox potential. The imbalance of the intracellular redox potential in HTLV-I (+) T-cell lines may be strongly associated with the sensitivity to RA, and exogenous thiol compounds may cause the intracellular environment to become resistant to ATRA (Miyatake et al., 1998, 2000). In one of our recent studies, the mechanism by which ATRA acts on ATL cells was examined. The results showed that the mechanism could be dichotomized into inhibition of the transcriptional activity of NF-�B related to HTLV-I and inhibition of reverse transcriptase (Yamaguchi et al., 2005). This dichotomy

assay revealed that telomerase activity was not inhibited in retinoid-treated HTLV-I (+) Tcell lines; this indicated premature senescence (data not shown). We observed cellular senescence in HTLV-I (+) T-cell lines and in fresh primary cells obtained from patients with acute ATL. The grade of cellular senescence was greater for the HUT102, MT-2, MT-4, and ATL-2 cells than the ED40515 cells, which do not express *Tax* mRNA because of a nonsense mutation. This is an additional report pointing to *Tax* as an oncogene, and oncogene induced senescence (OIS) was possibly induced. These cells cannot re-enter the cell cycle or undergo tumorigenesis once senescence is triggered. OIS is caused by the accumulation of DNA damage. This DNA damage is, in turn, caused by oncogene-driven accumulation of reactive oxygen species (ROS) (Maeda et al., 2011). Chemotherapy using antineoplastic agents that decrease OIS and reduce cellular senescence may rejuvenate these cells and finally induce chemotherapy resistance. In conclusion, retinoids may be a reasonable agent

We confirmed the clinical effects of ATRA in 20 ATL patients (Maeda et al., 2008). The median age was 56 years (range, 35–73). In total, 7 men and 13 women were enrolled in the study. Of these, 7 patients presented with the acute type; 3, lymphoma; 4, chronic; and 6, smoldering. The performance status (PS) of the patients ranged between 0 and 2, and 10 patients (50%) had skin involvement and 7 (35%), liver dysfunction. The treatment efficacy was as follows: CR, 0% of the patients; PR, 40%; NC, 45%; and PD, 15%. In the 7 acute patients, a PR was achieved in 2 (28.5%); NC, 2 (28.5%); and a PD, 3 (42.8%). In all the 3 lymphoma-type patients, a PR (100%) was achieved. In the 4 chronic-type patients, a PR was achieved in 1 (25%) and NC was observed in the remaining 3 (75%). Among the 6 smoldering-type patients, a PR was achieved in 2 (33.3 %) and NC was observed in 4 (66.6%). Adverse effects were noted in 10 of the 20 patients (50%). These effects were generally mild (headache in 5 patients; liver dysfunction, 2; hyperlipidemia, 2; and anorexia, 1). No hematological toxicity was observed. Considering the results described above, we indicated that ATRA has a therapeutic effect on ATL and should be the first choice for treating ATL. However, in fact, the present study showed no CR, which is not consistent with the results obtained in previous *in vitro* studies (Miyatake & Maeda 1997, Nawata et al., 2001). Interestingly, in the analysis among subtypes, ATL of the lymphoma-type showed a better PR rate than ATL of the acute-type (Maeda et al., 2008). In conclusion, the causes leading to a favorable response for ATRA treatment remain unknown. However, our clinical trial of ATRA for skin involvement demonstrated that ATRA was effective in the treatment of skin involvement in 6 of 8 patients (74%) (Maeda et al., 2004). Taken together, these results show that ATRA may have potential in the treatment of tumor formation with ATL cells than intravascular ATL cells. The present study showed that some patients are sensitive to ATRA while some are resistant. To elucidate the mechanism of resistance to ATRA, we focused on the intracellular redox potential. The imbalance of the intracellular redox potential in HTLV-I (+) T-cell lines may be strongly associated with the sensitivity to RA, and exogenous thiol compounds may cause the intracellular environment to become resistant to ATRA (Miyatake et al., 1998, 2000). In one of our recent studies, the mechanism by which ATRA acts on ATL cells was examined. The results showed that the mechanism could be dichotomized into inhibition of the transcriptional activity of NF-�B related to HTLV-I and inhibition of reverse transcriptase (Yamaguchi et al., 2005). This dichotomy

for ATL with facilitating cellular senescence (Maeda et al., 2011).

**6. Clinical application** 

model means multi-target therapy, and indicated that if one pathway is blocked by some factors, the other one will be available. Furthermore, we should recognize the differences between the clinical outcome and experimental results *in vitro*. We examined the differences in several clinical parameters (LDH, AL-P, sIL-2R, and age) between cases of NC and PR. However, no significant difference was observed (data not shown). Other intrinsic factors (i.e., retinoic acid receptor (RAR)-� expression, cellular retinoic acid binding protein (CRABP expression etc.) need to be investigated carefully. We previously established a myeloid cell line with retinoid resistance. The cells expressed multi drug resistance 1 (MDR-1) mRNA and p-glycoprotein cell surface protein, we assessed whether verapamil and ATRA would induce the differentiation of the cells, however, they did not. An increased expression of cellular retinoic acid-binding protein (CRABP)- was also detected on the cells compared with that of HL-60. These results suggest that high level of expression of CRABP-may contribute to be the mechanism of ATRA resistance (Sumimoto et al., 2000). Further, serum concentration of ATRA would be an important factor, especially trough level should be measured in each case. In the present study, the common adverse effects of ATRA were temporal and generally mild (5 patients had headaches, 2 had liver dysfunction, 2 had hyperlipidemia, and 1 had anorexia). Moreover, the adverse effects ranged between CTC grade 1 and 3. As mentioned above, ATRA may be useful in treating some ATL patients and may also be used in combination with other chemical agents. When ATRA used with conventional chemotherapy, we suggested that dose of anti-neoplastic agents could be reduced significantly. Further, the nonmyeloablative chemotherapy will be able to reduce the opportunities of severe infection and hemorrhagic disorder in the clinical course. In conclusion, we firmly believe that treatment with ATRA can provide some benefits to clinicians and ATL patients.

#### **7. Effects of HIV infection**

Finally, we concluded that the mechanism of ATRA's action may be dichotomized into the inhibition of NF-κB's transcriptional activity related to HTLV-1 and inhibition of RT (Yamaguchi et al., 2005). It was reported that vitamin A supplementation reduced HIVassociated disease and slowed the progression toward AIDS (Fawzi et al., 2002). Maciaszek et al. reported that ATRA repressed HIV-1 long terminal repeat-directed expression in THP-1 monocytes (Maciaszek et al., 1998). Furthermore, Hanley et al. reported that a synthetic pan-retinoic acid receptor antagonist, BMS-204 493, activated replication of HIV-1 in a dosedependent manner (Hanley et al., 2004). This phenomenon suggested that ATRA-induced transactivation of cellular gene expression is required for the viral replication (Recio et al., 2000). On the other hand, it was reported that RA stimulates transcription of HIV in human neuronal cells. The HIV-1 proviral DNA load in 8E5 cells (HIV positive T-cell line) was significantly reduced by ATRA as well as AZT. Furthermore, ATRA affected viral replication in the three HIV patients. Further, HIV proviral DNA load on treatment with AZT, 10-5 M ATRA or 10-7 M ATRA. Interestingly, ATRA could reduce viral replication not only in the 8E5 cell line but in the primary lymphocytes from HIV patients. Regarding ATRA and HIV infection, there are several interesting reports (Calvo et al., 1997, Kudva et al., 2004). Briefly, four patients were diagnosed with HIV infection and APL at the same time. The use of HAART was not reported in three of these cases. All three patients with APL and HIV infection treated with ATRA achieved a complete remission (Calvo et al., 1997, Kudva et al., 2004). Furthermore, the CD4+ cell count decreased during therapy, but

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increased once the treatment was completed, and the patient did not suffer any HIVassociated complications (Kudva et al., 2004). This phenomenon may explain why ATRA affects both APL and HIV infection. Furthermore, a case of APL and ATL associated with HTLV-I infection treated with ATRA was reported (Tsukasaki et al., 1995). The patient was diagnosed with APL and smoldering ATL simultaneously, and treated with ATRA (60mg/day p.o.). At day 17 of ATRA treatment, the WBC count was normal with less than 1% APL and ATL cells. Monoclonal integration of HTLV-I was undetectable at that time. Hematological findings showed no abnormality on morphological, phenotypical, cytogenetic and molecular biologic analyses at day 50, when ATRA therapy was discontinued. Moreover, we examined the effects of ATRA on RT activity. RT activity decreased significantly on treatment with ATRA as well as AZT. The mechanism by which ATRA inhibited HIV replication may be inhibition of RT activity (data not shown). Taken together, ATRA may be a useful therapeutic tool for HIV infection.

#### **8. Conclusion**

We have believed that treatment with ATRA can provide some benefits to clinicians and ATL patients as having based on several evidences. Finally, we hope that ATRA is a useful agent for other HTLV-I-associated disorders, including HAM (HTLV-I-associated myelopathy), HAAP (HTLV-I-associated arthropathy), HAB (HTLV-I- associated bronchopathy) and HAU (HTLV-I-associated uveitis).

#### **9. Acknowledgements**

Author thanks to Mrs. K. Furukawa and Mrs. K. Niki for for technical assistance and Ms. S. Yoshida and Ms. S. Nagayama for preparing the manuscript.

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