**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.