Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis

Structure and biologic function of Ikaros

The IKZF1 gene encodes several Ikaros isoforms via alternate splicing.6 Ikaros protein contains two separate zinc finger domains. There are four zinc fingers in the amino half of the protein that take part in sequence-specific DNA binding. The two zinc fingers at the C-terminus of the protein are responsible for protein–protein interaction. This enables Ikaros proteins to form dimers or multimers with different Ikaros isoforms, as well as with other members of Ikaros family genes that share the same protein-interaction domain.7 Small Ikaros isoforms lack DNA-binding zinc fingers resulting in a functionally inactive complex when associated with full-length Ikaros.8

Ikaros binds to the upstream regulatory element (URE) of its target genes and aids in their recruitment to pericentromeric heterochromatin (PC-HC), resulting in repression or activation of transcription of those genes via chromatin remodeling.9 The ability of Ikaros to localize to PC-HC is essential for Ikaros’ function as an activator or repressor of transcription.10 ,11 Several gene disruption experiments have shown that Ikaros is a master regulator of lymphoid development and suggest its involvement in lymphocyte activation and tumor suppression.12

Role of Ikaros as a tumor suppressor in leukemia

The role of Ikaros in tumor suppression was first described in Ikaros knockout mice. The absence of Ikaros has a severe impact on normal hematopoiesis with the absence of B, natural killer cell (NK) and dendritic cells, along with reduced T cells. However, Ikaros haplo knockout mice that lack one copy of IKZF1 develop an early T cell leukemia with 100% penetrance.12 ,13 Reintroduction of Ikaros into T-cell leukemia with Ikaros deficiency resulted in cellular arrest with partial T-cell differentiation.13

Altered Ikaros expression has been associated with the development of malignancies including childhood ALL, infant T-cell ALL, adult B cell ALL, myelodysplastic syndrome, AML and adult and juvenile CML.2 ,14 IKZF1 defects leading to a loss of Ikaros activity have been detected in 30% of pediatric B-cell ALL, in >80% of BCR-ABL1 ALL and BCR-ABL-like ALL, and approximately 5% of T-cell ALL.14 Overall, 90% of the Ikaros gene defects involve IKZF1 gene deletion and the rest are nonsense or functionally inactivating mutations of a single allele.15 Thus, Ikaros haploinsufficiency is sufficient to result in malignant transformation and the development of ALL. Deletion of IKZF1 confers poor prognosis and is known to be an independent negative prognostic marker in childhood ALL.3 ,16

Phosphorylation of Ikaros by CK2 controls Ikaros activity

Ikaros protein is abundant during the most stages of hematopoiesis. Although it has been shown that Ikaros transcription is controlled by a complex regulatory network.17 post-translational modifications are hypothesized to play an important role in the regulation of Ikaros function in hematopoietic cells. In vivo phosphopeptide mapping of Ikaros has shown that Ikaros is phosphorylated at multiple sites.18 The first evidence that phosphorylation is one of the major mechanisms regulating Ikaros function came with the discovery that Ikaros DNA binding to target genes is controlled in a cell cycle-dependent manner, during mitosis. This control occurs via direct phosphorylation at a linker sequence that connects the DNA binding zinc finger units.19 Studies from the Georgopoulos group identified several additional phosphorylated amino acids in Ikaros protein that are directly phosphorylated by CK2. These studies suggested that CK2-mediated phosphorylation of Ikaros can regulate its ability to control G1-to-S cell cycle progression in mice.20 Subsequent studies in human leukemia cells revealed that phosphorylation by CK2 regulates Ikaros DNA-binding in human leukemia cells in S phase of the cell cycle.20 ,21

Gurel et al,22 identified four novel sites in the N terminus of Ikaros that are phosphorylated by CK2 in vivo. Functional analysis of Ikaros phosphomimetic mutants (where phosphosites are mutated to aspartate to mimic phosphorylation) and phosphoresistant mutants (where phosphosites are mutated to alanine to mimic the dephosphorylated state) were used to study effects of CK2-mediated phosphorylation on Ikaros function. These analyses demonstrated that phosphorylation of Ikaros at specific amino acids by CK2 regulates two important Ikaros functions:

1. Pericentromeric localization: In the nucleus, Ikaros exhibits a punctate staining pattern in normal hematopoietic cells and leukemia cells. Confocal microcopy showed that this punctate staining pattern is due to the localization of Ikaros to PC-HC. Ikaros ability to localize to PC-HC is thought to be important for its function in chromatin remodeling and transcriptional regulation of its target genes. Ikaros phosphomimetic mutants at amino acids in the N-terminal region show severely reduced DNA binding affinity for probes that are derived from PC-HC. In contrast, phopshoresistant mutants were able to bind probes derived from PC-HC at a rate similar to wild-type Ikaros. These data suggested that Ikaros phosphorylation by CK2 at specific sites regulates DNA binding of Ikaros to PC-HC.22 Confocal microscopy analysis showed that Ikaros protein with phosphomimetic mutations at CK2 phospho acceptor sites loses its ability to localize to PC-HC, resulting in a diffuse nuclear distribution of Ikaros mutant protein. These results demonstrated that CK2-mediated phosphorylation regulates the ability of Ikaros to bind DNA at PC-HC, but also its subcellular localization and function in chromatin remodeling.22

2. DNA binding ability to Ikaros target genes: Ikaros binds to the upstream regulatory sequence of the terminal deoxytransferase (TdT) (dntt) gene and negatively regulates expression of TdT.23 However, molecular mechanisms that results in increased binding of Ikaros to the TdT D’ regulatory sequence during thymocyte differentiation were unknown. The aforementioned study by Gurel et al, showed that reversible phosphorylation of Ikaros at specific amino acids regulates the expression of TdT during thymocyte differentiation. Experiments using phosphoresistant mutants at CK2 phospho acceptor sites (amino acids #13 or #294) showed increased Ikaros’ DNA-binding affinity toward the TdT D’ regulatory sequence, while phosphomimetic mutants (at amino acid #294) had decreased Ikaros’ DNA binding by threefold as compared to wild type, suggesting that, phosphorylation of Ikaros controls its ability to bind to the regulatory sequence of the TdT gene. Furthermore, phosphopeptide mapping of endogenous Ikaros, in unstimulated thymocytes and in thymocytes induced to differentiate, showed that Ikaros undergoes dephosphorylation at amino acids #13 and #294 following the induction of thymocyte differentiation. These data suggest that the DNA-binding affinity of Ikaros for the TdT D’ regulatory element during thymocyte differentiation is controlled by phosphorylation at specific amino acids.

Treating cells in the above studies with a specific CK2 inhibitor, resulted in the loss of phosphorylation at several amino acid sites including #13 and #294, suggesting that high CK2 kinase activity is necessary for in vivo phosphorylation of these amino acids. Overall, this study provided an in-depth analysis of the molecular mechanism through which phosphorylation by CK2 controls the function of Ikaros in regulating gene transcription and chromatin remodeling.

Dephosphorylation of Ikaros by protein phosphatase 1

The above studies demonstrated the strong connection between the CK2 signal transduction pathway and transcriptional regulation by the Ikaros tumor suppressor. The discovery that Ikaros is a substrate for protein phosphatase 1 (PP1) provided new insights on the role of phosphorylation in regulating Ikaros function. Ikaros contains an evolutionarily-conserved PP1 recognition motif R/K-X_0-1-[V/I]-X-[F/W] at amino acids located at the C-terminal end of the protein.24 ,25 Interaction between Ikaros and PP1 was studied in detail by Popescu et al,26 using an Ikaros mutant that is unable to interact with PP1 (IK-A465/7, mutation of valine and alanine at amino acids 465 and 467). These studies determined that Ikaros dephosphorylation by PP1 regulates both Ikaros binding to pericentromeric DNA, as well as its subcellular localization. Importantly, the introduction of phosphoresistant mutations at CK2-phosphorylated residues restored DNA-binding affinity and PC-HC localization of this mutant. These results show that PP1 is important in preventing hyperphosphorylation of Ikaros by CK2 kinase, and the loss of Ikaros DNA binding activity. Hence, CK2-mediated phosphorylation is one of the major mechanisms regulating Ikaros function in DNA binding and chromatin remodeling and dephosphorylation of Ikaros is essential for preserving its function in hematopoietic cells.26

Phosphorylation regulates stability of Ikaros protein

An unexpected finding was that the loss of interaction with PP1 results in a severely shortened Ikaros half-life. The aforementioned study26 showed that, protein levels of the PP1-nonbinding Ikaros mutant (Ikaros A465/7) was more than fivefold decreased compared to wild type Ikaros, despite the presence of similar levels of Ikaros mRNA. The introduction of phosphoresistant mutations at CK2 phosphoacceptor sites stabilized the Ikaros A465/7 mutant protein and extended its half-life by fivefold. This demonstrated that phosphorylation of Ikaros by CK2 kinase at specific amino acids promotes its degradation while dephosphorylation by PP1 stabilises the Ikaros protein and extends its half-life.26

CK2 and PP1 Regulate Ikaros-mediated repression of TdT and cellular proliferation in leukemia

The above data showed that Ikaros activity can be regulated in an opposing manner by the CK2 and PP1 signaling pathways. Subsequent studies extended the analysis of CK2 and PP1 pathways in regulation of Ikaros function as regulator of TdT transcription.

Targeting CK2-Ikaros pathway in B cell Leukemia

Our recently published data showed that Ikaros controls cellular proliferation by repressing genes that regulate cell cycle progression, PI3K pathway and histone methylation. CK2-mediated phosphorylation of Ikaros impairs its ability to regulate its target genes in B-cell ALL (B-ALL).27–30 CK2 inhibition restores Ikaros tumor suppressor function in B-ALL, as well as in high-risk B-ALL with IKZF1 deletion and showed strong therapeutic effect in vivo .These data provided validation and the rationale for targeting CK2 as a novel therapeutic approach for high-risk B-cell ALL.