Chandrika Gowda

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

Genetic Targets in Pediatric Acute Lymphoblastic Leukemia

Acute leukemia represents 31% of all cancers diagnosed in children and 80% of it is of Lymphoblastic type. Multiple genetic lesions in the hematopoietic progenitor cells prior to or during differentiation to B and T cell lead to development of leukemia. There are several subtypes of Acute Leukemia based on chromosome number changes, the presence of certain translocations and gene mutations, each of which has different clinical, biological and prognostic features. High throughput genomic technologies like array-based comparative genomic hybridization (array-CGH) and single nucleotide polymorphism microarrays (SNP arrays), have given us insight through a very detailed look at the genetic changes of leukemia, specifically, ALL. Here, we discuss various genetic mutations identified in Acute Lymphoblastic Leukemia. We also explore various genetic targets and currently available as well as upcoming targeted therapies for ALL.

Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia.

IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a master regulatory of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL).1, 2, 3, 4 The deletion and/or mutation of Ikaros is associated with the development of B-cell acute lymphoblastic leukemia (B-ALL) with poor outcome.5, 6, 7, 8, 9, 10, 11 Ikaros directly associates with components of the histone deacetylase complex (NuRD), HDAC1, HDAC2 and Mi-2.12, 13, 14 Although Ikaros is hypothesized to regulate the transcription of target genes by recruiting the NuRD complex, the mechanism of Ikaros-mediated transcriptional regulation in leukemia is still unknown. Here we use a systems biology approach to determine the mechanism through which Ikaros and HDAC1 regulate gene expression in human B-ALL.

Casein Kinase II (CK2), Glycogen Synthase Kinase-3 (GSK-3) and Ikaros mediated regulation of leukemia

Signaling networks that regulate cellular proliferation often involve complex interactions between several signaling pathways. In this manuscript we review the crosstalk between the Casein Kinase II (CK2) and Glycogen Synthase Kinase-3 (GSK-3) pathways that plays a critical role in the regulation of cellular proliferation in leukemia. Both CK2 and GSK-3 are potential targets for anti-leukemia treatment. Previously published data suggest that CK2 and GSK-3 act synergistically to promote the phosphatidylinositol-3 kinase (PI3K) pathway via phosphorylation of PTEN. More recent data demonstrate another mechanism through which CK2 promotes the PI3K pathway - via transcriptional regulation of PI3K pathway genes by the newly-discovered CK2-Ikaros axis. Together, these data suggest that the CK2 and GSK-3 pathways regulate AKT/PI3K signaling in leukemia via two complementary mechanisms: a) direct phosphorylation of PTEN and b) transcriptional regulation of PI3K-promoting genes. Functional interactions between CK2, Ikaros and GSK3 define a novel signaling network that regulates proliferation of leukemia cells. This regulatory network involves both direct posttranslational modifications (by CK and GSK-3) and transcriptional regulation (via CK2-mediated phosphorylation of Ikaros). This information provides a basis for the development of targeted therapy for leukemia.

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

Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros’ DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL.

Abstract A21: Epigenetic regulation of cell cycle progression at the G2/M transition and mitosis in high-risk leukemia

High-risk acute lymphoblastic leukemia (ALL) is a clinical challenge due to drug resistance and poor prognosis. A characteristic molecular defect of most high-risk ALL is the deletion or inactivating mutation of one allele of the IKZF1 (Ikaros) tumor suppressor. Ikaros encodes a DNA-binding protein that regulates transcription of its target genes via chromatin remodeling. The mechanisms through which Ikaros regulates cellular proliferation in high-risk leukemia, are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that are critical in the control of G2/M transition (CDC2) and mitotic progression (ANAPC1 and ANAPC7) in leukemia. Gain- and loss-of-function experiments demonstrate that Ikaros represses the transcription of CDC2, ANAPC1 and ANAPC7. Overexpression of Ikaros in leukemia also results in cell cycle arrest. We studied the mechanism through which Ikaros represses CDC2, ANAPC1 ad ANAPC7. The use of serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes demonstrated that Ikaros can repress transcription of its target genes by two different mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and loss of H3K9ac (for ANAPC1 and CDC2); and 2) via an HDAC1-independent mechanism which is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac (for ANAPC7). In high-risk ALL that is characterized by deletion of one Ikaros allele, the function of Ikaros as a transcriptional regulator is impaired due to reduced binding to promoters of Ikaros target genes. We showed previously that Ikaros DNA-binding affinity is regulated via direct phosphorylation by pro-oncogenic Casein Kinase II (CK2). CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, which further reduces Ikaros function in this disease. In vivo CK2 inhibition with the CK2 specific inhibitor, CX-4945, results in a strong therapeutic effect in primary high-risk ALL xenografts. Analysis of primary high-risk B-ALL (that have deletion of one Ikaros allele) showed that treatment with CX-4945, restored Ikaros function as a transcriptional regulator of CDC2, ANAPC1 and ANAPC7, and was associated with cell cycle arrest. Epigenetic analysis of promoters of CDC2, ANAPC1 and ANAPC7 genes revealed that restoration of Ikaros binding to the promoters of these genes is associated with epigenetic alterations that are consistent with Ikaros overexpression and formation of repressive heterochromatin. In conclusion, our results reveal that: 1) Ikaros functions as a tumor suppressor by repressing transcription of genes that are critical for G/M transition (CDC2) and mitotic progression (ANAPC1 and ANAPC7); 2) Ikaros represses transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator of CDC2, ANAPC1 and ANAPC7 in high-risk leukemia. These results provide novel insights into the control of cell cycle progression in high-risk leukemia and the mechanisms by which CK2 inhibitors exert their therapeutic effects. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Citation Format: Chunhua Song, Chandrika Gowda, Yali Ding, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of cell cycle progression at the G2/M transition and mitosis in high-risk leukemia. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A21.

Abstract PR09: CK2 inhibition exerts a therapeutic effect in high-risk ALL by restoring IKZF1-mediated repression of cell cycle progression and the PI3K pathway

The IKZF1 (Ikaros) gene encodes a DNA-binding protein that acts as a tumor suppressor in leukemia. Deletion of one Ikaros allele results in B-cell acute lymphoblastic leukemia (B-ALL) with a high rate of relapse and poor outcome. The mechanisms through which Ikaros suppresses leukemogenesis and that regulate Ikaros activity as a tumor suppressor in leukemia are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that control two pathways crucial for proliferation of leukemia cells: 1) cell cycle progression and 2) the phosphatidylinositol 3-kinase (PI3K) pathway. Using gain-of-function and loss-of-function experiments we demonstrate that Ikaros transcriptionally represses genes that promote cell cycle progression and the PI3K pathway and activates transcription of a gene that suppresses the PI3K pathway. In high-risk B-ALL with deletion of one Ikaros allele, we show that the function of Ikaros as a transcriptional regulator is impaired due to reduced binding at promoters of its target genes. Previous work shows that Ikaros DNA-binding affinity is regulated via direct phosphorylation by the pro-oncogenic kinase, CK2 (Casein Kinase II). We show that CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, further reducing Ikaros function. Treatment of primary high-risk B-ALL (with deletion of one IKZF1allele) using the CK2 specific inhibitor, CX-4945, restored Ikaros function as a transcriptional regulator of genes that control cell cycle progression and the PI3K pathway. Treatment with CK2 inhibitor was also associated with cell cycle arrest and reduced phosphorylation of the AKT kinase, a downstream PI3K pathway target. Using serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes, we demonstrated that Ikaros can repress transcription of its target genes through two distinct mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and the loss of H3K9ac; and 2) by an HDAC1-independent mechanism that is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac. The therapeutic efficacy of CK2 inhibition using CX-4945 against high-risk B-ALL was demonstrated in vivo using 4 different xenografts: 3 different high-risk primary pre-B-ALL patient-derived xenografts and Nalm6 xenografts. CX-4945 showed strong therapeutic effects in all 4 xenografts, as evidence by reduced leukemia cell numbers in bone marrow and in spleen, together with prolonged survival of treated xenograft animals. Expression analysis of Ikaros target genes in leukemia cells treated in vivo with CX-4945 revealed an expression pattern cell cycle regulatory and PI3K pathway genes that was highly similar to that observed with Ikaros overexpression. These data suggest that CK2 inhibition in vivo exerts its therapeutic effect on high-risk B-ALL by restoring Ikaros function as a transcriptional regulator of genes that promote cell cycle progression and the PI3K pathway. In summary, our results reveal that: 1) Ikaros functions as a tumor suppressor by suppressing cell cycle progression and the PI3K pathway; 2) Ikaros regulates transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator in vivo, and has a strong therapeutic effect in primary xenografts of high-risk B-ALL. These results provide support for the use of CK2 inhibitors in clinical trials for high-risk B-ALL. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. This abstract is also presented as Poster B10. Citation Format: Chunhua Song, Chandrika Gowda, XiaoKang Pan, Kimberly J. Payne, Sinisa Dovat. CK2 inhibition exerts a therapeutic effect in high-risk ALL by restoring IKZF1-mediated repression of cell cycle progression and the PI3K pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR09.

Pediatric High Risk Leukemia — Molecular Insights

Acute leukemia comprises of 31% of all cancers in children making it the most com‐ mon childhood malignancy. Significant strides have been made in treatment, partly through risk stratification and intensified therapy. A number of subtypes remain at high risk for relapse and poor outcome, despite current therapies. Here we describe risk stratification and molecular diagnosis used to identify high risk leukemias and guide treatment. Specific cytogenetic alterations that contribute to high risk B and T cell acute lymphoblastic leukemia (ALL), as well as infant leukemia are discussed. Particular attention is given to genetic alterations in IKZF1, CRLF2, and JAK, that have been identified by whole genome sequencing and recently associated with Phlike ALL. Ongoing studies of disease mechanisms and challenges in developing pre-clinical patient-derived xenograft models to evaluate therapies are discussed.

Abstract 3504: Regulation of cell cycle progression by Ikaros in leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Control of cell cycle progression is achieved by the coordinated function of a large set of genes that are highly conserved in eukaryotic organisms. Malignant cells have impaired regulation of cell cycle progression which results in uncontrolled cellular proliferation. Thus, understanding the regulation of cell cycle progression in malignant cells is essential to advance our knowledge of the process of malignant transformation and for designing novel treatments. Ikaros is a zinc finger protein that acts as a tumor suppressor in leukemia. The loss of Ikaros activity due to deletion or mutation has been associated with the development of high-risk B-cell acute lymphoblastic leukemia (B-ALL), as well as with T-cell ALL and acute myelogenous leukemia (AML). Ikaros binds DNA and regulates transcription of its target genes via chromatin remodeling. The mechanism of Ikaros tumor suppressor activity is largely unknown. Here, we present evidence that Ikaros regulates cell cycle progression in leukemia. Using quantitative Chromatin Immunoprecipitation assay (qChIP), we demonstrate that Ikaros binds in vivo to promoter regions of several genes that regulate cell cycle progression in B-ALL cell lines and in primary cells from patients with B-ALL. To study how Ikaros regulates transcription of these genes, luciferase reporter assays were performed. The promoter regions of three Ikaros target genes were cloned into luciferase reporter constructs. Each of these constructs has been co-transfected with Ikaros or an empty vector (as a negative control) into HEK 293T cells. Results showed that Ikaros represses transcription of the three genes that promote cell cycle progression. Overexpression of Ikaros in leukemia cells by retroviral transduction results in reduced transcription of the cell cycle promoting genes, as evidenced by quantitative real-time PCR (qRT-PCR), as well as cell cycle arrest. These data suggest that Ikaros regulates cell cycle progression in leukemia by direct repression of the transcription of the genes that promote cell cycle progression, and identifies one mechanism of Ikaros function as a tumor suppressor in leukemia. Citation Format: Elanora Dovat, Jonathon Payne, Carlos M. Casiano, Justin Sloane, Chandrika Gowda, Kimberly J. Payne, Sinisa Dovat, Chunhua Song. Regulation of cell cycle progression by Ikaros in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2014-3504