Ikaros Function Research Articles

Ikaros antagonizes DNA binding by STAT5 in pre-B cells.

The IKZF1 gene, which encodes the Ikaros transcription factor, is frequently deleted or mutated in patients with B-cell precursor acute lymphoblastic leukemias that express oncogenes, like BCR-ABL, which activate the JAK-STAT5 pathway. Ikaros functionally antagonizes the transcriptional programs downstream of IL-7/STAT5 during B cell development, as well as STAT5 activity in leukemic cells. However, the mechanisms by which Ikaros interferes with STAT5 function is unknown. We studied the genomic distribution of Ikaros and STAT5 on chromatin in a murine pre-B cell line, and found that both proteins colocalize on >60% of STAT5 target regions. Strikingly, Ikaros activity leads to widespread loss of STAT5 binding at most of its genomic targets within two hours of Ikaros induction, suggesting a direct mechanism. Ikaros did not alter the level of total or phosphorylated STAT5 proteins, nor did it associate with STAT5. Using sequences from the Cish, Socs2 and Bcl6 genes that Ikaros and STAT5 target, we show that both proteins bind overlapping sequences at GGAA motifs. Our results demonstrate that Ikaros antagonizes STAT5 DNA binding, in part by competing for common target sequences. Our study has implications for understanding the functions of Ikaros and STAT5 in B cell development and transformation.

Ikaros‐Mediated Regulation of the Rab20 GTPase in B‐ALL is PP1 Dependent

Rab20 is a member of the Rab GTPase family, a group of over 60 small GTPases that play critical roles in membrane trafficking and vesicular transport in all eukaryotic cells. Rab20 has been found to play a critical role in the immune response and is highly expressed in macrophages where it plays a role in phagosome and endosome formation. RAB20 overexpression has been observed in various human malignancies. However, the role of RAB20 in leukemia is unknown. Here we present data that RAB20 expression is repressed by the Ikaros tumor suppressor protein in B‐cell acute lymphoblastic leukemia (B‐ALL). Additionally, we report that protein phosphatase 1 (PP1) regulates Ikaros’ function as a repressor of RAB20 transcription. Global chromatin immunoprecipitation coupled with next‐generation sequencing (ChIP‐Seq) demonstrated that Ikaros binds to the promoter of RAB20 in B‐ALL. Ikaros binding to the RAB20 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in both B‐ALL cell lines and primary B‐ALL. The role of Ikaros in regulating RAB20 expression in B‐ALL was dissected using gain‐of‐function and loss‐of‐function experiments. Ikaros overexpression resulted in increased Ikaros binding to the RAB20 promoter and decreased RAB20 expression. shRNA‐mediated knockdown of Ikaros abolished Ikaros binding at the RAB20 promoter and resulted in increased RAB20 expression. A luciferase reporter assay demonstrated that Ikaros directly represses RAB20 promoter activity. The role of Ikaros in regulating the chromatin signature at the RAB20 promoter was examined by combining Ikaros overexpression and knockdown in B‐ALL with qChIP against markers of active chromatin (H3K9ac & H3K4me3) and repressive chromatin (H3K9me3). Ikaros overexpression results in an increase in H3K9me3 and a decrease in both H3K9ac and H3K4me3 at the RAB20 promoter—indicating formation of repressive chromatin. Ikaros knockdown resulted in an increase in H3K9ac and H3K4me3—indicating formation of active chromatin. Since our previous studies showed that protein phosphatase 1 (PP1) can regulate Ikaros function, we tested whether PP1 affects Ikaros‐mediated regulation of RAB20 expression. Treatment of B‐ALL cells with the calyculin and tautomycin PP1 inhibitors resulted in decreased Ikaros binding at the RAB20 promoter and increased RAB20 expression. qChIP demonstrated that mutating the PP1‐interacting site of Ikaros—IKZF1(465‐7A)—ablated Ikaros’ RAB20 promoter binding. Consequently, using an Ikaros mutant with both a mutated PP1‐interacting site and phosphoresistant mutants (simulating active PP1 dephosphorylation) restored Ikaros binding to the RAB20 promoter. Together, these data demonstrate that Ikaros transcriptionally represses RAB20 expression in B‐ALL in a PP1‐dependent manner. The results provide a novel insight into intersection of PP1 and Rab GTPase signaling pathways and suggest a potential mechanism by which PP1 regulates Rab20 activity in leukemia.Support or Funding InformationNIH R01 CA213912; NIH F30 CA221109; The Four Diamonds Fund; St. Baldrick’s Foundation; Hyundai Hope on Wheels; Alex’s Lemonade Stand; Bear Necessities Pediatric Cancer Foundation

CK2 Inhibitor CX4945 Shows Strong In Vivo Anti Leukemia Effect in AML Via Augmented Ikaros-Mediated Regulation of Global Epigenetic Landscape

Over-expression of Casein Kinase II (CK2) pro-oncogenic kinase in Acute Myelogenous Leukemia (AML) is associated with poor prognosis. Inhibition of CK2 by siRNAs or with the specific inhibitor, CX-4945, shows strong cytotoxic effects. CK2 is a ubiquitous, constitutively active serine/ threonine kinase which has been implicated in leukemia progression via multiple mechanisms. One of the well-established mechanism is CK2 mediated phosphorylation and impairment of tumor suppressor function of Ikaros transcription factor. Impaired function of Ikaros has been associated with the development of leukemia including AML. In B-cell lymphoblastic leukemia, CK2 inhibition has been shown to exert a therapeutic effect via restoration and/or enhancement of the tumor suppressor activity of the Ikaros protein. The mechanism of therapeutic action of CK2 inhibition in AML is largely unknown and in vivo efficacy CK2 inhibitors in AML has never been established. Here we report anti-leukemia effect of CX-4945 in preclinical models of AML and demonstrate mechanism of action of CK2 inhibitor via restoration of Ikaros driven global regulation of epigenetic landscape. In order to study the effect of CK2 inhibition on Ikaros function in AML, we used U937 - a human myelomonocytic leukemia cell line with high baseline CK2 expression. U937 cells were treated with specific CK2 inhibitor, CX4945 for 72 hours. CK2 activity and Ikaros phosphorylation was measured using CK2 kinase assay and in vivo labeling - radio immunoblot assay. Results showed significant decrease in CK2 activity and Ikaros phosphorylation with no change in overall expression of the Ikaros protein. Expression analysis using RNA sequencing showed that treatment with CX-4945 caused significant upregulation of genes controlling immunity, and inflammation; and downregulation of genes involved in nucleic acid metabolism, RNA processing, and translation. Analysis of global genome-wide Ikaros occupancy using Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) of CX4945 treated U937 cells demonstrated that CX-4945 treatment significantly increased the number of Ikaros binding sites as well as increased peak strength while minimally re-distributing Ikaros' global genomic occupancy. While increased binding to the Promoter, Gene Body, and Gene Desert elements in CX-4945-treated cells was similar, the increase in Ikaros' DNA binding to enhancers was particularly pronounced. Further analysis showed that enhanced Ikaros DNA binding following treatment with CX-4945, directly induces formation of de novo enhancers. In order to test whether enhanced DNA binding of Ikaros is accompanied by augmented Ikaros function in the global epigenetic regulation of gene expression. We determined and compared chromatin accessibility of U937 cells before and after treatment with CX-4945, using ATAC-seq. Results indicate that Ikaros-induced de novo open chromatin at distal regulatory regions controls genes involved in the negative regulation of biological processes and cellular metabolism. Overall, these data demonstrate that, augmented Ikaros DNA-binding following CK2 inhibition resulted in 1) Ikaros' pioneering activity, 2) Ikaros' ability to induce the de novo formation of enhancers and super-enhancers, and 3) Ikaros' ability to induce the de novo formation of active enhancers and to activate poised enhancers. Together, these data uncover novel Ikaros functions in regulating the epigenetic landscape and identifies CK2 as a critical regulator of Ikaros activity. Next, we treated AML xenograft model of luciferase labelled U937 cells with CX4945 via oral gavage at dose 200mg/kg/day for 3 weeks and demonstrated significantly lower leukemia burden in treated group as measured by decreased bio-luminescence imaging. In summary, these results demonstrate for the first time that CK2 inhibitor, CX-4945 has strong anti-leukemia effect in AML preclinical models. One of the mechanisms by which CX4945 exert a therapeutic effect in AML involves enhancing Ikaros' function as regulator of global epigenomic landscape. These results provide strong mechanistic basis to develop novel targeted combination therapies using CK2 inhibitors for treatment of AML. Further studies evaluating combination therapies using patient derived xenograft (PDX) models of AML are underway. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

CK2 Inhibitor CX4945 Shows Strong In Vivo Synergistic Anti-Leukemia Activity with Rapamycin in B-ALL Due to Enhanced Ikaros Mediated Repression of mTOR Pathway

Casein Kinase II (CK2) is a pro-oncogenic protein that is overexpressed in various cancers including leukemia. Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros activity (genetic alteration and functional inactivation) in high-risk B-ALL is associated with resistance to conventional chemotherapy and a poor prognosis. Ikaros binds to the promoters and/or upstream regulatory sequences of its target genes and regulates their transcription via chromatin remodeling. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions and interferes with Ikaros' ability to repress cell cycle progression and the PI3K pathway. Targeted inhibition of CK2 using specific inhibitor CX4945 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. CX4945 showed strong in vivo anti-leukemia effect in high risk B-ALL patient derived xenograft models (Song et al, Blood 2015). CX4945 is currently in clinical trial for solid tumors. mTOR is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Rapamycin is a specific inhibitor of mTOR that is currently in clinical trial for the treatment of relapsed/refractory leukemia. Here we report in vivo efficacy of combination therapy of rapamycin with CX4945 using high risk B-ALL pre-clinical models and describe mechanism of synergistic action of CX4945 and rapamycin. Methods and results: The therapeutic effect of the CX-4945, in combination with rapamycin was tested using three different patient-derived xenografts (PDX) from patients with high-risk B-ALL and its efficacy will be compared to single-drug treatment. All these leukemia samples were obtained from high risk pediatric B-ALL patients with high CK2 expression and one had IKZF1 haplo - deficiency. After engraftment following tail vein inoculation of leukemia cells into immunocompromised (NRG) mice, mice were treated with vehicle, rapamycin or CX4945 alone or combination of both CX4945 and rapamycin. Anti-tumor effect was measured by quantifying leukemia burden in bone marrow, spleen and peripheral blood by cell count and immune-phenotyping using flow cytometry. Overall survival was measured using Kaplan -Myers analysis in another cohort after similar treatment regimen. The analysis of Ikaros' global genome-wide occupancy in the human B-ALL cell line, Nalm6, and in primary cells from patient with B-ALL, showed that Ikaros binds to the upstream regulatory element (URE) of MTOR gene. Further, Ikaros binding to MTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating mTOR transcription in Nalm-6 B-ALL cell line was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA results in increased transcription of mTOR whereas overexpression of Ikaros by transduction of Nalm-6 cells with Ikaros-containing retrovirus resulted in sharp reduction of mTOR expression. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR. Over-expression of CK2 via retroviral transduction resulted in increased transcription of the mTOR mRNA, as measured by qRT-PCR, as well as increased overall expression of mTOR protein, as measured by western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the MTOR gene promoter. CK2 inhibition was also associated with reduced histone H3K9ac and H3K4me3 marks at the mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In summary, these results establish in vivo anti-leukemia effect of combination therapy using rapamycin and CX4945 in high risk B-ALL PDX models. CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for evaluating clinical use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway. The results of our study establish a new paradigm for targeting the transcriptional network that regulates expression of the gene in hyperactive oncogenic signaling pathways such as mTOR. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Spontaneous Remission of Secondary Acute Lymphoblastic Leukemia Associated with Lenalidomide Therapy for Multiple Myeloma

Introduction: Multiple myeloma (MM) is an incurable malignancy of B-cell lineage. Introduction of immunomodulatory drugs such as lenalidomide has significantly improved overall survival of patients with MM. Lenalidomide maintenance is currently the standard of care for maintaining remission in MM. However, second primary malignancies are known to arise in patients on lenalidomide though their pathogenesis is not known. We report a patient who developed B-lymphoblastic leukemia (B-ALL) while on lenalidomide, which went into spontaneous remission after stopping lenalidomide. Whole exome sequencing (WES) was performed to examine the mutational landscape and clonal evolution of the different malignant clones. We also aimed to postulate a mechanism for lenalidomide-induced ALL. Case history and methods: A 59-year-old female with history of rheumatoid arthritis was diagnosed with IgG-κ MM and treated with 8 cycles of bortezomib, Lenalidomide and dexamethasone followed by lenalidomide-maintenance (10 mg/day). At 36 months after initiation of treatment for MM, she developed lymphopenia and her bone marrow (BM) biopsy showed 38% leukemic B lymphoblasts. Lenalidomide was discontinued and a follow-up BM biopsy done 2 months later showed spontaneous complete remission of ALL, which was confirmed at 8 months. She remains in complete remission of ALL and MM without any specific treatment at 12 months after the diagnosis of ALL. BM aspirate samples at diagnosis of MM, on lenalidomide-maintenance with MM in remission, at diagnosis of ALL, and after stopping lenalidomide with MM and ALL in remission were used to perform WES with 2x150 bp reads and 100x coverage utilizing the Illumina Hiseq. The raw reads were mapped to reference genome (hg19) and compared with peripheral blood T-lymphocytes as germ line control. Variants were annotated using dbSNP, CLINVAR and COSMIC through Mutect. Clonal evolution was analyzed by SciClone. The study was approved by Institutional Review Board. Results: The burden of somatic mutations was significantly higher at diagnosis of MM when compared to the three other time points. Analysis of clonal architecture revealed the distinct clustering of mutations at specific time points (Figure). Most mutations detected at diagnosis of MM (e.g. NOTCH2, BTG1, BCLAF1) disappeared after treatment for MM. Similarly, most mutations detected only at diagnosis of ALL (e.g.PIK3CD, CDK16) became undetectable at spontaneous remission. Interestingly, clones with mutations of IGSF3 (immunoglobulin superfamily) and CXXC4 (Wnt signaling pathway) were detectable while the patient was on lenalidomide and at diagnosis of ALL but disappeared after stopping lenalidomide, which suggests that these clones gained pro-survival advantage from lenalidomide. Only a few mutations (GSDMC, NBPF20, ANAPC1) persisted in both MM and ALL stages. GSDMC is a gasdermin family member which may modulate function of MYC. NBPF20 has been described in relapsed pediatric ALL. ANAPC1 is a cell cycle gene whose transcription is regulated by Ikaros. Loss of repressor function of Ikaros was recently reported to deregulate ANAPC1 expression and cause mitotic progression of ALL in vitro. As lenalidomide is known to induce degradation of Ikaros, we hypothesize that lenalidomide may create a favorable selection pressure for B-cell clones harboring mutations in Ikaros-dependent genes. Conclusions: Clonal evolution analysis suggests that MM and ALL arose from different B-cell sub-clones, which was consistent with previous observation. However, there are a few shared mutations between MM and pre-B ALL, which may be responsible for leukemogenesis in our case. Lenalidomide may affect intracellular protein interactions to induce selection of rare B-cell clones evolving into secondary ALL. Also, our case demonstrated that simply stopping lenalidomide may lead to spontaneous and durable regression of ALL. Transcriptomic and proteomic analysis including Ikaros expression is required to further understand the mechanism of appearance of ALL and its regression after stopping lenalidomide. Figure Disclosures Shlomchik: BlueSphere Bio: Other: Founder and Equity Interest.

Cellular signaling and epigenetic regulation of gene expression in leukemia.

Alterations in normal regulation of gene expression is one of the key features of hematopoietic malignancies. In order to gain insight into the mechanisms that regulate gene expression in these diseases, we dissected the role of the Ikaros protein in leukemia. Ikaros is a DNA-binding, zinc finger protein that functions as a transcriptional regulator and a tumor suppressor in leukemia. The use of ChIP-seq, RNA-seq, and ATAC-seq—coupled with functional experiments—revealed that Ikaros regulates both the global epigenomic landscape and epigenetic signature at promoter regions of its target genes. Casein kinase II (CK2), an oncogenic kinase that is overexpressed in leukemia, directly phosphorylates Ikaros at multiple, evolutionarily-conserved residues. Phosphorylation of Ikaros impairs the protein's ability to regulate both the transcription of its target genes and global epigenetic landscape in leukemia. Treatment of leukemia cells with a specific inhibitor of CK2 restores Ikaros function, resulting in cytotoxicity of leukemia cells. Here, we review the mechanisms through which the CK2-Ikaros signaling axis regulates the global epigenomic landscape and expression of genes that control cellular proliferation in leukemia.

Abstract 4400: Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC16 is one of several subunits of the anaphase-promoting complex (APC) with crucial role in cell division. Here, we show evidence that expression of CDC16 in T-ALL is modulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a tumor suppressor protein and transcription factor. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both cell lines and primary human ALL, shows that Ikaros binds to the promoter of the CDC16 gene. Ikaros functions as a tumor suppressor protein and its deletion is associated with development of T-ALL. Ikaros binding to CDC16 promoter was confirmed via quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. The role of Ikaros in regulating CDC16 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA resulted in increased transcription of CDC16 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strong reduction in transcription of CDC16. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of CDC16. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC16 expression. Since Ikaros function in T-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of CDC16 in T-ALL. Overexpression of CK2 via retroviral transduction resulted in increased CDC16 gene transcription as measured by qRT-PCR, as well as increased overall expression of CDC16, as analyzed by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the CDC16 gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor led to reduced expression of CDC16 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC16. Ikaros knock-down restored high expression of CDC16 in T-ALL cells that were treated with CK2 inhibitors. These data demonstrate that CK2 and Ikaros are major transcriptional regulators of CDC16 transcription in T-ALL and that inhibition of CK2 represses CDC16 transcription via Ikaros-mediated repression. In summary, these results provide evidence that expression of the CDC16 gene in T-ALL is regulated by the CK2 signaling pathway which modulates Ikaros funtion. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in preclinical models of T-ALL. Presented data reveals a novel mechanism of therapeutic action of CK2 inhibitors – repression of CDC16 expression via Ikaros. These results provide a rationale for the use of novel CK2 inhibitors in T-ALL to target CDC16. Citation Format: Pavan Kumar Dhanyam Raju, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jonathan Payne, Soumya Iyer, Nathalia Moreno Curry, Mary McGrath, Yevgeniya Bamme, Bihua Tan, Mario Soliman, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CDC16 expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4400.

Abstract 2620: Global epigenetic regulation of gene expression and tumor suppression in T-cell leukemia by Ikaros

Abstract Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros function results in the development of high-risk B-cell and T-cell acute lymphoblastic leukemia. Previous studies suggested that Ikaros controls expression of its target genes via chromatin remodeling, and by forming complexes with the histone deacetylase, HDAC1. However, the mechanisms through which Ikaros regulates the global epigenetic signature in leukemia is unknown. We studied the role of Ikaros in regulating tumor suppression in Ikaros-defficient T-cell acute lymphoblastic leukemia (T-ALL) cells. The re-introduction of Ikaros at physiological levels via retroviral transduction into T-ALL cells, results in a cessation of cellular proliferation and cell cycle arrest within 4 days. To determine the dynamics of Ikaros-mediated epigenetic regulation of gene expression in T-ALL, we performed ChIP-seq of Ikaros, HDAC1, and histone modifications, along with microarray and ATAC-seq in wild-type T-ALL (that lacks Ikaros), and during each of 3 days following the re-introduction of Ikaros. Our results demonstrated that Ikaros directly regulates the global epigenomic landscape including genome wide de novo formation of enhancers in T-ALL. Further analysis showed that Ikaros induces the re-distribution of HDAC1 via direct recruitment of HDAC1 to promoter and enhancer regions of different target genes resulting in their transcriptional repression. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and HDAC1, and are responsible for the cessation of proliferation and the cell cycle arrest of T-ALL cells. Our results identified novel functions of Ikaros in the epigenetic regulation of gene expression and tumor suppression in T-ALL. Citation Format: Yali Ding, Bo Zhang, Jonathon L. Payne, Kimberly J. Payne, Chunhua Song, Chandrika Gowda, Soumya Iyer, Pavan K. Dhanyamraju, Dhimant Desai, Suming Huang, Feng Yue, Sinisa Dovat. Global epigenetic regulation of gene expression and tumor suppression in T-cell leukemia by Ikaros [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2620.

Abstract 286: Synergistic efficacy of CK2 inhibitor with common chemotherapy drugs by restoring Ikaros function in high-risk ALL

Abstract Objective: IKZF1 gene-coding protein, Ikaros functions as a leukemia suppressor. Casein Kinase II activity is overexpressed in acute lymphoblastic leukemia (ALL) and CK2-mediated-dysfunction of Ikaros is one of the key reason for high-risk ALL and CK2 inhibitor -CX4945 treatment shows high therapeutic efficacy on high-risk ALL. The anti-apoptotic factors are highly expressed in leukemia and the commonly-used 1st-line chemotherapy drugs exerts the anti-tumor effect by suppression of anti-apoptosis signaling. Ikaros binding peaks was identified in the promoter of anti-apoptotic genes by ChIP-seq, suggesting Ikaros regulation on their expression. These observations also suggest the synergistic effect of restoring Ikaros function with common chemotherapy durgs in ALL. Methods: The ChIP-seq and qChIP assays were performed to determine the enrichment of Ikaros and H3K4me3 in promotor of the genes. Lentiviral Ikaros or IKZF1 shRNA were used for functional analysis. WST-1 cell proliferation assay, Annexin-V staining plus flow cytometry and Patients-derived xenograft mouse (PDX) model were used for observing the anti-tumor effect in vitro and in vivo, respectively. Results: ChIP-seq and qChIP assays identified Ikaros binding peaks in the promoter of anti-apoptotic genes in cell-lines and patients’ samples. Ikaros overexpression suppresses but IKZF1 knockdown promotes the gene expression. CX-4945 suppresses the expression of the genes by decreasing the H3k27me3 enrichment in an Ikaros and HDAC1-dependent manner in B-ALL cells. The anti-apoptotic gene is significantly up-regulated in ALL patients. CX-4945+chemoterhapy drugs significantly induces the cell proliferation arrest and apoptosis compared to single drugs in vitro and also show the synergistic effect analyzed by CalcuSyn software. CX-4945+chemotherapy drugs significantly reduced the total leukemia cells and % leukemic cells in the three high-risk B-ALL Patient Derived Xenograft (PDX) mice model compared to that of single drugs, which indicated that their synergistic therapeutic efficacy on leukemia development. Conclusion: Ikaros suppressed anti-apoptotic gene expression through histone modification in ALL. CK2 inhibitor, CX-4945 by restoring Ikaros function have synergistic efficacy with common chemotherapy drugs on high-risk B-ALL. Citation Format: chunhua song, Zheng Ge, Chandrika Gowda, Yali Ding, Jonathon Payne, Bihua Tan, Nathalia M. Cury, Elanora Dovat, Zhijun Zhao, Xiaoguang Lyu, Mary McGrath, Dhimant Desai, Soumya lyer, Pavan K. DhanyamRaju, Kimberly J. Payne, Sinisa Dovat. Synergistic efficacy of CK2 inhibitor with common chemotherapy drugs by restoring Ikaros function in high-risk ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 286.

Abstract 1297: Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML)

Abstract Casein Kinase II (CK2) is a pro-oncogenic kinase that is overexpressed in a majority of human malignancies, including leukemia. One third of AML cases without detectable high risk molecular alteration have high expression of CK2 which is strongly associated with poor outcome. Previously published data showed that CK2 directly phosphorylates several transcription factors including Ikaros (encoded by IKZF1) resulting in reduced DNA-binding affinity and loss of Ikaros function as a tumor suppressor. Here we present the evidence demonstrating role of Ikaros and CK2 in regulation of apoptosis in AML. We show that CK2 inhibitor, CX4945 has strong anti-leukemic effect in AML and one of the novel mechanisms of action involves restoration of Ikaros function as a transcriptional regulator of genes involved in apoptosis. Results: Treatment of AML cell lines and primary cells with CK2 inhibitor, CX4945 show cytotoxicity (IC50 3-5uM), increased apoptosis, cell cycle arrest and poor colony formation. Treatment of murine xenografts of AML with CX4945 showed significant anti-tumor effect. Further studies dissecting the mechanism of action of CX4945 are as follows: Genome-wide binding studies using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) demonstrated that treatment of AML cell line, U937 with CX4945 at IC50 concentration enhances binding affinity of Ikaros transcription factor at the promoter regions of several target genes. Among the significantly affected genes (more than two fold change) are anti-apoptotic genes such as BCL2A1 (B Cell Lymphoma 2 related protein A1) and Bcl-xL (B Cell Lymphoma extra-Large). Dysregulation of apoptotic pathway is hallmark of cancer cells. These findings were confirmed qChIP where CX4945 treated AML cells showed increased binding of Ikaros to the BCL2A1 and Bcl-xL promoters. We used gain-of-function and loss-of-function experiments to determine how Ikaros regulates BCL2A1 and Bcl-xL genes in AML. Ikaros overexpression using lentiviral transduction results in reduced expression of BCL2A1 and Bcl-xL both at mRNA and protein level. Similar results were seen with silencing of CK2 using CK2 SiRNA as well as CX4945. Increased expression of BCL2A1 and Bcl-xL was noted after Ikaros silencing using SiRNA which did not revert with CX4945 treatment.In conclusion, (1) Ikaros represses transcription of anti-apoptotic genes BCL2A1and Bcl-xL, (2) CK2 directly phosphorylates tumor suppressor Ikaros and inhibits its function as transcriptional regulator, (3) Inhibition of CK2 enhances Ikaros-mediated repression of BCL2A1 and Bcl-xL genes resulting in increased apoptosis, (4) CX4945 has strong anti-leukemia effect in AML xenograft models. CK2 is a promising therapeutic target in AML. Our results unveal a novel mechanism of action of CK2 inhibitor, CX4945 in AML which involves restoration of Ikaros mediated regulation of apoptosis. Citation Format: Badhauria Preeti, Soumya Iyer, Elanora Dovat, Pavan Kumar Dhanyam Raju, Jonathan Payne, Chunhua Song, Yali Ding, Claxton David, Sharma Arati, Chandrika Gowda. Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1297.

Ikaros tumor suppressor function includes induction of active enhancers and super-enhancers along with pioneering activity

Ikaros encodes a transcription factor that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which Ikaros regulates gene expression and cellular proliferation in T-ALL are unknown. Re-introduction of Ikaros into Ikaros-null T-ALL cells resulted in cessation of cellular proliferation and induction of T-cell differentiation. We performed dynamic, global, epigenomic, and gene expression analyses to determine the mechanisms of Ikaros tumor suppressor activity. Our results identified novel Ikaros functions in the epigenetic regulation of gene expression: Ikaros directly regulates de novo formation and depletion of enhancers, de novo formation of active enhancers and activation of poised enhancers; Ikaros directly induces the formation of super-enhancers; and Ikaros demonstrates pioneering activity by directly regulating chromatin accessibility. Dynamic analyses demonstrate the long-lasting effects of Ikaros DNA binding on enhancer activation, de novo formation of enhancers and super-enhancers, and chromatin accessibility. Our results establish that Ikaros’ tumor suppressor function occurs via global regulation of the enhancer and super-enhancer landscape and through pioneering activity. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and Ikaros-induced enhancers, and that are responsible for the cessation of proliferation and induction of T-cell differentiation in T-ALL cells.

Regulation of mTOR expression by Casein Kinase II (CK2) signaling pathway in leukemia

The mammalian target of rapamycin(mTOR) is a kinase encoded by the MTOR gene. mTOR is a critical component of PI3K/mTOR/AKT signaling pathway that positively regulates proliferation of leukemia cells. Targeting mTOR and PI3K/mTOR/AKT pathway has been used to treat T‐cell acute lymphoblastic leukemia (T‐ALL). However, little is known about mechanisms that regulate transcription and expression of mTOR gene in T‐ALL. Here, we present the evidence that expression of mTOR in T‐ALL is regulated at transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of the Ikaros, a transcription factor and a tumor suppressor protein. Global chromatin immunoprecipitation coupled with the next‐generation sequencing (ChIP‐seq) studies in primary human T‐ALL, demonstrated that Ikaros binds of the promoter of mTOR gene. Ikaros binding to mTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T‐ALL cells. The role of Ikaros in regulation of mTOR transcription in T‐ALL was tested using gain‐of‐function and loss‐of‐function experiments. Ikaros knock‐down with shRNA, results in increased transcription of mTOR in T‐ALL. Overexpression of Ikaros in human T‐ALL was associated with strongly reduced transcription of mTOR. In mice, T‐ALL cells that are derived from Ikaros‐knockout mice express high level of mTOR. Transduction of these cells with Ikaros‐containing retrovirus, results in sharp reduction of mTOR expression. Since Ikaros function in T‐ALL is negatively regulated by pro‐oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR in T‐ALL. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the MTOR gene, as well as increased overall expression of mTOR, as measured by Western blot. Increased expression of CK2 was associated with loss of Ikaros binding to the promoter of the MTOR gene. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of mTOR in primary T‐ALL. Inhibition of CK2 was associated with increased Ikaros binding at promoter of mTOR. Ikaros knock‐down restored high expression of mTOR in T‐ALL cells that are treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of mTOR transcription in T‐ALL and that CK2 inhibition represses mTOR transcription via Ikaros‐mediated repression. CK2 inhibition, along with an increased Ikaros binding was associated with reduced histone H3K9ac and H3K4me3 marks at mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In conclusion, results presented the evidence that expression of mTOR oncogene is regulated by CK2 signaling pathway that involves Ikaros activity in T‐ALL. Results provide the rationale for the use of CK2 inhibitors in T‐ALL to target PI3K/mTOR/AKT pathway.Support or Funding InformationR01CA209829‐02 (SD and KP) and R01CA213912‐01A1 (SD)St. Baldrick's foundation ( SD and CG) and Hyundai Hope on wheels (SD and CG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aberrant ARID5B expression and its association with Ikaros dysfunction in acute lymphoblastic leukemia

Mutations and single nucleotide polymorphisms of AT-rich interactive domain-containing protein 5B (ARID5B) are involved in the oncogenesis of acute lymphoblastic leukemia (ALL) and treatment outcomes. However, ARID5B expression and clinical significance in ALL remain unclear. We found ARID5B is significantly down-regulated in ALL compared to healthy bone marrow controls. ARID5B also interacts with PHD finger protein 2 (PHF2). Low expression of ARID5B (ARID5Blow) or ARID5B and PHF2 (ARID5BlowPHF2low) is correlated with the markers of cell proliferation and poor prognosis in ALL patients. Ikaros directly regulates ARID5B expression in ALL. Restoring Ikaros function by Casein Kinase II inhibition also promotes ARID5B expression through recruitment of trimethylation of lysine 4 on histone H3 (H3K4me3) at its promoter region. In summary, our data show that aberrant expression of ARID5B and PHF2 is related to leukemic cell proliferation and several poor prognostic markers. Our data indicate ARID5Blow expression, particularly ARID5BlowPHF2low expression, is linked to Ikaros dysfunction and involved in the oncogenic effect of high-risk ALL, which may represent a high-risk subgroup of ALL.

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1-/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

Abstract C44: Epigenetic regulation of CRLF2 oncogene expression by Casein Kinase II (CK2) signaling in B-cell acute lymphoblastic leukemia that occurs at high frequency in Hispanic children

Abstract B-cell acute lymphoblastic leukemia (B-ALL) occurs more frequently in Hispanic children as compared to non-Hispanic whites. The specific subtype of B-ALL that is caused by overexpression of CRLF2 (CRLF2 B-ALL), occurs 5 times more frequently in Hispanic children as compared to the others. Since this type of B-ALL is associated with poor prognosis, the death rate of B-ALL is 39% higher in Hispanic children than in non-Hispanic whites. Thus, understanding the molecular mechanisms that regulate CRLF2 expression in CRLF2 B-ALL is essential for the development of targeted therapy for this disease. Our previous work determined that transcription of CRLF2 is negatively regulated by a tumor-suppressor protein, Ikaros. Ikaros deregulation is a feature of over 80% of CRLF2 B-ALL. Here we present evidence that Ikaros-mediated repression of CRLF2 transcription in B-ALL in Hispanic children is regulated by Casein Kinase II (CK2). CK2 is an oncogenic kinase that is overexpressed in B-ALL. We have previously shown that CK2 can directly phosphorylate Ikaros and that phosphorylation by CK2 can impair Ikaros function as transcriptional regulator. We tested whether inhibition of CK2 affects the ability of Ikaros to regulate CRLF2 transcription. Molecular inhibition by shRNA that targets the catalytic subunit of CK2, as well as pharmacologic inhibition of CK2 by the specific inhibitor, CX-4945, resulted in reduced expression of CRLF2 as measured by qRT-PCR. This was associated with increased Ikaros binding to the CRLF2 promoter as measured by quantitative chromatin immunoprecipitation (qChIP). To determine whether Ikaros function is essential for CRLF2 repression following CK2 inhibition, we compared expression of CRLF2 in cells that have Ikaros knocked-down by shRNA vs. cells with control shRNA, following treatment with CX-4945. Results showed that Ikaros knockdown abolished the ability of CK2 inhibitors to repress transcription of CRLF2 in B-ALL. These results demonstrate that Ikaros is an essential component of CK2 signaling that regulates CRLF2 expression. Analysis of the epigenetic signature at the CRLF2 promoter performed by serial qChIP assays showed that increased Ikaros binding to the CRLF2 promoter, following CK2 inhibition, is associated with enrichment for the H3K9me3 histone modification, which is a marker of repressive chromatin. In conclusion, we demonstrate that expression of the CRLF2 oncogene in acute leukemia that disproportionally occurs in Hispanic children is epigenetically regulated by the CK2-Ikaros axis. In CRLF2 B-ALL, Ikaros-mediated repression of CRLF2 is impaired due to overexpression of CK2. Treatment of CRLF2 B-ALL with CK2 inhibitors restores Ikaros tumor suppressor function, resulting in CRLF2 repression. Results identified a signaling network that regulates CRLF2 expression and suggest that restoration of Ikaros activity with CK2 inhibitors can be a therapeutic approach for CRLF2 B-ALL to reduce the health disparity for Hispanic children with ALL. Supported by 1R01CA209829. Citation Format: Chunhua Song, Zheng Ge, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of CRLF2 oncogene expression by Casein Kinase II (CK2) signaling in B-cell acute lymphoblastic leukemia that occurs at high frequency in Hispanic children [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr C44.

Abstract 1512: Regulation of the CDC42 signaling pathway by IKZF1 in T-cell acute lymphoblastic leukemia

Abstract The IKZF1 gene encodes Ikaros—a DNA-binding, Kruppel-like zinc finger protein that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). Ikaros regulates transcription of a large number of genes that are involved in control of cellular proliferation. However, the molecular mechanisms through which Ikaros regulates proliferation of T-ALL cells are still unknown. The use of quantitative chromatin immunoprecipitation (qChIP) showed that Ikaros binds to the promoter regions of CDC42 in vivo in primary T-ALL patient samples as well as in T-ALL cell lines. Ikaros overexpression in B-ALL cells via retroviral transduction results in decreased transcription of CDC42, as evidenced by qRT-PCR and Western blot. The luciferase reporter assay showed that Ikaros represses CDC42 transcription by directly binding to its promoter. The shRNA-mediated knock-down of Ikaros in T-ALL resulted in increased expression of CDC42. These results suggest that Ikaros functions as a transcriptional repressor of CDC42 in T-ALL. Next, we studied the upstream signaling pathways that regulate Ikaros-mediated control of CDC42 transcription in T-ALL. Because Ikaros activity in leukemia is regulated via direct phosphorylation by Casein Kinase II (CK2), we tested the effect of CK2 inhibition on Ikaros-mediated regulation of CDC42 expression. Treatment of T-ALL cells with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of CDC42 in T-ALL cells. This was associated with an increase in DNA-binding of Ikaros to the promoter of the CDC42 gene. Ikaros knock-down restored high expression of CDC42 in T-ALL cells that were treated with CK2 inhibitors. These data suggest that transcriptional repression of the CDC42 gene by Ikaros is impaired in T-ALL due to increased CK2 activity and reduced Ikaros DNA-binding affinity toward the CDC42 gene promoter. Inhibition of CK2 restores Ikaros function and results in transcriptional repression of the CDC42 gene. In conclusion, our results suggest that Ikaros regulates CDC42 signaling pathway via transcriptional repression of the small GTPase, CDC42, in T-ALL. Ikaros-mediated repression of CDC42 in T-ALL is negatively regulated by CK2. These results suggest that regulation of CDC42 expression is one of the mechanisms through which Ikaros regulates cellular proliferation in T-ALL. Citation Format: Mario Soliman, Chunhua Song, Jonathon L. Payne, Zheng Ge, Chandrika Gowda, Yali Ding, Kimberly J. Payne, Sinisa Dovat. Regulation of the CDC42 signaling pathway by IKZF1 in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1512.

Ikaros regulates B-cell tolerance

Abstract B-cell development and activation are tightly regulated at multiple steps to ensure a protective immune response, and at the same time avoiding harmful self-reactivity and autoimmunity. Ikaros is a transcription factor that is critical for B-cell development, as demonstrated by the complete lack of B-cells in Ikaros-null mice. Furthermore, Ikaros is shown to play important roles also at later stages of B-cell development, but the precise roles of Ikaros at different stages of B-cell development is still not fully understood. In recent years, Ikaros (encoded by the IKZF1 gene) has been linked to autoimmune disease in humans through both genome-wide association studies (GWAS) as well as recent reports of germline IKZF1 mutations in patients with autoimmune disease. However, the mechanisms underlying altered Ikaros function and the development of autoimmunity are not known. We previously developed Ikaros-mutant mouse models with targeted deletions of the exons encoding the DNA-binding zinc finger 1 (ZnF1) or ZnF4, and found that both mutants have B cells, but display selective partial defects at different stages of B-cell development. We recently found that the Ikzf1-ZnF4-mutant strain displays very high levels of serum Anti-Nuclear Antibodies (ANA) at young age, a hallmark of autoimmune disease. This indicates that Ikaros (and specifically exon 6 encoding ZnF4) is required to regulate B-cell tolerance, and we hypothesize that Ikaros regulates this, at least in part, by establishing a chromatin structure that sets restrictions on B-cell responses to limit autoreactive B cells. We are using our Ikaros-mutant mice to further study the role of Ikaros in B-cell development and tolerance, and will present our results to-date at the meeting.

A kindred with mutant IKAROS and autoimmunity

IKAROS (encoded by IKZF1) is an important hematopoietic transcription factor critical for early B cell differentiation, with major defects known to lead to low B cell numbers and hypogammaglobulinemia. More perplexing is the link between IKZF1 variants and autoimmunity, including polymorphisms associated with susceptibility to SLE, and recently, rare variants driving monogenic autoimmunity. We identified a novel p.L188V mutation in IKZF1 in the index patient and her father and found this mutation to lead to loss of DNA binding. Peripheral B cells lacking a full complement of IKAROS function show upregulation of molecules accentuating B cell activation, while CD22, a key negative feedback circuit, is suppressed. The resulting hyperresponsiveness of peripheral B cells, in combination with elevated follicular helper T cell (Tfh) numbers, provides a putative mechanistic explanation for the association of IKZF1 variants with the emergence of autoimmune manifestations in this kindred.

The Ikaros family in lymphocyte development.

The IKZF family of transcription factors are essential regulators of lymphopoiesis. Ikaros, Helios, Aiolos and Eos function as transcriptional repressors and activators during T and B cell differentiation and in mature cell function, depending on the stage of development and/or cell type. Their potential mechanisms of action are varied. Ikaros family proteins partner with multiple complexes, including NuRD, PRC2 and transcription elongation factors, to modulate gene expression and the chromatin state. In humans, mutations in the IKZF genes are associated with B cell deficiency, leukemias and autoimmunity. In this review, we focus on the function of Ikaros family proteins in early T and B lymphocyte development, and discuss the molecular and physiological activities of this family.

Lack of Ikaros cripples expression of Foxo1 and its targets in naive T cells.

Ikaros is a transcription factor that regulates lymphocyte development from the level of the haematopoietic stem cell. Lack of Ikaros reduces the ability of progenitor cells to commit to the T-cell lineage, resulting in reduced numbers of early thymic T-cell progenitors and mature T cells. Mature CD4 T cells that lack Ikaros have defects in proliferation, T helper cell differentiation, cytokine expression and the ability to become anergic. A role for Ikaros in the naive T cell has not yet been identified. The receptors interleukin-7 receptor α (IL-7Rα) and l-selectin are important for ensuring survival and proper homing of naive T cells, respectively. Here we show that lack of Ikaros leads to reduced expression of these receptors in naive T cells, which impacts their ability to home and survive in response to IL-7. We define the mechanism underlying this phenotype as a requirement for Ikaros in maintenance of expression of Foxo1, a transcriptional regulator that is required for their expression. We also demonstrate that CD4 T cells lacking Ikaros are significantly crippled in their ability to become induced regulatory T cells, a phenotype also linked to reduced Foxo1 expression. Finally, we show that restoring Ikaros function to Ikaros-deficient CD4 T cells increases levels of Foxo1 message. Together, these studies define, for the first time, a role for Ikaros in naive T cells and establish it as the first transcriptional regulator required for maintaining levels of Foxo1 gene expression in these cells.