Protein Kinase CK2 (Casein Kinase 2 or CK2) is a constitutively active serine-threonine kinase overactive in human malignancies. Increased expression and activity of CK2 in Acute Myeloid Leukemia (AML) is associated with a poor outcome. CK2 promotes AML cell survival by impinging on multiple oncogenic signaling pathways. The selective small-molecule CK2 inhibitor CX-4945 has shown in vitro cytotoxicity in AML. Here, we report that CX-4945 has a strong in vivo therapeutic effect in preclinical models of AML. The analysis of genome-wide DNA-binding and gene expression in CX-4945 treated AML cells shows that one mechanism, by which CK2 inhibition exerts a therapeutic effect in AML, involves the revival of IKAROS tumor suppressor function. CK2 phosphorylates IKAROS and disrupts IKAROS’ transcriptional activity by impairing DNA-binding and association with chromatin modifiers. Here, we demonstrate that CK2 inhibition decreases IKAROS phosphorylation and restores IKAROS binding to DNA. Further functional experiments show that IKAROS negatively regulates the transcription of anti-apoptotic genes, including BCL-XL (B cell Lymphoma like–2 like 1, BCL2L1). CX-4945 restitutes the IKAROS-mediated repression of BCL-XL in vivo and sensitizes AML cells to apoptosis. Using CX-4945, alongside the cytotoxic chemotherapeutic drug daunorubicin, augments BCL-XL suppression and AML cell apoptosis. Overall, these results establish the in vivo therapeutic efficacy of CX-4945 in AML preclinical models and determine the role of CK2 and IKAROS in regulating apoptosis in AML. Furthermore, our study provides functional and mechanistic bases for the addition of CK2 inhibitors to AML therapy.

Regulation of oncogenic gene expression by transcription factors that function as tumor suppressors is one of the major mechanisms that regulate leukemogenesis. Understanding this complex process is essential for explaining the pathogenesis of leukemia as well as developing targeted therapies. Here, we provide an overview of the role of Ikaros tumor suppressor and its role in regulation of gene transcription in acute leukemia. Ikaros (IKZF1) is a DNA-binding protein that functions as a master regulator of hematopoiesis and the immune system, as well as a tumor suppressor in acute lymphoblastic leukemia (ALL). Genetic alteration or functional inactivation of Ikaros results in the development of high-risk leukemia. Ikaros binds to the specific consensus binding motif at upstream regulatory elements of its target genes, recruits chromatin-remodeling complexes and activates or represses transcription via chromatin remodeling. Over the last twenty years, a large number of Ikaros target genes have been identified, and the role of Ikaros in the regulation of their expression provided insight into the mechanisms of Ikaros tumor suppressor function in leukemia. Here we summarize the role of Ikaros in the regulation of the expression of the genes whose function is critical for cellular proliferation, development, and progression of acute lymphoblastic leukemia.

IKAROS, encoded by the IKZF1 gene, is a DNA-binding protein that functions as a tumor suppressor in T cell acute lymphoblastic leukemia (T-ALL). Recent studies have identified IKAROS’s novel function in the epigenetic regulation of gene expression in T-ALL and uncovered many genes that are likely to be directly regulated by IKAROS. Here, we report the transcriptional regulation of two genes, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) and phosphoinositide kinase, FYVE-type zinc finger containing (PIKFYVE), by IKAROS in T-ALL. PIK3CD encodes the protein p110δ subunit of phosphoinositide 3-kinase (PI3K). The PI3K/AKT pathway is frequently dysregulated in cancers, including T-ALL. IKAROS binds to the promoter regions of PIK3CD and PIKFYVE and reduces their transcription in primary T-ALL. Functional analysis demonstrates that IKAROS functions as a transcriptional repressor of both PIK3CD and PIKFYVE. Protein kinase CK2 (CK2) is a pro-oncogenic kinase that is overexpressed in T-ALL. CK2 phosphorylates IKAROS, impairs IKAROS’s DNA-binding ability, and functions as a repressor of PIK3CD and PIKFYVE. CK2 inhibition results in increased IKAROS binding to the promoters of PIK3CD and PIKFYVE and the transcriptional repression of both these genes. Overall, the presented data demonstrate for the first time that in T-ALL, CK2 hyperactivity contributes to PI3K signaling pathway upregulation, at least in part, through impaired IKAROS transcriptional regulation of PIK3CD and PIKFYVE. Targeting CK2 restores IKAROS’s regulatory effects on the PI3K oncogenic signaling pathway.

Ikaros is a DNA-binding protein that regulates gene expression and functions as a tumor suppressor in B-cell acute lymphoblastic leukemia (B-ALL). The full cohort of Ikaros target genes have yet to be identified. Here, we demonstrate that Ikaros directly regulates expression of the small GTPase, Rab20. Using ChIP-seq and qChIP we assessed Ikaros binding and the epigenetic signature at the RAB20 promoter. Expression of Ikaros, CK2, and RAB20 was determined by qRT-PCR. Overexpression of Ikaros was achieved by retroviral transduction, whereas shRNA was used to knockdown Ikaros and CK2. Regulation of transcription from the RAB20 promoter was analyzed by luciferase reporter assay. The results showed that Ikaros binds the RAB20 promoter in B-ALL. Gain-of-function and loss-of-function experiments demonstrated that Ikaros represses RAB20 transcription via chromatin remodeling. Phosphorylation by CK2 kinase reduces Ikaros’ affinity toward the RAB20 promoter and abolishes its ability to repress RAB20 transcription. Dephosphorylation by PP1 phosphatase enhances both Ikaros’ DNA-binding affinity toward the RAB20 promoter and RAB20 repression. In conclusion, the results demonstrated opposing effects of CK2 and PP1 on expression of Rab20 via control of Ikaros’ activity as a transcriptional regulator. A novel regulatory signaling network in B-cell leukemia that involves CK2, PP1, Ikaros, and Rab20 is identified.