Targeting Casein Kinase II Exerts a Therapeutic Effect in Pediatric High Risk Leukemia Via Inhibition of Cell Cycle Progression and the PI3K Pathway
Authors: Gowda Chandrika S. , Song Chunhua , Ding Yali , Muthusami Sunil , Payne Kimberly , Ge Zheng , Dovat Sinisa 
Published: October 2019

Abstract
IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a tumor suppressor in acute lymphoblastic leukemia. Deletion of one Ikaros allele results in the development of high-risk B-cell acute lymphoblastic leukemia (B-ALL) with a high incidence of relapse and poor prognosis. The mechanisms through which Ikaros suppresses leukemogenesis and that regulate Ikaros tumor suppressor activity in leukemia are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that control two pathways that are crucial in leukemia cell proliferation: 1) cell cycle progression and 2) the phosphatidylinositol 3-kinase (PI3K) pathway. Gain- and loss-of-function experiments demonstrate that Ikaros represses the transcription of genes that promote cell cycle progression and the PI3K pathway and activates transcription of a gene that suppresses the PI3K pathway. We show that in high-risk B-ALL with deletion of one Ikaros allele, the function of Ikaros as a transcriptional regulator is impaired due to reduced DNA-binding affinity for promoters of its target genes. It has been shown 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 high-risk B-ALL. Treatment of primary high-risk B-ALL (with deletion of one Ikaros allele) using the CK2 specific inhibitor, CX-4945, restored Ikaros function as a transcriptional regulator of the genes that regulate cell cycle progression and the PI3K pathway, and was associated with cell cycle arrest and loss of phosphorylation of the AKT kinase - a downstream target of the PI3K pathway. 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; 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. The therapeutic effect of CK2 inhibition by CX-4945 on high-risk B-ALL was demonstrated in vivo using 4 different xenografts: 3 different high-risk primary pre-B-ALL xenografts and Nalm6 xenografts. Treatment with CX-4945 showed a strong therapeutic effect in all 4 xenografts, as evidence by reduced leukemia cell number in bone marrow and in spleen, along with prolonged survival of all xenografts. Expression analysis of Ikaros target genes that regulate cell cycle progression and the PI3K pathway in leukemia cells treated in vivo with CX-4945 revealed an expression pattern that was highly similar to that observed with Ikaros overexpression. This suggests that CK2 inhibition in vivo exerts its therapeutic effect on high-risk B-ALL via restoration of Ikaros function as 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.

Disclosures
No relevant conflicts of interest to declare.