Regulation of Ikaros Function by CK2 Kinase During Lymphocyte Differentiation and Leukemia

Abstract 4196

Ikaros encodes a DNA-binding zinc finger protein that functions as a master regulator of lymphocyte differentiation and acts as a tumor suppressor in leukemia. The loss of Ikaros activity has been associated with both B and T cell leukemia and deletion of Ikaros has been associated with poor outcome in childhood ALL. Ikaros function depends on its ability to localize to pericentromeric heterochromatin (PC-HC). Ikaros protein binds to the upstream regulatory elements of target genes, aids in their recruitment to PC-HC, and regulates their transcription. Ikaros protein interacts with histone deacetylation complex and represses transcription of its target genes via chromatin remodeling. We have previously reported that Ikaros' activity and protein stability is regulated by pro-oncogenic CK2 kinase (Popescu et al. J Biol Chem 2009 284:13869). Here we studied the effect of CK2-mediated phosphorylation on Ikaros function in primary thymocytes. Using Ikaros phosphomimetic and phosphoresistant mutants of CK2 phosphorylation sites we demonstrate that Ikaros proteins with phosphomimetic mutations at CK2 phosphorylation sites 1) have decreased DNA-binding affinity for the promoter of the terminal deoxynucleotidetransferase (TdT) gene, and other Ikaros target genes involved in lymphocyte differentiation; 2) lose the ability to associate with Sin3a, a component of the NuRD histone deacetylase complex, as indicated by co-immunoprecipitation assays and 3) fail to repress genes involved in thymocyte differentiation as indicated by luciferase reporter assay. The introduction of phosphoresistant mutations at five N-terminal CK2 phosphorylation sites on the Ikaros protein restored Ikaros' association with Sin3a, as well as wild-type levels of repressor activity. Treatment of primary thymocytes with specific inhibitors of CK2 kinase (TBB or DMAT) resulted in increased Ikaros' DNA-binding affinity to the promoter regions of its target genes as measured by quantitative chromatin immunoprecipitation, similar to the results with phosphoresistant mutants. We tested the effect of CK2 kinase inhibition on Ikaros' activity in human leukemia cells. Inhibition of CK2 kinase with TBB led to increased Ikaros' DNA-binding affinity and increased repression of Ikaros target genes. Increased Ikaros activity following inhibition of CK2 kinase was associated with increased sensitivity of human leukemia cells to Doxorubicin or radiation. In summary, these results demonstrate that CK2 kinase regulates thymocyte differentiation by controlling Ikaros' association with chromatin remodeling complexes and its ability to repress the transcription of developmentally regulated genes. Results suggest that CK2 kinase exerts its pro-oncogenic activity in human leukemia cells by inhibiting Ikaros' function as a tumor suppressor. Inhibition of CK2 kinase restores Ikaros function in thymocyte differentiation, along with its tumor suppressor activity and led to increased sensitivity of human leukemia cells to chemotherapy and/or radiation treatment. Thus, inhibition of the CK2 kinase pathway is a promising therapeutic target for human leukemia.