Epigenetic Regulation of c-Kit Expression in Acute Myeloid Leukemia By Oncogenic Casein Kinase II (CK2)

Abstract The c-kit gene encodes the type III receptor tyrosine kinase, CD117, which functions as a receptor for stem cell factor (SCF). Overexpression and/or activating mutations of c-kit have been detected in a large number of acute myeloid leukemias (AML), systemic mastocytosis, as well as other types of human malignancies. Several studies suggest that activating mutations of c-kit, as well as its overexpression, are associated with worse outcomes in some types of AML. However, very little is known about mechanisms that regulate c-kit expression in AML. Here we report that expression of c-kit in AML is regulated on the transcriptional level by oncogenic Casein Kinase II (CK2) and the Ikaros protein. Global genome-wide binding studies using chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq), as well as quantitative chromatin immunoprecipitation (qChIP), demonstrate that the Ikaros tumor suppressor protein binds to the promoter of the c-kit gene in AML. Overexpression of Ikaros in AML cells results in reduced expression of c-kit at both the mRNA and protein levels. Ikaros knock-down with shRNA results in increased transcription of c-kit in AML. These data suggest that Ikaros represses transcription of c-kit. Previous studies suggested that CK2 impairs the ability of Ikaros to regulate transcription of its target genes in leukemia. Since CK2 is overexpressed in AML, we tested whether CK2 can regulate expression of c-kit in this leukemia. Results showed that molecular inhibition of CK2 with shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor, CX-4945, results in reduced transcription of c-kit in AML cell lines and in primary AML cells. This was associated with increased binding of Ikaros to the c-kit promoter, as evidenced by qChIP. Ikaros knock-down with shRNA abolished the ability of CK2 inhibitors to repress c-kit transcription in AML. These data demonstrate that Ikaros is a key regulator of c-kit transcription in AML, and that CK2 inhibitors repress transcription of c-kit by increasing Ikaros binding to the c-kit promoter in AML. These results suggest that increased expression and/or activity of CK2 in AML positively regulates c-kit expression by inhibiting Ikaros-mediated repression of the c-kit gene. Since it has been previously shown that Ikaros can regulate transcription of its target genes via epigenetic mechanisms, we tested whether CK2 inhibition in AML affects the epigenetic signature at the c-kit promoter. Results showed that treatment with a specific CK2 inhibitor CX-4945 results in enrichment for the repressive chromatin markers, H3K9me3 and H3K27me3, and reduced the H3K9ac marker of active chromatin at the c-kit promoter in both AML cell lines and in primary AML cells. In conclusion, the presented results demonstrate that expression of c-kit in AML is controlled by CK2 and Ikaros via epigenetic regulation of c-kit transcription. These results support the use of CK2 inhibitors for the treatment of AML cases with overexpression and/or activating mutations of the c-kit gene. Disclosures Payne: ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees; ELF Zone Inc: Equity Ownership. Dovat: ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees.