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.