Targeting CK2 And NOTCH1/MYC Signaling In T-cell Acute Lymphoblastic Leukemia

Background:T-cell acute lymphoblastic leukemia(T-ALL)is an aggressive cancer derived from developing thymocytes.Despite the advance of modern therapy,T-ALL remains fatal in 20%of pediatric and 50%of adult patients.Moreover,frequent application of multi-agent cytotoxic drugs often leads to high toxicities and drug resistance,underscoring the need for targeted therapy.In the majority of T-ALL cases,the NOTCH]signaling is aberrantly activated due to gain-of-function NOTCH1 mutations.Suppression of aberrant NOTCH1 signaling in T-ALL cells by gamma secretase inhibitors(GSIs)has been met with much enthusiasm in the past decade.However,the gastrointestinal toxicities and drug resistance of GSIs restrain their clinical applications.The proto-oncogene MYC is a transcriptional target of NOTCH 1 and a dominant driver of T-ALL pathogenesis.Targeting MYC-mediated transcriptional programs through BET bromodomain inhibitor JQ1 exhibits anti-leukemic efficacy in vitro and in vivo.However,global repression of transcription by MYC inhibition is predicted to cause toxicities.Identification of drug(s)synergizing with JQ1 to kill T-ALL cells may enhance the efficacy while reducing toxicities.Protein kinase CK2 is a tetrameric serine-threonine kinase composed of two catalytic(α or α’)and one regulatory(β)subunits.CK2 can phosphorylate NOTCH1 and is overexpressed in a variety of solid and hematologic tumors.The activity of CK2 and protein levels of CK2a and CK2β are elevated in primary T-ALL cells.CK2 inhibition by CX-4945,a potent and specific inhibitor tested in clinical trials for breast cancer and multiple myeloma,significantly reduces growth and survival of human T-ALL cells,and down-regulates NOTCH1 in lung cancer cells.However,it remains unclear whether the cytotoxic effect of CX-4945 on T-ALL cells is associated with the repression of the NOTCH 1 signaling.Objective:This study focuses on investigation of the regulatory role of CK2 on the NOTCH1 signaling pathway in the context of T-ALL,with the goal to understand the molecular connections between CK2 and NOTCH1/MYC and to identify novel therapeutic strategies for T-ALL treatment.Methods:We first analyzed databases that are publically available to determine CK2 subunit expression in subsets of developing T cells versus primary patient T-ALL cells.Next,western blotting was used to detect protein levels of CK2 and two major components of the NOTCH1 pathway,cleaved-NOTCH1 and MYC,in T-ALL cell lines and human primary T-ALL samples.Levels of cleaved-PARP(an apoptotic marker)and CK2 activity protein(phospho AKT ser129)were also analyzed by western blotting.RNA interference(RNAi)technology by shRNA targeting CK2a was used to knock down CK2a in JURKAT BCL-2-overexpressing cells and the suppression efficiency of CK2a shRNA was examined by western blotting.The half-life of cleaved-NOTCH1 was measured by pulse-chase analysis upon genetic and pharmacological inhibition of CK2 in T-ALL cells.MYC transcript alterations upon CX-4945 treatment were examined by Quantitative RT-PCR analysis.CellTiter-Blue(?)cell viability assay was used to test the cell viability change upon drug treatment in T-ALL cells.Flow cytometry analysis was performed to detect apoptosis and cell cycle distribution after drug treatment.Mouse xenograft model was established to grow and harvest human primary T-acute lymphoblastic leukemia cells.Results:The expression of protein kinase CK2 is elevated in human patient T-ALL cells,compared with normal thymus tissue and developing T cells.Protein levels of both cleaved-NOTCH1 and its transcriptional target MYC are upregulated in human patient T-ALL cells and positively correlate with expression levels of CK2.CK2 inhibitor CX-4945 suppresses CK2 enzymatic activity,downregulates protein levels of cleaved-NOTCH1 and MYC,and suppresses transcript levels of c-MYC.CK2 inhibition destabilizes NOTCH1 and inactivates NOTCH1 signaling pathway.CX-4945 synergizes with JQ1 to reduce human T-ALL cell viability,but antagonizes with JQ1 in normal peripheral blood mononuclear cells(PBMC)thus conferring minimal sensitivity to these normal cells.The combination treatment of CX-4945 and JQ1 in human T-ALL cells significantly induces apoptosis,compared with either drug alone,while minimally affecting cell cycle phase distribution.Conclusion and Significance:Our studies demonstrate that CK2 inhibitor CX-4945 destabilizes NOTCH1.CX-4945 exhibits striking synergism with JQ1 to kill human T-ALL cells with elevated CK2,cleaved-NOTCH1 and MYC expression.The combination of CX-4945 and JQ1 may offer a better approach to target the NOTCH1 signaling in refractory/relapsed T-ALL.Our studies provide a rationale to test the combination treatment of CX-4945 and JQ1 on human refractory/relapsed T-ALL using pre-clinical in vivo models.Given the wide involvement of CK2 and NOTCH1/MYC in cancers,the combination treatment of JQ1 and CX-4945 should be investigated in other cancer types as well.