Clinical-grade CK2 inhibitor CX-4945 synergistically enhances venetoclax-mediated antileukemic activity in preclinical acute myeloid leukemia models

Introduction: Dysregulated apoptotic machinery and signaling contribute to acquire resistance to regimens containing venetoclax (VEN), a BCL2 (B-cell lymphoma-2) inhibitor, in acute myeloid leukemia (AML) and pose a clinical challenge. Casein kinase 2 (CK2) is a serine/threonine kinase with 300+ substrates and regulate cell cycle, survival, differentiation, and apoptosis. Its aberrant activity promotes disease progression, poor prognosis, and drug resistance. CX-4945 (CX), a selective CK2 inhibitor, shows potent anticancer activity in leukemia and other solid tumors. Here, we tested combined CK2 and BCL2 targeting with CX and VEN in pre-clinical AML models. Methods: Cytotoxic and pro-apoptotic effects of the CX+VEN combo were tested in AML cell lines and primary samples by WST and annexin V assays. The ZIP synergy score was determined by SynergyFinder tool. Flow cytometry and immunoblotting assessed cell surface markers, various apoptosis regulators along with CK2 target levels after drug treatment. Dynamic BH3 profiling assessed priming of VEN-resistant (VR) cells to apoptosis after CX treatment. Transcriptome of VR-AML cells with and without drug treatment was analyzed by RNA sequencing. In vivo efficacy of CX+VEN combo was tested in cell line and patient-derived xenograft (PDX) mouse models. Results: CK2alpha ( CSNK2A1 ) expression exhibited negative correlation similar to that of BCL2 levels with VEN activity in BeatAML cohort and VR-AML cells showed higher CK2 activity. CX+VEN combo showed synergistic cytotoxicity and augmented apoptosis in VEN-sensitive (MOLM13, HL60, THP1), VR-AML (U937, MOLM13/VR, HL60/VR) cell lines, and PDX cells in vitro . CX treatment enhanced VR-AML cells priming to BH3 peptides and increased cytochrome c release. Also, CX+VEN combo effectively induced apoptosis and decreased leukemia stem cells (CD34+CD38-) and chemo-resistant (CD47+CD123+) subpopulations in VR-AML cells. AML cells showed downregulation of CK2 activity and other pro-survival BCL2 member family proteins with increased PARP activity after CX+VEN combo treatment. Functional enrichment analysis of transcriptome after CX+VEN combo treatment showed upregulation of cell cycle arrest, TP53 and apoptotic signature genes that were repressed in VR-AML cells. Lastly, CX+VEN combo effectively decreased leukemia burden and prolonged overall median survival of xenograft (CDX/PDX) mice in vivo . Conclusions: CX+VEN combo showed a superior antileukemic activity in different pre-clinical AML models and CK2 inhibition overcome VEN resistance. CX-4945 (Silmitasertib) has favorable pharmacokinetics with good tolerability in human studies and is being evaluated in early phases of clinical trial. Our findings provide a rationale for CK2 and BCL2 co-targeting as an effective approach for AML treatment and to overcome VEN resistance.