IDENTIFICATION OF GENETIC PATHWAYS CONTROLLING RESISTANCE TO STANDARD COMBINATION CHEMOTHERAPY IN ACUTE MYELOID LEUKAEMIA

Abstract

Genetic aberrations and clonal evolution underpin chemoresistant and relapsed acute myeloid leukaemia (AML). We used a genome-wide CRISPR knock-out screen to identify genes which mediate resistance to Cytarabine (AraC) and Doxorubicin (Dox) in AML, informing combination therapies to potentially circumvent resistance. Cas9-expressing human AML cell lines, OCI-AML3 and MV4-11, were transduced with the Brunello gRNA library and treated with continuous or intermittent Dox/AraC (D/A), at synergistic doses sufficient to eliminate non-transduced controls, but allow outgrowth of resistant library-transduced populations. In resistant populations, gRNAs targeting AraC metabolism components, including deoxycytidine kinase (DCK) were enriched. Additionally, cyclin-dependent kinase inhibitor 2a (CDKN2A) and checkpoint kinase 2 (CHEK2) were major hits. CDKN2A and CHEK2 mediate apoptosis and cell cycle arrest in response to DNA damage and are clinically relevant in AML. In competition assays, CDKN2A and CHK2 inactivation by CRISPR led to a survival advantage over empty-vector controls in the presence of D/A, confirming their contribution to chemoresistance. This resistance was mediated by evasion of cell cycle arrest in response to chemotherapy, with minimal reductions in apoptosis seen. Given convergence of CDKN2A and CHEK2 pathways on cell cycle G1S transition, we hypothesised that the CDK4/6 inhibitor, palbociclib could enhance efficacy of D/A therapy in AML with deregulation of CDKN2A or CHEK2. Palbociclib induced a rapid G1 arrest in empty vector, CDKN2A- and CHEK2-inactivated cells by 24 hours. Further, it synergised with D/A over longer treatments (4-11 days), demonstrating the therapeutic potential of cell cycle inhibitors in combination with conventional agents to improve chemotherapy response in AML.