Abstract 325: A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition
Authors: Tamilla Nechiporuk, Stephen E. Kurtz, Olga Nikolova, Amanda d'Almeida, Kevin Watanabe-Smith, Mara Rosenberg, Brian Druker, Jeffrey W. Tyner, Shannon K. McWeeney
Journal: Cancer Research (2019)

Abstract Therapeutics targeted to specific proteins known to drive or modulate progression of acute myeloid leukemia (AML) have shown limited effectiveness as resistance frequently develops during their clinical use. Similar to many cancers, a stalled process of programmed cell death in apoptosis-primed cells is one of the prevalent mechanisms of drug resistance in AML. The intrinsic mitochondrial apoptotic pathway is controlled by a rheostat of anti-apoptotic and pro-apoptotic proteins and perturbing the balance toward pro-apoptotic responses remains a focus of many small molecular therapeutics. Venetoclax, an inhibitor of anti-apoptotic gene BCL2, approved for treatment of a certain subtypes of chronic lymphocytic leukemia (CLL), shows limited success as a monoagent in AML clinical trials due to an inherent resistance in approximately 37% of all patients (IC50 &amp;gt; 5μM). To identify mechanisms underlying intrinsic and acquired resistance to the BCL2 inhibitor, venetoclax, we used a genome-wide CRISPR/Cas9 screen to identify genes whose inactivation results in loss of venetoclax sensitivity in MOLM13 cells, derived from an AML patient that harbored mutation in FLT3-ITD, one of the major genetic landscapes of AML. Interrogation with two independently derived genome-wide libraries yielded identical top candidates TP53, BAX and PMAIP1. We also observed other gene targets that implicate the mitochondrial intrinsic, TP53-controlled pro-apoptotic pathway in the acquisition of venetoclax resistance. Correlatively, in a large AML cohort (Beat AML dataset), patients with loss of function mutations in TP53, or either low expression of TP53 or BAX exhibited statistically significant lower levels of responses to venetoclax ex vivo. Resultant venetoclax resistant cells with inactivation of TP53 or BAX had significantly reduced capacity for apoptosis when treated with venetoclax and exhibited elevated pro-survival signaling exemplified by increased levels of MAPK and AKT. In addition, loss of TP53 led to changes in the ratio of anti-apoptotic proteins BCL2, BCL-xL and MCL1 implicating TP53 as a major transcriptional regulator of the apoptotic rheostat in MOLM13 cells. Evaluation of the TP53 and BAX knockout cells for sensitivities to a panel of small molecule inhibitors revealed loss of sensitivities to a wide range of additional inhibitors including FLT3, AKT, multi-kinase inhibitors as well as a surprising gain of sensitivity to NTRK inhibitors relying on alternative pathways of cell death. Our results provide an independent confirmation of TP53 and its apoptotic network involvement to the venetoclax response in AML cells and suggest strategies to overcome resistance. Citation Format: Tamilla Nechiporuk, Stephen E. Kurtz, Olga Nikolova, Amanda d'Almeida, Kevin Watanabe-Smith, Mara Rosenberg, Brian Druker, Jeffrey W. Tyner, Shannon K. McWeeney. A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 325.