Abstract A048: Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy

Essential epigenetic dependencies have become evident in many cancers. Based on functional antagonism between BAF/SWI/SNF and PRC2 in SMARCB1-deficient sarcomas, we and colleagues recently completed the clinical trial of the EZH2 inhibitor tazemetostat, leading to its FDA approval. However, the principles of tumor response to epigenetic therapy in general, and tazemetostat in particular, remain unknown. First, using functional genomics of patient tumors and diverse experimental models, we define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient sarcomas and rhabdoid tumors. We found distinct classes of acquired mutations that converge on the RB1/E2F axis and decouple EZH2-dependent differentiation and cell cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest despite EZH2 inhibition, and suggests a general mechanism for effective EZH2 therapy. We provide prospective biomarkers for therapy stratification, including the cell cycle-regulated PRICKLE1 gene. To circumvent tazemetostat resistance and improve response in vitro and in vivo we develop the rational combination of tazemetostat with barasertib, to target the downstream cell cycle kinase AURKB. Second, through transcriptomic inference of SMARCB1-deficient tumor cells, we predicted ATR as a target to overcome resistance to EZH2 inhibition. We found that EZH2 inhibition induces DNA damage, at least in part via upregulation of the transposase-derived PGBD5 nuclease. Finally, we establish a second rational combination strategy for increased anti-tumor efficacy between tazemetostat and the ATR inhibitor, elimusertib, by leveraging the synthetic lethal interaction with PGBD5-dependent DNA damage repair signaling via ATR. Thus, the epigenetic therapy, EZH2 inhibition, induces a targetable collateral vulnerability through activation of the PGBD5-ATR axis. This establishes a mechanistically rational combination strategy for targeting EZH2 and ATR in SMARCB1-deficient cancers based on an EZH2-PGBD5-ATR synthetic lethal axis and is immediately translatable to clinical trials for patients with these aggressive malignancies. Citation Format: Helen S Mueller. Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2): nr A048.