Abstract 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. 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. Thus, we develop combination strategies to circumvent tazemetostat resistance using cell cycle bypass and synthetic lethal targeting, and provide prospective biomarkers for therapy stratification. This offers a paradigm for rational epigenetic combination therapy suitable for immediate translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers. Significance Genomic studies of patient tumors and cell lines identify mutations converging on a common pathway that is essential for response to EZH2 inhibition. Resistance mutations decouple drug-induced differentiation from cell cycle control. We identify complementary epigenetic combination strategies to overcome resistance and improve response, supporting their investigation in clinical trials.

Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy