Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy

Recent work has revealed essential epigenetic dependencies in many cancers. Based on the functional antagonism between BAF/SWI/SNF and Polycomb repressive complex 2 chromatin remodeling 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 response and resistance to epigenetic therapy in general and tazemetostat in particular remain unknown. Using comparative functional genomics of clinical tumor specimens and diverse experimental model systems, we have now defined molecular mechanisms of resistance to tazemetostat in epithelioid sarcomas and rhabdoid tumors. We found distinct classes of acquired mutations that converge on the RB/E2F axis and decouple EZH2 inhibition-dependent tumor cell differentiation and cell cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest, despite an active transcriptional response, and provides a general mechanism for effective EZH2 inhibitor therapy. Thus, we develop combination strategies to circumvent tazemetostat resistance by using cell cycle bypass and synthetic lethal targeting, and provide prospective biomarkers for future therapy stratification. This offers a paradigm for rational epigenetic combination therapy suitable for immediate translation to clinical trials for patients. Overall design: We used CRISPR/Cas9 genome editing to generate biallelic RB1del G401 cells, as compared to isogenic RB1-wild type control cells produced by targeting the safe harbor locus AAVS1. We confirmed absence of RB1 protein expression in two independent clones using Western immunoblotting. Two RB1del clones were tested, termed "E1" and"F2." Cells were treated with 10µM tazemetostat or equivalent volume of DMSO for 11 days, using biological triplicates.