Histone deacetylase activity limits the response to EZH2 inhibition-based therapy in epithelioid sarcoma and is targetable by epigenetic combination

Abstract Background. Epithelioid sarcoma (EpS) is an ultra-rare, aggressive soft tissue sarcoma (STS) driven by loss of the tumor suppressor INI1, encoded by SMARCB1 , which triggers hyperactivation of the chromatin-modifying enzyme EZH2. However, objective responses to the EZH2 inhibitor (EZH2i) tazemetostat (TZM) remain limited to approximately 15% of patients. Combination strategies incorporating EZH2i with doxorubicin (DOX), the first-line standard for advanced STSs, may enhance the activity of either agent alone, warranting investigation of determinants of response and resistance. Methods. Two INI1-deficient in-house generated proximal-type EpS (P-EpS) patient-derived xenograft (PDX) models, EpS-1 and EpS-2, and their matched cell lines were established. EpS-1 and EpS-2 models harbored a homozygous SMARCB1 deletion and underwent comprehensive genomic, epigenomic, transcriptomic, protein-level, and histological characterization. Both models were used to evaluate TZM and DOX, alone and in combination, and to investigate determinants of response and resistance. Results. TZM + DOX greatly inhibited tumor growth in EpS-1 in vivo, though regrowth occurred approximately three weeks after treatment cessation. EpS-2 displayed intrinsic resistance to all treatments. RNA sequencing of post-treatment PDXs revealed divergent transcriptional responses: in EpS-1 but not in EpS-2 TZM + DOX downregulated gene sets linked to chromatin organization and histone deacetylases (HDACs). Consistently, H3K27 acetylation (H3K27ac) accumulated exclusively in EpS-1, suggesting HDAC activity limits EZH2i-induced chromatin remodeling in resistant EpS. In EpS-2, co-treatment with TZM, DOX, and the pan-HDAC inhibitor vorinostat (SAHA) restored H3K27ac, induced apoptosis, and showed tendency toward synergism. Conclusions. This study identifies distinct resistance profiles to EZH2i-based therapy in P-EpS PDXs. HDAC activity limits chromatin remodeling induced by EZH2i (± DOX), and its inhibition with SAHA restores sensitivity, supporting HDAC inhibition as a rational strategy to overcome EZH2i resistance.