Tazemetostat and doxorubicin in patient-derived preclinical models of epithelioid sarcoma (ES)

Background: ES is an ultra-rare sarcoma characterized by the absence of INI1/SMARCB1. The activation of EZH2, consequent to loss of INI1, has been identified as relevant for therapeutic targeting. We exploited ES patient-.derived xenograft (PDX) models and paired cell lines to test the activity tazemetostat plus doxorubicin and identify modifiers of tumor response and resistance. Material and methods: We established two PDXs in SCID mice from two proximal-type ES characterized by INI1-deficiency due to epigenetic silencing (ES-1) or gene deletion (ES-2). Histo-morphology, IHC, and transcriptomic profile of clinical ES and paired PDXs were consistent. 2D cell lines were generated from each PDX. Tazemetostat and doxorubicin, as single-agents or in combination, were tested and maximum tumor volume inhibition percentage (max TVI%) was assessed to measure treatment activity in PDXs. Cell proliferation (Ki67-index) was evaluated at IHC and expression of proteins involved in apoptotic and autophagic response was analyzed at western blotting. Results: Tazemetostat plus doxorubicin had higher effectiveness (max TVI: 95%) than single-agent doxorubicin (max TVI: 50%) or tazemetostat (max TVI: 56%). We established a post-treatment PDX model (ES-1/R) from tumors that started to re-grow roughly 3 weeks after the end of doxorubicin plus tazemetostat. After a re-challenge with the same schedules, doxorubicin was as effective as observed in the ES-1 model while a slightly lower susceptibility to tazemetostat, alone and in combination with doxorubicin, was detected (max TVI: 40 and 84%, respectively). Inherited resistance to either doxorubicin or tazemetostat or their combination was observed in the ES-2 PDX. We observed a reduction of the Ki67-index and a persistent activation of apoptosis in ES-1 but not ES-2 tumors explanted from mice after exposure to the combined treatment as well as in combination-treated in vitro cell lines. Tazemetostat with or without doxorubicin induced an autophagic response in ES-1 and ES-2 cell models. Drug combination with the autophagy inhibitor bafilomycin A, suggested a cytoprotective role for autophagy in ES-2 only. Transcriptomic analysis of untreated ES-1 and ES-2 PDXs, revealed higher expression of EMT- angiogenesis- inflammatory response- TGFα- and TGFβ-related signaling in ES-2 compared to ES-1. ES-2 also showed down-regulation of NSD1, a H3K36 methyltransferase whose loss was reported to confer resistance to EZH2 inhibition in SMARCB1-deficient rhabdoid tumor cells. Conclusions: Tazemetostat plus doxorubicin showed synergistic antitumor activity in an ES tumor model, an effect that was maintained at a re-challenge in the derived post-treatment PDX. Possible determinants of resistance to this drug combination were also identified in an inherited resistant tumor model. Conflict of interest: Advisory Board: S Stacchiotti: Bayer, Bavarian Nordic, Deciphera, Daiichi, Eli Lilly, Epizyme, Karyopharm, MaxiVax, Pharmamar, Takeda Other Substantive Relationships: S Stacchiotti: honoraria: Eli Lilly, Pharmamar travel grants: Pharmamar institutional research funding: Advenchen, Amgen Dompé, AROG, Bayer, Blueprint Medicines, Daiichi Sankyo Pharma, Deciphera, Eli Lilly, Epizyme, GSK, Karyopharm, Novartis, Pfizer, PharmaMar, SpringWorks. AM Frezza: institutional research funding: Advenchen, Amgen Dompé, AROG, Bayer, Blueprint Medicines, Daiichi Sankyo Pharma, Deciphera, Eli Lilly, Epizyme, GSK, Karyopharm, Novartis, Pfizer, PharmaMar, SpringWorks. A Gronchi: sponsorship or research funding: PharmaMar remuneration: Bayer, Lilly, Nanobiotix, Novartis, Pfizer, PharmaMar and SpringWorks.