(P 170) ANTITUMOR ACTIVITY OF TAZEMETOSTAT AND DOXORUBICIN COMBINATION ON PATIENT-DERIVED PRECLINICAL MODELS OF EPITHELIOID SARCOMA

Objective: Epithelioid sarcoma (ES) is an ultra-rare and aggressive type of soft-tissue sarcoma marked by the absence of INI1/SMARCB1, due to either gene deletion or epigenetic silencing. The two different morphologic variant of ES, distal- and proximal-type ES, are characterized by differences in epidemiological features, natural history and patient outcomes, being the proximal variant the most aggressive, with a dismal prognosis. Based on the notion that INI1 loss leads to the activation of EZH2, the catalytic component of PRC2 complex, this enzyme has been proposed as a relevant therapeutic target for ES. In a phase II study, tazemetostat, a new oral compound able to inhibit EZH2, achieved an overall response rate of approximately 15% of INI1-negative ES patients, with durable responses. The possibility to increase tazemetostat activity by combining it with other drugs, such as doxorubicin, is currently being explored. In the present study, we investigated the activity of the tazemetostat-doxorubicin combination in patient-derived xenograft (PDX) models and paired cell lines established from ES patients with the aim to identify possible determinants of resistance.

Methods: Two PDXs of proximal ES characterized by INI1-deficiency due to epigenetic silencing (ES-1) or gene deletion (ES-2) were developed in SCID mice and characterized in terms of histo-morphology (by IHC) and transcriptomic profile (by RNASeq). Corresponding 2D cell lines were also generated. PDXs were exposed to tazemetostat and doxorubicin, as single-agents and in combination, and drug activity was evaluated as maximum tumor volume inhibition percentage (max TVI%). The effects of treatments on cell proliferation (Ki67-index) and expression of proteins involved in apoptotic and autophagic response were assessed by immuno-histochemistry and western blotting, respectively.

Results: In the ES-1 PDX model, a moderate antitumor effect was achieved with single-agent doxorubicin and tazemetostat (max TVI: 50 and 56%, respectively), while the combined treatment showed a significantly higher activity (max TVI: 95%). From tumors starting to re-grow about 3 weeks after the end of the combined treatment, we generated a post-treatment PDX model (ES-1R) that, at re-challenge with the same drugs, maintained the original sensitivity to doxorubicin and a slightly lower susceptibility to tazemetostat, alone and in combination with doxorubicin (max TVI: 40 and 84%, respectively). The ES-2 PDX was inherently completely resistant to both drugs, singly administered or in combination. The assessment of drug-induced effects in tumors explanted from mice after in vivo treatment and in vitro cell lines, showed a significantly reduced Ki67-index as well as a persistent activation of apoptosis in ES-1 but not ES-2 models after exposure to the combined treatment. Tazemetostat (±doxorubicin) induced an autophagy response in both cell models, having a cytoprotective role in ES-2 as demonstrated by combined exposure with the autophagy inhibitor bafilomycin A. Comparative analysis of basal PDX transcriptomic profiles revealed an enrichment of EMT-, angiogenesis-, inflammatory response-, TGFα- and TGFβ-related signaling in ES-2 compared to ES-1. Moreover, a down-regulated expression of NSD1, a H3K36 methyltransferase whose loss was recently reported to confer resistance to EZH2 inhibition in SMARCB1-deficient rhabdoid tumor cells, was observed in ES-2.

Conclusion: Results of the present preclinical study i) show a synergistic antitumor activity of the tazemetostat-doxorubicin combination, which was also maintained at re-challenge, in an ES PDX model and its derived post-treatment variant, and ii) provide preliminary evidence on possible determinants of resistance to the combined treatment.