Abstract 1200: FET fusion oncoproteins disrupt physiologic DNA repair and create a targetable opportunity for ATR inhibitor therapies

Abstract The FET family of intrinsically disordered proteins (FUS, EWSR1, TAF15) are frequent 5’ oncogenic transcription factor (TF) fusion partners in a diversity of sarcomas and leukemias. These TF fusion oncoproteins are often the sole oncogenic driver alteration in these cancers and due to the difficulty in pharmacologic targeting of TFs, precision medicine approaches have been lacking. Here we focus on the pediatric bone tumor Ewing sarcoma (ES) as a model for the class of FET rearranged cancers. ES patients with relapsed or metastatic disease continue to have dismal outcomes, yet decades of clinical experience and laboratory testing of ES cancer cell lines have shown ES to be among the most chemo- and radiosensitive cancers, at least initially. In this study, we address the long-standing hypothesis that ES tumors harbor an intrinsic DNA repair defect by studying the role of the ES fusion oncoprotein EWSR1-FLI1 in regulating the DNA damage response (DDR). Native FET family members are among the earliest factors that localize to DNA double-strand breaks (DSBs). Here we discover that EWSR1-FLI1 (and other FET) fusion oncoproteins are aberrantly recruited to DNA DSBs and interfere with native FET (EWSR1) protein function in activating the DNA damage sensor ATM. We demonstrate the functional importance and therapeutic relevance of ATM suppression in ES through increased reliance on a compensatory DDR kinase, ATR, and FET fusion oncoprotein-dependent synthetic lethality with ATR inhibitors (nanomolar range IC50’s). Lastly, we tested ATR inhibition in PDX models across the class of FET rearranged cancers as a synthetic lethal therapeutic strategy to specifically target functional ATM defects induced by FET fusion oncoproteins themselves. We demonstrate significant anti-tumor responses to ATR inhibitor monotherapy in all 5 PDX models including those derived from multiply relapsed ES and clear cell sarcoma patients. In summary, our study provides a new mechanism for how an oncogene can impair physiologic DNA repair (i.e., aberrant recruitment of an oncogenic fusion protein to sites of DNA damage) to create functional states akin to genetic DDR pathway loss, and rationale for a new targeted therapeutic strategy (i.e., ATM/ATR synthetic lethality) in ES and the broader class of undruggable FET fusion-driven cancers. Citation Format: Shruti Menon, Daniel Gracilla, Marcus Breese, Filemon Dela Cruz, Asmin Tulpule. FET fusion oncoproteins disrupt physiologic DNA repair and create a targetable opportunity for ATR inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1200.