Abstract A028: Novel Synthetic Lethality Opportunity for Ewing Sarcoma

Abstract Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. The EWS-FLI1 protein, the oncogenic driver in EwS, is a fusion of the FET protein, EWSR1, at its amino terminus, with the carboxy terminus of the transcription factor, FLI1. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of EWS-FLI1 in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POLQ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POLQ protein expression, impaired MMEJ, and cellular sensitivity to inhibitors of NHEJ repair and HR repair, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POLQ mitotic foci and increases MMEJ activity. Based on this mechanism, inhibitors of NHEJ or HR may provide a targeted therapy for patients with Ewing sarcoma and a novel Synthetic Opportunity. Highlights: 1) Ewing sarcoma tumors have a deficiency in POLQ expression and a corresponding loss of MMEJ activity 2) EWSR1 is a splicing factor that interacts with other splicing factors such as FUBP1 and KHSRP/FUBP2 to accurately splice the POLQ mRNA 3) The EWS-FLI1 fusion oncoprotein, or loss of EWSR1, causes a splicing defect, leading to exon25 skipping of the POLQ pre-mRNA and loss of POLQ expression 4)The MMEJ deficiency of EwS cells results in cellular sensitivity to inhibitors of Homologous Recombination or Non-Homologous End-Joining 5) Exon 25 skipping of POLQ mRNA is a predictive biomarker for HR inhibitors in human cancers 6) Ewing Sarcoma is the first example of a human cancer with a defect in the MMEJ DNA Repair pathway. Citation Format: Shuhei Asada, Guangli Zhu, Alan D. D'Andrea. Novel Synthetic Lethality Opportunity for Ewing Sarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A028.