Abstract 7035: Targeting Ewing sarcoma with a novel RBM39 degrader: DNA damage repair pathway effects

Abstract Patients with metastatic Ewing Sarcoma have a five-year survival rate of 15-30%. For 30 years, there are limited approved therapies, including chemotherapy, radiotherapy and surgery, which are associated with significant morbidities and secondary cancers, supporting the need for new impactful therapies. Analysis of the RNA splicing factor RNA Binding Motif Protein 39 (RBM39) in the Cancer Target Discovery and Development (CTD2) Network showed that Ewing sarcoma cell lines are especially sensitive to RBM39 loss. Here we demonstrate effects on mediators of DNA damage repair in association with significant anti-cancer effects of an orally administered novel RBM39 degrader, ST-00937. Methods and Results: A next generation RBM39 degrader ST-00937 was designed through structure-based drug design and prioritized based on in vitro anti-cancer potency, metabolic stability and oral pharmacokinetics in mice. Human Ewing sarcoma A673 cells were utilized to explore the effects of ST-00937 in this cancer type. Tumor cell viability was evaluated by CellTiter-Glo® Luminescent Cell Viability Assay (CTG) and anti-tumor efficacy was evaluated in nude mice bearing tumors established with A673 cells. Protein levels in cell lysates and tumor homogenate were measured by Western Blot. Target gene RNA splicing was evaluated by RT-PCR.ST-00937 degraded RBM39 by 6 hours of treatment and reduced in vitro A673 Ewing sarcoma cell viability with an IC50 of 170 nM. These in vitro effects translated to complete A673 tumor regressions in mice by the 11th day of twice daily ST-00937 oral dosing at 30mg/kg, without an effect on body weight. By the third day of treatment, RBM39 was undetectable in A673 tumors. Mechanically both the EWS-FLI1 driver gene fusion in A673 cells and RBM39 degradation are reported to reduce DNA damage repair pathways. Here we report RBM39 degradation in A673 cells significantly increases phosphorylated ʏH2Ax, a marker of DNA double strand breaks. This DNA damage is thought to contribute to the induction of cell apoptosis detected as increased cleaved caspase-3. Increased DNA double strand breaks is furthermore associated with negative effects on 3 proteins involved in homologous recombination repair of DNA double strand breaks; mis-splicing of BRCA1 (Breast cancer type 1) and ATM (ATM Serine/Threonine Kinase), and reduced protein levels of RAD51D (recombination repairing regulator Recombinase homolog D). Conclusion: The RBM39 molecular glue degrader ST-00937 therefore demonstrates total tumor regression in a model of Ewing sarcoma. Mechanistic studies indicate that the robust activity of ST-00937 involves deficient homologous recombination repair. Continued research in our lab will add further insights into the mechanism of action of RBM39 degraders in this cancer of high unmet need. Citation Format: Fei Liu, Yunkai Zhang, Baiyun Wang, James Finn, James Tonra, Lan Huang, Dong Liu, Haihong Jin, Xing Liu, Dan Lu. Targeting Ewing sarcoma with a novel RBM39 degrader: DNA damage repair pathway effects [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 7035.