Abstract 6865: Discovery and characterization of small molecule inhibitors targeting EXO1 for BRCAness cancer therapy

Abstract Human exonuclease 1 (EXO1), a member of the structure specific nuclease family, plays a critical role in maintaining genome stability by processing DNA double-strand breaks (DSBs), nicks, and replication intermediates during DNA replication and repair. As its exonuclease activity is essential for homology-directed repair and replication fork processing, EXO1 has emerged as a compelling therapeutic target, especially in cancers marked by heightened DNA damage and replication stress. Through high-throughput screening of 45, 000 compounds, we identified eight distinct scaffolds of EXO1 inhibitors that demonstrated effective and selective inhibition. Two lead compounds, C200 and F684, underwent structural and mutagenesis studies, which confirmed their binding mechanisms. Biochemical assays further validated their potent and selective inhibition of EXO1 activity. Tumor cell profiling revealed that these inhibitors exploit synthetic lethality in BRCA1-deficient cells, emphasizing their specificity and therapeutic potential for targeting BRCAness cancers. Mechanistically, EXO1 inhibition suppressed DNA end resection, increased the accumulation of DSBs, and triggered S-phase PARylation, effectively disrupting DNA repair pathways essential for cancer cell survival. These findings establish EXO1 inhibitors as promising candidates for the treatment of BRCAness cancers and lay the groundwork for further optimization and development of these compounds as targeted therapeutics. Citation Format: Yixing Wang, Jessica Hess, Li Zheng, Binghui Shen. Discovery and characterization of small molecule inhibitors targeting EXO1 for BRCAness cancer therapy [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 6865.