Abstract 5475: Leveraging LC-MS workflows to characterize a novel WRN covalent inhibitor

Werner protein (WRN) is a helicase with functions critical to DNA repair pathways, and as such is an active target for the development of cancer therapeutics. Notably, WRN has been demonstrated as a synthetic lethality target in DNA mismatch repair deficient (dMMR)/microsatellite instable (MSI-H) tumors owing to its role in the DNA damage response. Accordingly, the development of small-molecule WRN inhibitors is an active and attractive area of cancer research and has the potential to be a powerful new therapeutic modality in several cancers including colorectal, gastric, and endometrial. Here, we report the application of targeted and discovery LC-MS workflows towards the characterization of M6021, a small molecule covalent inhibitor of WRN. A custom targeted LC-MS assay has been developed and optimized for detection and quantification of covalent compound engagement to WRN in preclinical models, including several cancer cell lines and mouse xenograft models. This assay enables reproducible, precise and accurate quantitative characterization of near-complete WRN engagement in clinically relevant biological matrices, despite low endogenous WRN expression and treatment-induced reduction in WRN levels. Plate-based sample preparation and analysis enables high-throughput evaluation of multiple experimental parameters (dose, treatment time, matrix, etc) across meaningful cohorts of replicates. Further, we employed a discovery LC-MS based covalent inhibitor selectivity assay to demonstrate that M6021 is highly selective for WRN, proteome-wide. Using this assay, we demonstrate that the novel WRN inhibitor M6021 is a potent and selective covalent inhibitor of WRN helicase, suitable for use as a high-quality chemical probe for understanding WRN biology in dMMR/MSI-H tumors. Citation Format: Kurt Baltier, Anthony Tubbs, Doug McLaughlin, Giulia Bottoni, Allison Drew, Brian Erickson, Craig Braun. Leveraging LC-MS workflows to characterize a novel WRN covalent inhibitor [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 5475.