Abstract 4211: Studying WRN inhibitors with dSTRIDE-WRN assay

Abstract The Werner syndrome helicase (WRN) is a critical enzyme involved in resolving replication stress and repairing double-strand DNA breaks, particularly in microsatellite instability-high (MSI-H) cancers. WRN inhibitors, such as HRO761, exploit the synthetic lethality of MSI-H cancers by trapping WRN on DNA, inducing genomic instability, and selectively killing tumor cells. Understanding the precise molecular mechanisms of WRN inhibition and its role in DNA repair pathways is essential for developing targeted therapies. Here, we introduce a newly developed assay for measuring WRN activity, specifically focusing on its exonuclease domain. This novel assay leverages the STRIDE platform technology, enabling direct and sensitive detection of single- and double-strand DNA breaks in situ within fixed cells. The dSTRIDE-WRN assay detects double-strand DNA breaks in close proximity to the WRN protein, providing a direct readout of its enzymatic activity. HCT116, a colorectal carcinoma cell line displaying high-level microsatellite instability (MSI-H) was chosen for optimization and validation of the assay. Two antibodies were tested initially, both producing satisfactory immunofluorescence staining; however, only one provided reliable readouts once incorporated into the STRIDE assay. The selected antibody generated consistent results across technical replicates and exhibited minimal signal in negative controls, confirming the assay's specificity and reproducibility. The assay’s ability to inform about WRN inhibition was evaluated by treating cells with HRO761, a selective inhibitor of WRN, with readouts collected at 1, 2, 4, and 6 hours post-treatment. A marked decrease in the dSTRIDE-WRN assay signal was observed at 4 and 6 hours, suggesting that HRO761 also influences WRN activity during DNA strand-breaks repair. These findings establish the dSTRIDE-WRN assay as a sensitive and reliable tool for quantifying WRN activity and its inhibition in the context of DNA damage repair. By providing a direct readout of WRN’s enzymatic function, the assay offers valuable insights into the molecular mechanisms underlying WRN-targeting therapies. This platform holds the potential for advancing the development and evaluation of WRN inhibitors in MSI-H cancers and beyond. Citation Format: Olga Wójcikowska, Magdalena Bartyńska, Maja Białecka, Magdalena Kordon-Kiszala, Kamil Solarczyk. Studying WRN inhibitors with dSTRIDE-WRN assay [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 4211.