Abstract 4207: Discovery of WRN helicase inhibitors that covalently engage a novel, induced allosteric site

The RecQ helicase WRN is a synthetic lethal dependency in microsatellite instability-high (MSI-H) cancer and a therapeutic target of interest in that context. The two clinical-stage WRN inhibitors, HRO761 and RO7589831, both bind in an allosteric pocket near the interface of the two helicase domains (D1 and D2). To discover novel WRN inhibitors, we conducted a high-throughput phenotypic screen using γH2AX immunofluorescence imaging in MSI-H HCT116 colorectal cancer cells and identified a singleton hit, Compound A. Compound A phenocopied WRN siRNA knockdown in multiparametric analysis and was confirmed to engage and inhibit purified WRN protein. X-ray co-crystal structures with and without ATP revealed that Compound A binds to a novel allosteric site in the D1 domain which is absent in apo-WRN. The allosteric site was induced by localized rearrangement, including displacement of the DEAH-box upon ligand binding. Electron density was indicative of covalent bond formation via addition of Cys672 to the imine C=N bond of Compound A. Mechanistically, Compound A was ATP-noncompetitive, exhibited time-dependent inhibition of WRN ATPase and helicase activity, and did not inhibit the paralogous RecQ helicases BLM, RECL and RECQL4. Exploration of structure-activity relationships resulted in Compound B and Compound C that were characterized for therapeutic pharmacology. Both Compounds B and C demonstrated high potency and selectively induced γH2AX phosphorylation, cell cycle arrest, apoptosis, and clonogenic killing in MSI-H cells. The cytotoxicity of Compound C in HCT116 cells was unaffected by isogenic, CRISPR-mediated knockout of BLM, RECQL, RECQL4, RECQL5 or BRIP1 (FANCJ). In global proteomics and cell signaling studies, Compound C caused concentration-dependent degradation of WRN and upregulation of DNA damage response pathways in HCT116 but not microsatellite stable (MSS) SW620 colorectal cancer cells. Compound B was highly selective for MSI-H models and recapitulated the profile of sensitivity to WRN perturbation in a PRISM screen of 906 cell lines. Co-administration of Compound C with a CYP inhibitor to mice bearing HCT116 xenografts produced dose-dependent γH2AX phosphorylation in tumor tissue. Whole-genome CRISPR modifier screens with Compound C and a close analog of HRO761 in HCT116 and KM12 cells showed generally concordant functional genomic profiles, with perturbations in SMARCAL1, HELLS, and WDR76 enriched and POLQ and RBM6 depleted by WRN inhibition. In conclusion, we report a series of WRN inhibitors that engage a novel cryptic binding site and show a distinct mechanism of inhibition to WRN inhibitors currently in clinical development. Citation Format: Francis W. Hunter, Lindsey DeRatt, Kenneth Maksimchuk, Tatsuya Maehigashi, Seong Joo Koo, Liesbeth Micholt, Ranjeet P. Dash, Faraz Kazmi, Zhengyu Jiang, Albi Francis, Neetu Shukla, Aaron Patrick, Trpta Bains, Romy Perez-Abraham, Nikit Kumar, Zhijie Liu, Brian Reiss, Caitlin Morgan, Inha Heo, Werner Eyckmans, Gerald Chu, Anna Kuhn, Lorraine Angelillo, Lin-Chien Huang, Yu Sun, Ashley M. Rouleau, Yongkang Kim, John J. Shin, Harris Bell-Temin, Victoria Wong, Ruth Steele, Paul Shaffer, Kurtis E. Bachman, David Pocalyko, Scott Kuduk. Discovery of WRN helicase inhibitors that covalently engage a novel, induced allosteric site [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 4207.