Abstract Werner syndrome helicase (WRN), a member of the RecQ family of DNA helicases, is a synthetic-lethal dependency in DNA mismatch repair-deficient cancers that exhibit high microsatellite instability (MSI-H). Here, we characterize Compound 1, a potent and selective small molecule WRN inhibitor. Compound 1 binds within an allosteric pocket in the helicase domain of WRN and forms an irreversible covalent interaction with cysteine 727 (C727), a residue not conserved in other RecQ helicases. In vitro, Compound 1 potently inhibited WRN’s helicase activity and was confirmed to be a highly selective covalent inhibitor of WRN C727 by mass spectrometry-based cysteine profiling in human MSI-H cancer cells. Consequently, Compound 1 potently impacted the viability of a broad panel of MSI-H cell lines, including colorectal SW48 and RKO (IC50 = 10 nM and 100 nM, respectively), and ovarian TOV-21G (IC50 300 nM) cells. Treatment with Compound 1 elicited robust induction of pharmacodynamic (PD) biomarkers including sensors of DNA damage and the proteasome-dependent degradation of WRN across the panel. Notably, these responses were selective for MSI-H cells and were not observed in microsatellite-stable (MSS) cells nor in cell lines derived from normal tissues up to the highest concentration tested (5 μM). In vivo, Compound 1 treatment resulted in a dose-dependent modulation of PD biomarkers, which correlated with strong anti-tumor efficacy in SW48, RKO, and TOV-21G MSI-H cell line-derived xenograft (CDX) models. Covalent engagement of WRN by Compound 1 was measurable in tumor tissue and in peripheral blood monocytes. The exposure and target occupancy of Compound 1 required for efficacy strongly correlated with the expansion of repetitive thymine-adenine (TA) nucleotide DNA microsatellites. These repeats form non-canonical secondary structures that interfere with DNA replication in MSI-H cells and require unwinding by WRN. Long-read DNA sequencing confirmed that the extent of TA-repeat expansion was associated with Compound 1 activity in MSI-H CDX models. An analysis of primary MSI-H tumor cohorts revealed that the majority of patient tumor samples exhibited TA-repeat expansions within the range of Compound 1 efficacy observed in cell-based models. Importantly, dosing of Compound 1 was sufficient to elicit tumor regression in low-TA repeat CDX MSI-H models (RKO, TOV-21G), which have proven to be refractory to WRN inhibitors advanced previously. Together, these data demonstrate that covalent inhibition of WRN by Compound 1 is an effective strategy to selectively kill MSI-H tumor cells with expanded TA repeats. Citation Format: Pablo E. Hollstein, Daniel A. Aiello, Anne Y. Saiki, Keegan S. Cooke, Petia Mitchell, Xiang Yi, Ishwar N. Kohale, Matthew J. Rardin, John Rodgers, Mauro Poggio, Chun Su, Sigurgeir Olaffson, Doruk Beyter, Jennifer A. Roth, Andrew J. Holland, Paul E. Hughes. Preclinical characterization of a potent and selective Werner helicase (WRN) inhibitor with activity in low-TA repeat microsatellite instability-high (MSI-H) cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7086.
Hollstein et al. (Fri,) studied this question.