Preclinical characterization of a potent and selective Werner helicase (WRN) inhibitor with activity in low-TA repeat microsatellite instability-high (MSI-H) cancers

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.