WRN under the scalpel: Helicase-domain hotspot & splice-site knock-ins in HCT116 and RKO

The WRN helicase is a synthetic-lethal vulnerability in microsatellite-instability-high (MSI-H) tumors, yet allele-specific resistance is a foreseeable risk as small-molecule WRN inhibitors advance. Endogenous point-mutation models, built in the native regulatory context, more faithfully capture drug-target engagement and pathway compensation than overexpression systems. Such models enable rigorous mechanism-of-action confirmation, resistance-liability mapping, chemotype backup selection, patient-stratification/biomarker hypotheses, and faster SAR and combination design.Across isogenic HCT116 and RKO models, two WRN-targeting chemotypes diverged at specific liability nodes. WT HCT116 was highly sensitive (VVD-214 48.47 nM; HRO761 86.89 nM), and baseline RKO remained responsive (366/720 nM). C727 dictated VVD-214 resistance: C727A drove VVD-214 to >10 µM while preserving near-WT HRO761 (153-179 nM); C727S pushed HRO761 to 2.1-3.5 µM and VVD-214 to >10 µM; in RKO-C727S, both exceeded 10 µM. I852F showed the reciprocal pattern-VVD-214 stayed potent (62-77 nM) but HRO761 was inactive (>10 µM). G729D and F730L caused bilateral right-shifts, larger for HRO761 (VVD-214 0.71-1.34 µM; HRO761 2.9-8.0 µM); combining G729D+I852F abolished HRO761 (>10 µM) while VVD-214 remained sub-µM (0.78-0.82 µM). The splice variant c.1577-1G>C was hypersensitive to VVD-214 (36-37 nM) yet right-shifted for HRO761 (~1.11 µM). Collectively, C727 → VVD-214 resistance and I852 →HRO761 resistance, while G729/F730/splice confer a broader HRO761 bias-guiding chemotype switching and backup selection.Alleles were introduced by CRISPR-Cas9 HDR using ~200-nt ssODN donors; single-cell clones were sequence-verified (Sanger/NGS), STR-authenticated, and mycoplasma-free banked. Dose-response viability assays (72-96 h, 10-point curves) were fit with four-parameter logistic models to derive IC₅₀; each clone included n≥2-3 biological replicates. This WRN allele panel is immediately deployable to (1) build allele-resolved resistance maps, (2) prioritize chemotype backups based on the C727/I852 complementarity, (3) generate patient-selection/biomarker hypotheses for MSI-H settings, and (4) accelerate SAR and combination strategies-thereby front-loading chemical and clinical derisking for VVD-214- and HRO761-class WRN inhibitors.