Mitotic error-tolerant survival following WRN inhibition is overcome by sustained spindle checkpoint activation

Background: Microsatellite instability-high (MSI-H) cancers are known to depend on the Werner (WRN) helicase, which resolves repetitive TA sequences. Recently, a selective WRN inhibitor, HRO-761, has shown to be highly effective in MSI-H cells, validating WRN as a promising therapeutic target. However, the cellular mechanisms by which WRN inhibition fails to completely eliminate tumor cells remain unclear. In this study, we uncovered a mitotic survival pathway activated upon WRN inhibition and propose a drug combination strategy with Vinblastine to suppress it. Method: To examine the cellular state following WRN inhibition, we performed confocal microscopy after durg treatment and confirmed spindle assembly checkpoint (SAC) activation by Western blot. Drug sensitivity profiles were analyzed using GDSC2, and OmicsExpression and CRISPRGeneEffect from DepMap (25Q2) were analyzed. Combination effects were assessed using CCK-8 and colony formation assays in MSI-H colorectal cancer cell lines (HCT-116, LOVO, SNU-C2B). Results: WRN inhibition in MSI-H colorectal cancer cells induced robust cell-cycle arrest and activated SAC, as evidenced by increased MAD2 re-localization (IF) and accumulation of Cyclin B1 and Securin (WB). Despite the initial SAC activation, we also observed subsequent SAC deactivation accompanied by abnormal morphological changes characteristic of mitotic error. These cells continued to survive while bearing persistent mitotic errors, representing an error-tolerant survival phenotype following WRN inhibition. Transcriptomic analyses revealed that genes involved in kinetochore-microtubule attachment were significantly downregulated after WRN inhibition (NES < -2, adjusted P < 0.05). Using whole cell line expression and CRISPR Gene Effect data, we found that cells with reduced kinetochore gene expression exhibited increased dependency on alpha-tubulin and beta-tubulin (P < 0.05), suggesting a complementary and potentially synthetic-lethal relationship between WRN loss and microtubule function. We combined WRN inhibition with Vinblastine, a microtubule-targeting agent(MTA) known to prolong SAC activation, and observed reduced cell viability in CCK-8 and colony-formation assays. In HCT-116 cells, co-treatment with HRO-761 and Vinblastine (0.1 nM) decreased the IC₅₀ from 1.058µM to 0.382 µM, demonstrating a clear synergistic effect of the combination. Conclusion: Based on our findings, we identified a previously unrecognized mitotic error-tolerant survival pathway induced by WRN inhibition. We further demonstrated that Vinblastine, by sustaining spindle checkpoint activation, synergized with WRN inhibition to reduce cell viability and suppress escape mechanism. These results suggest that combining MTA with WRN inhibitors represents a promising therapeutic strategy in MSI-H cancers.