Microsatellite Instable Cancer Cells Acquire On-target Resistance Mutations to WRN Helicase Inhibitors.

The Werner syndrome helicase (WRN) is a promising target for cancers with microsatellite instability (MSI), leading to the initiation of at least five phase I clinical trials. Acquired resistance is a substantial obstacle to obtaining lasting benefits from targeted therapies in oncology and may be particularly acute in the setting of mismatch repair-deficient (dMMR) tumors, which can sample increased fitness landscapes owing to a higher mutational burden. In this study, we characterized resistance mechanisms using the clinical candidate HRO761 and two novel inhibitors in MSI cell lines and xenograft models. We observed the rapid emergence of resistance both in vitro and in vivo, with sequencing revealing clustered mutations within the WRN helicase domain. Computational structural analyses indicated that these mutations either directly interfere with inhibitor binding or alter the protein conformation required for inhibitor engagement. Notably, although most mutations conferred broad resistance across all three compounds, we identified specific alterations (L528S, C727R, and F730L) that exhibited selectivity between chemical scaffolds. This chemotype-specific resistance profile suggests opportunities for developing next-generation inhibitors that retain activity against resistant variants and for implementing rational treatment strategies with existing inhibitors. Overall, our findings demonstrate that on-target resistance to WRN inhibitors emerges rapidly in dMMR backgrounds but also highlight potential approaches to overcome resistance, supporting the continued development of WRN-targeted therapies for MSI cancers.