Werner helicase inhibitors (WRNi) are in clinical development for treating microsatellite instability (MSI) tumors, which depend on WRN due to defective mismatch repair. However, acquired resistance may limit their efficacy. Here, we systematically investigate resistance mechanisms to WRNi in cancer cells, focusing on on-target WRN mutations, cross-resistance among clinical-grade inhibitors, and potential therapeutic strategies to overcome resistance.

We performed genome-wide CRISPR screens, pharmacogenomic profiling, and time-to-progression (TTP) assays to identify WRNi resistance mechanisms. Semi-saturation mutagenesis of WRN and long-term drug exposure studies were used to track resistance emergence. Structural modeling predicted the impact of mutations on drug binding, while viability assays and DNA damage markers validated cross-resistance. Patient-derived organoids (PDOs) from MSI colorectal cancer were used to assess clinical relevance.

CRISPR screens confirmed WRN’s essential role in MSI cells, with no genetic bypass mechanisms identified. Pharmacogenomic profiling of WRNi revealed modulators of sensitivity, including SMARCAL1, a chromatin remodeler linked to WRN-MSI synthetic lethality. TTP assays and mutagenesis identified recurrent on-target WRN mutations driving resistance, including G729D, which disrupts WRNi binding, causing broad cross-resistance, and I852F, which selectively confers resistance to HRO761 while preserving sensitivity to VVD-133214. A non-canonical splice site mutation led to exon skipping but retained WRN function, allowing continued MSI dependency. These findings were validated in PDOs, where resistance to HRO761 emerged through the same exon-splicing mutation. Notably, WRNi-resistant cells remained sensitive to ATR inhibitors and irinotecan, supporting a WRN-specific resistance mechanism.

Our study identifies on-target WRN mutations as key drivers of three clinical grade WRNi resistance and highlights strategies to overcome it. By characterizing cross-resistance across clinical-grade WRNi, we propose switching inhibitors with different mechanisms of action to restore sensitivity. These findings provide a framework for biomarker-driven patient stratification, resistance monitoring through ctDNA, and combinatorial approaches to improve WRNi efficacy in MSI cancers.