TrAEL-seq with WRN inhibitors in sensitive and resistant cells

Werner helicase inhibitors (WRNi) show promise for treating patients with microsatellite-unstable (MSI) tumors characterized by defective DNA mismatch repair. Multiple WRNi recently entered Phase 1 clinical trials. Here, we investigate the impact of cancer cell evolutionary adaptation on WRN pharmacological inhibition with implications for understanding drug selectivity and potential clinical resistance. Coupling genome-wide CRISPR screens with WRN gene knockout, no suppressors of WRN dependency were identified, underscoring WRN’s essential non-redundant function in MSI cells. Pharmacogenomic screens pinpointed modulators of WRNi sensitivity, including SMARCAL1, linking WRN-MSI synthetic lethality with chromatin remodelling and DNA repair pathways. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving spontaneous secondary resistance to multiple WRNi in vitro and in vivo. Specific resistance mutations preserve sensitivity to alternative WRNi, whereas others induce cross-resistance. Our work guides next-generation strategies for targeting MSI cancers, enabling cross-resistance studies to evaluate current and novel WRNi efficacy and informing future clinical trial design. We employed Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution, to identify the genomic sites of DNA double-strand breaks (DSBs) in MSI cells treated with WRN inhibitors. We used SW48 cells with or without SMARCAL1, which confers resistance to WRN inhibitors