CRISPR Screening Identifies SMARCAL1 and MRN as Modulators of WRN Dependency in MSI-H Colorectal Cancer

Microsatellite instability-high (MSI-H) colorectal cancer cells depend on the Werner syndrome helicase (WRN) to resolve cruciform DNA structures that arise from expanded TA-dinucleotide repeats. Loss of WRN induces replication stress and double-strand breaks (DSBs), a vulnerability that can be recapitulated by the selective WRN inhibitor HRO761 in MSI cancer cells. To uncover the mechanisms governing sensitivity to WRN inhibition, we conducted genome-wide CRISPR/Cas9 screens in colorectal cancer cell lines treated with or without HRO761. These screens identified SMARCAL1 as a key modulator of WRN dependency. Depletion of SMARCAL1 rendered cells resistant to WRN inhibition, and rescue of this effect required the ATPase/translocase activity of SMARCAL1. Mechanistically, SMARCAL1 antagonized WRN and supported cruciform DNA structures, thereby enhancing cellular reliance on WRN. In addition, the MRE11-RAD50-NBS1 (MRN) complex, rather than MUS81 or ERCC1/XPF, was the principal mediator of cruciform DNA processing following WRN inhibition. Acute disruption of the MRN complex conferred profound resistance to WRN inhibition, whereas ATM deficiency produced a more modest resistant phenotype. Further genetic and pharmacological epistasis analyses demonstrated that the MRN complex regulates WRN inhibitor sensitivity through both ATM-dependent signaling and MRE11 nuclease-dependent functions. Importantly, the key resistance mechanisms identified in this study were independently validated using a structurally distinct clinical-stage WRN inhibitor VVD-214. Collectively, these findings identify the SMARCAL1-MRN-ATM axis as a critical regulator of WRN dependency and provide mechanistic insight into resistance to WRN-targeted therapy.