Extra-S-phase DNA damage in MSI cells evades cell cycle arrest and leads to lethality upon inhibition of WRN

WRN helicase plays an instrumental role in DNA repair, replication fork stability and genome maintenance. Recently, WRN has emerged as a synthetic lethal target in microsatellite instability (MSI) cancers, which exhibit deficiencies in the DNA mismatch repair pathway. While WRN’s helicase activity is critical for MSI cell survival, the underlying mechanism remains only partially understood. Previous studies suggest that WRN resolves cruciform DNA structures that otherwise induce replication stress. Here, we examine the cell cycle-resolved kinetics of DNA damage following WRN inhibition in MSI cells. We show that inhibition of WRN leads to the accumulation of DNA double-strand breaks throughout the S- and G2-phases, supporting and expanding on the cruciform-resolution model. We further show that the resulting G2 arrest is ATM-dependent and mechanistically distinct from canonical DSB-induced arrest, sharing features with the DNA decatenation checkpoint triggered by topoisomerase II (TOPOII) inhibition. Comparative analysis of MSI cell lines with varying sensitivity to WRN inhibition reveals differences in DNA damage kinetics and response to combined WRN and TOPOII inhibition. Our findings suggest that the dependency of MSI cells to WRN involves multiple DNA damage pathways and provide mechanistic insight that could guide future therapeutic strategies exploiting WRN-MSI synthetic lethality.