共价与非共价WRN抑制剂的细胞学比较揭示了共有及独特的应答生物标志物

Werner Syndrome ATP-dependent helicase (WRN) is crucial for preserving genome integrity through its involvement in key cellular processes such as DNA repair and DNA replication. The WRN helicase has emerged as a promising target for cancer therapy since the discovery of the synthetic lethal interaction between WRN deficiency and microsatellite instability. Although no WRN inhibitors have been approved to date, several candidates, including HRO761 and VVD-214 (RO7589831), have entered clinical trials. HRO761 inhibits WRN non-covalently by interacting with its D1 and D2 helicase domains, while VVD-214 covalently binds to cysteine 727 within an allosteric pocket of WRN. This study aimed to find shared and unique predictive response biomarkers for these two WRN inhibitors. HRO761 and VVD-214 were profiled on a panel of 114 cancer cell lines in cell viability assays using ATP as readout. Response data were related to the microsatellite status of the cell lines as well as to publicly available basal gene expression levels, protein expression levels and WRN dependency based on CRSIPR knock-out and RNAi knock-down screens. Additionally, a selection of microsatellite stable (MSS) and microsatellite instable (MSI) cell lines was used to assess sensitivity to the WRN inhibitors in colony formation unit assays and to evaluate DNA damage induction using fluorescent imaging. Despite their distinct binding mechanisms, the two WRN inhibitors showed nearly identical response patterns across the 114 cancer cell lines. Notably, VVD-214 was 1.7 times more potent than HRO761. The response to the two inhibitors did not correlate well with basal WRN gene expression and WRN protein expression, but showed a significant positive correlation with WRN dependency from a CRISPR knock-out screen and RNAi knock-down screen. The gene expression analysis identified MLH1 expression as a potential resistance marker for both WRN inhibitors. After splitting the 114 cell lines into an MSS and an MSI group, all cell lines in the MSS group were found to be insensitive to the WRN inhibitors, as expected. Surprisingly, the MSI group included both sensitive and non-sensitive cell lines for the two inhibitors. Within the MSI group, VVD-214 was 2.9-fold more potent than HRO761, whereas in the MSS group the difference was only 1.5-fold. Colony formation unit assays and DNA damage imaging further confirmed the differential responses to the WRN inhibitors between MSS and MSI cell lines. The WRN inhibitors HRO761 and VVD-214 showed similar response profiles in the tested cancer cell lines, although some differences were observed. This study highlights potential shared and unique response biomarkers for the WRN inhibitors HRO761 and VVD-214.