Brian T. Jones

This file contain information on the Cell lines and organoids used in this study

This file contains details of the BE sgRNA library

Supplementary Figure 1. Generating base editor-expressing MSI cell lines for screening. Supplementary Figure 2. Quality control for base-editor screens in MSI cell lines. Supplementary Figure 3. Discovery and characterization of novel inhibitors of WRN helicase. Supplementary Figure 4. Correlation of WRN inhibitor sensitivity with genetic and molecular markers in MSI models. Supplementary Figure 5. Comparative analysis of DNA damage in MSI and MSS cells treated with WRN inhibitors and genetic inactivation. Supplementary Figure 6: Effects of GSK_WRN4 on MSI and MSS colorectal cancer xenografts.

This table lists the results from Base editing screen

This table lists the results from Base editing screen

Supplementary Figure 1. Generating base editor-expressing MSI cell lines for screening. Supplementary Figure 2. Quality control for base-editor screens in MSI cell lines. Supplementary Figure 3. Discovery and characterization of novel inhibitors of WRN helicase. Supplementary Figure 4. Correlation of WRN inhibitor sensitivity with genetic and molecular markers in MSI models. Supplementary Figure 5. Comparative analysis of DNA damage in MSI and MSS cells treated with WRN inhibitors and genetic inactivation. Supplementary Figure 6: Effects of GSK_WRN4 on MSI and MSS colorectal cancer xenografts.

This file contain information on the Cell lines and organoids used in this study

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR–Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology.See related commentary by Wainberg, p. 1369

This file contains details of the BE sgRNA library

This table lists the results from Base editing screen