RESUMO
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 tumours, and WRN inhibitors are in development. Here, 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 (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) 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.
RESUMO
Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive T cells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.
