Disrupting Mismatch Repair Induces Immune-Sensitive Phenotype in Colorectal Cancer

Immune checkpoint blockade (ICB) has transformed cancer therapy by overcoming tumor immune evasion, particularly in advanced solid tumors resistant to conventional treatments. However, clinical responses are highly variable and strongly associated with mismatch repair (MMR) status: patients with MMR-deficient/microsatellite instability-high (dMMR/MSI-H) tumors respond more robustly than those with MMR-proficient/microsatellite stable (pMMR/MSS) tumors. While ICB responsiveness has been attributed to neoantigen-driven immunogenicity or increased susceptibility to inflammatory cell death (e.g., PANoptosis), it remains unclear whether MMR deficiency alone can confer both advantages. Here, we investigated this question by transiently knocking down MLH1 in pMMR/MSS colorectal cancer cells to induce a dMMR/MSI-Hlike phenotype. MLH1 knockdown resulted in MSI at 2 of 5 tested loci. When co-cultured with pre-activated CD8+T cells (E:T = 1:5), reprogrammed cells triggered significant pro-inflammatory cytokine release, with IFN- levels approximately double those of the dMMR benchmark, indicating hyper-enhanced immunogenicity. Separately, MLH1-deficient cells displayed intermediate susceptibility to cytokine-mediated inflammatory cell death (50.95%), compared with low responses in pMMR cells (8.68%) and near-complete death in dMMR cells (99.09%). These findings demonstrate that transient MLH1 knockdown can simultaneously enhance tumor immunogenicity and susceptibility to inflammatory cell death, supporting both neoantigen-based and PANoptotic models of ICB responsiveness. This approach offers a controllable, reversible strategy to sensitize immune-cold pMMR tumors to ICB, potentially reducing the risk of chronic side effects associated with prolonged interventions. By establishing MLH1 knockdown as a flexible adjunct to multiple therapies, from ICB to cytokine-based treatments, our work lays a foundation for translational strategies that unify treatment paradigms for pMMR and dMMR colorectal cancers.