Pharmacological Mismatch Repair Inhibition and Colibactin-Induced Mutagenesis in Colorectal Cancer: Therapeutic and Biological Implications

Genomic instability is a hallmark of cancer, driving tumorigenesis through the accumulation of genetic alterations. While it promotes malignant transformation, it also influences tumor immunogenicity by generating neoantigens that can enhance immune recognition. Tumor mutational burden (TMB) therefore plays a dual role in cancer progression and response to immunotherapy. In colorectal cancer (CRC), the gut microbiota is an important source of genomic instability. Chronic exposure to colibactin, a genotoxin produced by certain Escherichia coli strains, induces DNA damage in colonic epithelial cells and contributes to the early stages of tumorigenesis. However, the impact of colibactin-induced mutations on tumor immunogenicity remains unexplored. In contrast, mismatch repair deficiency (MMRd) represents a well-characterized mechanism of genomic instability in CRC. MMRd tumors exhibit microsatellite instability (MSI) and high TMB, making them responsive to immune checkpoint inhibitors (ICIs). However, the majority of CRCs are mismatch repair proficient (MMRp), microsatellite stable (MSS), and largely unresponsive to immunotherapy. In this work, we investigated how the induction of genomic instability might influence tumor immunogenicity and open new therapeutic avenues in CRC. We first examined the effects of colibactin exposure in CRC cells. Colibactin treatment induced DNA double-strand breaks, and tumor cell sensitivity correlated with homologous recombination (HR) deficiency. Prolonged exposure selected for a tolerant phenotype with restored HR activity. Of note, however, this adaptation occurred without a significant increase in TMB, thus limiting our ability to evaluate the immunogenic contribution of colibactininduced mutations. We then explored a pharmacological strategy to promote genomic instability. We characterized NP1867, a first-in-class covalent inhibitor of PMS2, a core MMR component. Cell-based assays confirmed specific target engagement and functional inhibition of MMR activity. Long-term NP1867 treatment induced a switch to an MSI-high genotype, with enrichment of MMRd-associated mutational signatures and increased TMB. In vivo, NP1867- pretreated tumor cells showed enhanced immune infiltration and responded to ICIs, 7 providing proof-of-concept that pharmacological MMR inhibition can convert immunologically cold tumors into immune-responsive ones. Together, these findings highlight the therapeutic potential of inducing genomic instability to enhance antitumor immunity and suggest that MMR inhibition may represent a promising strategy to extend the benefit of immunotherapy to a broader subset of CRC patients.