Determining the mechanism of pks+ E.coli-associated carcinogenesis in early onset colorectal cancer.

Sloan-Kettering Inst Can Research, New York NY

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Abstract

PROJECT SUMMARY Colorectal cancer (CRC) is now the leading cause of cancer deaths in individuals under 50, yet the causes behind this rapid rise in early onset CRC (EOCRC) remain largely unknown. The microbiome has emerged as potential environmental driver, altering the colon microenvironment through inflammatory/tumorigenic signals, or genotoxins that accelerate carcinogenesis, but why these would disproportionately affect younger people remains elusive. Escherichia coli (E.coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin and have been linked to CRC pathogenesis. While pks+ E.coli are found as commensal bacteria, they can become pathogenic in dysbiosis and inflammation, and are enriched in IBD, polyposis, and CRC patients. Colibactin directly alkylates DNA, leading to mutational A-T rich signatures (SBS-pks) detectable by whole genome sequencing (WGS). Since WGS is not routinely used clinically, we developed an approach to identify SBS-pks signatures in a targeted exon capture assay, MSK-IMPACT. We identify in our institutional pan- cancer cohort of 78,905 tumors 149/1845 samples with MSS CRC with >10% mutations attributable to SBS-pks (Gerstberger et al., in submission). We find that SBS-pks+ CRCs are associated with EOCRC, consistent with a recent WGS study (Diaz-Gay et al., Nature, 2025). To mechanistically investigate why pks+ E. coli preferentially induces CRC in the young, we developed a novel patient-derived organoid (PDO)-microbe co-culture model that leverages reversal of epithelial polarity. Three-month co-culture with human CRC PDOs and NC101 pks+ E.coli or its isogenic ∆pks strain selectively induces SBS-pks signatures in pks+ E.coli co-cultured PDOs. We find that pks+ E.coli activate DNA replication and DNA damage repair (DDR) pathways coupled with cell cycle delay in S phase, indicative of replication stress, and induce a phenotypic switch into more proliferative and regenerative intestinal stem cell (ISC) states that are poised for tumorigenesis. Ageing is associated with delayed cell cycle progression, reduced DNA repair proficiency, proliferation, and stem cell fitness. Using young/old PDO co-culture and mouse CRC models with pks+ and ∆pks E. coli, we will test the hypothesis that (1) the young epithelium has greater DDR and ISC plasticity, enabling error prone repair and dynamic colibactin induced entry into ISC states, while in aged epithelia, colibactin injury induces synthetic lethality due to deficient DNA repair and decreased plasticity. Alternatively, (2) young intestinal epithelia may harbor an epigenetic memory of inflammation from emerging environmental stressors, rendering them poised to tumorigenesis upon colibactin injury, which we will test through epigenomic and inflammatory stimulus-response assays of our existing biobank of young/old normal colon/CRC organoids from patients with/without CRC. Outcomes of this research will inform our understanding of EOCRC mechanisms and have potential implications for exposures and preventative screening.