Sporadic colorectal cancer (CRC) is a public health problem, affecting over a million people each year globally and, in the United States, consistently ranks in the top 2-3 causes of cancer-related death in men and women despite the wide adoption of colonoscopy. Metachronous pre-cancers are also high following polypectomy. However, only a small percent of colon pre-cancers (conventional adenomas or sessile serrated lesions) progress to CRC, and we lack both molecular markers to identify these individuals and an understanding of the mechanisms fostering pre-cancer. The large disease burden of CRC, co-localized with the densely populated colon microbiota, and the recognition that many CRC risk factors (e.g., smoking, obesity, carnivorous diet) modify the colon microbiota has spurred investigations into ‘if and how’ the microbiota contributes to CRC pathogenesis. Accrued data now strongly support the hypothesis that the microbiota is a key environmental contributor to colon carcinogenesis. Nonetheless, little is yet known about ‘if and how’ the microbiota contributes to colon pre- cancers. Our preliminary data identify Escherichia coli that release the non-ribosomal, metabolite genotoxin, colibactin, known as pks+ E. coli (Ecpks), as strongly associated with detection of colon pre-cancers. Further, colibactin is reported to induce specific mutational signatures in colon epithelial cell DNA, in particular, mutations in APC, a CRC driver gene particularly important in adenoma development. Notably, these Ecpks mutations have been associated with CRC. In our COLON MAP study, we have also identified that sessile serrated lesions which account for a large proportion of interval CRC, are likely a result of microbial insult. However, it remains unknown whether Ecpks contributes to SSL development and progression. The few previous human studies, including our preliminary studies, are based on a cross-sectional design. Thus, to address this gap and understand the temporal sequence, we will also include a longitudinal design and human colonoids models to test our core hypothesis that a subset of Ecpks associate with and contribute to driving human pre-cancer progression. We will use a wide array of iterative approaches and conceptual and experimental integration of this project with Projects 1 and 3 and the cores of this U54. Our specific aims in this translational project are: 1) To evaluate the association of Ecpks colonization with human colorectal pre-cancers with greater progressive vs. lower (“indolent”) potential using in-hand human cohorts, clinical predictors, and longitudinal data; 2) To examine mechanisms associated with pre-cancer colon lesions stratified by disease state (indolent or progressive) and Ecpks status using whole exome sequencing, single cell RNAseq and multiplex immunofluorescence (MxIF) approaches; and 3) To identify Ecpks virulence determinants associated with progressive (vs indolent) colon pre-cancer and disease mechanisms using h...