Project Summary Emerging data have linked the gut microbiome to colorectal adenomas, the established early lesions in colorectal cancer (CRC) and de facto targets during screening colonoscopy. However, deficit in our knowledge of the basic science of the gut microbiome and the specific driver mechanisms for inducing adenoma formation and progression has impeded translation. We hypothesize that the microbiome is an adenoma nonautonomous inducer of colorectal “priming,” the term we use in this application to refer to molecular/cellular alterations critical for adenoma formation and progression. The near-term objective of this proposal is to define whether and how the microbiome and the primed colon act in concert to drive adenoma formation. The long-term objective of this proposal is to develop microbiome-based strategies for precision prevention of adenomas and ultimately CRC. In Aim 1, we will identify interdependencies of the microbiome and the primed colon. We will leverage insights from a novel, validated metagenomic analysis. Using gnotobiotic mice, we will study interactions between a primed colon, tumorigenic gut bacteria, and tumorigenesis. We use analytic approaches such as mediation analysis to quantify the extent to which the microbiome and a primed colon each mediate the other’s tumorigenic effects. In Aim 2, we will identify the adenoma-associated gut microbiome features that characterize aggressive vs indolent adenomas in humans. We will analyze a unique cohort of longitudinally monitored adenomas through deep sequencing, in situ bacterial imaging, rigorously defined genetic/molecular profiles generated in Project 1, and machine learning to identify adenoma-associated microbiome features that interact with primed colon attributes to drive adenoma progression. In Aim 3, we will determine if microbiome- induced senescent fibroblasts drive progression behavior in adenoma cells in ex vivo adenoma organoids through specific CRC-associated senescence-associated secretory phenotype (SASP) factors. Using primary colon fibroblasts derived from gnotobiotic mice colonized with a CRC-associated bacterial consortium, we will assess oncogenic effects on adenoma progression behavior in an ex vivo organoid model. We will determine whether tumorigenic effects of the microbiome are mediated through induction of the senescent colon primed state, and we will mechanistically test roles for candidate CRC-associated SASP factors in driving progression behavior of adenoma cells in ex vivo human adenoma organoids. Our findings could serve as the basis for biology-backed, intervenable, precision prevention of adenomas and ultimately CRC.