PROJECT SUMMARY/ABSTRACT The proposed study seeks to determine if DNA methyltransferases from members of the microbiota contribute to bacterial pathoadaptation in the tumor niche, direct xenogeneic epi-modifications on the human genome, and can be exploited as therapeutic targets in cancer prevention/treatment. Within a patient’s tumor, malignant cells are surrounded by a complex microenvironment encompassing a range of non-transformed cells and also a diverse collection of microorganisms. For example, Fusobacterium nucleatum (Fn) is significantly enriched in colorectal adenocarcinoma compared to adjacent normal tissue. Although cellular and animal models have supported a role for this bacterium in cancer initiation and progression, we still have little information as to how they directly contribute to cancer. In published work, our group has demonstrated that Fn, which is usually part of the oral microbiome and not found in the lower gastrointestinal tract, is an invasive intracellular bacterium that effectively colonizes the colorectal cancer (CRC) niche. In preliminary studies, we isolated and characterized the genomes and epigenomes of >150 Fn strains from CRC tumors and the oral cavity and discovered that within the Fn subspecies Fn subsp. animalis, there are two distinct clades that differ in their enrichment in CRC, which we named Fn oral-clade and Fn CRC-clade. We show that Fna CRC-clade is the only Fn group significantly enriched in human tumors and fecal specimens. Of the many differences between these two clades, perhaps one of the most striking is their distinct DNA methylome signatures, with CRC-clade uniquely harboring Gm6ANTC methyl-modifications. We, therefore, hypothesize that the DNA methyl-modifying enzyme (M.FnI) responsible for m6A methylation at this motif contributes to Fn CRC-clade virulence during carcinogenesis. We propose to test this hypothesis using genetically engineered CRC-clade clinical isolates in cell culture and animal model experiments to determine if M.FnI regulates bacterial gene expression to promote their pathoadaptation (Aim 1). Additionally, we speculate that M.FnI can act as a nucleomodulin to directly interfere with the human host epigenome and gene regulation. We will investigate this possibility through ectopic expression of M.FnI in human CRC cell lines and pre-cancer organoid models to delineate its nucleomodulin potential (Aim 2). Finally, we seek to develop chemical probes specific for M.FnI to aid in authenticating it as a therapeutic target in CRC prevention and progression (Aim 3). While we focus here on a single pathogen and single disease, successful completion of these aims will provide fundamental knowledge on the role of microbial epigenetic systems in tumor colonizing microbes. Further, this work has potential to reveal a hidden paradigm of host microbe-epigenetic crosstalk underlying the oncogenic process in bacterial-colonized, hypermethylated tumors. As such, if our core hypothesis is...