Understanding and Harnessing Microbial Reprogramming to Combat Carcinogenic Colibactin in Colorectal Cancer - Abstract In alignment with the FOA PAR-22-085, which emphasizes microbial-based cancer therapies, our objectives are twofold: (1) to develop a novel strategy to enzymatically inhibit colibactin, a procarcinogenic metabolite from enteric bacteria associated with colorectal cancer (CRC), and (2) to understand how these engineered microbes reduce the in vivo colonization of colibactin-producing bacteria but spare other microbes. The rising incidence of colonization by pathogenic gut bacteria producing procarcinogenic molecules has garnered increasing attention, with colibactin representing one of the most extensively studied microbial metabolites linked to CRC. Colibactin induces DNA damage, senescence, and genomic mutations in the host intestinal epithelium. Colibactin-producing bacteria carry the polyketide synthase (pks) gene island, and recent studies have demonstrated a high prevalence of pks+ bacteria in 55–67% of CRC patients. Despite efforts to understand the mechanisms underlying colibactin-induced CRC, few therapeutic strategies have been developed to target colibactin directly. Interestingly, pks+ bacteria possess an intracellular resistance protein, ClbS, which inactivates colibactin and shields the bacteria from colibactin-induced self-DNA damage. Inspired by this natural defense mechanism, we hypothesize that engineering nonpathogenic living microbes to deliver ClbS via bacterial surface display can safely and effectively inactivate colibactin in the gut. We base our hypothesis on a synthesis of our recent publication and exciting preliminary data, demonstrating that (1) surface- displayed ClbS prevents colibactin-mediated genotoxicity in cell lines and colon organoids of mouse and human origins. (2) ClbS-displaying commensal E. coli reduces tumorigenesis in a colitis-associated CRC model infected with pks+ bacteria without adverse effects. (3) ClbS-displaying E. coli inhibits pks+ bacteria but spares the isogenic strains lacking the pks island in conventional mice containing the native gut microbiome. While our preliminary data demonstrate an innovative solution to targeting colibactin through engineered bacteria, it raises several questions to address mechanistically and therapeutically. Aim 1 will understand ‘if and how’ ClbS-displaying commensal E. coli inhibits pks+ bacteria directly or indirectly by abrogating colibactin- mediated niche competition with the gut microbiome. In parallel, Aim 2 will enhance mechanistic understanding and translational potential by investigating how ClbS-displaying E. coli impacts the tumor initiation, progression, colibactin-specific mutational signatures, and responses to immune checkpoint blockade in mouse CRC models. The expected outcomes are (1) to elucidate the molecular mechanisms through which engineered E. coli inhibits pks+ bacteria and neutralizes colibactin and (2) to offer a novel therapeutic approach to CRC intervention. Although this work focuses on targeting colibactin in pks+ bacteria, a long-term goal of this proposal is to engineer microbial therapeutics targeting other cancer-promoting bacterial metabolites via the bacterial surface display in conjunction with the standard care of treatment for CRC.
