Abstract 1215: Synthetic colibactins reveal the structural and biological mode-of-action of a microbial carcinogen

Abstract Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as “pks” encoding the biosynthesis of a secondary metabolite, colibactin, that putatively alkylates host DNA via nucleophilic cyclopropane ring addition. While evidence suggests colibactin’s genotoxicity promotes mutagenesis and cancer, it has been impossible to directly test this hypothesis due to compound instability and sparse production. Recently synthesized stable colibactin analogs facilitate the first studies investigating the compounds structural mode of action. We tested the genotoxic capacity of two compounds: colibactin 742 and an analogous molecule with modified proposed DNA-binding residues (colibactin 746), in human cell lines and colonic organoids. To assess transcriptional pathways associated with acute colibactin response, we compared transcriptional profiles in a normal human colonic cell line (FHC) after short-term exposure to colibactin 742 or 746 and pks+ K. pneumoniae or an isogenic ΔclbP mutant. To determine the mutagenic and transformative potential of colibactin 742, we exposed the human colonic epithelial cell line HCT 116 to 742 or 746 for 32-51 days followed by a 30-day recovery period. We assessed changes in gene expression by RNAseq and mutagenesis by whole-genome sequencing followed by mutational signature fitting. 742 but not 746 caused DNA damage in cell lines and human colonoids, quantified by γH2AX phosphorylation. 742 recapitulated transcriptomic responses to pks+ K. pneumoniae infection, and significantly upregulated pathways associated with p53 signaling, senescence, and IL-1 signaling, relative to 746 treated cells. Moreover, chronic cyclical exposure significantly increased expression of BRCA1-associated DNA repair pathways and genes involved in cross-link repair or replication-associated DNA damage response, consistent with colibactin’s putative cross-linking activity. Whole-genome sequencing revealed an increase in single-base substitutions (SBS) and indels (ID) in 742 treated cells, with a higher proportion of [T>N] SBS and 1-4 bp insertion/deletions in T-rich homopolymers, respectively. We found that 742-induced mutations could be attributed to the proposed colibactin signature (SBS88), reactive oxygen species (ROS; SBS17), or mismatch-repair deficiency (MMRd; SBS44). Colibactin 742 treatment caused an increase in H2DCFDA staining in intestinal epithelial cells, and an increase in transcriptomic pathways associated with peroxisome biogenesis or oxidative phosphorylation, supporting the theory that colibactin 742 treatment caused ROS-induced mutations. ID signatures primarily recapitulated pks-associated mutations. These findings provide the first direct evidence supporting colibactin’s proposed mode-of-action. Furthermore, our study suggests colibactin may indirectly cause mutations attributed to ROS or MMRd. Citation Format: Michael W. Dougherty, Rafael Valdés-Mas, Kevin M. Wernke, Raad Z. Gharaibeh, Alberto Riva, Ye Yang, Marcus Muehlbauer, Eran Elinav, Jens Puschhof, Seth B. Herzon, Christian Jobin. Synthetic colibactins reveal the structural and biological mode-of-action of a microbial carcinogen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1215.