Abstract 5900: Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer
Authors: Sogari Alberto , Rovera Emanuele , Reilly Nicole Megan , Lamba Simona , Russo Mariangela , Lorenzato Annalisa , Durinikova Erika , Trusolino Livio , Arena Sabrina , Donalisio Manuela , Nicolantonio Federica Di , Lembo David , Bardelli Alberto 
Published: April 2023

Abstract
Introduction: The bacterial genotoxin colibactin is enriched in colorectal cancer (CRC) and promotes the accumulation of mutations that drive tumorigenesis. However, systematic assessment of its impact on DNA damage response is lacking and the effect of colibactin exposure on response to other genotoxic agents (such as chemotherapy) is missing.
Materials and Methods: We implemented an in vitro bacteria-coculture system to assess the effect of colibactin on a representative subset of 40 molecularly and pharmacologically annotated CRC cell lines and in a panel of isogenic DDR KO cell lines we generated. We further validated our results in patient-derived organoids. Finally, we recapitulated prolonged exposure to colibactin occurring during tumorigenesis by chronically infecting sensitive cells until the emergence of a tolerant phenotype.}
Results: We found that different cell lines display specific sensitivity to colibactin’s genotoxic stress: while colibactin-tolerant cells are capable to quickly and efficiently repair colibactin-induced DNA damage, sensitive cells lack this ability. Moreover, we found that homologous recombination (HR) proficiency discriminates colibactin-tolerant cells, which display higher levels of RAD51 foci (as marker of activation of HR) compared to sensitive cells upon infection with colibactin. Screening of isogenic DDR KO cell lines revealed that genetic inactivation of the intertwined pathways of HR (through KO of ATM) and replication stress (RS) response (through KO of ATRIP) significantly sensitized cells to colibactin. In addition, we found that restoration of HR activity was sufficient to induce a colibactin-tolerant phenotype in initially sensitive cell lines. Notably, thanks to a previous effort of pharmacological characterization of CRC cell lines in our lab, we found a significant correlation between sensitivity to colibactin and irinotecan active metabolite SN38, but not oxaliplatin. We validated the same correlation in patient-derived organoids annotated for response to SN38. While colibactin, SN38 and oxaliplatin all induced RS in treated cells, we found that colibactin and SN38 showed a similar DNA damage response which involved activation of ATM. Finally, chronic re-infection of sensitive, HR-deficient CRC cells with colibactin selected a tolerant phenotype characterized by restoration of HR activity. Of translational relevance, colibactin-tolerant derivative cells acquired cross-resistance to SN38 and PARP inhibitor olaparib but not to oxaliplatin.
Conclusion: Our results shed novel insight into colibactin’s genotoxic mechanism and support a model in which colibactin both promotes tumorigenesis and acts as an evolutionary bottleneck which selects HR proficient CRC cells. Furthermore, our study provides preclinical evidence on colibactin’s role in promoting chemoresistance in colorectal cancer.
Citation Format: Alberto Sogari, Emanuele Rovera, Nicole Megan Reilly, Simona Lamba, Mariangela Russo, Annalisa Lorenzato, Erika Durinikova, Livio Trusolino, Sabrina Arena, Manuela Donalisio, Federica Di Nicolantonio, David Lembo, Alberto Bardelli. Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer. [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 5900.