Abstract 6127: Genomic instability regulates STING mediated anti-tumor immunity through distinctive cytosolic DNA structures

Abstract Background. Cancer genomes are characterized by instability, with significant levels of DNA damage and mutation due to DNA repair defects. In colorectal cancer (CRC), defects in mismatch repair define the microsatellite instable (MSI) subtype, with stronger immunogenicity and better prognosis than the more common chromosomally instable (CIN) subtype. The mechanisms behind these differences are unknown, but their understanding could identify strategies to improve CIN CRC prognosis. The STING pathway detects foreign DNA in the cytosol, leading to induction of innate and adaptive immune responses. In cancer cells, STING also recognizes self cytosolic DNA (cyDNA) in the form of micronuclei or diffuse fragments generated by endogenous or treatment induced DNA damage. Our lab previously identified this pathway as a key player in the increased cytotoxic T cell infiltration characteristic of MSI CRCs. As MSI CRCs do not contain more cyDNA than CIN, we hypothesize that specific sequence or structural patterns exist within cyDNA from different CRC subtypes that lead to differential STING activation and thereby induction of overall anti-tumor immunity. Methodology. We first aimed to examine whether cyDNA produced in MSI vs CIN CRC cells differentially induce STING and thereby anti-tumor immunity. CyDNA isolated from MC38 CRC cells with mutations in Mlh1 (modeling MSI) or Kras (CIN) were used to stimulate dendritic cells (DCs), key mediators of anti-tumor immunity. We found equal concentrations of MSI cyDNA more strongly activates STING, and thereby cytotoxic T cells, than CIN cyDNA. CyDNA from MSI CRC patient organoids showed similar results. Next, we aimed to characterize specific changes in cyDNA composition in MSI vs CIN CRC cells and examine how these alterations affect STING signalling. Sequencing identified patterns prevalent in highly stimulatory cyDNAs, including microsatellites. Stimulation with oligos and cyDNA enriched for microsatellites indicate enhanced STING and cytotoxic T cell activation through improved cGAS binding. Likewise, adoptive transfer of DCs activated by microsatellite rich DNAs induced stronger DC and T cell activation in orthotopic CIN tumors. Preliminary data suggests secondary DNA structures may influence this process. Finally, we aim to evaluate the contribution of micronuclei on STING activation in MSI vs CIN CRCs. DC stimulations with micronuclei isolated from MSI and CIN cells indicate MSI micronuclei also lead to enhanced STING activation over CIN micronuclei. We will evaluate micronuclei composition to identify underlying mechanisms. Significance. CRC is the second most deadly cancer worldwide, and immune recognition is crucial to treatment efficacy. This project will further our understanding of immune differences between MSI and CIN CRCs that may be used to develop therapeutic strategies, such as STING agonists, to improve CIN patient prognosis. Citation Format: Shayla Mosley, Natalie Lapa, Courtney Mowat, Kristi Baker. Genomic instability regulates STING mediated anti-tumor immunity through distinctive cytosolic DNA structures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6127.