Cytosolic DNA structures produced by mismatch repair deficiency coordinate anti-tumor immunity in colorectal cancer

• MSI CRCs produce cyDNA enriched with G-quadruplexes, leading to high STING activation • STING activation by micronuclei causes immunosuppression via IL-10 production by Tregs • Micronuclei are a previously unrecognized inducer of pro-tumorigenic STING activation MSI CRCs produce cyDNA enriched with G-quadruplexes, leading to high STING activation STING activation by micronuclei causes immunosuppression via IL-10 production by Tregs Micronuclei are a previously unrecognized inducer of pro-tumorigenic STING activation Patients with the microsatellite instable (MSI) subtype of colorectal cancer (CRC) have a better prognosis and immunotherapy response than patients with the chromosomally instable (CIN) subtype due to improved cytotoxic T cell responses. This is in part due to high production of the chemokines CXCL10 and CCL5 from constitutive activation of the cytosolic DNA (cyDNA) sensor cGAS/STING by specific features of MSI cyDNA that lead to more effective cGAS/STING pathway activation. Here, we investigate MSI and CIN cyDNA structure and show that MSI cyDNA is enriched in G-quadruplexes that improve cGAS/STING and CD8 + T cell activation. We also show that micronuclei are less effective at inducing anti-tumor immunity and instead increase Treg activation and IL-10 production. Overall, these data highlight the role of specific cyDNA structures in anti-tumor immunity and provide knowledge for improved design of therapeutic DNA-based cGAS/STING agonists to improve the prognosis of poorly immunogenic tumors like CIN CRCs.