Abstract 3281: CRISPR activation of endogenous STING as an innovative strategy for targeted immunotherapy

Abstract Background: Stimulator of Interferon Genes (STING) has long been of interest within the field of cancer immunology, as the cyclic GMP-AMP synthase (cGAS)-STING pathway can drive inflammation in the tumor microenvironment through the sensing of cytosolic DNA and production of 2’3’-cGAMP. Importantly, in cancer cells with high levels of chromosomal instability (CIN), we and others have observed selective suppression of STING expression, which we hypothesize benefit cancer cells to avoid activating an immune response and to promote immune escape. Method: The murine triple-negative breast cancer model 4T1, which presents with CIN was explored. We conducted both in vitro and in vivo experiments to characterize cGAS-STING pathway signaling in response to exo- as well as endogenous DNA stimulation. Utilizing a CRISPR-dCas9 activation (CRISPRa) approach we selectively regulated STING expression in cancer cells and evaluated immune responsiveness in vitro. The anti-tumoral effect of activating the cGAS-STING signaling cascade was tested in vivo using aEpCAM-labelled lipid-nanoparticles (LNPs) for delivering mRNA to support selective uptake in 4T1 cells. Results: Initially, 4T1 cellswere found to have low STING expression, in conjunction with high levels of micronuclei and cGAS activity, supporting endogenous accumulation of 2’3’-cGAMP. Using CRISRPa of STING, we observed a rapid cGAS dependent type-I interferon and inflammatory cytokine response correlating with STING expression in a time-dependent manner. Using EpCAM-targeted LNPs we enabled specific mRNA delivery to 4T1 cells in vitro and in vivo. Our current efforts focus on exploring the in vivo anti-tumoral effects of this STING-targeting immunotherapy which will be presented at AACR. Conclusion: Our data reveal that reestablishing endogenous STING expression in cancer cells that exhibit elevated CIN, may result in an anti-tumoral inflammatory response. We believe that the altered immune profile determined by STING reactivation presents a therapeutic potential to promote tumor regression. Using this specifically targeted LNP-mRNA system, this type of epigenetic regulation may become a cornerstone of future personalized cancer therapies in addition to traditional immunotherapy. Citation Format: Emil L. Lauridsen, Stine H. Godsk, Anders Etzerodt, Martin R. Jakobsen. CRISPR activation of endogenous STING as an innovative strategy for targeted immunotherapy [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 3281.