Abstract B036: cGAS-STING activation promotes anti-tumor inflammatory response in osteosarcoma: Implications for tumor microenvironment reprogramming and tumor progression in patients
Authors: Young Elizabeth P , Johnson Christine A , Lee Alex G , Schott Courtney R , Sweet-Cordero E. Alejandro 
Published: December 2023

Abstract
A “cold” tumor microenvironment (TME) is often seen in pediatric patients with osteosarcoma (OS). Genomic instability, characteristic of OS, can stimulate the cGAS-STING pathway and activate the innate immune system. However, tumor-intrinsic STING pathway silencing mechanisms link genomic instability to immune evasion in OS. We hypothesized that dysregulation of the cGAS-STING pathway suppresses an innate immune response to genomic instability and plays a key role in tumor progression by shaping an immunosuppressive TME in pediatric OS patients. Using a panel of 11 OS patient-derived xenograft (PDX)-derived cell lines, we characterized cGAS-STING pathway function and observed heterogeneity in STING responsiveness. Comprehensive biochemical evaluation of the inflammatory signaling response to STING agonism revealed two groups (6 responsive and 5 unresponsive) among our panel of 11 OS PDX-derived cell lines. To investigate the effects of OS tumor-intrinsic STING activation on macrophage polarization, we used tumor conditioned media (TCM) assays. We hypothesized that cGAS-STING activation in OS cell lines would be sufficient to shift macrophage phenotype ratios, enriching for a higher proportion of inflammatory macrophages, through the secretion of inflammatory factors that stimulate innate immune cells. A cGAS agonist, G3-YSD, was used to stimulate the STING pathway in murine OS lines. Addition of the TCM to macrophages resulted in a decrease in CD206+ “M2-like” macrophages and an increase in MHC II+ “M1-like” macrophages, measured via flow cytometry, in a manner dependent on tumor STING. Lastly, we performed bulk RNA sequencing of OS PDX cell lines treated with a STING agonist, defining for the first time the OS-specific STING activation signature. We then evaluated primary OS patient samples for evidence of this signature by gene set enrichment analysis and performed Kaplan-Meier survival analysis after stratifying samples as high or low for this signature, which demonstrated a significant protective effect of the STING activation signature on progression-free and overall survival. Together, we have shown that dysregulation of the cGAS-STING pathway represses the innate immune response to the genomic instability present in many OS cells and represents a key factor in the establishment of aggressive behavior in OS tumors. We have defined two subtypes of OS in terms of responsiveness to STING activation and demonstrated that STING activation has a protective effect in the human disease, which is the foundation of current efforts to explore targetable mechanisms to reverse STING unresponsiveness and activate immune surveillance in this cancer.
Citation Format: Elizabeth P Young, Christine A Johnson, Alex G Lee, Courtney R Schott, E. Alejandro Sweet-Cordero. cGAS-STING activation promotes anti-tumor inflammatory response in osteosarcoma: Implications for tumor microenvironment reprogramming and tumor progression in patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B036.