cGAS-STING Agonist as a Therapeutic Target for Pediatric High Grade GliomadissertationsHawkins, William MoriartycreatorLawler, SeanAdvisorOancea, ElenaReaderMathiowitz, EdithReaderBrown University. Biology and Medicine: Biotechnologysponsor2025, 49 p.born digitalThesis (Sc. M.)--Brown University, 2025thesesObjective: Pediatric high-grade gliomas are among the most aggressive pediatric malignancies, associated with poor prognosis and limited therapeutic options. This study evaluates the therapeutic potential of ADU-S100, a cGAS-STING agonist, aiming to enhance innate immune responses in these typically immunologically cold tumors. Methods: Patient-derived pHGG cell lines were treated in vitro with the cGAS-STING agonist ADU-S100. The activation of the cGAS-STING pathway was evaluated using Western blot analysis, while the viability of tumor cells post-treatment was measured using CellTiter-Glo assays. The functional response of the pathway was further confirmed by quantifying secretion of canonical STING-induced cytokines C-C Chemokine Ligand 5 (CCL5) and interferon gamma-induced protein 10 (CXCL10) via ELISA. Following the identification of a responsive cell line, co-culture experiments were conducted with peripheral blood mononuclear cells (PBMCs) at various ratios and ADU-S100 concentrations to model immune-mediated tumor cell killing. Finally, to establish an in vivo disease model, a murine-derived H3K27M mutant cell line was intracranially implanted in mice for validation and future therapeutic testing. Results: cGAS-STING signaling pathway components were detected in pHGG cell lines by western blot. ADU-S100 treatment resulted in robust activation of the cGAS-STING pathway in the SU-DIPG-36 cell line, demonstrated by increased secretion of CCL5 and CXCL10, indicative of a type I interferon response.ADU-S100 treatment resulted in a decrease in viability for SU-DIPG-4 and HCMEC cells. However, co-culture experiments did not demonstrate significant immune-mediated tumor cell death, irrespective of ADU-S100 dosage or PBMC-to-tumor-cell ratio. An in vivo murine model of H3K27M-mutant pHGG was successfully established, confirmed by intracranial tumor growth expressing the H3K27M mutation. Conclusions: This study identifies SU-DIPG-36 cells as a responsive pHGG cell line to ADU-S100, and SU-DIPG-4 cell lines as having a significant reduction in ATP post treatment with ADU-S100, providing critical models to further investigate cGAS-STING activation and its potential as a biomarker-driven therapeutic approach. Although the co-culture assay highlighted challenges in modeling immune-mediated cytotoxicity in vitro, the successful development of a relevant murine tumor model offers valuable opportunities for future preclinical therapeutic investigations.PediatricsBrain--CancerEnglishRPB20250707