EXTH-22. Targeting therapy-resistant SOX9-positive cells with a cytotoxic gene therapy in glioblastoma

Abstract Glioblastoma (GBM) is an aggressive and incurable brain tumor, with treatment resistance and recurrence posing persistent clinical challenges. The transcription factor SOX9 has recently emerged as a key regulator of therapy resistance. Still, no specific SOX9 drugs exist, likely due to the inherent difficulty of targeting transcription factors. Here, we delineate a critical role for SOX9 in driving resistance to standard-of-care radiation and temozolomide (TMZ) therapy. Using a panel of 35 patient-derived GBM cell lines of varying subgroups and MGMT status we defined TMZ sensitivities and demonstrated that SOX9 is selectively activated in resistant lines following cytotoxic stress. Elevated SOX9 expression correlated with poor patient survival by attenuating TMZ-induced DNA damage. CRISPR-based depletion of SOX9 sensitized GBM cells to radiation and TMZ, underscoring its functional importance in mediating resistance. To therapeutically exploit this vulnerability, we developed a SOX9-responsive suicide gene therapy. This system couples the Herpes Simplex Virus Thymidine Kinase (HSV-TK) gene to a SOX9-inducible enhancer, enabling selective ablation of SOX9-positive GBM cells upon ganciclovir (GCV) treatment. Delivery via AAV2 or AAV9 vectors effectively eliminated resistant tumors and significantly extended survival in both immunodeficient and immunocompetent orthotopic GBM mouse models. SOX9-directed gene therapy synergized with radiation to enhance DNA damage and even led to long term survival due to a reprogrammed tumor immune microenvironment. We establish SOX9 as a central driver of therapeutic resistance in GBM and present a first-in-class strategy to selectively target resistant tumor populations. Clinical translation of this strategy holds promise for improving outcomes in patients with SOX9-driven tumor relapse.