Abstract 1819: Inhibition of the cell-scaffolding protein DLG5 sensitizes glioblastoma stem cells to radiotherapy

Abstract Glioblastoma (GBM) is a fatal CNS tumor that responds poorly to conventional chemo- and radiotherapy. Although radiation achieves a significant adjuvant effect after surgical debulking, radioresistant tumor cells repopulate the tumor bed, resulting in unavoidable recurrence. GBM radioresistance has been largely associated with a population of GBM stem cells (GSCs) driven by Sonic Hedgehog (SHH) and Hippo pathways that promote metabolic reprogramming, DNA repair, and cell proliferation. We have previously described that GBM stemness pathways such as SHH are regulated by a cell-scaffolding member of the Scribble/DLG family, DLG5, which is a "high hierarchy" target that maintains a persistent GSC population. Given the related identity of stemness and post-treatment resistance in GSCs, we have investigated here if DLG5 can be targeted to increase the sensitivity of GSCs to conventional radiotherapy. Our results in cultured GSCs confirmed that DLG5 not only regulates the SHH pathway -as we have previously shown- but also the Hippo pathway. Specifically, DLG5 knockdown increased the expression of the Hippo-repressor kinases, MST-1/MST-2, and phosphorylation of the Hippo effector YAP; these alterations were accompanied by downregulation of Hippo-downstream genes (CTGF, BIRC5) as well as genes involved in DNA-damage repair (BRCA1, RAD51) and stemness (OCT4, NANOG). In agreement with these results, DLG5-deficient GSCs subjected to different irradiation regimes (2 - 8 Gy) showed decreased cell viability, reduced survival fraction in clonogenic assays, and reduced self-renewal ability compared to control GSCs. Irradiated DLG5-deficient cells had elevated cleaved Caspase-3 and PARP1 compared to control cells, increased long-term DNA damage, and accumulation in G2/M phase suggesting mitotic catastrophe. The sensitizing effects of DLG5 deficiency were also tested in intracranial GSC-derived tumors subjected to fractionated radiotherapy (5 x 2Gy q24h). Radiotherapy significantly increased the survival of animals implanted with DLG5-deficient tumor cells compared to controls. Overall, our work suggests that targeting DLG5 increases the efficacy of radiotherapy in GBM by disabling the ability of this cell-scaffolding protein to regulate signaling pathways that drive tumor stemness. Work in progress is currently focused on identifying critical functional domains of DLG5 and developing inhibitors to block DLG5-driven mechanisms in GBM tumors. Citation Format: Somanath Kundu, Soham Mitra, Abigail Venskus, Harish Babu, Jason Horton, Mariano Viapiano. Inhibition of the cell-scaffolding protein DLG5 sensitizes glioblastoma stem cells to radiotherapy [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 1819.