PROJECT SUMMARY Glioblastoma (GBM) is an aggressive brain cancer type that responds poorly to standard treatment and ultimately develops chemo- and radioresistance. This is due, in large part, to the presence of a sizable and heterogeneous population of GBM stem-like cells (GSCs) that escape conventional therapies and replenish the tumor mass. This project addresses the dire need of attacking these resilient GSCs in order to improve GBM therapy. We will pursue a novel approach focused on targeting cell-scaffolding proteins that are essential for cellular functions. Because GSCs rely on these scaffolding proteins to maintain signaling mechanisms that are critical to radioresistance, we expect that our approach will create an inescapable vulnerability to the effects of radiation, achieving significantly increased tumor lethality. We will focus on a cell-scaffolding protein of the DLG (Disc Large Homologs) family, which are protein-carriers that are critical to keep signaling mechanisms active in their correct locations within the cell. We recently discovered that an unusual member of this family, DLG5, is highly upregulated in GBM and is necessary to maintain the GSC population in the tumor. Our published and preliminary work shows that DLG5 keeps tumor stemness and radioresistance mechanisms, such as Sonic Hedgehog (Shh) and Hippo, in a persistently active state. Accordingly, our central hypothesis is that DLG5 maintains redundant mechanisms that contribute to tumor stemness and the resistance of GSCs to radiation. We predict that targeting of DLG5 will create a non-recoverable vulnerability that can be combined with radiotherapy for improved attack of GBM. To validate this hypothesis, our first Aim is to characterize how the genetic targeting of DLG5 sensitizes GBM cells to radiotherapy. We will investigate the regulation of complementary Shh/Hippo signaling by DLG5 in GSCs and will determine if DLG5 deficiency causes a dominant negative effect that synergizes with radiation to kill these tumor cells. Phenotypic and mechanistic studies will be pursued in GSC cultures, followed by studies in tumor organoids and in vivo orthotopic GBM models. Our second Aim is to validate new agents to disrupt DLG5 functions and radioresistance in GBM. We will engineer cells with DLG5 deletion constructs to identify DLG5 domains that are critical to keep active Shh/Hippo signaling and to maintain the radioresistant features of GSCs. Next, we will focus on our described interaction of DLG5 with the ubiquitin-ligase cullin-3, which is "sequestered" by DLG5 in order to keep stemness pathways in a persistently active state. We will test novel cell-penetrating peptides designed to disrupt the interaction of these two proteins, with the expectation that releasing cullin-3 from DLG5 will result in dominant negative effects on radioresistance. Successful completion of this exploratory project will demonstrate the importance of cell- scaffolding proteins as high-level targe...