PROJECT SUMMARY Dys-regulated signaling through the Ras/ERK pathway, initiated by receptors including EGFR and MET, is a common driver of resistance to therapy in glioblastoma multiforme (GBM), and other cancers. Despite intensive efforts to develop robust inhibitors of this pathway, Ras/ERK signaling remains an elusive target across oncology. Here, we propose a new way to target Ras/ERK signaling in GBM through an ERK- dependent “suicide gene” approach. The aim is to introduce exogenous genes that drive the selective conversion of non-toxic prodrugs to lethal substances. The HSVtk/GCV (Herpes simplex virus thymidine kinase/ganciclovir) prodrug system is one such strategy. Integration of the HSVtk gene into the host (cancer) cell genome enables phosphorylation of GCV, an acyclic analog of the 2’-deoxyguanosine nucleotide, which competes with guanine during DNA synthesis. We recently developed a new strategy in which HSVtk expression is regulated by ERK activity. Specifically, we engineered a viral vector that expresses a fusion of HSVtk with a domain from the transcription factor fos-related antigen 1 (FRA1) and a nuclear localization sequence. ERK phosphorylation of the FRA1 fragment slows the turnover of the HSVtk fusion, which becomes lethal at sufficient expression levels in the presence of GCV. Preliminary data demonstrate that the GBM cell expression of the new suicide gene construct can indeed drive DNA damage-dependent death in an ERK activity-dependent manner. Here, we propose to advance this preliminary work through two complementary specific aims. In our first aim, we will demonstrate the ability of the suicide gene product (termed HSVtk-FIRE) to selectively kill specific GBM tumor cell types (versus other cell types found in GBM tumors) based on high Ras activity and to test its ability to cooperate with approved or investigational therapeutics. In our second aim, we will determine whether HSVtk-FIRE can be used as an effective therapy in mouse models of GBM. The second aim will feature the application of convection-enhanced delivery to promote delivery of viral vectors that will transduce tumor cells with the suicide gene either with or without focused ultrasound, which has been shown in preliminary studies to promote convection-enhanced delivery in the brain. Ultimately, these studies will advance the possibility of adding a powerful new approach for targeting elevated Ras activity in glioblastoma and other cancers. Such approaches are desperately needed—particularly in GBM, where new improvements in patient survival have not occurred in many years despite knowledge of targetable molecular processes such as Ras/ERK signaling that should provide opportunities for improved patient outcomes.