Project Summary Glioblastoma multiforme (GBM) is one of the most therapy-resistant tumors, with a dismal 5-year survival rate of <10%. Intrinsic and acquired resistance to radiation therapy contributes significantly to the refractory nature of these tumors. Ionizing radiation (IR) exerts its cytotoxic effects primarily by generating free radicals, in particular reactive oxygen species (ROS). Moderating redox is therefore critical to mitigating the lethal effects of IR and increase the effectiveness of RT. In this proposal we postulate that GBM tumors can generate an antioxidant response to RT by rewiring their metabolism and this is a major mechanism leading to their survival and treatment failure. As yet, little is known about the metabolic response of GBM undergoing RT and the molecular drivers of metabolic plasticity are unknown. In the parent award we provide evidence for radiation-induced metabolic reprogramming in GBM, which includes enhanced consumption of glucose and glutamine by irradiated GBM cells and diversion of the flow of glycolytic intermediates into the antioxidant, NADPH-producing pentose phosphate pathway (PPP). Based on preliminary studies, we hypothesize that the IR-induced metabolic reprogramming in GBM is orchestrated in part by the activation of the transcription factor NRF2, which turns on the transcription of metabolic enzymes that drive the PPP. We also hypothesize that diversion of glycolytic intermediates into the PPP is further amplified by the IR-induced inhibition of the redox-sensitive, glycolytic enzyme PKM2. Here, we propose to expand our current working model based on new compelling data that point to serine synthesis pathway (SSP) as an additional radiation-induced metabolic pathway that also contributes to metabolic rewiring in GBM. The radiation-enhanced SSP activity is driven by NRF2, in keeping with NRF2 being the orchestrator of IR-induced metabolic reprogramming in GBM. However, we have also shown that IR activates the HIF-1 pathway independently of hypoxia and IR-induced upregulation of SSP enzyme expression is completely prevented by HIF-1a inhibition, suggesting dual roles for these two redox- sensitive factors in coordinating the metabolic rewiring of GBM following RT that drives antioxidant pathways and IR resistance through the PPP and de novo SSP, with assistance from PKM2 blockade. Together with the body of data from the parent award, these studies will build a comprehensive picture of radiation-induced metabolic rewiring in GBM that will illuminate interventional strategies aimed at improving RT outcomes in this dreadful disease.