Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a uniformly fatal pediatric brainstem tumor and the leading cause of brain-tumor related deaths in children. It is therefore imperative to identify novel treatment strategies for this aggressive and devastating disease. Metabolic reprogramming in tumors and in the tumor microenvironment contribute to evasion of therapy and tumor recurrence. The goal of this study was to identify and therapeutically target metabolic vulnerabilities in DIPG that mediate aggressiveness and treatment resistance. METHODS DIPG tumors are marked by cellular heterogeneity and are driven by a population of cells with stem cell properties. We took a comprehensive metabolomics and transcriptomic screening approach to determine the operative pathways in the tumor driving stem cell compartment. To demonstrate efficacy and potential therapeutic window of activity, we treated DIPG tumors with clinically available and brain-penetrant inhibitors of the identified dysregulated metabolic pathways. RESULTS Our multi-omics analyses revealed that tumorigenic patient-derived DIPG cells significantly upregulate metabolic programs including cholesterol biosynthesis and mitochondrial oxidative phosphorylation (OXPHOS) compared to DIPG cells that were induced to undergo differentiation (events associated with a loss of tumorigenic capabilities). The therapeutic targeting of DIPG tumors with clinically available and brain penetrant inhibitors of OXPHOS and cholesterol biosynthesis resulted in tumor cell killing and growth inhibition both in vitro and in vivo. Moreover, there was a therapeutic window of activity in tumorigenic DIPG cells compared with differentiated gliomas and non-malignant cells. CONCLUSION Our findings demonstrate that DIPG harbor perturbations in metabolic programs that can be exploited for therapeutic benefits. The results from this study defined the metabolic pathways operative in the tumor-driving population in DIPG and demonstrated efficacy of targeting these pathways.