Project Summary/ Abstract Despite significant advances in our understanding of the molecular drivers of Diffuse Midline Gliomas (DMGs) including Diffuse Intrinsic Pontine Gliomas (DIPGs), there are no viable treatment options resulting in certain fatality of children with these brain tumors. The lack of understanding of pathogenesis of these pediatric brain tumors is a significant barrier to developing effective treatments. More than 80% of DMGs bear a histone H3 mutation at lysine 27 to methionine (H3K27M). H3K27M causes global reduction in the repressive mark H3K27me3, which is the preferred epigenetic state of these tumor cells. Our premise is based on reprograming of cellular metabolism by oncogenes which is one of the fundamental mechanisms driving cancer cell survival and growth. Our group has shown that H3K27M mutations drive tricarboxylic acid (TCA) cycle metabolism to enhance production of the metabolite α-ketoglutarate (αKG), a critical cofactor for the H3K27 histone lysine demethylases. We have reported that αKG is required to maintain the preferred epigenetic state of H3K27 hypomethylation and interruption of this integrated pathway is potently therapeutic by increases H3K27me3 to reverse the favored epigenetic state of H3K27M tumor cells. Our proposal is based on studies from a clinical trial with the imipridone ONC201 in children with H3K27M DMGs at the University of Michigan. ONC201 shows remarkable survival benefit with near doubling in overall survival in a subset of ONC201 treated H3K27M DMGs. Our premise is also based on ONC201’s known effect to suppress mitochondrial oxidative phosphorylation. Response to ONC201 in H3K27M DMGs was associated with suppression of key TCA cycle- related genes. Moreover, ONC201 reversed the preferred H3K27 hypomethylated state by increasing global and genomic H3K27me3 levels. These supporting data have led to hypothesize that ONC201 disrupts integrated metabolic/ epigenetic pathways to reverse the preferred epigenetic state of H3K27M tumor cells. Three specific aims address this hypothesis. Aim 1 will define the molecular mechanisms by which ONC201 inhibits combined metabolic/ epigenetic pathways and interrogate downstream epigenetic alterations in H3K27M tumor cells. Aim 2. Will understand the effect of hypoxic microenvironment on H3K27M cells and their response to ONC201 treatment. Aim 3. Will delineate the mechanisms that govern intrinsic resistance to ONC201 and elucidate the epigenetic state of ONC201 sensitive and resistant H3K27M DMGs, and begin to define strategies to overcome ONC201 resistance. Our work is immediately translatable as it is based on our ongoing clinical trial and will further our knowledge of mechanisms the govern response and resistance to ONC201 to directly impact the treatment of children with lethal H3K27M DMGs.