PROJECT SUMMARY Lower grade gliomas, as well as the high grade gliomas that arise from them, are now diagnostically defined by the presence of mutations in genes encoding isocitrate dehydrogenase (IDH) enzymes. Mutant IDH enzymes exhibit the neomorphic ability to synthesize the oncometabolite (R)-2HG. (R)-2HG accumulates to millimolar levels in IDH-mutant glioma cells and competes with a structurally similar metabolite, 2-oxoglutarate (2OG), for binding to 2OG-dependent enzymes. These enzymes include dioxygenases that promote DNA and histone demethylation. Consequently, (R)-2HG accumulation in IDH-mutant cells causes chromatin hypermethylation and epigenetic reprogramming, ultimately leading to oncogenic signaling, a block in differentiation, and malignant transformation. Recently, an inhibitor of mutant IDH (IDHi) oncoproteins, vorasidenib, was shown to dramatically enhance progression-free survival of patients with grade 2 IDH-mutant glioma in a large-scale multi-center phase 3 trial, setting the stage for promulgation of vorasidenib (and similar IDHi drugs) as new “standard-of-care” therapies for these brain tumors. Patients in this study did, however, experience progression on IDHi therapy, indicating that widespread use of these agents will create a new molecular brain tumor cohort: post-IDHi recurrent IDH-mutant glioma. Developing new treatments tailored to the unique biology of this glioma cohort is an emergent, unmet clinical need. Against this backdrop, our group has developed two treatment strategies that hold promise for combating acquired resistance to IDHi therapy. These strategies seek to exploit, rather than inhibit, mutant IDH activity by targeting synthetic lethal vulnerabilities conferred by the (R)- 2HG oncometabolite. We discovered that (R)-2HG accumulation sensitizes IDH-mutant glioma cells to inhibitors of de novo pyrimidine nucleotide synthesis and NAD+ metabolism. These dependencies can be targeted with orludodstat, an inhibitor of the pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH), or with temozolomide (TMZ) and poly(ADP-ribose) glycohydrolase (PARG) inhibitor combination therapy, respectively. Our central hypothesis is that these synthetic lethal treatments will serve as effective second line therapies for IDH-mutant gliomas with IDHi acquired resistance. We will test this hypothesis through three specific aims. In Specific Aim #1, we will create neurosphere, organoid, and genetically engineered mouse models of IDH-mutant lower grade glioma with IDHi acquired resistance and use them to test the antitumor activity of orludodstat. In Specific Aim #2, we will use these models and others to test the impact of IDHi resistance on the antitumor activity of combined TMZ and PARG inhibitor therapy. In Specific Aim #3, we will use primary samples from a window-of-opportunity trial of orludodstat in grade 2 IDH-mutant glioma patients who have progressed on IDHi therapy to assess molecular markers of target engagem...