Dissecting Mechanisms of Pyrimidine Synthesis Dependence in IDH Mutant Glioma

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PROJECT SUMMARY Driver mutations in genes encoding the metabolic enzyme isocitrate dehydrogenase (IDH) are present in >80% of lower-grade gliomas and the high-grade tumors that arise from them. To identify new therapeutic targets for this incurable disease, our laboratory recently conducted an unbiased drug screen and discovered that IDH oncogenes confer dependence on the de novo pyrimidine nucleotide synthesis pathway for glioma cell survival. Despite our identification of this vulnerability, the molecular mechanism linking IDH mutations and dependence on de novo pyrimidine nucleotide synthesis is unknown. Therefore, I developed a platform to comprehensively profile nitrogen metabolism in patient-derived IDH mutant glioma stem-like cells (GSCs) treated with either vehicle or an inhibitor of mutant IDH, identifying the contribution of glutamine to pyrimidine nucleotides as among the most differentially regulated metabolic networks between these conditions. I subsequently found evidence of disjunction between the two main routes for pyrimidine nucleotide production: de novo synthesis and salvage pathways. Both pathways contribute to synthesis of uridine monophosphate (UMP), the metabolite from which all other pyrimidine nucleotides are derived. My research revealed that although IDH mutant GSCs use both pathways to produce UMP, these cells preferentially use UMP derived from the de novo pathway to synthesize pyrimidine nucleotides downstream of UMP. This phenotype was not observed in human astrocytes, suggesting that it may be tumor specific. I hypothesize that IDH mutant glioma cells are dependent on de novo pyrimidine synthesis because they harbor a novel metabolic enzyme complex that channels UMP from the de novo synthesis pathway to downstream pyrimidines. I will test this hypothesis through three studies. First, I will evaluate whether this pyrimidine synthesis partitioning phenotype is unique to IDH mutant GSCs by performing stable isotope tracing studies across a panel of IDH mutant and IDH wild-type patient-derived GSCs. Second, I will test whether pyrimidine biosynthesis enzymes form a complex in IDH mutant GSCs but not in human astrocytes using immunofluorescence microscopy and immunoprecipitation with Western blotting. Third, I will test the relevance of pyrimidine synthesis pathway disjunction for de novo pyrimidine synthesis inhibitor therapy with in vivo stable isotope tracing. I will perform these experiments in conjunction with treatment with a de novo pyrimidine synthesis inhibitor in a patient-derived orthotopic xenograft model of IDH mutant glioma. The proposed research has the potential to uncover new modes of regulation of nucleotide metabolism and answer vital mechanistic questions surrounding a new synthetic lethality-based treatment strategy that is poised to enter clinical testing in patients with IDH mutant glioma.