Dissecting Mechanisms of Pyrimidine Synthesis Dependence in IDH Mutant Glioma

项目摘要 >80%的人存在编码代谢酶异柠檬酸脱氢酶（IDH）的基因中的驱动突变。 低级别胶质瘤和高级别胶质瘤的区别。为了确定新的治疗靶点， 对于这种不治之症，我们的实验室最近进行了一项无偏见的药物筛选，发现IDH 癌基因赋予神经胶质瘤细胞存活对从头嘧啶核苷酸合成途径的依赖性。 尽管我们发现了这种脆弱性，但联系IDH突变和依赖性的分子机制 嘧啶核苷酸的从头合成是未知的。因此，我开发了一个平台， 用以下任一种处理的患者来源的IDH突变胶质瘤干细胞（GSC）中的氮代谢谱 载体或突变IDH的抑制剂，鉴定谷氨酰胺对嘧啶核苷酸的贡献， 这些条件之间最差异调节的代谢网络。我后来发现了 嘧啶核苷酸生产的两种主要途径之间的分离：从头合成和补救 途径。这两种途径都有助于合成尿苷一磷酸（UMP）， 衍生所有其它嘧啶核苷酸。我的研究表明，尽管IDH突变体GSC同时使用 由于UMP是通过从头途径产生的，因此这些细胞优先使用从头途径产生的UMP来合成 UMP下游的嘧啶核苷酸。在人类星形胶质细胞中没有观察到这种表型，表明 它可能是肿瘤特异性的我假设IDH突变胶质瘤细胞依赖于从头嘧啶 合成，因为它们具有一种新的代谢酶复合物， 下游嘧啶的合成途径。我将通过三项研究来验证这一假设。首先我会 通过以下方法评估这种嘧啶合成分配表型是否是IDH突变GSC所特有的： 在一组IDH突变体和IDH野生型患者来源的GSC中进行稳定同位素示踪研究。二我 将测试嘧啶生物合成酶是否在IDH突变GSC中形成复合物，而在人类中不形成复合物。 星形胶质细胞的免疫荧光显微镜和免疫沉淀与蛋白质印迹。第三，我会 检测嘧啶合成途径中断与从头嘧啶合成抑制剂治疗的相关性 体内稳定同位素示踪。我将在进行这些实验的同时， 在IDH突变型胶质瘤的患者来源的原位异种移植模型中使用嘧啶合成抑制剂。的 拟议中的研究有可能揭示核苷酸代谢调节的新模式， 围绕一种新的合成致死性治疗策略的重要机制问题， IDH突变型胶质瘤患者的临床试验。

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.