Dissecting Mechanisms of Pyrimidine Synthesis Dependence in IDH Mutant Glioma

项目总结 代谢酶异柠檬酸脱氢酶(IDH)编码基因的驱动突变存在于80% 低级别胶质瘤和由此产生的高级别肿瘤。寻找新的治疗靶点 对于这种不治之症，我们实验室最近进行了无偏药物筛选，发现IDH 癌基因依赖于从头开始的嘧啶核苷酸合成途径来维持胶质瘤细胞的生存。 尽管我们发现了这种脆弱性，但将IDH突变和依赖联系起来的分子机制 从一开始，嘧啶核苷酸的合成是未知的。因此，我开发了一个平台来全面地 两种药物对IDH突变型胶质瘤干细胞(GSCs)氮代谢的影响 载体或突变型IDH的抑制剂，鉴定谷氨酰胺对嘧啶核苷酸的贡献如下 这些疾病之间调控差异最大的新陈代谢网络。后来我找到了证据 嘧啶核苷酸生产的两条主要途径：从头合成和回收 小路。这两条途径都有助于尿苷一磷酸(UMP)的合成，UMP是尿苷一磷酸的代谢物 所有其他的嘧啶核苷酸都是衍生出来的。我的研究表明，尽管IDH突变的GSC使用两者 产生UMP的途径，这些细胞优先使用来自从头途径的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，这表明 它可能是肿瘤特异性的。我推测IDH突变的胶质瘤细胞依赖于从头开始的嘧啶。 合成，因为它们含有一种新的代谢酶复合体，可以从从头开始引导UMP 合成下游嘧啶类化合物的途径。我将通过三项研究来检验这一假设。首先，我会 评估这种嘧啶合成分配表型是否是IDH突变的GSCs所特有的 一组IDH突变型和IDH野生型患者来源的GSCs的稳定同位素示踪研究。第二，我 将测试嘧啶生物合成酶是否在IDH突变的GSCs中形成复合体，但在人类中不形成 用免疫荧光显微镜观察星形胶质细胞，用Western blotting进行免疫沉淀。第三，我会 检测嘧啶合成途径脱节与从头治疗的相关性 体内稳定的同位素示踪。我将在进行这些实验的同时使用一种新的治疗方法 患者来源的IDH突变型胶质瘤原位异种移植模型中的嘧啶合成抑制剂。这个 拟议中的研究有可能发现核苷酸新陈代谢的新调节模式并回答 围绕着一种新的基于合成致命性的治疗策略的关键机制问题即将进入 IDH突变型胶质瘤患者的临床检测。

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.