Dissecting Mechanisms of Pyrimidine Synthesis Dependence in IDH Mutant Glioma

IDH 突变胶质瘤中嘧啶合成依赖性的剖析机制

基本信息

批准号：
10612820
负责人：
Milan Rashmin Savani
金额：
$ 3.9万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-20 至 2026-04-19
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612820
关键词：
Affect Astrocytes Biochemical Pathway Biology Brain Brain Neoplasms Cell Line Cell Survival Cells Clinic Clinical Trials Complex DHODH gene Dependence Development Dihydroorotate dehydrogenase Disease Drug Screening Drug Targeting Enzyme Interaction Enzymes Equilibrium Fellowship Genes Glioma Glutamine Human Immunofluorescence Microscopy Immunoprecipitation Isocitrate Dehydrogenase Laboratories Link Malignant Neoplasms Mass Spectrum Analysis Measures Metabolic Metabolic Pathway Metabolism Modeling Molecular Multienzyme Complexes Mutation Nitrogen Normal Cell Oncogenes Oncoproteins Pathway interactions Patient Selection Patients Pediatric Oncologist Pharmaceutical Preparations Phenotype Physiological Pre-Clinical Model Production Pyrimidine Pyrimidine Nucleotides Pyrimidines Regulation Repression Research Resistance Role Route Scientist Starvation Testing Therapeutic Training Uridine Uridine Monophosphate Western Blotting Work Xenograft Model cancer cell career driver mutation enzyme biosynthesis experimental study in vivo inhibitor inhibitor therapy liquid chromatography mass spectrometry mutant neoplastic cell nerve stem cell new therapeutic target nitrogen metabolism novel novel therapeutics nucleotide metabolism research clinical testing response response biomarker stable isotope stem-like cell targeted treatment treatment strategy tumor

项目摘要

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.

项目概要编码代谢酶异柠檬酸脱氢酶 (IDH) 的基因驱动突变存在于 >80% 低级别神经胶质瘤和由其产生的高级别肿瘤。确定新的治疗靶点对于这种不治之症，我们实验室最近进行了公正的药物筛选，发现IDH 癌基因赋予神经胶质瘤细胞存活依赖于从头嘧啶核苷酸合成途径。尽管我们发现了这一漏洞，但连接 IDH 突变和依赖性的分子机制关于嘧啶核苷酸的从头合成尚不清楚。因此，我开发了一个平台来全面分析患者来源的 IDH 突变神经胶质瘤干细胞 (GSC) 中的氮代谢情况载体或突变 IDH 抑制剂，确定谷氨酰胺对嘧啶核苷酸的贡献这些条件之间调节差异最大的代谢网络。我随后找到了证据嘧啶核苷酸生产的两种主要途径之间的分离：从头合成和回收途径。两种途径都有助于合成尿苷单磷酸 (UMP)，尿苷单磷酸的代谢产物所有其他嘧啶核苷酸都是衍生的。我的研究表明，虽然 IDH 突变型 GSCs 同时使用产生 UMP 的途径，这些细胞优先使用源自 de novo 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，表明它可能是肿瘤特异性的。我假设 IDH 突变神经胶质瘤细胞依赖从头嘧啶合成，因为它们含有一种新型代谢酶复合物，可以从头开始引导 UMP 下游嘧啶的合成途径。我将通过三项研究来检验这个假设。首先，我会通过执行评估这种嘧啶合成分配表型是否是 IDH 突变体 GSC 所独有的对一组 IDH 突变体和 IDH 野生型患者来源的 GSC 进行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 GSC 中形成复合物，但在人类中不形成复合物使用免疫荧光显微镜和蛋白质印迹法进行免疫沉淀的星形胶质细胞。第三，我会测试嘧啶合成途径分离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。我将结合从头治疗来进行这些实验 IDH 突变神经胶质瘤患者来源的原位异种移植模型中的嘧啶合成抑制剂。这拟议的研究有可能揭示核苷酸代谢调节的新模式并给出答案围绕一种新的基于合成致死性的治疗策略的重要机制问题即将进入 IDH 突变神经胶质瘤患者的临床测试。