Identification of enzymes inhibited by oncometabolites in Succinate Dehydrogenase mutant tumors

琥珀酸脱氢酶突变肿瘤中癌代谢物抑制的酶的鉴定

• 批准号: 10579575
• 负责人: Lawrence S Kirschner
• 金额: $ 7.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10579575
• 关键词: Acute Myelocytic Leukemia; Aerobic; Affect; Biological Models; CRISPR interference; CRISPR/Cas technology; Cancer Etiology; Cell Line; Cell model; Cell physiology; Cells; Cellular Metabolic Process; Characteristics; Citric Acid Cycle; Clear Cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Correlative Study; DNA; DNA Modification Process; Data; Data Set; Disease; Elderly; Endocrine; Enzyme Inhibition; Enzymes; Epithelium; Eukaryotic Cell; Familiarity; Family; Family member; Fumarate Hydratase; Future; Gastrointestinal Stromal Tumors; Generations; Genes; Genetic; Glioblastoma; Glioma; Hereditary Paraganglioma; Histones; Human; Hydroxylation; In Vitro; Inherited; Investigation; Isocitrate Dehydrogenase; Knowledge; Lead; Lesion; Light; Localized Disease; Malate Dehydrogenase; Malignant Neoplasms; Malignant neoplasm of thyroid; Mediating; Metabolic; Missense Mutation; Mitochondrial Proteins; Mixed Function Oxygenases; Modeling; Molecular; Mutation; Neoplasms; Nuclear; Oncogenic; Operative Surgical Procedures; Oxygenases; Paraganglioma; Patients; Phenotype; Pheochromocytoma; Process; Production; Property; Protein Isoforms; Proteins; Quality of life; RNA; Reactive Oxygen Species; Renal Cell Carcinoma; Renal carcinoma; Reporting; Research Project Grants; Role; Somatic Mutation; Source; Structural Protein; Study models; Succinate Dehydrogenase; Succinates; Survival Rate; Syndrome; System; Thyroid Gland; Time; Tissues; Tumor Promotion; United States; United States National Institutes of Health; Validation; Variant; Work; aldehyde dehydrogenases; alpha ketoglutarate; cofactor; gene repression; high risk; histone demethylase; in silico; in vivo; in vivo Model; knock-down; leukemia; member; mouse model; mutant; neoplastic; small hairpin RNA; stem; stem cell biomarkers; stem cells; therapy development; transcription factor; tumor; tumorigenesis

PROJECT SUMMARY The tricarboxylic acid cycle (TCA cycle, also known as the Krebs cycle) is a fundamental process in eukaryotic cells, serving as the source for ATP generation and producing reducing equivalents for cell metabolism under aerobic conditions. In recent years, it has become clear that genes encoding the enzymes in the TCA cycle can be causative genetic lesions in human cancers, including in inherited tumor syndromes associated with renal cancer, paragangliomas/pheochromocytomas (PPGL), and Gastrointestinal Stromal Tumors (GISTs) among others, including epithelial thyroid cancer. They are also well described in sporadic tumors from these same tissues, as well as in a spectrum of other cancers, including acute myeloid leukemia (AML) and glioblastoma. Mutations in Krebs cycle enzymes and their related cofactors are thought to cause tumor formation through the oncogenic effects of excess metabolite accumulation (oncometabolites). These intermediate metabolites act by interference with the function of enzymes requiring the metabolic cofactor alpha-ketoglutarate (aKG, also known as 2- oxoglutarate). There are approximately 70 aKG-dependent enzymes in humans, and they perform a variety of essential cellular functions, including mediating modification of DNA, RNA, and histone proteins. Enzymes of this class are also responsible for oxidative hydroxylation of proteins, including the structural protein collagen. We have previously demonstrated using both in vitro and in vivo models for thyroid neoplasia that loss of the Succinate Dehydrogenase D subunit (SDHD) causes phenotypic changes indicative of early stages of cancer. Further, this genetic changes causes cells to gain a stem-like phenotype, as evidenced by expression of the stem cell associated transcription factors Nanog and Oct4 and production of the stem cell marker Aldehyde Dehydrogenase (ALDH). Despite the fact that interference with aKG-dependent oxygenases has been proposed as a neoplastic mechanism, no prior efforts have been made to identify family members which are responsible for the neoplastic change. To fill this knowledge gap, we propose to use a high throughput CRISPR-based transcriptional repression screen to identify aKG-dependent enzymes whose inhibition leads to a recapitulation of the stem-like phenotype. Identified hits will be validated using a combination of cellular models and in silico analysis of tumor-based omics data. The Aims for this R03 pilot proposal are as follows: 1) To use a CRISPRi screen to identify alpha-ketoglutarate (aKG) dependent enzyme(s) whose inhibition leads to the acquisition of a stem-like phenotype 2) To validate identified enzymes in cell line models and in human tumor datasets Identification of the enzymes whose functions are affected by accumulation of the oncometabolite succinate will shed important new light on the molecular mechanism of disease associated with Sdhx and other TCA cycle mutations, and will provide a stepping stone for future research grants that will allow detailed delineation of genetic and/or protein targets that drive tumor formation in the thyroid and other tissues (e.g. glioma, leukemia).

项目总结 三羧酸循环(TCA循环，又称Krebs循环)是真核生物中的一个基本过程 细胞，作为ATP生成的来源，并在以下条件下产生细胞代谢的还原当量 有氧条件。近年来，编码三氯乙酸循环中的酶的基因已经变得清晰起来。 是人类癌症的致病基因损害，包括与肾脏相关的遗传性肿瘤综合征 癌症、副神经节瘤/嗜铬细胞瘤(PPGL)和胃肠道间质瘤(GIST) 其他，包括上皮性甲状腺癌。它们在散发性肿瘤中也有很好的描述， 在其他癌症中，包括急性髓系白血病(AML)和胶质母细胞瘤也是如此。 Krebs循环酶及其相关辅助因子的突变被认为是通过 过度代谢物积累的致癌效应(代谢物)。这些中间代谢产物通过 干扰需要代谢辅因子α-酮戊二酸(AKG)的酶的功能 作为2-羟基戊二酸)。人类体内大约有70种依赖AKG的酶，它们的功能多种多样 基本的细胞功能，包括介导DNA、RNA和组蛋白的修饰。酶的作用 这一类还负责蛋白质的氧化羟化，包括结构蛋白胶原蛋白。 我们以前已经使用体外和体内甲状腺肿瘤模型证明了 琥珀酸脱氢酶D亚单位(SDHD)可引起早期癌症的表型变化。 此外，这种遗传变化导致细胞获得干细胞样的表型，这从 干细胞相关转录因子Nanog和Oct4与干细胞标志物乙醛的产生 脱氢酶(ALDH)。尽管对依赖AKG的加氧酶的干扰已被提出 作为一种肿瘤机制，以前没有做出任何努力来确定哪些家庭成员应对此负责 肿瘤性的改变。为了填补这一知识空白，我们建议使用基于CRISPR的高吞吐量 转录抑制筛选鉴定抑制AKG导致重述的AKG依赖酶 属于茎状表型。识别的命中将使用蜂窝模型和硅胶的组合进行验证 基于肿瘤的组学数据分析。这项R03试验计划的目的如下： 1)用CRISPRi筛选鉴定抑制α-酮戊二酸依赖性酶(S) 导致获得一种茎状表型 2)在细胞系模型和人类肿瘤数据集中验证已识别的酶 琥珀酸失碳酯积累对酶功能影响的鉴定 为SDHx和其他TCA循环相关疾病的分子机制提供了重要的新线索 突变，并将为未来的研究拨款提供垫脚石，这将允许详细描述 基因和/或蛋白质靶点，推动甲状腺和其他组织(如胶质瘤、白血病)的肿瘤形成。