Identification and characterisation of Krebs cycle gene mutations in endocrine tumours and beyond

内分泌肿瘤及其他领域克雷布斯循环基因突变的鉴定和表征

• 批准号: 277178119
• 负责人: Dr. Barbara Klink
• 金额: --
• 依托单位: Institut für Klinische Chemie und Laboratoriumsmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277178119?language=en
• 关键词: Identification; characterisation; Krebs; cycle; gene

Mutations in Krebs cycle related genes cause a number of different tumours, including gastrointestinal stromal tumours (GIST), renal cell carcinomas (RCC), gliomas, phaeochromocytomas and paragangliomas (PPGL). Mutations of succinate dehydrogenase subunit B (SDHB) are especially known to cause cancers with high metastatic potential. This project combines the investigation of upstream pathogenic abnormalities in patient tissue and downstream effects in cell line models. Specifically, it will be tested whether Krebs cycle metabolites stratify patients with PPGL to offer useful prognostic biomarkers. The second objective examines how inactivation of tumour suppressors, SDHB and SDHD, in two cell line models impacts cellular energy metabolism and signalling networks, thereby identifying a possible Achilles heel for treatment of these cancers. Thirdly, the project aims to identify pathogenic aberrations in Krebs cycle related genes in a range of patient tumours. These objectives will be addressed by characterising PPGLs and other tumour entities by Krebs cycle metabolite profiling using our already established LC-MS/MS method. Metabolite concentrations in PPGLs will be analysed in relation to clinical characteristics and patient outcome. Moreover, metabolite profiling will be utilised as a screening tool for mutations in Krebs cycle related genes in PPGLs, GISTs, RCCs and other neoplasms. Tumours with aberrant profiles will be analysed using a next-generation panel or by exome sequencing to identify causative mutations. Addionally, a rat phaeochromocytoma and a human neuroblastoma line will be used to generate knockout SDHB and SDHD lines by CRISPR/Cas 9 gene editing technology. These cell lines will be investigated for changes in cell growth, migration, DNA methylation, gene expression and metabolite profiles. Additionally, a new method based on our existing LC-MS/MS assay will be developed to assess changes in metabolic fluxes. Potential biomarkers for metastatic progression or for indicating the dependency of tumours on key factors/ pathways identified in cell line models will be validated in patient samples. The proposed project will provide information about the incidence of Krebs cycle gene mutations in several tumour types, and aims to further expand on personalising cancer treatment and improving clinical diagnostics by extending the list of known pathogenic gene mutations. Identification of key energy and signalling pathways active in oncometabolite-driven tumours will provide the possibility for targeted interventions.

克雷伯循环相关基因的突变导致许多不同的肿瘤，包括胃肠道间质瘤（GIST）、肾细胞癌（RCC）、胶质瘤、嗜铬细胞瘤和副神经节瘤（PPGL）。琥珀酸脱氢酶亚基B （SDHB）的突变尤其被认为会导致具有高转移潜力的癌症。该项目结合了对患者组织上游致病异常和细胞系模型下游影响的研究。具体来说，将测试克雷布斯循环代谢物是否能对PPGL患者进行分层，以提供有用的预后生物标志物。第二个目标是研究两种细胞系模型中肿瘤抑制因子SDHB和SDHD的失活如何影响细胞能量代谢和信号网络，从而确定治疗这些癌症的可能致命弱点。第三，该项目旨在确定一系列患者肿瘤中克雷布斯循环相关基因的致病性畸变。这些目标将通过使用我们已经建立的LC-MS/MS方法通过克雷布斯循环代谢物分析来表征PPGLs和其他肿瘤实体来解决。将分析ppgl中代谢物浓度与临床特征和患者预后的关系。此外，代谢物谱分析将被用作PPGLs、gist、rcc和其他肿瘤中克雷布斯循环相关基因突变的筛选工具。将使用下一代面板或外显子组测序来分析具有异常特征的肿瘤，以确定致病突变。此外，将利用大鼠嗜铬细胞瘤和人神经母细胞瘤细胞系，通过CRISPR/ cas9基因编辑技术产生敲除SDHB和SDHD细胞系。这些细胞系将被研究细胞生长、迁移、DNA甲基化、基因表达和代谢物谱的变化。此外，基于我们现有的LC-MS/MS分析，我们将开发一种新的方法来评估代谢通量的变化。转移进展的潜在生物标志物或指示肿瘤对细胞系模型中确定的关键因素/途径的依赖性将在患者样本中进行验证。拟议的项目将提供关于几种肿瘤类型中克雷布斯循环基因突变发生率的信息，并旨在通过扩展已知致病基因突变列表进一步扩展个性化癌症治疗和改进临床诊断。鉴定在肿瘤代谢物驱动的肿瘤中活跃的关键能量和信号通路将为靶向干预提供可能性。