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

Krebs周期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B亚单位(SDHB)的突变尤其被认为会导致高转移潜能的癌症。该项目结合了对患者组织中上游致病异常和细胞系模型中下游影响的调查。具体地说，将测试Krebs循环代谢产物是否对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个细胞系模型中肿瘤抑制基因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阿喀琉斯之踵。第三，该项目旨在确定一系列患者肿瘤中与Krebs周期相关的基因的致病异常。这些目标将通过使用我们已经建立的LC-MS/MS方法通过Krebs循环代谢物分析来表征PPGL和其他肿瘤实体来实现。PPGLS中的代谢物浓度将根据临床特征和患者预后进行分析。此外，代谢物图谱将被用作筛查PPGLS、GIST、RCC和其他肿瘤中与Krebs周期相关的基因突变的工具。具有异常特征的肿瘤将使用下一代专家小组或外显子组测序进行分析，以确定致病突变。此外，还将利用CRISPR/Cas 9基因编辑技术，利用大鼠嗜铬细胞瘤和人神经母细胞瘤细胞系产生SDHB和SDHD基因敲除株。这些细胞系将被研究细胞生长、迁移、DNA甲基化、基因表达和代谢物图谱的变化。此外，将在我们现有LC-MS/MS分析的基础上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用于指示肿瘤对细胞系模型中确定的关键因素/途径的依赖性的潜在生物标志物将在患者样本中得到验证。拟议的项目将提供有关几种肿瘤类型中Krebs周期基因突变的发生率的信息，并旨在通过扩大已知致病基因突变的列表，进一步扩大癌症治疗的个人化和改善临床诊断。确定在肿瘤中活跃的关键能量和信号通路将为有针对性的干预提供可能性。

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.