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

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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.