Development and application of analytical tools to study the origin, fate and impact of the oncometabolite 2-hydroxyglutarate and its lactone

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This thesis investigated the impact and metabolism of the known oncometabolite D 2 hydroxyglutarate and its recently recognized lactone R-5-oxo-2-tetrahydro-furancarboxylic acid. Production and biological effects of 2 hydoxyglutarate are well described in the literature. However, accumulation of this metabolite as a result of insufficient degradation was studied for the first time in this thesis. Therefore, a cell-based enzyme assay was developed to measure D 2-hydoxyglutarate degradation by D 2 hydroxyglutarate dehydrogenase. Quantification of 2-hydroxyglutarate was accomplished by LC-MS/MS. Applying this assay, it was found that D 2 hydroxyglutarate dehydrogenase activity differs across cell lines but is not upregulated in response to elevated 2-hydroxyglutarate levels. Moreover, in preliminary investigations on 2 hydroxyglutarate in serum of AML-patients with mutations in isocitrate dehydrogenase, 2 hydroxyglutarate-lactone was discovered. The HCT116 panel with IDH1/2 mutation served as a model to learn more about the novel metabolite as both metabolites – 2 HG and 2 HG lactone - can be detected in extracts of these cells. Thereby, 2 hydroxyglutarate was verified as the precursor by performing 13C5-glutamine tracing experiments. However, 2 hydroxyglutarate lactonization does not take place whenever the precursor is abundant, as for some biological specimens with elevated 2 hydroxyglutarate the lactone was not detectable. Further experiments provided hints that 2 hydroxyglutarate-lactone is of enzymatic origin but did not lead to the identification of this enzyme. In doing all these experiments, interconversion of 2 hydroxyglutarate and its lactone needed to be controlled and required development of new protocols using different approaches of hyphenated mass spectrometry. For instance, set up of an enantioselective analysis of 2 hydroxyglutarate and its lactone was an objective of this thesis but was not achieved with the possibilities at the institute. Finally, this thesis provided insights into origin, fate and impact of 2-HG and 2-HG-lactone in the context of tumor metabolism.

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