Investigating isocitrate dehydrogenase mutations in cancer using new metabolomics methods

Altered cellular metabolism, as a result of oncogenic mutations, has been observed in a range of cancer types. A detailed understanding of how cancers modify cellular metabolism, which includes changes to the genome, proteome and metabolome, is therefore integral for the development of effective and targeted therapeutic treatments.
Isocitrate dehydrogenase mutations lead to a change of enzyme function, which causes the production of high intracellular concentrations of 2 hydroxyglutarate, and are found in a range of cancer types including leukaemia, chondrosarcoma, T cell lymphoma and glioma. Although a range of epigenetic and metabolic effects of isocitrate dehydrogenase 1 and 2 mutations have been identified, which suggest a pro oncogenic role, the tumorigenic mechanism is not well understood. Over 70 % of low grade glioma (grade 2 and 3) in humans have isocitrate dehydrogenase 1 or 2 mutations, and have one of the lowest 5 year survival rates of all cancer types. Consequently, there is a need for better diagnostic and therapeutic approaches. Small molecule inhibitors, which are specific to isocitrate dehydrogenase mutations and reduce the abundance of 2 hydroxyglutarate, have been developed; however, resistance to treatment has since been reported, including the manifestation of second site mutations. Increased understanding of the metabolic changes associated with isocitrate dehydrogenase mutations has the potential to improve therapeutic efficacy, and elucidate new therapeutic pathways.
Metabolomics, the comprehensive analysis and identification of low molecular weight compounds in biological systems, allows the discovery of metabolic adaptations to disease and changes in environment, enabling hypothesis generation and directing further investigations. Untargeted metabolomics is typically used for the identification of biomarkers, in a discovery-led approach using statistical analysis of experimental groups. A challenge for untargeted metabolomics is the comprehensive analysis of all metabolites present in a biological sample, due to the range of chemical functionalities and matrix complexity. Analysis of polar and ionic metabolites, especially those which represent central carbon metabolism, are not well characterised by the most common hyphenated analytical methods including reversed phase chromatography, ion pair chromatography and hydrophobic interaction liquid chromatography, coupled to mass spectrometry. Development of selective eluent suppression technology has enabled the hyphenation of conventional ion chromatography to mass spectrometry, providing a potentially effective method for the analysis of polar metabolites. Anion exchange chromatography has recently been applied to targeted analysis, demonstrating reproducible analysis of some polar and ionic metabolites. Comprehensive untargeted metabolomics using ion chromatography mass spectrometry has not previously been explored to any significant extent.
In this thesis, an in depth study of the metabolic effects associated an isocitrate dehydrogenase 1 mutation in cancer cells will be undertaken, which will include the development of new anion exchange chromatography and ion mobility spectrometry approaches coupled to existing state of the art mass spectrometry. The aims are to both develop and validate new methods, and use these to comprehensively explore how metabolism is altered in association with an isocitrate dehydrogenase 1 mutation. Human glioblastoma and embryonic kidney derived cell lines with and without a transduced isocitrate dehydrogenase 1 mutation will be used as model systems.
It is demonstrated that anion exchange chromatography mass spectrometry provides increased sensitivity and comprehensive coverage of central carbon metabolites compared with an existing hydrophobic interaction liquid chromatography mass spectrometry method. A new ion mobility spectrometry mass spectrometry method is also developed and used for the analysis of derivatised samples, offering improved sensitivity for metabolites with primary and secondary amine functionality, and increasing confidence in identification using collisional cross section measurements. These analyses revealed that lysine and tryptophan degradation products are altered in isocitrate dehydrogenase 1 mutant cells and the possibility for inhibition of 2 oxoglutate dependent pathways by 2 hydroxyglutarate is explored. Treatment of isocitrate dehydrogenase 1 mutant cells with 2 oxoglutarate and 2 hydroxyglutarate is investigated and the results suggest additional regulatory metabolic effects in isocitrate dehydrogenase 1 mutant cells, beyond the enrichment of 2 hydroxyglutarate and the depletion of 2 oxoglutarate. The relationship between an isocitrate dehydrogenase 1 mutation and the concentration of 2 hydroxyglutarate and 2 oxoglutarate, in transduced cell lines, demonstrated the potential for metabolic therapies, in combination with existing pharmacological treatment. The alteration of immunosuppressive metabolites such as kynurenine also provides a possible explanation for different median survival times of patients with isocitrate dehydrogenase mutation positive and wild type tumours, and their distinct responses to treatment.
Anion exchange chromatography mass spectrometry and ion mobility spectrometry mass spectrometry were both shown to be robust and sensitive methods for untargeted analysis of cell extracts and provide benefits, beyond current methods, for hypothesis driven research. The sensitivity of isocitrate dehydrogenase 1 mutant cells to changes in redox state and 2 oxoglutarate concentration elucidates new targets for combination therapies, to develop more selective and effective treatments. New potential biomarkers are also suggested for clinical classification of tumours with isocitrate dehydrogenase 1 mutations.

Keywords: Metabolomics
IC-MS
Cancer
LC-MS
Advisor: McCullagh, J