The metabolic and epigenetic effects of the isocitrate dehydrogenase-1 mutation in glioma

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

The dehydrogenation reactions between LiH and organic amines for solid state hydrogen storage systems

Hydrogen is considered as an appealing alternative to fossil fuels in the pursuit of sustainable, secure and prosperous growth in the UK and abroad. However there exists a persisting bottleneck in the effective storage of hydrogen for mobile applications in order to facilitate a wide implementation of hydrogen fuel cells in the fossil fuel dependent transportation industry. To address this issue, new means of solid state chemical hydrogen storage are proposed in this thesis. This involves the coupling of LiH with three different organic amines: melamine, urea and dicyandiamide. In principle, thermodynamically favourable hydrogen release from these systems proceeds via the deprotonation of the protic N-H moieties by the hydridic metal hydride. Simultaneously hydrogen kinetics is expected to be enhanced over heavier hydrides by incorporating lithium ions in the proposed binary hydrogen storage systems. Whilst the concept has been successfully demonstrated by the results obtained in this work, it was observed that optimising the ball milling conditions is central in promoting hydrogen desorption in the proposed systems. The theoretical amount of 6.97 wt% by dry mass of hydrogen was released when heating a ball milled mixture of LiH and melamine (6:1 stoichiometry) to 320 °C. It was observed that ball milling introduces a disruption in the intermolecular hydrogen bonding network that exists in pristine melamine. This effect extends to a molecular level electron redistribution observed as a function of shifting IR bands. It was postulated that stable phases form during the first stages of dehydrogenation which contain the triazine skeleton. Dehydrogenation of this system yields a solid product Li2NCN, which has been rehydrogenated back to melamine via hydrolysis under weak acidic conditions. On the other hand, the LiH and urea system (4:1 stoichiometry) desorbed approximately 5.8 wt% of hydrogen, from the theoretical capacity of 8.78 wt% (dry mass), by 270 °C accompanied by undesirable ammonia and trace amount of water release. The thermal dehydrogenation proceeds via the formation of Li(HN(CO)NH2) at 104.5 °C; which then decomposes to LiOCN and unidentified phases containing C-N moieties by 230 °C. The final products are Li2NCN and Li2O (270 °C) with LiCN and Li2CO3 also detected under certain conditions. It was observed that ball milling can effectively supress ammonia formation. Furthermore results obtained from energetic ball milling experiments have indicated that the barrier to full dehydrogenation between LiH and urea is principally kinetic. Finally the dehydrogenation reaction between LiH and dicyandiamide system (4:1 stoichiometry) occurs through two distinct pathways dependent on the ball milling conditions. When ball milled at 450 RPM for 1 h, dehydrogenation proceeds alongside dicyandiamide condensation by 400 °C whilst at a slower milling speed of 400 RPM for 6h, decomposition occurs via a rapid gas desorption (H2 and NH3) at 85 °C accompanied by sample foaming. The reactant dicyandiamide can be generated by hydrolysis using the product Li2NCN.