Coupling Krebs cycle metabolites to signalling in immunity and cancer

Abstract

Metabolic reprogramming has become a key focus for both immunologists and cancer biologists, with exciting advances providing new insights into underlying mechanisms of disease. Metabolites traditionally associated with bioenergetics or biosynthesis have been implicated in immunity and malignancy in transformed cells, with a particular focus on intermediates of the mitochondrial pathway known as the Krebs cycle. Among these, the intermediates succinate, fumarate, itaconate, 2-hydroxyglutarate isomers (D-2-hydroxyglutarate and L-2-hydroxyglutarate) and acetyl-CoA now have extensive evidence for "non-metabolic" signalling functions in both physiological immune contexts and in disease contexts, such as the initiation of carcinogenesis. This review will describe how metabolic reprogramming, with emphasis placed on these metabolites, leads to altered immune cell and transformed cell function. The latest findings are informative for new therapeutic approaches which could be transformative for a range of diseases.

Figure 1. The Krebs cycle

The Krebs cycle is a metabolic pathway operating in the mitochondrial matrix of all aerobic organisms. Breakdown of nutrients, such as glucose, generates acetyl-CoA which can then be funnelled into this pathway. For a full cycle to be completed, a series of 10 enzymatic reactions are required. These reactions are catalysed by the mitochondrial enzymes citrate synthase (CS), isocitrate dehydrogenase (IDH), aconitase (ACO2), α-ketoglutarate dehydrogenase (OGDH), succinyl-CoA synthetase, succinate dehydrogenase (SDH), fumarase (FH) and malate dehydrogenase (MDH). The primary function of the TCA cycle is to generate reducing equivalents, such as NADH and FADH₂ (produced by SDH). NADH and FADH₂ can then transfer electrons to the ETC to drive oxidative phosphorylation (OXPHOS) and the production of the high energy nucleoside triphosphate, ATP, via ATP synthase.

Figure 2. The diverse signalling roles of succinate

Succinate levels are elevated in response to LPS stimulation, in synovial fluids from rheumatoid arthritis (RA) patients, in the circulation in models of diet-induced obesity and in adipose tissue in response to hypoxia and hyperglycaemia. ii) Succinate oxidation (as well as direct product inhibition) and reactive oxygen species (ROS) stabilizes hypoxia-inducible factor-1alpha (HIF-1α) which binds to the hypoxia-response element (HRE) in the IL-1β promoter iii) Succinate is the ligand for the G-protein coupled receptor succinate receptor 1 (SUCNR1). Ligand binding induces Gi and Gq signalling cascades. iv) SUCNR1 ligation on dendritic cells induces cell migration v) and acts in synergy with TLR ligands to increase interleukin-1 beta (IL-1β) expression and IL-1β and LPS further enhances SUCNR1 expression. v) Endogenously generated succinate is released, e.g. via Slc13a3/5 in neural stem cells (NSCs) and binds to SUCNR1 on the same or nearby SUCNR1-expressing cells. vi) Activation of SUCNR1 on NSCs induces the secretion of anti-inflammatory prostaglandin E2 which is anti-inflammatory. vii) Succinate regulates the activity the Jumonji C-domain containing histone demethylases (JMJDs) and the Ten eleven translocation (TET) family of 5mC hydroxylases, which play a role in histone and DNA demethylation, respectively and can thereby remodel the epigenome. viii) Another consequence of succinate accumulation is the modification of proteins by lysine succinylation.

Figure 3. Itaconate is a thiol reactive anti-inflammatory metabolite

In LPS-activated macrophages, mitochondrial IRG1/CAD synthesises itaconate from cis-aconitate, whereby it can inhibit SDH or is exported out of the mitochondria via the oxoglutarate carrier (OGC). In the cytosol, itaconate alkylates KEAP1, a novel PTM termed 2,3-dicarboxypropylation, and GSH to form an itaconate-GSH adduct, or 2,3-dicarboxypropyl-GSH. This in turn activates the anti-inflammatory and anti-oxidant transcription factor Nrf2 and ATF3. Levels of the metabolite 2,3-dicarboxypropyl cysteine (Itaconate-cysteine adduct) increase, which is indicative of the turnover of 2,3-dicarboxypropylated targets. Activation of Nrf2 acts to negatively regulate the pro-inflammatory cytokine IL-1β, whilst activation of ATF3 inhibits IκBζ and IL-6.

Figure 4. Fumarate is an oncometabolite and epigenetic modifier

Under conditions of FH-deficiency, fumarate levels markedly increase leading to perturbations in mitochondrial OXPHOS. Elevated fumarate acts as a competitive inhibitor of SDH and inhibits Complex 1 via succination of key [Fe-S] cluster biogenesis proteins. In the cytosol, increased fumarate can succinate KEAP1 and GSH to activate Nrf2, whilst fumarate also acts to competitively inhibit PHDs and stabilise HIF1α. In the nucleus, fumarate acts as an epigenetic modifier, whereby it can inhibit the KDM family of histone demethylases and the TET family of DNA demethylases, which acts as a signal to induce EMT.

Figure 5. 2-hydroxyglutarate is an oncometabolite and epigenetic modifier

2-hydroxyglutarate exists as two enantiomers L-2-HG and D-2-HG. Under conditions whereby IDH1/2 are mutated (mIDH1/2), this mutant enzyme preferentially acts on α-KG, as opposed to isocitrate, to generate D-2-HG. In this context, D-2-HG accumulates to high levels, however, under normal conditions D-2HG levels are maintained at low concentrations via D2HGDH. In response to hypoxia or acidic pH, MDH2 can promiscuously generate L-2-HG from α-KG, whose levels are usually maintained at low concentrations via L2HGDH. Accumulation of D-2-HG and/or L-2-HG in the nucleus results in the competitive inhibition of the KDM family of histone demethylases and the TET family of DNA demethylases, thus acting as an important epigenetic modifier driving tumorigenesis and as a regulator of T cell immunity.

Figure 6. Therapeutic opportunities targeting metabolic signalling pathways

i) The SDH inhibitor malonate and the malonate prodrug, DMM (hydrolysed to release malonate), ameliorate damage associated with succinate accumulation during ischaemia. ii) SUCNR1 receptor inhibitors, such as compound “5g” are in development. iii) Enasidinib, which targets mutated IDH2 to reduce 2-HG production, is the leading to the treatment of AML. iv) 4-OI, DI and DMF are anti-inflammatory are may offer therapeutic potential. v) Inhibitors of α-KGDDs are an emerging target, but selectivity is an issue. Dimethyloxaloylglycine (DMOG) acts on TETs but also PHDs. vi) Specific inhibition of histone lysine demethylases (KDMs) has also been challenging but EPT-103182 has shown promise in early pre-clinical work. vii) The specific PHD inhibitor, Vadadustat, has shown promise in chronic kidney disease.