Perturbation of Intermediary Metabolism Inhibits Glycolysis, Oxidative Phosphorylation, Amino Acid Synthesis and Prevents Lymphoma Development in Mice

Abstract

Alpha-ketoglutarate (αKG) is an essential intermediate metabolite. The sources of cellular αKG are the oxidation of isocitrate by IDHs (isocitrate dehydrogenases 1-3), glutaminolysis and the action of the metabolic repair enzymes D-2/L-2- hydroxyglutarate dehydrogenase (D2/L2HGDH), which oxidizes 2-HG into αKG. As a TCA cycle intermediate, αKG participates in the generation of reducing equivalents for energy production, is a source of precursors for synthesis of macromolecules, acts as a nitrogen scavenger, and is an obligatory co-substrate for multiple dioxygenases. Earlier, we discovered loss-of-function mutation of D2HGDH in diffuse large B cell lymphomas (DLBCL; Lin et al, Nat Commun 2015) and found that the main metabolic consequence of these mutations was depletion of αKG, rather than accumulation of D-2-HG, which typifies IDH1/2 mutant tumors. Here, we examined how D-2-HG/αKG homeostasis influence DLBCL metabolism and if exogenous modulation of these intermediate metabolites plays a role in lymphoma biology. We first created KO models of D2HGDH in DLBCL cell lines and queried its impact on oxidative phosphorylation and glycolysis using a seahorse assay. Deletion of D2HGDH increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), reflecting elevated mitochondrial respiration and glycolysis, and ATP production. This gain in energy production translated in increased proliferation of multiple (n=10) DLBCL cell lines exposed to the cell permeable octyl-D-2-HG metabolite. Considering that D-2-HG competitively inhibits αKG-dependent activities, and that αKG has been recently shown to suppress epithelial cancers (Tran et al, Nat Cancer, 2020), we examined the effect of αKG on energy metabolism in DLBCL. We exposed 12 DLBCL cell lines to various cell-permeable formulations of αKG (dimethyl-αKG [DMαKG] 5mM, or octyl-αKG 1mM) and detected a rapid (1h) and significant suppression of OCR (mean = 89%, 81-93%) and ECAR (mean = 58%, 35-66%) in all models. This collapse in energy generation had severe repercussions on cell growth and viability; exposure to αKG suppressed DLBCL proliferation (mean = 72% vs. vehicle-treated cells, 47%-99%) and induced apoptosis (mean = 60% vs. vehicle-treated cells, 28%-93%). To gain insight into how αKG supplementation drives the collapse of both cell respiration and glycolysis we used mass spectrometry (MS) to quantify central carbon metabolites in 12 DLBCL cell lines exposed to DMαKG for 30' and 24h. Following the 30' pulse, we detected a significant elevation in all measured metabolites (αKG, succinate, fumarate, malate, citrate, pyruvate and lactate). Conversely, at 24h, while the levels of αKG, succinate, fumarate, malate, citrate remained significantly elevated, the ability to generate lactate and/or pyruvate was blunted in all models, possibly secondary to the inhibition of glycolysis detected with the seahorse assays. We also used MS to measure the impact of αKG supplementation on amino acid levels. We detected a rapid and sustained (30'-24h) depletion of the branched-chain amino acids (BCAAs) isoleucine, leucine and valine (Log2 fold change, 1.37, 1.04 and 0.44 respectively, p<0.001). We attributed the depletion of BCAAs to increased activity of the αKG-dependent BCAA transaminases, BCAT1/2. Importantly, this hitherto unrecognized effect of αKG resulted in broad inhibition of MTORC1 function (secondary to leucine depletion) in DLBCL cell lines and primary lymphomas. Next, we tested the relevance of these findings in vivo. Herein, starting at 5 weeks of age, we randomized Eµ-Myc mice (n=45,) to receive drinking water supplemented with 15mg/ml DMαKG or normal water. The primary endpoint of this trial was time to lymphoma development (and death). Mice exposed to αKG supplementation had a significantly longer survival and protracted lymphoma diagnosis (Figure 1). Remarkably, OCR and ECAR were significantly suppressed (37% and 26%, respectively, p<0.01) in splenocytes of αKG-supplemented vs control mice (n=11). Further, at 12 months of αKG exposure, the mice displayed no clinical toxicity and had normal blood counts and biochemistry. We concluded that αKG displays anti-lymphoma activity, which mechanistically associates with a collapse in energy generation, depletion of BCAAs and suppression of MTORC1. The benefit of αKG supplementation in the context of cancer deserves further examination in humans. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal