Paulo A. Gameiro

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel–Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

Oncogenic K‐Ras decouples glucose and glutamine metabolism to support cancer cell growth

Oncogenes such as K‐ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K‐Ras‐dependent metabolic alterations, we employed 13C metabolic flux analysis (MFA), non‐targeted tracer fate detection (NTFD) of 15N‐labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope‐labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K‐Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non‐canonical labeling of TCA cycle‐associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K‐Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K‐Ras in the metabolic reprogramming of cancer cells.

Loss of RBF1 changes glutamine catabolism

Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo (13)C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H(2)O(2) preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.

Cofactor Balance by Nicotinamide Nucleotide Transhydrogenase (NNT) Coordinates Reductive Carboxylation and Glucose Catabolism in the Tricarboxylic Acid (TCA) Cycle

Background: The role of cofactors in regulating the TCA cycle is poorly understood. The source(s) of NADPH contributing to reductive carboxylation (RC) in the mitochondrion are unknown. Results: Knockdown of nicotinamide nucleotide transhydrogenase (NNT) decreases RC and stimulates glucose catabolism in the TCA cycle. Conclusion: NNT produces NADPH for RC and modulates glucose catabolism. Significance: NNT coordinates glutamine and glucose metabolism in the TCA cycle. Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)+ cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)+ ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle.

13C isotope-assisted methods for quantifying glutamine metabolism in cancer cells.

Glutamine has recently emerged as a key substrate to support cancer cell proliferation, and the quantification of its metabolic flux is essential to understand the mechanisms by which this amino acid participates in the metabolic rewiring that sustains the survival and growth of neoplastic cells. Glutamine metabolism involves two major routes, glutaminolysis and reductive carboxylation, both of which begin with the deamination of glutamine to glutamate and the conversion of glutamate into α-ketoglutarate. In glutaminolysis, α-ketoglutarate is oxidized via the tricarboxylic acid cycle and decarboxylated to pyruvate. In reductive carboxylation, α-ketoglutarate is reductively converted into isocitrate, which is isomerized to citrate to supply acetyl-CoA for de novo lipogenesis. Here, we describe methods to quantify the metabolic flux of glutamine through these two routes, as well as the contribution of glutamine to lipid synthesis. Examples of how these methods can be applied to study metabolic pathways of oncological relevance are provided.

Glutaminase and poly(ADP-ribose) polymerase inhibitors suppress pyrimidine synthesis and VHL-deficient renal cancers

Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel–Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of &agr;-ketoglutarate (&agr;KG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL–/– RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL–/– cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor–induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra–S phase checkpoint, and suppressed the growth of VHL–/– RCC cells. These effects were rescued by administration of glutamate, &agr;KG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL–/– cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.

Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins

Significance The PI3K-Akt-mTOR pathway integrates signaling information from various mitogen and nutrient sensors to regulate cell growth and proliferation. Hyperactivation of this pathway has been observed in a number of diseases, including many cancers. Pharmacological inhibition of mechanistic target of rapamycin (mTOR) causes immediate suppression of cell-wide protein synthesis; interestingly, this effect is not uniform across all transcripts, but is biased toward mRNAs encoding ribosomal proteins. In this study, we developed a rapid, scalable, and targeted assay for RNA translation that enabled us to identify translation modulators of ribosomal proteins that act independent of mTOR signaling. These modulators include chemical compounds, acting via their “off-target” effects, as well as certain metabolic perturbations, and together, they represent a noncanonical mode of regulating ribosome biogenesis and protein synthesis capacity. The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.

Systems-Level Analysis of Cancer Metabolism

The complexity of cancer requires systems-level approaches to examine uncontrolled proliferation, with many analytical tools now providing massive information on distinct cellular processes. In contrast to the genetic anchors founded in cancer biology that underpin tumor suppressors and oncogenes as units of malignant function, we now see a shift of attention towards metabolism. This trend calls for the increased use of stable isotopic tracers to dissect effects in metabolic fluxes that arise from gene deregulation. When combined with analytical techniques such as mass spectrometry or nuclear magnetic resonance (NMR) and computational tools to interpret such datasets, isotopic tracers can allow for the determination of various metabolic events involved in tumorigenesis at a fine resolution. As such, the interplay between fluxes and signaling warrants a thorough investigation that will lead to targeted therapies rooted on metabolic targets. This chapter describes stable isotopic methods to determine fluxes and identify switches, illustrating how metabolic activity can be quantitatively interpreted to address fundamental questions in cancer.

Nutrient Deprivation Elicits a Transcriptional and Translational Inflammatory Response Coupled to Decreased Protein Synthesis

SUMMARY Nutrient deprivation inhibits mRNA translation through mTOR and eIF2α signaling, but it is unclear how the translational program is controlled to reflect the degree of a metabolic stress. In a model of breast cellular transformation, various forms of nutrient deprivation differentially affect the rate of protein synthesis and its recovery over time. Genome-wide translational profiling of glutamine-deprived cells reveals a rapid upregulation of mRNAs containing uORFs and downregulation of ribosomal protein mRNAs, which are followed by selective translation of cytokine and inflammatory mRNAs. Transcription and translation of inflammatory and cytokine genes are stimulated in response to diverse metabolic stresses and depend on eIF2α phosphorylation, with the extent of stimulation correlating with the decrease in global protein synthesis. In accord with the inflammatory stimulus, glutamine deprivation stimulates the migration of transformed cells. Thus, pro-inflammatory gene expression is coupled to metabolic stress, and this can affect cancer cell behavior upon nutrient limitation.

Abstract 1123: Glutaminase inhibitors suppress pyrimidine synthesis and promote DNA replication stress in VHL-deficient human renal cancer cells

Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of a-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease associated Von Hippel-Lindau (VHL) mutants and showed that HIF2a expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. Reductive carboxylation can be detected in vivo. HIF2a-mediated glutamine metabolism is induced by low intracellular citrate levels, which promote the reductive flux by mass action to maintain lipogenesis. In addition to being a source of lipogenic acetyl-CoA, glutamine can be used in the Krebs cycle to generate aspartate - the carbon source for pyrimidine biosynthesis. We showed that VHL-deficient RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Pharmacological inhibition of glutaminase (GLS) by the commercially available glutaminase inhibitor BPTES and a glutaminase inhibitor which is in Phase I clinical study (CB-839, Calithera Biosciences Inc) depleted the levels of pyrimidines in VHL-deficient cells but not in VHL-reconstituted cells, which utilized glucose oxidation to supply the aspartate pool. Nucleoside depletion activates an intra-S-phase checkpoint corresponding to DNA replication stress and suppressed S-phase transition in VHL-deficient RCC cells. DNA replication stress was rescued by administration of glutamate or dimethyl-alpha-ketoglutarate. Addition of nucleobases partially rescued the effect of glutaminase inhibitors on cell growth. Our data suggest that glutaminase inhibitors suppress de novo pyrimidine synthesis, leading to DNA replication stress in VHL-deficient RCC cells. We currently test the hypothesis that this mechanistic insight may allow the development of novel therapeutic strategies for RCC by combining glutaminase inhibitors with drugs promoting DNA replication stress. Citation Format: Arimichi Okazaki, Paulo Gameiro, Gregory Stephanopoulos, Othon Iliopoulos. Glutaminase inhibitors suppress pyrimidine synthesis and promote DNA replication stress in VHL-deficient human renal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2015-1123