Seth J. Parker

Tracing Compartmentalized NADPH Metabolism in the Cytosol and Mitochondria of Mammalian Cells

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.

Reductive carboxylation supports redox homeostasis during anchorage-independent growth

Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier

Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied (13)C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.

IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models

Continuous de novo fatty acid synthesis is a common feature of cancer that is required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain the de novo fatty acid synthesis needed for growth and viability of non-small-cell lung cancer (NSCLC) cells. We describe the ability of ND-646—an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization—to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53−/− (also known as KRAS p53) and Kras;Stk11−/− (also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology.

Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism

The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.

Metabolic consequences of oncogenic IDH mutations

Specific point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) occur in a variety of cancers, including acute myeloid leukemia (AML), low-grade gliomas, and chondrosarcomas. These mutations inactivate wild-type enzymatic activity and convey neomorphic function to produce d-2-hydroxyglutarate (d-2HG), which accumulates at millimolar levels within tumors. d-2HG can impact α-ketoglutarate-dependent dioxygenase activity and subsequently affect various cellular functions in these cancers. Inhibitors of the neomorphic activity of mutant IDH1 and IDH2 are currently in Phase I/II clinical trials for both solid and blood tumors. As IDH1 and IDH2 represent key enzymes within the tricarboxylic acid (TCA) cycle, mutations have significant impact on intermediary metabolism. The loss of some wild-type metabolic activity is an important, potentially deleterious and therapeutically exploitable consequence of oncogenic IDH mutations and requires continued investigation in the future. Here we review how IDH1 and IDH2 mutations influence cellular metabolism, epigenetics, and other biochemical functions, discussing these changes in the context of current efforts to therapeutically target cancers bearing these mutations.

Distinct metabolic states can support self-renewal and lipogenesis in human pluripotent stem cells under different culture conditions

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here, we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using (13)C/(2)H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth, chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration, altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development.

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C > 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR-IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer. Cancer Res; 77(16); 4460-71. ©2017 AACR.

Chasing One-Carbon Units to Understand the Role of Serine in Epigenetics

In this issue of Molecular Cell, Maddocks et al. (2016) use stable isotope tracing, mass spectrometry, and nutrient modulation in cancer cells to highlight the role of serine in supporting methylation through maintenance of nucleotide levels.