John M. Asara

Phosphorylation of ULK1 (hATG1) by AMP-Activated Protein Kinase Connects Energy Sensing to Mitophagy

A protein kinase links energy stores to control of autophagy. Adenosine monophosphate–activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.

Pyruvate kinase M2 is a phosphotyrosine-binding protein.

Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

Glutamine supports pancreatic cancer growth through a Kras-regulated metabolic pathway

Cancer cells have metabolic dependencies that distinguish them from their normal counterparts. Among these dependencies is an increased use of the amino acid glutamine to fuel anabolic processes. Indeed, the spectrum of glutamine-dependent tumours and the mechanisms whereby glutamine supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of glutamine use in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumour growth. Whereas most cells use glutamate dehydrogenase (GLUD1) to convert glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle, PDAC relies on a distinct pathway in which glutamine-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate by aspartate transaminase (GOT1). Subsequently, this oxaloacetate is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP+ ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as glutamine deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of glutamine metabolism is mediated by oncogenic KRAS, the signature genetic alteration in PDAC, through the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumours.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses

The glycolytic metabolism of cancers differs from normal tissues, allowing tumor cells to survive under oxidative stress. Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.

Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis

Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer.

Evidence for an Alternative Glycolytic Pathway in Rapidly Proliferating Cells

Glucose Metabolism Revisited Cancer cells are revved up to reproduce rapidly and typically consume glucose rapidly by glycolysis. Why then do cancer cells express an isoform of a rate-limiting enzyme in glycolysis, pyruvate kinase M2, which has decreased activity? Vander Heiden et al. (p. 1492) propose that consequent accumulation of phosphoenolpyruvate, with the help of an enzymatic activity that remains to be characterized, can lead to phosphate transfer to phosphoglycerate mutase, another glycolytic enzyme, providing the cell with a different way to make pyruvate. This may allow cancer cells to produce pyruvate without generating excess adenosine triphosphate, which can act through feedback to inhibit glycolyis. Characterization of cancer cell metabolism provides evidence for a previously uncharacterized metabolic pathway. Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression of PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1 and may provide an alternate glycolytic pathway that decouples adenosine triphosphate production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support the anabolic metabolism observed in many proliferating cells.

Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (KrasG12D, herein KRas) in a p53LoxP/WT background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.

The Crohn's Disease Protein, NOD2, Requires RIP2 in Order to Induce Ubiquitinylation of a Novel Site on NEMO

BACKGROUND Crohns disease is an autoimmune inflammatory disorder of the gastrointestinal tract and is characterized clinically by dysregulation of both pro-inflammatory and anti-inflammatory cytokine signaling networks. The function of the Crohns disease protein, NOD2, highlights the biphasic nature of the pathology of Crohns disease. NOD2 can both strongly activate and negatively attenuate NF-kB signaling. The biochemical mechanism for this dual function of NOD2 is unknown. RESULTS We demonstrate that NOD2 activation leads to ubiquitinylation of NEMO, a key component of the NF-kB signaling complex. This ubiquitinylation is agonist dependant, and it does not regulate proteosomal destruction of NEMO. We show the NOD2-dependent ubiquitinylation of NEMO is dependent on the scaffolding protein kinase RIP2. Crohns disease-associated polymorphisms of NOD2 show a decreased ability to bind RIP2, and this decreased ability to bind RIP2 correlates with a decreased ability to ubiquitinylate NEMO. We map the site of NEMO ubiquitinylation to a novel NEMO ubiquitinylation site (Lysine 285) and show that this ubiquityinylation occurs in vivo. Lastly, we show functionally that RIP2-induced ubiquitinylation of NEMO is at least in part responsible for RIP2-mediated NF-kB activation. CONCLUSIONS These data suggest that this novel mode of regulation of the NF-kB signaling pathway could be a factor underlying the pathogenesis of Crohns disease.

A positive/negative ion–switching, targeted mass spectrometry–based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue

The revival of interest in cancer cell metabolism in recent years has prompted the need for quantitative analytical platforms for studying metabolites from in vivo sources. We implemented a quantitative polar metabolomics profiling platform using selected reaction monitoring with a 5500 QTRAP hybrid triple quadrupole mass spectrometer that covers all major metabolic pathways. The platform uses hydrophilic interaction liquid chromatography with positive/negative ion switching to analyze 258 metabolites (289 Q1/Q3 transitions) from a single 15-min liquid chromatography–mass spectrometry acquisition with a 3-ms dwell time and a 1.55-s duty cycle time. Previous platforms use more than one experiment to profile this number of metabolites from different ionization modes. The platform is compatible with polar metabolites from any biological source, including fresh tissues, cancer cells, bodily fluids and formalin-fixed paraffin-embedded tumor tissue. Relative quantification can be achieved without using internal standards, and integrated peak areas based on total ion current can be used for statistical analyses and pathway analyses across biological sample conditions. The procedure takes ∼12 h from metabolite extraction to peak integration for a data set containing 15 total samples (∼6 h for a single sample).

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.