INVESTIGATING THE HETEROGENEITY OF GLUCOSE AND GLUTAMINE METABOLISM IN CANCER

Rapidly proliferating cancer cells have increased biosynthetic and bioenergetic needs compared to quiescent cells. Hence, they undergo a reprogramming of intermediary metabolism, mainly by upregulating the uptake and catabolism of certain nutrients. The classical example of this is the ‘Warburg effect’ or aerobic glycolysis, defined as an increase in glucose uptake coupled to lactate secretion, regardless of oxygen availability in cancer cells. One consequence of this is that the majority of glucose carbon is diverted away from the mitochondria and the tricarboxylic acid (TCA) cycle, and secreted out of the cell as lactate. This prompts some cancer cells to exhibit an increased dependence on glutamine metabolism to refill the TCA cycle. However, recent studies have uncovered widespread heterogeneity on the roles of glucose and glutamine metabolism in cancer cells, prompting a need for further clarification. In order to examine these metabolic pathways in cancer cells, I first helped develop high-resolution LC-MS metabolomic workflows. The initial objective of my thesis focused on the Warburg effect, and specifically, the increased rate of glycolytic flux that occurs in colon cancer cells. I demonstrated that changes in glycolytic flux could modify specific histone acylation marks in a dose-dependent manner, suggesting that a possible function of the Warburg effect is to confer specific signaling effects on cancer cells. The second objective of my thesis focused on defining the contribution of the mitochondrial glutaminase isoenzyme GLS2 on the observed variability in glutamine dependence in various breast cancer cell types. I identified GLS2 as an important metabolically active enzyme in breast cancer cells that can feed TCA cycle anaplerosis. This finding has important clinical implications due to the fact that a glutaminase inhibitor, which fails to block GLS2 activity, is currently in Phase II trials. In summary, my dissertation work further contributes to our fundamental understanding of the metabolic programs operating in cancer cells, by uncovering novel aspects of both glucose and glutamine metabolism. This work identifies new underlying causes of the metabolic heterogeneity observed in cancer cells and may prove relevant to future improvements in cancer therapies

Quantitative determinants of aerobic glycolysis identify flux through the enzyme GAPDH as a limiting step

Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective. DOI: http://dx.doi.org/10.7554/eLife.03342.00

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment

Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αβ T cell numbers. MPC1-deficient T cells have defective thymic development at the β-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αβ T cell development and that its deficiency results in reduced but activated peripheral T cell populations