Brian Prescott Fiske

Metabolic pathway alterations that support cell proliferation.

Proliferating cells adapt metabolism to support the conversion of available nutrients into biomass. How cell metabolism is regulated to balance the production of ATP, metabolite building blocks, and reducing equivalents remains uncertain. Proliferative metabolism often involves an increased rate of glycolysis. A key regulated step in glycolysis is catalyzed by pyruvate kinase to convert phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, there is strong selection for expression of the less active M2 isoform of pyruvate kinase (PKM2) in tumors and other proliferative tissues. Cell growth signals further decrease PKM2 activity, and cells with less active PKM2 use another pathway with separate regulatory properties to convert PEP to pyruvate. One consequence of using this alternative pathway is an accumulation of 3-phosphoglycerate (3PG) that leads to the diversion of 3PG into the serine biosynthesis pathway. In fact, in some cancers a substantial portion of the total glucose flux is directed toward serine synthesis, and genetic evidence suggests that glucose flux into this pathway can promote cell transformation. Environmental conditions can also influence the pathways that cells use to generate biomass with the source of carbon for lipid synthesis changing based on oxygen availability. Together, these findings argue that distinct metabolic phenotypes exist among proliferating cells, and both genetic and environmental factors influence how metabolism is regulated to support cell growth.

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.

PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells.

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.

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.

Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development.

Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.

A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use a quantitative high-throughput screen to identify small molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Lack of Evidence for PKM2 Protein Kinase Activity

The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [(32)P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [(32)P]-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase.

An epitope tag alters phosphoglycerate dehydrogenase structure and impairs ability to support cell proliferation

BackgroundThe gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein.ResultsThe addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure.ConclusionsPurification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.

Seeing the Warburg effect in the developing retina

Proliferating cells of the Xenopus laevis retina facultatively use aerobic glycolysis instead of oxidative phosphorylation. This demonstrates that the metabolic rewiring usually associated with the Warburg effect in tumorigenesis may be a more widespread feature of proliferative metabolism than generally appreciated.

Increased PHGDH expression uncouples hair follicle cycle progression and promotes inappropriate melanin accumulation

Copy number gain of the PHGDH gene that encodes the first enzyme of the serine biosynthesis pathway is found in some human cancers, including a subset of melanomas. In order to study the effect of increased PHGDH expression in tissues in vivo, we generated mice harboring a PHGDHtetO allele that allows tissue-specific, doxycycline-inducible PHGDH expression. Tissues and cells derived from PHGDHtetO mice exhibit increased serine biosynthesis. Histological examination of skin tissue from PHGDHtetO mice reveals the presence of melanin granules in anagen II hair follicles, despite the fact that melanin synthesis is normally closely coupled to the hair follicle cycle and does not begin until later in the cycle. This phenotype occurs in the absence of any global change in hair follicle cycle timing. The inappropriate presence of melanin early in the hair follicle cycle following PHGDH expression is also accompanied by increased melanocyte abundance in anagen II skin. Together, these data support a model in which PHGDH expression affects melanocyte proliferation and/or differentiation and may provide insight into how PHGDH expression impacts normal melanocyte biology to promote melanoma. SIGNIFICANCE The significance behind copy number gain of PHGDH in human cancers is unclear. In this study, we generate a mouse model that mimics PHGDH gene copy number gain and characterize its effect on normal tissues. Increased PHGDH expression yields a phenotype of aberrant melanin production, which indicates that PHGDH expression may play a role in normal melanocyte biology. This result may provide insight into why PHGDH copy number gain is observed in melanoma more frequently than in most other tumor types.