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Sepsis is the leading cause of death in hospitalized children worldwide. Despite its hypothesized immune-mediated mechanism, targeted immunotherapy for sepsis is not available for clinical use.To determine the association between longitudinal cytometric, proteomic, bioenergetic, and metabolomic markers of immunometabolic dysregulation and pathogen type in pediatric sepsis.Serial peripheral blood mononuclear cell (PBMC) samples were obtained from 14 sepsis patients (34 total samples) and 7 control patients for this observational study. Flow cytometry was used to define immunophenotype, including T cell subset frequency and activation state, and assess intracellular cytokine production. Global immune dysfunction was assessed by tumor necrosis factor-α (TNF-α) production capacity and monocyte human leukocyte antigen DR (HLA-DR) expression. Mitochondrial function was assessed by bulk respirometry. Plasma cytokine levels were determined via Luminex assay. Metabolites were measured by liquid chromatography-mass spectrometry. Results were compared by timepoint and pathogen type.Sepsis patients were older (15.9 years vs. 10.4 years, P = 0.02) and had higher illness severity by PRISM-III (12.0 vs. 2.0, P 0.001) compared to controls; demographics were otherwise similar, though control patients were predominately male. Compared to controls, sepsis patients at timepoint 1 demonstrated lower monocyte HLA-DR expression (75% vs. 92%, P = 0.02), loss of peripheral of non-naïve CD4+ T cells (62.4% vs. 77.6%, P = 0.04), and reduced PBMC mitochondrial spare residual capacity (SRC; 4.0 pmol/s/106 cells vs. 8.4 pmol/s/106 cells, P = 0.01). At sepsis onset, immunoparalysis (defined as TNF-α production capacity 200 pg/mL) was present in 39% of sepsis patients and not identified among controls. Metabolomic findings in sepsis patients were most pronounced at sepsis onset and included elevated uridine and 2-dehydrogluconate and depleted citrulline. Loss of peripheral non-naïve CD4+ T cells was associated with immune dysfunction and reduced cytokine production despite increased T cell activation. CD4+ T cell differentiation and corresponding pro- and anti-inflammatory cytokines varied by pathogen.Pediatric sepsis patients exhibit a complex, dynamic physiologic state characterized by impaired T cell function and immunometabolic dysregulation which varies by pathogen type.

Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated-cimA strains produced 2.0g/L citramalate in 48h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production.

Ethanol and lactate are typical waste products of glucose fermentation. In mammals, glucose is catabolized by glycolysis into circulating lactate, which is broadly used throughout the body as a carbohydrate fuel. Individual cells can both uptake and excrete lactate, uncoupling glycolysis from glucose oxidation. Here we show that similar uncoupling occurs in budding yeast batch cultures of Saccharomyces cerevisiae and Issatchenkia orientalis. Even in fermenting S. cerevisiae that is net releasing ethanol, media

Plasma metabolite concentrations and labeling enrichments are common measures of organismal metabolism. In mice, blood is often collected by tail snip sampling. Here, we systematically examined the effect of such sampling, relative to gold-standard sampling from an in-dwelling arterial catheter, on plasma metabolomics and stable isotope tracing. We find marked differences between the arterial and tail circulating metabolome, which arise from two major factors: handling stress and sampling site, whose effects were deconvoluted by taking a second arterial sample immediately after tail snip. Pyruvate and lactate were the most stress-sensitive plasma metabolites, rising ~14 and ~5-fold. Both acute handling stress and adrenergic agonists induce extensive, immediate production of lactate, and modest production of many other circulating metabolites, and we provide a reference set of mouse circulatory turnover fluxes with noninvasive arterial sampling to avoid such artifacts. Even in the absence of stress, lactate remains the highest flux circulating metabolite on a molar basis, and most glucose flux into the TCA cycle in fasted mice flows through circulating lactate. Thus, lactate is both a central player in unstressed mammalian metabolism and strongly produced in response to acute stress.

Kelly cells stably expressing non-targeting shRNA (shNT) or SHMT2 shRNA (shSHMT2) were exposed to normoxia (Nor) or 0.5% O2 (Hyp) for 48 hours incubated with glucose concentrations indicated, % cell viability was determined using Trypan Blue staining (Data represent Mean {plus minus} S.D from three biological repeats).

MOV file - 7511KB, Acute autophagy ablation causes progressive motor and behavioral deficits.

Supplementary Data from Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism

PDF file - 2587KB, Comparison of collapsed expression of all muscle genes during fasting.

PDF file - 145KB, Differential expression analysis for significant biological processes by GO term DAVID Analysis for liver tissues during fasting.

The correlation of SHMT2 and PHGDH expression in the samples from patients who died from the disease (top panel, pPHGDHvsSHMT1=0.092, pPHGDHvsSHMT2=7.3×10-5) and the correlation found in the samples from patients where the tumor regressed or matured spontaneously (bottom panel, pPHGDHvsSHMT1=0.3, pPHGDHvsSHMT2=0.0084).

The de novo synthesis of the nonessential amino acid serine is often upregulated in cancer. In this study, we demonstrate that the serine catabolic enzyme, mitochondrial serine hydroxymethyltransferase (SHMT2), is induced when MYC-transformed cells are subjected to hypoxia. In mitochondria, SHMT2 can initiate the degradation of serine to CO2 and NH4+, resulting in net production of NADPH from NADP+. Knockdown of SHMT2 in MYC-dependent cells reduced cellular NADPH:NADP+ ratio, increased cellular reactive oxygen species, and triggered hypoxia-induced cell death. In vivo, SHMT2 suppression led to impaired tumor growth. In MYC-amplified neuroblastoma patient samples, there was a significant correlation between SHMT2 and hypoxia-inducible factor-1 α (HIF1α), and SHMT2 expression correlated with unfavorable patient prognosis. Together, these data demonstrate that mitochondrial serine catabolism supports tumor growth by maintaining mitochondrial redox balance and cell survival.Significance: In this study, we demonstrate that the mitochondrial enzyme SHMT2 is induced upon hypoxic stress and is critical for maintaining NADPH production and redox balance to support tumor cell survival and growth. Cancer Discov; 4(12); 1406–17. ©2014 AACR.See related commentary by Martínez-Reyes and Chandel, p. 1371This article is highlighted in the In This Issue feature, p. 1355

The hypoxic induction of SHMT2 depends on HIF-1alpha. SK-N-BE(2) cells stably expressing non-targeting shRNA or a shRNA against HIF-1alpha were exposed to normoxia (Nor) or 0.5% O2 (Hyp) for 8 hours. mRNA levels were measured using Q-PCR and normalized to normoxia control.

(A) Kelly cells stably expressing non-targeting shRNA (shNT) or SHMT2 shRNA (shSHMT2) were exposed to normoxia (Nor) or 0.5% O2 (Hyp) for 24 hours. Total cellular DNA were extracted and relative mitochondria/nuclear DNA ratio were measured using Q-PCR. (Data represent Mean {plus minus} S.D of four independent experiments). (B) Kelly cells were exposed to normoxia (Nor) or 0.5% O2 (Hyp) for 6h. ROS levels were measured using a flow cytometry-based MitoTracker Red CM-H2Ros assay. All values are normalized to normoxic shNT control cells (Data represent Mean {plus minus} S.D of three biological repeats, ***p

Knockdown of SHMT2 reduces xenograft growth in vivo.

(A) Immunoblots of N-Myc and c-Myc in the six cell lines. (B, C) H293T and HT1080 cells were exposed to normoxia (Nor) or 0.5% O2 (Hyp) for 6 or 16 hours, (D) H1299 cells were exposed to normoxia (Nor) or 0.5% O2 (Hyp) for 16 hours. mRNA levels were measured using Q-PCR and normalized to normoxia control.

Supplementary Method Descriptions Molecular modeling and computational studies Enzyme expression and purification. In vitro enzymatic assays and Ki determinations In vivo efficacy trials with MIA PaCa-2 pancreatic cancer xenografts. Cytochrome c oxidase assay Synthesis of AGF94 and 5-substituted pyrrolo[3,2-d]pyrimidine compounds

Folate-dependent one-carbon (C1) metabolism is compartmentalized into the mitochondria and cytosol and supports cell growth through nucleotide and amino acid biosynthesis. Mitochondrial C1 metabolism, including serine hydroxymethyltransferase (SHMT) 2, provides glycine, NAD(P)H, ATP, and C1 units for cytosolic biosynthetic reactions, and is implicated in the oncogenic phenotype across a wide range of cancers. Whereas multitargeted inhibitors of cytosolic C1 metabolism, such as pemetrexed, are used clinically, there are currently no anticancer drugs that specifically target mitochondrial C1 metabolism. We used molecular modeling to design novel small-molecule pyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1 metabolism at SHMT2. In vitro antitumor efficacy was established with the lead compounds (AGF291, AGF320, AGF347) toward lung, colon, and pancreatic cancer cells. Intracellular targets were identified by metabolic rescue with glycine and nucleosides, and by targeted metabolomics using a stable isotope tracer, with confirmation by in vitro assays with purified enzymes. In addition to targeting SHMT2, inhibition of the cytosolic purine biosynthetic enzymes, β-glycinamide ribonucleotide formyltransferase and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, and SHMT1 was also established. AGF347 generated significant in vivo antitumor efficacy with potential for complete responses against both early-stage and upstage MIA PaCa-2 pancreatic tumor xenografts, providing compelling proof-of-concept for therapeutic targeting of SHMT2 and cytosolic C1 enzymes by this series. Our results establish structure–activity relationships and identify exciting new drug prototypes for further development as multitargeted antitumor agents.

PDF file - 116KB, Differential expression analysis for significant biological processes by GO term DAVID Analysis for muscle tissues during fasting.

PDF file - 3190KB, Supplementary Figures (S1-S6) provide additional characterization about effects of systemic autophagy ablation in normal adult tissues and tumors. Supplementary Methods supply detailed information of experiments.

Supplementary Figure S1 shows in vitro antiproliferative activity and targeted pathways of previously reported AICARFTase and GARFTase inhibitors. Supplementary Figure S2 shows docking of AGF291, AGF320, and AGF347 (along with 5-formyl-THF) in human SHMT2 and rabbit SHMT1. Supplementary Figure S3 shows plasma membrane folate transporter expression levels in H460, HCT116, and MIA PaCa-2 human tumor cell lines as compared to those in the IGROV-1 epithelial ovarian cancer cell line. Supplementary Figure S4 shows in vitro antiproliferative activity and targeted pathways of AGF291, AGF320, and AGF347 along with previously reported GARFTase inhibitor AGF94 in H460, HCT116, and MIA PaCa-2 cell lines. Supplementary Figure S5 shows targeted metabolomics data on total serine, serine isotope labeling patterns, total GAR, total AICAR, total adenine nucleotides, and total dTTP along with isotope labeling patterns of H460, HCT116, and MIA PaCa-2 cell lines treated with AGF291, AGF320, and AGF347 not shown in main text Figure 4. Supplementary Figure S6 shows a Western blot confirming knockdown of SHMT2 in H460 SHMT2 KD cell line and knockout of SHMT2 in HCT116 SHMT2 KO cell line. Supplementary Figure S7 shows in vivo efficacies of AGF347 and gemcitabine towards MIA PaCa-2 early and late stage tumor xenograft models where mouse serum folate levels were not depleted to approximate those found in humans. Supplementary Figure S8 shows a cytochrome c oxidase assay on tumors harvested from the metabolomics arm of the late stage in vivo AGF347 trial. Table S1 shows docking scores of novel compounds in human SHMT2 and rabbit SHMT1 (corresponding to Supplementary Figure S2). Table S2 shows quantitative data from in vivo early- and late-stage trials of AGF347 and gemcitabine against MIA PaCa-2 tumor xenografts in NCR SCID mice.

PDF file - 2193KB, Comparison of collapsed expression of all liver genes during fasting.

SF 1. Heat map showing the tumor growth inhibition (TGI) of six metabolic inhibitors in PDX panel. SF 2. Tumor growth curves of PDXs treated with phenformin. SF 3. Tumor doubling time of 12 individual PDXs. SF 4. Aggregate tumor doubling time of PDXs treated with vehicle or phenformin.

This file contains additional figures related to findings from the described subcutaneous xenograft experiments, the primary murine NSCLC experiments and the metastatic breast cancer xenograft experiments. In addition, it contains a table listing the oligonucleotide sequences utilized in our genetic studies.

Supplementary Data from Selenium Modulates Cancer Cell Response to Pharmacologic Ascorbate

Supplementary Figure from Selenium Modulates Cancer Cell Response to Pharmacologic Ascorbate

The enzyme glucose-6-phosphate dehydrogenase (G6PD) is a major contributor to NADPH production and redox homeostasis and its expression is upregulated and correlated with negative patient outcomes in multiple human cancer types. Despite these associations, whether G6PD is essential for tumor initiation, growth, or metastasis remains unclear. Here, we employ modern genetic tools to evaluate the role of G6PD in lung, breast, and colon cancer driven by oncogenic K-Ras. Human HCT116 colorectal cancer cells lacking G6PD exhibited metabolic indicators of oxidative stress, but developed into subcutaneous xenografts with growth comparable with that of wild-type controls. In a genetically engineered mouse model of non–small cell lung cancer driven by K-Ras G12D and p53 deficiency, G6PD knockout did not block formation or proliferation of primary lung tumors. In MDA-MB-231–derived human triple-negative breast cancer cells implanted as orthotopic xenografts, loss of G6PD modestly decreased primary site growth without ablating spontaneous metastasis to the lung and moderately impaired the ability of breast cancer cells to colonize the lung when delivered via tail vein injection. Thus, in the studied K-Ras tumor models, G6PD was not strictly essential for tumorigenesis and at most modestly promoted disease progression.Significance:K-Ras–driven tumors can grow and metastasize even in the absence of the oxidative pentose pathway, a main NADPH production route.

High-dose ascorbate (vitamin C) has shown promising anticancer activity. Two redox mechanisms have been proposed: hydrogen peroxide generation by ascorbate itself or glutathione depletion by dehydroascorbate (formed by ascorbate oxidation). Here we show that the metabolic effects and cytotoxicity of high-dose ascorbate in vitro result from hydrogen peroxide independently of dehydroascorbate. These effects were suppressed by selenium through antioxidant selenoenzymes including glutathione peroxidase 1 (GPX1) but not the classic ferroptosis-inhibiting selenoenzyme GPX4. Selenium-mediated protection from ascorbate was powered by NADPH from the pentose phosphate pathway. In vivo, dietary selenium deficiency resulted in significant enhancement of ascorbate activity against glioblastoma xenografts. These data establish selenoproteins as key mediators of cancer redox homeostasis. Cancer sensitivity to free radical-inducing therapies, including ascorbate, may depend on selenium, providing a dietary approach for improving their anticancer efficacy.Significance:Selenium restriction augments ascorbate efficacy and extends lifespan in a mouse xenograft model of glioblastoma, suggesting that targeting selenium-mediated antioxidant defenses merits clinical evaluation in combination with ascorbate and other pro-oxidant therapies.

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTEN.Conclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639–47. ©2017 AACR.

Supplementary Figure 2 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Figure Legends 1-7 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Methods from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Table S1. Amino acid concentrations in pancreatic tumor and benign adjacent tissues Figure S1. Quantification of macropinocytosis in human pancreatic tumors. Figure S2. Few macropinosomes are present in the normal pancreatic tissue adjacent to PDAC tumors. Figure S3. Ras-mutant pancreatic cells are capable of indefinite growth in medium lacking leucine supplemented with physiological levels of serum protein. Figure S4. Ras-mutant pancreatic cells are capable of indefinite growth in medium lacking lysine or phenylalanine supplemented with physiological levels of serum protein. Figure S5. Growth of KRPC cells in leucine-free medium with 50 g/L serum protein cannot be explained by the presence of contaminating leucine. Figure S6. Stable isotope tracing reveals significant contribution of serum protein to amino acid pools in human PDAC cells. Figure S7. EIPA, an inhibitor of macropinocytosis, reduces intracellular (A) and extracellular (B) serum protein-derived amino acid pools in a dose-dependent fashion. Figure S8. Fractional contribution of serum protein-derived nitrogen to intracellular amino acid pools. Figure S9. Amino acid frequency in bovine serum albumin relative to human proteome.

Supplementary Figure 6 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplemental data. Human metabolomics data (raw and processed peak intensities and lists of samples and metabolites).

Supplementary Figure 7 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Figures and Table legends

Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC. Cancer Res; 75(3); 544–53. ©2014 AACR.

Supplementary Figure 1 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Figure 3 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Figure 4 from Inhibition of Glutaminase Preferentially Slows Growth of Glioma Cells with Mutant IDH1

Supplementary Figure 1. Baseline TIGAR mRNA expression in tumors.