Pawel Lorkiewicz

Glucose-Independent Glutamine Metabolism via TCA Cycling for Proliferation and Survival in B Cells

Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

Loss of FBP1 by snail-mediated repression provides metabolic advantages in basal-like breast cancer

The epithelial-mesenchymal transition (EMT) enhances cancer invasiveness and confers tumor cells with cancer stem cell (CSC)-like characteristics. We show that the Snail-G9a-Dnmt1 complex, which is critical for E-cadherin promoter silencing, is also required for the promoter methylation of fructose-1,6-biphosphatase (FBP1) in basal-like breast cancer (BLBC). Loss of FBP1 induces glycolysis and results in increased glucose uptake, macromolecule biosynthesis, formation of tetrameric PKM2, and maintenance of ATP production under hypoxia. Loss of FBP1 also inhibits oxygen consumption and reactive oxygen species production by suppressing mitochondrial complex I activity; this metabolic reprogramming results in an increased CSC-like property and tumorigenicity by enhancing the interaction of β-catenin with T-cell factor. Our study indicates that the loss of FBP1 is a critical oncogenic event in EMT and BLBC.

Stable isotope-resolved metabolomics and applications for drug development

Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), during the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly cancer. The metabolome is the functional readout of the genome, functional genome, and proteome; it is also an integral partner in molecular regulations for homeostasis. The interrogation of the metabolome, or metabolomics, is now being applied to numerous diseases, largely by metabolite profiling for biomarker discovery, but also in pharmacology and therapeutics. Recent advances in stable isotope tracer-based metabolomic approaches enable unambiguous tracking of individual atoms through compartmentalized metabolic networks directly in human subjects, which promises to decipher the complexity of the human metabolome at an unprecedented pace. This knowledge will revolutionize our understanding of complex human diseases, clinical diagnostics, as well as individualized therapeutics and drug response. In this review, we focus on the use of stable isotope tracers with metabolomics technologies for understanding metabolic network dynamics in both model systems and in clinical applications. Atom-resolved isotope tracing via the two major analytical platforms, NMR and MS, has the power to determine novel metabolic reprogramming in diseases, discover new drug targets, and facilitates ADME studies. We also illustrate new metabolic tracer-based imaging technologies, which enable direct visualization of metabolic processes in vivo. We further outline current practices and future requirements for biochemoinformatics development, which is an integral part of translating stable isotope-resolved metabolomics into clinical reality.

Titania microparticles and nanoparticles as matrixes for in vitro and in situ analysis of small molecules by MALDI-MS.

TiO(2) microparticles (TMs) and nanoparticles (TNs), prepared by hydrolysis of Ti butoxide and maintained in aqueous solution, were evaluated as matrixes for the detection of small molecules. With aqueous suspensions of TMs, positive-ion spectra collected for phospholipids (PLs) extracted from soybeans showed high sensitivity and no matrix interferences. Negative-ion traces had a suitable signal-to-noise ratio (S/N) but showed high levels of phosphatidic acid caused by the loss of the headgroup in other PLs. The hydrolysis was more pronounced when TNs were used. In situ studies performed by applying TM suspensions directly onto 50-microm thick soybean slices did not yield satisfactory results as TMs formed a crusty layer that precluded desorption/ionization. When a TN suspension was used instead, PLs and triacylglycerols were detected. Only minor PL hydrolysis was observed. TNs also enabled the coverage of rose petals and the detection of different levels of flavonols/anthocyanins and their glycosides as the color changed from yellow to orange-red. Although the hydrolysis of PLs limits the use of TMs or TNs for in vitro PL analysis, TNs offer a suitable alternative for the analysis of other molecules of low molecular weight, and their small size (average of approximately 200 nm) facilitates their penetration into tissue for in situ imaging.

Knockdown of Malic Enzyme 2 Suppresses Lung Tumor Growth, Induces Differentiation and Impacts PI3K/AKT Signaling

Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to yield CO2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD+ or NADP+. We find that ME2 is highly expressed in many solid tumors. In the A549 non-small cell lung cancer (NSCLC) cell line, ME2 depletion inhibits cell proliferation and induces cell death and differentiation, accompanied by increased reactive oxygen species (ROS) and NADP+/NADPH ratio, a drop in ATP, and increased sensitivity to cisplatin. ME2 knockdown impacts phosphoinositide-dependent protein kinase 1 (PDK1) and phosphatase and tensin homolog (PTEN) expression, leading to AKT inhibition. Depletion of ME2 leads to malate accumulation and pyruvate decrease, and exogenous cell permeable dimethyl-malate (DMM) mimics the ME2 knockdown phenotype. Both ME2 knockdown and DMM treatment reduce A549 cell growth in vivo. Collectively, our data suggest that ME2 is a potential target for cancer therapy.

Role in Tumor Growth of a Glycogen Debranching Enzyme Lost in Glycogen Storage Disease

BACKGROUND Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. METHODS Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. RESULTS We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. CONCLUSIONS Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.

2‐(2‐Aminoethylamino)‐5‐nitropyridine as a basic matrix for negative‐mode matrix‐assisted laser desorption/ionization analysis of phospholipids

Fast and easy analysis of phospholipids (PLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been well demonstrated. However, when using common organic matrices, such as 2,5-dihydroxybenzoic acid (DHB), the detection of most PL classes in positive-ion mode is difficult when PLs containing zwitterionic groups, such as phosphatidylcholines (PCs) and sphingomyelins (SMs) are present. To reduce this limitation, 2-(2-aminoethyloamino)-5-nitropyridine (AAN), a basic compound, was evaluated as an alternative matrix. Negative-ion spectra showed enhanced detection of phosphatidyl ethanolamines (PEs), phosphatidyl serines (PSs), phosphatidyl glycerols (PGs), and phosphatidyl inositols (PIs) in simple mixtures and in a crude methanolic soybean extract. The relative ionization efficiency (RIE) was highest for PIs and lowest for PGs, PSs, and PEs. Compared to DHB and para-nitroaniline, AAN resulted in greater sensitivity for the detection of PL classes in the negative mode. Indeed, the S/N ratio was nearly an order of magnitude higher than that reported for similar PI concentrations but with DHB. MALDI spots produced with AAN were homogeneous thus allowing automation and improved reproducibility. Positive-mode traces could also be acquired with AAN as the matrix, but with lower sensitivity than in the negative mode.

Stable Isotope Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics are poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, stable isotope-resolved metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks in a wide range of experimental systems, including human subjects. MS offers a wide range of instrumental capabilities involving different levels of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS that is affordable by many individual laboratories to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter focuses on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM.

Glutamine Regulates Cardiac Progenitor Cell Metabolism and Proliferation

Autologous transplantation of cardiac progenitor cells (CPCs) alleviates myocardial dysfunction in the damaged heart; however, the mechanisms that contribute to their reparative qualities remain poorly understood. In this study, we examined CPC metabolism to elucidate the metabolic pathways that regulate their proliferative capacity. In complete growth medium, undifferentiated CPCs isolated from adult mouse heart proliferated rapidly (Td = 13.8 hours). CPCs expressed the Glut1 transporter and their glycolytic rate was increased by high extracellular glucose (Glc) concentration, in the absence of insulin. Although high Glc concentrations did not stimulate proliferation, glutamine (Gln) increased CPC doubling time and promoted survival under conditions of oxidative stress. In comparison with Glc, pyruvate (Pyr) or BSA‐palmitate, Gln, when provided as the sole metabolic substrate, increased ATP‐linked and uncoupled respiration. Although fatty acids were not used as respiratory substrates when present as a sole carbon source, Gln‐induced respiration was doubled in the presence of BSA‐palmitate, suggesting that Gln stimulates fatty acid oxidation. Additionally, Gln promoted rapid phosphorylation of the mTORC1 substrate, p70S6k, as well as retinoblastoma protein, followed by induction of cyclin D1 and cdk4. Inhibition of either mTORC1 or glutaminolysis was sufficient to diminish CPC proliferation, and provision of cell permeable α‐ketoglutarate in the absence of Gln increased both respiration and cell proliferation, indicating a key role of Gln anaplerosis in cell growth. These findings suggest that Gln, by enhancing mitochondrial function and stimulating mTORC1, increases CPC proliferation, and that interventions to increase Gln uptake or oxidation may improve CPC therapy. Stem Cells 2015;33:2613—2627

Type 2 Diabetes Dysregulates Glucose Metabolism in Cardiac Progenitor Cells.

Type 2 diabetes is associated with increased mortality and progression to heart failure. Recent studies suggest that diabetes also impairs reparative responses after cell therapy. In this study, we examined potential mechanisms by which diabetes affects cardiac progenitor cells (CPCs). CPCs isolated from the diabetic heart showed diminished proliferation, a propensity for cell death, and a pro-adipogenic phenotype. The diabetic CPCs were insulin-resistant, and they showed higher energetic reliance on glycolysis, which was associated with up-regulation of the pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). In WT CPCs, expression of a mutant form of PFKFB, which mimics PFKFB3 activity and increases glycolytic rate, was sufficient to phenocopy the mitochondrial and proliferative deficiencies found in diabetic cells. Consistent with activation of phosphofructokinase in diabetic cells, stable isotope carbon tracing in diabetic CPCs showed dysregulation of the pentose phosphate and glycero(phospho)lipid synthesis pathways. We describe diabetes-induced dysregulation of carbon partitioning using stable isotope metabolomics-based coupling quotients, which relate relative flux values between metabolic pathways. These findings suggest that diabetes causes an imbalance in glucose carbon allocation by uncoupling biosynthetic pathway activity, which could diminish the efficacy of CPCs for myocardial repair.