Acute myeloid leukemia (AML) is a hematological malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional genotoxic chemotherapy and a diagnosis of AML is usually fatal.More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. Here, we show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating acute lymphoblastic leukemia cells in vitro and in vivo. Thus, targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia. Key Points• Glutaminase inhibition commonly impairs glutathione metabolism and induces mitochondrial oxidative stress in acute myeloid leukemia cells • A glutaminase inhibitor synergizes with pro-oxidant drugs in inducing apoptosis and eliminating leukemia cells in vitro and in vivo Homoharringtonine (HHT; omacetaxine mepesuccinate), arsenic trioxide (ATO), cell-permeable glutathione reduced ethyl ester (GSH-MEE) and dimethyl 2oxoglutarate (α-ketoglutarate) were purchased from Millipore Sigma. Metabolic tracing experimentsCells were seeded at 3 x 10 5 /ml (replicates of 3) and treated with vehicle (DMSO) or CB-839 at 500 nM for 8 h, followed by incubation in glutamine-free RPMI 1640 supplemented with 13 C 5 , 15 N 2 -labeled L-glutamine (Cambridge Isotope Laboratories) for up to 12 h, in the presence of vehicle or drug. Flash frozen cell pellets (~ 1 x 10 6 cells) or supernatants (50 µl) were extracted and subjected to analysis by ultra-high pressure liquid chromatography and mass spectrometry (UHPLC/MS) as was previously described 20 . Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of 13 C and 15 N isotopes were performed using MAVEN (Princeton University, Princeton, NJ) and manually validated. Cell viability assaysCells were seeded at 0.5-1.0 x10 5 /ml in triplicate wells of 48-well tissue culture plates. Where indicated, the cells were treated with drug for a period of 48-72 h.After treatment, a sample of cells from each well was stained with PI (10 µg/ml) and viable cells (PI -) were counted with a flow cytometer (Millipore Guava

Altered metabolism in pulmonary artery smooth muscle cells (pASMcs) and endothelial cells (pAecs) contributes to the pathology of pulmonary hypertension (pH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human pASMcs and pAecs from pH versus control specimens, and that tGf-β treatment would phenocopy these metabolic changes. We used 13 c-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify 13 c-labeled metabolites. We found pH pASMcs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased pAecs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid β-oxidation. tGf-β treatment increased glycolysis in pASMcs, but did not recapitulate the pAec disease phenotype. in tGf-β-treated pASMcs, glucose, glutamine and fatty acids all contributed carbons to the tcA cycle. in conclusion, pASMcs and pAecs collected from pH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-β treatment. Changes in cellular metabolism are increasingly recognized as a hallmark of pulmonary hypertension (PH) pathobiology 1-4. Shifts in the uptake of metabolic substrates and how they are utilized downstream enables the disease phenotype of vascular cells in PH, including increased proliferation, apoptosis resistance, hypertrophy and vasoconstriction 3. One critical metabolic shift observed in PH is an increase in glycolysis, which is thought to occur in resident vascular wall cells including pulmonary artery smooth muscle cells (PASMCs), endothelial cells (PAECs) and fibroblasts 5-7. Increased glucose uptake can be demonstrated in vivo by increased uptake of the glucose analog 18 F-fluorodeoxyglucose in the lung parenchyma of PH subjects 6,8. The concept that glycolysis in PH is detrimental has led to investigation of the potential utility of dichloroacetate (DCA), which by blocking pyruvate dehydrogenase kinase causes increased glucose flux into the TCA cycle, and less glycolysis 9. Glutamine uptake and metabolism by PAECs has also been shown to contribute to their disease phenotype 10. However, comprehensive assessment of substrate uptake and how the substrates are utilized by pulmonary vascular cells in PH is lacking. A potential driver of altered cellular metabolism is transforming growth factor β (TGF-β) signaling, which underlies many forms of heritable (through mutations in BMPR2 and other members of the TGF-β signaling superfamily) and idiopathic PAH, and PAH etiologies associated with other conditions such as autoimmune

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Metabolomics is the comprehensive characterization of endogenous small molecules metabolites, xenometabolites and their metabolisms. The recent introduction of high-throughput metabolomics approaches has fostered strides related to the capacity to in silico elaborate metabolomics data by means of system biology. Recent progresses in bioanalytical technologies assisted with algorithms enabling large-scale data analysis potentiate application of metabolomics approaches in biomarker- based disease diagnosis, therapeutic target identification, personalized medicine, and the monitoring of clinical outcomes. In this review article we will focus on recent applications of metabolomics approaches in the identification of potential therapeutic candidates based on our and others' confirmed experience with this cutting-edge technology.

Background: Red blood cell (RBC) storage solutions, also known as additive solutions (ASs), first developed in the 1970s, enable extended storage of RBCs. Unfortunately, the advancements in this field have been limited, due to labor intensive and time-consuming serial in vitro and in vivo testing, coupled with very high commercialization hurdles. This study examines the utility of deep 96-well plates for preliminary screenings of novel ASs through comparison of RBC storage with the standard PVC bags in terms of hemolysis and ATP levels, under both normoxic (N) and hypoxic/hypocapnic (H) storage conditions. The necessity for the presence of DEHP, normally provided by PVC bags, is also examined.Materials and methods: A pool of 2 ABO compatible RBC units was split between a bag and a plate. Each plate well contained either 1, 2 or 0 PVC strips cut from standard storage bags to supply DEHP. The H bags and plates were processed in an anaerobic glovebox and stored in O2 barrier bags. Hemolysis and ATP were measured bi-weekly using standard methods.Results: Final ATP and hemolysis values for the plate-stored RBCs were comparable to the typical values observed for 6-week storage of leukoreduced AS-3 RBCs in PVC bags under both N and H conditions. Hemolysis was below FDA and EU benchmarks of 1% and 0.8%, respectively, and excluding DEHP from plates during storage, resulted in an inconsequential increase when compared to bag samples.Discussion: In combination with high-throughput metabolomics workflow, this platform provides a highly efficient preliminary screening platform to accelerate the initial testing and consequent development of novel RBC ASs.