Glutamine Uptake Research Articles

Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions

BackgroundMacrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.MethodsImmunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM.ResultsSM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM.ConclusionsThese findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA.2D7p_hrucSDMCj1WNQG2ziVideo

Glutamine Metabolism in Osteoprogenitors Is Required for Bone Mass Accrual and PTH-Induced Bone Anabolism in Male Mice.

Skeletal homeostasis critically depends on the proper anabolic functioning of osteolineage cells. Proliferation and matrix synthesis are highly demanding in terms of biosynthesis and bioenergetics, but the nutritional requirements that support these processes in bone-forming cells are not fully understood. Here, we show that glutamine metabolism is a major determinant of osteoprogenitor function during bone mass accrual. Genetic inactivation of the rate-limiting enzyme glutaminase 1 (GLS1) results in decreased postnatal bone mass, caused by impaired biosynthesis and cell survival. Mechanistically, we uncovered that GLS1-mediated glutamine catabolism supports nucleotide and amino acid synthesis, required for proliferation and matrix production. In addition, glutamine-derived glutathione prevents accumulation of reactive oxygen species and thereby safeguards cell viability. The pro-anabolic role of glutamine metabolism was further underscored in a model of parathyroid hormone (PTH)-induced bone formation. PTH administration increases glutamine uptake and catabolism, and GLS1 deletion fully blunts the PTH-induced osteoanabolic response. Taken together, our findings indicate that glutamine metabolism in osteoprogenitors is indispensable for bone formation. © 2020 American Society for Bone and Mineral Research (ASBMR).

Treatment Strategies and Metabolic Pathway Regulation in Urothelial Cell Carcinoma: A Comprehensive Review.

For a long time, cisplatin-based chemotherapy had been viewed as first-line chemotherapy for advanced and metastatic urothelial carcinoma (UC). However, many patients with UC had been classified as cisplatin-ineligible who can only receive alternative chemotherapy with poor treatment response, and the vast majority of the cisplatin-eligible patients eventually progressed, even those with objective response with cisplatin-based chemotherapy initially. By understanding tumor immunology in UC, immune checkpoint inhibitors, targeting on programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) pathways, had been proven as first-line treatment for cisplatin-ineligible metastatic UC and as second-line treatment for patients with platinum-refractory metastatic UC by the U.S Food and Drug Administration (FDA). In 2020, JAVEIN bladder 100 further reported that PD-L1 inhibitors showed benefits on prolonged survival and progression-free survival as maintenance therapy. Besides targeting on immune checkpoint, manipulation of the tumor microenvironment by metabolic pathways intervention, including inhibition on tumor glycolysis, lactate accumulation and exogenous glutamine uptake, had been investigated in the past few years. In this comprehensive review, we start by introducing traditional chemotherapy of UC, and then we summarize current evidences supporting the use of immune checkpoint inhibitors and highlight ongoing clinical trials. Lastly, we reviewed the tumor metabolic characteristic and the anti-tumor treatments targeting on metabolic pathways.

Targeting Glutamine Metabolism Overcomes Resistance to Targeted Therapies in Refractory Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment. Despite the encouraging initial MCL tumor response to ibrutinib (IBN), relapse occurs only after few months of treatment due to multiple resistance mechanisms. Thus, the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of IBN-R cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their grow and survival. The aim of this work was to uncover potential targets responsible for the upregulation of OXPHOS pathway in the refractory/relapsed (R/R) MCL by using multiple biochemical and biological strategies. We focused the present study on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate (α-KG) that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. To further demonstrate the reliance of OXPHOS on glutamine anaplerosis, we have further tested the combinatory effects of targeting GLS and OXPHOS using their respective inhibitors, CB-839 and IACS-010759, on tumor killing activity in R/R MCL. Methods:Primary MCL cells from patient leukapheresis or whole blood specimens, as well as established MCL cell lines were used as experimental models of MCL. Metabolomic profiling was used to determine intracellular metabolite fluxes and levels. Cell Titer Glo assay was used to measure cell proliferation/viability after treatment with inhibitors. Annexin V and propidium iodide were used to measure cell apoptosis and cell cycle arrestviaflow cytometry analysis. Magnetic microbeads-based B-cell isolation method were used for the purification of malignant B cells from patient samples. Western blot analysis was used to evaluate protein level expression. Patient-derived Xenograft (PDX) mouse model created from patients with MCL was used to evaluate the in vivo anti-tumor activity and potential clinical value of GLS and OXPHOS inhibitors. Results:Our recent metabolomic profiling studies have demonstrated that glutaminolysis and OXPHOS are upregulated in IBN-R MCL, manifested by increased glutamine uptake in the ibrutinib-resistant MCL cell lines (p=0.03).Inhibition of glutamine metabolism with the allosteric GLS1-selective inhibitor BPTES resulted in inhibition of cell viability (0.2381uM-9.98uM), indicating that MCL cells are dependent on glutamine metabolism for their proliferation. To corroborate with the above finding, we also presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples and cell lines confirmed by immunoblotting. Inhibiting of GLS would lead to significant reduction in OXPHOS, mitochondria membrane potential and ATP production, as either single drug or in combination with other targeting agents. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigation on solid tumors. Inhibiting GLS1 with CB-839 leads to the decreased cell viability in MCL (0.5626nM-308.4nM). Of note, the treatment with CB-839 to MCL cell lines induces S phase reduction in both Jeko-1 (17.23%) and Z-138 (14.01%), as well as induces significant apoptosis (p=0.013 and p=0.002 in Jeko-1 and Z-138 cells). GLS inhibition will be further explored in the context of mitochondria defect or hypoxia, where OXPHOS maybe deficient. Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. Conclusion:In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the new GLS inhibitor, GCB-839, may present a clinical potential for a new indication and warrants more in-depth investigation. Deciphering the mechanisms involved in MCL metabolic heterogeneity and adaptability during drug resistance development would be crucial to identify key actors enabling MCL cells to escape from therapy. Disclosures Wang: Acerta Pharma:Research Funding;Molecular Templates:Research Funding;InnoCare:Consultancy;Oncternal:Consultancy, Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Targeted Oncology:Honoraria;MoreHealth:Consultancy;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Lu Daopei Medical Group:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Verastem:Research Funding;Nobel Insights:Consultancy;BioInvent:Research Funding;Guidepoint Global:Consultancy;AstraZeneca:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Pharmacyclics:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Janssen:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Loxo Oncology:Consultancy, Research Funding;Pulse Biosciences:Consultancy;OncLive:Honoraria;Beijing Medical Award Foundation:Honoraria;VelosBio:Research Funding.

Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study.

Oxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown. This study managed to investigate this issue by conducting in vitro experiments. Oxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions. The retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H2O2) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H2O2 induced oxidative damages in ARPE-19 cells. Mechanistically, H2O2 induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H2O2 stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H2O2 increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3' untranslated region (3'UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H2O2 treatment on ARPE-19 cells. In general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H2O2 stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.

Macrophage-derived glutamine boosts satellite cells and muscle regeneration

Muscle regeneration is sustained by infiltrating macrophages and consequent satellite cell (SC) activation1–4. Macrophages and SC communicate in different ways1–5 but their metabolic interplay was never investigated so far. Here, we found that muscle injuries and aging are characterized by intratissutal glutamine restriction. Low glutamine levels endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity at the expense of glutamate dehydrogenase-1 (GLUD1)-mediated glutamine oxidation. Glud1 knockout (KO) macrophages display constitutively high GS activity which prevents glutamine shortage. Import of macrophage-derived glutamine by SC through the glutamine-transporter SLC1A5 activates mTOR and promotes SC proliferation and differentiation. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischemia, or aging. Conversely, SLC1A5 blockade in SC or GS inactivation in macrophages negatively affects SC functions and muscle regeneration. These results highlight a metabolic cross-talk between SC and macrophages whereby macrophage-derived glutamine sustains SC functions. Thus, GLUD1 targeting offers new therapeutic opportunities for the regeneration of injured or aged muscles.

Topotecan induces hepatocellular injury via ASCT2 mediated oxidative stress

BackgroundTopotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. Materials and methodsMethyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. ResultsMTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. ConclusionTopotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production.

Human placental uptake of glutamine and glutamate is reduced in fetal growth restriction

Fetal growth restriction (FGR) is a significant risk factor for stillbirth, neonatal complications and adulthood morbidity. Compared with those of appropriate weight for gestational age (AGA), FGR babies have smaller placentas with reduced activity of amino acid transporter systems A and L, thought to contribute to poor fetal growth. The amino acids glutamine and glutamate are essential for normal placental function and fetal development; whether transport of these is altered in FGR is unknown. We hypothesised that FGR is associated with reduced placental glutamine and glutamate transporter activity and expression, and propose the mammalian target of rapamycin (mTOR) signaling pathway as a candidate mechanism. FGR infants [individualised birth weight ratio (IBR) < 5th centile] had lighter placentas, reduced initial rate uptake of 14C-glutamine and 14C-glutamate (per mg placental protein) but higher expression of key transporter proteins (glutamine: LAT1, LAT2, SNAT5, glutamate: EAAT1) versus AGA [IBR 20th–80th]. In further experiments, in vitro exposure to rapamycin inhibited placental glutamine and glutamate uptake (24 h, uncomplicated pregnancies) indicating a role of mTOR in regulating placental transport of these amino acids. These data support our hypothesis and suggest that abnormal glutamine and glutamate transporter activity is part of the spectrum of placental dysfunction in FGR.

Abstract PR06: The glutaminase inhibitor CB-839 potentiates antimelanoma activity of standard-of-care targeted therapies and immunotherapies

Abstract Nearly all metastatic melanoma patients who respond to targeted therapies will relapse with the disease within a year. Although more durable responses are seen with immune therapies, about half of the melanoma patients do not respond to them, and a significant number of responders eventually relapse. Most relapsed melanomas also exhibit post-treatment resistance to these treatments. Hence, there is a clear and present need to develop therapeutics that counteract resistance associated with relapse. We and others earlier showed that melanomas with elevated mitochondrial activity possess improved cellular rigor and are intrinsically resistant to the antitumor effects of BRAF and MEK inhibitors. In many other instances, melanomas that initially respond to these inhibitors acquire resistance by elevating mitochondrial activity. Mitochondrial activity is elevated in part by increased cellular uptake of glutamine, and its conversion to alpha-ketoglutarate in the TCA cycle, with glutaminase enzyme playing a rate-limiting role. In this study, we show that BRAFV600E-mutant melanomas with intrinsic or acquired resistance to MAPK pathway inhibitors have lower glucose uptake and increased glutamine uptake compared to those that are sensitive. Treatment of these resistant melanomas with single-agent glutaminase inhibitor, CB-839, moderately inhibited their growth. However, a more robust inhibition of their growth was achieved when CB-839 was combined with BRAF and MEK inhibitors. In addition, CB-839 increased the in vivo activity of tumor-infiltrating lymphocytes (TILs) in a mouse vaccine model and also enhanced the proapoptotic effect of human autologous patient-derived TILs on their cognate melanoma cells. Seahorse bioenergetics stress tests showed that CB-839 inhibited mitochondrial OxPhos in tumor cells to a greater extent than in activated TILs. Additional molecular studies are currently in progress. A recent clinical trial in melanoma patients showed that combination treatment with CB-839 and the immune checkpoint blocker, nivolumab, caused an objective response in three melanoma patients who had earlier progressed on treatment with immune checkpoint blockade. Our preclinical results complement this clinical finding and suggest that CB-839 combination could potentiate the efficacy of targeted and immune therapies in refractory melanomas. Citation Format: Sruthy Varghese, Snigdha Pramanik, Rishika Prasad, Hannah Hodges, Leila Williams, Weiyi Peng, Hussein Tawbi, Vashisht Yennu Nanda. The glutaminase inhibitor CB-839 potentiates antimelanoma activity of standard-of-care targeted therapies and immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR06.

Macrophage Stimulated by Low Ambient Temperature Hasten Tumor Growth via Glutamine Production.

Ambient temperature can regulate the immune response and affect tumor growth. Although thermoneutral caging reduces tumor growth via immune activation, little attention has been paid to the tumorigenic effect of low temperature. In the present study, tumor growth was higher at low ambient temperature (4 °C for 8 h/d) than at the standard housing temperature (22 °C) in allograft models. Low temperature-stimulated tumor growth in mice was reduced by monocyte depletion using clodronate liposomes. Proliferation was considerably greater in cancer cells treated with 33 °C-cultured RAW264.7 cell-conditioned media (33CM) than in cells treated with 37 °C-cultured RAW264.7 cell-conditioned media (37CM). Additionally, glutamine levels were markedly higher in 33CM-treated cells than in 37CM-treated cells. We further confirmed that the addition of glutamine into 37CM enhanced its effects on cancer cell proliferation and glutamine uptake inhibition ameliorated the accelerated proliferation induced by 33CM. Consistently, the inhibition of glutamine uptake in the allograft model exposed to low temperature, effectively reduced tumor volume and weight. Collectively, these data suggest that the secretion and utilization of glutamine by macrophages and cancer cells, respectively, are key regulators of low temperature-enhanced cancer progression in the tumor microenvironment.

Targeting glutamine metabolism as an effective means to promote allograft acceptance while inhibit tumor growth

Recently the role of metabolic signaling pathways has emerged as playing a critical role in dictating the outcome of T cell responses. The uptake and metabolism of the amino acid glutamine is essential for effector T cell activation. Since the growth and expansion of tumor cells relies on similar anabolic and metabolic requirements, we hypothesized that glutamine blockage might represent a promising strategy to promote allograft survival while inhibit tumor growth. 6-Diazo-5-oxo-L-norleucine (DON) was used as a glutamine antagonist. First, an in vitro study of T cell proliferation was performed to examine the ability of glutamine antagonism to inhibit T cell proliferation. Then we investigated whether DON could prolong allograft survival and inhibit tumor growth by using a fully MHC-mismatched mice full thickness skin transplantation model and a mice TC-1 tumor-bearing model. The proliferation study demonstrated that DON inhibited effector T cells proliferation in a dose-dependent manner. We found a marked prolonged graft median survival time and significant tumor inhibition for mice that received DON compared to those that received no treatment.These results highlight that targeting glutamine metabolism can promote allograft acceptance in a long tumor-free period.

Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes.

Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY.IMPORTANCEL. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

Abstract 3801: Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells

Abstract Breast cancer stem cells (BCSCs) have been shown to be associated with tumor recurrence, metastasis and drug resistance. We reported that the ganglioside GD2 selectively identifies BCSCs and its over-expression in Triple-Negative Breast Cancer (TNBC). However, the factors regulating GD2+ BCSCs in a growing tumor are not known. Here we hypothesize that nutrient deprivation-induced oxidative stress upregulates GD2+ BCSCs in TNBC. TNBC cell lines including SUM159 and MDA-MB-231 were cultured under different nutrient-deprived conditions and analyzed for GD2 expression by flow cytometry. We have identified a steady increase of GD2+ cells from 5%±2% on day 3 to 20%±5% on day 6 in cells cultured in nutrient deprived condition. We further performed the in vivo by injecting GFP+ MDA-MB-231 cells into NSG mice and measured GD2+ expression in the growing tumor once a week. Interestingly, we also noticed a positive correlation between the percentage of GD2+ cells and tumor volume in vivo with r=0.9829 and p&amp;lt;0.0004 suggesting nutrient deprived conditions in the growing tumors results in higher GD2 expression. Next, the cells were cultured in medium with or without glucose or media containing 2-deoxy glucose (2DG, glycolysis inhibitor). We found that the percentage of GD2+ cells increased 2-fold in the absence of glucose or in the presence of 2DG (10mM) compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 biosynthesis in TNBC cells. Global metabolomics profiling using liquid chromatography-mass spectrometry followed by KEGG pathway analysis identified signatures such as glutathione mediated detoxification and glutathione biosynthesis to be most highly upregulated in GD2+ compared to GD2- cells. As glutamine is a key precursor for glutathione biosynthesis, we cultured SUM159 and MDA-MB-231 cells in media with or without glutamine and found that the percentage of GD2+ cells positively correlates with glutamine concentration in the medium. Next, to target glutamine metabolism, we treated TNBC cells with glutaminase inhibitor, CB-839 or glutamine transporter inhibitor, V9302. This resulted in a 70-80% reduction of GD2 expression in a dose dependent manner. CB-839 and V9302 also inhibited mammosphere formation by 40-50% and increased reactive oxygen species (ROS) in a dose-dependent manner in TNBC cells. In conclusion, oxidative stress results in GD2+ BCSC phenotype in TNBC cells. Inhibition of glutamine uptake or its utilization by V9302 or CB-839 inhibits GD2+ BCSC phenotype and alters redox homeostasis in BCSCs by inducing ROS levels. Targeting glutamine transporter or glutaminase could complement conventional chemotherapy by targeting BCSCs in TNBC. Citation Format: Appalaraju Jaggupilli, Stanely J. Ly, Roshan Borkar, Khoa Nguyen, Bin Yuan, Nagireddy Putluri, Michael Andreeff, V. Lokesh Battula. Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3801.

Glutamine uptake and utilization of human mesenchymal glioblastoma in orthotopic mouse model

BackgroundGlioblastoma (GBM) are highly heterogeneous on the cellular and molecular basis. It has been proposed that glutamine metabolism of primary cells established from human tumors discriminates aggressive mesenchymal GBM subtype to other subtypes.MethodsTo study glutamine metabolism in vivo, we used a human orthotopic mouse model for GBM. Tumors evolving from the implanted primary GBM cells expressing different molecular signatures were analyzed using mass spectrometry for their metabolite pools and enrichment in carbon 13 (13C) after 13C-glutamine infusion.ResultsOur results showed that mesenchymal GBM tumors displayed increased glutamine uptake and utilization compared to both control brain tissue and other GBM subtypes. Furthermore, both glutamine synthetase and transglutaminase-2 were expressed accordingly to GBM metabolic phenotypes.ConclusionThus, our results outline the specific enhanced glutamine flux in vivo of the aggressive mesenchymal GBM subtype.

Reprogramming of host glutamine metabolism during Chlamydia trachomatis infection and its key role in peptidoglycan synthesis.

Obligate intracellular bacteria such as Chlamydia trachomatis undergo a complex developmental cycle between infectious, non-replicative elementary-body and non-infectious, replicative reticulate-body forms. Elementary bodies transform to reticulate bodies shortly after entering a host cell, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell, it is unknown how the replicative part of the developmental cycle is initiated. Here we show, using a cell-free approach in axenic media, that the uptake of glutamine by the bacteria is crucial for peptidoglycan synthesis, which has a role in Chlamydia replication. The increased requirement for glutamine in infected cells is satisfied by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine is effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limits the growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5-knockout organoids and mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infection strategies.

Fluoride Exposure Affects Glutamine Uptake in Müller Glia Cells.

Fluoride, a pollutant present in contaminated ground water, oral care products, food, and pesticides, has deleterious effects in the structure and function of the central nervous system. Among the established neurological defects described in the exposed population, a reduced score in intelligence quotient tests in children of contaminated areas has gained attention over the past years. Maternal fluoride exposure during gestation decreases learning and memory abilities that correlate with a significant diminution of glutamate receptors expression. Since the involvement of glia cells in the maintenance and regulation of glutamatergic synapses is well-documented, in this contribution, we characterized the effect of fluoride exposure in the regulation of glia glutamine transporters. To this end, we used the Müller glia cell line, Mio-M1, and through the use of [3H]L-Glutamine uptake experiments and a Western blot approach, we demonstrate here the functional expression of system N of glutamine transporters, SNAT3 and SNAT5, in this model of human retina radial glia cells. Furthermore, these transporters interact with the glutamate transporter excitatory amino acid transporter 1, in an activity-dependent manner. Fluoride treatment reduces glutamine uptake and cell membrane [3H]glutamine surface binding, in good correlation with a decrease in SNAT3 and 5 protein levels. These results demonstrate that glia cells respond to the presence of fluoride reducing glutamine mobilization and by these means decreases glutamate turnover suggesting a disruption of glutamatergic transmission.

Abstract 308: Reductive Carboxylation Contributes to Cardiac Adaptation in Response to the Oncometabolite D2-hydroxyglutarate

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

The Tumor Suppressor PTEN as Molecular Switch Node Regulating Cell Metabolism and Autophagy: Implications in Immune System and Tumor Microenvironment.

Recent studies conducted over the past 10 years evidence the intriguing role of the tumor suppressor gene Phosphatase and Tensin Homolog deleted on Chromosome 10 PTEN in the regulation of cellular energy expenditure, together with its capability to modulate proliferation and survival, thus expanding our knowledge of its physiological functions. Transgenic PTEN mice models are resistant to oncogenic transformation, present decreased adiposity and reduced cellular glucose and glutamine uptake, together with increased mitochondrial oxidative phosphorylation. These acquisitions led to a novel understanding regarding the role of PTEN to counteract cancer cell metabolic reprogramming. Particularly, PTEN drives an “anti-Warburg state” in which less glucose is taken up, but it is more efficiently directed to the mitochondrial Krebs cycle. The maintenance of cellular homeostasis together with reduction of metabolic stress are controlled by specific pathways among which autophagy, a catabolic process strictly governed by mTOR and PTEN. Besides, a role of PTEN in metabolic reprogramming and tumor/stroma interactions in cancer models, has recently been established. The genetic inactivation of PTEN in stromal fibroblasts of mouse mammary glands, accelerates breast cancer initiation and progression. This review will discuss our novel understanding in the molecular connection between cell metabolism and autophagy by PTEN, highlighting novel implications regarding tumor/stroma/immune system interplay. The newly discovered action of PTEN opens innovative avenues for investigations relevant to counteract cancer development and progression.

Slc38a1 Conveys Astroglia-Derived Glutamine into GABAergic Interneurons for Neurotransmitter GABA Synthesis.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA—i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)—which is enriched on parvalbumin+ GABAergic neurons—and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

Dietary glutamine supplementation suppresses epigenetically-activated oncogenic pathways to inhibit melanoma tumour growth

Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.