Absence Of Glutamine Research Articles

Asparagine Availability Is a Critical Limiting Factor for Infectious Spleen and Kidney Necrosis Virus Replication.

Infectious spleen and kidney necrosis virus (ISKNV) has brought huge economic loss to the aquaculture industry. Through interfering with the viral replication and proliferation process that depends on host cells, its pathogenicity can be effectively reduced. In this study, we investigated the role of asparagine metabolites in ISKNV proliferation. The results showed that ISKNV infection up-regulated the expression of some key enzymes of the asparagine metabolic pathway in Chinese perch brain (CPB) cells. These key enzymes, including glutamic oxaloacetic transaminase 1/2 (GOT1/2) and malate dehydrogenase1/2 (MDH1/2) associated with the malate-aspartate shuttle (MAS) pathway and asparagine synthetase (ASNS) involved in the asparagine biosynthesis pathway, were up-regulated during ISKNV replication and release stages. In addition, results showed that the production of ISKNV was significantly reduced by inhibiting the MAS pathway or reducing the expression of ASNS by 1.3-fold and 0.6-fold, respectively, indicating that asparagine was a critical limiting metabolite for ISKNV protein synthesis. Furthermore, when asparagine was added to the medium without glutamine, ISKNV copy number was restored to 92% of that in the complete medium, indicating that ISKNV could be fully rescued from the absence of glutamine by supplementing asparagine. The above results indicated that asparagine was a critical factor in limiting the effective replication of ISKNV, which provided a new idea for the treatment of aquatic viral diseases.

Role of L-glutamine in the regulation of rat Sertoli cell proliferation by FSH.

FSH leads to glutamine dependence, which is required for mTORC1 activation and in consequence Sertoli cell proliferation. The spermatogenic capacity of adult individuals depends on, among other factors, the number of Sertoli cells (SCs) that result from the proliferative waves during development. FSH upregulates SC proliferation at least partly, through the activation of the PI3K/Akt/mTORC1 pathway, among other mechanisms. It is widely known that mTORC1 is a sensor of amino acids. Among amino acids, glutamine acquires relevance since it might contribute to cell cycle progression through the modulation of mTORC1 activity. It has not been studied yet whether glutamine intervenes in FSH-mediated regulation of SC proliferation and cell cycle progression, or if FSH has any effect on glutamine metabolism. Eight-day-old rat SCs were incubated in culture media without glutamine or with glutamine in the absence or presence of a glutamine transporter inhibitor or a glutaminase activity inhibitor under basal conditions or stimulated with FSH. The results obtained show that FSH does not promote SC proliferation and mTORC1 activation in the absence of glutamine. Also, FSH modulates glutamine metabolism increasing glutaminase isoform 2 and reducing glutamine synthetaseexpression. FSH did not promote SC proliferation and mTORC1 activation when glutaminase activity was inhibited. The results suggest that glutamine or its metabolites might cooperate with FSH in the upregulation of SC proliferation through mTORC1. In addition, as FSH modulates glutamine metabolism through the induction of glutaminase isoform 2, the hormonal control of glutamine metabolism might be part of the intricate signaling network triggered by FSH, which is crucial to establish the population of mature SCs that supports the reproductive function.

Mitochondrial adaptation decreases drug sensitivity of persistent triple negative breast cancer cells surviving combinatory and sequential chemotherapy

Triple negative breast cancer (TNBC) is an aggressive malignancy for which chemotherapy remains the standard treatment. However, between 3 and 5 years after chemotherapy, about half patients will relapse and it is essential to identify vulnerabilities of cancer cells surviving neoadujuvant therapy. In this study, we established persistent TNBC cell models after treating MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide, and then with paclitaxel, for a total of 18 weeks. The resulting chemo-persistent cell lines were more proliferative, both in vitro and in xenografted mice. Interestingly, MDA-MB-231 persistent cells became less sensitive to chemotherapeutic drugs, whereas SUM159-PT persistent cells kept similar sensitivity compared to control cells. The reduced sensitivity to chemotherapy in MDA-MB-231 persistent cells was found to be associated with an increased oxidative phosphorylation (OXPHOS) and modified levels of tricarboxylic acid cycle (TCA) intermediates. Integration of data from proteomics and metabolomics demonstrated TCA cycle among the most upregulated pathways in MDA-MB-231 persistent cells. The absence of glucose and pyruvate impeded OXPHOS in persistent cells, while the absence of glutamine did not. In contrast, OXPHOS was not modified in control cells independently of TCA substrates, indicating that MDA-MB-231 persistent cells evolved towards a more pyruvate dependent profile. Finally, the inhibition of pyruvate entry into mitochondria with UK-5099 reduced OXPHOS and re-sensitized persistent cells to therapeutic agents. Together, these findings suggest that targeting mitochondrial pyruvate metabolism may help to overcome mitochondrial adaptation of chemo-persistent TNBC.

MYC sensitises cells to apoptosis by driving energetic demand

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.

Metformin Treatment or PRODH/POX-Knock out Similarly Induces Apoptosis by Reprograming of Amino Acid Metabolism, TCA, Urea Cycle and Pentose Phosphate Pathway in MCF-7 Breast Cancer Cells.

It has been considered that proline dehydrogenase/proline oxidase (PRODH/POX) is involved in antineoplastic activity of metformin (MET). The aim of this study is identification of key metabolites of glycolysis, pentose phosphate pathway (PPP), tricarboxylic acids (TCA), urea cycles (UC) and some amino acids in MET-treated MCF-7 cells and PRODH/POX-knocked out MCF-7 (MCF-7crPOX) cells. MCF-7crPOX cells were generated by using CRISPR-Cas9. Targeted metabolomics was performed by LC-MS/MS/QqQ. Expression of pro-apoptotic proteins was evaluated by Western blot. In the absence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to similar inhibition of glycolysis (drastic increase in intracellular glucose and pyruvate) and increase in the utilization of phospho-enol-pyruvic acid, glucose-6-phosphate and some metabolites of TCA and UC, contributing to apoptosis. However, in the presence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to utilization of some studied metabolites (except glucose), facilitating pro-survival phenotype of MCF-7 cells in these conditions. It suggests that MET treatment or PRODH/POX-knock out induce similar metabolic effects (glucose starvation) and glycolysis is tightly linked to glutamine metabolism in MCF-7 breast cancer cells. The data provide insight into mechanism of anticancer activity of MET as an approach to further studies on experimental breast cancer therapy.

An enhanced mitochondrial function through glutamine metabolism in plasmablast differentiation in systemic lupus erythematosus.

To evaluate the dysfunction of B-cell metabolism and its involvement in SLE pathology. We assessed the expression of metabolic markers of B cells in the peripheral blood of healthy controls (HCs) and SLE patients by using flow cytometry. In vitro, peripheral B cells were isolated from HCs and SLE patients to investigate the metabolic regulation mechanisms involved in their differentiation. The expression level of DiOc6 (mitochondrial membrane hyperpolarization) was higher in B cells from SLE patients than in HCs, and correlated to the percentage of plasmablasts in CD19+ cells and with SLEDAI, a disease activity score. Stimulation of CD19+ cells with the Toll-like receptor 9 (TLR9) ligand CpG and IFN-α enhanced glycolysis, oxidative phosphorylation (OXPHOS), DiOc6 expression, and plasmablast differentiation in vitro. In the absence of glutamine, both glycolysis and OXPHOS were reduced, and plasmablast differentiation was suppressed, whereas there was no change in the absence of glucose. As glutamine is an important nutrient for protein synthesis, we further investigated the effect of the glutaminase inhibitor BPTES, which inhibits glutamine degradation, on metabolic regulation. BPTES reduced DiOc6 expression, OXPHOS, reactive oxygen species (ROS) production, adenosine triphosphate (ATP) production, plasmablast differentiation without affecting glycolysis. Metformin inhibited CpG- and IFN-α-induced glutamine uptake, mitochondrial functions and suppressed plasmablast differentiation. Mitochondrial dysfunction in B cells is associated with plasmablast differentiation and disease activity in SLE. Enhanced mitochondrial functions mediated by glutamine metabolism are important for plasmablast differentiation, which may be a potential therapeutic target for SLE.

NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions

SUMMARYCitrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.

GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri

N-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC–MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.

Abstract 2015: Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic tumor microenvironment

Abstract Solid tumors are characterized by poor vasculature, thereby causing insufficient oxygen and nutrient supply within the tumor. To thrive in such conditions, hypoxic tumor cells stimulate the expression of nutrient transporters to support cell metabolism and pH regulators to buffer the intracellular pH changes caused by metabolic by-products. Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in the pH regulation of hypoxic tumor cells. CAIX is highly expressed in several solid tumors, and is an indicator of poor prognosis and response to therapy. Although the importance of CAIX's role in mediating tumor progression is well-known, the underlying mechanism remains unclear. To identify the interactors of CAIX in the hypoxic tumor microenvironment, we performed a proteomic analysis using proximity dependent biotin identification (BioID) and found the Alanaine, Serine, Cysteine transporter (ASCT2) to be a high confidence interactor of CAIX from this analysis. ASCT2, which predominantly acts as a glutamine transporter is upregulated in several cancers and is shown to support tumor growth by facilitating increased glutamine uptake. In this study, using a panel of solid tumor cell lines, we validate the interaction of CAIX with ASCT2 in the hypoxic microenvironment. Functionally, loss of CAIX causes an increased glutamine uptake via ASCT2 and an increased capacity of cells to utilize glutamine for mitochondrial respiration. Concomitantly, ROS and glutathione (GSH) levels also increase upon CAIX inhibition. Cytotoxicity studies show a robust cell death in the absence of glutamine, following treatment with the CAIX/XII small molecule inhibitor SLC-0111. A similar effect on cell death is observed by blocking glutamine metabolism using inhibitors such as V-9302 (ASCT2 inhibitor) or CB-839 (Glutaminase-1 inhibitor) with SLC-0111. Furthermore, blocking GSH synthesis using BSO (Glutamylcysteine synthatase inhibitor) with SLC-0111 causes significant cell death. These results suggest that CAIX depletion causes increased cellular stress and to overcome this, cells increase glutamine uptake in part for GSH synthesis. In summary, we show that CAIX and ASCT2 coordinate their functions in hypoxic cancer cells to support tumor growth, and co-targeting them could be beneficial in treating solid tumors. Citation Format: Geetha Venkateswaran, Shawn Chafe, Shannon Awrey, Oksana Nemirovsky, Shoukat Dedhar. Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2015.

Abstract 2873: Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions

Abstract For women, breast cancer is the most diagnosed cancer, with metastasis being the primary cause of most breast cancer-related deaths. During metastasis, cells detach from the extracellular matrix (ECM) to move to a secondary site. Unlike normal cells, metastatic cells are resistant to cell death upon ECM detachment. The amino acid glutamine is used as an energy source during metastatic progression via replenishing the TCA cycle. Breast cancer cells have decreased viability in the absence of glutamine, but the effect of glutamine in ECM detached conditions in metastatic cells is not known. We hypothesize that metastatic and non-metastatic cells have a decreased viability in ECM detachment in glutamine deprived conditions. In this study, metastatic MCF10CA1a cells and non-metastatic Harvey-ras oncogene transfected MCF10A-ras cells were employed. As expected, in ECM detached conditions, the MCF10A-ras cells had a greater decrease in viability (58.4%) in comparison to the MCF10CA1a cells (32.7%). However, in glutamine deprived conditions, the MCF10CA1a cells decrease in viability was 15.2% greater compared to the MCF10A-ras cells. mRNA expression of the major glutamine transporter, SLC1A5, was decreased in detached conditions in both cell lines, but to a greater extent in the MCF10A-ras cells (70.04%) compared to the MCF10CA1a cells (20.15%). Additionally, mRNA expression in detached conditions of glutamate dehydrogenase, the enzyme that metabolizes the downstream glutamine metabolite glutamate to α-ketoglutarate (αKG), was decreased by 77% in the non-metastatic cells, but increased by 23% in the metastatic cells. Additionally, mRNA expression of GOT1, a gene which encodes for the transaminase enzyme which synthesizes αKG and aspartate from glutamate and oxaloacetate, decreased 73% in detached conditions in the non-metastatic cells but was unchanged in the metastatic cells. Moreover, following detachment the glutamine synthesizing enzyme, glutamine synthetase mRNA expression was increased by 81.92% in the non-metastatic cells and 88.43% in the metastatic cells. In summary, the greater effect of glutamine depletion on viability, and the maintained expression of glutamine metabolizing enzymes in the metastatic cells suggest greater dependence on glutamine metabolism in ECM detached conditions compared to non-metastatic cells. Comparison of metastatic and non-metastatic breast cancer cells will provide insights to direct the development of new targets for the prevention of breast cancer metastasis. Citation Format: Kanika K. Garg, Madeline Sheeley, Dorothy Teegarden. Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2873.

Asparagine sustains cellular proliferation and c‑Myc expression in glutamine‑starved cancer cells.

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c‑Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c‑Myc protein expression, leading to a reduction in the levels of the canonical c‑Myc isoform and an increase in the expression of c‑Myc1, a c‑Myc isoform translated from an in‑frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c‑Myc expression as in glutamine‑fed cells, with high levels of canonical c‑Myc and c‑Myc1 almost undetectable. In asparagine‑fed cells, global protein translation was higher than in glutamine‑starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine‑starved asparagine‑fed cells, the inhibition of c‑Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c‑Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.

Glucose Favors Lipid Anabolic Metabolism in the Invasive Breast Cancer Cell Line MDA-MB-231.

Metabolic reprogramming in tumor cells is considered one of the hallmarks of cancer. Many studies have been carried out in order to elucidate the effects of tumor cell metabolism on invasion and tumor progression. However, little is known about the immediate substrate preference in tumor cells. In this work, we wanted to study this short-time preference using the highly invasive, hormone independent breast cancer cell line MDA-MB-231. By means of Seahorse and uptake experiments, our results point to a preference for glucose. However, although both glucose and glutamine are required for tumor cell proliferation, MDA-MB-231 cells can survive two days in the absence of glucose, but not in the absence of glutamine. On the other hand, the presence of glucose increased palmitate uptake in this cell line, which accumulates in the cytosol instead of going to the plasma membrane. In order to exert this effect, glucose needs to be converted to glycerol-3 phosphate, leading to palmitate metabolism through lipid synthesis, most likely to the synthesis of triacylglycerides. The effect of glucose on the palmitate uptake was also found in other triple-negative, invasive breast cancer cell lines, but not in the non-invasive ones. The results presented in this work suggest an important and specific role of glucose in lipid biosynthesis in triple-negative breast cancer.

Identification and functional analysis of glutamine transporter in Streptococcus mutans

ABSTRACT Background Streptococcus mutans, a biofilm-forming bacterium, possesses several transporters that function as import/export molecules. Among them, the PII protein family is composed of members that regulate glutamine synthesis in bacterial species. Objective In this study, we characterized the function of the glutamine transporter in S. mutans MT8148. Methods The SMU.732 gene, corresponding to glnP in S. mutans, is homologous to the glutamine transporter gene in Bacillus subtilis. We constructed a glnP-inactivated mutant strain (GEMR) and a complement strain (comp-GEMR) and evaluated their biological functions. Results Growth of GEMR was similar in the presence and absence of glutamine, whereas the growth rates of MT8148 and comp-GEMR were significantly lower in the presence of glutamine as compared to its absence. Furthermore, biofilms formed by MT8148 and comp-GEMR were significantly thicker than that formed by GEMR, while the GEMR strain showed a significantly lower survival rate in an acidic environment than the other strains. Addition of n-phenyl-2-naphthylamine, used to label of the membrane, led to increased fluorescence intensity of MT8148 and GEMR, albeit that was significantly lower in the latter. Conclusions These results suggest that glnP is associated with glutamine transport in S. mutans, especially the import of glutamine involved in biofilm formation.

Asparagine Is a Critical Limiting Metabolite for Vaccinia Virus Protein Synthesis during Glutamine Deprivation.

Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.

1731-P: The Involvement of Glutaminolysis in ß-Cell Differentiation and Its Clinical Application for Type 1 Diabetes

Objective: B cells play a crucial role in type 1 diabetes (1DM). Recently, “Immunometabolism” attract much attention. In this study, we examined the role of mitochondrial function and glutaminolysis in B cell differentiation and its relevance to 1DM. Methods: Peripheral blood mononuclear cells were obtained from healthy controls (HCs) and 1DM patients and assessed by FACS. In addition, CD19+ cells were isolated from HCs and change of mitochondrial function and glutaminolysis were assessed in the absence of glucose or glutamine, by metformin in vitro. Results: Initially, mitochondrial function in B cells from 1DM patients was assessed using DiOc6 as a marker of depolarization-activated mitochondrial membrane. CD24-DiOc6+ cells in IgD-CD27- memory B cell from 1DM patients were significantly higher than those of HCs. Next, we assessed the role of mitochondrial function and glutaminolysis in human B cell differentiation in vitro. Stimulation with CpG (TLR9 ligand) and IFN-α, 1) increased the area of cytoplasm including many expanded mitochondrial cristae with slightly wider and loosely organized intermembrane space in electric microscopy, accompanied with ROS production and DiOc6 up-regulation, 2) induced CD27hiCD38hi plasmablasts differentiation and immunoglobulin production. Interestingly, ROS production and DiOc6 expression were significantly decreased in the absence of glutamine, leading to inhibition of plasmablasts differentiation and immunoglobulin production. This tendency was not shown in the absence of glucose. Metformin, which is known as AMPK activator, abrogated glutamine uptake, resulting in suppression of ROS production, DiOc6 expression, plasmablast differentiation and immunoglobulin production. Conclusion: These results suggest that mitochondrial activation via glutaminolysis may play an important role in the differentiation from IgD-CD27- double negative B cells to plasmablasts and production of immunoglobulins in 1DM patients. Disclosure M. Hajime: None. S. Iwata: None. M. Zhang: None. S. Nakayamada: None. Y. Okada: None. Y. Tanaka: None.

3071 Cell Survival in Corneal Endothelial Dystrophies

OBJECTIVES/SPECIFIC AIMS: Purpose - The goal of this study is to understand how loss of the membrane protein SLC4A11 alters endothelial cell metabolism thereby producing Corneal Endothelial Dystrophy. Studies from our lab indicated that glutamine-dependent mitochondrial dysfunction is one of the outcomes of SLC4a11 loss. In the current study, we ask if autophagy and mitophagy pathways and the signaling pathways that regulate these processes are altered in SLC4a11 KO cells. METHODS/STUDY POPULATION: Methods – Immortalized mouse WT and SLC4a11 KO cell lines were incubated in DMEM with and without 0.5mM glutamine for 6 hours. In order to assess mitophagy, cells were stained using Lysotracker Red and Mitotracker Green. Colocalization co-efficients of red and green channels were obtained for at least 35 cells using Zeiss-Zen Pro software. Student’s t-test was used to determine statistical significance. For Western Blots, antibodies against LC3b, AMPK, pAMPK, and b-actin were used to examine autophagy flux and potential signaling pathways that regulate autophagy. RESULTS/ANTICIPATED RESULTS: Results – In the presence of glutamine, the colocalization co-efficient of Lysotracker Red and Mitotracker Green channels was significantly increased in KO cells (0.74 ±0.18) relative to WT (0.58±0.20) with a p-value ≤0.0024. In the absence of glutamine, the colocalization co-efficient was reversed, for KO cells 0.54 ±0.14 and for WT cells 0.77±0.0.16 with a p-value ≤0.0001, suggesting increased mitophagy by glutamine in KO cells. Western Blots indicated that glutamine increased autophagy flux, as indicated by increased levels of LC3b following bafilomycin A treatment in KO cells. Concomitantly, there was an increase in pAMPK/AMPK levels suggesting a potential mechanism for increased mitophagy. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion and Future studies –Our data indicates enhanced mitophagy as well as autophagy in SLC4a11 KO cells. Future studies will determine whether these processes regulate cell survival in mouse models of corneal endothelial dystrophies.

Glutaminolysis Epigenetically Regulates Antiapoptotic Gene Expression in Idiopathic Pulmonary Fibrosis Fibroblasts.

Fibrotic responses involve multiple cellular processes, including epigenetic changes. Epigenetic changes are sensitive to alterations in the tissue microenvironment such as the flux of tricarboxylic acid (TCA) cycle metabolites. TCA metabolites directly regulate epigenetic states, in part by regulating histone modification-related enzymes. Glutaminolysis is a critical metabolic process by which glutamine is converted to glutamate by glutaminase and then to α-ketoglutarate (α-KG), a TCA cycle metabolite. Idiopathic pulmonary fibrosis (IPF) is a disease characterized by aberrant metabolism, including enhanced glutaminolysis. IPF fibroblasts are apoptosis resistant. In this study, we explored the relationship between glutaminolysis and the resistance to apoptosis of IPF fibroblasts. Inhibition of glutaminolysis decreased expression of XIAP and survivin, members of the inhibitor of apoptosis protein (IAP) family. α-KG is a cofactor for JMJD3 histone demethylase, which targets H3K27me3. In the absence of glutamine, JMJD3 activity in fibroblasts is significantly decreased, whereas H3K27me3 levels are increased. Chromatin immunoprecipitation assays confirmed that JMJD3 directly interacts with XIAP and survivin promoter regions in a glutamine-dependent manner. Exogenous α-KG partially restores JMJD3 function and its interaction with the XIAP and survivin promoter regions under glutamine-deficient conditions. Interestingly, α-KG upregulates XIAP, but not survivin, suggesting differential α-KG-dependent and -independent mechanisms by which glutamine regulates these IAPs. Our data demonstrate a novel mechanism of metabolic regulation in which glutaminolysis promotes apoptosis resistance of IPF fibroblasts through epigenetic regulation of XIAP and survivin.

Glutaminase-1 stimulates the proliferation, migration, and survival of human endothelial cells

Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G0/G1 phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.

Abstract 1439: Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a syndrome characterized by leiomyomas and an aggressive form of papillary kidney cancer. The genetic driver of HLRCC is a loss-of-function mutation of the Krebs cycle enzyme fumarate hydratase (FH), resulting in increased levels of the Krebs cycle intermediate fumarate, and resulting impaired oxidative phosphorylation. as well as in stabilization of HIF1α protein, which is the final common pathway of many different types of renal cell carcinomas (RCCs). To better understand the energy sources utilized by FH-deficient cells, we examined the nutrient requirements of FH-deficient RCC UOK262 (UOK262D) cell line in comparison to UOK262 cells transfected with an FH transgene (UOK262R). Experiments with selective addition of nonessential amino acids (NEAA) from the media revealed that both UOK262D and UOK262R cell lines required asparagine (A), but not other NEAA, for growth after 96 hours of incubation in the absence of glutamine (G). A (100 uM) and G (2 mM) demonstrated an approximately equal effect on the growth rate of both FH deficient and repleted cells, which was only observed after a minimum of 96 hrs of incubation. The growth effects of A and G were additive in both cell lines. While G increased the oxygen consumption rate (OCR) and decreased glycolysis (ECAR) in both cell lines, addition of A had no additive effect on both parameters, suggesting that G is used for energy production and A is required for biomass production. Similar effects of A and G, alone and in combination, on proliferation were seen in FH deficient and repleted UOK268 cell lines and in the SDH deficient and repleted UOK269 cells. Both A and G activated the mTOR pathway in both UOK262D and UOK262R cell lines as indicated by increased phosphorylation of mTOR, S6K and S6RP at 48h. However, mTOR pathway activation was not observed after A and/or G exposure in UOK268 or UOK269 pairs of cell lines. To determine the genomic response of UOK262 cell lines to A and/or G, we conducted RNASeq analysis in cells incubated for 96 hours to A, G, both or neither. In UOK262D cells, A+G, but not A or G individually, increased RNA levels of many genes associated with endoplasmic reticulum stress response. These gene expression changes were not seen in the FH repleted cell line UOK262R incubated in A+G. Therefore, although the growth effects of A+G in UOK262R and UOK262D cells were similar, and the effects on mTOR signaling pathway effects were similar between the two cell lines, the RNASeq experiment revealed a fundamentally different response to the combination of the 2 amino acids in the FH deficient cells vs. the FH repleted cells. In summary, we have identified asparagine as a key nutrient in HLRCC, establishing it as a potential target for a metabolically rational treatment approach. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH. Citation Format: Rony Panarsky, Youfeng Yang, W. Marston Linehan, Jeffrey A. Moscow. Asparagine stimulates growth and endoplasmic reticulum stress response in a fumarate hydratase-deficient renal cell carcinoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1439.

Glutamine-utilizing transaminases are a metabolic vulnerability of TAZ/YAP-activated cancer cells.

The transcriptional regulators TAZ and YAP (TAZ/YAP) have emerged as pro-tumorigenic factors that drive many oncogenic traits, including induction of cell growth, resistance to cell death, and activation of processes that promote migration and invasion. Here, we report that TAZ/YAP reprogram cellular energetics to promote the dependence of breast cancer cell growth on exogenous glutamine. Rescue experiments with glutamine-derived metabolites suggest an essential role for glutamate and α-ketoglutarate (AKG) in TAZ/YAP-driven cell growth in the absence of glutamine. Analysis of enzymes that mediate the conversion of glutamate to AKG shows that TAZ/YAP induce glutamic-oxaloacetic transaminase (GOT1) and phosphoserine aminotransferase (PSAT1) expression and that TAZ/YAP activity positively correlates with transaminase expression in breast cancer patients. Notably, we find that the transaminase inhibitor aminooxyacetate (AOA) represses cell growth in a TAZ/YAP-dependent manner, identifying transamination as a potential vulnerable metabolic requirement for TAZ/YAP-driven breast cancer.