GLUL Expression Research Articles

The Aging Bone Marrow Niche Disrupts Glutamine Metabolism By Upregulating HSC Derived Glutamine Synthetase

As our aging population grows it is becoming increasingly important to better understand the physiological changes that drive that drive the aging process. Aging has long been known to drive haematopoietic stem cell (HSCs) dysfunction and therefore has been implicated in promoting the development of age-related haematopoietic diseases, such as leukaemia and myeloma. It is likely that a combination of intrinsic changes within the HSCs and extrinsic changes within the bone marrow (BM) microenvironment contribute to HSC aging. We have previously shown that the bone marrow niche accumulates senescent cells which contribute to impaired HSC response to stress 1. Here we investigate the intrinsic changes that can be observed in aged HSCs and how these are influenced by the bone marrow niche. In order to investigate the intrinsic changes that drive metabolic HSC aging we BM was isolated from young (8-12 weeks) and aged (18-24 months). Single HSCs were then FACS purified and sequenced using the SmartSeq2 protocol. Three transcriptionally distinct populations were identified, HSCs from young mice (Y), HSCs from aged mice with an aging transcriptional signature (O) and HSCs from aged mice that are transcriptionally similar to HSCs from young mice (OY). Assessment of differentially expressed genes between these three populations revealed that glutamine synthetase ( glul) is significantly upregulated in the O population compared to both the Y and OY population. Glul is responsible for the conversion of glutamate to glutamine and therefore levels of both were next measured in FACS purified HSCs from aged and young mice. Results show that whilst there was no difference in glutamate levels, the glutamine levels were significantly reduced in HSCs from aged mice. These data suggest that glutamine metabolism is impaired in aged HSC. Next, to determine the impact of the aged BM niche on glul expression in aged HSCs, HSCs from young and aged PepCboy (CD45.1+) mice were FACS purified and adoptively transferred into young C57Bl/6 mice. After 12 weeks the mice were sacrificed and engraftment of CD45.1+ cells was confirmed by flow cytometry. In addition CD45.1+ HSCs were FACS purified to assess glul expression and measure glutamine and glutamate levels. Results confirm that the mechanism for changes in glul expression and glutamine levels is driven by the aging BM niche and transplantation into a young niche reverses this change. To understand the importance of dysregulated glutamine metabolism in aged humans we collected BM from older patients undergoing elective hip replacement surgeries. All samples were collected following informed consent and under approval of the UK NHS Health Research Authority (ref07/H0310/146). CD34+ cells were isolated and RNA extracted to measure glul expression. Similarly, to data from aged mice glul expression was elevated in aged CD34+ cells compared to young CD34+ controls. Furthermore, glutamine levels were also low in aged CD34+ cells. This suggests that the changes in HSCs from aged mice correlate with finding in human stem cells and mice are therefore a suitable species to study dysregulated glutamine metabolism in aged HSCs. Together this work demonstrates that aging causes a disruption of glutamine and glutamate metabolism in HSCs. The cause for this disruption and its direct consequences on overall HSC metabolism remains to be explored. However, it is likely that these metabolic changes will have a knock-on effect on HSC function and may elucidate some of the drivers of metabolic HSC aging.

GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progresses of gastric cancer

Glutamate-ammonia ligase (GLUL, also known as glutamine synthetase) is a crucial enzyme that catalyzes ammonium and glutamate into glutamine in the ATP-dependent condensation. Although GLUL plays a critical role in multiple cancers, the expression and function of GLUL in gastric cancer remain unclear. In the present study, we have found that the expression level of GLUL was significantly lower in gastric cancer tissues compared with adjacent normal tissues, and correlated with N stage and TNM stage, and low GLUL expression predicted poor survival for gastric cancer patients. Knockdown of GLUL promoted the growth, migration, invasion and metastasis of gastric cancer cells in vitro and in vivo, and vice versa, which was independent of its enzyme activity. Mechanistically, GLUL competed with β-Catenin to bind to N-Cadherin, increased the stability of N-Cadherin and decreased the stability of β-Catenin by alerting their ubiquitination. Furthermore, there were lower N-Cadherin and higher β-Catenin expression levels in gastric cancer tissues compared with adjacent normal tissues. GLUL protein expression was correlated with that of N-Cadherin, and could be the independent prognostic factor in gastric cancer. Our findings reveal that GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progress of gastric cancer.

Targeted metabolomics analysis identified the role of FOXA1 in the change in glutamate-glutamine metabolic pattern of BaP malignantly transformed 16HBE cells

The carcinogenic mechanism of benzo[a]pyrene (BaP) is far from being elucidated. FOXA1 has been confirmed to play an oncogenic role in BaP-transformed cell THBEc1. To explore the changes in amino acid metabolic patterns, especially glutamate-glutamine (Glu-Gln) metabolic pattern caused by BaP-induced transformation and the possible role FOXA1 might play in it, we compared amino acid metabolic characteristics between THBEc1 cells and control 16HBE cells using a targeted metabolomics method and determined the effects of FOXA1 knockout on the amino acid metabolic pattern using FOXA1 knockout cell THBEc1-ΔFOXA1-c34. The amino acid metabolic patterns of THBEc1 and 16HBE cells were different, which was manifested by the differential consumption of 18 amino acids and the difference in the intracellular content of 21 amino acids. The consumption and intracellular content of Glu and Gln are different between the two types of cells, accompanied by upregulation of FOXA1, GLUL, SLC1A3, SLC1A4, SLC1A5 and SLC6A14, and downregulation of FOXA2 and GPT2 in THBEc1 cells. FOXA1 knockout changed the consumption of 19 amino acids and the intracellular content of 21 amino acids and reversed the metabolic pattern of Glu and the changes in FOXA2, GLUL, SLC1A3 and SLC6A14 in THBEc1 cells. Additionally, FOXA1 knockout inhibited cell proliferation and further increased the dependence of THBEc1 cells on Glu. In conclusion, FOXA1 knockout partially reversed the change in Glu-Gln metabolism caused by BaP-induced transformation by upregulating the expression of GLUL and SLC1A3. Our findings provide a clue for the possible role of FOXA1 in amino acid metabolism regulation.

Single-cell and spatial analyses reveal the association between gene expression of glutamine synthetase with the immunosuppressive phenotype of APOE+CTSZ+TAM in cancers.

An immunosuppressive state is regulated by various factors in the tumor microenvironment (TME), including, but not limited to, metabolic plasticity of immunosuppressive cells and cytokines secreted by these cells. We used single-cell RNA-sequencing (scRNA-seq) data and applied single-cell flux estimation analysis to characterize the link between metabolism and cellular function within the hypoxic TME of colorectal (CRC) and lung cancer. In terms of metabolic heterogeneity, we found myeloid cells potentially inclined to accumulate glutamine but tumor cells inclined to accumulate glutamate. In particular, we uncovered a tumor-associated macrophage (TAM) subpopulation, APOE+CTSZ+TAM, that was present in high proportions in tumor samples and exhibited immunosuppressive characteristics through upregulating the expression of anti-inflammatory genes. The proportion of APOE+CTSZ+TAM and regulatory T cells (Treg) were positively correlated across CRC scRNA-seq samples. APOE+CTSZ+TAM potentially interacted with Treg via CXCL16-CCR6 signals, as seen by ligand-receptor interactions analysis. Notably, glutamate-to-glutamine metabolic flux score and glutamine synthetase (GLUL) expression were uniquely higher in APOE+CTSZ+TAM, compared with other cell types within the TME. GLUL expression in macrophages was positively correlated with anti-inflammatory score and was higher in high-grade and invasive tumor samples. Moreover, spatial transcriptome and multiplex immunofluorescence staining of samples showed that APOE+CTSZ+TAM and Treg potentially colocalized in the tissue sections from CRC clinical samples. These results highlight the specific role and metabolic characteristic of the APOE+CTSZ+TAM subpopulation and provide a new perspective for macrophage subcluster-targeted therapeutic interventions or metabolic checkpoint-based cancer therapies.

Large-scale analysis of circulating glutamate and adipose gene expression in relation to abdominal obesity.

Circulating levels of the amino acid glutamate are associated with central fat accumulation, yet the pathophysiology of this relationship remains unknown. We aimed to (i) refine and validate the association between circulating glutamate and abdominal obesity in a large twin cohort, and (ii) investigate whether transcriptomic profiles in adipose tissue could provide insight into the biological mechanisms underlying the association. First, in a cohort of 4665 individuals from the TwinsUK resource, we identified individuals with abdominal obesity and compared prevalence of the latter across circulating glutamate quintiles. Second, we used transcriptomic signatures generated from adipose tissue, both subcutaneous and visceral, to investigate associations with circulating glutamate levels. Individuals in the top circulating glutamate quintile had a sevenfold higher prevalence of abdominal obesity compared to those in the bottom quintile. The adipose tissue transcriptomic analyses identified GLUL, encoding Glutamate-Ammonia Ligase, as being associated with circulating glutamate and abdominal obesity, with pronounced signatures in the visceral depot. In conclusion,circulating glutamate is positively associated with the prevalence of abdominal obesity which relates to dysregulated GLUL expression specifically in visceral adipose tissue.

Glutamine synthetase in human carotid plaque macrophages associates with features of plaque vulnerability: An immunohistological study

Background and aimsGlutamine synthetase (GLUL), the sole generator of glutamine, is a metabolic nexus molecule also involved in atherosclerosis. We recently demonstrated a 2.2-fold upregulation of GLUL mRNA in stroke-causing carotid plaques when compared with plaques from asymptomatic patients. Here we compared in the same cohort GLUL mRNA expression with plaque gross morphology, and the colocalization of immunodetectable GLUL protein with histopathological changes and molecular and mechanical mediators linked to plaque development. MethodsEndarterectomy specimens from 19 asymptomatic and 24 stroke patients were sectioned longitudinally and immunostained for GLUL, CD68, α-smooth muscle actin, iron, heme oxygenase-1 and CD163, and graded semiquantitatively in every 1 mm2. The amounts of cholesterol clefts and erythrocytes were graded. The fibrous cap thickness within each 1 mm2 area was measured. The association between the local pathological findings was analyzed by a hierarchical mixed modelling approach. ResultsThe previously found correlation between GLUL mRNA and clinical symptomatology was supported by the increased GLUL mRNA in diseased tissue and increased local GLUL immunoreactivity in areas with multiple different atherosclerotic changes. A longer symptom-to-operation time correlated with lower GLUL mRNA (Rs = −0.423, p=0.050) but few outliers had a significantly higher GLUL mRNA levels, which persisted throughout the post-symptomatic period. Plaque ulceration associated with 1.8-fold higher GLUL mRNA (p=0.006). Macrophages were the main GLUL immunoreactive cells. GLUL immunostaining colocalized with erythrocytes, iron, CD163, and heme oxygenase-1. The correlations between local variables were consistent in both asymptomatic and stroke-causing plaques. An inverse correlation was found between the fibrous cap thickness and local GLUL immunoreactivity (p=0.012). Considerable variability in interplaque expression pattern of GLUL was present. ConclusionsOur results link connect macrophage GLUL expression with carotid plaque features characterizing plaque vulnerability.

Maintenance of glutamine synthetase expression alleviates endotoxin-induced sepsis via alpha-ketoglutarate-mediated demethylation.

Glutamine synthetase (Glul) is the enzyme that synthesizes endogenous glutamine, which is responsible for critical metabolic pathways and the immune system. However, the role of Glul in regulating endotoxin (lipopolysaccharide, LPS)-induced sepsis remains unclear. Here, we found that Glul expression in macrophages was significantly inhibited in endotoxemia, and that Glul deletion induced macrophages to differentiate into the pro-inflammatory type and aggravated sepsis in mice. Mechanistically, TLR4/NF-κB-induced alpha-ketoglutarate (α-KG) depletion inhibits Glul expression through H3K27me3-mediated methylation in septic mice. Both Glul overexpression with adeno-associated virus (AAV) and restoration by replenishing α-KG can alleviate the severity of sepsis. In conclusion, the study demonstrated that Glul can regulate LPS-induced sepsis and provides a novel strategy for the treatment of this disease.

Hepatoblastoma: glutamine depletion hinders cell viability in the embryonal subtype but high GLUL expression is associated with better overall survival

PurposeGlutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival.MethodsWe correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations.ResultsHigh GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival.ConclusionGrowth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

Abstract 2466: Stochastic loss of GLUL expression correlates with STK11-loss-dependent glutamine addiction and may impact anti-PD1 therapy resistance in NSCLC

Abstract Our work aims to delineate the molecular interactions linking somatic disruption of Serine-Threonine Kinase 11 (STK11) with anti-PD1 therapy resistance in KRAS-driven lung adenocarcinoma (LUAD). Data presented here suggests glutamine synthetase (GLUL) may modulate this phenotype. Immune checkpoint inhibitors (ICIs), including anti-PD1 monoclonal antibody therapy, represent a promising therapeutic option for non-small cell lung cancer (NSCLC) patients. Unfortunately, efficient use of these drugs is hampered by an inability to accurately predict which patients will respond. Clinical observations have correlated anti-PD1 therapy resistance in KRAS-driven LUAD with somatic STK11 disruption, but the mechanism(s) responsible remains unknown. To investigate this connection we knocked out STK11 in three different human KRAS-driven LUAD cell lines and performed RNA-seq analysis to identify STK11-loss-dependent differentially expressed genes. Intriguingly, GLUL expression was reproducibly lost in a subset of KRAS-driven/STK11 null clones across two different human lung adenocarcinoma cell lines. GLUL is an enzyme that converts glutamate to glutamine. Glutamine is a critical metabolite supplying carbon and nitrogen necessary for amino acid, nucleotide, and lipid synthesis. Additionally, glutamine plays an important role in redox homeostasis, chromatin organization, and intra-tumoral signaling. Glutamine dependency is a common phenotype across many solid tumor types, and has previously been linked with STK11-deficiency in the context of polycystic kidney disease. Our current work suggests loss of GLUL expression correlates with an altered tumor-intrinsic response to nutrient stress in KRAS-driven LUAD culminating in immune evasion and anti-PD1 therapy resistance. Ongoing experiments aimed to define the relationship between STK11 loss and GLUL expression suggest this phenomenon is stochastic in nature and may provide tumor cells with a selective advantage. In conclusion, understanding the biology underlying the molecular connection between STK11 and GLUL may reveal reliable biomarkers for anti-PD1 therapy response and suggest a method for restoring sensitivity to current therapies. Citation Format: Sean M. Lenahan, Hailey M. Sarausky, David Joseph Seward. Stochastic loss of GLUL expression correlates with STK11-loss-dependent glutamine addiction and may impact anti-PD1 therapy resistance in NSCLC [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 2466.

Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

Abstract A09: The role of asparagine in glutamine-independent proliferation of breast cancer cells

Abstract The majority of proliferating cells utilize the carbon backbone of glutamine as a major anaplerotic input into the TCA cycle. In addition, glutamine serves as a donor of reduced nitrogen for the biosynthesis of purine and pyrimidine nucleotides, as well as for a number of non-essential amino acids. Thus, cells rely on external sources of glutamine for biomass accumulation and growth. However, recent data indicate that some cancer cell types – luminal breast cancer lines in particular – are able to proliferate in the absence of exogenous glutamine. Furthermore, luminal breast cancer lines display increased expression of GLUL (glutamine synthetase) and reduced expression of GLS1 (glutaminase), when compared to basal subtype. We sought to identify key metabolic determinants of glutamine-independent proliferation. As our preliminary data indicate, the presence of amino acid asparagine is required for the proliferation of luminal breast cancer cells as well as normal breast epithelial cells in the setting of glutamine deprivation. Both asparagine and glutamine contain amide groups in their respective side chains and are thus structurally similar to each other. However, most normal mammalian tissues – with the exception of the liver and the kidney – are unable to catabolize asparagine, making it essentially metabolically inert for the majority of cell types. Our group has previously shown that asparagine suppresses cell death in cells that depend on glutamine for survival. In this study, we attempt to elucidate the mechanism by which asparagine facilitates the biomass accumulation and proliferation of glutamine-independent cell types. First, we investigate whether luminal breast cancer cells may possess an ability to catabolize asparagine in a manner similar to glutaminolysis and thus use it as an alternative source of reduced nitrogen and carbon in a setting of glutamine deprivation. Second, we explore a potential regulatory metabolic function of asparagine, whereby it may regulate cellular glutamine synthesis and thus enable proliferation in conditions of glutamine deprivation. Citation Format: Natalya N. Pavlova, Craig B. Thompson. The role of asparagine in glutamine-independent proliferation of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A09.

Glioblastoma cells induce differential glutamatergic gene expressions in human tumor-associated microglia/macrophages and monocyte-derived macrophages

Glioblastoma cells produce and release high amounts of glutamate into the extracellular milieu and subsequently can trigger seizure in patients. Tumor-associated microglia/macrophages (TAMs), consisting of both parenchymal microglia and monocytes-derived macrophages (MDMs) recruited from the blood, are known to populate up to 1/3 of the glioblastoma tumor environment and exhibit an alternative, tumor-promoting and supporting phenotype. However, it is unknown how TAMs respond to the excess extracellular glutamate in the glioblastoma microenvironment. We investigated the expressions of genes related to glutamate transport and metabolism in human TAMs freshly isolated from glioblastoma resections. Quantitative real-time PCR analysis showed (i) significant increases in the expressions of GRIA2 (GluA2 or AMPA receptor 2), SLC1A2 (EAAT2), SLC1A3 (EAAT1), (ii) a near-significant decrease in the expression of SLC7A11 (cystine-glutamate antiporter xCT) and (iii) a remarkable increase in GLUL expression (glutamine synthetase) in these cells compared to adult primary human microglia. TAMs co-cultured with glioblastoma cells also exhibited a similar glutamatergic profile as freshly isolated TAMs except for a slight increase in SLC7A11 expression. We next analyzed these genes expressions in cultured human MDMs derived from peripheral blood monocytes for comparison. In contrast, MDMs co-cultured with glioblastoma cells compared to MDMs co-cultured with normal astrocytes exhibited decreased expressions in the tested genes except for GLUL. This is the first study to demonstrate transcriptional changes in glutamatergic signaling of TAMs in a glioblastoma microenvironment, and the findings here suggest that TAMs and MDMs might potentially elicit different cellular responses in the presence of excess extracellular glutamate.

Abstract 1416: CB-839, a novel potent and selective glutaminase inhibitor, has broad antiproliferative activity in cell lines derived from both solid tumors and hematological malignancies

Abstract Many tumor cells utilize altered metabolic pathways to meet the bioenergetic and biosynthetic demands of rapid and sustained growth. One of the key nutrients that fuels tumor growth is the amino acid glutamine. It has long been recognized that the growth and survival of many tumor cell lines in vitro is dependent on extracellular glutamine. A critical step in the utilization of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase. Glutamate and glutamate-derived metabolites in turn support a number of crucial cellular pathways including the citric acid cycle, redox balance and amino acid synthesis. CB-839 is a novel and selective inhibitor of glutaminase that has antitumor activity in preclinical models of triple negative breast cancer (TNBC), a tumor type that is particularly dependent on glutamine. Across a panel of breast cancer cell lines, the activity of CB-839 correlates with high expression of glutaminase, specifically the GAC splice variant but not the KGA splice variant, and low expression of glutamine synthetase (GLUL), an enzyme that converts glutamate to glutamine. This expression pattern is found in primary TNBC tumors suggesting a reliance on exogenous glutamine and glutaminase activity in vivo. To determine if other tumor types have a similar expression pattern, we undertook a systematic evaluation of GAC, KGA and GLUL expression across a diverse set of primary tumors using a normalized microarray dataset allowing comparison across a range of tumor and normal tissue samples. Elevated GAC and decreased GLUL expression relative to other tumor types or corresponding normal tissue was identified in a number of tumor types including non-small cell lung cancer, multiple myeloma, and non-Hodgkin's lymphoma. To explore whether this expression pattern predicts a reliance on glutamine and glutaminase, we tested the glutamine dependence and the activity of CB-839 on a panel of 72 cell lines representing 5 tumor types indicated by the primary tumor expression analysis. Across this cell line panel, the majority were dependent on glutamine showing either cell death, growth arrest or slowed growth after glutamine withdrawal. Similarly, CB-839 had antiproliferative activity in the majority of cell lines with IC50s in the range of 10-300 nM. Importantly, there was a strong correlation between glutamine dependence and response to 1 µM CB-839 as measured by relative cell growth or death (correlation coefficient 0.72, p<0.0001), suggesting that the glutamine requirement for tumor cell growth or survival is mediated by glutaminase and that this phenotype is consistent across numerous tumor types. These data suggest that CB-839 may have therapeutic activity in a range of tumor types that are glutamine dependent and motivate the clinical evaluation of this compound targeting a novel tumor survival pathway. Citation Format: Francesco Parlati, Susan D. Demo, Matthew I. Gross, Julie R. Janes, Evan R. Lewis, Andy L. MacKinnon, Mirna L.M. Rodriguez, Peter J. Shwonek, Taotao Wang, Jinfu Yang, Dong Zhang, Frances Zhao, Mark K. Bennett. CB-839, a novel potent and selective glutaminase inhibitor, has broad antiproliferative activity in cell lines derived from both solid tumors and hematological malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1416. doi:10.1158/1538-7445.AM2014-1416

Abstract P2-09-03: Antitumor activity of the glutaminase inhibitor, CB-839, in triple-negative breast cancer

Abstract Glutamine (Gln) serves as an important source of energy and building blocks for many tumor cells. The first step in Gln utilization is its conversion to glutamate (Glu) by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 inhibits recombinant GAC with an IC50 of <15 nM. Treatment of the triple-negative breast cancer (TNBC) cell line MDA-MB-231 in vitro with CB-839 reduced cell viability and suppressed cellular Glu pools with comparable potency (IC50 ∼15 nM). TNBC breast cancer cell lines have been reported to have a heightened dependence on exogenous Gln for growth relative to estrogen receptor (ER) positive cells (Kung et al, 2011). Consistent with this observation, an analysis of 58 breast cancer cell lines in the Cancer Cell Line Encyclopedia revealed that low ER and/or Her2 expression is correlated with high glutaminase expression (particularly the GAC isoform) and low expression of glutamine synthetase (GLUL), an enzyme that converts Glu to Gln. This reciprocal pattern of GAC and GLUL expression suggests that TNBC may be particularly sensitive to glutaminase inhibition. To test this hypothesis, the anti-tumor activity of CB-839 was evaluated on a panel of breast tumor cell lines (n>25) that included a mixture of TNBC and ER/Her2-positive subtypes. The TNBC subtype displayed the greatest sensitivity to CB-839 treatment (IC50s ranging from 5-100 nM) and this sensitivity was correlated with: i) dependence on extracellular Gln for growth (r = 0.83, p<0.0001); ii) GAC (but not KGA) expression (r = -0.76, p<0.0001); iii) glutaminase activity in cell lysates (r = -0.75, p = 0.0002); and iv) the ratio of cellular Glu to Gln (r = 0.53, p = 0.014) indicative of efficient Gln to Glu conversion. CB-839 also displayed significant anti-tumor activity in two TNBC xenograft models: 1) as a single agent in a patient-derived tumor chosen based on Gln utilization biomarkers and 2) in a cell line model (JIMT1) both as a single agent and in combination with paclitaxel. To determine if potential markers of Gln utilization and CB-839 sensitivity are reflected in primary tumors, an analysis of large mRNA expression and metabolomic datasets was undertaken. Consistent with the observations in cell lines, there was significantly elevated expression of GAC and reduced expression of GLUL in TNBC tumors relative to the ER+ population in The Cancer Genome Atlas mRNA expression dataset (n = 756). Likewise, analysis of the metabolite levels in 262 primary breast tumors showed that the Glu to Gln ratio in the ER- populations was significantly elevated relative to the ER+ population (fold increase = 3.2, p = 8.0E-09). This elevated metabolite ratio in the ER- population was also significantly correlated with the mRNA expression level of GLUL (r = -0.27, p = 0.001). These observations suggest that biomarkers of Gln utilization and CB-839 sensitivity extend to primary TNBC tumors. Together with the in vitro and in vivo preclinical anti-tumor activity of CB-839 in TNBC models, this primary tumor biomarker evaluation provides a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in TNBC patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-03.

Antitumor Activity Of The Glutaminase Inhibitor CB-839 In Hematological Malignances

Many tumor cells are dependent on glutamine (Gln) for growth and survival. Gln is the most abundant amino acid in plasma and tumor cells can utilize Gln for energy production and generation of building blocks for the synthesis of macromolecules. A key step in Gln utilization is its conversion to glutamate (Glu) by the mitochondrial enzyme glutaminase (GLS). Suppressing GLS activity via either siRNA knockdown or small molecule inhibitor treatment has anti-proliferative activity in several tumor types. GLS mRNA levels are elevated in several hematological malignances, particularly those derived from the B-cell lineage including multiple myeloma (MM), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and several types of non-Hodgkin's lymphoma (NHL). Interestingly, these B-cell derived tumors express low levels of glutamine synthetase (GLUL), an enzyme that reverses the action of GLS by converting Glu to Gln. A high GLS/low GLUL mRNA expression pattern may be indicative of tumor-dependence on extracellular Gln for growth. This expression pattern is observed in triple negative breast cancer (TNBC), a tumor type that we and others have shown to be highly dependent on exogenous Gln for cell proliferation and sensitive to GLS inhibitor treatment. CB-839 is a novel, potent, orally bioavailable GLS inhibitor that inhibits recombinant human GLS with an IC50 of 15 nM. The anti-proliferative activity of CB-839 on a panel of breast cell lines is correlated with a high ratio of GLS to GLUL expression, a pattern associated with the TNBC subtype. Given the similarity of the GLS/GLUL expression pattern observed in TNBC with some hematologic malignancies, we evaluated the anti-tumor activity of CB-839 in cell culture and xenograft models of hematological malignancies predicted to be dependent on Gln for growth. Across a panel of 18 cell lines derived from MM, ALL and diffuse large B-cell lymphoma (DLBCL), Gln deprivation resulted in a minimum of 70% reduction in growth for 16 cell lines and induced cell death in 5 cell lines. Across the same panel of cell lines, the majority were sensitive to CB-839 treatment with 16 cell lines exhibiting an antiproliferative IC50 of<100nM (range 2-72 nM). Importantly, there was a strong correlation between sensitivity to Gln withdrawal and CB-839 sensitivity (r=0.63, p=0.005), suggesting that the dominant pathway by which Gln supports tumor cell growth and survival is through its conversation to Glu by GLS. Consistent with this notion, CB-839 treatment caused a decrease in the intracellular pool of Glu and an increase in the intracellular pool of Gln. The extent of decrease in Glu was highly correlated with the effect of CB-839 on cell proliferation (r=-0.64, p=0.006). The in vivo antitumor activity of CB-839 was also evaluated in a xenograft model using the multiple myeloma RPMI-8226 model. Oral, twice daily dosing of CB-839 resulted in a significant decrease in tumor volume (70% tumor growth inhibition). As observed in vitro, CB-839 dosing caused an increase in tumor Gln levels and a decrease in the level of tumor Glu. In a dose dependent fashion, there was a correlation between tumor Gln accumulation, tumor GLS inhibition and antitumor activity. Efficacious doses of CB-839 were well tolerated with no effect on hematological cell counts or body weight. These data suggest that GLS inhibition with CB-839 may provide therapeutic benefit in hematological malignances and motivate the evaluation of this compound in clinical trials that include patients with B-cell malignancies. Disclosures:Parlati:Calithera BIosciences: Employment. Bromley-Dulfano:Calithera Biosciences: Employment. Demo:Calithera Biosciences: Employment. Janes:Calithera Biosciences: Employment. Gross:Calithera Biosciences: Employment. Lewis:Calithera Biosciences: Employment. MacKinnon:Calithera Biosciences: Employment. Rodriguez:Calithera Biosciences: Employment. Yang:Calithera Biosciences: Employment. Zhao:Calithera Biosciences: Employment. Bennett:Calithera Biosciences: Employment.

Gene expression deficits in pontine locus coeruleus astrocytes in men with major depressive disorder

Norepinephrine and glutamate are among several neurotransmitters implicated in the neuropathology of major depressive disorder (MDD). Glia deficits have also been demonstrated in people with MDD, and glia are critical modulators of central glutamatergic transmission. We studied glia in men with MDD in the region of the brain (locus coeruleus; LC) where noradrenergic neuronal cell bodies reside and receive glutamatergic input. The expression of 3 glutamate-related genes (SLC1A3, SLC1A2, GLUL) concentrated in glia and a glia gene (GFAP) were measured in postmortem tissues from men with MDD and from paired psychiatrically healthy controls. Initial gene expression analysis of RNA isolated from homogenized tissue (n = 9-10 pairs) containing the LC were followed by detailed analysis of gene expressions in astrocytes and oligodendrocytes (n = 6-7 pairs) laser captured from the LC region. We assessed protein changes in GFAP using immunohistochemistry and immunoblotting (n = 7-14 pairs). Astrocytes, but not oligodendrocytes, demonstrated robust reductions in the expression of SLC1A3 and SLC1A2, whereas GLUL expression was unchanged. GFAP expression was lower in astrocytes, and we confirmed reduced GFAP protein in the LC using immunostaining methods. Reduced expression of protein products of SLC1A3 and SLC1A2 could not be confirmed because of insufficient amounts of LC tissue for these assays. Whether gene expression abnormalities were associated with only MDD and not with suicide could not be confirmed because most of the decedents who had MDD died by suicide. Major depressive disorder is associated with unhealthy astrocytes in the noradrenergic LC, characterized here by a reduction in astrocyte glutamate transporter expression. These findings suggest that increased glutamatergic activity in the LC occurs in men with MDD.

In vitro differentiation of cultured human CD34+ cells into astrocytes

Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbecco's modified eagle's medium. Thus, cultured cells were induced with astrocyte defined medium (ADM) and in the differentiated astrocytes serine/threonine protein kinases (STPK) and glutamine synthetase (GLUL) activities were estimated. The expression of glial fibrillary acidic protein (GFAP) and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb) and GFAP (2.9 kb) in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.

Abstract P4-06-10: Epigenetic silencing of glutamine synthetase (Glul) defines glutamine depletion therapy

Abstract Background: Methylation-dependent transcriptional silencing of genes involved in amino acid synthesis can provide potential targets for novel synthetic lethality strategies. Glutamine synthetase (Glul) is the key enzyme in the biosynthesis of glutamine. We identified Glul as a novel gene subject to methylation-dependent transcriptional silencing in breast cancer cell lines using a combined functional screen with methylation reversal assays and methylation arrays. Methods: Methylation reversal assays were performed using 5-aza-2-deoxycytidine and/or trichostatin treatment coupled with whole genome mRNA microarrays (Illumina HT-12 v4 Expression BeadChip Kit). Expression of Glul with and without pharmacological methylation reversal with azacytidine and/or trichostatin was validated using qRT-PCR and Western Blot. Methylation of Glul was analysed using methylation microarrays (Illumina 450K Methylation BeadChip), bisulphite sequencing and pyrosequencing. Sensitivity to glutamine deprivation was assessed using an MTT assay after culturing cells in media with various glutamine concentrations or in complete absence of glutamine. We used a panel of 55 breast cancer cell lines and formalin-fixed paraffin-embedded tissue from a series of 116 stage I-III primary breast cancers with linked mature clinical outcome data that were randomly selected from the Cuneo Tissue Bank. Tissue samples were subject to histopathological review to ensure adequate representation of cancer cells. Results: Dense methylation of the CpG-island of Glul was detected in 45% of cell lines across all subtypes. Methylation of the CpG island was linked with absent or down-regulated expression of Glul in some but not all cell lines, and Glul expression could be reactivated by azacytidine and trichostatin in these cell lines. Methylation of shore areas was detected in several cell lines but was not associated with transcriptional silencing. Cells with methylation-dependent low or absent Glul expression were highly sensitive to glutamine deprivation, whereas cell lines without Glul methylation were rescued by compensatory up-regulation of Glul. Using pyrosequencing, dense methylation of the CpG island of Glul was found in 32.8% of patients, with an additional 17.2% of patients showing partial methylation. No significant association with a specific breast cancer subtype or outcome was found. Conclusions: This is the first report of methylation-dependent transcriptional silencing of Glul expression in cancer. Our data demonstrate that a significant proportion of primary breast cancers show methylation of Glul and suggest that glutamine deprivation could be a novel synthetic lethality strategy for these cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-10.

Overexpression of α-methylacyl-CoA racemase is associated with CTNNB1 mutations in hepatocellular carcinomas

α-Methylacyl-CoA racemase (AMACR) is expressed in the majority of hepatocellular carcinomas (HCCs) at variable levels, but the significance of AMACR overexpression remains elusive. The aim of this study was to investigate the relationship between AMACR expression and the presence of CTNNB1 mutations in HCCs. The expression of AMACR and GLUL, an established downstream target of β-catenin was examined in HCCs, by quantitative reverse transcription polymerase chain reaction (PCR), and the expression of their protein products by immunohistochemistry. The quantitative reverse transcription PCR analysis showed that the expression of AMACR was significantly higher in HCCs with CTNNB1 mutations than in mutation-negative HCCs or normal livers, like the expression of GLUL. Immunohistochemistry also showed that strong AMACR protein expression was closely correlated with the presence of CTNNB1 mutations. HCCs with CTNNB1 mutations and those with AMACR overexpression frequently exhibited bile production. The overexpression of AMACR was closely correlated with the presence of CTNNB1 mutations in HCCs. AMACR is a putative target of β-catenin as well as an excellent immunohistochemically detectable marker of HCCs with CTNNB1 mutations. As AMACR is physiologically involved in bile acid synthesis, the current observation implies a regulatory role of β-catenin in bile acid metabolism.

Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2

Hepatoblastoma is characterized by a diversity of differentiation patterns, some resembling stages of liver development, and occasionally associated with clinical behavior. Our hypothesis is that histologic microheterogeneity in hepatoblastoma correlates with molecular heterogeneity and reflects different stages of developmental arrest. We studied the activation status of the Wnt and Notch pathways and the differential expression of hepatocyte nuclear factor 4α, EGFR, and IGF2 genes, relevant to liver development and malignant transformation in histologic variants of hepatoblastoma. Eighty-seven percent of 32 hepatoblastoma cases studied carried CTNNB1 mutations within the ubiquitination domain. Large deletions were seen only in pure fetal cases, also characterized by CCND1 and GLUL (GS) overexpression. Hepatoblastomas with small-cell type appeared clearly distinct and were the only ones with negative GLUL expression. HES1 expression and HES1/AXIN2 used to measure Notch versus Wnt activation ratio were particularly elevated in pure fetal cases and were lowest in hepatoblastomas with small-cell component. Hepatocyte nuclear factor 4α was relatively elevated only in embryonal hepatoblastomas. DLK1, DKK, AXIN2, IGF2, and EGFR were increased in all subtypes. Our results support the hypothesis that hepatoblastoma microheterogeneity correlates with molecular heterogeneity. DLK1, a marker of bipotential oval cells, is consistently up-regulated in hepatoblastoma. Therefore, we speculate that hepatoblastomas may arise from a proliferating bipotential precursor. Wnt activation is prevalent in hepatoblastomas, most significantly in predominantly embryonal and mixed types, whereas Notch activation, needed for cholangiocytic differentiation at a more differentiated state, is highest in pure fetal hepatoblastomas. The relative Wnt versus Notch activation appears useful in stratifying different subtypes.