Distinct Regulation of Th17 and Th1 Cell Differentiation by Glutaminase-Dependent Metabolism.

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

Filamentous GLS1 promotes ROS-induced apoptosis upon glutamine deprivation via insufficient asparagine synthesis.

GLS1 orchestrates glutaminolysis and promotes cell proliferation when glutamine is abundant by regenerating TCA cycle intermediates and supporting redox homeostasis. CB-839, an inhibitor of GLS1, is currently under clinical investigation for a variety of cancer types. Here, we show that GLS1 facilitates apoptosis when glutamine is deprived. Mechanistically, the absence of exogenous glutamine sufficiently reduces glutamate levels to convert dimeric GLS1 to a self-assembled, extremely low-Km filamentous polymer. GLS1 filaments possess an enhanced catalytic activity, which further depletes intracellular glutamine. Functionally, filamentous GLS1-dependent glutamine scarcity leads to inadequate synthesis of asparagine and mitogenome-encoded proteins, resulting in ROS-induced apoptosis that can be rescued by asparagine supplementation. Physiologically, we observed GLS1 filaments in solid tumors and validated the tumor-suppressive role of constitutively active, filamentous GLS1 mutants K320A and S482C in xenograft models. Our results change our understanding of GLS1 in cancer metabolism and suggest the therapeutic potential of promoting GLS1 filament formation.

SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells.

Glutaminase regulates glutaminolysis to promote cancer cell proliferation. However, the mechanism underlying glutaminase activity regulation is largely unknown. Here, we demonstrate that kidney-type glutaminase (GLS) is highly expressed in human pancreatic ductal adenocarcinoma (PDAC) specimens with correspondingly upregulated glutamine dependence for PDAC cell proliferation. Upon oxidative stress, the succinyl-coenzyme A (CoA) synthetase ADP-forming subunit ß (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 dissociates from GLS, resulting in enhanced GLS K311 succinylation, oligomerization, and activity. Activated GLS increases glutaminolysis and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby counteracting oxidative stress and promoting tumor cell survival and tumor growth in mice. In addition, the levels of SUCLA2 pS79 and GLS K311 succinylation, which were mutually correlated, were positively associated with advanced stages of PDAC and poor prognosis for patients. Our findings reveal critical regulation of GLS by SUCLA2-coupled GLS succinylation regulation and underscore the regulatory role of metabolites in glutaminolysis and PDAC development.

Norovirus NS1/2 protein increases glutaminolysis for efficient viral replication.

Viruses are obligate intracellular parasites that rely on host cell metabolism for successful replication. Thus, viruses rewire host cell pathways involved in central carbon metabolism to increase the availability of building blocks for successful propagation. However, the underlying mechanisms of virus-induced alterations to host metabolism are largely unknown. Noroviruses (NoVs) are highly prevalent pathogens that cause sporadic and epidemic viral gastroenteritis. In the present study, we uncovered several strain-specific and shared host cell metabolic requirements of three murine norovirus (MNV) strains, MNV-1, CR3, and CR6. While all three strains required glycolysis, glutaminolysis, and the pentose phosphate pathway for optimal infection of macrophages, only MNV-1 relied on host oxidative phosphorylation. Furthermore, the first metabolic flux analysis of NoV-infected cells revealed that both glycolysis and glutaminolysis are upregulated during MNV-1 infection of macrophages. Glutamine deprivation affected the viral lifecycle at the stage of genome replication, resulting in decreased non-structural and structural protein synthesis, viral assembly, and egress. Mechanistic studies further showed that MNV infection and overexpression of the non-structural protein NS1/2 increased the enzymatic activity of the rate-limiting enzyme glutaminase. In conclusion, the inaugural investigation of NoV-induced alterations to host glutaminolysis identified NS1/2 as the first viral molecule for RNA viruses that regulates glutaminolysis either directly or indirectly. This increases our fundamental understanding of virus-induced metabolic alterations and may lead to improvements in the cultivation of human NoVs.

Repeat expansions nested within tandem CNVs: a unique structural change in GLS exemplifies the diagnostic challenges of non-coding pathogenic variation.

Glutaminase deficiency has recently been associated with ataxia and developmental delay due to repeat expansions in the 5'UTR of the glutaminase (GLS) gene. Patients with the described GLS repeat expansion may indeed remain undiagnosed due to the rarity of this variant, the challenge of its detection and the recency of its discovery. In this study, we combined advanced bioinformatics screening of ~3000 genomes and ~1500 exomes with optical genome mapping and long-read sequencing for confirmation studies. We identified two GLS families, previously intensely and unsuccessfully analyzed. One family carries an unusual and complex structural change involving a homozygous repeat expansion nested within a quadruplication event in the 5'UTR of GLS. Glutaminase deficiency and its metabolic consequences were validated by in-depth biochemical analysis. The identified GLS patients showed progressive early-onset ataxia, cognitive deficits, pyramidal tract damage and optic atrophy, thus demonstrating susceptibility of several specific neuron populations to glutaminase deficiency. This large-scale screening study demonstrates the ability of bioinformatics analysis-validated by latest state-of-the-art technologies (optical genome mapping and long-read sequencing)-to effectively flag complex repeat expansions using short-read datasets and thus facilitate diagnosis of ultra-rare disorders.

Hyperpolarized [5-13C,4,4-2H2,5-15N]-L-glutamine provides a means of annotating in vivo metabolic utilization of glutamine.

Glutamine is consumed by rapidly proliferating cells and can provide the carbon and nitrogen required for growth through various metabolic pathways. However, delineating the metabolic fate of glutamine is challenging to interrogate in vivo. Hyperpolarized magnetic resonance, by providing high transient nuclear magnetic resonance signals, provides an approach to measure fast biochemical processes in vivo. Aminohydrolysis of glutamine at carbon-5 plays an important role in providing nitrogen and carbon for multiple pathways. Here, we provide a synthetic strategy for isotope-enriched forms of glutamine that prolongs glutamine-C5 relaxation times and thereby reveals in vivo reactions involving carbon-5. We investigate multiple enrichment states, finding [5-13C,4,4-2H2,5-15N]-L-glutamine to be optimal for hyperpolarized measurement of glutamine conversion to glutamate in vivo. Leveraging this compound, we explore pancreatic cancer glutamine metabolism in vivo. Taken together, this work provides a means for studying glutamine metabolic flux in vivo and demonstrates on-target effects of metabolic enzyme inhibitors.

TAp73 is a marker of glutamine addiction in medulloblastoma.

Medulloblastoma is the most common solid primary brain tumor in children. Remarkable advancements in the understanding of the genetic and epigenetic basis of these tumors have informed their recent molecular classification. However, the genotype/phenotype correlation of the subgroups remains largely uncharacterized. In particular, the metabolic phenotype is of great interest because of its druggability, which could lead to the development of novel and more tailored therapies for a subset of medulloblastoma. p73 plays a critical role in a range of cellular metabolic processes. We show overexpression of p73 in a proportion of non-WNT medulloblastoma. In these tumors, p73 sustains cell growth and proliferation via regulation of glutamine metabolism. We validated our results in a xenograft model in which we observed an increase in survival time in mice on a glutamine restriction diet. Notably, glutamine starvation has a synergistic effect with cisplatin, a component of the current medulloblastoma chemotherapy. These findings raise the possibility that glutamine depletion can be used as an adjuvant treatment for p73-expressing medulloblastoma.

Glutamine metabolism improves left ventricular function but not macrophage-mediated inflammation following myocardial infarction.

Glutamine is a critical amino acid that serves as an energy source, building block, and signaling molecule for the heart tissue and the immune system. However, the role of glutamine metabolism in regulating cardiac remodeling following myocardial infarction (MI) is unknown. In this study, we show in adult male mice that glutamine metabolism is altered both in the remote (contractile) area and in infiltrating macrophages in the infarct area after permanent left anterior descending artery occlusion. We found that metabolites related to glutamine metabolism were differentially altered in macrophages at days 1, 3, and 7 after MI using untargeted metabolomics. Glutamine metabolism in live cells was increased after MI relative to no MI controls. Gene expression in the remote area of the heart indicated a loss of glutamine metabolism. Glutamine administration improved left ventricle (LV) function at days 1, 3, and 7 after MI, which was associated with improved contractile and metabolic gene expression. Conversely, administration of BPTES, a pharmacological inhibitor of glutaminase-1, worsened LV function after MI. Neither glutamine nor BPTES administration impacted gene expression or bioenergetics of macrophages isolated from the infarct area. Our results indicate that glutamine metabolism plays a critical role in maintaining LV contractile function following MI and that glutamine administration improves LV function. Glutamine metabolism may also play a role in regulating macrophage function, but macrophages are not responsive to exogenous pharmacological manipulation of glutamine metabolism.NEW & NOTEWORTHY Glutamine metabolism is altered in both infarct macrophages and the remote left ventricle (LV) following myocardial infarction (MI). Supplemental glutamine improves LV function following MI while inhibiting glutamine metabolism with BPTES worsens LV function. Supplemental glutamine or BPTES does not impact macrophage immunometabolic phenotypes after MI.

IGF2BP3 promotes glutamine metabolism of endometriosis by interacting with UCA1 to enhances the mRNA stability of GLS1.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) has been implicated in numerous inflammatory and cancerous conditions. However, its precise molecular mechanisms in endometriosis (EMs) remains unclear. The aim of this study is to examine the influence of IGF2BP3 on the occurrence and progression of EMs and to elucidate its underlying molecular mechanism. METHODS: Efects of IGF2BP3 on endometriosis were confrmed in vitro and in vivo. Based on bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assays and Fluorescent in situ hybridization (FISH) were used to show the association between IGF2BP3 and UCA1. Single-cell spatial transcriptomics analysis shows the expression distribution of glutaminase 1 (GLS1) mRNA in EMs. Study the effect on glutamine metabolism after ectopic endometriotic stromal cells (eESCs) were transfected with Sh-IGF2BP3 and Sh-UCA1 lentivirus. RESULTS: Immunohistochemical staining have revealed that IGF2BP3 was upregulated in ectopic endometriotic lesions (EC) compared to normal endometrial tissues (EN). The proliferation and migration ability of eESCs were greatly reduced by downregulating IGF2BP3. Additionally, IGF2BP3 has been observed to interact with urothelial carcinoma associated 1 (UCA1), leading to increased stability of GLS1 mRNA and subsequently enhancing glutamine metabolism. Results also demonstrated that IGF2BP3 directly interacts with the 3' UTR region of GLS1 mRNA, influencing its expression and stability. Furthermore, UCA1 was able to bind with c-MYC protein, stabilizing c-MYC mRNA and consequently enhancing GLS1 expression through transcriptional promotion. CONCLUSION: These discoveries underscored the critical involvement of IGF2BP3 in the elevation and stability of GLS1 mRNA in the context of glutamine metabolism by interacting with UCA1 in EMs. The implications of our study extended to the identification of possible therapeutic targets for individuals with EMs.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Improved Photodynamic Therapy Based on Glutaminase Blockage via Tumor Membrane Coated CB-839/IR-780 Nanoparticles.

Photodynamic therapy (PDT) has promising applications. However, the lethal function of reactive oxygen species (ROS) produced during PDT is typically limited. This restriction is induced by oxygen shortage in the tumor microenvironment due to tumor cell hypermetabolism and reductive chemicals overexpression in tumor tissues. Glutamine (Gln) metabolism is crucial for malignancy development and is closely associated with redox. Herein, a novel nanoparticle (NP) named IRCB@M is constructed to boost PDT through dual effects. This NP simultaneously blocks aerobic respiration and inhibits cellular reduced substances by blocking the Gln metabolic pathway. Within the nanocomplex, a photosensitizer (IR-780) and a glutaminase inhibitor (CB-839) are self-assembled and then encapsulated by cancer cell membranes for homologous targeting. The Gln metabolism intervention relieves hypoxia and decreases the levels of nicotinamide adenine dinucleotide phosphate (NADPH) as well as reduced glutathione (GSH) in vitro and in vivo, which are the dual amplification effects on the IR-780-mediated lethal PDT. The antitumor effects against gastric cancer are ultimately evoked in vivo, thus offering a novel concept for enhancing PDT and other ROS-dependent therapeutic approaches.

Glutaminase potentiates the glycolysis in esophageal squamous cell carcinoma by interacting with PDK1.

Increasing evidence has demonstrated that glutaminase (GLS) as a key mitochondrial enzyme plays a pivotal role in glutaminolysis, which widely participates in glutamine metabolism serving as main energy sources and building blocks for tumor growth. However, the roles and molecular mechanisms of GLS in esophageal squamous cell carcinoma (ESCC) remains unknown. Here, we found that GLS was highly expressed in ESCC tissues and cells. GLS inhibitor CB-839 significantly suppressed cell proliferation, colony formation, migration and invasion of ESCC cells, whereas GLS overexpression displayed the opposite effects. In addition, CB-839 markedly suppressed glucose consumption and lactate production, coupled with the downregulation of glycolysis-related proteins HK2, PFKM, PKM2 and LDHA, whereas GLS overexpression exhibited the adverse results. In vivo animal experiment revealed that CB-839 dramatically suppressed tumor growth, whereas GLS overexpression promoted tumor growth in ESCC cells xenografted nude mice. Mechanistically, GLS was localized in mitochondria of ESCC cells, which interacted with PDK1 protein. CB-839 attenuated the interaction of GLS and PDK1 in ESCC cells by suppressing PDK1 expression, which further evoked the downregulation of p-PDHA1 (s293), however, GLS overexpression markedly enhanced the level of p-PDHA1 (s293). These findings suggest that interaction of GLS with PDK1 accelerates the glycolysis of ESCC cells by inactivating PDH enzyme, and thus targeting GLS may be a novel therapeutic approach for ESCC patients.

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

In brief: FSH leads to glutamine dependence, which is required for mTORC1 activation and in consequence Sertoli cell proliferation. Abstract: 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.

Analysis of anti-tumor effect and mechanism of GLS1 inhibitor CB-839 in colorectal cancer using a stroma-abundant tumor model.

BACKGROUND: Glutaminase 1 (GLS1), a key enzyme in glutamine metabolism in cancer cells, acts as a tumor promoter and could be a potential therapeutic target. CB-839, a GLS1-specific inhibitor, was developed recently. Herein, we aimed to elucidate the anti-tumor effects and mechanism of action of CB-839 in colorectal cancer (CRC). METHODS: Using the UCSC Xena public database, we evaluated GLS1 expression in various cancers. Immunostaining for GLS1 was performed on 154 surgically resected human CRC specimens. Subsequently, we examined the GLS1 mRNA expression levels in eight CRC cell lines and evaluated the association between GLS1 expression and CB-839 efficacy. To create a reproducible CRC model with abundant stroma and an allogeneic immune response, we co-transplanted CT26 and stem cells into BALB/c mice and treated them with CB-839. Finally, RNA sequencing of mouse tumors was performed. RESULTS: Database analysis showed higher GLS1 expression in CRC tissues than in normal colon tissues. Clinical samples from 114 of the 154 patients with CRC showed positive GLS1 expression. GLS1 expression in clinical CRC tissues correlated with vascular invasion. CB-839 treatment inhibited cancer cell proliferation depending on GLS1 expression in vitro and inhibited tumor growth and metastasis in the CRC mouse model. RNA sequencing revealed that CB-839 treatment inhibited stromal activation, tumor growth, migration, and angiogenesis. These findings were validated through in vitro and in vivo experiments and clinical specimen analysis. CONCLUSIONS: GLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

Role of ammonia and glutamine in the pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease: A systematic review.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects over 30% of the global population, with a significant risk of advancing to liver cirrhosis and hepatocellular carcinoma. The roles of ammonia and glutamine in MASLD's pathogenesis are increasingly recognized, prompting this systematic review. This systematic review was conducted through a meticulous search of literature on December 21, 2023, across five major databases, focusing on studies that addressed the relationship between ammonia or glutamine and MASLD. The quality of the included studies was evaluated using CASP checklists. This study is officially registered in the PROSPERO database (CRD42023495619) and was conducted without external funding or sponsorship. Following PRISMA guidelines, 13 studies were included in this review. The studies were conducted globally, with varying sample sizes and study designs. The appraisal indicated a mainly low bias, confirming the reliability of the evidence. Glutamine's involvement in MASLD emerged as multifaceted, with its metabolic role being critical for liver function and disease progression. Variable expressions of glutamine synthetase and glutaminase enzymes highlight metabolic complexity whereas ammonia's impact through urea cycle dysfunction suggests avenues for therapeutic intervention. However, human clinical trials are lacking. This review emphasizes the necessity of glutamine and ammonia in understanding MASLD and identifies potential therapeutic targets. The current evidence, while robust, points to the need for human studies to corroborate preclinical findings. A personalized approach to treatment, informed by metabolic differences in MASLD patients, is advocated, alongside future large-scale clinical trials for a deeper exploration into these metabolic pathways.

L-asparaginase anti-tumor activity in pancreatic cancer is dependent on its glutaminase activity and resistance is mediated by glutamine synthetase.

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

Combined thioredoxin reductase and glutaminase inhibition exerts synergistic anti-tumor activity in MYC-high high-grade serous ovarian carcinoma.

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.

Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

Targeting glutamine metabolism exhibits anti-tumor effects in thyroid cancer.

BACKGROUND: Effective treatment for patients with advanced thyroid cancer is lacking. Metabolism reprogramming is required for cancer to undergo oncogenic transformation and rapid tumorigenic growth. Glutamine is frequently used by cancer cells for active bioenergetic and biosynthetic needs. This study aims to investigate whether targeting glutamine metabolism is a promising therapeutic strategy for thyroid cancer. METHODS: The expression of glutaminase (GLS) and glutamate dehydrogenase (GDH) in thyroid cancer tissues was evaluated by immunohistochemistry, and glutamine metabolism-related genes were assessed using real time-qPCR and western blotting. The effects of glutamine metabolism inhibitor 6-diazo-5-oxo-l-norleucine (DON) on thyroid cancer cells were determined by CCK-8, clone formation assay, Edu incorporation assay, flow cytometry, and Transwell assay. The mechanistic study was performed by real time-qPCR, western blotting, Seahorse assay, and gas chromatography-mass spectrometer assay. The effect of DON prodrug (JHU-083) on thyroid cancer in vivo was assessed using xenograft tumor models in BALB/c nude mice. RESULTS: GLS and GDH were over-expressed in thyroid cancer tissues, and GLS expression was positively associated with lymph-node metastasis and TNM stage. The growth of thyroid cancer cells was significantly inhibited when cultured in glutamine-free medium. Targeting glutamine metabolism with DON inhibited the proliferation of thyroid cancer cells. DON treatment did not promote apoptosis, but increased the proportion of cells in the S phase, accompanied by the decreased expression of cyclin-dependent kinase 2 and cyclin A. DON treatment also significantly inhibited the migration and invasion of thyroid cancer cells by reducing the expression of N-cadherin, Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-9. Non-essential amino acids, including proline, alanine, aspartate, asparagine, and glycine, were reduced in thyroid cancer cells treated with DON, which could explain the decrease of proteins involved in migration, invasion, and cell cycle. The efficacy and safety of DON prodrug (JHU-083) for thyroid cancer treatment were verified in a mouse model. In addition to suppressing the proliferation and metastasis potential of thyroid cancer in vivo, enhanced innate immune response was also observed in JHU-083-treated xenograft tumors as a result of decreased expression of cluster of differentiation 47 and programmed cell death ligand 1. CONCLUSIONS: Thyroid cancer exhibited enhanced glutamine metabolism, as evidenced by the glutamine dependence of thyroid cancer cells and high expression of multiple glutamine metabolism-related genes. Targeting glutamine metabolism with DON prodrug could be a promising therapeutic option for advanced thyroid cancer.

A paradigm shift in cancer research based on integrative multi-omics approaches: glutaminase serves as a pioneering cuproptosis-related gene in pan-cancer.

BACKGROUND: Cuproptosis is a newly identified form of unprogrammed cell death. As a pivotal metabolic regulator, glutaminase (GLS) has recently been discovered to be linked to cuproptosis. Despite this discovery, the oncogenic functions and mechanisms of GLS in various cancers are still not fully understood. METHODS: In this study, a comprehensive omics analysis was performed to investigate the differential expression levels, diagnostic and prognostic potential, correlation with tumor immune infiltration, genetic alterations, and drug sensitivity of GLS across multiple malignancies. RESULTS: Our findings revealed unique expression patterns of GLS across various cancer types and molecular subtypes of carcinomas, underscoring its pivotal role primarily in energy and nutrition metabolism. Additionally, GLS showed remarkable diagnostic and prognostic performance in specific cancers, suggesting its potential as a promising biomarker for cancer detection and prognosis. Furthermore, we focused on uterine corpus endometrial carcinoma (UCEC) and developed a novel prognostic model associated with GLS, indicating a close correlation between GLS and UCEC. Moreover, our exploration into immune infiltration, genetic heterogeneity, tumor stemness, and drug sensitivity provided novel insights and directions for future research and laid the foundation for high-quality verification. CONCLUSION: Collectively, our study is the first comprehensive investigation of the biological and clinical significance of GLS in pan-cancer. In our study, GLS was identified as a promising biomarker for UCEC, providing valuable evidence and a potential target for anti-tumor therapy. Overall, our findings shed light on the multifaceted functions of GLS in cancer and offer new avenues for further research.