Inhibition Of Glutamine Metabolism Research Articles

Alveolar and Bone Marrow-derived Macrophages Differ in Metabolism and Glutamine Utilization.

Changes in metabolic activity are key regulators of macrophage activity. Pro-inflammatory macrophages upregulate glycolysis, which promotes an inflammatory phenotype, whereas pro-repair macrophages rely upon oxidative metabolism and glutaminolysis to support their activity. Work to understand how metabolism regulates macrophage phenotype has been done primarily in macrophage cell lines and bone marrow-derived macrophages (BMDM). Our study sought to understand changes in metabolic activity of murine tissue-resident alveolar macrophages (AM) in response to LPS stimulation and to contrast them to BMDM. These studies also determined the contribution of glutamine metabolism using the glutamine inhibitor, DON. We found that compared to BMDM, AM have higher rates of oxygen consumption and contain a higher concentration of intracellular metabolites involved in fatty acid oxidation. In response to LPS, BMDM but not AM increased rates of glycolysis. Inhibition of glutamine metabolism using DON altered the metabolic activity of AM but not BMDM. Within AM, glutamine inhibition led to increases in intracellular metabolites involved in glycolysis, the TCA cycle, fatty acid oxidation, and amino acid metabolism. Glutamine inhibition also altered the metabolic response to LPS within AM but not BMDM. Our data reveal striking differences in the metabolic activity of AM and BMDM.

Association of glutaminase expression with immune-suppressive tumor microenvironment, clinicopathologic features, and clinical outcomes in endometrial cancer

ObjectiveIncreased glutamine metabolism by cancer cells via upregulation of the drug-targetable enzyme glutaminase may contribute to an immune-suppressive tumor microenvironment. Inhibiting glutamine metabolism can not only suppress tumor growth, but...

KDM4A promotes malignant progression of breast cancer by down-regulating BMP9 inducing consequent enhancement of glutamine metabolism

BackgroundRecent studies have found that histone-modified genes play an increasingly important role in tumor progression. Lysine(K) specific demethylase 4A (KDM4A) is a histone lysine-specific demethylase highly expressed in a variety of malignant tumors, data showed that KDM4A was negatively correlated with the Bone Morphogenetic Protein 9 (BMP9) in breast cancer. And previous experiments have demonstrated that exogenous BMP9 significantly inhibits breast cancer development.Materials and methodsWe detected the expression of KDM4A in breast cancer and the relationship between KDM4A and BMP9 using real-time quantitative PCR (RT-qPCR) and Western blot, and verified the interaction between KDM4A and BMP9 by ChIP experiments. At the same time, we also detected whether KDM4A had effects on the RNA and protein stability of BMP9 using actinomycin D and cycloheximide. Measurement of alpha-ketoglutarate (α-KG) level by ELISA to observe the effect of BMP9 on glutamine metabolism in breast cancer cells. Nucleoplasmic distribution of KDM4A after exogenous BMP9 treatment in breast cancer cells were observed by immunofluorescence staining and Western blot. A subcutaneous xenograft tumor model in nude mice was used to study the therapeutic effects of exogenous BMP9 and KDM4A inhibitor (JIB-04) in breast cancer. CCK-8, conoly formation, Transwell, wound healing, and immunohistochemistry were used to monitor the growth of tumor and cell function.ResultsWe found that KDM4A was abnormally highly expressed in breast cancer, and silenced BMP9 expression by removing histone methyl groups from the BMP9 gene region. Meanwhile, KDM4A could also reduce the stability of BMP9 protein. BMP9 inhibit glutamine metabolism in breast cancer, resulting in a decrease in its product α-KG, is confirmed by ELISA. Altered nucleoplasmic distribution of KDM4A due to decreased α-KG was confirmed by immunofluorescence staining and Western blot. Animal experiments confirm that the combination of exogenous BMP9 and JIB-04 shows significantly better results in breast cancer.ConclusionsKDM4A silences BMP9 expression by removing histone methyl groups from the BMP9 gene region, leading to further enhancement of glutamine metabolism, which contributes to malignant tumor progression. In addition, using JIB-04 in combination with exogenous BMP9 could inhibit the malignant progression of breast cancer cells and the growth of tumors more significantly.

Self-assembled metal-polyphenolic based multifunctional nanomedicine to improve therapy treatment of pancreatic cancer by inhibition of glutamine metabolism

Cancer poses a significant threat to human health and life. Chemotherapy, immunotherapy and chemodynamic therapy (CDT) are effective treatments for cancer. However, the presence of metabolic reprogramming via glutamine in tumor cells limits their therapeutic effectiveness. Herein, we propose an effective assembly strategy to synthesize a novel metal-polyphenolic based multifunctional nanomedicine (Fe-DBEF) containing Pluronic F127 stable ferric ion crosslinked epigallocatechin gallate (EGCG) nanoparticles loaded with GLS1 inhibitor bis-2-(5-phenylacetamino-1,3,4-thiadiazole-2-yl) ethyl sulfide (BPTES) and chemotherapy drug doxorubicin (DOX). Our study demonstrates that Fe-DBEF nanomedicine exhibits high efficiency anti-proliferation properties in pancreatic cancer through a combination of in vitro cell experiments, human organoid experiments and KPC animal experiments. Notably, Fe-DBEF nanomedicine can reduce the production of glutathione (GSH) in tumor cells, thereby reducing their resistance to ROS therapy. Additionally, excessive ROS production also aggravates DNA damage caused by DOX, synergistically sensitizing chemotherapy and promoting apoptosis for efficient treatment of pancreatic cancer. Overall, our findings suggest that inhibiting glutamine metabolism to increase the sensitivity of chemotherapy/CDT using metal-polyphenolic based multifunctional nanomedicine provides a promising combination of multiple therapeutic means for treating pancreatic cancer.

Targeting YAP Activity and Glutamine Metabolism Cooperatively Suppresses Tumor Progression by Preventing Extracellular Matrix Accumulation.

Cancer cells use multiple mechanisms to evade the effects of glutamine metabolism inhibitors. The pathways that govern responses to alterations in glutamine availability within the tumor may represent therapeutic targets for combinatorial strategies with these inhibitors. Here, we showed that targeting glutamine utilization stimulated Yes-associated protein (YAP) signaling in cancer cells by reducing cyclic adenosine monophosphate/protein kinase A (PKA)-dependent phosphorylation of large tumor suppressor (LATS). Elevated YAP activation induced extracellular matrix (ECM) deposition by increasing the secretion of connective tissue growth factor that promoted the production of fibronectin and collagen by surrounding fibroblasts. Consequently, inhibiting YAP synergized with inhibition of glutamine utilization to effectively suppress tumor growth in vivo, along with a concurrent decrease in ECM deposition. Blocking ECM remodeling also augmented the tumor suppressive effects of the glutamine utilization inhibitor. Collectively, these data reveal mechanisms by which targeting glutamine utilization increases ECM accumulation and identify potential strategies to reduce ECM levels and increase the efficacy of glutamine metabolism inhibitors. Significance: Blocking glutamine utilization activates YAP to promote ECM deposition by fibroblasts, highlighting the potential of YAP inhibitors and antifibrotic strategies as promising approaches for effective combination metabolic therapies in cancer.

A Ketogenic Diet Sensitizes Pancreatic Cancer to Inhibition of Glutamine Metabolism.

Pancreatic cancer is the third leading cause of cancer death in the United States, and while conventional chemotherapy remains the standard treatment, responses are poor. Safe and alternative therapeutic strategies are urgently needed 1 . A ketogenic diet has been shown to have anti-tumor effects across diverse cancer types but will unlikely have a significant effect alone. However, the diet shifts metabolism in tumors to create new vulnerabilities that can be targeted (1). Modulators of glutamine metabolism have shown promise in pre-clinical models but have failed to have a marked impact against cancer in the clinic. We show that a ketogenic diet increases TCA and glutamine-associated metabolites in murine pancreatic cancer models and under metabolic conditions that simulate a ketogenic diet in vitro. The metabolic shift leads to increased reliance on glutamine-mediated anaplerosis to compensate for low glucose abundance associated with a ketogenic diet. As a result, glutamine metabolism inhibitors, such as DON and CB839 in combination with a ketogenic diet had robust anti-cancer effects. These findings provide rationale to study the use of a ketogenic diet with glutamine targeted therapies in a clinical context.

L-[5-11C]Glutamine PET imaging noninvasively tracks dynamic responses of glutaminolysis in non-alcoholic steatohepatitis

Inhibiting glutamine metabolism has been proposed as a potential treatment strategy for improving non-alcoholic steatohepatitis (NASH). However, effective methods for assessing dynamic metabolic responses during interventions targeting glutaminolysis have not yet emerged. Here, we developed a positron emission tomography (PET) imaging platform using l-[5-11C]glutamine ([11C]Gln) and evaluated its efficacy in NASH mice undergoing metabolic therapy with bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a glutaminase 1 (GLS1) inhibitor that intervenes in the first and rate-limiting step of glutaminolysis. PET imaging with [11C]Gln effectively delineated the pharmacokinetics of l-glutamine, capturing its temporal-spatial pattern of action within the body. Furthermore, [11C]Gln PET imaging revealed a significant increase in hepatic uptake in methionine and choline deficient (MCD)-fed NASH mice, whereas systemic therapeutic interventions with BPTES reduced the hepatic avidity of [11C]Gln in MCD-fed mice. This reduction in [11C]Gln uptake correlated with a decrease in GLS1 burden and improvements in liver damage, indicating the efficacy of BPTES in mitigating NASH-related metabolic abnormalities. These results suggest that [11C]Gln PET imaging can serve as a noninvasive diagnostic platform for whole-body, real-time tracking of responses of glutaminolysis to GLS1 manipulation in NASH, and it may be a valuable tool for the clinical management of patients with NASH undergoing glutaminolysis-based metabolic therapy.

SIRT4 Has an Anti-Cancer Role in Cervical Cancer by Inhibiting Glutamine Metabolism via the MEK/ERK/C-Myc Axis.

Glutamine metabolism is crucial in cell proliferation, aging, and apoptosis across various cancer types. Existing research indicates that Sirtuin 4 (SIRT4), primarily located in mitochondria, modulates this process. This study aimed to clarify the regulatory relationship between SIRT4 and glutamine metabolism in cervical cancer. SIRT4 mRNA levels and their clinical correlation to cervical cancer were analyzed using the UALCAN database. Immunohistochemistry (IHC) was performed to assess SIRT4 protein expression in tissue samples from cervical cancer patients. Transient transfection was employed to create Hela and Siha cell lines with overexpressed SIRT4, mitogen-activated extracellular signal-regulated kinase (MEK), and glutaminase 1 (GLS1). The impact on cellular functions was studied using MTT, soft agar, transwell, and western blotting assays. Glutamate and ATP levels were also measured to evaluate metabolic changes. Low levels of SIRT4 mRNA in cervical cancer tissues correlated with tumor metastasis and poor survival rates. Overexpression of SIRT4 led to suppressed cell proliferation, colony growth, and motility, along with significant down-regulation of GLS expression, a key contributor to glutamine metabolism. Additionally, SIRT4 overexpression resulted in the inactivation of the MEK/ERK/c-myc signaling pathway, while overexpression of MEK reversed these effects. Notably, the inhibitory effects of SIRT4 on cell proliferation, colony formation, migration, and invasion in Hela and Siha cells were significantly attenuated following GLS1 overexpression. SIRT4 acts as an anti-cancer agent in cervical cancer by inhibiting glutamine metabolism through the MEK/ERK/c-myc signaling pathway, providing a novel sight for cervical cancer therapy.

Necroptosis-Mediated Synergistic Photodynamic and Glutamine-Metabolic Therapy Enabled by a Biomimetic Targeting Nanosystem for Cholangiocarcinoma.

Targeted delivery of glutamine metabolism inhibitors holds promise for cholangiocarcinoma therapy, yet effective delivery vehicles remain a challenge. This study reports the development of a biomimetic nanosystem, termed R-CM@MSN@BC, integrating mesoporous organosilicon nanoparticles with reactive oxygen species-responsive diselenide bonds for controlled release of the glutamine metabolism inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and the photosensitizer Ce6. Erythrocyte membrane coating, engineered with Arg-Gly-Asp (RGD) peptides, not only enhanced biocompatibility but also improved tumor targeting and tissue penetration. Upon laser irradiation, R-CM@MSN@BC executed both photodynamic and glutamine-metabolic therapies, inducing necroptosis in tumor cells and triggering significant immunogenic cell death. Time-of-flight mass cytometry analysis revealed that R-CM@MSN@BC can remodel the immunosuppressive tumor microenvironment by polarizing M1-type macrophages, reducing infiltration of M2-type and CX3CR1+ macrophages, and decreasing T cell exhaustion, thereby increasing the effectiveness of anti-programmed cell death ligand 1 immunotherapy. This strategy proposed in this study presents a viable and promising approach for the treatment of cholangiocarcinoma.

D-mannose alleviates intervertebral disc degeneration through glutamine metabolism

BackgroundIntervertebral disc degeneration (IVDD) is a multifaceted condition characterized by heterogeneity, wherein the balance between catabolism and anabolism in the extracellular matrix of nucleus pulposus (NP) cells plays a central role. Presently, the available treatments primarily focus on relieving symptoms associated with IVDD without offering an effective cure targeting its underlying pathophysiological processes. D-mannose (referred to as mannose) has demonstrated anti-catabolic properties in various diseases. Nevertheless, its therapeutic potential in IVDD has yet to be explored.MethodsThe study began with optimizing the mannose concentration for restoring NP cells. Transcriptomic analyses were employed to identify the mediators influenced by mannose, with the thioredoxin-interacting protein (Txnip) gene showing the most significant differences. Subsequently, small interfering RNA (siRNA) technology was used to demonstrate that Txnip is the key gene through which mannose exerts its effects. Techniques such as colocalization analysis, molecular docking, and overexpression assays further confirmed the direct regulatory relationship between mannose and TXNIP. To elucidate the mechanism of action of mannose, metabolomics techniques were employed to pinpoint glutamine as a core metabolite affected by mannose. Next, various methods, including integrated omics data and the Gene Expression Omnibus (GEO) database, were used to validate the one-way pathway through which TXNIP regulates glutamine. Finally, the therapeutic effect of mannose on IVDD was validated, elucidating the mechanistic role of TXNIP in glutamine metabolism in both intradiscal and orally treated rats.ResultsIn both in vivo and in vitro experiments, it was discovered that mannose has potent efficacy in alleviating IVDD by inhibiting catabolism. From a mechanistic standpoint, it was shown that mannose exerts its anti-catabolic effects by directly targeting the transcription factor max-like protein X-interacting protein (MondoA), resulting in the upregulation of TXNIP. This upregulation, in turn, inhibits glutamine metabolism, ultimately accomplishing its anti-catabolic effects by suppressing the mitogen-activated protein kinase (MAPK) pathway. More importantly, in vivo experiments have further demonstrated that compared with intradiscal injections, oral administration of mannose at safe concentrations can achieve effective therapeutic outcomes.ConclusionsIn summary, through integrated multiomics analysis, including both in vivo and in vitro experiments, this study demonstrated that mannose primarily exerts its anti-catabolic effects on IVDD through the TXNIP-glutamine axis. These findings provide strong evidence supporting the potential of the use of mannose in clinical applications for alleviating IVDD. Compared to existing clinically invasive or pain-relieving therapies for IVDD, the oral administration of mannose has characteristics that are more advantageous for clinical IVDD treatment.

Abstract 3161: PCTK1 driven glutamine metabolic adaptation in brain metastasis

Abstract Brain metastasis (BrM) is the most common malignancy of the central nervous system and the median survival time of patients with BrM is less than 1 year. Although recent impressive advances in targeted therapy and immunotherapy enable more effective control of the systemic diseases, the incidence of BrM upon disease recurrence is steadily increasing. Unfortunately, there is no effective treatments for BrM. To surmount this challenge, we have performed an in vivo human kinome screening to uncover novel kinases that promote breast cancer (BC) BrMs in mice and may serve as therapeutic targets. We identified >20 different kinases enriched in BrM lesions of our mouse models and PCTK1 is one of the top “hits”. We transduced PCTK1 expression vector and the control vector into MDA-MB-231 human BC cells to generate 231.PCTK1 and 231.vector cells, respectively. Intra-carotid injection of the 231.PCTK1 cells into mice induced BrM significantly faster with shortened mouse survival than that of the control 231.vector cells, confirming PCTK1 as a BrM-promoting kinase. However, when 231.vector or 231.PCTK1 cells were injected into the mouse’ mammary glands, PCTK1 overexpression did not lead to enhanced primary tumor growth, suggesting that its pro-tumoral functions are BrM-specific. To uncover how PCTK1 promotes BrM but not primary tumor, we examined whether PCTK1 may confer growth advantage in BrM by utilizing the unique metabolic microenvironment of the brain. We analyzed the relationship between PCTK1 gene expression and gene expression of members of the glutamine metabolism pathway in TCGA database. We found that in the brain tumors, such as low-grade glioma, PCTK1 positively correlated with the expression of glutamine metabolizing genes, including glutaminase 1 and 2 (GLS and GLS2). To evaluate whether PCTK1-overexpressing cells can uptake glutamine from the media directly, we cultured 231.PCTK1 and 231.vector cells in low-nutrient BME media plus 5% d.FBS, with addition of different concentrations of glutamine. The PCTK1-overexpressing cells showed a clear growth advantage at glutamine concentrations ranging from 400 μM to 2 mM. Then, we knocked down PCTK1 in MDA-MB-231 cells by shRNAs and found that PCTK1 knocking down reduced the cell proliferation in 5% d.FBS.BME with 500 µM glutamine, a physiological glutamine level in the brain. We measured the expression of GLS, an enzyme that converts glutamine to glutamate, one of the key steps in the glutamine metabolism, and found that knockdown PCTK1 in MDA-MB-231 cells dramatically reduced the GLS expression, which indicates that PCTK1 can modulate glutamine metabolism in cancer cells. In summary, our data suggest that PCTK1 promotes BC BrM, at least partly, through increasing glutamine metabolism. Currently, we are exploring whether targeting PCTK1 or inhibiting glutamine metabolism can deter BrM progression. Our studies may reveal novel therapeutic targets for BrM inhibition. Citation Format: Lin Zhang, Frank Lowery, Chenyu Zhang, Yu-Wen Huang, Ya-Jing Jiang, Yimin Duan, Dihua Yu. PCTK1 driven glutamine metabolic adaptation in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3161.

Abstract 2871: Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer

Abstract Purpose Statement: Alterations in the PI3K/AKT signaling pathway are common in cervical cancer. The most commonly observed mutations are in PIK3CA (26%) and PTEN (8%). However, the biological and metabolic implications of acquiring these mutations in the cervical cancer genome are poorly characterized. Materials/Methods: Isogenic cell lines with PIK3CA E545K and PTEN null mutations were generated using Crispr/CAS9. Cell viability and clonogenic cell survival assays were performed in the presence and absence of inhibitors of glutamine and asparagine metabolism. Glucose and glutamine metabolism was evaluated by stable isotope labeling with U13C glucose and13C5 glutamine in presence of glutamine antagonist DON. RNA-seq was employed to evaluate transcriptomic alterations. Results: Introduction of the PIK3CA E545K mutation and PTEN mutation in cervical tumor cells leads to an increase in AKT signaling compared to the parental (wild-type) cells. Notably, RNASeq and mass spectrometric analysis of both the tumor cells and their culture media revealed heightened dependencies on glucose and glutamine in the tumor cells carrying the PIK3CA E545K and PTEN mutations as compared to the wild-type cells. Treatment with glutamine antagonists (DON, JHU-083) and GLS inhibitors (CB-839 and IACS-6274), resulted in reduced cell viability and clonogenic survival in tumor cells with PI3K/PTEN mutation compared to wild-type. Additionally, we observed a metabolic reliance on the amino acid asparagine in tumors with PI3K/PTEN alterations when treated with DON. Asparaginase in combination with DON effectively inhibited the proliferation of human and mouse cell lines derived from primary tumors with PTEN mutations. Furthermore, the incorporation of a glutamine antagonist and asparaginase into conventional chemoirradiation further enhanced the therapeutic efficacy in cervical tumors with PI3K/PTEN alterations which are otherwise treatment resistant. Conclusions: Orthogonal evaluations using glutamine agonists, GLS inhibitors, and mass spectrometric point to a significant role played by PI3K/PTEN mutations in altering cervix tumor cell metabolism towards a more glutamine dependent phenotype. This study suggests a potential strategy for improving the treatment of PI3K-AKT-altered cervical tumors by targeting glutamine and asparagine metabolism in combination with standard therapies. These treatment strategies are currently being evaluated in vivo using a patient derived xenograft model with PIK3CA E545K mutation as well as an immunocompetent cervical tumor model in mice with PTEN deletion. Citation Format: Nishanth Noel Gabriel, Naoshad Muhammad, Kevin Cho, Kay Jayachandran, Jin Zhang, Gary Patti, Julie Schwarz. Co-targeting asparagine and glutamine metabolism is a viable treatment strategy for PI3K/PTEN mutated cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2871.

Abstract 4397: Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression

Abstract Most cases of Pancreatic Ductal Adenocarcinoma (PDAC) are diagnosed after metastasis has occurred, making it crucial to find effective treatments for late-stage disease. Previously, our group found widespread epigenetic reprogramming during PDAC metastasis in the absence of additional driver mutations. These cells develop dependence on glucose metabolism through the oxidative pentose phosphate pathway (oxPPP). Inhibition of oxPPP with 6-aminonicotinamide (6AN) reverses the chromatin changes and restores cells to a less malignant state (McDonald et al. Nat Gen, 2017). To identify a clinically actionable mechanism for restoration, we hypothesized that oxPPP dependence leads to downstream metabolic changes that mediate chromatin state. Using untargeted metabolomic analysis, we found that alpha-ketoglutarate (AKG), a cofactor of lysine and histone demethylases, was downregulated by oxPPP inhibition (p=0.033) in a patient-derived xenograft lung metastasis cell line, A13Lg. Treatment of cells with 6AN and a cell permeable precursor of AKG largely reversed the chromatin changes seen on a western blot. Given that AKG is mainly produced by glutamine metabolism, we inhibited glutaminolysis with glutamine deprivation (Gln(-)) or the glutamine antagonist JHU083. Both treatments decreased AKG (Gln(-) p=0.002, JHU083 p=0.038) and prevented cell growth (Gln(-) p<0.0001, JHU083 p=0.0002). Glutamine-deprived cells showed decreased ability to form colonies (p=0.004), and JHU083 treated cells showed impaired migration in a Boyden chamber assay (p=0.003). Western blots showed a decrease in H3K27 acetylation, a marker of euchromatin, and an increase in H3K9 and H3K27 methylation, markers of heterochromatin, in response to both treatments. Further, homogenized time-resolved fluorescence (HTRF) assays on glutamine-deprived cells showed that H3K9 di- (p=0.0016,) and tri- (p=0.0003) methylation was increased. Adding cell permeable AKG to media without glutamine largely negated both the phenotypic and epigenetic changes. Using CUT&RUN against H3K27Ac, we found that acetylation was depleted at promoters involved in tumor cell proliferation, invasion, and migration. Bulk RNA-sequencing also showed decreased expression of genes involved in these pathways. Epigenetic and phenotypic changes were not as apparent after treatment with CB839, a glutaminase 1 inhibitor, suggesting that broad inhibition of glutamine metabolism is necessary for chromatin restoration. Together, these data suggest that decreasing AKG in PDAC distant metastasis cells, through glutamine deprivation or JHU083, leads to chromatin changes that specify less malignant phenotypes, providing a rationale for targeting glutamine metabolism as a therapy for late-stage PDAC. *APF and MM are co-corresponding authors. Citation Format: Kenna Sherman, Jiaqi Cheng, Weiqiang Zhou, Hongkai Ji, Masahiro Maeda, Andrew P. Feinberg. Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4397.

Progesterone inhibits endometrial cancer growth by inhibiting glutamine metabolism through ASCT2.

Endometrial carcinoma (EC) is a common malignancy that originates from the endometrium and grows in the female reproductive system. Surgeries, as current treatments for cancer, however, cannot meet the fertility needs of young women patients. Thus, progesterone (P4) therapy is indispensable due to its effective temporary preservation of female fertility. Many cancer cells are often accompanied by changes in metabolic phenotypes, and abnormally dependent on the amino acid glutamine. However, whether P4 exerts an effect on EC via glutamine metabolism is unknown. In the present study, we found that P4 could inhibit glutamine metabolism in EC cells and down-regulate the expression of the glutamine transporter ASCT2. This regulation of ASCT2 affects the uptake of glutamine. Furthermore, the in vivo xenograft studies showed that P4 inhibited tumor growth and the expression of key enzymes involved in glutamine metabolism. Our study demonstrated that the direct regulation of glutamine metabolism by P4 and its anticancer effect was mediated through the inhibition of ASCT2. These results provide a mechanism underlying the effects of P4 therapy on EC from the perspective of glutamine metabolism.

Inhibition of glutamine metabolism increases sensitivity to plasma-activated medium-induced cytotoxicity

Non-thermal atmospheric pressure plasma (NTP), an ionized gas containing electrons, ions, radicals, and photons, has various biological effects, including wound healing and anticancer effects. Plasma-activated medium (PAM), which is prepared by irradiating medium with NTP, preferentially kills cancer cells. Large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) included in PAM are closely related to its anticancer effects. The precise mechanism of PAM-induced cytotoxicity is not fully understood; however, PAM exposure has been reported to reduce cellular energy metabolism. Glutamine (Gln) is an important amino acid as an energy source in cancer cells. Gln is converted to glutamate by glutaminase (GLS), and is utilized to synthesize ATP and glutathione (GSH). Expression levels of GLS have been shown to be higher in certain types of cancers. In this study, we examined the effects of GLS inhibition on PAM cytotoxicity using breast cancer MDA-MB-231 cells. Pretreatment with BPTES, a glutaminase 1 (GLS1) inhibitor, dose-dependently enhanced PAM-induced cell death. PAM-induced ROS production and γ-H2AX formation, a DNA damage marker, were increased in cells pretreated with BPTES compared with PAM alone. BPTES pretreatment enhanced a PAM-induced decrease in intracellular GSH, indicating the possibility that BPTES reduces the antioxidant capacity of MDA-MB-231 cells. In addition, BPTES pretreatment enhanced PAM-induced loss of the mitochondrial membrane potential and reduction of ATP production. Moreover, GLS1 knockdown promoted PAM-induced cell death. Taken together, the combination of GLS1 inhibitors such as BPTES is considered to be useful for enhancing the cytotoxic effects of PAM against cancer cells.

Fumarate induces LncRNA-MIR4435-2HG to regulate glutamine metabolism remodeling and promote the development of FH-deficient renal cell carcinoma

Fumarate hydratase (FH) deficient renal cell carcinoma (RCC) is a type of tumor with definite metabolic disorder, but the mechanism of metabolic remodeling is still unclear. LncRNA was reported to closely correlate with cancer metabolism, however the biological role of LncRNA in the development of progression of FH-deficent RCC was not well studied either. FH-deficient RCC samples were collected in my hospital and used for RNA-sequencing and Mass spectrometry analysis. FH-deficient RCC cell line UOK262 and control pFH cells were used for in vitro experiments, including proliferation assay, transwell assay, western-blot, mass spectrometry and so on. PDX mouse model was used for further drug inhibition experiments in vivo. In this study, we analyzed the profiles of LncRNA and mRNA in FH-deficienct RCC samples, and we found that the LncRNA-MIR4435-2GH was specifically highly expressed in FH-deficient RCC compared with ccRCC. In vitro experiments demonstrated that MIR4435-2HG was regulated by Fumarate through histone demethylation, and the deletion of this gene could inhibit glutamine metabolism. RNA-pulldown experiments showed that MIR4435-2HG specifically binds to STAT1, which can transcriptionally activate GLS1. GLS1 inhibitor CB-839 could significantly suppress tumor growth in PDX tumor models. This study analyzed the molecular mechanism of MIR4435-2HG in regulating metabolic remodeling of FH-deficient RCC in clinical samples, cells and animal models by combining transcriptional and metabolic methods. We found that that GLS1 was a therapeutic target for this tumor, and MIR4435-2HG can be used as a drug sensitivity marker.

LncRNA FEZF1-AS1 facilitates cisplatin resistance in non-small cell lung cancer through modulating the miR-32-5p-glutaminase axis.

Non-small cell lung cancer (NSCLC) is one of the prevalent malignancies. Cisplatin (CDDP) is a conventional chemotherapeutic agent against NSCLC. However, inherent and acquired chemoresistance limited the effectiveness of cisplatin in treatment of NSCLC. This study aimed to investigate the roles and underlying mechanisms of lncRNA-FEZF1-AS1 in mediating cisplatin sensitivity in NSCLC. We found that FEZF1-AS1 levels were significantly higher in lung cancer patients and cell lines. Blocking FEZF1-AS1 sensitized lung cancer cells to cisplatin. Additionally, both glutamine metabolism and FEZF1-AS1 were significantly elevated in cisplatin resistant NSCLC cell lines, A549/CDDP R and SK-MES-1 CDDP/R. Analysis using bioinformatics, RNA pull-down assay and luciferase assay demonstrated that FEZF1-AS1 sponged miR-32-5p, which acted as a tumor suppressor in NSCLC. Glutaminase (GLS), a key enzyme in the glutamine metabolism, was predicted and validated as the direct target of miR-32-5p in NSCLC cells. Inhibiting glutamine metabolism or reducing glutamine supply effectively resensitized cisplatin-resistant cells. Furthermore, restoring miR-32-5p in FEZF1-AS1-overexpressing cisplatin resistant cells successfully overcame FEZF1-AS1-mediated cisplatin resistance by targeting GLS. These findings were further supported by in vivo xenograft mice experiments. This study uncovered the roles and molecular mechanisms of lncRNA FEZF1-AS1 in mediating cisplatin resistance in NSCLC, specifically through modulating the miR-32-5p-GLS axis, providing support for the development of new therapeutic approaches against chemoresistant lung cancer.

DON of Hope: Starving Pancreatic Cancer by Glutamine Antagonism.

A promising approach to treat solid tumors involves disrupting their reliance on glutamine, a key component for various metabolic processes. Traditional attempts using glutamine inhibitors like 6-diazo-5-oxo-L-norleucine (DON) and CB-839 were unsuccessful, but new hope arises with DRP-104, a prodrug of DON. This compound effectively targets tumor metabolism while minimizing side effects. In a recent study published in Nature Cancer, Encarnación-Rosado and colleagues demonstrated in preclinical models that pancreatic ductal adenocarcinoma (PDAC) responds well to DRP-104, although tumors adapt through the MEK/ERK signaling pathway, which can be countered by the MEK inhibitor trametinib. In a related study, Recouvreux and colleagues found that DON is effective against pancreatic tumors, revealing that PDAC tumors upregulate asparagine synthesis in response to DON, making them susceptible to asparaginase treatment. Both studies underscore the potential of inhibiting glutamine metabolism and adaptive pathways as a promising strategy against PDAC. These findings pave the way for upcoming clinical trials utilizing DRP-104 and similar glutamine antagonists in the battle against solid tumors.

A PD-L1-targeting Regulator for Metabolic Reprogramming to Enhance Glutamine Inhibition-Mediated Synergistic Antitumor Metabolic and Immune Therapy.

Inhibition of glutamine metabolism in tumor cells can cause metabolic compensation-mediated glycolysis enhancement and PD-L1 upregulation-induced immune evasion, significantly limiting the therapeutic efficacy of glutamine inhibitors. Here, inspired by the specific binding of receptor and ligand, a PD-L1-targeting metabolism and immune regulator (PMIR) are constructed by decorating the glutaminase inhibitor (BPTES)-loading zeolitic imidazolate framework (ZIF) with PD-L1-targeting peptides for regulating the metabolism within the tumor microenvironment (TME) to improve immunotherapy. At tumor sites, PMIR inhibits glutamine metabolism of tumor cells for elevating glutamine levels within the TME to improve the function of immune cells. Ingeniously, the accompanying PD-L1 upregulation on tumor cells causes self-amplifying accumulation of PMIR through PD-L1 targeting, while also blocking PD-L1, which has the effects of converting enemies into friends. Meanwhile, PMIR exactly offsets the compensatory glycolysis, while disrupting the redox homeostasis in tumor cells via the cooperation of components of the ZIF and BPTES. These together cause immunogenic cell death of tumor cells and relieve PD-L1-mediated immune evasion, further reshaping the immunosuppressive TME and evoking robust immune responses to effectively suppress bilateral tumor progression and metastasis. This work proposes a rational strategy to surmount the obstacles in glutamine inhibition for boosting existing clinical treatments.

Tripartite motif containing 33 demonstrated anticancer effect by degrading c‑Myc: Limitation of glutamine metabolism and proliferation in endometrial carcinoma cells.

Tripartite motif containing 33 (TRIM33) has been reported to be involved in various tumor progression. However, its role in endometrial carcinoma (EC) remains to be elucidated. By mining the publicly available databases UALCAN and TIMER, low expression of TRIM33 was found in tumor tissues of EC patients. Clinically, downregulation of TRIM33 in EC tissues was positively correlated with the extensive muscle invasion and poor differentiation grade. Invitro, experiments performed on human HEC‑1‑A and AN3CA cells showed that overexpression of TRIM33 inhibited the proliferation, migration and invasion of EC cells, whereas TRIM33 knockdown resulted in the opposite results. Furthermore, upregulation of TRIM33 significantly inhibited the glutamine uptake and decreased the intracellular glutamate in EC cells, which is evidenced by the reduction of solute carrier family 1 member 5 and glutaminase. Invivo, TRIM33 also dramatically inhibited tumor growth and glutamine metabolism. Additionally, co‑immunoprecipitation assay confirmed the interaction between TRIM33 and c‑Myc. Overexpression of TRIM33 could reduce the protein stability of c‑Myc by promoting its degradation. In addition, upregulation of c‑Myc could reverse the effects of TRIM33 on EC cells. Together, the present study demonstrated that TRIM33 acted as a tumor suppressor in EC, which is manifested in its inhibition of glutamine metabolism and cell growth via promoting c‑Myc protein degradation.