Inhibition Of Glutamine Metabolism Research Articles

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.

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.

Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma.

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Here, we elucidate how 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism, blocks PDAC tumor growth and metastasis. We find that DON significantly reduces asparagine production by inhibiting asparagine synthetase (ASNS), and that the effects of DON are rescued by asparagine. As a metabolic adaptation, PDAC cells upregulate ASNS expression in response to DON, and we show that ASNS levels are inversely correlated with DON efficacy. We also show that L-asparaginase (ASNase) synergizes with DON to affect the viability of PDAC cells, and that DON and ASNase combination therapy has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of cotargeting adaptive responses to control PDAC progression.

Monocarboxylate Transporter-1 (MCT1)-Mediated Lactate Uptake Protects Pancreatic Adenocarcinoma Cells from Oxidative Stress during Glutamine Scarcity Thereby Promoting Resistance against Inhibitors of Glutamine Metabolism.

Metabolic compartmentalization of stroma-rich tumors, like pancreatic ductal adenocarcinoma (PDAC), greatly contributes to malignancy. This involves cancer cells importing lactate from the microenvironment (reverse Warburg cells) through monocarboxylate transporter-1 (MCT1) along with substantial phenotype alterations. Here, we report that the reverse Warburg phenotype of PDAC cells compensated for the shortage of glutamine as an essential metabolite for redox homeostasis. Thus, oxidative stress caused by glutamine depletion led to an Nrf2-dependent induction of MCT1 expression in pancreatic T3M4 and A818-6 cells. Moreover, greater MCT1 expression was detected in glutamine-scarce regions within tumor tissues from PDAC patients. MCT1-driven lactate uptake supported the neutralization of reactive oxygen species excessively produced under glutamine shortage and the resulting drop in glutathione levels that were restored by the imported lactate. Consequently, PDAC cells showed greater survival and growth under glutamine depletion when utilizing lactate through MCT1. Likewise, the glutamine uptake inhibitor V9302 and glutaminase-1 inhibitor CB839 induced oxidative stress in PDAC cells, along with cell death and cell cycle arrest that were again compensated by MCT1 upregulation and forced lactate uptake. Our findings show a novel mechanism by which PDAC cells adapt their metabolism to glutamine scarcity and by which they develop resistance against anticancer treatments based on glutamine uptake/metabolism inhibition.

Critical role of antioxidant programs in enzalutamide-resistant prostate cancer.

Therapy resistance to second-generation androgen receptor (AR) antagonists, such as enzalutamide, is common in patients with advanced prostate cancer (PCa). To understand the metabolic alterations involved in enzalutamide resistance, we performed metabolomic, transcriptomic, and cistromic analyses of enzalutamide-sensitive and -resistant PCa cells, xenografts, patient-derived organoids, patient-derived explants, and tumors. We noted dramatically higher basal and inducible levels of reactive oxygen species (ROS) in enzalutamide-resistant PCa and castration-resistant PCa (CRPC), in comparison to enzalutamide-sensitive PCa cells or primary therapy-naive tumors respectively. Unbiased metabolomic evaluation identified that glutamine metabolism was consistently upregulated in enzalutamide-resistant PCa cells and CRPC tumors. Stable isotope tracing studies suggest that this enhanced glutamine metabolism drives an antioxidant program that allows these cells to tolerate higher basal levels of ROS. Inhibition of glutamine metabolism with either a small-molecule glutaminase inhibitor or genetic knockout of glutaminase enhanced ROS levels, and blocked the growth of enzalutamide-resistant PCa. The critical role of compensatory antioxidant pathways in maintaining enzalutamide-resistant PCa cells was validated by targeting another antioxidant program driver, ferredoxin 1. Taken together, our data identify a metabolic need to maintain antioxidant programs and a potentially targetable metabolic vulnerability in enzalutamide-resistant PCa.

Differential Effects of Glutamine Inhibition Strategies on Antitumor CD8 T Cells.

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes.

Detachable MOF-Based Core/Shell Nanoreactor for Cancer Dual-Starvation Therapy With Reversing Glucose and Glutamine Metabolisms.

Tumor-dependent glucose and glutamine metabolisms are essential for maintaining survival, while the accordingly metabolic suppressive therapy is limited by the compensatory metabolism and inefficient delivery efficiency. Herein, a functional metal-organic framework (MOF)-based nanosystem composed of the weakly acidic tumor microenvironment-activated detachable shell and reactive oxygen species (ROS)-responsive disassembledMOF nanoreactor core is designed to co-load glycolysis and glutamine metabolism inhibitors glucose oxidase (GOD) and bis-2-(5-phenylacetmido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES) for tumor dual-starvation therapy. The nanosystem excitingly improves tumor penetration and cellular uptake efficiency via integrating the pH-responsive size reduction and charge reversal and ROS-sensitive MOF disintegration and drug release strategy. Furthermore, the degradation of MOF and cargoes release can be self-amplified via additional self-generation H2 O2 mediated by GOD. Last, the released GOD and BPTES collaboratively cut off the energy supply of tumors and induce significant mitochondrial damage and cell cycle arrest via simultaneous restriction of glycolysis and compensatory glutamine metabolism pathways, consequently realizing the remarkable triple negative breast cancer killing effect in vivo with good biosafety via the dual starvation therapy.

Abstract 286: Targeting metabolic vulnerabilities in acute myeloid leukemia

Abstract Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Importantly, Gln depletion has emerged as a therapeutic approach for cancers that have undergone metabolic reprogramming and have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Asparaginases are bacterial enzymes that hydrolyze plasma glutamine to glutamate and ammonia, depriving glutamine-dependent AML cells from one of their vital nutrients. Preliminary data and literature have shown that following treatment with asparaginases in vitro there is a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase cytotoxicity in AML cells. We will test this by targeting, phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, which is a very common target for serine biosynthesis. We determined that crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in the proliferation, growth, and expansion of AML cells, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media as well as following crisantaspase treatment. We set out to explore the effects of PHGDH knockout and observed that the PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from, ~250-fold lower compared to the parental lines. Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Indicating enhanced oxidative stress and cytotoxicity when both Gln and Ser are targeting in combination. Additionally, silencing of PHGDH reinforced anti-leukemic effects of glutamine modulation by different glutamine metabolism inhibitors, including V9302, CB-839 and BPTES. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (BI4916) demonstrated an inhibition of AML cell proliferation in multiple cell lines. It has also demonstrated potential synergism and increase in cellular cytotoxicity when used in combination with Rylaze. Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Citation Format: Kanwal Mahmood Hameed, Ashkan Emadi. Targeting metabolic vulnerabilities in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 286.

Rethinking glutamine metabolism and the regulation of glutamine addiction by oncogenes in cancer.

Glutamine, the most abundant non-essential amino acid in human blood, is crucial for cancer cell growth and cancer progression. Glutamine mainly functions as a carbon and nitrogen source for biosynthesis, energy metabolism, and redox homeostasis maintenance in cancer cells. Dysregulated glutamine metabolism is a notable metabolic characteristic of cancer cells. Some carcinogen-driven cancers exhibit a marked dependence on glutamine, also known as glutamine addiction, which has rendered the glutamine metabolic pathway a breakpoint in cancer therapeutics. However, some cancer cells can adapt to the glutamine unavailability by reprogramming metabolism, thus limiting the success of this therapeutic approach. Given the complexity of metabolic networks and the limited impact of inhibiting glutamine metabolism alone, the combination of glutamine metabolism inhibition and other therapeutic methods may outperform corresponding monotherapies in the treatment of cancers. This review summarizes the uptake, transport, and metabolic characteristics of glutamine, as well as the regulation of glutamine dependence by some important oncogenes in various cancers to emphasize the therapeutic potential of targeting glutamine metabolism. Furthermore, we discuss a glutamine metabolic pathway, the glutaminase II pathway, that has been substantially overlooked. Finally, we discuss the applicability of polytherapeutic strategies targeting glutamine metabolism to provide a new perspective on cancer therapeutics.

Glutamine metabolism targeting liposomes for synergistic chemosensitization and starvation therapy in ovarian cancer.

Platinum-based chemotherapy is a first-line therapeutic regimen against ovarian cancer (OC); however, the therapeutic potential is always reduced by glutamine metabolism. Herein, a valid strategy of inhibiting glutamine metabolism was proposed to cause tumor starvation and chemosensitization. Specifically, reactive oxygen species-responsive liposomes were developed to co-deliver cisplatin (CDDP) and bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) [C@B LPs]. The C@B LPs induced effective tumor cell starvation and significantly sensitized OC cells to CDDP by reducing glutathione generation to prevent CDDP detoxification, suppressing ATP production to avoid CDDP efflux, hindering nucleotide synthesis to aggravate DNA damage induced by CDDP, and blocking mammalian target of rapamycin (mTOR) signaling to promote cell apoptosis. More importantly, C@B LPs remarkably inhibited tumor growth in vivo and reduced the side effects. Taken together, this study provided a successful strategy of synergistic chemosensitization and starvation therapy escalating the rate of therapeutic success in OCs. STATEMENT OF SIGNIFICANCE: This work proposed a valid strategy of inhibiting glutamine metabolism to cause tumor starvation and chemosensitization. Specifically, ROS-responsive liposomes were developed to co-deliver cisplatin CDDP and BPTES [C@B LPs]. The C@B LPs induced effective tumor cell starvation and significantly sensitized OC cells to cisplatin by reducing glutathione generation to prevent cisplatin detoxification, suppressing ATP production to avoid cisplatin efflux, hindering nucleotide synthesis to aggravate DNA damage induced by cisplatin, and blocking mTOR signaling to promote cell apoptosis. More importantly, C@B LPs remarkably inhibited tumor growth in vivo and reduced the side effects. Taken together, this study provided a successful strategy of synergistic chemosensitization and starvation therapy escalating the rate of therapeutic success in OCs.

Abstract P6-11-11: Breast cancer stem cell marker, CD24 regulates metabolic reprogramming in triple negative breast cancer

Abstract Breast cancer stem cell (BCSC) is a subset of cancer cells that can dictate the tumor initiation, metastatic progression, and therapeutic resistance in BC. The eradication of this unusual population is emerging as a new paradigm in cancer treatment. Molecularly, CD24 negativity in conjunction with high expression of CD44 is considered a hallmark for the BCSCs. While extensive studies have been performed to delineate the role of CD44 in BCSCs, the regulation of CD24 and its functional role have not been fully understood. In this study, we investigated the regulatory mechanisms responsible for low CD24 expression in BCSCs and their functional relevance in BCSCs. Analysis of DNA from tumor tissues and blood from BC patients as well as BCSCs sorted from BC cell lines suggest that CD24 is epigenetically regulated via DNMT-1/HDAC1-dependent increased methylation of CpG islands in the CD24 proximal promoter region. To understand the role of CD24 in triple-negative BC (TNBC), an aggressive subgroup of BC, we knocked down (CD24-KD) and overexpressed (CD24-OE) CD24 in metastatic TNBC cells. While CD24-KD resulted in increased proliferation and stemness, CD24-OE diminished the proliferative and stem-like potential. To further identify the signaling cascade underpinning these effects, we performed phospho-proteome analysis using Reverse Phase Protein Array (RPPA). Remarkably, we observed an enhanced activation of AMPK and NF-kB signaling cascades, and reduced PDGFRβ signaling upon depletion of CD24. As signaling cascades are intricately linked to cellular metabolism, we performed a metabolomic analysis of CD24-KD and CD24-OE cells. Our comprehensive analysis revealed heightened activation of mitochondrial fatty acid β-oxidation (FAO) in CD24-KD and increased glutamine metabolism in CD24-OE cells. In coherence with these findings, we observed significant regulation of genes related to fatty acid metabolism in the TNBC patient cohort expressing low levels of CD24. Consistently, the CD24-KD cells demonstrated increased sensitivity towards FAO inhibitors while CD24-OE cells were more sensitive to glutamine metabolism inhibitors. In vivo studies to further understand the translational significance of this metabolic axis is underway. Taken together, our study demonstrates, for the first time, that CD24 presents a novel metabolic vulnerability that can target BCSCs to gain a therapeutic advantage in the treatment of drug-resistant TNBC patients. Citation Format: Divya Murthy, Debasmita Dutta, Sukjin Yang, Junhyoung Park, Benny Kaipparettu. Breast cancer stem cell marker, CD24 regulates metabolic reprogramming in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-11.

Liver X Receptor Inverse Agonist GAC0001E5 Impedes Glutaminolysis and Disrupts Redox Homeostasis in Breast Cancer Cells.

Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors which regulate the expression of lipid and cholesterol metabolism genes. Moreover, LXRs and their ligands have been shown to inhibit tumor growth in a variety of cancers. We have previously identified the small molecule compound GAC0001E5 (1E5) as an LXR inverse agonist and a potent inhibitor of pancreatic cancer cells. Transcriptomic and metabolomic studies showed that 1E5 disrupts glutamine metabolism, an essential metabolic pathway commonly reprogrammed during malignant transformation, including in breast cancers. To determine the role of LXRs and potential application of 1E5 in breast cancer, we examined LXR expression in publicly available clinical samples, and found that LXR expression is elevated in breast tumors as compared to normal tissues. In luminal A, endocrine therapy-resistant, and triple-negative breast cancer cells, 1E5 exhibited LXR inverse agonist and "degrader" activity and strongly inhibited cell proliferation and colony formation. Treatments with 1E5 downregulated the transcription of key glutaminolysis genes, and, correspondingly, biochemical assays indicated that 1E5 lowered intracellular glutamate and glutathione levels and increased reactive oxygen species. These results indicate that novel LXR ligand 1E5 is an inhibitor of glutamine metabolism and redox homeostasis in breast cancers and suggest that modulating LXR activity and expression in tumor cells is a promising strategy for targeting metabolic reprogramming in breast cancer therapeutics.