Glutaminase Inhibitor Research Articles

Blockade of glutamine-dependent cell survival augments antitumor efficacy of CPI-613 in head and neck cancer

BackgroundAlterations in metabolism are one of the emerging hallmarks of cancer cells and targeting dysregulated cancer metabolism provides a new approach to developing more selective therapeutics. However, insufficient blockade critical metabolic dependencies of cancer allows the development of metabolic bypasses, thus limiting therapeutic benefits.MethodsA series of head and neck squamous cell carcinoma (HNSCC) cell lines and animal models were used to determine the efficacy of CPI-613 and CB-839 when given alone or in combination. Glutaminase 1 (GLS1) depletion was achieved by lentiviral shRNAs. Cell viability and apoptosis were determined in HNSCC cells cultured in 2D culture dish and SeedEZ™ 3D scaffold. Molecular alterations were examined by Western blotting and immunohistochemistry. Metabolic changes were assessed by glucose uptake, lactate production, glutathione levels, and oxygen consumption rate.ResultsWe show here that HNSCC cells display strong addiction to glutamine. CPI-613, a novel lipoate analog, redirects cellular activity towards tumor-promoting glutaminolysis, leading to low anticancer efficacy in HNSCC cells. Mechanistically, CPI-613 inhibits the tricarboxylic acid cycle by blocking the enzyme activities of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which upregulates GLS1 and eventually promotes the compensatory role of glutaminolysis in cancer cell survival. Most importantly, the addition of a GLS1 inhibitor CB-839 to CPI-613 treatment abrogates the metabolic dependency of HNSCC cells on glutamine, achieving a synergistic anticancer effect in glutamine-addicted HNSCC.ConclusionsThese findings uncover the critical role of GLS1-mediated glutaminolysis in CPI-613 treatment and suggest that the CB-839 and CPI-613 combination may potentiate synergistic anticancer activity for HNSCC therapeutic gain.

A Combination of Glutaminase Inhibitor 968 and PD-L1 Blockade Boosts the Immune Response against Ovarian Cancer.

Programmed cell death 1 ligand (PD-L1) blockade has been used therapeutically in the treatment of ovarian cancer, and potential combination treatment approaches are under investigation to improve the treatment response rate. The increased dependence on glutamine is widely observed in various type of tumors, including ovarian cancer. Kidney-type glutaminase (GLS), as one of the isotypes of glutaminase, is found to promote tumorigenesis. Here, we have demonstrated that the combined treatment with GLS inhibitor 968 and PD-L1 blockade enhances the immune response against ovarian cancer. Survival analysis using the Kaplan–Meier plotter dataset from ovarian cancer patients revealed that the expression level of GLS predicts poor survival and correlates with the immunosuppressive microenvironment of ovarian cancer. 968 inhibits the proliferation of ovarian cancer cells and enhances granzyme B secretion by CD8+ T cells as detected by XTT assay and flow cytometry, respectively. Furthermore, 968 enhances the apoptosis-inducing ability of CD8+ T cells toward cancer cells and improves the treatment effect of anti-PD-L1 in treating ovarian cancer as assessed by Annexin V apoptosis assay. In vivo studies demonstrated the prolonged overall survival upon combined treatment of 968 with anti-PD-L1 accompanied by increased granzyme B secretion by CD4+ and CD8+ T cells isolated from ovarian tumor xenografts. Additionally, 968 increases the infiltration of CD3+ T cells into tumors, possibly through enhancing the secretion of CXCL10 and CXCL11 by tumor cells. In conclusion, our findings provide a novel insight into ovarian cancer cells influence the immune system in the tumor microenvironment and highlight the potential clinical implication of combination of immune checkpoints with GLS inhibitor 968 in treating ovarian cancer.

Circ-CREBBP promotes cell tumorigenesis and glutamine catabolism in glioma by regulating miR-375/glutaminase axis

Circular RNA CREB-binding protein (circ-CREBBP) has been reported to involve in the tumorigenesis of glioma. However, the role and underlying molecular mechanism of circ-CREBBP in glioma glutamine catabolism remain unclear. The expression of circ-CREBBP, microRNA (miR)-375 and glutaminase (GLS) was detected using quantitative real-time polymerase chain reaction and western blot. The 3‑(4, 5‑dimethylthiazol‑2‑y1)‑2, 5‑diphenyl tetrazolium bromide (MTT), colony formation, flow cytometry and transwell assays were used to determine the effects of them on glioma cell malignant biological behaviors in vitro. Glutamine metabolism was analyzed using assay kits. Murine xenograft model was established to investigate the role of circ-CREBBP in vivo. The binding interactions between miR-375 and circ-CREBBP or GLS were confirmed by the dual-luciferase reporter assay. Circ-CREBBP was highly expressed in glioma tissues and cells, and high expression of circ-CREBBP predicted poor prognosis. Circ-CREBBP knockdown suppressed cell proliferation, migration, invasion and glutamine metabolism while expedited cell apoptosis in glioma in vitro, as well as impeded tumor growth in vivo. Circ-CREBBP directly targeted miR-375, which was demonstrated to restrain glioma cell growth, motility and glutamine metabolism. Moreover, miR-375 inhibition reverted the anticancer effects of circ-CREBBP knockdown on glioma cells. GLS was a target of miR-375, GLS silencing or the treatment of GLS inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) impaired glioma cell malignant phenotypes and glutamine metabolism. Importantly, GLS up-regulation weakened the tumor-suppressive functions of miR-375 on glioma cells. Mechanistically, circ-CREBBP indirectly regulated GLS expression through sponging miR-375. In all, circ-CREBBP expedited glioma tumorigenesis and glutamine metabolism through miR-375/GLS axis, suggesting a promising target for combined glioma therapy.

Activation of the NRF2 antioxidant program sensitizes tumors to G6PD inhibition.

The KEAP1/NRF2 pathway promotes metabolic rewiring to support redox homeostasis. Activation of NRF2 occurs in many cancers, often due to KEAP1 mutations, and is associated with more aggressive disease and treatment resistance. To identify metabolic dependencies in cancers with NRF2 activation, we performed a metabolism-focused CRISPR screen. Glucose-6-phosphate dehydrogenase (G6PD), which was recently shown to be dispensable in Ras-driven tumors, was a top dependency. G6PD catalyzes the committed step of the oxidative pentose phosphate pathway that produces NADPH and nucleotide precursors, but neither antioxidants nor nucleosides rescued. Instead, G6PD loss triggered tricarboxylic acid (TCA) intermediate depletion because of up-regulation of the alternative NADPH-producing enzymes malic enzyme and isocitrate dehydrogenase. In vivo, G6PD impairment markedly suppressed KEAP1 mutant tumor growth, and this suppression was further augmented by TCA depletion by glutaminase inhibition. Thus, G6PD inhibition–induced TCA depletion is a therapeutic vulnerability of NRF2-activated cancer.

An Investigation into the Impact of a Glutaminase Inhibitor, Compound 968, on Nrf2 Signaling

Glutaminase is a critical enzyme that catalyzes the process of glutaminolysis for energy synthesis. Meanwhile, glutaminase also contributes to the pathological process of various diseases, such as cancer, neurodegenerative diseases, and inflammation. This leads to the discovery of glutaminase inhibitors for therapeutical uses. However, the mechanisms of the beneficial therapeutical effect of glutaminase inhibitors are still unclear. This pilot study aimed to determine the impact of a well-characterized glutaminase inhibitor, compound 968 (C968), on Nrf2 signaling. We performed molecular docking, luciferase assay, and quantitative PCR to determine the activation of Nrf2 and the expression of several Nrf2-related genes. These experiments found that C968 induced the Nrf2 activation and promoted the expression of Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H Quinone Dehydrogenase-1 (NQO-1). All findings provide evidence that Nrf2 activation could be one of the mechanisms contributing to the therapeutical activity of C968, but more studies are warranted to further confirm this mechanism.

Identification and characterization of a novel glutaminase inhibitor.

In humans, there are two forms of glutaminase (GLS), designated GLS1 and GLS2. These enzymes catalyse the conversion of glutamine to glutamate. GLS1 exists as two isozymes: kidney glutaminase (KGA) and glutaminase C (GAC). Several GLS inhibitors have been identified, of which DON (6‐diazo‐5‐oxonorleucine), BPTES (bis‐2‐(5‐phenylacetamido‐1, 3, 4‐thiadiazol‐2‐yl) ethyl sulphide), 968 (5‐(3‐Bromo‐4‐(dimethylamino)phenyl)‐2,2‐dimethyl‐2,3,5,6‐tetrahydrobenzo[a]phenanthridin‐4(1H)‐one) and CB839 (Telaglenastat) are the most widely used. However, these inhibitors have variable efficacy, specificity and bioavailability in research and clinical settings, implying the need for novel and improved GLS inhibitors. Based on this need, a diverse library of 28,000 compounds from Enamine was screened for inhibition of recombinant, purified GAC. From this library, one inhibitor designated compound 19 (C19) was identified with kinetic features revealing allosteric inhibition of GAC in the µm range. Moreover, C19 inhibits anti‐CD3/CD28‐induced CD4+ T‐cell proliferation and cytokine production with similar or greater potency as compared to BPTES. Taken together, our data suggest that C19 has the potential to modulate GLS1 activity and alter metabolic activity of T cells.

The Pivotal Role of Glutaminolysis in Multiple Myeloma: Novel Strategies for Target Therapies Against Myeloma

Introduction:Multiple myeloma (MM) is a uniformly fatal disorder of B cells characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Metabolic reprogramming is recognized as one of the hallmarks of cancer cells. Glutamine is the most abundant circulating amino acid in blood, glutamine metabolism through glutaminolysis may be associated with myeloma cell maintenance and survival.Materials and Methods:In this study, we investigated whether glutaminolysis was involved the proliferation in myeloma cells. We also investigated whether glutaminase (GLS) inhibitor, CB-839 could suppress myeloma cells and enhance the sensitivity of myeloma cells to histone deacetylase (HDAC) inhibition.Results:We first investigated the relationship between glutamine transporter or GLS gene expression and MM patients by microarray gene expression data from the online Gene Expression Omnibus (GEO). Glutamine transporter genes such as SLC38A1 and SLC1A5 were increased in myeloma and plasma cell leukemia cells (GSE13591). In contrast, GLS1 expression was not changed. We next investigated the glutaminolysis in myeloma cells. Deprivation of glutamine in culture medium revealed that cellular growth inhibition and cell cycle arrest at G0/G1 phase. Gene expression of AURKA (aurora kinase A), AURKB (aurora kinase B), HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A Member 1) and CCNB1 (cyclin B1) were reduced from the public microarray datasets (GSE59931) and protein expressions were also reduced by immunoblot analysis. We next evaluated the effect of GLS inhibitor, CB-839. 72 h treatment of MM cells were inhibited by CB-839 in a dose dependent manner. Cellular cytotoxicity was also increased. Glutamine is converted by GLS into glutamate and alpha-ketoglutarate (α-KG), and related nicotinamide adenine dinucleotide phosphate (NADP) production. Intracellular α-KG and NADPH were reduced by CB-839. As metabolites are the substrates used to generate chromatin modification including acetylation of histone, we investigated HDAC inhibitor, panobinostat in myeloma cells. 72 h treatment of MM cells were inhibited by panobinostat and histone acetylation was increased. Combined treatment with panobinostat and CB-839 caused more cytotoxicity than each drug alone. Panobinostat and CB-839 also inhibited bortezomib resistant cells. Caspase 3/7 activity and cellular cytotoxicity were also increased. Proteasomal activity was reduced. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by panobinostat and CB-839 on myeloma cells. Immunoblot analysis revealed that protein expression of aurora kinase A, aurora kinase B, HSP90 and cyclin B1 were reduced, and cleaved caspase 3 and γ- H2AX were increased by panobinostat and CB-839 treatment. GLS shRNA transfectant cells were inhibited cellular proliferation and sub-G1 phase was increased by cell cycle analysis. GLS shRNA transfectant cells were increased the sensitivity of panobinostat compared to control cells.Conclusion:The glutaminolysis is involved myeloma cell proliferation and GLS inhibitor is effective to myeloma cells and enhance cytotoxic effects of HDAC inhibitors. We also provide the promising clinical relevance as a candidate drug for treatment of myeloma patients. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition.A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis.To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells.Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. DisclosuresMinden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The glutaminase inhibitor telaglenastat enhances the antitumor activity of signal transduction inhibitors everolimus and cabozantinib in models of renal cell carcinoma.

Dysregulated metabolism is a hallmark of cancer that manifests through alterations in bioenergetic and biosynthetic pathways to enable tumor cell proliferation and survival. Tumor cells exhibit high rates of glycolysis, a phenomenon known as the Warburg effect, and an increase in glutamine consumption to support the tricarboxylic acid (TCA) cycle. Renal cell carcinoma (RCC) tumors express high levels of glutaminase (GLS), the enzyme required for the first step in metabolic conversion of glutamine to glutamate and the entry of glutamine into the TCA cycle. We found that RCC cells are highly dependent on glutamine for proliferation, and this dependence strongly correlated with sensitivity to telaglenstat (CB-839), an investigational, first-in-class, selective, orally bioavailable GLS inhibitor. Metabolic profiling of RCC cell lines treated with telaglenastat revealed a decrease in glutamine consumption, which was concomitant with a decrease in the production of glutamate and other glutamine-derived metabolites, consistent with GLS inhibition. Treatment of RCC cells with signal transduction inhibitors everolimus (mTOR inhibitor) or cabozantinib (VEGFR/MET/AXL inhibitor) in combination with telaglenastat resulted in decreased consumption of both glucose and glutamine and synergistic anti-proliferative effects. Treatment of mice bearing Caki-1 RCC xenograft tumors with cabozantinib plus telaglenastat resulted in reduced tumor growth compared to either agent alone. Enhanced anti-tumor activity was also observed with the combination of everolimus plus telaglenastat. Collectively, our results demonstrate potent, synergistic, anti-tumor activity of telaglenastat plus signal transduction inhibitors cabozantinib or everolimus via a mechanism involving dual inhibition of glucose and glutamine consumption.

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

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

Recent Advances in Development of Glutaminase Inhibitors as Anticancer Agents

Glutaminolysis is closely related to uncontrolled tumor growth and metastasis in various glutamine-dependent cancers. As such, rewiring cancer metabolism using a glutaminolysis intervention has attracted much attention with recent progress in developing glutaminase inhibitors as anticancer agents. This review paper described the current understanding of glutaminases and their roles in cancers and small-molecule glutaminase inhibitors. In particular, recent developments of natural and synthetic glutaminase inhibitors were covered, focusing on their binding sites and isoform selectivity and briefly, on their structure-activity relationship. Some of the glutaminase inhibitors are in clinical trials, and if successful, it would lead to novel pharmacological intervention to effectively treat cancers.

Glutamine anaplerosis is required for amino acid biosynthesis in human meningiomas.

We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unraveling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells. 1H NMR spectroscopy of tumor tissues from meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using 13C/15N glutamine tracer, in 5 patient-derived meningioma cells. Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75%, and 33%, respectively, with alanine and glutamine levels being statistically significant (P ≤ .02). 13C/15N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells. Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) is very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Gilteritinib Inhibits Glutamine Uptake and Utilization in FLT3-ITD-Positive AML.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's antileukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. In addition, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Finally, we found that the glutaminase inhibitor CB-839 enhanced antileukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Inhibition of Metabolism as a Therapeutic Option for Tamoxifen-Resistant Breast Cancer Cells

Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential.

Simple SummaryIn chronic myeloid leukemia (CML), a neoplasm brilliantly taken care of by a molecularly targeted therapeutic approach, the achievement of cure is nevertheless prevented by the maintenance of a small subset of treatment-resistant leukemia stem cells (LSCs), sustaining the so-called minimal residual disease of CML. The phenotypical and functional characterization of this LSC subset is, therefore, crucial to aim at the eradication of disease. Such a characterization includes the acquisition of information relative to the metabolic profile of treatment-resistant LSCs, which is functional to their maintenance in bone marrow. A number of metabolic features of LSCs were shown to determine their sensitivity or resistance to therapy. Glutamine metabolism emerged from this study as a potential target to overcome the persistence of therapy-resistant LSCs.This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.

Reproducible Lipid Alterations in Patient-Derived Breast Cancer Xenograft FFPE Tissue Identified with MALDI MSI for Pre-Clinical and Clinical Application.

The association between lipid metabolism and long-term outcomes is relevant for tumor diagnosis and therapy. Archival material such as formalin-fixed and paraffin embedded (FFPE) tissues is a highly valuable resource for this aim as it is linked to long-term clinical follow-up. Therefore, there is a need to develop robust methodologies able to detect lipids in FFPE material and correlate them with clinical outcomes. In this work, lipidic alterations were investigated in patient-derived xenograft of breast cancer by using a matrix-assisted laser desorption ionization mass spectrometry (MALDI MSI) based workflow that included antigen retrieval as a sample preparation step. We evaluated technical reproducibility, spatial metabolic differentiation within tissue compartments, and treatment response induced by a glutaminase inhibitor (CB-839). This protocol shows a good inter-day robustness (CV = 26 ± 12%). Several lipids could reliably distinguish necrotic and tumor regions across the technical replicates. Moreover, this protocol identified distinct alterations in the tissue lipidome of xenograft treated with glutaminase inhibitors. In conclusion, lipidic alterations in FFPE tissue of breast cancer xenograft observed in this study are a step-forward to a robust and reproducible MALDI-MSI based workflow for pre-clinical and clinical applications.

Targeting Metabolic Reprogramming to Improve Breast Cancer Treatment: An In Vitro Evaluation of Selected Metabolic Inhibitors Using a Metabolomic Approach.

Characteristic metabolic adaptations are recognized as a cancer hallmark. Breast cancer, like other cancer types, displays cellular respiratory switches—in particular, the Warburg effect—and important fluctuations in the glutamine and choline metabolisms. This cancer remains a world health issue mainly due to the side effects associated with chemotherapy, which force a reduction in the administered dose or even a complete discontinuation of the treatment. For example, Doxorubicin is efficient to treat breast cancer but unfortunately induces severe cardiotoxicity. In the present in vitro study, selected metabolic inhibitors were evaluated alone or in combination as potential treatments against breast cancer. In addition, the same inhibitors were used to possibly potentiate the effects of Doxorubicin. As a result, the combination of CB-839 (glutaminase inhibitor) and Oxamate (lactate dehydrogenase inhibitor) and the combination of CB-839/Oxamate/D609 (a phosphatidylcholine-specific phospholipase C inhibitor) caused significant cell mortality in both MDA-MB-231 and MCF-7, two breast cancer cell lines. Furthermore, all inhibitors were able to improve the efficacy of Doxorubicin on the same cell lines. Those findings are quite encouraging with respect to the clinical goal of reducing the exposure of patients to Doxorubicin and, subsequently, the severity of the associated cardiotoxicity, while keeping the same treatment efficacy.

Compound 968 reverses adriamycin resistance in breast cancer MCF-7ADR cells via inhibiting P-glycoprotein function independently of glutaminase

Adriamycin (ADR) is a chemotherapeutic drug widely utilized to treat multiple types of cancers; however, the clinical efficacy of ADR is compromised due to the development of drug resistance in patients. The combination of drugs with ADR may provide a better therapeutic regimen to overcome this obstacle. Glutaminase (GLS) has been explored as a therapeutic cancer target, and its inhibition also results in increased sensitivity of tumor cells to chemotherapeutic agents. This study aimed to investigate whether GLS inhibition could reverse ADR resistance. We treated the ADR-resistant MCF-7 (MCF-7ADR) cells with a GLS inhibitor, compound 968 or CB-839, in combination with ADR. We found that compound 968, rather than CB-839, together with ADR synergistically inhibited the cell viability. These results indicated that compound 968 reversed ADR resistance in MCF-7ADR cells independently of GLS. Moreover, we modified the structure of compound 968 and finally obtained a compound 968 derivative, SY-1320, which was more potent than compound 968 in eliminating the drug resistance in MCF-7ADR cells. Furthermore, using drug affinity responsive target stability and streptavidin–biotin immunoprecipitation assays, we demonstrated that SY-1320 could specifically target P-glycoprotein (P-gp) and increase ADR accumulation through inhibition of P-gp, thereby resulting in cell death in MCF-7ADR cells. Together, our findings indicate that compound 968 or SY-1320 might be a promising drug for new combination chemotherapy in breast cancer to overcome the drug resistance.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

A Phase I Dose-Escalation and Expansion Study of Telaglenastat in Patients with Advanced or Metastatic Solid Tumors.

Glutamine is a critical fuel for solid tumors. Interference with glutamine metabolism is deleterious to neoplasia in preclinical models. A phase I study of the oral, first-in-class, glutaminase (GLS) inhibitor telaglenastat was conducted in treatment-refractory solid tumor patients to define recommended phase II dose (RP2D) and evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity. Dose escalation by 3 + 3 design was followed by exploratory tumor-/biomarker-specific cohorts. Among 120 patients, fatigue (23%) and nausea (19%) were the most common toxicity. Maximum tolerated dose was not reached. Correlative analysis indicated >90% GLS inhibition in platelets at plasma exposures >300 nmol/L, >75% tumoral GLS inhibition, and significant increase in circulating glutamine. RP2D was defined at 800 mg twice-daily. Disease control rate (DCR) was 43% across expansion cohorts (overall response rate 5%, DCR 50% in renal cell carcinoma). Telaglenastat is safe, with a favorable PK/PD profile and signal of antitumor activity, supporting further clinical development.