CB-839 Treatment Research Articles

CB-839 induces reversible dormancy in lung tumor-cells

Glutaminase inhibitors are currently being explored as potential treatments for cancer. This study aimed to elucidate the molecular mechanisms underlying the effects of CB-839 on lung tumor cell lines compared to non-tumor cell lines. Viability assays based on NADPH-dependent dehydrogenases activity, ATP energy production, or mitochondrial reductase activity were used to determine that CB-839 caused significant tumor cell specific inhibition of cellular functions. Clonogenic survival assay revealed a dose dependent reduction in clonogenic survival of various lung tumor cells presenting estimated IC50 values between 10 and 90 nM, while no effect on non-tumor cells was observed. CB-839 led to a 20% reduction in glutaminase (GLS1, a mitochondrial enzyme that catalyzes the conversion of glutamine to glutamate) activity, and a dose-dependent reduced glutamine consumption in tumor cells and had no effect on non-tumor cells. Cell cycle analysis showed the CB-839 did not lead to cell cycle arrest. Apoptosis and necrosis assays revealed an only slight increase in apoptosis in tumor cells. Furthermore, a trypan blue exclusion assay revealed about 40% growth reduction in tumor cells at 0.1–1 μM CB-839 treatment. Surprisingly, treated cells resumed normal growth when re-plated in a drug-free medium, demonstrating reversibility. In hypoxic conditions, CB-839's effect on clonogenic survival was amplified in a dose dependent manner consistent with increased role of GLS1 for energy production under hypoxic conditions. In conclusion, these results suggest CB-839 efficacy is linked to temporary and reversible reduction in glutamine utilization suggesting induction of dormancy.

Therapeutic Targeting of the GLS1-c-Myc Positive Feedback Loop Suppresses Glutaminolysis and Inhibits Progression of Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is addicted to glutaminolysis. Targeting this metabolic dependency has emerged as a potential therapeutic approach for HNSCC. In this study, we conducted a bioinformatic analysis of The Cancer Genome Atlas HNSCC cohort that revealed a robust correlation between expression of MYC (encoding the protein c-Myc) and glutaminase 1 (GLS1), which catalyzes the first step in glutaminolysis. Intriguingly, disruption of GLS1 signaling in HNSCC cells by genetic depletion or CB-839 treatment resulted in a reduction in c-Myc protein stability via a ubiquitin-specific peptidase 1-dependent ubiquitin-proteasome pathway. On the other hand, c-Myc directly binds to the promoter region of GLS1 and upregulates its transcription. Notably, the GLS1-c-Myc pathway enhanced acetyl-coenzyme A carboxylase-dependent Slug acetylation, prompting cancer cell invasion and metastasis. Thus, the GLS1-c-Myc axis emerged as a positive feedback loop critical for driving the aggressiveness of HNSCC. Therapeutically, combining CB-839 with the c-Myc inhibitor MYCi975 strongly suppressed GLS1-c-Myc signaling, resulting in a superior antitumor effect compared with either single agent in an orthotopic mouse model of HNSCC. These findings hold promise for the development of effective therapies for patients with HNSCC, addressing an urgent need arising from the significant incidence and high metastatic rate of the disease. Significance: GLS1 and c-Myc form a positive feedback loop that promotes head and neck cancer metastasis and can be targeted as a promising therapeutic strategy for this disease.

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

BackgroundGlutaminase 1 (GLS1), a key enzyme in glutamine metabolism in cancer cells, acts as a tumor promoter and could be a potential therapeutic target. CB-839, a GLS1-specific inhibitor, was developed recently. Herein, we aimed to elucidate the anti-tumor effects and mechanism of action of CB-839 in colorectal cancer (CRC). MethodsUsing the UCSC Xena public database, we evaluated GLS1 expression in various cancers. Immunostaining for GLS1 was performed on 154 surgically resected human CRC specimens. Subsequently, we examined the GLS1 mRNA expression levels in eight CRC cell lines and evaluated the association between GLS1 expression and CB-839 efficacy. To create a reproducible CRC model with abundant stroma and an allogeneic immune response, we co-transplanted CT26 and stem cells into BALB/c mice and treated them with CB-839. Finally, RNA sequencing of mouse tumors was performed. ResultsDatabase analysis showed higher GLS1 expression in CRC tissues than in normal colon tissues. Clinical samples from 114 of the 154 patients with CRC showed positive GLS1 expression. GLS1 expression in clinical CRC tissues correlated with vascular invasion. CB-839 treatment inhibited cancer cell proliferation depending on GLS1 expression in vitro and inhibited tumor growth and metastasis in the CRC mouse model. RNA sequencing revealed that CB-839 treatment inhibited stromal activation, tumor growth, migration, and angiogenesis. These findings were validated through in vitro and in vivo experiments and clinical specimen analysis. ConclusionsGLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

Abstract PO2-15-11: Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models

Abstract Background: Glutamine is an important metabolic substrate in many aggressive tumors, with comparable importance to glucose metabolism. Utilizing human breast cancer mouse xenograft models, we studied the kinetics of the PET imaging agent, [5-11C] glutamine, a biochemical authentic substrate for glutamine metabolism, to further characterize the metabolism of glutamine and downstream labeled metabolites. Studies were performed with and without inhibition of the enzyme, glutaminase (GLS), the first step in glutamine catabolism that generates glutamate, and key target for therapy directed to glutamine-metabolizing cancers. Methods: The study used xenograft mouse models for two breast cancer cell lines, HCC1806, a highly glutaminolytic triple-negative cell line, and MCF-7, a hormone receptor positive line with only low levels of glutaminolysis. Mice were injected with 5-[11C]L-glutamine, and the contributions of individual metabolites to the total [11C] signal in blood and tumor tissue were estimated at 10, 20, and 30 minutes after injection by assaying the fractional contributions of [5-11C] glutamine and labeled metabolites, specifically [5-11C] glutamate, [11C] CO2 and all other metabolites with [11C], in both blood and tumor tissue. This generated estimated time activity curves for [5-11C] glutamine and downstream metabolites in both HCC1806 and MCF-7 mouse models with and without CB-839 treatment, an inhibitor of GLS. We also investigated a compartment model that describes these data, including model simulations that fit the model to match the measured curves. Results: We found that, out to 30 minutes post-injection, the vast majority of tumor radioactivity was in the form of either 5-11C] glutamine or [5-11C] glutamate, with smaller amounts of radioactivity in the form of more downstream metabolites, including [11C]CO2. Systemic administration of the glutaminase inhibitor alters the arterial input curve by reducing the systemic rate of conversion of [5-11C] glutamine to [5-11C] glutamate. However, in GLS active tumors, [5-11C] glutamate retained in the large cellular glutamate pool represents a greater fraction of total radioactivity than in the blood and leads to a total tumor time-activity curve that is only marginally different than the total tumor time-activity curve after GLS inhibition. Data from blood and tissue analysis were used to test a hypothetical kinetic model for [5-11C] glutamine, which provided estimates of glutamate and glutamate tumor tissue concentrations and tumor GLS activity that agree with direct measurements in the animal models. Conclusion: The study of [ 5-11C] glutamine in breast cancer models leads to kinetic insight that are in line with biochemical studies and direct measurement taken for tumor tissue obtained from the animal models. Retention of metabolized [5-11C] glutamine as [5-11C] glutamate in a large tissue pool in tumors with high GLS activity makes non-invasive inference of GLS activity challenging without additional ex vivo analysis, supporting the use of non-metabolized glutamine analogs such as such as [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) to infer in vivo glutamine metabolism. Citation Format: Christopher Hensley, Prashanth Padakanti, Hoon Choi, Christina Dulal, Hsiaojiu Lee, Austin Pantel, Rong Zhou, David Mankoff. Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-11.

Abstract 3061: A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness

Abstract Both GLS1 and c-Myc are upregulated in head and neck squamous cell carcinoma (HNSCC) primary tumors and further increased in metastatic tumors. However, the regulation between these two molecules remains largely unknown. GLS1 is a critical enzyme that regulates glutamate, which plays an important role in cancer metabolism that supports cancer growth and survival. Our bioinformatic analysis of the TCGA HNSCC cohort revealed a strong correlation between c-Myc and GLS1 expression. We describe the importance of c-Myc in regulating GLS1 and vice versa. c-Myc protein directly binds to the promoter of the GLS1 gene and upregulates its expression at the transcriptional level. Interestingly, blocking GLS1 signaling in HNSCC cells by lentiviral shRNA knockdown or CB-839 treatment downregulates USP1, one of the best characterized human DUBs, which in turn reduces c-Myc protein stability via the ubiquitin-proteasome pathway. The GLS1-c-Myc axis thus represents a novel positive feedback loop that is critical for driving the aggressiveness of HNSCC. As the treatment of HNSCC remains a major challenge, these novel findings provide the molecular basis for combining GLS1-specific inhibitors with c-Myc-targeted therapy for the treatment of HNSCC patients. Citation Format: Jianqiang Yang, Fanghui Chen, Fan Yang, Yong Teng. A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness [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 3061.

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.

Metabolic Adjustments following Glutaminase Inhibition by CB-839 in Glioblastoma Cell Lines.

Most tumor cells can use glutamine (Gln) for energy generation and biosynthetic purposes. Glutaminases (GAs) convert Gln into glutamate and ammonium. In humans, GAs are encoded by two genes: GLS and GLS2. In glioblastoma, GLS is commonly overexpressed and considered pro-oncogenic. We studied the metabolic effects of inhibiting GLS activity in T98G, LN229, and U87MG human glioblastoma cell lines by using the inhibitor CB-839. We performed metabolomics and isotope tracing experiments using U-13C-labeled Gln, as well as 15N-labeled Gln in the amide group, to determine the metabolic fates of Gln carbon and nitrogen atoms. In the presence of the inhibitor, the results showed an accumulation of Gln and lower levels of tricarboxylic acid cycle intermediates, and aspartate, along with a decreased oxidative labeling and diminished reductive carboxylation-related labeling of these metabolites. Additionally, CB-839 treatment caused decreased levels of metabolites from pyrimidine biosynthesis and an accumulation of intermediate metabolites in the de novo purine nucleotide biosynthesis pathway. The levels of some acetylated and methylated metabolites were significantly increased, including acetyl-carnitine, trimethyl-lysine, and 5-methylcytosine. In conclusion, we analyzed the metabolic landscape caused by the GLS inhibition of CB-839 in human glioma cells, which might lead to the future development of new combination therapies with CB-839.

Effects of Glutaminase Inhibition on Venetoclax, Ibrutinib, and AZD-5991 Resistance in Chronic Lymphocytic Leukemia Cell Lines

Introduction: Bruton tyrosine kinase and BCL-2 inhibitors are widely used in CLL, but resistance problem is highly relevant. One of the resistance mechanisms may be metabolic rewiring followed by activation of oxidative phosphorylation. Glutamine plays a major role in cancer metabolism, and glutamine limitation rather than glucose contributes to lower oxygen uptake and mediates apoptosis of cancer cells. It was shown that glutamine metabolism changes contribute to venetoclax (Chen Z. et al, Blood, 2021; Guièze R. et al. Cancer Cell, 2019) and ibrutinib (Galicia-Vázquez G. et al. Frontiers in Oncology, 2018) resistance. We investigated in vitro effects of glutaminase-1 (GLS-1) enzyme inhibition and its potential synergistic effects with venetoclax, ibrutinib, and MCL-1 inhibitor AZD-5991 on resistant cell lines. CB-839 (telaglenastat) is a potent, reversible, and selective GLS-1 inhibitor with 24 nM IC50 for recombinant human mitochondrial glutaminase C. The compound was tested in the clinic for hematological malignancies and solid tumors. Methods: We have tested CB-839 alone or in combination with venetoclax, ibrutinib, and AZD-5991 on resistant HG-3 and MEC-1 CLL cell lines. Cells were incubated with 0.01 to 100 μM of CB-839 for 72 hours. The inhibition of glutamine, glutamate, and glutathione induced by CB-839 alone was measured using a luminescence assay. DCFDA/ H2DCFDA and MitoSOX Red stainings were utilized to evaluate cellular reactive oxygen species (Total ROS) and mitochondrial superoxide (MitoSOX) accumulation, respectively. Intracellular nucleotides including ATP level were measured using high-pressure liquid chromatography. For drug combinations, MTS assay and Annexin V/Propidium Iodide staining were used to assess cell proliferation and apoptosis, respectively. The combination index (CI) for synergy was calculated using Compusyn software. Results: HG3 and MEC1 cell lines were resistant to venetoclax, ibrutinib, and AZD-5991 single agents. The IC50 values for HG3 are 8.1 μM, 1.3 μM, 9.9 μM, and for MEC1 are 2.9 μM, 1.2 μM, and 1.9 μM for venetoclax, ibrutinib, and AZD-5991, respectively. In these cell lines, CB-839 caused a dose-dependent decrease in GLS-1 activity, measured as intracellular glutamate concentration, and inhibited glutathione synthesis (Figure 1A). CB-839 treated cells showed increased MitoSOX, but not Total ROS accumulation. However, as the endpoint effect, the decrease of cell proliferation rather than apoptosis was detected. CB-839 treatment caused a mild decrease in ATP pool in both cell lines. According to MTS assay, venetoclax demonstrated only the particular synergism with CB-839 (CI <1.0 at all effective doses (ED50, ED75, ED90, ED95) for MEC-1, and at ED50, ED75 for HG-3 cell line). In addition, CB-839 increased pro-apoptotic effect of venetoclax in both cell lines. Notably, that CB-839 did not improve any ibrutinib-mediated effects on cell proliferation and apoptosis in CLL cell lines. The most distinct synergism was observed in CB-839 and AZD-5991 combination group (CI <1.0 at all ED for MEC-1 and HG-3 cell lines) with 5- and 19.8-fold shifts in IC50 (Figure 1B). To extend these results, we are performing these combinations in primary CLL cells that are sensitive or resistant to targeted agents such as ibrutinib or venetoclax. Conclusions: Combinations of CB-839 with other agents targeting multiple mechanisms have the potential to overcome metabolic adaptation. Our findings suggest that CB-839 has limited efficacy in overcoming ibrutinib CLL resistance. However, the synergism with apoptosis inducers such as venetoclax and AZD-5991 require further investigations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of glutaminolysis ameliorates lupus by regulating T and B cell subsets and downregulating the mTOR/P70S6K/4EBP1 and NLRP3/caspase-1/IL-1β pathways in MRL/lpr mice

Background and Aim of the StudySystemic lupus erythematosus (SLE) is an autoimmune disease characterized by lymphocyte imbalance. The differentiation and function of T and B cells receive regulation from intracellular energy metabolism. Herein, we aimed to investigate glutamine metabolism levels in SLE and explore the effects of modulating glutamine metabolism on T and B cell subsets and related signaling pathways in MRL/lpr lupus mice. MethodsWe assessed intracellular glutamine metabolism in SLE patients and MRL/lpr mice by measuring intracellular glutamate and Glutaminase 1 (GLS1) protein levels. Intraperitoneal injection of the GLS1 inhibitor CB839 was performed to reduce glutamine metabolism and lupus-like manifestations in MRL/lpr mice were evaluated. The proportions and numbers of T and B cell subsets were determinedvia flow cytometry. Pathway-related proteins were detected using western blotting. ResultsIn this study, we reported that glutamine metabolism levels were aberrantly elevated in splenic mononuclear cells from MRL/lpr lupus mice, as well as in peripheral blood mononuclear cells (PBMCs) of SLE patients. Inhibition of glutamine metabolism by CB839 treatment for 8 weeks alleviated the lupus-like manifestations in MRL/lpr mice, including the kidney lesions, urinary protein/creatinine ratio, spleen index, and serum IgG1. Meanwhile, CB839 treatment ameliorated the depletion of IL-10 producing B cells (B10) and adjusted the Th1/TH2 and TH17/Treg imbalance. The inhibition of GLS1 by CB839 reduced the numbers of follicular helper T (TfH) cells and activated B cells in lupus mice. The proportions of mature B cells and plasma cells were not affected. Furthermore, the hyperactivated mTOR/P70S6K/4EBP1 and NLRP3/caspase-1/IL-1β pathways in MRL/lpr mice were reversed by CB839 treatment. ConclusionOur study confirmed the presence of abnormal intracellular glutamine metabolism in SLE and revealed potential therapeutic targets for this disease.

18F-Fluciclovine PET Imaging of Glutaminase Inhibition in Breast Cancer Models.

Aggressive cancers such as triple-negative breast cancer (TNBC) avidly metabolize glutamine as a feature of their malignant phenotype. The conversion of glutamine to glutamate by the glutaminase enzyme represents the first and rate-limiting step of this pathway and a target for drug development. Indeed, a novel glutaminase inhibitor (GLSi) has been developed and tested in clinical trials but with limited success, suggesting the potential for a biomarker to select patients who could benefit from this novel therapy. Here, we studied a nonmetabolized amino acid analog, 18F-fluciclovine, as a PET imaging biomarker for detecting the pharmacodynamic response to GLSi. Methods: Uptake of 18F-fluciclovine into human breast cancer cells was studied in the presence and absence of inhibitors of glutamine transporters and GLSi. To allow 18F-fluciclovine PET to be performed on mice, citrate in the tracer formulation is replaced by phosphate-buffered saline. Mice bearing triple-negative breast cancer (TNBC) xenografts (HCC38, HCC1806, and MBA-MD-231) and estrogen receptor-positive breast cancer xenografts (MCF-7) were imaged with dynamic PET at baseline and after a 2-d treatment of GLSi (CB839) or vehicle. Kinetic analysis suggested reversible uptake of the tracer, and the distribution volume (VD) of 18F-fluciclovine was estimated by Logan plot analysis. Results: Our data showed that cellular uptake of 18F-fluciclovine is mediated by glutamine transporters. A significant increase in VD was observed after CB839 treatment in TNBC models exhibiting high glutaminase activity (HCC38 and HCC1806) but not in TNBC or MCF-7 exhibiting low glutaminase. Changes in VD were corroborated with changes in GLS activity measured in tumors treated with CB839 versus vehicle, as well as with changes in VD of 18F-(2S,R4)-fluoroglutamine, which we previously validated as a measure of cellular glutamine pool size. A moderate, albeit significant, decrease in 18F-FDG PET signal was observed in HCC1806 tumors after CB839 treatment. Conclusion: 18F-fluciclovine PET has potential to serve as a clinically translatable pharmacodynamic biomarker of GLSi.

Abstract 6163: Targeting glucose metabolism through inhibition of pyruvate dehydrogenase kinase to improve response to immune-checkpoint inhibition in melanoma

Abstract Immune-checkpoint inhibitors have improved survival of patients with advanced melanoma, however, 40-50% of patients do not benefit sufficiently. Melanoma tumor lesions have high glucose uptake due to metabolic reprogramming, which supports tumor cell growth and is partially mediated through elevated expression of mitochondrial pyruvate dehydrogenase kinases (PDK1-4). Resultant decreased glucose levels and low pH in the tumor microenvironment are detrimental to antitumor immune cell function. Here, we aimed to reverse metabolic reprogramming in melanoma through PDK inhibition, thereby inhibiting tumor cell growth while maintaining or potentially enhancing antitumor immunity. We used a panel of four melanoma cell lines with genetic backgrounds similar to those most frequently found in patients, namely the BRAF-mutant A375 and S-MEL-28 cell lines, the NRAS-mutant SK-MEL-2 cell line and the BRAF/NRAS wild-type MeWo cell line. These were then treated with PDK inhibitor dichloroacetate (DCA) to determine effects on viability and metabolic phenotype. Furthermore, we investigated whether DCA synergized with treatment with the glutaminase inhibitor CB-839. Finally, we determined the effect of DCA on viability, interferon-γ (IFN-γ) secretion and antitumor activity of CD8+ T cells. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC50 values ranging from 13.3 to 27.0 mM. DCA led to an up to 6-fold increase in oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio in all cell lines. SK-MEL-28 cells were not sensitive to CB-839, while the IC50 values in the other cell lines ranged from 7.9 nM in MeWo to 139.2 nM in SK-MEL-2 cells. DCA synergized with CB-839 in 2D and 3D culture, ranging from 2-fold sensitization compared to either drug alone in MeWo up to 5-fold sensitization in SK-MEL-2. In activated CD8+ T cells, viability was not affected by DCA treatment of up to 21 mM, whereas proliferation was only mildly inhibited. These cells also showed a 2.5-fold increase in OCR:ECAR ratio after DCA treatment. Interestingly, IFN-γ secretion by CD8+ T cells was increased 2.8-fold after DCA treatment and tumor cell killing by CD8+ T cells in a coculture with A375 cells was not impaired by DCA or CB-839 treatment. We conclude that DCA can indeed reprogram cellular metabolism in melanoma and synergizes with other metabolically targeted drugs in antitumor activity, while keeping the antitumor reactivity of CD8+ T cells intact. Supported by the Dutch Cancer Foundation (10913/2017-1). Citation Format: Jiske F. Tiersma, Bernardus Evers, Barbara M. Bakker, Steven de Jong, Mathilde Jalving. Targeting glucose metabolism through inhibition of pyruvate dehydrogenase kinase to improve response to immune-checkpoint inhibition in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6163.

Preclinical studies for improving radiosensitivity of non-small cell lung cancer cell lines by combining glutaminase inhibition and senolysis

Glutamine metabolism, known as glutaminolysis, is abnormally activated in many cancer cells with KRAS or BRAF mutations or active c-MYC. Glutaminolysis plays an important role in the proliferation of cancer cells with oncogenic mutations. In this study, we characterized radiation-induced cell death, which was enhanced by glutaminolysis inhibition in non-small cell lung cancer A549 and H460 cell lines with KRAS mutation. A clonogenic survival assay revealed that treatment with a glutaminase inhibitor, CB839, enhanced radiosensitivity. X-irradiation increased glutamate production, mitochondrial oxygen consumption, and ATP production, whereas CB839 treatment suppressed these effects. The data suggest that the enhancement of glutaminolysis-dependent energy metabolism for ATP production is important for survival after X-irradiation. Evaluation of the cell death phenotype revealed that glutaminolysis inhibitory treatment with CB839 or a low-glutamine medium significantly promoted the proliferation of β-galactosidase-positive and IL-6/IL-8 secretory cells among X-irradiated tumor cells, corresponding to an increase in the senescent cell population. Furthermore, treatment with ABT263, a Bcl-2 family inhibitor, transformed senescent cells into apoptotic cells. The findings suggest that combination treatment with a glutaminolysis inhibitor and a senolytic drug is useful for efficient radiotherapy.

Pharmacological Inhibition of Glutaminase 1 Attenuates Alkali-Induced Corneal Neovascularization by Modulating Macrophages.

Corneal neovascularization (CoNV) in response to chemical burns is a leading cause of vision impairment. Although glutamine metabolism plays a crucial role in macrophage polarization, its regulatory effect on macrophages involved in chemical burn-induced corneal injury is not known. Here, we elucidated the connection between the reprogramming of glutamine metabolism in macrophages and the development of alkali burn-induced CoNV. Glutaminase 1 (GLS1) expression was upregulated in the mouse corneas damaged with alkali burns and was primarily located in F4/80-positive macrophages. Treatment with a selective oral GLS1 inhibitor, CB-839 (telaglenastat), significantly decreased the distribution of polarized M2 macrophages in the alkali-injured corneas and suppressed the development of CoNV. In vitro studies further demonstrated that glutamine deprivation or CB-839 treatment inhibited the proliferation, adhesion, and M2 polarization of bone marrow-derived macrophages (BMDMs) from C57BL/6J mice. CB-839 treatment markedly attenuated the secretion of proangiogenic factors, including vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) from interleukin-4- (IL-4-) regulated M2 macrophages. Our findings revealed that GLS1 inhibition or glutamine deprivation prevented alkali-induced CoNV by inhibiting the infiltration and M2 polarization of macrophages. This work suggests that pharmacological GLS1 inhibition is a feasible and effective treatment strategy for chemical burn-related CoNV in humans.

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.

The Glutaminase Inhibitor CB-839 (Telaglenastat) Enhances the Antimelanoma Activity of T-Cell-Mediated Immunotherapies.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen-specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)-associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

Abstract PO-021: Targeting glutamine dependencies in pancreatic cancers

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a 5-year survival rate of less than 10%. PDAC cells activate a non-canonical glutamine metabolic pathway essential to increase the level of NADPH, an important metabolite for redox balance, promoting tumor growth. Targeting glutamine metabolism using CB-839, a small molecule that targets glutaminase, the first step of glutaminolysis failed in PDAC. CB839 treatment only blocks the carbon utilization of glutamine and not the nitrogen portion which is critical for anabolic processes. We have previously shown that PDAC cells can rewire their metabolism and overcome GLS inhibition within days. We hypothesize that using a glutamine analog, such as 6-Diazo-5-oxo-L-norleucine (DON), which broadly inhibits all of the metabolic reactions that require glutamine, will provide a new therapeutic avenue for PDAC. Here, we investigate the role of DON in PDAC metabolism and progression. DON treatment leads to a profound decrease in proliferation, TCA cycle metabolites, and mitochondrial basal respiration in a panel of PDAC cells. In vivo studies using a syngeneic, orthotopically transplantation model in the pancreas demonstrated a significant decrease in tumor growth. Bulk RNA-seq studies demonstrated a decrease in the transcriptional signature of ECM and hypoxia, which was confirm by histology, suggesting the role of the TME in the response to glutamine blockage. Currently, we are testing the role of this drug and potential mechanisms of resistance using an autochthonous PDAC mouse model. Citation Format: Alec Kimmelman, Joel Encarnacion-Rosado. Targeting glutamine dependencies in pancreatic cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-021.

AZA + Glutaminase Inhibition with Telaglenastat (CB-839) for Advanced MDS: An Updated Interim Analysis

AZA + Glutaminase Inhibition with Telaglenastat (CB-839) for Advanced MDS: An Updated Interim Analysis

5-Fluorouracil Enhances the Antitumor Activity of the Glutaminase Inhibitor CB-839 against PIK3CA-Mutant Colorectal Cancers

PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. SIGNIFICANCE: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.

A powerful drug combination strategy targeting glutamine addiction for the treatment of human liver cancer.

The dependency of cancer cells on glutamine may be exploited therapeutically as a new strategy for treating cancers that lack druggable driver genes. Here we found that human liver cancer was dependent on extracellular glutamine. However, targeting glutamine addiction using the glutaminase inhibitor CB-839 as monotherapy had a very limited anticancer effect, even against the most glutamine addicted human liver cancer cells. Using a chemical library, we identified V-9302, a novel inhibitor of glutamine transporter ASCT2, as sensitizing glutamine dependent (GD) cells to CB-839 treatment. Mechanically, a combination of CB-839 and V-9302 depleted glutathione and induced reactive oxygen species (ROS), resulting in apoptosis of GD cells. Moreover, this combination also showed tumor inhibition in HCC xenograft mouse models in vivo. Our findings indicate that dual inhibition of glutamine metabolism by targeting both glutaminase and glutamine transporter ASCT2 represents a potential novel treatment strategy for glutamine addicted liver cancers.

Knock-down of PSAT1 Enhances Sensitivity of NSCLC Cells to Glutamine-limiting Conditions.

Phosphoserine aminotransferase 1 (PSAT1) is an enzyme implicated in serine biosynthesis, and its overexpression has been linked to cancer cell proliferation. Therefore, targeting PSAT1 is considered to be an anticancer strategy. The viability of non-small cell lung cancer (NSCLC) cells was measured by MTT assay. Protein and mRNA expression were determined by western blot and reverse transcription polymerase chain reaction, respectively. Glutamine-limiting conditions were generated through glutamine deprivation or CB-839 treatment, which induced PSAT1 expression in NSCLC cells. PSAT1 expression induced by glutamine-limiting conditions was regulated by activating transcription factor 4. Knock-down of PSAT1 enhanced the sensitivity of NSCLC cells to glutamine-limiting conditions. Interestingly, ionizing radiation induced PSAT1 expression, and knocking down PSAT1 increased cell sensitivity to ionizing radiation. Inhibiting PSAT1 might aid in the treatment of lung cancer, and PSAT1 may be a therapeutic target for lung cancer.