Glutaminase Inhibitor Research Articles

Abstract 1112: Exploiting oncogene-conferred glutamine addiction as a therapeutic vulnerability in resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment, and the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of ibrutinib resistant (IBN-R) cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their growth and survival. Recently, glutamine has been proposed to be the essential amino acid in the malignant metabolism, confers those specific tumors with glutamine addiction. Accordingly, such addiction is interplayed with several oncogenic pathways including Myc and mTOR. Our previous studies indicate the increased Myc copies in IBN-R MCL. Therefore, our aim of this work was to uncover the relationship between glutamine addiction with MCL resistance. In the work presented, we focused on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. Targeting glutamine addiction and glutamine-fueled OXPHOS would act as a potential therapeutic strategy in conquering drug resistance in glutamine-addicted MCL. We presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples by immunoblotting. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigations on solid tumors. Inhibiting GLS1 with CB-839 decreases the cell viability in MCL (IC50=0.56nM-308.4nM). Of note, the combinatory treatment of GLS and OXPHOS inhibitor, IACS-010759, induces apoptosis (53% compared with control, and 33%-50% with single agent). Furthermore, the combinatory treatment also shows synergistic effects in decreasing cell viability in IBN-R MCL cell line, Z-138 (CI=0.44), as well as in decreasing mitochondrial membrane potential (≈16% compared with single agent, and 32% with control) and increasing ROS generation (6 times to control, and 3-4 times to single agent). Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the GLS inhibitor, CB-839, may present a clinical potential for a new indication and the combinatory treatment with our in-house inhibitor, IACS-010759, warrants more in-depth investigation as a novel therapeutic regimen. Citation Format: Lingzhi Li, Changying Jiang, Lucy J. Navsaria, Yang Liu, Joseph M. McIntosh, Michael Wang, Yinxin Yao. Exploiting oncogene-conferred glutamine addiction as a therapeutic vulnerability in resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1112.

Abstract 2015: Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic tumor microenvironment

Abstract Solid tumors are characterized by poor vasculature, thereby causing insufficient oxygen and nutrient supply within the tumor. To thrive in such conditions, hypoxic tumor cells stimulate the expression of nutrient transporters to support cell metabolism and pH regulators to buffer the intracellular pH changes caused by metabolic by-products. Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in the pH regulation of hypoxic tumor cells. CAIX is highly expressed in several solid tumors, and is an indicator of poor prognosis and response to therapy. Although the importance of CAIX's role in mediating tumor progression is well-known, the underlying mechanism remains unclear. To identify the interactors of CAIX in the hypoxic tumor microenvironment, we performed a proteomic analysis using proximity dependent biotin identification (BioID) and found the Alanaine, Serine, Cysteine transporter (ASCT2) to be a high confidence interactor of CAIX from this analysis. ASCT2, which predominantly acts as a glutamine transporter is upregulated in several cancers and is shown to support tumor growth by facilitating increased glutamine uptake. In this study, using a panel of solid tumor cell lines, we validate the interaction of CAIX with ASCT2 in the hypoxic microenvironment. Functionally, loss of CAIX causes an increased glutamine uptake via ASCT2 and an increased capacity of cells to utilize glutamine for mitochondrial respiration. Concomitantly, ROS and glutathione (GSH) levels also increase upon CAIX inhibition. Cytotoxicity studies show a robust cell death in the absence of glutamine, following treatment with the CAIX/XII small molecule inhibitor SLC-0111. A similar effect on cell death is observed by blocking glutamine metabolism using inhibitors such as V-9302 (ASCT2 inhibitor) or CB-839 (Glutaminase-1 inhibitor) with SLC-0111. Furthermore, blocking GSH synthesis using BSO (Glutamylcysteine synthatase inhibitor) with SLC-0111 causes significant cell death. These results suggest that CAIX depletion causes increased cellular stress and to overcome this, cells increase glutamine uptake in part for GSH synthesis. In summary, we show that CAIX and ASCT2 coordinate their functions in hypoxic cancer cells to support tumor growth, and co-targeting them could be beneficial in treating solid tumors. Citation Format: Geetha Venkateswaran, Shawn Chafe, Shannon Awrey, Oksana Nemirovsky, Shoukat Dedhar. Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2015.

Abstract 1572: Broad glutamine pathway inhibition by DRP-104 results in anti-tumor activity in hypermetabolic lung tumors resistant to PD-1 or osimertinib therapy

Abstract Recent breakthroughs with checkpoint inhibitors (CPI) or the third generation tyrosine kinase inhibitor (TKI) osimertinib (OSI) for patients with specific mutations to the epidermal growth factor receptor (EGFR) gene are now able to induce durable responses in non-small cell lung cancer (NSCLC) patients1,2. However, the majority of patients who receive CPI or OSI will be either non-responsive to treatment or develop therapy resistance. We sought to identify alternative treatment strategies to overcome therapy resistance by targeting metabolism in aggressive tumors. The fast rate of growth in therapy resistant hyper-metabolic tumors (HMTs) makes them dependent on nutrients to sustain anabolic growth. To demonstrate this dependency, we coupled PET imaging using 18F-FDG and 11C-Glutamine (11C-GLN) with in vivo metabolomics and identified a conserved metabolic signature in which lung squamous cell carcinomas (LUSC) and EGFR mutant lung adenocarcinomas (LUAD) were marked by dependence upon both glucose and glutamine3,4. Importantly, this metabolic signature is predictive of either response or resistance to targeted therapies that inhibit glucose and glutamine metabolism that may be exploited in a clinical setting.We previously reported that combined targeting of glucose and glutamine metabolism with mTOR kinase inhibitor TAK228 or TKI in combination with the selective glutaminase (GLS) inhibitor CB-839 was required to significantly suppress glucose and glutamine metabolism in both LUSC and EGFR mutant LUADs resulting in metabolic crisis and tumor cell death3,5. Here, we tested the novel broad acting glutamine antagonist, DRP-104, as a single agent or in combination with anti-PD-1 in CPI-resistant LUSC and in EGFR mutant LUAD tumor models. DRP-104 significantly inhibited tumor progression across genetically engineered mouse models (GEMMs) with biallelic deletion of lkb1, pten and p53 (Lkb1-/-;Pten-/-;P53-/-) and patient derived xenograft (PDX) models of LUSC. Importantly, LUSC showed significant single agent response to DRP-104 and the combination of DRP-104 and anti-PD-1 treatment demonstrated therapeutic synergy, suggesting that broad inhibition of glutamine metabolism by DRP-104 induced metabolic remodeling of the tumor immune microenvironment and permissiveness to CPI blockade6. Likewise, we showed that DRP-104 induced a significant response in an OSI-resistant PDX model of EGFR mutant LUAD. Overall, DRP-104 showed great potential to treat hypermetabolic therapy-resistant LUSC and EGFR mutant LUAD as a single-agent therapy and in combination with immune CPI. Further clinical development of DRP-104 in this patient population is warranted and clinical trials are currently ongoing. Citation Format: Morgan Brady, Milica Momcilovic, Chiara Montemurro, Yumi Yokoyama, Heather Christofk, Katerina Politi, Margaret Dugan, Aaron Lisberg, Robert Wild, David B. Shackelford. Broad glutamine pathway inhibition by DRP-104 results in anti-tumor activity in hypermetabolic lung tumors resistant to PD-1 or osimertinib therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1572.

Abstract 1102: Integrated approach towards defining mechanism based combinations to guide clinical development of glutaminase inhibitors

Abstract Metabolic dysregulation is a hallmark of cancer, and recently, increasing evidence has shown a critical role for glutamine metabolism to support the bioenergetics and biosysnthetic needs of tumor and immune cells. We have previously disclosed the development of the GLS1 inhibitor, IPN60090, which is currently advancing through Phase 1 clinical studies (NCT03894540). Metabolic plasticity has been suggested to confer adaptive resistance to metabolic inhibitors and defines mechanisms that could be exploited to enhance therapeutic benefit. To address the clinical problems of innate drug resistance and adaptation, we interrogated metabolic and adaptive responses to IPN60090 in vitro and in vivo. We and others have previously identified KEAP1/NFE2L2 mutant non-small cell lung cancer models as sensitive to GLS1 inhibition. Through an integrated approach, including metabolic, transcriptomic, and proteomic analysis, we have identified molecular pathways that confer resistance to GLS1 inhibition. Nodes in these pathways that drive aquired resistance, may serve as additional patient stratification biomarkers in subsets on NSCLC or provide opportunites for further drug development. Additionally, unbiased in vivo functional genomics screening identified mulitple signaling pathways that act as critical nodes governing resistance to GLS1 inhibition. Drug combinations were tested in vitro and in vivo to identify those that are most synergistic with IPN60090. We found that PI3K/AKT/mTOR signaling is a major contributor to IPN60090 resistance, and we demonstrate that dual targeting of GLS1 and PI3K/AKT/mTOR signaling in tumors with KEAP1/NFE2L2 mutations results in synergistic anti-tumor efficacy. IPN60090, dosed in combination with inhibitors of these pathways yields regressions and off-treatment, durable responses in preclinical models of KEAP1-mutant NSCLC. Based on these data, combination strategies are being developed for Phase 1b expansion cohorts. Citation Format: Jeffrey J. Kovacs, Nakia D. Spencer, Christopher A. Bristow, Alessandro Carugo, Virginia Giluiani, Meredith A. Miller, Angela Harris, Ningping Feng, Michael L. Soth, Kang Le, Elisa de Stanchina, Charles M. Rudin, Giulio Draetta, Timothy A. Yap, Philip Jones, Timothy Heffernan. Integrated approach towards defining mechanism based combinations to guide clinical development of glutaminase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1102.

Impaired anaplerosis is a major contributor to glycolysis inhibitor toxicity in glioma

BackgroundReprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839.MethodsWe analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models.ResultsMetabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors.ConclusionTogether, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.

Prostate cancer cells survive anti-androgen and mitochondrial metabolic inhibitors by modulating glycolysis and mitochondrial metabolic activities.

Most cancer cells are more glycolytic even under aerobic conditions compared with their normal counterparts. Recent evidence of tumor cell metabolism, however, shows that some tumors also increase mitochondrial oxidative phosphorylation (ox-phos) at some disease states during progression and/or development of drug resistance. Our data show that anti-androgen enzalutamide (ENZA) resistant prostate cancer (PCa) cells use more mitochondrial metabolism leading to higher ox-phos as compared to the ENZA-sensitive cells and can become vulnerable to mitochondrial metabolism targeted therapies. Seahorse assay, mass spectrometry and high resolution fluorescence confocal microscopy coupled with image analysis has been used to compare mitochondrial metabolism in ENZA-treated and -untreated anti-androgen-sensitive LNCaP and -resistant C4-2, CWR22ν1, and PCa2b cells. Ex vivo fluorescence microscopy and image analysis has been standardized to monitor mitochondrial electron transport (ETS) activity that likely increases ox-phos in circulating tumor cells (CTCs) isolated fom patients undergoing AR-targeted therapies. Our data show that PCa cells that are resistant to anti-androgen ENZA switch from glycolysis to ox-phos leading to an increased ETS activity. ENZA pretreated cells are more vulnerable to ETS component complex I inhibitor IACS-010759 (IACS) and mitochondrial glutaminase inhibitor CB-839 that reduces glutamate supply to tricarboxylic acid cycle. CTCs isolated from 6 of 20 patient blood samples showed relatively higher ETS activity than the rest of the patients. All six patients have developed ENZA resistance within less than 6 months of the sample collection. The enhanced growth inhibitory effects of mitochondrial metabolic inhibitors IACS and CB-839 in ENZA pretreated PCa cells provides a rationale for designing a drug combination trial. Patients can be selected for such trials by monitoring the mitochondrial ETS activities in their CTCs to maximize success.

Glutaminolysis is involved in the activation of mTORC1 in in vitro-produced porcine embryos.

Glutamine supplementation to porcine embryo culture medium improves development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the activation of mechanistic target of rapamycin complex 1 (mTORC1) to support rapid proliferation. The objective of this study was to determine if glutamine metabolism, known as glutaminolysis, was involved in mTORC1 activation in porcine embryos. Culture with 3.75 mM GlutaMAX improved development to the blastocyst stage compared to culture with 1 mM GlutaMAX, and culture with 0 mM GlutaMAX decreased development compared to all groups with GlutaMAX. Ratios of phosphorylated to total MTOR were increased when embryos were cultured with 3.75 or 10 mM GlutaMAX, which was enhanced by the absence of leucine, but ratios for RPS6K were unchanged. As another indicator of mTORC1 activation, colocalization of MTOR and a lysosomal marker was increased in embryos cultured with 3.75 or 10 mM GlutaMAX in the absence of leucine. Culturing embryos with glutaminase inhibitors decreased development and the ratio of phosphorylated to total MTOR, indicating reduced activation of the complex. Therefore, glutaminolysis is involved in the activation of mTORC1 in porcine embryos, but further studies are needed to characterize downstream effects on development.

Simultaneous Pharmacologic Inhibition of Yes-Associated Protein 1 and Glutaminase 1 via Inhaled Poly(Lactic-co-Glycolic) Acid-Encapsulated Microparticles Improves Pulmonary Hypertension.

BackgroundPulmonary hypertension (PH) is a deadly disease characterized by vascular stiffness and altered cellular metabolism. Current treatments focus on vasodilation and not other root causes of pathogenesis. Previously, it was demonstrated that glutamine metabolism, as catalyzed by GLS1 (glutaminase 1) activity, is mechanoactivated by matrix stiffening and the transcriptional coactivators YAP1 (yes‐associated protein 1) and transcriptional coactivator with PDZ‐binding motif (TAZ), resulting in pulmonary vascular proliferation and PH. Pharmacologic inhibition of YAP1 (by verteporfin) or glutaminase (by CB‐839) improved PH in vivo. However, systemic delivery of these agents, particularly YAP1 inhibitors, may have adverse chronic effects. Furthermore, simultaneous use of pharmacologic blockers may offer additive or synergistic benefits. Therefore, a strategy that delivers these drugs in combination to local lung tissue, thus avoiding systemic toxicity and driving more robust improvement, was investigated.Methods and ResultsWe used poly(lactic‐co‐glycolic) acid polymer‐based microparticles for delivery of verteporfin and CB‐839 simultaneously to the lungs of rats suffering from monocrotaline‐induced PH. Microparticles released these drugs in a sustained fashion and delivered their payload in the lungs for 7 days. When given orotracheally to the rats weekly for 3 weeks, microparticles carrying this drug combination improved hemodynamic (right ventricular systolic pressure and right ventricle/left ventricle+septum mass ratio), histologic (vascular remodeling), and molecular markers (vascular proliferation and stiffening) of PH. Importantly, only the combination of drug delivery, but neither verteporfin nor CB‐839 alone, displayed significant improvement across all indexes of PH.ConclusionsSimultaneous, lung‐specific, and controlled release of drugs targeting YAP1 and GLS1 improved PH in rats, addressing unmet needs for the treatment of this deadly disease.

First-in-human biomarker-driven phase I trial of the potent and selective glutaminase-1 (GLS1) inhibitor IACS-6274 (IPN60090) in patients (pts) with molecularly selected advanced solid tumors.

3001 Background: Glutamine metabolism is frequently deregulated in different cancers, including tumors harboring KEAP1/ NFE2L2 mutations or those expressing low Asparagine Synthetase (ASNS) levels. IACS-6274 is a potent oral GLS1 inhibitor discovered at MD Anderson Cancer Center with excellent pharmacokinetics (PK) and antitumor activity in biomarker-defined preclinical models. Methods: Pts with advanced solid tumors received IACS-6274 BID at escalating doses using a phase 1 BOIN design. PK and pharmacodynamic (PD) studies were conducted in serial tumor and/or blood samples. Peripheral glutamine metabolism was assessed in peripheral blood mononuclear cells (PBMC) to assess glutamine metabolism via 13C-isotope labelling. Predictive biomarker studies included tumor analyses for KEAP1, NFE2L2, STK11, NF1 mutations and IHC for ASNS loss. Results: 22 pts with advanced ovarian (n=8), NSCLC (n=7), melanoma (n=2), leiomyosarcoma, gastric, anal, endometrial and HNSCC (all n=1) received IACS-6274 at 20 (n=1), 40 (n=1), 80 (n=1), 120 (n=4), 180 (n=11) or 240 (n=4) mg BID. Molecular alterations assessed included pts with ASNS loss (n=6), STK11 (n=5), KEAP1 (n=5), NFE2L2 (n=4) and NF1 (n=1). Prior lines of therapies: 2-4 (n=12); ≥5 (n=10). Common IACS-6274-related adverse events included G1-2 photopsia (n=7), photophobia (n=7), increased creatinine (n=4) and AST (n=4). Less common G3 toxicities at 180 and 240 mg included reversible nausea (n=3), vomiting and fatigue (n=2). Dose-limiting toxicities of G3 acute renal failure and PRES syndrome were seen in one patient at 240mg BID, which fully resolved. Plasma exposures showed a dose-dependent increase across doses with observed half-life ̃12 hrs. Patients at 180mg displayed steady-state exposures at C1D14 with Cmax of 45.8 μM +/- 18.6 μM and average AUC(0-12hrs) of 382.48 h*μM +/- 159.27 h*μM. Glutamate to glutamine ratios decreased in PBMC samples in pts at C1D14 vs baseline; pts at 120, 180 and 240 mg had inhibition of 82.5% (P<0.0001), 83.9% (P<0.0001) and 85.3% (P<0.0001), respectively, exceeding doses predicted to be efficacious in preclinical models. A robust PK/PD relationship was established across doses (P<0.0001). The recommended phase 2 dose was 180mg BID. Best RECISTv1.1 response was stable disease (SD) in 17 of 20 evaluable pts. Disease control rate at 12 weeks was 60%. Durable RECISTv1.1 SD ≥6 months +/- tumor regression were seen in pts with advanced ASNS-loss ovarian cancer (n=2), PD-1/L1-exposed melanoma (n=2) and NF1 mutant leiomyosarcoma (n=1). Conclusions: IACS-6274 was well tolerated at biologically active doses with good human PK, significant PD target modulation and preliminary antitumor activity observed. The clinical trial assessment of rational combinations to maximize benefit in molecularly-selected pts is initiating. Clinical trial information: NCT03894540.

Genomic profiling of esophageal squamous cell carcinoma to reveal actionable genetic alterations.

e16042 Background: Esophageal cancer is the eighth most common cancer in the world and more than half of global cases occur in China. Studies demonstrated that esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) are two distinct disease entities. Due to the lack of effective therapies, the five-year survival rates of ESCC patients remain dismal. Therefore, there is an urgent need to establish a framework through genomic profiling to facilitate the development of precision therapies for ESCC. Methods: To characterize therapeutic targets in 118 Chinese ESCC patients, deep panel sequencing of 831 cancer genes (OncoPanscan, Genetronhealth) was performed on their tumor tissues and paired genomic DNA samples. Results: The most frequently mutated genes in our ESCC cohort were TP53 (97%), PIK3CA (19%), CDKN2A (18%), NOTCH1 (17%), KMT2D (15%), LRP1B (15%) NOTCH3 (15%) NFE2L2 (13%), and EP300 (12%). Consistent with previous reports, we found significantly elevated mutations in cancer-related genes including NOTCH1 (16.9%), NOTCH2 (3.2%), NOTCH3 (15.3%) and RB1 (9.3%). Importantly, 17.8% (21/118) patients in our cohort harbored the 11q13 amplicon ( CCND1, FGF3, FGF4 and FGF19). The median copy number was 8.19 (range 6.07-42.3). These patients can participate in clinical trials with FGFR inhibitor alone or in combination with CDK4/6 inhibitors. Additionally, we also observed frequent genetic alterations in the KEAP1 (Kelch-like ECH-associated protein 1)-NFE2L2 (nuclear factor erythroid 2 like 2)-CUL3 (cullin 3) pathway. 80% (12/15) of missense mutations in NFE2L2 were located at the KEAP1 binding domain of NRF2 protein. These mutations were either around the ETGE motif (D77G, E79Q, G81V/D and E82D) or the DLG motif (D27V, I28T, D29G, L30F, G31E, V32E, R34G). We also identified four missense mutations of KEAP1 and one alternation of CUL3 in splicing site. Taken together, 17% (20/118) of ESCC patients harbored mutations in the NFE2L2/KEAP1/CUL3 pathway, which may be eligible for clinical trials of glutaminase inhibitor telaglenastat. Two patients had high level of ERBB2 amplification which can be targeted with anti-HER2 therapy. Furthermore, 11.9% (14/118) patients carried activating PIK3CA mutations including N345K, E542K, E545K, M1043I and H1047R which may be targeted by PIK3CA inhibitor alpelisib. Lastly, patients with loss-of-function mutation in NF1 (n = 4), STK11 (n = 1) and PTEN (n = 3) can be respectively targeted with MEK inhibitor and mTOR inhibitor. Overall, 43% of patients in our ESCC cohort had actionable genetic mutations with corresponding precision therapy options. Conclusions: Our findings indicated that amplification of the 11q13 amplicon and dysfunction of the KEAP1-NRF2-CUL3 axis are the major driving events of ESCC. The results of genomic profiling can guide physicians to enroll a significant portion of ESCC patients into genomically matched clinical trials.

CANTATA: Primary analysis of a global, randomized, placebo (Pbo)-controlled, double-blind trial of telaglenastat (CB-839) + cabozantinib versus Pbo + cabozantinib in advanced/metastatic renal cell carcinoma (mRCC) patients (pts) who progressed on immune checkpoint inhibitor (ICI) or anti-angiogenic therapies.

4501 Background: Dysregulated metabolism is a hallmark of RCC, driven by overexpression of glutaminase (GLS), a key enzyme of glutamine metabolism. Telaglenastat (Tela) is an investigational, first-in-class, selective, oral GLS inhibitor that blocks glutamine utilization and critical downstream pathways. Preclinically, Tela synergized w/ cabozantinib (Cabo), a VEGFR2/MET/AXL inhibitor, against RCC tumors. In a Ph 1 study cohort, Tela+Cabo showed encouraging safety/efficacy as 2L+ therapy for mRCC. This trial compared Tela+Cabo vs Pbo+Cabo in previously treated pts w/ clear-cell mRCC (NCT03428217). Methods: Eligible pts had 1-2 prior lines of systemic therapy for mRCC, including ≥1 anti-angiogenic therapy or nivolumab + ipilimumab (nivo/ipi), KPS ≥70%, measurable disease (RECIST 1.1), no prior Cabo or other MET inhibitor. Pts were randomized 1:1 to receive Cabo (60 mg PO QD) with either Tela (800 mg PO BID) or Pbo, until disease progression/unacceptable toxicity, and were stratified by prior PD-(L)1 inhibitor therapy (Y/N) and IMDC prognostic risk group. Primary endpoint was progression-free survival (PFS; RECIST 1.1) by blinded independent radiology review. The study was designed to detect a PFS hazard ratio (HR) of 0.69 w/ alpha 0.05 and 85% power. Data cutoff date: August 31, 2020. Results: 444 pts were randomized (221 Tela+Cabo; 223 Pbo+Cabo). Baseline characteristics were balanced between arms. Median follow-up was 11.7 mo; 276 pts received prior ICI, including 128 w/ prior nivo/ipi. Median PFS (mPFS) was 9.2 mo for Tela+Cabo vs 9.3 mo for Pbo+Cabo (HR = 0.94; 95% CI: 0.74, 1.21; stratified log-rank P= 0.65) with overall response rates (ORR; confirmed) of 31% with Tela+Cabo vs 28% Pbo+Cabo, respectively. Overall survival was not mature at data cutoff. In a prespecified subgroup analysis in pts w/ prior ICI, mPFS was numerically longer w/ Tela+Cabo than Pbo+Cabo (11.1 vs 9.2 mo, respectively; unstratified HR = 0.77; 95% CI: 0.56, 1.06). In the Pbo+Cabo arm, mPFS was 9.2 mo for pts w/ prior ICI exposure and 9.5 mo for pts without, and ORR was 32% and 20%, respectively; if ICI included nivo/ipi, ORR was 37%. Rates of adverse events (AEs) were similar between arms.Grade 3-4 AEs occurred in 71% of Tela+Cabo pts and 79% of Pbo+Cabo pts and included hypertension (17% vs 18%) and diarrhea (15% vs 13%). Cabo was discontinued due to AEs in 10% of Tela+Cabo pts and 15% of Pbo+Cabo pts. Conclusions: The addition of Tela did not improve the efficacy of Cabo in mRCC in this study. Tela+Cabo was well tolerated with AEs consistent with known risks of both agents. The study provides valuable insight on efficacy outcomes of a contemporary population of pts w/ mRCC who receive Cabo in the 2/3L setting. Clinical trial information: NCT03428217.

Epithelial Ovarian Cancer microenvironment regulates Myeloid cell metabolism to confer immunosuppression via glutamine metabolism.

Abstract Myeloid Cells are a major constituent of epithelial ovarian cancer (EOC) microenvironment that confer immunosuppression and promote tumor growth. Using the syngeneic ID8 EOC model we show that its microenvironment is enriched in myeloid cell recruiting factors like GM-CSF, G-CSF, VEGF, TNF-a, IL-1b and IL-6. Tumor microenvironment (TME) exposed myeloid cells are highly immunosuppressive as seen by increased expression of CSF-R, S100a9, STAT-3, VEGF, TNF-a, iNOS, STAT-3 and T-cell suppression activity. TME exposed myeloid cells exhibited an activated metabolic phenotype that was fueled by glutamine dependent oxidative phosphorylation as assessed by XFe seahorse analyzer and targeted metabolomics. Deprivation of glutamine or use of a glutaminase inhibitor, BPTES reduced oxidative phosphorylation, decreased immunosuppressive markers and protected from T-cell suppression. Targeted energy metabolism gene expression arrays revealed that the mitochondrial enzyme, dihydrolipoamide succinyl transferase (DLST), a component of alpha-ketoglutarate dehydrogenase (a-KG) complex in TCA cycle, was elevated in TME exposed myeloid cells. Glutamine deprivation reduced DLST expression, suggesting that DLST expression is dependent on availability of glutamine. A novel small molecule inhibitor of a-KG complex, 3-methyl 2-oxalo valeric acid, regressed EOC growth, reversed myeloid cell energy phenotype and alleviated T-cell suppression. We propose that EOC induced expression of DLST metabolically regulates the immunosuppressive function of myeloid cells and targeting DLST may represent a new strategy to reduce immunosuppressive environment plaguing ovarian cancer.

Inhibition of eEF2K synergizes with glutaminase inhibitors or 4EBP1 depletion to suppress growth of triple-negative breast cancer cells

The eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the ‘Collagen containing extracellular matrix’ pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.

Glutamine deficiency promotes stemness and chemoresistance in tumor cells through DRP1-induced mitochondrial fragmentation.

Glutamine is essential for maintaining the TCA cycle in cancer cells yet they undergo glutamine starvation in the core of tumors. Cancer stem cells (CSCs), responsible for tumor recurrence are often found in the nutrient limiting cores. Our study uncovers the molecular basis and cellular links between glutamine deprivation and stemness in the cancer cells. We showed that glutamine is dispensable for the survival of ovarian and colon cancer cells while it is required for their proliferation. Glutamine starvation leads to the metabolic reprogramming in tumor cells with enhanced glycolysis and unaltered oxidative phosphorylation. Production of reactive oxygen species (ROS) in glutamine limiting condition induces MAPK-ERK1/2 signaling pathway to phosphorylate dynamin-related protein-1(DRP1) at Ser616. Moreover, p-DRP1 promotes mitochondrial fragmentation and enhances numbers of CD44 and CD117/CD45 positive CSCs. Besides the established features of cancer stem cells, glutamine deprivation induces perinuclear localization of fragmented mitochondria and reduction in proliferation rate which are usually observed in CSCs. Treatment with glutaminase inhibitor (L-DON) mimics the effects of glutamine starvation without altering cell survival in in vitro as well as in in vivo model. Interestingly, the combinatorial treatment of L-DON with DRP1 inhibitor (MDiVi-1) reduces the stem cell population in tumor tissue in mouse model. Collectively our data suggest that glutamine deficiency in the core of tumors can increase the cancer stem cell population and the combination therapy with MDiVi-1 and L-DON is a useful approach to reduce CSCs population in tumor.

Abstract PO-094: Glutaminase inhibition increases ionizing radiation induced oxidative stress and DNA damage in head and neck squamous cell carcinoma models

Abstract Ionizing radiation (IR) is a pillar of head and neck cancer treatment, the 6th most common cancer worldwide. However, its efficacy is limited by radioresistance, resulting in locoregional failure rates of ~50%. The mechanisms that tumor cells use to evade IR-induced cellular damage are poorly understood. Our initial proteomics analysis indicated that IR induced metabolic pathways, including those involving the Krebs cycle. Glutamate is a key substrate of the Krebs cycle. Glutaminase is the enzyme that converts glutamine to glutamate. Analysis of The Cancer Genome Atlas’s transcriptome database revealed that glutaminase overexpression (upper 25%) in patients with HNSCC was associated with reduced patient survival (p≤0.03). In this study, we therefore aimed to identify whether glutaminase inhibition with CB-839 (currently in Phase 1/2 trials) could enhance cellular response to IR. To do this, we used three representative human-derived HNSCC cell lines: CAL-27, FaDu, and HN5. First, we established that proliferation of all 3 HNSCC cell lines was glutamine dependent (20-50% increase, n=3 per line, p≤0.01). Second, a synergistic decrease in cell survival was observed in clonogenic assays with HNSCC cell lines treated with IR and CB-839 relative to either treatment alone (n=3 per line, p≤0.05). Thirdly, in 3D cell culture, which better represents the host microenvironment, combinatorial treatment significantly reduced HNSCC spheroid size (11-26% decrease, n=3 per line, p≤0.0001). The functional effect of CB-839 on cellular metabolism was examined using Seahorse MitoStress test assays. CB-839 significantly reduced spare respiratory capacity and OCR/ECAR ratio in HNSCC cell lines examined (p≤0.001, p≤2.0 × 10−6, n>3). To understand the mechanism behind this reduced cell growth and survival, we examined whether CB-839 enhanced oxidative stress and DNA damage when combined with IR using immunofluorescence. Oxidative stress marker, 8-oxoguanine, was ~2 fold higher with combined treatment as compared to CB-839 or IR alone in CAL-27 cells (p≤0.01, p≤0.05; respectively). Additionally, increased levels of y-H2AX were observed in CAL-27 cells treated with both CB-839 and IR relative to independent treatment. Combination of CB-839 with IR significantly inhibited tumor growth in CAL-27 xenograft mice relative to vehicle (p≤0.01), CB-839 (p≤0.05) or IR (p≤0.01) (p values, day 19). To summarize, successful treatment of HNSCC is impeded by adaptive resistance to IR and locoregional failure. Overall, these data suggest that the rational combination of IR and glutaminase inhibitor (CB-839) enhances cellular response and tumor control relative to independent treatment and provides pre-clinical data in support of additional investigation in patients with HNSCC. Citation Format: Christina A. Wicker, Brian G. Hunt, Sarah Palackdharry, William R. Elaban, Trisha M. Wise-Draper, Gordon B. Mills, Susan E. Waltz, Vinita Takiar. Glutaminase inhibition increases ionizing radiation induced oxidative stress and DNA damage in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-094.

Structure-Enabled Discovery of Novel Macrocyclic Inhibitors Targeting Glutaminase 1 Allosteric Binding Site.

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule 13b (LL202) was identified with robust GLS1 inhibitory activity (IC50 = 6 nM) and high GLS1 binding affinity (SPR, Kd = 24 nM; ITC, Kd = 37 nM). The X-ray crystal structure of the 13b-GLS1 complex was resolved, revealing a unique binding mode and providing a novel structural scaffold for GLS1 allosteric inhibitors. Importantly, 13b clearly adjusted the cellular metabolites and induced an increase in the ROS level by blocking glutamine metabolism. Furthermore, 13b exhibited a similar in vivo antitumor activity as CB839. This study adds to the growing body of evidence that macrocyclization provides an alternative and complementary approach for the design of small-molecule inhibitors, with the potential to improve the binding affinity to the targets.

DDRE-01. METABOLIC PLASTICITY AND HETEROGENEITY IN IDH1MUT CELL LINES PRODUCES RESISTANCE TO GLUTAMINASE INHIBITION BY CB839

BACKGROUNDMutant IDH1 (IDH1mut) gliomas have characteristic genetic and metabolic profiles and exhibit phenotype that is distinct from their wild-type counterparts. The glutamine/glutamate pathway has been hypothesized as a selective therapeutic target in IDH1mut gliomas. However, little information exists on the contribution of this pathway to the formation of D-2-hydroxyglutarate (D-2HG), a hallmark of IDHmut cells, and the metabolic consequences of inhibiting this pathway.METHODSWe employed an untargeted metabolic profiling approach in order to detect metabolic changes arising from glutaminase (GLS) inhibition treatment. Subsequently, 13C metabolic tracing analysis through a combined Nuclear Magnetic Resonance and Liquid Chromatography-Mass Spectrometry approach, we explored the fate of glutamine and glucose under treatment with CB839 a glutaminase-GLS-inhibitor and their respective contributions to D-2HG formation.RESULTS AND CONCLUSIONSThe effects of CB839 on cellular proliferation differed among the cell lines tested, leading to designations of GLS-inhibition super-sensitive, -sensitive or -resistant. Our data indicates a decrease in the production of downstream metabolites of glutamate, including those involved in the TCA cycle, when treating the sensitive cells with CB839 (glutaminase -GLS- inhibitor). Notably, CB839-sensitive IDH1mutcells respond to GLS inhibition by upregulating glycolysis and lactate production. In contrast, CB839-resistantIDH1mut cell lines do not rely only on glutamine for the sustenance of TCA cycle. In these cells, glucose contribution to TCA is enough to compensate the downregulation of glutamine-derived TCA metabolites. This investigation reveals that the glutamine/glutamate pathway contributes differentially to D-2HG in a cell-line dependent fashion on a panel of IDHmut cell lines. Further, these results demonstrate that there is a heterogeneous landscape of IDH1mut metabolic phenotypes. This underscores the importance of detailed metabolic profiling of IDH1mut patients prior to the decision to target glutamine/glutamate pathway clinically.

Expression of activated VEGFR2 by R1051Q mutation alters the energy metabolism of Sk-Mel-31 melanoma cells by increasing glutamine dependence.

Vascular endothelial growth factor receptor 2 (VEGFR2) activating mutations are emerging as important oncogenic driver events. Understanding the biological implications of such mutations may help to pinpoint novel therapeutic targets. Here we show that activated VEGFR2 via the pro-oncogenic R1051Q mutation induces relevant metabolic changes in melanoma cells. The expression of VEGFR2R1051Q leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2WT. Furthermore, activated VEGFR2R1051Q augments the dependence on glutamine (Gln) of melanoma cells, thus increasing Gln uptake and their sensitivity to Gln deprivation and to inhibitors of glutaminase, the enzyme initiating Gln metabolism by cells. Overall, these results highlight Gln addiction as a metabolic vulnerability of tumors harboring the activating VEGFR2R1051Q mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2.

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.

Immunohistochemical evaluation of glutaminase expression in prostate adenocarcinoma and correlation with clinicopathologic parameters.

251 Background: Glutaminolysis plays a significant role in the metabolic reprogramming of cancer cell growth and proliferation. Glutaminase (GLS1), the rate-limiting enzyme of the glutamine pathway, is frequently dysregulated in cancer. High GLS1 expression is reported in colorectal and breast cancers and has been found to correlate with the tumor stage and disease progression. Furthermore, a new orally bioavailable glutaminase inhibitor (CB-839) is in early phase clinical trials in select tumors. The purpose of this study is to investigate the status of GLS1 expression in prostate cancer (PCa) and to correlate expression levels with clinicopathologic parameters. Methods: Radical prostatectomy samples from 154 patients with prostate adenocarcinoma were retrospectively reviewed and used to evaluate GLS1 expression by immunohistochemistry (IHC). The IHC expression score was calculated by multiplying the intensity of the stain by the proportion of cells staining; cases were then segmented into negative, low, or high expression groups. In addition, 41 samples of benign prostate tissue were used as a control. Associations between GLS1 levels and clinicopathologic parameters were analyzed by Pearson’s chi-squared and Log-rank tests. Results: GLS1 expression in the benign controls were negative, low, and high in 59%, 41%, 0% of cases, respectively, compared to 53%, 21.5%, 25.5%, respectively, in PCa (p < 0.003). Most PCa patients were age < 60 (55.8%), white (78.6%), stage T2 (52.9%), node negative (80.5%), Grade Group 3 (44%), and non-smokers (63.6%). There was no difference between GLS1 expression and age, race, Gleason score, stage, node status, and smoking status by univariate analysis. The median biochemical-progression free survival for negative, low, and high expression was 10, 9, and 10 years, respectively (p = 0.7). Conclusions: In our study, PCa samples were more likely to have GLS1 expression compared to benign controls. Although GLS1 expression did not appear to be a prognostic marker, our cohort was enriched for cases with localized disease and low-to-intermediate grade PCa. As a result, future studies are warranted to evaluate the expression levels in high grade and advanced PCa cases to determine a role for prognostic and or therapeutic implication to justify future preclinical studies with CB-839.