Glutamine Antagonist Research Articles

Abstract 440: Potentiation of EGFR peptide cancer vaccine by an orally bioavailable glutamine antagonist prodrug JHU-083

Abstract Lung cancer is the leading cause of cancer mortality worldwide. More than 85% of lung cancers are non-small cell lung cancer (NSCLC). EGFR mutations occur in 47.9% of Asia-Pacific patients with NSCLC and 19.2% of Western patients. The most common EGFR mutations (>90%) are deletions in exon 19 and/or point mutations in exon 21 (L858R). Th1 helper cellular immunity is critical for immunotherapy-mediated tumor regression. We have previously characterized an MHC-II-restricted EGFR multi-peptide vaccine (two peptides: “SCVRACGADSYEMEEDGVRK” and “VWSYGVTVWELMTFGSKPY”) (EGFR-V) that targets the EGFR protein and decreases EGFR-driven lung tumorigenesis by ~80% in EGFRL858R transgenic mice that were vaccinated before doxycycline-induction of the EGFR protein [1]. However, diminished efficacy was observed when this MHC-II-restricted EGFR multi-peptide vaccine was given two weeks after doxycycline induction of the EGFR protein, suggesting that expression of the EGFR oncoprotein significantly increased the immunosuppressive microenvironment. JHU083, an orally bioavailable glutamine antagonist, has recently been shown to not only inhibit tumor growth but also boost anti-cancer immunity [2]. To determine if JHU083 could potentiate the efficacy of EGFR vaccine in a post-initiation setting, we evaluated in vivo antitumor efficacy of EGFR vaccine combined with JHU083 using an EGFRL858R transgenic mouse model. JHU083 inhibited tumor burden by 31% and EGFR vaccine suppressed tumor burden by 33%, whereas combining JHU083 with anti-EGFR peptide vaccine had an additive effect (54% tumor inhibition). Mechanistically, JHU083 by itself, markedly reduced the immunosuppressive monocytic myeloid-derived suppressor cells (MDSCs) in lung tissues. The combination of JHU083 and the vaccine significantly reduced Tregs in lung tissues. The anti-EGFR vaccine primarily induced expansion of antigen specific antitumor CD4+ effector T cells. Long term administration of JHU-083 did not decrease bodyweight in mice. Together with previous results, these data suggest that JHU083 could be used to reshape the tumor microenvironment toward one that enhances antitumor T cell responses and could further enhance the efficacy of the EGFR anticancer vaccine.

Abstract 1563: DRP-104, a broad acting glutamine antagonist, synergizes with immune checkpoint blockade in vivo

Abstract We previously reported that DRP-104, a novel broad acting glutamine antagonist, has significant therapeutic potential in cancer by directly targeting tumor metabolism and simultaneously inducing a potent antitumor immune response. We also reported the immunomodulatory effect of DRP-104 on tumor growth inhibition (TGI) as a single agent and in combination with PD-1/PD-L1 checkpoint inhibitors in the MC38 and CT26 colon cancer syngeneic models(1). DRP-104 mediated TGI is associated with increased tumor-infiltrating leukocytes (TIL) including T, NKT, and NK cells; M1-polarized tumor associated macrophages; and decreased immune-suppressive cells such as MDSCs.Here we further elucidate the immunomodulatory effect of DRP-104 on tumor growth inhibition. In the MC38 mouse model, CD8+, CD4+, or NK cells were depleted by anti-CD8+Ab, anti-CD4+Ab, or anti-asialoGM1 Ab respectively to assess the contribution of these immune cells towards DRP-104 efficacy. CD8+T cell depletion significantly reduced anti-tumor efficacy for DRP-104 (73.9% TGI in CD8+T depletion setting vs 120% TGI with 57.8% regression in undepleted animals). NK cell depletion delayed early anti-tumor response to DRP-104 treatment, while in undepleted mice DRP-104 treatment showed TGI as early as 2 days after dose initiation. Interestingly, CD4+depleted mice showed enhanced efficacy to DRP-104 with TGI of 213% (regressions) and 6/8 tumor free mice 29 days after the end of treatment vs 1/8 mice tumor free in the CD4+ undepleted group with DRP-104 treatment. As such, we tested the combination of DRP-104 with anti-TIGIT antibody which has been reported to deplete Treg cells in the TME(2). In MC38 (anti-TIGIT insensitive) and CT26 (anti-TIGIT sensitive), DRP-104 showed significant single agent TGI. Furthermore, combination of DRP-104 with anti-TIGIT Ab demonstrated enhanced tumor growth inhibition and resulted in improvement in survival. In summary, immune cell depletion experiments confirmed DRP-104's immuno-oncology mechanism of action. CD8+ or NK cell depletion reduced DRP-104 efficacy while CD4+ cell depletion enhanced efficacy. Furthermore, combination therapy of DRP-104 with anti-PD-1 or anti-TIGIT Ab achieved significantly enhanced anti-tumor efficacy even in checkpoint inhibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in combination with immune checkpoint inhibitors. The first in human clinical study of DRP-104 is ongoing.

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 2321: Comprehensive metabolic profiling of high MYC medulloblastoma revealed key differences between in vitro and in vivo in glucose and glutamine usage

Abstract Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. In other cancers, high-level expression of MYC increase glutamine transport and fuels the TCA cycle, supporting cell growth and proliferation. However, the metabolism of high MYC amplified medulloblastoma subgroup remains underexplored. We performed comprehensive metabolic studies of xenografts of human MYC-amplified medulloblastoma tumors. We found that glucose was the main carbon source for TCA cycle. We also found that glutaminase ii pathway was the main pathway utilizing glutamine. In orthotopic xenografts, glutathione was the most abundant upregulated metabolite in tumor compared to normal brain. Glutamine derived glutathione was synthesized through glutamine transaminase K (GTK) enzyme in vivo, and it was significantly inhibited by glutamine analog 6-diazo-5-oxo-l-norleucine (DON). We engineered a DON prodrug with higher lipophilicity (JHU395) and found that JHU395 suppressed medulloblastoma growth in vitro and induced apoptosis. A single dose of JHU395 induced apoptosis in orthotopic D425 MED tumors but not in normal cerebellum or cortex. Twice weekly 15mg/kg dosing of JHU395 significantly extended the survival of the mice with D425 MED orthotopic xenografts (p<0.001 by log-rank test comparing treated vs vehicle control). Glutamine antagonists exploit MYC-amplified medulloblastoma's reliance on glutamine metabolism and may have therapeutic applications in human patients. Citation Format: Khoa Dang Pham, Bradley Poore, Allison Hanaford, Micah J. Maxwell, Heather Sweeney, Akhila Parthasarathy, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Comprehensive metabolic profiling of high MYC medulloblastoma revealed key differences between in vitro and in vivo in glucose and glutamine usage [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 2321.

Glutamine metabolism is essential for the production of IL-17A in γδ T cells and skin inflammation

γδ T cell is one of the most important pathogenic immune cells in autoimmunity, especially in mucosal and epithelial diseases. Metabolism is essential for the maintenance of immune homeostasis. However, unlike αβ T cells, the metabolic regulation of γδ T cell activation still remain unclear. Here, we identified glutamine metabolism as a critical regulator for the generation of IL-17-producing γδ T cells. Metabolic screening uncovered that amino acids related to glutamine metabolism increased most obviously during γδ T cell activation. Pharmaceutical blocking of glutamine impaired IL-17 production in γδ T cells both in vitro and in vivo. Mechanism studies further revealed that genes downregulated upon glutamine deprivation enriched in IL-17 and IL-23/STAT3 signaling pathways. Consistent with this, the activation of STAT3 was suppressed after glutamine blocking. More importantly, application of glutamine antagonist in vivo alleviated the progression of IL-23 induced psoriatic mice model. In addition, both the glutamine level and the expression of glutamine related enzymes were found higher in psoriasis patients when compared with healthy controls. Therefore, our work identified an important metabolic regulatory pathway in γδ T cell activation and suggested that glutamine metabolism could be used as a target for the treatment of γδ T cell related diseases.

Inhibitory Effect of a Glutamine Antagonist on Proliferation and Migration of VSMCs via Simultaneous Attenuation of Glycolysis and Oxidative Phosphorylation.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.

Phase 1 and phase 2a, first-in-human (FIH) study, of DRP-104, a broad glutamine antagonist, in adult patients with advanced solid tumors.

TPS3149 Background: Dependence of cancer cells on glutamine has made glutaminolysis an attractive therapeutic target in cancer. Prior clinical trials evaluating glutamine analogues for the treatment of cancer were abandoned due to lack of efficacy and/or tolerability. DON (6-Diazo-5-oxo-L-norleucine) is an irreversible inhibitor of several enzymes that utilize glutamine as a metabolic substrate. In addition to direct anti-tumor efficacy, inhibition of glutamine metabolism in the tumor microenvironment has been shown to improve T-cell activation and tumor infiltration, increasing anti-tumor immune responses. As such, combining DON with an immune checkpoint inhibitor (ICI), has strong preclinical rationale. The investigational product DRP-104 (sirpiglenastat) is an inactive prodrug of DON designed to limit systemic DON exposure while targeting glutamine dependence in tumor cells. Methods: A phase 1/2a, FIH, multi-center, non-randomized, multi-cohort, open-label study of DRP-104 is currently open to accrual for patients with advanced solid tumors. This study will be conducted in 4 parts: A) Dose Escalation of IV and subQ DRP-104 (Run-In phase followed by modified Continual Reassessment Method) to define MTD/RP2D. Primary objective of dose escalation is to assess the safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary antitumor activity of DRP-104 as a single agent; B) Dose Expansion of IV and subQ DRP-104 for safety assessment while primary objective is to select and recommend phase 2 DRP-104 route of administration; C) Phase 2a at recommended MTD/RP2D of selected route of DRP-104 in 2 patient cohorts: patients with locally advanced/metastatic NSCLC with KEAP1, NFE2L2 and/or STK11 mutation and patients with unresectable or metastatic SCCHN, in order to assess the safety, tolerability and preliminary antitumor activity of DRP-104 as a single agent; D) Phase 2a at recommended MTD/RP2D of selected route of DRP-104 in combination with atezolizumab in adult patients with advanced solid tumors previously treated with an ICI, in order to assess the safety, tolerability and preliminary antitumor activity of DRP-104 in combination with atezolizumab; DRP-104 IV is infused TIW over 1 hour infusion for 2 consecutive weeks followed by 1 week off. DRP-104 subQ is administered BIW weekly. Study is currently open with 6 IV patients (Run-In Phase completed and at Dose Level 4) and 3 subQ patients at Dose Level 1 at time of submission. Clinical trial information: NCT04471415.

FSMP-18. COMPREHENSIVE METABOLIC PROFILING OF HIGH MYC MEDULLOBLASTOMA REVEALS KEY DIFFERENCES BETWEEN IN VITRO AND IN VIVO GLUCOSE AND GLUTAMINE USAGE

Reprograming of cellular metabolism is a hallmark of cancer. Altered metabolism can overcome unfavorable conditions, allowing cancer cells to proliferate and invade in different tumor microenvironments. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. However, the metabolism of high MYC amplified medulloblastoma subgroup remains underexplored. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in different environments – in vitro, in flank xenografts and in orthotopic xenografts. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and the high-MYC medulloblastoma cells in culture. Compared to normal brain, MYC-amplified medulloblastoma orthotopic brain tumor xenografts showed upregulation of nucleotide, amino acid and glutathione pathways. Glucose was the main carbon source for the nucleotide synthesis and the TCA cycle in vivo. The glutaminase ii pathway was the main pathway utilizing glutamine in MYC-amplified medulloblastoma. In brain and flank xenografts, glutathione was the most abundant upregulated metabolite. Glutamine derived glutathione was synthesized through glutamine transaminase K (GTK) enzyme in vivo. The glutamine analog 6-diazo-5-oxo-l-norleucine (DON) significantly inhibited glutathione, amino acid, and nucleotide synthesis. In conclusion, we found that MYC-amplified medulloblastoma relied on glutamine metabolism in synthesizing glutathione in vivo. Glutamine antagonists may have therapeutic applications in human patients.

Novel Glutamine Antagonist JHU395 Suppresses MYC-Driven Medulloblastoma Growth and Induces Apoptosis.

Medulloblastoma is the most common malignant pediatric brain tumor. Amplification of c-MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism across many types of cancer. We modified the naturally occurring glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) by adding 2 promoeities to increase its lipophilicity and brain penetration creating the prodrug isopropyl 6-diazo-5-oxo-2-(((phenyl (pivaloyloxy) methoxy) - carbonyl) amino) hexanoate, termed JHU395. This prodrug was shown to have a 10-fold improved CSF-to-plasma ratio and brain-to-plasma ratio relative to DON. We hypothesized that JHU395 would have superior cell penetration compared with DON and would effectively and more potently kill MYC-expressing medulloblastoma. JHU395 treatment caused decreased growth and increased apoptosis in multiple human high-MYC medulloblastoma cell lines at lower concentrations than DON. Parenteral administration of JHU395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain. Treatment of mice bearing orthotopic xenografts of human MYC-amplified medulloblastoma with JHU395 increased median survival from 26 to 45 days compared with vehicle control mice (p < 0.001 by log-rank test). These data provide preclinical justification for the ongoing development and testing of brain-targeted DON prodrugs for use in medulloblastoma.

Glutamine Inhibition Reduces Iatrogenic Laryngotracheal Stenosis.

Glutamine inhibition has been demonstrated an antifibrotic effect in iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts in vitro. We hypothesize that broadly active glutamine antagonist, DON will reduce collagen formation and fibrosis-associated gene expression in iLTS mice. Prospective controlled animal study. iLTS in mice were induced by chemomechanical injury of the trachea using a bleomycin-coated wire brush. PBS or DON (1.3 mg/kg) were administered by intraperitoneal injection (i.p.) every other day. Laryngotracheal complexes were harvested at days 7 and 14 after the initiation of DON treatment for the measurement of lamina propria thickness, trichrome stain, immunofluorescence staining of collagen 1, and fibrosis-associated gene expression. The study demonstrated that DON treatment reduced lamina propria thickness (P=.025) and collagen formation in trichrome stain and immunofluorescence staining of collagen 1. In addition, DON decreased fibrosis-associated gene expression in iLTS mice. At day 7, DON inhibited Col1a1 (P < .0001), Col3a1 (P=.0046), Col5a1 (P < .0001), and Tgfβ (P=.023) expression. At day 14, DON reduced Co1a1 (P =.0076) and Tgfβ (P =.023) expression. Broadly active glutamine antagonist, DON, significantly reduces fibrosis in iLTS mice. These results suggest that the concept of glutamine inhibition may be a therapeutic option to reduce fibrosis in the laryngotracheal stenosis. N/A Laryngoscope, 131:E2125-E2130, 2021.

Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens

γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site.

The glutamine antagonist prodrug JHU-083 slows malignant glioma growth and disrupts mTOR signaling

BackgroundMetabolic reprogramming is a common feature in cancer, and it is critical to facilitate cancer cell growth. Isocitrate Dehydrogenase 1/2 (IDH1 and IDH2) mutations (IDHmut) are the most common genetic alteration in glioma grade II and III and secondary glioblastoma and these mutations increase reliance on glutamine metabolism, suggesting a potential vulnerability. In this study, we tested the hypothesis that the brain penetrant glutamine antagonist prodrug JHU-083 reduces glioma cell growth.Material and MethodsWe performed cell growth, cell cycle, and protein expression in glutamine deprived or Glutaminase (GLS) gene silenced glioma cells. We tested the effect of JHU-083 on cell proliferation, metabolism, and mTOR signaling in cancer cell lines. An orthotopic IDH1R132H glioma model was used to test the efficacy of JHU-083 in vivo.ResultsGlutamine deprivation and GLS gene silencing reduced glioma cell proliferation in vitro in glioma cells. JHU-083 reduced glioma cell growth in vitro, modulated cell metabolism, and disrupted mTOR signaling and downregulated Cyclin D1 protein expression, through a mechanism independent of TSC2 modulation and glutaminolysis. IDH1R132H isogenic cells preferentially reduced cell growth and mTOR signaling downregulation. In addition, guanine supplementation partially rescued IDHmut glioma cell growth, mTOR signaling, and Cyclin D1 protein expression in vitro. Finally, JHU-083 extended survival in an intracranial IDH1 mut glioma model and reduced intracranial pS6 protein expression.ConclusionTargeting glutamine metabolism with JHU-083 showed efficacy in preclinical models of IDHmut glioma and measurably decreased mTOR signaling.

Targeting glutamine metabolism as an effective means to promote allograft acceptance while inhibit tumor growth

Recently the role of metabolic signaling pathways has emerged as playing a critical role in dictating the outcome of T cell responses. The uptake and metabolism of the amino acid glutamine is essential for effector T cell activation. Since the growth and expansion of tumor cells relies on similar anabolic and metabolic requirements, we hypothesized that glutamine blockage might represent a promising strategy to promote allograft survival while inhibit tumor growth. 6-Diazo-5-oxo-L-norleucine (DON) was used as a glutamine antagonist. First, an in vitro study of T cell proliferation was performed to examine the ability of glutamine antagonism to inhibit T cell proliferation. Then we investigated whether DON could prolong allograft survival and inhibit tumor growth by using a fully MHC-mismatched mice full thickness skin transplantation model and a mice TC-1 tumor-bearing model. The proliferation study demonstrated that DON inhibited effector T cells proliferation in a dose-dependent manner. We found a marked prolonged graft median survival time and significant tumor inhibition for mice that received DON compared to those that received no treatment.These results highlight that targeting glutamine metabolism can promote allograft acceptance in a long tumor-free period.

Glutamine Antagonist JHU-083 Normalizes Aberrant Hippocampal Glutaminase Activity and Improves Cognition in APOE4 Mice

Given the emergent aging population, the identification of effective treatments for Alzheimer's disease (AD) is critical. We investigated the therapeutic efficacy of JHU-083, a brain-penetrable glutamine antagonist, in treating AD using the humanized APOE4 knock-in mouse model. Cell culture studies were performed using BV2 cells and primary microglia isolated from hippocampi of adult APOE4 knock-in mice to evaluate the effect of JHU-083 treatment on LPS-induced glutaminase (GLS) activity and inflammatory markers. Six-month-old APOE4 knock-in mice were administered JHU-083 or vehicle via oral gavage 3x/week for 4-5 months and cognitive performance was assessed using the Barnes maze. Target engagement in the brain was confirmed using a radiolabeled GLS enzymatic activity assay, and electrophysiology, gastrointestinal histology, blood chemistry, and CBC analyses were conducted to evaluate the tolerability of JHU-083. JHU-083 inhibited the LPS-mediated increases in GLS activity, nitic oxide release, and pro-inflammatory cytokine production in cultured BV2 cells and primary microglia isolated from APOE4 knock-in AD mice. Chronic treatment with JHU-083 in APOE4 mice improved hippocampal-dependent Barnes maze performance. Consistent with the cell culture findings,postmortem analyses of APOE4 mice showed increased GLS activity in hippocampal CD11b+ enriched cells versus age-matched controls, which was completely normalized by JHU-083 treatment. JHU-083 was well-tolerated, showing no weight loss effect or overt behavioral changes. Peripheral nerve function, gastrointestinal histopathology, and CBC/clinical chemistry parameters were all unaffected by chronic JHU-083 treatment. These results suggest that the attenuation of upregulated hippocampal glutaminase by JHU-083 represents a new therapeutic strategy for AD.

Abstract 2569: Uncovering metabolic bottlenecks in KEAP1 mutant lung cancer

Abstract During tumorigenesis cancer cells continuously encountermetabolic bottlenecks as a result of accelerated growth, overall increased metabolic demand and increased oxidative stress due to the formation of reactive oxygen species. We use a combination of genetically-engineered mouse models, an accelerated CRISPR/Cas9-based experimentalplatformand biochemical approaches to identify metabolic liabilities that can be exploited using novel targeted therapies.Lung cancer, the leading cause of cancer-related deaths worldwide,is the most common cancer type to acquire mutually exclusive gain-of-function mutations in the anti-oxidant transcription factor NRF2or loss-of-function mutations in its negative regulator KEAP1. Loss of Keap1activates Nrf2, increases antioxidant production and dramatically accelerates KRAS-driven lung cancer. Our data are in line with mounting evidence demonstrating that antioxidants promote cancer progression. Our studies focus on elucidating the mechanisms underlying novel metabolic dependencies in KEAP1mutant tumors and explore the therapeutic potential of targeting metabolism in highly relevant pre-clinical mouse and human lung cancer models. We observe that the ability of KEAP1mutant tumors to divert their metabolism towards antioxidant production comes with a cost, generating multiple metabolic vulnerabilities, including a dependency on glutamine metabolism that can be therapeutically exploited using DRP-104, a novel broad acting glutamine antagonist which inhibits all glutamine-consuming reactions. In addition, we have extended our findings to demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system xc-(XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs. Citation Format: Sarah E. LeBoeuf, Warren Wu, Triantafyllia Karakousi, Robert Wild, Thales Papagiannakopoulos. Uncovering metabolic bottlenecks in KEAP1 mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2569.

Abstract 5607: Broad acting glutamine antagonism remodels the tumor microenvironment, induces distinctive immune modulation, and synergizes with immune checkpoint blockade

Abstract We previously reported that DRP-104, a novel broad acting glutamine antagonist, has significant therapeutic potential in cancer via directly targeting tumor metabolism and inducing a potent antitumor immune response. Here we sought to further elucidate the immunomodulatory effect of DRP-104 on tumor growth inhibition as a single agent and in combination with PD-1/PD-L1 checkpoint inhibitors in the MC38 and CT26 colon cancer syngeneic models. We utilized multiple technologies to assess mechanistic effects on immune cells in vivoincluding flowcytometry, multiplex immunoassay, gene expression profiling and GeoMx™ digital spatial profiling. DRP-104 mediated tumor growth inhibition was associated with increased tumor-infiltrating leukocytes (TIL) including T, NKT, and NK cells, M1-polarized tumor associated macrophages, and decreased immune-suppressive cells such as MDSCs. Nanostring® IO360 analysis revealed broad immunological modulation such as increase in cytotoxicity/antigen presentation score, increase in tumor metabolic stress/apoptotic score and decrease in cell proliferation score. GeoMx™ profiling also showed increased tumoral T cells and granzyme B expression. Luminex analysis of tumor lysates revealed DRP-104 treatment decreased pro-tumorigenic proteins, such as VEGF and IL-8. DRP-104 showed significant dose dependent tumor growth inhibition and regressions as a single agent in mouse syngeneic tumor models, including those resistant to immune checkpoint inhibitors. Lastly, combination of DRP-104 with anti-PD-1 Ab or anti-PD-L1 Ab further reduced tumor growth resulting in significant improvement in survival with long-term durable cures. In summary, DRP-104 treatment resulted in broad remodeling of the tumor microenvironment including enhanced infiltration and function of multiple immune cells distinct from activities obtained by a checkpoint inhibitor treatment. Combination therapy of DRP-104 with anti-PD-1/PD-L1 achieved significantly enhanced anti-tumor efficacy including long-term durable cures even in checkpoint inhibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in combination with PD-1/PD-L-1 checkpoint inhibitors. Citation Format: Yumi Yokoyama, Robert Wild. Broad acting glutamine antagonism remodels the tumor microenvironment, induces distinctive immune modulation, and synergizes with immune checkpoint blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5607.

Abstract 510: Novel glutamine antagonist JHU-395 suppresses MYC-driven medulloblastoma growth and induces apoptosis

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism, and medulloblastoma tumors with evidence of high glutamate have a poor prognosis. We modified the naturally occurring glutamine analog 6-diazo-5-oxo-l-norleucine (DON) to increase its lipophilicity and its stability in plasma, creating a prodrug termed JHU-395. We hypothesized that this prodrug would have enhanced cell penetration compared to DON and would kill MYC-expressing medulloblastoma. JHU-395 treatment caused decreased growth and increased apoptosis in human MYC-expressing medulloblastoma cell lines at lower concentrations than DON. In D283MED for example IC50 for DON is 20 uM while JHU 395 IC50 is 1 uM (p value &amp;lt; 0.05). Similar results were obtained with other high-MYC medulloblastoma cell lines, including D425MED and D341MED. JHU-395 induced apoptosis at low micromolar concentrations as measured by cleaved PARP Western blot and cleaved caspase 3 (CC3) immunofluorescence (60% CC3+ cells compared to 30% CC3+ in control p&amp;lt;0.05). Parenteral administration of JHU-395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain as measured by mass spectrometry. These data suggest that JHU-395 may have activity against high MYC medulloblastoma in vivo, and testing this prodrug against MYC-driven medulloblastoma orthotopic xenografts is currently underway. Citation Format: Khoa Pham, Micah Maxwell, Heather Sweeney, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Novel glutamine antagonist JHU-395 suppresses MYC-driven medulloblastoma growth and induces apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 510.

Abstract IA10: Targeting glutamine metabolism as a means of enhancing immunotherapy for cancer

Abstract Tumors possess specialized metabolic programming that facilitates their rapid growth. We have developed a novel glutamine antagonist that not only demonstrates single-agent efficacy in a variety of mouse tumor models but also synergizes with anti-PD-1, adoptive cellular therapy (ACT), and A2aR antagonism to overcome resistance to immunotherapy and promote durable cures. Our data demonstrate that targeting tumor glutamine metabolism alters the tumor microenvironment by making it less acidic, less hypoxic, and more nutrient replete. These alterations lead to enhanced endogenous antitumor responses (in the absence of additional immunotherapy). Further analysis of tumor-infiltrating lymphocyte (TIL) reveals markedly increased infiltration of CD8+ T cells with increased proliferative index (Ki67) and the generation of robust memory phenotypes in the treated mice. Further, RNA sequencing data revealed that CD8+ TIL from vehicle-treated mice showed significantly enhanced expression of apoptotic transcriptional programs compared with TIL from treated mice. Additionally, targeting glutamine metabolism leads to decreased MDSC with an increase in inflammatory TAMs. Furthermore, we observe a downregulation of IDO and a marked decrease in kynurenine in the tumors of treated mice. These changes in macrophage subsets also correlated with decreased metastasis. Overall our findings suggest a novel “metabolic” approach to enhancing immunotherapy for cancer. Citation Format: Jonathan Powell, Robert Leone, Min-Hee Oh, Rana Rais, Pavel Majer, Barbara Slusher. Targeting glutamine metabolism as a means of enhancing immunotherapy for cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA10.

Pathways from glutamine to apoptosis: ROS and PP2A in SKW6.4

Abstract Altered glutamine metabolism is emerging target of cancer therapy. Glutamine deprivation has been proposed to induce apoptosis in multiple cancers. Dependency on glutamine metabolism of skw 6.4 lymphoma cell was investigated in vitro and in vivo. Glutamine deprivation induces apoptosis of skw 6.4 cell. Intracellular concentration of metabolites in tricarboxylic acid cycle and glutathione concentration were reduced and concentration of reactive oxygen species (ROS) and expression of PP2A were increased in glutamine-deprived skw6.4 cell. Reduction of ROS by antioxidants and knockdown of PP2A inhibited apoptosis induced by glutamine deprivation. Glutamine antagonist reduces skw6.4 tumor size in mouse xenograft model in PP2A-dependent manner. Overall, these results suggest survival of skw6.4 cells depends on the supply of glutamine in PP2A-dependent manner.

The Novel Glutamine Antagonist Prodrug JHU395 Has Antitumor Activity in Malignant Peripheral Nerve Sheath Tumor.

The carbon and nitrogen components of glutamine are used for multiple biosynthetic processes by tumors. Glutamine metabolism and the therapeutic potential of glutamine antagonists (GA), however, are incompletely understood in malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma observed in patients with neurofibromatosis type I. We investigated glutamine dependence of MPNST using JHU395, a novel orally bioavailable GA prodrug designed to circulate inert in plasma, but permeate and release active GA within target tissues. Human MPNST cells, compared with Schwann cells derived from healthy peripheral nerve, were selectively susceptible to both glutamine deprivation and GA dose-dependent growth inhibition. In vivo, orally administered JHU395 delivered active GA to tumors with over 2-fold higher tumor-to-plasma exposure, and significantly inhibited tumor growth in a murine flank MPNST model without observed toxicity. Global metabolomics studies and stable isotope-labeled flux analyses in tumors identified multiple glutamine-dependent metabolites affected, including prominent effects on purine synthesis. These data demonstrate that glutamine antagonism is a potential antitumor strategy for MPNST.