Glutamine Analog Research Articles

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.

Energy deficiency caused by CTPS downregulation in decidua may contribute to pre-eclampsia by impairing decidualization.

Pre-eclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation. It seriously affects the health of maternity and the fetus. However, the pathogenesis of PE is still unknown. Decidualization deficiency is considered a contributing factor to the development of PE. CTP synthetase (CTPS) which is the rate-limiting enzyme in the CTP de novo biosynthesis, is essential for nucleic acid synthesis and cellular energy metabolism, and often appears as cytoophidium in many cell types. Here, we found that the expression of CTPS was significantly downregulated in decidual tissues of patients with severe PE compared with healthy pregnant women. During in vitro decidualization, changes in CTPS were accompanied by opposite fluctuation of the AMPK signaling pathway. Moreover, the downregulation of CTPS by glutamine analogs or CTPS small interfering RNA inhibited the decidualization process and the AMPK signaling pathway. Investigating the underlying mechanism of action by co-immunoprecipitation coupled with mass spectrometry showed that CTPS interacted with ATP synthase (ATPS) and maintained the content of ATP on Day 3 of decidualization. However, when combined with mitochondrial stress protein STRESS-70 instead of ATPS, the concentration of ATP on Day 6 of induction was reduced. Corresponding to this, CTPS was mainly distributes in the cytoplasm on Day 3 of induction, while it appeared both in the cytoplasm and the nucleus on Day 6 in decidualized cells, which was similar to that in cells before induction. In summary, we believe that CTPS plays an important role in decidualization by participating in energy metabolism. Abnormal expression of CTPS in decidualization would lead to abnormal decidualization and consequently result in the occurrence of PE.

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.

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 < 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<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.

Nuliglucaemia Lucidae: Extreme Deprivation of Blood Glucose as Organic Context for Cancer Treatment with Antimetabolites

The present report describes our clinical findings regarding the use of high dose intravenous insulin in cancer patients, as a means to deplete the blood compartment of glucose molecules. The purpose of this intervention is to create a favorable physiological state for the competitive inhibition of several rate-limiting enzymes within cancer cells, with structural analogues that behave as antimetabolites. Regardless of their histological origin, virtually all solid tumors reported on to date (February 2020) are found to be hypercaptant in PET-CT scans following the intravenous injection of 2-¹⁸fluoro-deoxy-D-glucose. Most solid tumors display a hypermetabolic phenotype (SUVmax ≥ 3), with marked overexpression of glucose transporters (GLUTs) in the outer membrane of their anaplastic cells subpopulations. The fact that neoplastic cells also overexpress glycolytic, fermentative and glutaminolytic enzymes up to an order of magnitude relative to healthy cells further strengthens the argument for a competitive inhibition with antimetabolites. The rationale for a deep, systemic deprivation of glucose was suggested by classical enzymological work concerning competitive inhibition (the Woods principle), and our group has shown that a metabolic intervention with structural analogues of glucose and pyruvate is strongly enhanced by a systemic suppression of the natural substrates of hexokinase 2 (HK-2) and lactic dehydrogenase isozyme A (LDH-A), followed by the timely introduction of several non- metabolizable analogues. Sustained, deep hypoglycemia (<10mg/dl) under physiological ketosis provides an advantageous context for antitumor treatment with structural analogues of glucose, pyruvate and glutamine. Data provided in this report demonstrates the feasibility and safety of the procedure.

Glutamine Analogues Impair Cell Proliferation, the Intracellular Cycle and Metacyclogenesis in Trypanosoma cruzi.

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.

Abstract A59: Targeting the glutamine dependent hexosamine biosynthesis pathway sensitizes pancreatic tumors to anti-PD1 therapy

Abstract Background: Pancreatic cancer is notoriously difficult to treat owing to its late detection, metastasis and increased therapeutic resistance, leading to a 5-year survival rate of 9%. Immune evasive microenvironment further contributes to challenges in developing viable therapy options. As a result, anti-PD-1 therapy that has shown promising results in various cancers like melanoma, non-small cell lung cancer and kidney cancer is ineffective against pancreatic cancer patients. Hexosamine biosynthesis pathway (HBP), a glutamine-dependent metabolic shunt pathway responsible for producing UDP-GlcNAc and thus glycosylation, is overexpressed in pancreatic cancer. Our study shows that targeting HBP by a glutamine analog DON in pancreatic cancer can sensitize it to anti-PD1 immune therapy. Methods: All experiments were done using primary cells isolated from spontaneous pancreatic cancer mice, KrasLSL.G12D/+; p53R172H/+; PdxCretg/+ (KPC), which are the epithelial cancer cells and the cancer-associated fibroblasts (CAFs) isolated from the same mice. 6-Diazo-5-oxo-L-norleucine (DON) was used as an inhibitor of GFAT, the rate-limiting enzyme of HBP. DON was used at a dose of 1mg/kg alone or 0.5mg/kg in combination with PD1 (100 microgm). Results: Our study showed mice orthotopically implanted with KPC and CAF cells at a ratio of 1:9 showed a marked decrease in tumor volume when treated with DON. In addition to a decrease in tumor volume, these mice also had a profound decrease in their circulating tumor cells and a significant reduction in the collagen content in the tumor while the stromal content remained unaffected. Mice treated with low-dose DON in combination with low-dose anti-PD1 therapy showed a pronounced decrease in tumor volume as compared to either treatments alone. As expected, mice treated with the DON+PD1 combination had an increased infiltration of CD4 and CD8 cells. Conclusion: Our study highlights for the first time an impressive response to PD1 therapy in pancreatic cancer when given in combination with DON, a glutamine analog. Since PDL1 depends on the HBP pathway for its glycosylation, inhibition by DON and the subsequent internalization of PDL1 leads to an increased immune response against pancreatic cancer that can be targeted therapeutically. Note: This abstract was not presented at the conference. Citation Format: Nikita S. Sharma, Vineet K. Gupta, Roey Hadad, Bhuwan Giri, Anthony Ferrantella, Vikas Dudeja, Ashok K. Saluja, Sulagna Banerjee. Targeting the glutamine dependent hexosamine biosynthesis pathway sensitizes pancreatic tumors to anti-PD1 therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A59.

CTP synthase forms cytoophidia in archaea

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.

L-asparaginase and 6-diazo-5-oxo-L-norleucine synergistically inhibit the growth of glioblastoma cells.

Glioblastoma is an aggressive central nervous system tumor with a 5-year survival rate of < 10%. The standard therapy for glioblastoma is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide. New approaches to treatment of glioblastoma, such as targeting metabolism, have been studied. The object of this study is to evaluate whether asparagine could be a new target for treatment of glioblastoma. We investigated a potential treatment for glioblastoma that targets the amino acid metabolism. U251, U87, and SF767 glioblastoma cells were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase hydrolyzes asparagine into aspartate and depletes asparagine. L-asparaginase has been used for the treatment of acute lymphoblastic leukemia. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase. Cell viability was measured after 72h of treatment. MTS assay showed that L-asparaginase suppressed the proliferation of U251, U87, and SF767 cells in a dose-dependent manner. DON also inhibited the proliferation of these cell lines in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. Exogenous asparagine rescued the effect of inhibition of proliferation by L-asparaginase and DON. The expression of asparagine synthetase mRNA was increased in cells treated with a combination of L-asparaginase and DON. This combined treatment also induced greater apoptosis and autophagy than did single-drug treatment. The results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with glioblastoma.

(2S, 4R)-4-[18F]Fluoroglutamine for In vivo PET Imaging of Glioma Xenografts in Mice: an Evaluation of Multiple Pharmacokinetic Models

PurposeThe glutamine analogue (2S, 4R)-4-[18F]fluoroglutamine ([18F]FGln) was investigated to further characterize its pharmacokinetics and acquire in vivo positron emission tomography (PET) images of separate orthotopic and subcutaneous glioma xenografts in mice.Procedures[18F]FGln was synthesized at a high radiochemical purity as analyzed by high-performance liquid chromatography. An orthotopic model was created by injecting luciferase-expressing patient-derived BT3 glioma cells into the right hemisphere of BALB/cOlaHsd-Foxn1nu mouse brains (tumor growth monitored via in vivo bioluminescence), the subcutaneous model by injecting rat BT4C glioma cells into the flank and neck regions of Foxn1nu/nu mice. Dynamic PET images were acquired after injecting 10–12 MBq of the tracer into mouse tail veins. Animals were sacrificed 63 min after tracer injection, and ex vivo biodistributions were measured. Tumors and whole brains (with tumors) were cryosectioned, autoradiographed, and stained with hematoxylin-eosin. All images were analyzed with CARIMAS software. Blood sampling of 6 Foxn1nu/nu and 6 C57BL/6J mice was performed after 9–14 MBq of tracer was injected at time points between 5 and 60 min then assayed for erythrocyte uptake, plasma protein binding, and plasma parent-fraction of radioactivity to correct PET image-derived whole-blood radioactivity and apply the data to multiple pharmacokinetic models.ResultsOrthotopic human glioma xenografts displayed PET image tumor-to-healthy brain region ratio of 3.6 and 4.8 while subcutaneously xenografted BT4C gliomas displayed (n = 12) a tumor-to-muscle (flank) ratio of 1.9 ± 0.7 (range 1.3–3.4). Using PET image-derived blood radioactivity corrected by population-based stability analyses, tumor uptake pharmacokinetics fit Logan and Yokoi modeling for reversible uptake.ConclusionsThe results reinforce that [18F]FGln has preferential uptake in glioma tissue versus that of corresponding healthy tissue and fits well with reversible uptake models.

Targeting tumor-intrinsic hexosamine biosynthesis sensitizes pancreatic cancer to anti-PD1 therapy.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine [DON]). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.

The Distinctive Metabolic Environment of the Bone Marrow Niche Drives Leukemia Chemoresistance

Chemoresistance is the difference between remission and cure in cancer patients and is particularly evident in acute myeloid leukemia (AML) where complete remissions are common, but few are cured. Genetic analysis in patients points to residual founder clones persisting through therapy and ultimately causing relapse. However, most of these genetic alterations are currently not amenable to targeted therapies. We hypothesized that residual chemoresistant cells must pass through extreme metabolic challenges when under selection from chemotherapy and surrounded by massive cell death. Our goal was to define the distinctive metabolic profile of AML cells as they pass through that time of extreme selection pressure and determine whether metabolic pathway manipulation would reveal an exploitable vulnerability for therapy. We used in vivo imaging to define the kinetics of leukemic growth, response to chemotherapy and relapse in the mouse. We reasoned that the moment of maximal response immediately following chemotherapy represents a time when the metabolic milieu is maximally hostile and therefore a distinctive selection moment that the cells ultimately causing relapse must pass through. We captured cells at that time and conducted unbiased metabolic profiling, which revealed a metabolite profile that was distinct from AML cells during pre-chemo growth or late post-chemo relapse. Metabolite enrichment analysis revealed hyperactivation of glutamine metabolism during the extreme selection period, with chemoresistant cells showing high levels of glutamine, glutamate and aspartate. Inhibition of glutamine metabolism using the glutamine analog 6-diazo-5-oxo-L-norleucine specifically during the window of maximal selection pressure reduced the number of chemoresistant cells and improved survival in mouse models of AML. Unexpectedly, we found that residual AML cells were not sensitive to inhibition of the enzyme glutaminase, a target currently under clinical investigation for AML. In vivo metabolic tracer analysis instead revealed that chemoresistant AML cells mainly depend on glutamine nitrogen to fuel pyrimidine synthesis. Aspartate, another essential metabolite for pyrimidine synthesis, can be made from glutamine through the Krebs cycle. Interestingly, we detected almost no glutamine carbon entering the Krebs cycle in vivo even though labeled aspartate was found, indicating a different aspartate source. We found that aspartate levels in peripheral blood plasma are very low compared to those in bone marrow plasma, and explored the possibility of a local bone marrow source. Single cell RNA sequencing of bone marrow stromal cells (BMSC) revealed that CXCL12-positive BMSC express high levels of aspartate synthesis genes and transporters, which further increased in the presence of AML cells. In vitro co-cultures showed that BMSC convert glutamine to aspartate, and transfer this to AML cells. Genetic inhibition of aspartate synthesis in BMSC sensitized AML cells to chemotherapy, showing the importance of this metabolic crosstalk. We propose that induction of a timed metabolic collapse targeting AML cells both directly and indirectly through the bone marrow niche can prevent development of chemoresistance and improve treatment outcomes. Figure Disclosures Scadden: Novartis: Other: Sponsored research; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Unbiased Metabolic Profiling Predicts Sensitivity of High MYC-Expressing Atypical Teratoid/Rhabdoid Tumors to Glutamine Inhibition with 6-Diazo-5-Oxo-L-Norleucine.

Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the MYC proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT. Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT. Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT. Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.

Orally bioavailable glutamine antagonist prodrug JHU-083 penetrates mouse brain and suppresses the growth of MYC-driven medulloblastoma

A subset of poor-prognosis medulloblastoma has genomic amplification of MYC. MYC regulates glutamine metabolism in multiple cellular contexts. We modified the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) to mask its carboxylate and amine functionalities, creating a prodrug termed JHU-083 with increased oral bioavailability. We hypothesized that this prodrug would kill MYC-expressing medulloblastoma. JHU-083 treatment caused decreased growth and increased apoptosis in human MYC-expressing medulloblastoma cell lines. We generated a mouse MYC-driven medulloblastoma model by transforming C57BL/6 mouse cerebellar stem and progenitor cells. When implanted into the brains of C57BL/6 mice, these cells formed large cell/anaplastic tumors that resembled aggressive medulloblastoma. A cell line derived from this model was sensitive to JHU-083 in vitro. Oral administration of JHU-038 led to the accumulation of micromolar concentrations of DON in the mouse brain. JHU-083 treatment significantly increased the survival of immune-competent animals bearing orthotopic tumors formed by the mouse cerebellar stem cell model as well as immune-deficient animals bearing orthotopic tumors formed by a human MYC-amplified medulloblastoma cell line. These data provide pre-clinical justification for the ongoing development and testing of orally bioavailable DON prodrugs for use in medulloblastoma patients.

Abstract 5267: Unbiased metabolic profiling of atypical teratoid/rhabdoid tumors predicts sensitivity to the glutamine metabolic inhibitor 6-diazo-5-oxo-L-norleucine

Abstract Atypical teratoid rhabdoid tumors (AT/RT) are deadly tumors of infancy in need of new, targeted therapies. Molecular analyses have revealed 3 epigenetically distinct subgroups of AT/RT. High MYC-expressing tumors are a particularly aggressive subgroup, with a 5-year median survival of 18.5%. MYC is difficult to directly target given large, flat, featureless protein-protein binding sites. We performed the first unbiased metabolic profile of AT/RT cell models by high-performance liquid chromatography-mass spectrometry. This study revealed that high MYC-expressing AT/RT have a unique metabolic profile (Partial Least Squares-Discriminant Analysis). Pathway analysis highlights a dependence on glutamine metabolism in high-MYC expressing AT/RT. High MYC-expressing cell lines grow poorly in glutamine-free media compared to low-MYC cell lines (MTS assay, glutamine-free media vs normal media). Due to this dependence on glutamine metabolism, we hypothesized that high-MYC expressing AT/RT would be especially sensitive to glutamine metabolic inhibitors. We show that the glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON) slows high-MYC expressing AT/RT cell growth, induces high rates of apoptosis, and improves survival in orthotopic mouse models of high-MYC expressing AT/RT (p =0.0027 by log-rank test). In contrast, low-MYC expressing models are relatively insensitive to DON. In uniformly labeled glutamine metabolic analyses of AT/RT cells, DON treatment led to depletion of glutathione levels by preventing the production of glutamate. DON treatment synergized with carboplatin to kill AT/RT cells in vitro and extend the life of mice bearing AT/RT orthotopic xenografts (p&amp;lt; 0.001 by log rank test). We aim to translate these findings into a new clinical trial to improve survival in this deadly disease. Citation Format: Sabrina Z. Wang, Brad Poore, Jesse Alt, Antoinette Price, Sariah Allen, Brent Orr, Rana Rais, Barbara Slusher, Charles Eberhart, Eric H. Raabe, Jeffrey H. Rubens. Unbiased metabolic profiling of atypical teratoid/rhabdoid tumors predicts sensitivity to the glutamine metabolic inhibitor 6-diazo-5-oxo-L-norleucine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5267.

Abstract 5277: Glutamine antagonists synergize with L-asparaginase in MYCN-driven rhabdomyosarcoma

Abstract Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Chromosomal translocations producing fusion transcription factors (PAX-FOXO1) are associated with poor prognosis. A subset of fusion-positive rhabdomyosarcomas have downstream activation of the transcription factor MYCN, a known oncogenic driver. Attempts to inhibit directly either PAX-FOXO1 or MYCN have been unsuccessful. MYCN is known to drive tumor cell reliance on glutamine metabolism. 6-diazo-5-oxo-L-norleucine (DON) is a well-characterized glutamine analog that irreversibly inhibits glutamine-utilizing enzymes. DON was well tolerated in a phase I pediatric clinical trial, but it has never been systematically tested in rhabdomyosarcoma. We show that high MYCN confers sensitivity to DON therapy in vitro. We have also developed a targeted glutamine antagonist compound, JHU083, which is similarly effective against MYCN-driven rhabdomyosarcoma cell lines. Furthermore, DON administered by intraperitoneal injection twice weekly significantly reduces flank tumor volume in a murine xenograft MYCN-driven rhabdomyosarcoma model (mean tumor volume 757 mm3 vs. 2167 mm3 in control animals, p-value 0.0000197 by t-test). In stable isotope resolved metabolomics experiments, DON primarily prevents asparagine synthesis, depleting intracellular asparagine levels by at least 50% in MYCN-driven cell lines (p-value = 0.0003). Finally, DON combined with L-asparaginase synergistically inhibits growth of MYCN-driven rhabdomyosarcoma cell lines (CI &amp;lt;&amp;lt; 1 by the Chou-Talalay method, indicating strong synergy; p-value = 0.0001). We conclude that DON depletes cellular pools of asparagine, and combination therapy with DON and L-asparaginase synergistically inhibits the growth of MYCN-driven rhabdomyosarcoma. These studies provide the preclinical justification for potential clinical trials for the use DON or DON prodrugs in combination with L-asparaginase as new therapeutic options for patients with MYCN-driven rhabdomyosarcoma. Citation Format: Micah J. Maxwell, Brad A. Poore, Allison Hanaford, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Glutamine antagonists synergize with L-asparaginase in MYCN-driven rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5277.

Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders.

HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .

Conformational changes in glutaminyl-tRNA synthetases upon binding of the substrates and analogs using molecular docking and molecular dynamics approaches

Aminoacyl-tRNA synthetases (aaRSs) are considered as important components in protein translation as they facilitate the attachment of specific transfer RNA (tRNA) to form aminoacyl-tRNAs. Our study focused on understanding the crystal structure of Glutaminyl-tRNA synthetase (GlnRS) from Thermus thermophilus HB8 (PDB ID:5ZDO) and mechanism of formation of enzyme–substrate complex using substrates and its analogs by applying molecular dynamics simulation (MDS) to investigate the conformational changes. Least energy structure of TtGlnRS was considered to dock the enzyme substrates such as glutamine (Gln), glutamic acid (Glu), adenosine monophosphate (AMP), adenosine triphosphate (ATP), QSI and various substrate analogs (2MA, 4SU and 5MU) onto the active site of the enzyme. We focused on comparative analysis of binding specificity between Gln and Glu; similarly, ATP and AMP. Active site organization as observed by MDS analysis showed interactive changes associated with substrate and catalytically important loops. Study found that when tRNAGln specific for GlnRS was docked into the active site of the TtGlnRS enzyme it interacts with 2' OH on the ribose acceptor end of the tRNA. Upon validation with 50 ns MDS, the maximum deviations and conformational changes of secondary structural elements were observed to be high in the loop regions of enzyme–substrate complexes. Binding affinity of ATP to TtGlnRS was further proved by isothermal titration calorimetry.AbbreviationsaaRSsaminoacyl-tRNA synthetasesAMPadenosine monophosphateATPadenosine triphosphateGlideGrid-based LIgand Docking with EnergeticGlnRSglutaminyl-tRNA synthetaseGRAVYGRand AVerage of hydropathicitYGROMACSGROingen Machine for Chemical SimulationsHADDOCKHigh Ambiguity Driven protein–protein DOCKingITCisothermal titration calorimetry2MA2-methyladenosine 5'-(dihydrogen phosphate)MDSmolecular dynamics simulation5MU5-methyluridine 5'-monophosphateNPTnumber of particles, pressure and temperatureNVTnumber of particles, volume and temperatureOPLS-AAoptimized potential for liquid simulation all atomPDBBrookhaven Protein DatabankPMEParticle-Mesh EwaldQSI5'-o-[n-(l-Glutaminyl)-sulfamoyl]adenosineRgradius of gyrationRMSDroot mean square deviationRMSFroot mean square fluctuation4SU4-thiouracil 5'-monophosphateSPCsimple point chargetRNAtransfer ribo nucleic acidTtThermus thermophilusXPextra precisionCommunicated by Ramaswamy H. Sarma

Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate Synthase Inhibitors upon Nanoparticle-Mediated Delivery.

Numerous glutamine analogues have been reported as irreversible inhibitors of the glucosamine-6-phosphate (GlcN-6-P) synthase in pathogenic Candida albicans in the last 3.5 decades. Among the reported inhibitors, the most effective N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) has been extensively studied in order to develop its more active analogues. Several peptide-FMDP conjugates were tested to deliver FMDP to its subcellularly located GlcN-6-P synthase target. However, the rapid development of fungal resistance to FMDP-peptides required development of different therapeutic approaches to tackle antifungal resistance. In the current state of the global antifungal resistance, subcellular delivery of FMDP via free diffusion or endocytosis has become crucial. In this study, we report on in vitro nanomedical applications of FMDP and one of its ketoacid analogues, N3- trans-4-oxo-4-phenyl-2-butenoyl-l-2,3-diaminopropanoic acid (BADP). FMDP and BADP covalently attached to polyethylene glycol-coated iron oxide/silica core-shell nanoparticles are tested against intrinsically multidrug-resistant C. albicans. Three different human cancer cell lines potentially overexpressing the GlcN-6-P synthase enzyme are tested to demonstrate the immediate inhibitory effects of nanoparticle conjugates against mammalian cells. It is shown that nanoparticle-mediated delivery transforms FMDP and BADP into strong anticancer agents by inhibiting the growth of the tested cancer cells, whereas their anti-Candidal activity is decreased. This study discusses the emerging inhibitory effect of the FMDP/BADP-nanoparticle conjugates based on their cellular internalization efficiency and biocompatibility.

Inhibition of glutamine metabolism accelerates resolution of acute lung injury.

Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying excessive lung inflammation or enhance resolution of injury. Metabolic programming plays a critical role in regulating inflammatory responses. Due to their high metabolic needs, neutrophils, macrophages, and lymphocytes rely upon glutamine metabolism to support activation and function. Additionally, during times of physiologic stress, nearly all cells, including fibroblasts and epithelial cells, require glutamine metabolism. We hypothesized that inhibiting glutamine metabolism reduces lung inflammation and promotes resolution of acute lung injury. Lung injury was induced by instilling lipopolysaccharide (LPS) intratracheally. To inhibit glutamine metabolism, we administered a glutamine analogue, 6‐diazo‐5‐oxo‐L‐norleucine (DON) that binds to glutamine‐utilizing enzymes and transporters, after injury was well established. Treatment with DON led to less lung injury, fewer lung neutrophils, lung inflammatory and interstitial macrophages, and lower levels of proinflammatory cytokines and chemokines at 5 and/or 7 days after injury. Additionally, DON led to earlier expression of the growth factor amphiregulin and more rapid recovery of LPS‐induced weight loss. Thus, DON reduced lung inflammation and promoted resolution of injury. These data contribute to our understanding of how glutamine metabolism regulates lung inflammation and repair, and identifies a novel target for future therapies for ARDS and other inflammatory lung diseases.