Glutamine Antagonist Research Articles

Abstract 5182: Targeting abnormal metabolism downstream of MYC in atypical teratoid/rhabdoid tumors

Abstract Atypical teratoid rhabdoid tumors (AT/RT) are deadly infantile brain tumors in dire need of new, targeted therapies. Recent molecular analysis revealed considerable tumor heterogeneity subdividing AT/RT into 3 distinct groups. The MYC subgroup has a dismal 5 year survival of 18.5%. MYC is known to drive reliance on glutamine for cellular metabolism suggesting that high-MYC expressing neoplasms may be sensitive to glutamine metabolic inhibitors. 6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist that decreases the tumorigenicity of high MYC expressing models of medulloblastoma and neuroblastoma . We hypothesize that DON will reduce the tumorigenicity of the MYC subgroup of AT/RT. High levels of c-MYC protein are expressed in about 1/3 of human AT/RT (Immunohistochemistry for c-MYC on 22 human AT/RT, H-score > 1 standard deviation over the median considered high c-MYC expression). Similarly two of six of AT/RT cell lines expressed high levels of c-MYC protein by western blot. DON treatment slowed cell growth of high c-MYC expressing AT/RT cell lines BT12 and CHLA06 (MTS assay p<0.005 10uM DON vs DMSO control; t-test), whereas cell lines BT37 and CHLA05, which had low c-MYC expression were not affected. Cell cycle analysis demonstrated that DON caused cell cycle arrest in the G2/M phase for the high c-MYC expressing lines BT12 and CHLA06 (31.2% of BT12 DMSO controls in G2/M phase vs 54.5% after 10uM DON treatment and 27.4% vs 48.9% in CHLA06). In contrast the cell cycle was not affected by DON treatment in the cell lines with low c-MYC expression (21.3% DMSO vs 20.2% after 10uM DON in BT37). DON also increased apoptosis in cell lines with high c-MYC and induced DNA strand breaks (Western blot for c-PARP and p-H2A.X respectively). DON significantly improved survival in BT-12 high c-MYC expressing orthotopic mouse xenografts (median survival increased from 21 to 36 days, p<0.005; log-rank test). These data develop the framework for future clinical trials using glutamine antagonists to target the MYC subgroup of AT/RT. Citation Format: Sabrina Wang, Jeffrey Rubens, Sariah Allen, Brent Orr, Charles Eberhart, Eric Raabe. Targeting abnormal metabolism downstream of MYC in atypical teratoid/rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2017-5182

Abstract SY33-01: Targeting metabolism to enhance immunotherapy

Abstract The extraordinary proliferative capacity of cancer necessitates specialized metabolic programming on the part of cancer cells. Indeed, this specialized metabolism not only facilitates growth but also creates a hypoxic, acidic, nutrient-deprived tumor microenvironment able to thwart immune destruction. We hypothesized that by targeting tumor metabolism we could not only slow down tumor growth but also “condition” tumors to be more susceptible to immunotherapy. Inherent to the specialized metabolism of tumors is a markedly increased requirement for glutamine. To this end we have devised a novel glutamine antagonist that is delivered as an inert prodrug that is converted to the active drug by enzymatic activity, which is enriched in tumors. In syngeneic mouse models of cancer our prodrug (JHU-083) markedly inhibited tumor growth, leading to cure of mice after only 14 days of monotherapy. Importantly, targeting glutamine metabolism led to a decreased extracellular acidification rate (ECAR), decreased hypoxia, and increased nutrient availability. Furthermore, we observed decreases in MDSC and fibrocytes with a concomitant increase in inflammatory (TNF-producing) tumor-associated macrophages (TAMs). Likewise, there was an increase in the CD8:Treg ratio and in some tumors a decrease in PDL1 expression on both tumors and infiltrating immune cells. Most importantly, JHU-083 markedly enhanced the efficacy of immunotherapy in the form of checkpoint blockade, adoptive cellular therapy (ACT), and A2aR blockade. Overall our studies define a novel compound for targeting metabolism as a means of enhancing immunotherapy for cancer. Citation Format: Jonathan D. Powell, Robert Leone, Judd Englert, Rana Rais, Barbara Slusher. Targeting metabolism to enhance immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY33-01. doi:10.1158/1538-7445.AM2017-SY33-01

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON)

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Glutamine antagonist-mediated immune suppression decreases pathology but delays virus clearance in mice during nonfatal alphavirus encephalomyelitis

Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.

Riluzole: real-world evidence supports significant extension of median survival times in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the commonest form of motor neuron disease and is a fatal, degenerative, multisystem disorder affecting upper and/or lower motor neurons in the motor cortex, brain stem, and spinal cord. ALS is characterized by progressive atrophy of associated bulbar, limb, thoracic, and abdominal muscles and supporting cells manifesting in a range of muscular symptoms such as weakness and wasting and eventual paralysis; the majority of patients will die from respiratory failure within 2–5 years of onset. Riluzole, a synthetic benzothiazole drug with glutamine antagonist activity, is indicated for the treatment of patients with ALS and is the only drug that has been shown to slow the course of the disease and extend survival in ALS patients. The original analyses, and subsequent meta-analyses, of data obtained from randomized controlled trials (RCTs) suggest that riluzole typically extends survival by 2–3 months and increases the chance of an additional year of survival by ~9%. However, published real-world evidence (RWE) from 10 clinical ALS databases indicates that riluzole therapy may afford much greater extension of survival, and improvements in median survival times of more than 19 months have been reported in the overall ALS patient population. This article will review the available data from RCTs and RWE on riluzole therapy.

Discovery of 6-Diazo-5-oxo-l-norleucine (DON) Prodrugs with Enhanced CSF Delivery in Monkeys: A Potential Treatment for Glioblastoma.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

Protective Effects of Glutamine Antagonist 6-Diazo-5-Oxo-l-Norleucine in Mice with Alphavirus Encephalomyelitis.

Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease. Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.

Abstract 1035: Targeting glutamine metabolism with the novel inhibitor JHU-083 inhibits tumor growth and alters the tumor immune microenvironment

Abstract Glutamine metabolism plays a critical role in promoting multiple biosynthetic pathways necessary for tumor growth. Therefore we endeavored to create a small molecule inhibitor that would block multiple pathways of glutamine metabolism as opposed to blocking one enzyme. Previous studies employing the non-specific glutamine antagonist 6-diazo-5-oxo-L-norlecuine (DON) have achieved this goal however, with much toxicity. To this end we devised a novel prodrug of DON (JHU-083) with distinct distribution and cellular partitioning properties. When dosed on an equimolar basis, JHU-083 retains the antitumor efficacy of DON but is devoid of the gut toxicity and morbidity/mortality. To this end Wild-type mice were injected subcutaneously EL4 T-cell lymphoma cells. The tumors were allowed to engraft and treatment was initiated on day 5 with either vehicle, DON or JHU-083. After 7 days of treatment there was complete tumor regression in both the DON and JHU-083 treated mice, while tumors continued to grow in the vehicle treated mice. Importantly, after 7 days of treatment the DON treated mice appeared ill with lethargy and ruffled fur and even death as a result of treatment toxicity. Alternatively, the JHU-083 treated mice remained healthy and vibrant. Based on these studies in a lymphoma model we next tested JHU-083 in 2 models of colon cancer. To this end we injected wild-type mice subcutaneously with MC38 or CT26 cells. Again, similar to the EL4 lymphoma experiments, we observed that JHU-083 resulted in complete regression of MC38 and CT26 tumors after 4 and 7 days of treatment respectively. The tumor microenvironment, which promotes immune evasion, is very much influenced by tumor metabolism. Thus, we wanted to determine the effect of inhibiting glutamine metabolism on the tumor immune microenvironment. To this end mice injected with B16 melanoma cells were treated for 3 days with DON during which time the tumors decreased in size. Interestingly, there was also a concomitant decrease in the percentage of Foxp3+ cells and an increase in the CD8:Foxp3+ T cell ratio amongst the TILS. Overall our studies demonstrate the principle of cellular selectivity based upon demand. That is, a non-selective inhibitor of glutamine metabolism can robustly inhibit tumor growth while demonstrating minimal side effects based on the differential demand of cancer cells versus normal cells. Furthermore, our studies suggest that sequencing anti-metabolic therapy with immunotherapy might prove to be a novel means of broadening and enhancing the role of immunotherapy for the treatment of cancer. Citation Format: Judson Englert, Chih-Hsien Cheng, Robert Leone, Pavel Majer, Rana Rais, Barbara Slusher, Jonathan Powell. Targeting glutamine metabolism with the novel inhibitor JHU-083 inhibits tumor growth and alters the tumor immune microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1035.

Alphavirus Encephalomyelitis: Mechanisms and Approaches to Prevention of Neuronal Damage.

Mosquito-borne viruses are important causes of death and long-term neurologic disability due to encephalomyelitis. Studies of mice infected with the alphavirus Sindbis virus have shown that outcome is dependent on the age and genetic background of the mouse and virulence of the infecting virus. Age-dependent susceptibility reflects the acquisition by neurons of resistance to virus replication and virus-induced cell death with maturation. In mature mice, the populations of neurons most susceptible to infection are in the hippocampus and anterior horn of the spinal cord. Hippocampal infection leads to long-term memory deficits in mice that survive, while motor neuron infection can lead to paralysis and death. Neuronal death is immune-mediated, rather than a direct consequence of virus infection, and associated with entry and differentiation of pathogenic T helper 17 cells in the nervous system. To modulate glutamate excitotoxicity, mice were treated with an N-methyl-D-aspartate receptor antagonist, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonists or a glutamine antagonist. The N-methyl-D-aspartate receptor antagonist MK-801 protected hippocampal neurons but not motor neurons, and mice still became paralyzed and died. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonists GYKI-52466 and talampanel protected both hippocampal and motor neurons and prevented paralysis and death. Glutamine antagonist 6-diazo-5-l-norleucine protected hippocampal neurons and improved memory generation in mice surviving infection with an avirulent virus. Surprisingly, in all cases protection was associated with inhibition of the antiviral immune response, reduced entry of inflammatory cells into the central nervous system, and delayed virus clearance, emphasizing the importance of treatment approaches that include prevention of immunopathologic damage.

Novel lymphoid targeted prodrugs of the glutamine antagonist DON for the treatment of hematological malignancies

ObjectiveThe glutamine antagonist 6‐diazo‐5‐oxo‐l‐norleucine (DON) has shown robust efficacy in both lymphoma and leukemia animal models and exploratory clinical studies. DON broadly blocks glutamine utilizing reactions critical for the synthesis of nucleic acids/proteins and the generation of alpha‐ketoglutarate for energy metabolism. Unfortunately its severe toxicity hampered its development. Using a prodrug approach, we aimed to synthesize lymphoid‐targeted prodrugs of DON designed to circulate intact in blood and preferentially activate inside PBMCs affording reduced systemic exposure and improved tolerability, while maintaining its robust efficacy.methodsSynthetic efforts for DON prodrugs were particularly challenging due to DON's inherent instability. We found it was imperative to mask both the carboxylate as well as the amine functionality of DON to achieve sufficient chemical stability. We concealed the carboxylate and amine with various alkyl esters (e.g. methyl, ethyl and isopropyl), amino acids (e.g. leucine, alanine), and other bulkier promoeities used in FDA approved drugs (e.g. POC, POM, ODOL). We then evaluated the prodrugs for in vitro plasma stability across multiple species (mouse, dog, monkey, pig, human). Ex vivo whole blood experiments were further conducted to determine selective conversion in PBMCs. Lastly in vivo experiments using pigs were carried out and PBMC to plasma ratios were calculated.resultsOur novel DON prodrugs showed species specific plasma stability with rodents metabolizing the prodrug more rapidly compared to higher species. Of the various modifications on the carboxylate, the isopropyl ester afforded significant lymphoid‐targeting in pigs, monkeys, dog, and humans. In human whole blood ex vivo studies several of the isopropyl ester containing prodrugs delivered DON into PBMCs versus plasma with a >30‐fold differential, in contrast to DON which delivered a small percentage (<10%) of drug to PBMCs. Lastly in vivo studies in pigs confirm this PBMC selectivity with favorable pharmacokinetics.conclusionsWe have successfully synthesized lymphoid‐targeted DON prodrugs which deliver >30‐fold more DON to lymphoid cells in human whole blood versus plasma. Comparative efficacy and toxicity studies are ongoing in lymphoma animal models. These novel prodrugs may pave a path for the development of a new and safer therapeutic for hematological malignancies.

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance.

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance.

Abstract B37: Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma

Abstract Oncogenic Myc is known to alter cellular metabolism resulting in glutamine addiction, and the MYCN-amplified subclass of neuroblastoma is associated with extremely poor prognosis. Successful identification of therapeutic agents that are effective in neuroblastoma, particularly in tumors with MYCN amplification, could significantly increase patient survival. To explore the effect of inhibiting glutamine metabolism in neuroblastoma, we used the glutamine antagonist DON (6-diazo-5-oxo-norleucine). DON is an irreversible competitive inhibitor which alkylates enzymes that utilize glutamine. Although first studied in the 1950s as a possible cancer chemotherapeutic, DON was associated with severe nausea which limited its use in patients. DON remains a potent inhibitor, and a useful tool to study the effect of modulating glutamine metabolism in vivo. We tested the DON sensitivity of six human neuroblastoma cell lines (Kelly, IMR-32, SK-N-BE2, SK-N-AS, SK-N-FI, and SH-SY5Y) compared to immortalized BJ fibroblast control cells. DON effectively inhibited all neuroblastoma cell lines tested with minimal effect on BJ cells. Furthermore, MycN overexpression in SK-N-AS cells, a cell line which is not MYCN-amplified and expresses low levels of cMyc, was sufficient to make cells significantly more sensitive to DON. Next, we tested the effect of DON treatment on subcutaneous xenografts. For these experiments we used the cell lines SK-N-AS (not MYCN-amplified), SK-N-BE2 (MYCN-amplified with moderate MycN levels), and IMR-32 (MYCN-amplified with high MycN levels). Mice were treated twice per week by intraperitoneal injection with DON (50 or 100mg/kg) or water control. Treatment with 50mg/kg DON significantly inhibited tumor growth of all three cell lines, suggesting neuroblastoma tumors may be broadly sensitive to glutamine inhibitors. IMR-32 tumors were the most sensitive to DON treatment, leading to a 6-fold reduction in tumor size after 2.5 weeks of treatment. Tumor BrdU incorporation decreased following DON treatment, and IMR-32 tumors showed increased levels of cleaved caspase 3, a marker of apoptosis. At the higher 100mg/kg dose, DON treatment led to a more substantial decrease in BrdU incorporation, and at this dose BE2 tumors also showed significantly increased levels of cleaved caspase 3, which were not detected in AS tumors. These results suggest that MYCN-amplified tumors may be particularly sensitive to glutamine inhibition. To examine the mechanism of DON-induced cell death, we treated cells with the pan-caspase inhibitor QVD. QVD treatment for 48 or 72hr significantly rescued both MYCN-amplified cell lines (Kelly, IMR-32) and cells that express high levels of cMyc (SY5Y), while having minimal effect in the remaining neuroblastoma cell lines with low Myc levels. These results confirm that DON induces cell death partially by activating apoptosis in high Myc expressing cell lines. Next, we used shRNA to show that in Kelly cells DON-induced apoptosis occurs by signaling through Bax, but not Bak. Lastly, we tested DON in combination with pro-apoptotic compounds in vitro. We found that DON treatment combined with the Bcl-2 family inhibitor ABT-263 had synergistic effects on cell death in the MYCN-amplified cell lines Kelly and BE2. Our results suggest that combined targeting of glutamine metabolism while reducing the threshold for tumor cell apoptosis may be beneficial for neuroblastoma treatment. Citation Format: Rachelle R. Olsen, Michelle N. Mary-Sinclair, Zhirong Yin, Kevin W. Freeman. Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B37.

Abstract 972: Targeting glucose and glutamine regulated BCL2 family members for multiple myeloma therapy

Abstract Multiple myeloma (MM) is a plasma cell malignancy accounting for approximately 11000 deaths annually in the US. Despite use of immunomodulatory drugs and proteasome inhibitors, MM is largely incurable with a median survival of 5 years due to the development of intrinsic and acquired resistance to these drugs. Targeting metabolic dependencies in a cancer cell may provide an effective strategy to target the molecular heterogeneity and chemo resistance characteristic of MM. Resistance is in part mediated by ineffective regulation of Bcl-2 family members. Myeloid cell leukemia factor 1 (MCL-1) is a key resistance promoting anti-apoptotic protein expressed in MM cells. We and others have previously established that glucose regulates expression of MCL-1. MM cells are heavily reliant upon glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. While glucose withdrawal was found to suppress MCL-1 expression uniformly, this did not always correlate with the extent of apoptosis induced. One potential mechanism for continued survival despite suppression of MCL-1 could be due to a shift in binding of pro-apoptotic BCL2 proteins to alternate pro-survival BCL2 members. Our investigation through co-immunoprecipitation experiments revealed that glucose or glutamine withdrawal enhances a shift in binding of pro-apoptotic BIM from MCL-1 to BCL2/ BCLxL. This shift in the association of BIM to BCL2 and BCLxL increased sensitivity to BH3 mimetics ABT-199/737. Induction of NOXA, that has a greater affinity for MCL-1 than BIM, is known to promote the redistribution of BIM to BCL2 and BCLxL. Indeed, glucose and glutamine withdrawal were found to increase NOXA expression associated with activation of the upstream GCN2/PERK/eIF2α/ATF4 axis in a cell type specific manner. Knock down of NOXA in MM cells successfully increased cell survival and reduced sensitivity towards ABT-199 in glucose or glutamine deprived cells. We have extended these observations to a panel of 7 myeloma cell lines and primary myeloma patient samples. Our study broadly divides MM cells into two subtypes on the basis of glucose or glutamine dependency and provides effective ways to their survival by targeting their specific metabolic dependencies in combination with the BH3 mimetic ABT-199. We also demonstrate that glucose transport and metabolism in MM can be targeted with the FDA approved GLUT4 inhibitor ritonavir and glutamine utilization can be inhibited by glutamine antagonist DON (6-Diazo-5-oxo-L-norleucine). Both ritonavir and DON treatments sensitized MM cell lines and primary patient samples to ABT-199, bolstering the utility of repurposing FDA-approved ritonavir and ABT-199 (in clinical trial) for MM therapy. Citation Format: Richa Bajpai, Shannon M. Matulis, Changyong Wei, Ajay K. Nooka, Lawrence H. Boise, Mala Shanmugam. Targeting glucose and glutamine regulated BCL2 family members for multiple myeloma therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 972. doi:10.1158/1538-7445.AM2015-972

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

Evaluating the Toxicity of the Analgesic Glutaminase Inhibitor 6-Diazo-5-Oxo-L-Norleucine in vitro and on Rat Dermal Skin Fibroblasts.

6-diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist produced naturally by Streptomyces. It inhibits several glutamine-dependent enzyme pathways. Of particular note is its inhibitory effect on the mitochondrial enzyme, glutaminase (GLS), the primary producer of neuronal glutamate. Glutamate is an excitatory neurotransmitter released by primary sensory peripheral nerve terminals and spinal synaptic terminals during pain signaling. Previous work using the tail incision and inflammatory models of pain has demonstrated that a single application of the glutaminase inhibitor, DON, into a surgical incision or the paw of arthritic animals results in pain relief. Even though this compound shows promise as a therapeutic agent, limited data exist regarding its dermal toxicity. As a first approach, we evaluated the effect of several concentrations of DON, on the viability, mitochondrial oxidative capacity and proliferation of rat skin fibroblasts, and then examined the effect of DON after incubation with human liver microsomes on proliferation. Finally, we evaluated DON treated rat skin (tail and hind paw) for cellular necrosis, inflammation and mitotic bodies. No significant effects (p > 0.05) of DON were noted on apoptosis, necrosis, and mitochondrial activity in experiments with cultured rat skin fibroblasts. Flow cytometry revealed the absence of apoptosis in cells treated at the IC50 of 232.5 μM. Enhanced toxicity post-exposure to human microsomes was not observed when compared to DON alone. The H&E staining of the rat skin revealed no obvious pathology in the DON treatment group (10 mM). DON has no/minimal cellular toxicity in vitro on dermal fibroblasts at concentrations that effectively provide analgesia. The local application of concentrations greater than the in vitro IC50 for DON revealed no in vivo skin toxicity. These data provide results indicating zero-to-minimal cellular toxicity with DON and support the further investigation of DON as an analgesic.

Neurological sequelae induced by alphavirus infection of the CNS are attenuated by treatment with the glutamine antagonist 6-diazo-5-oxo-l-norleucine.

Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits. Infection of mice with the prototypic alphavirus, Sindbis virus, provides an animal model with which to study disease mechanisms and examine potential therapeutics. Infectious virus is cleared from the brain within a week after infection, but viral RNA is cleared slowly and persists for the life of the animal. However, no studies have examined the effect of infection on neurocognitive function over time. In the present study, we examined neurocognitive function at different phases of infection in 5-week-old C57BL/6 mice intranasally inoculated with Sindbis virus. At the peak of active virus infection, mice demonstrated hyperactivity, decreased anxiety, and marked hippocampal-dependent memory deficits, the latter of which persisted beyond clearance of infectious virus and resolution of clinical signs of disease. Previous studies indicate that neuronal damage during alphavirus encephalomyelitis is primarily due to inflammatory cell infiltration and glutamate excitotoxicity rather than directly by virus infection. Therefore, mice were treated with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist that can suppress both the immune response and excitotoxicity. Treatment with DON decreased inflammatory cell infiltration and cell death in the hippocampus and partially prevented development of clinical signs and neurocognitive impairment despite the presence of infectious virus and high viral RNA levels. This study presents the first report of neurocognitive sequelae in mice with alphavirus encephalomyelitis and provides a model system for further elucidation of the pathogenesis of virus infection and assessment of potential therapies.

Antagonizing Bcl-2 family members sensitizes neuroblastoma and Ewing's sarcoma to an inhibitor of glutamine metabolism.

Neuroblastomas (NBL) and Ewing’s sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become “addicted” to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists.

Abstract B67: Targeting glutamine metabolism in neuroblastoma and Ewing's sarcoma

Abstract Therapeutic targeting of aberrant cancer metabolism is currently an area of great interest in pediatric oncology. Previous reports have established that oncogenic Myc alters cellular metabolism resulting in cells dependent on exogenous glutamine. Therefore, we chose to target glutamine metabolism in the pediatric cancers Neuroblastoma and Ewing's Sarcoma. Neuroblastoma (NB) is a pediatric malignancy of the developing sympathetic nervous system, and approximately 20% of patients have amplified MYCN and very poor prognosis. Ewing's Sarcoma (EWS) is an aggressive tumor of the bone and soft tissue characterized by chromosome translocations resulting in EWSR1/ETS family fusion proteins. Approximately 85% of EWS cases contain an EWSR1/FLI fusion protein that targets c-Myc for overexpression and potentially cooperates with c-Myc in cell transformation. We chose to use the glutamine antagonist DON (6-diazo-5-oxo-norleucine) as a tool to examine the effect of inhibiting glutamine metabolism in NB and EWS cells. DON was first explored as a cancer chemotherapeutic in the 1950s and 1960s, but severe nausea in patients following treatment limited its use as a cancer therapeutic. DON is a competitive inhibitor which irreversibly alkylates glutamine-utilizing enzymes, including those involved in nucleotide biosynthesis and glutaminolysis, two important Myc-regulated metabolic pathways in cancer. DON effectively inhibited a panel of six NB and three EWS cell lines, while having minimal effect on BJ fibroblast control cells. In addition, we found a strong correlation between sensitivity to glutamine addiction and susceptibility to DON (R2 = 0.73). Next, we used human tumor xenografts in nude mice to examine the effect of inhibiting glutamine metabolism in the context of an established tumor. Following injection of neuroblastoma (SK-N-AS, SK-N-BE2) or Ewing's Sarcoma (SK-N-MC, SK-ES-1) cells, tumors were allowed to reach 200mm3 in size before being randomized into treatment groups. Mice were then treated with 100mg/kg DON or water control twice per week. DON treatment significantly inhibited both tumor growth and BrdU incorporation in all NB and EWS tumors tested, suggesting that targeting glutamine metabolism could be an effective therapeutic approach. Interestingly, although DON treatment was strongly cytostatic in all cell lines tested, only in the SK-N-BE2 cells did we detect increased cleavage of caspase-3 as a marker for apoptosis. These results suggest that perhaps combining glutamine inhibition with therapies to induce apoptosis may be more effective than targeting glutamine metabolism alone. To further examine the mechanism by which DON and glutamine withdrawal induce cell death, we treated NB and EWS cell lines with the pan-caspase inhibitor QVD. Treatment with 20uM QVD for either 48hr or 72hr significantly rescued cell number after DON treatment. This effect was most pronounced in NB cell lines with NMyc amplification, such as Kelly and IMR-32. Lastly, we performed an in vitro screen to identify pro-apoptotic compounds that increased the effects of DON on NB and EWS cell lines. We found three compounds which increased the effects of DON at clinically relevant concentrations: the synthetic retinoic acid derivative fenretinide, and the Bcl-2 family inhibitors obatoclax mesylate and navitoclax. Of these, navitoclax caused the greatest increase in DON activity across the entire panel of cell lines tested, suggesting that antagonizing Bcl-2 family members may be beneficial in combination with glutamine deprivation. Our results strongly suggest that combining therapeutic approaches to inhibit glutamine metabolism and induce tumor cell apoptosis may be a promising strategy for treatment of both NB and EWS tumors. Citation Format: Rachelle R. Olsen, Michelle N. Mary, Kevin W. Freeman. Targeting glutamine metabolism in neuroblastoma and Ewing's sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B67.

Crystal structures of γ-glutamyltranspeptidase in complex with inhibitors

γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) is involved in the degradation of γ-glutamyl compounds such as glutathione (GSH; γ-glutamyl-cysteinyl-glycine) . A major physiological role of this enzyme is to cleave the extracellular GSH as a source of cysteine for intracellular glutathione biosynthesis. Another crucial role of GGT is to cleave glutathione-S-conjugates as a key step in detoxification of xenobiotics and drug metabolism. In mammals, GGT has been implicated in physiological disorders such as Parkinson's disease, other neurodegenerative diseases including Alzheimer's disease and cardiovascular disease. The indispensable roles played by GGT in GSH-mediated detoxification and cellular response to oxidative stress suggest that GGT is an attractive pharmaceutical target. We here report the binding mode of acivicin, a well-known glutamine antagonist, to B. subtilis GGT at 1.8 Å resolution showing that acivicin is bound to the Oγ atom of Thr403, the catalytic nucleophile of the enzyme, through its C3 atom [1]. The observed electron density around the C3 atom was best fitted to the planar and sp2 hybridized carbon, consistent with a simple nucleophilic substitution of Cl at the imino carbon by Oγ atom of Thr403. Furthermore, comparison of three bacterial enzymes, the GGTs from E. coli, H. pylori and B. subtilis in complex with acivicin, showed significant diversity in the orientation of the dihydroisoxazole ring among three GGTs. The differences are discussed in terms of the recognition of the α-amino and α-carboxy groups in preference to the dihydroisoxazole ring as observed in time-lapse soaking crystal structures of B. subtilis GGT with acivicin.

Cardiac glutaminolysis: a maladaptive cancer metabolism pathway in the right ventricle in pulmonary hypertension

The rapid growth of cancer cells is permitted by metabolic changes, notably increased aerobic glycolysis and increased glutaminolysis. Aerobic glycolysis is also evident in the hypertrophying myocytes in right ventricular hypertrophy (RVH), particularly in association with pulmonary arterial hypertension (PAH). It is unknown whether glutaminolysis occurs in the heart. We hypothesized that glutaminolysis occurs in RVH and assessed the precipitating factors, transcriptional mechanisms, and physiological consequences of this metabolic pathway. RVH was induced in two models, one with PAH (Monocrotaline-RVH) and the other without PAH (pulmonary artery banding, PAB-RVH). Despite similar RVH, ischemia as determined by reductions in RV VEGFα, coronary blood flow, and microvascular density was greater in Monocrotaline-RVH versus PAB-RVH. A sixfold increase in (14)C-glutamine metabolism occurred in Monocrotaline-RVH but not in PAB-RVH. In the RV working heart model, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) decreased glutaminolysis, caused a reciprocal increase in glucose oxidation, and elevated cardiac output. Consistent with the increased glutaminolysis in RVH, RV expressions of glutamine transporters (SLC1A5 and SLC7A5) and mitochondrial malic enzyme were elevated (Monocrotaline-RVH > PAB-RVH > control). Capillary rarefaction and glutamine transporter upregulation also occurred in RVH in patients with PAH. cMyc and Max, known to mediate transcriptional upregulation of glutaminolysis, were increased in Monocrotaline-RVH. In vivo, DON (0.5 mg/kg/day × 3 weeks) restored pyruvate dehydrogenase activity, reduced RVH, and increased cardiac output (89 ± 8, vs. 55 ± 13 ml/min, p < 0.05) and treadmill distance (194 ± 71, vs. 36 ±7 m, p < 0.05) in Monocrotaline-RVH. Glutaminolysis is induced in the RV in PAH by cMyc-Max, likely as a consequence of RV ischemia. Inhibition of glutaminolysis restores glucose oxidation and has a therapeutic benefit in vivo. Patients with pulmonary artery hypertension (PAH) have evidence of cardiac glutaminolysis. Cardiac glutaminolysis is associated with microvascular rarefaction/ischemia. As in cancer, cardiac glutaminolysis results from activation of cMyc-Max. The specific glutaminolysis inhibitor DON regresses right ventricular hypertrophy. DON improves cardiac function and exercise capacity in an animal model of PAH.