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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.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON’s toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC(0–t) = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC(0–t) = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.

Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive soft tissue sarcomas that occur with significantly increased incidence in people with the neuro-genetic syndrome neurofibromatosis type I (NF1). These complex karyotype sarcomas are often difficult to resect completely due to the involvement of neurovascular bundles, and are relatively chemotherapy- and radiation-insensitive. The lifetime risk of developing MPNST in the NF1 population has led to great efforts to characterize the genetic changes that drive the development of these tumors and identify mutations that may be used for diagnostic or therapeutic purposes. Advancements in genetic sequencing and genomic technologies have greatly enhanced researchers’ abilities to broadly and deeply investigate aberrations in human MPNST genomes. Here, we review genetic sequencing efforts in human MPNST samples over the past three decades. Particularly for NF1-associated MPNST, these overall sequencing efforts have converged on a set of four common genetic changes that occur in most MPNST, including mutations in neurofibromin 1 (NF1), CDKN2A, TP53, and members of the polycomb repressor complex 2 (PRC2). However, broader genomic studies have also identified recurrent but less prevalent genetic variants in human MPNST that also contribute to the molecular landscape of MPNST and may inform further research. Future studies to further define the molecular landscape of human MPNST should focus on collaborative efforts across multiple institutions in order to maximize information gathered from large numbers of well-annotated MPNST patient samples, both in the NF1 and the sporadic MPNST populations.

Neurofibromatosis Type I (NF1) is a heritable tumor predisposition syndrome in which up to 10% of patients develop malignant peripheral nerve sheath tumor (MPNST), an aggressive sarcoma. For MPNST that is incompletely resected at diagnosis, traditional cytotoxic chemotherapeutic strategies offer a 5 year event-free survival of less than 40%; thus new therapeutic strategies are desperately needed. Reprogramming of energy metabolism, whereby tumor cells take up more glutamine than healthy cells and direct this substrate to replenish metabolites for proliferation, is a hallmark of several cancers that has not been effectively leveraged for treatment of MPNST. Our group has recently described JHU 395, a nervous system targeted prodrug of the glutamine antagonist 6-diazo-5-oxo-norleucine (DON) which delivers DON preferentially to the brain resulting in less gastrointestinal toxicity, which was the main toxicity of DON in past clinical trials. The primary goals of this study were to evaluate glutamine antagonism and JHU 395 activity in MPNST. Using multiple Schwann and MPNST cell lines we investigated cell proliferation in culture under glutamine deprivation and antagonism. Mass spectrometry (MS)-based metabolomic profiling was used to characterize differences between MPNST cells treated with vehicle versus DON. MS-based bioanalytical methods were also used to investigate DON delivery to tumor cells by JHU 395. We found that growth of MPNST cells in culture is preferentially inhibited by glutamine deprivation and DON treatment when compared to immortalized Schwann cells derived from non-tumored nerve (IC50 of 8-9 micromolar versus >30 micromolar). Targeted metabolomics analyses of DON treated human MPNST cells demonstrated multiple differences in downstream glutamine-dependent metabolites including intermediates in purine synthesis and amino acid synthesis, suggesting that DON acts broadly within the tumor cell to inhibit growth. While DON showed limited partitioning into MPNST cells versus plasma, JHU 395 preferentially delivered DON into MPNST with over 5-fold higher cell-to-plasma ratio. Prodrug pharmacokinetic studies in mouse demonstrated that oral administration of the prodrug safely delivered DON at micromolar concentrations to peripheral nerve. In conclusion, compared to healthy Schwann cells, MPNST cells have unique vulnerability to antagonizing glutamine utilization. The nervous system directed prodrug JHU 395 enhances DON delivery to MPNST and represents a novel potential therapeutic approach for these aggressive tumors. Based on these results we have initiated efficacy studies of JHU 395 in the murine NPcis (NF1+/-;p53+/-) model of MPNST. Citation Format: Kathryn Lemberg, Ying Wu, Jesse Alt, Liang Zhao, Alexandra J. Gadiano, Chabely Rodriguez, Rana Rais, Pavel Majer, Jaishri Blakeley, Barbara Slusher. Novel prodrugs of the glutamine antagonist 6-diazo-5-oxo-norleucine (DON) as treatment for malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3524.

SummaryNonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system xc−), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.PaperFlick