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Supplemental Figure 2: JHU395 DON delivery to P493B lymphoma cells and in vivo time-dependent pharmacokinetics of JHU395 in CD-1 mouse plasma and jejunum

Supplemental Figure 3: Ex vivo JHU395 vs vehicle treated tumor histological analyses and plasma glutamine from non-tumored mice

Supplemental Figure 5: 13C5-glutamine in vivo flux analysis in vehicle vs JHU395 treated tumors

Supplemental Table 1: List of significantly affected metabolites identified in volcano plot following JHU395 versus vehicle treatment of flank MPNST

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.

Supplemental Figure 1: JHU395 and DON treatment of sNF02.2 MPNST cells, evaluation of PARP cleavage following JHU395 treatment in sNF96.2 cells, and effects of DON and JHU395 on oxygen consumption rates in sNF96.2 MPNST cells.

Supplemental Figure 4: Weight changes in non-tumored NPcis mice over one month of daily JHU395 dosing.

6-Diazo-5-oxo- l -norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8 + T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl( S )-2-(( S )-2-acetamido-3-(1 H -indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104’s effect was CD8 + T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

Background: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. Objective: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. Methods: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. Results: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. Conclusion: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

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.

Early clinical investigations of a glutamine antagonist in combination with a purine antimetabolite in pediatric cancers had promise but excessive gastrointestinal toxicity. We recently described that the well-tolerated glutamine antagonist prodrug JHU395 impeded tumor growth in Ras-driven sarcoma models with significant effects on nucleotide metabolites. We therefore investigated the combination of the purine antimetabolite 6-mercaptopurine (6-MP) with JHU395 in NF1-mutant malignant peripheral nerve sheath tumor (MPNST) models. 6-MP (10 micromolar) inhibited growth of human MPNST cells as a single agent and in combination with low dose JHU395 (1 micromolar). However, at previously published doses in mice, single agent 6-MP (20 mg/kg i.p. daily) resulted in weight loss and elevations in serum alanine aminotransferase (ALT), a marker of hepatotoxicity. To limit GI and hepatotoxicity from methylated side products of 6-MP, we developed Protide-TGMP-905 (Pro-905), a phosphoramidate protide of the 6-MP active metabolite 6-TGMP, which enables delivery of active monophosphorylated nucleotide antimetabolite with minimal formation of hepatotoxic methylated 6-MP (Me6-MP) metabolites. When administered at 20% of the 6-MP dose, Pro-905 treated mice (10 mg/kg i.p. daily) experienced similar myelosuppression, an indication of active metabolite formation, without elevation of ALT. One hour following Pro-905 administration (10 mg/kg i.p.) 6-TGMP was detectable in mouse plasma (0.25 nmol/ml), whereas one hour following 6-MP administration 6-TGMP in plasma was below the limit of detection. When administered to flank MPNST mice at this dose, Pro-905 inhibited tumor growth as a single agent and abrogated tumor growth in combination with JHU395 (Day 10 log2-fold change tumor volume: Vehicle = 2.81, Pro-905 = 1.66, JHU395 = 1.76, Combination = 0.31) and improved survival compared to vehicle (Median survival: Vehicle = 15 days, Combination = 23 days). Tumor metabolomics studies were performed to investigate metabolic differences between single agent JHU395, Pro-905, and combination treated mice. Protide purine antimetabolites in combination with glutamine antagonists are tolerated and efficacious in Ras-driven sarcoma models in preclinical studies. Citation Format: Kathryn M. Lemberg, Joanna Marie H. Aguilar, Jesse Alt, Marcela Krecmerova, Pavel Majer, Rana Rais, Barbara Slusher. A novel protide purine antimetabolite combines with the prodrug glutamine antagonist JHU395 in preclinical models of Ras-driven sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1293.

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.

Meaningful engagement in quality improvement (QI) projects by trainees is often challenging. A fellow-led QI project aimed to improve adherence to a blood culture clinical decision algorithm and reduce unnecessary cultures in pediatric oncology inpatients.We visualized preintervention rates of blood cultures drawn on pediatric oncology inpatients using a control chart. Following the introduction of the algorithm to our division, an Ishikawa fishbone diagram of cause-and-effect identified two areas for improvement: prescriber education on the algorithm and targeted feedback on its use. We developed two interventions to support algorithm awareness and use: (1) bundled educational interventions and (2) targeted chart review and feedback. Fellows reviewed750 blood culture episodes and adjudicated each as "adherent" or "nonadherent" to the algorithm. In addition, fellows provided direct feedback to prescribers regarding nonadherent episodes and discussed strategies for algorithm adherence.Blood culture rates in preintervention, intervention, and follow-up periods were 33.35, 25.24, and 22.67 cultures/100 patient-days, respectively. The proportion of nonadherent culture episodes decreased from 47.14% to 11.11%. The use of the algorithm did not prolong the time to cultures drawn on patients with new fever. Seventy-five percent of fellows provided feedback to inpatient teams on algorithm use. Following this project, trainees reported feeling more qualified to apply QI principles to patient care.Implementation of a clinical decision algorithm reduced the rate of cultures drawn on pediatric oncology inpatients. Fellow-led education of the care team decreased the proportion of nonadherent culture episodes and provided active engagement in QI.

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.

Malignant peripheral nerve sheath tumor (MPNST) is a sarcoma that occurs in ~10% of patients with neurofibromatosis type I (NF1). Incomplete surgical resection at diagnosis leads to a 4-year event-free survival rate of 30 micromolar). JHU395 delivered GA to MPNST cells with >4-fold higher cell-to-plasma ratio compared to native GA and maintained ~2.5-fold higher tumor-to-plasma GA levels in vivo. Mice treated with JHU395 had >40% smaller mean tumor volume compared to controls with no overt toxicity. Quantitiative bioanalysis revealed that JHU395 treated tumors had >60% higher glutamine levels compared to controls 30 minutes after oral dosing. In vivo flux analysis demonstrated that JHU395 preferentially affects tumor glutamine utilization for nucleotide synthesis, while glutamine-derived substrates for the citric acid cycle appear unaffected. In summary, JHU395 inhibits growth of MPNST with inhibition of glutamine utilization in nucleotide synthesis and is well-tolerated. Nervous tissue penetrant glutamine antagonism is a feasible and effective therapeutic approach in preclinical models of MPNST. Future studies will investigate JHU395 in combination with nucleotide synthesis inhibitors in MPNST and investigate JHU395 efficacy in additional sarcoma models including patient-derived samples. Citation Format: Kathryn M. Lemberg, Liang Zhao, Ying Wu, Jesse Alt, Joanna Marie H. Aguilar, Jenny Lam, Alexandra J. Gadiano, Rana Rais, Pavel Majer, Jaishri Blakeley, Barbara S. Slusher. JHU395, a nervous tissue penetrant glutamine antagonist, restricts growth of malignant peripheral nerve sheath tumor with inhibition of nucleotide synthesis [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 3602.

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

DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration vs. disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the ‘joining’ configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and thus may provide a mechanism to antagonize formation of single-end transposition products.

HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence-specific (IHF) or non-specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site-specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles ( approximately 105-140 degrees ). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU-DNA and IHF-DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU-induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU's role as an architectural cofactor in many different systems that may require differing geometries.