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

Abstract

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.

Figure 1:

MPNST cells are sensitive to glutamine deprivation and competitive glutamine antagonists. A) Live cell counts from trypan blue exclusion assay comparing growth of human MPNST cells (sNF96.2) in control (>2mM glutamine) or low (50μM) glutamine media normalized to number of cells seeded. Cells were counted every 24 hours for five days. Data is +/− S.D. B) Percent growth of cells based on alamar blue fluorescence (590 nm) of human MPNST or immortalized Schwann cells (ipn02.32λ; imm SC) growing in glutamine-free media with added glutamine titrated at increasing concentrations. Viability was measured at 72 hours glutamine treatment. Data is +/− S.D. C) Structures of DON and JHU395 with clogP values indicated. D) Percent viable MPNST and immortalized Schwann cells based on alamar blue fluorescence normalized to untreated controls following treatment with DON or JHU395. Viability was measured at 72 hours drug treatment. Data is +/− S.D. *** 0.0001 < p ≤ 0.001, ** 0.001 < p ≤ 0.01, * 0.01 < p ≤ 0.05 by Student’s t-test (A).

Figure 2:

JHU395 is a lipophilic, plasma stable prodrug GA that delivers DON to MPNST in vitro and in vivo. A) Calculated apparent permeability (P_app) of DON, JHU395, or Verapamil (positive control) following LC-MS quantification of donor and acceptor samples in a 5 hour parallel artificial membrane permeation assay. Data is +/− S.D. B) Percentage of quantified intact JHU395 in plasma from multiple species following 60 minutes incubation at 37°C. Data is +/− S.D. C) Percentage of quantified intact JHU395 and DON in human plasma and MPNST cells (sNF96.2) following one hour incubation with 20 μM JHU395. Data is +/− S.E.M. D) DON quantification (nmol/g tissue or nmol/ml plasma) following oral administration of 1.2 mg/kg JHU395 to flank MPNST B6 mice. Chart indicates DON C_max, T_max, and area under the curve (AUC) in tumor and plasma. Error bars are +/− S.D. E) DON quantification (nmol/g tissue or nmol/ml plasma) in plasma and jejunum thirty minutes after 1.2 mg/kg JHU395 to B6 mice. Error bars are +/− S.D. *** 0.0001 < p ≤ 0.001 by Student’s t-test.

Figure 3:

Orally administered JHU395 decreases tumor growth in a flank MPNST model with concomitant increase in glutamine. A) Characterization of syngeneic murine flank MPNST by H&E stain (i), Ki67 immunohistochemistry (ii), and western blotting for phospho-ERK1/2 in the presence of MEK inhibitor (PD0325901 5μM for 3h). Scalebar on images = 100 μm. B) Mean tumor volumes in mice treated daily with vehicle (grey) or JHU395 (green; 1.2 mg/kg p.o. × 5 days, 0.5 mg/kg p.o. × 9 days). Data shown is mean +/− SEM; * p ≤ 0.05 by Student’s t-test on day 15 and is representative of three independent experiments. Right panels show log₂ (fold change) of individual animal tumor volumes for Day 15 versus Day 1. C) Tumor and plasma glutamine quantified in tissues from JHU395- and vehicle-treated animals. Data shown is mean +/− SD. **** p ≤ 0.0001, *** 0.0001 < p ≤ 0.001 by Student’s t-test.

Figure 4:

Orally administered JHU395 has minimal GI or hepatotoxicity. A) Average weight change in flank MPNST mice treated during efficacy study with vehicle or JHU395 (1.2 mg/kg/day p.o. × 5 days, then 0.5 mg/kg p.o. × 9 days; n=9). Change from mean starting weight did not exceed 10%. B) Jejunal histology in a subset of vehicle and JHU395 treated mice after 14 days oral dosing. No increase in apoptotic figures or disruption of crypt architecture was observed. Scalebar = 49 μm. C) Markers of liver toxicity from blood (AST, ALT, total bilirubin) were measured in vehicle or JHU395 treated animals at sacrifice (day 15). All data shown is mean +/− S.D. p > 0.05 for each lab value by t-test.

Figure 5:

Orally administered JHU395 has no overt neurotoxicity. A) Brain histology was evaluated in vehicle (PBS + 1% Tween-80 + 2.5% ethanol p.o. daily × 14 days; n=3) and JHU395 (1.2 mg/kg/day p.o. × 5 days, then 0.5 mg/kg p.o. × 9 days; n=3) treated mice. No cortical vacuolization was observed. Scalebar = 400 μm. B) Heatmap showing scoring on modified Irwin behavioral test of vehicle (n=11), JHU395 (n=9), or oxotremorine (n=5; assay positive control) B6 flank MPNST mice over a 14 day dosing period. Mean scores are normalized to day 0 vehicle group and shown per behavior per day. C) Time in seconds that vehicle, JHU395, and oxotremorine treated mice were able to hang from inverted wire in seconds on days 0, 8, and 15 as part of modified Irwin assay. Data shown is mean +/− S.D. p > 0.05 for vehicle and JHU395 grip times by multiple t-tests.

Figure 6:

Metabolomics analyses demonstrate JHU395 inhibition of tumor glutamine utilization in purine synthesis. A) Volcano plot of metabolites identified from murine flank MPNST (n = 8 tumors/group) harvested two hours after oral administration of JHU395 (1.2 mg/kg/dose) or vehicle. Metabolites selected by volcano plot with fold change threshold (x) 2 and t-tests threshold (y) 0.1 are shown in red circles. Both fold changes and p values are log transformed. B) Quantitative LC-MS analysis of FGAR from tumors (n = 8 per group) of vehicle or JHU395 treated murine flank MPNST. Data shown is mean +/− S.D.; p < 0.01 by t-test. C) ¹⁵N₂-glutamine labeled JHU395-treated tumors show significantly decreased m+1, m+2 isotopologue enrichment (inosine monophosphate, adenosine monophosphate) and significantly decreased m+2, m+3 isotopologue enrichment (guanosine monophosphate) compared to vehicle-treated tumors. Data shown is for 6–7 animals per treatment representative of two independent experiments. Graphed data is mean percent enrichment +/− S.D. Statistical testing was done by multiple t-tests where *** p ≤ 0.001, ** 0.001 < p ≤ 0.01, * 0.01 < p ≤ 0.05. D) Percent viable human MPNST cells (sNF96.2) based on alamar blue fluorescence normalized to untreated controls following treatment with JHU395 in standard media (control) or media containing 100μM guanosine monophosphate (GMP). Viability was measured at 72 hours JHU395 treatment. E) Schematic of JHU395 inhibition of glutamine utilization for nucleotide synthesis versus glutaminolysis in human MPNST.