The sNF96.2 cells (RRID:CVCL_K281) were received from American Type Culture Collection and grown in DMEM (Life Technologies #11965), 10% FBS (Hyclone), 2mM L-glutamine and 1% penicillin/streptomycin. JH-2-002 and JH-2-031 MPNST cells were received from the JH NF1 Biospecimen Repository (http://bit.ly/nf1bank) and grown in DMEM/F12 (Life Technologies 11320–033), 10% FBS (Gemini), 2 mM L-glutamine, and 1% penicillin/streptomycin [19, 21]. Cell lines were authenticated at the Johns Hopkins Genetic Resources DNA Services Core Facility by STR profiling using Promega GenePrint10 in comparison to available data [19, 21] and tested negative for Mycoplasma using a Captivate Bio EZ-PCR kit. For establishment of murine flank MPNST, cells harvested from a tumor in an NPcis (NF1^+/−;p53^+/−) mouse and frozen in 5% DMSO were cultured as previously described [6]. All cells were incubated at 37°C, in a humidified atmosphere with 5% CO₂. Confluency was monitored using an Axiovert 25 optical microscope. JHU395 was synthesized as previously described [22]. 6-mercaptopurine (Tocris #4103) was purchased commercially. Pro-905 was synthesized as described below and in the Supplemental Methods. For details of compound dose response and colony area assays [23] in cell culture please see Supplemental Methods.

sNF96.2 and JH-2-002 cells were plated in equal numbers and grown to 70% confluence per 10 cm² plate in media. Cells were treated in quadruplicate with JHU395 (10 μM) or DMSO for 6 hours, then media was aspirated and cells were washed with cold PBS. Polar metabolites were extracted in 2 mL ice cold 80% methanol/20% water following the published protocol of Yuan et. al. [24]. Dried metabolite pellets were resuspended in 20 uL LC/MS grade water and 5 uL was injected onto an AB/SCIEX 6500 QTRAP hybrid triple quadrupole mass spectrometer targeting >300 polar metabolites via selected reaction monitoring (SRM) with polarity switching. Metabolites were separated using amide XBridge HILIC (Waters Corp.) chromatography with a Shimadzu Prominence UFLC instrument. Data were integrated using MultiQuant 3.0 software to generate peak area intensities. For details of FGAR quantification please see Supplemental Methods.

Radiolabeled glycine, hypoxanthine, and guanine incorporation to DNA and RNA were measured in sNF96.2 cells and JH-2-002 cells respectively by a protocol similar to described in [4]. Briefly, cells (~85% confluent) were incubated in dialyzed FBS media for 15 hours, then treated with vehicle (DMSO), JHU395 (10μM), Pro-905 (10μM), or combination for 6 hours. At the same time of drug treatment, cells were also labeled with the given radionuclide (2-μCi of either U-¹⁴C-glycine, ³H-hypoxanthine or 8-³H-guanine) for 6 hours. After 6 hours, cells were harvested and RNA or DNA was isolated using Allprep DNA/RNA kits according to the manufacturer’s instructions and quantified using a spectrophotometer. 30 μl of eluted RNA or 70 μl of eluted DNA were added to scintillation vials and radioactivity was measured by liquid scintillation counter and normalized to the total RNA or DNA concentrations, respectively. All conditions were analyzed with biological triplicates and representative of at least two independent experiments.

Studies in laboratory mice were conducted under a protocol approved by the Johns Hopkins Animal Care and Use Committee. C57BL6/NHsd (B6) male mice, at ~12 weeks of age between 25 and 30 g were obtained from Envigo. NSG mice (#005557) were obtained from Jackson Labs and bred in house. NPcis (B6;129S2-Trp53^tm1Tyj Nf1^tm1Tyj/J, RRID:IMSR_JAX:008191) transgenic mice [25] were obtained from Jackson Labs, Inc, and bred in house. Animals were genotyped using previously validated primers on ear punches (Transnetyx).

To explore the anti-tumor efficacy of JHU395 in a genetically-engineered mouse model of MPNST, NPcis mice were bred to generate double heterozygote (NF1^+/−;p53^+/−) mice. Animals were monitored three times weekly for spontaneous tumor development. Once a palpable tumor reached a volume of ≥100 mm³ as measured by calipers and calculated by the formula: volume = (L × (W ²))/2, the animal was enrolled onto the treatment study and began treatment with either vehicle (PBS + 1% Tween-80 and 2.5% ethanol TIW p.o., n = 10) or JHU395 (2.4 mg/kg TIW p.o., n = 11). Animal weights, body condition scores (BCS), and tumor volumes were measured three times weekly during the study.

Reasons for animal euthanasia were defined by guidelines set forth by the Johns Hopkins University Animal Care and Use Committee including tumor size exceeding 2 cm in either dimension, poor body condition scoring (BCS), and other clinical signs of pain and distress. On the day of euthanasia, each animal received a final timed dose of vehicle or JHU395 and was euthanized two hours later by CO₂ inhalation. Whole body perfusion (10% neutral buffered formalin) was performed on a subset of animals (n = 3–4/treatment group) and specimens saved for necropsy and histopathology with H&E staining at IDEXX, Inc. Tumor and plasma were collected from the rest of the animals. Tumor was flash frozen in liquid nitrogen and all samples were stored at −80°C until further bioanalysis. For bioanalysis details, please see Supplemental Methods.

Pro-905 was synthesized in a four-step synthesis starting from 2´,3´-O-isopropylidene-guanosine. 2´,3´-O-isopropylideneguanosine was prepared based on previously described methods [26] and reacted with isopropyl [chloro(phenoxy)phosphoryl]-L-alaninate. The product (Cpd 902) was transformed to its 6-thioguanosine analog (Cpd 904) by reaction with anhydrous sodium hydrogen sulfide, followed by deprotection of the isopropylidene group to give Cpd 905 (Pro-905). Products were characterized by ¹H-NMR, ¹³C-NMR and high-resolution mass spectrometry. The final product Pro-905 was achieved with 99.5% purity. For detailed methods regarding synthetic techniques and molecular characterization please refer to Supplemental Methods, Detailed Pro-905 Synthesis and Characterization. Subsequent scale up to achieve gram quantities was performed with Redicius, Inc.

Mice (B6, ~12 weeks of age, bearing murine flank MPNST at ~500 mm³) were dosed with 6-MP, Pro-905, or vehicle at doses and routes indicated. The mice were euthanized at specified time points post-drug administration and blood samples (~0.8 mL) were collected in heparinized microtubes by cardiac puncture. Tumors and liver were removed and flash frozen on dry ice. Blood samples were centrifuged at a temperature of 4 °C at 3000 × g for 10 min. Plasma (~300 μL) was collected in polypropylene tubes, and all plasma and tissue samples were stored at −80 °C until bioanalysis.

For quantifying 6-MMP/6-TGMP levels, plasma samples (25 μL), were extracted using a protein precipitation method by addition of 125 μL of methanol containing internal standard (IS; 2-Chloroadenine, 1 μM), followed by vortex-mixing and then centrifugation at 16000 × g for 5 min at 4 °C. The tumor tissues were diluted 1:5 w/v with methanol containing the IS (1 μM), homogenized, then vortex-mixed and centrifuged at 16000 × g for 5 min at 4 °C. Supernatants were analyzed by LC-MS/MS using an UltiMate 3000 UHPLC coupled to Q Exactive Focus Orbitrap Mass Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The mobile phase used for chromatographic separation consisted of water + 0.01% formic acid (A), and acetonitrile + 0.01% formic acid (B), delivered at a flow rate of 0.4 mL/min.

For 6-MMP analysis, a gradient LC method (time (min)/%B: 0–0.25/2.5, 2.25–3.25/95, 3.25–4.25/2.5) was used for the analyses. Separation was achieved using an Atlantis dC18 2.1 × 150 mm, 3 μm particle size column (Waters, Milford, MA, USA). The mass spectrometer was operated with the capillary temperature setting at 350°C and a spray voltage of 3 kV. Nitrogen was used as the sheath and auxiliary gas set to 30 and 3 arbitrary units, respectively. The mass spectrometer operating in positive PRM mode dissociated the parent ion of 6-MMP at m/z 167.0386 with a collision energy (CE) setting of 23 quantifying product ions of 126.0119, 134.0585 and 152.0152. The parent ion of IS at m/z 170.0228 was dissociated with a CE setting of 23 quantifying product ion of 152.0566.

For TGMP analysis, a gradient LC method (time (min)/%B: 0–1.5/0, 2 –3/95, 3–4.5/0) was used for the analyses. Separation was achieved using an SB-Aq 2.1 × 100 mm, 1.3 μm particle size column (Agilent, Santa Clara, CA, USA). The mass spectrometer was operated with the capillary temperature setting at 350°C and a spray voltage of 3.5 kV. Nitrogen was used as the sheath and auxiliary gas set to 25 and 15 arbitrary units, respectively. The mass spectrometer operating in negative PRM mode dissociated the parent ion of TGMP at m/z 378.0279 with a CE setting of 21 quantifying product ions of 78.9591 and 211.0015. The parent ion of IS at m/z 168.0082 was dissociated with a CE setting of 17 quantifying product ions of 97.0237 and 125.0187.

JH-2-031 MPNST patient derived xenograft specimens at mouse passage 3 were obtained from the JH NF1 Biospecimen Repository [21]. Female NSG mice at 10 weeks of age were anesthetized with ketamine/xylazine (100 mg/kg ketamine + 10 mg/kg xylazine i.p.) until surgical plane confirmed. ~3 mm³ tumor xenograft pieces were implanted subcutaneously in the right flank in Matrigel (Corning Inc; 354230). When palpable tumors > ~500 mm³ formed, mice were euthanized and tumors harvested under sterile conditions, dissected to ~3 mm³ pieces, and immediately passaged to recipient experimental mice. For the experiment shown in Supplemental Figure 3, tumors at mouse passage 7 were used. To evaluate Pro-905 efficacy, mice with tumor volumes > 100 mm³ were randomized to receive vehicle (PBS + 1% Tween + 10% EtOH) or Pro-905 (20 mg/kg) i.p. 5d/week. Mean starting tumor volume per group was ~225 mm³. Mice were weighed and tumors measured with calipers three times per week. Tumor volume was calculated with the formula V = (L * (W²)) /2. At study endpoint, which was four weeks from treatment initiation, mice were euthanized by CO₂ inhalation and tumors were harvested and weighed. Data shown are representative of two independent experiments.

Male B6 mice (Envigo) approximately 10 weeks of age were injected in the right flank with 3 × 10⁶ cells that were originally harvested from the tumor of an NPcis mouse and grow in culture. Tumors were allowed to form for 3–4 weeks. Mice were randomized to treatment groups to achieve equivalent mean tumor volumes (based on caliper measurements) at start of treatment.

For 6-MP antitumor activity, the mean tumor volume for the groups at start of experiment was 450 mm³. Mice were treated with 6-MP (20 mg/kg i.p. daily 5d/week) or vehicle for two weeks. Animal weights and tumor volumes were measured three times per week. Blood and tumor were harvested at the conclusion of the study and evaluated for tumor weights and clinical chemistry/complete blood counts.

For Pro-905 and combination Pro-905+JHU395 efficacy studies: mice were randomized to 4 groups (n = 7–8 mice/group) for treatment with mean tumor volume ~ 350 mm³ at start of dosing. Mice were administered Pro-905 (10 mg/kg i.p. daily until day 10; dose reduced on days 10–12 to 5 mg/kg i.p. in both combination and single agent Pro-905 arms due to ~15% weight loss occurring in combination therapy arm); JHU395 (1.2 mg/kg p.o. daily 5 days /week); the combination or Vehicle. Animal weights and tumor volumes were measured 3x/week. Tumor volume was calculated with the formula V=(LxW²)/2. On the day of tissue harvest (day 12 since start of study), ~two hours following final dosing, mice were euthanized by CO₂ inhalation. Blood and tumor were harvested. Tumor was flash frozen in liquid nitrogen. A subset of mice (n=2–3 per group) were terminally perfused with 10% neutral buffered formalin and submitted for processing and histopathologic review by a veterinary pathologist (IDEXX Bioanalytics, Inc.)

For the polar metabolomics studies in single agent JHU395, Pro-905, and Combination treated tumors, the tumors were generated and animals treated similarly to above. Tumors were harvested after five days of treatment and flash frozen in liquid nitrogen. Metabolites were extracted in 80% cold methanol: water and separated from insoluble material by centrifugation. Supernatants were divided to equal aliquots based on tissue mass and dried at room temperature overnight under nitrogen gas. Dried metabolite pellets from 25 mg wet tumor tissue were analyzed by targeted LC-MS/MS via SRM with polarity switching on a 6500 QTRAP. Peak intensities were normalized to vehicle treated samples and analyzed further using Metaboanalyst [24, 27].

Tumor sections were stained for Ki67 to assess proliferation. Briefly, slides were deparaffinized and rehydrated through graded alcohols, followed by quenching of endogenous peroxidase activity in 3% H₂O₂ (30m, RT). Heated antigen retrieval was performed in 10 mM citric acid (20m, microwave). Slides were blocked with 2.5% BSA, followed by incubation with 1° anti-Ki67 (Abcam ab16667; 1:200, O/N, 4C) or rabbit IgG (Cell Signaling DA1E; concentration-matched, O/N, 4C) in 2.5% BSA. A polymer-based secondary detection system was employed according to manufacturer’s instructions (Vector, ImmPRESS-HRP Anti-Rabbit IgG, MP-7401). All slides were individually developed with Sigmafast^™ DAB (Sigma, D4293) for 90 seconds under microscopic control, followed by counterstaining with Hematoxylin QS (Vector, H-3404). Slides were then digitized with an Aperio CS2 slide scanner (Leica Biosystems) and analyzed by a trained blinded investigator using Aperio Imagescope^™ software. Representative images of each sample were photographed, and tumors were manually circumscribed to calculate tumor area. A representative area, equivalent in size to the smallest tumor (14 mm²), was then selected within each sample for quantification. Automated total nuclei counts and Ki67 positive nuclei counts were obtained using the Aperio Nuclear v9 algorithm, with thresholds set to detect moderate-to-strong nuclear DAB. Counts were converted to % proliferation ([Ki67 Nuclei Count/Total Nuclei Count]*100), plotted in GraphPad Prism (RRID:SCR_002798) and analyzed by ordinary one-way ANOVA with multiple comparisons (*p < 0.05).

The data generated in this study are available upon request from the corresponding author.

JHU395 is a nervous tissue-penetrant prodrug of the GA inhibitor DON [22, 28] which has been shown to inhibit three enzymes in de novo purine synthesis including phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosyl formylglyinamidine amidotransferase (PFAS), and guanosine monophosphate synthetase (GMPS) [29–31] (Fig 1A). Using quantitative liquid chromatography-mass spectrometry (LC-MS) methods recently described [32], we observed that 24h incubation of JHU395 (10 μM) in both sNF96.2 and JH-2-002 human MPNST cells dramatically increased the PFAS substrate formylglycinamide ribonucleotide (FGAR) over 100-fold (Fig 1B). Using polar metabolomics, we further demonstrated that GA substrates phosphoribosyl pyrophosphate (PRPP) and xanthosine monophosphate (XMP) were also significantly increased by JHU395 (10 μM) in sNF96.2 cells (Fig 1C). Radiolabeled glycine was used to monitor de novo purine synthesis and incorporation of glycine-containing metabolites into newly synthesized nucleic acids of DNA and RNA. JHU395 treatment of MPNST cells significantly reduced de novo purine synthesis, as evidenced by a >25% reduction of glycine label incorporation in DNA and RNA (Fig 1D). Human NF1-MPNST are genomically heterogenous with now well-established diversity in NF1 mutations, alterations in tumor suppressors (e.g. CDKN2A, TP53), epigenetic regulators (PRC2 complex components), and other variations [33, 34] [35]. Thus, we compared JHU395 inhibitory potency in three different MPNST cell lines including commercially available sNF96.2 cells, as well as two patient-derived cell lines (JH-2-002, JH-2-031) [19, 21, 36]. We found that JHU395 partially inhibited growth in all three cell lines with IC₅₀ values in the low micromolar range (Fig 1E).

JHU395 was evaluated for single agent antitumor activity in the transgenic NPcis spontaneous MPNST model [25]. In brief, male and female mice (Supplemental Fig 1A) were assigned to treatment (vehicle or JHU395 2.4 mg/kg p.o. t.i.w.) as they developed palpable tumors (defined as > 100 mm³). Animals continued treatment until necessitating euthanasia due to tumor volume or other parameter (weight change, body condition score) exceeding institutional standards (Fig 2A). Mice tolerated JHU395 treatment well with no significant weight loss over the treatment period (Supplemental Fig 1B). Similar to prior studies in this model [37–39], tumor volumes were assessed at day 10 relative to day 1 to evaluate drug activity. JHU395 displayed anti-tumor effects as demonstrated by a significant reduction in tumor volume (day 10/day 1) in the JHU395 treated mice relative to vehicle treated mice (JHU395 mean log₂ fold change 0.66 versus vehicle mean log₂ fold change 2.60; p = 0.045 by t-test) (Fig 2B). Notably, one JHU395-treated animal showed regression in tumor volume by day 10 and a second JHU395-treated animal had an impalpable tumor by day 10. JHU395 treatment slowed tumor growth compared to vehicle-treatment in mice over the first thirty days of dosing (Supplemental Fig 1C). There was also a significant survival difference between the JHU395- and vehicle-treated animals (22 versus 12 day median survival respectively; log rank p-value =0.014) (Fig 2C). At the time of euthanasia, tumors were excised, flash frozen, and metabolomic markers were analyzed (n=5–6 tumors/treatment). JHU395-treated tumors had elevated FGAR levels indicative of de novo purine synthesis inhibition, with corresponding decreases in the purine nucleotide monophosphates IMP and GMP (Fig 2D). Representative tumor histology from vehicle and JHU395 treated mice is shown in Supplemental Figure 1D.

Given the promising partial antitumor activity observed for JHU395 in MPNST, we hypothesized that combination treatment could lead to improved efficacy. Prior pediatric cancer clinical studies showed enhanced efficacy when GA inhibition was combined with the purine antimetabolite 6-MP, though the combination was limited by toxicity [9].

We initially investigated single agent 6-MP activity in MPNST cell and mouse models as proof-of-concept. Following administration, 6-MP is transformed to an active monophosphorylated nucleoside antimetabolite 6-thioguanosine monophosphate (TGMP) through multiple steps involving enzymes in the purine salvage pathway [7] (Supplemental Fig 2A). The active metabolites generated have myelosuppressive effects and monitoring of peripheral blood cell counts are used as a surrogate marker of antitumor activity [40]. It is also well-known that this 6-MP metabolic process generates inactive and hepatotoxic methylated metabolites (e.g. 6-methylmercaptopurine, 6-MMP). In the clinic 6-MP treatment requires consistent monitoring, to identify effects of these toxic metabolites, as well as titrate dosing appropriately and provide adequate supportive care [41].

In our studies, 6-MP dose-dependently inhibited proliferation of MPNST cells in culture (Supplemental Fig 2B) and tumor growth in a flank tumor model (Supplemental Figure 2C). Following 10 days of treatment (6-MP 20 mg/kg i.p. daily, the equivalent to 60 mg/m²/day in humans) [42, 43], the mean tumor volume was smaller in the 6-MP group compared to vehicle (1107 ± 654 mm³ vs 2038 ± 468 mm³, respectively; p = 0.0098). Consistent with clinical reports [44], some mice treated with 6-MP had increased alanine liver transaminases (46 U/L ± 13 versus 21 U/L ± 4; p=0.024; Supplemental Fig 2D). In addition, 6-MP’s hepatotoxic methylated metabolite 6-MMP was detectable in the liver of these mice up to 6 hours following treatment (Supplemental Fig 2E).

We hypothesized that generating a prodrug to more efficiently deliver TGMP to tumor cells, and bypass the metabolic processing required to activate 6-MP, would lead to a better tolerated antimetabolite devoid of 6-MP’s hepatotoxic methylated metabolites (Fig 3A). As TGMP is a nucleotide and negatively charged, a protide approach was taken. We synthesized a phosphoramidate protide of the nucleoside analog 6-thioguanosine termed Pro-905 (Fig 3B). The synthesis was accomplished in a three-step process starting from 2’,3’-O-isopropylidene-guanosine and isopropyl [chloro(phenoxy)phosphoryl]-L-alaninate. Pro-905 was purified to >99.5% by HPLC and characterized by ¹H and ¹³C NMR and mass spectrometry (see Supplemental Methods). Pro-905 inhibited purine salvage-dependent ³H-hypoxanthine incorporation to DNA and RNA in tumor cells (Fig 3C). Pro-905 also inhibited sNF96.2 and JH-2-002 human MPSNT colony formation in a dose dependent manner (Fig 3D), with a magnitude of effect similar to 6-MP (Fig 3E).

Based on the promising in vitro data with Pro-905, we next tested its pharmacokinetic profile and tumor delivery in mice. Given the more direct metabolic route of Pro-905 to active TGMP, we hypothesized that Pro-905 would be a more efficient purine antimetabolite versus 6-MP. In support of this, when dosed on an equimolar basis, Pro-905 had >2.5-fold enhanced tumor delivery of TGMP versus 6-MP (Pro-905 TGMP AUC_{0–> t} 268 nmol/g × hr versus 6-MP TGMP AUC_{0–> t} 114 nmol/g × hr; Fig 4A). Moreover, while 6-MP treatment resulted in exposure to the hepatotoxic metabolite MMP, Pro-905 did not (Fig 4B).

Having confirmed increased delivery of TGMP to tumor, we next evaluated Pro-905 for myelosuppression, as assessed by peripheral blood counts, to serve as a biomarker of antitumor activity in vivo [40]. White blood cell (WBC) counts and platelets from B6 mice treated with Pro-905 at two dose levels (1 and 10 mg/kg) were compared to those from mice treated with 6-MP (20 mg/kg) to assess activity. Following 12 days of treatment, WBC and platelet values from the 10 mg/kg i.p. Pro-905 were comparable to those from the 20 mg/kg 6-MP treated mice (Fig 4C). When Pro-905 at 10 mg/kg was administered daily to B6 mice bearing flank MPNST for three weeks, tumor growth was slowed compared to vehicle (Day 15 vehicle tumor volume 3049 ± 317 mm³ vs Day 15 Pro-905 tumor volume 1279 ± 777 mm³; p = 0.0047) (Fig 4D). Pro-905 treated animal weights remained within 10% of starting weight over this time (Fig 4E) with no elevations in liver transaminases or bilirubin (Fig 4F).

In parallel we evaluated tolerability and efficacy of Pro-905 in immunodeficient Nod scid gamma (NSG) mice, bearing patient-derived xenograft (PDX) MPNST. Unlike the B6 mice, NSG mice treated with 10 mg/kg Pro-905 i.p. did not have evidence of myelosuppression as a marker of TGMP activity (white blood cell counts: vehicle 1.002 K/μl ± 0.1899 versus Pro-905 0.856 K/μl ± 0.178. Absolute neutrophil counts: vehicle 0.710 K/μl ± 0.140 versus Pro-905 0.644K/μl ± 0.198). However in a second tolerability study, NSG mice treated with 20mg/kg i.p. Pro-905 did have evidence of myelosuppression. Compared to vehicle-treated mice, Pro-905-treated animals (20 mg/kg i.p.) exhibited lower white blood counts (vehicle 1.53 ± 0.64K/ μL versus Pro-905 0.970 ± 0.33K/μL) and absolute neutrophil counts (vehicle 1.190 K/ μL ± 0.629 versus Pro-905 0.688 K/ μL ± 0.269). Given that the 20 mg/kg i.p. Pro-905 dose had signs of activity it was chosen for efficacy studies.

NSG mice bearing the human MPNST PDX JH-2-031 were dosed with Pro-905 (20 mg/kg i.p. 5 days per week for 4 weeks) and tumor growth was monitored. Pro-905 slowed tumor growth over the duration of the study compared to vehicle (Supplemental Fig 3A). At sacrifice the Pro-905-treated mice had significantly smaller mean tumor masses (1322 mg versus 2733 mg respectively; p = 0.013 by t-test; Supplemental Fig 3B), Pro-905 as a single agent was well-tolerated in this model; animal body weights remained within 10% of starting weights over two weeks of dosing (Supplemental Fig 3C). Taken together these studies support the antitumor activity of Pro-905 against human MPNST in vivo.

Based on these promising results showing single agent Pro-905 activity against MPNST we hypothesized that more prominent antitumor activity would be observed when Pro-905 is combined with the de novo purine synthesis inhibitor JHU395 (Fig 5A). We tested the combination in cell culture using a colony forming assay in human MPNST cells. Using JH-2-002 cells, single agent Pro-905 (10 μM) or JHU395 (1 μM) each decreased colony area~20% compared to no treatment. When combined at these doses, the two agents reduced colony formation by ~80% compared to no treatment (Fig 5B). A similar trend was observed in sNF96.2 cells (Supplemental Fig 4A). We next sought to identify the effects Pro-905 and JHU395 on nucleic acids in MPNST cells. Pro-905 and the combination, but not JHU395 alone, inhibited guanine incorporation to newly synthesized DNA and RNA (Fig 5C). We also explored the effects of Pro-905, JHU395, and their combination, on markers of DNA damage (γH2AX) and apoptotic cell death (cleaved PARP). Single agent Pro-905 (10 μM), JHU395 (1 μM), and the combination induced DNA damage (γH2AX) following 120h of treatment, corresponding to more than one cell doubling time, but not at 72h of treatment (Fig 5D). Cleaved PARP as a marker of apoptosis was not observed at this dose of JHU395 at either time point, but was observed when MPNST cells were treated with JHU395 at higher doses or with the anthracycline doxorubicin as a positive control (Supplemental Fig 4B, Fig 5D, lanes 2 & 10). Full scan western blots are pictured in Supplemental Figure 5. Taken together, these results demonstrate that Pro-905 and JHU395 combine in MPNST cells to decrease cell proliferation with effects on nucleic acid synthesis and DNA damage, but without some markers of classical apoptosis.

B6 mice with flank MPNST were treated with JHU395 (1.2 mg/kg p.o. 5d/week), Pro-905 (10 mg/kg i.p. 5d/week), the combination, or vehicle. Following five days of treatment, tumors were harvested, flash frozen, and metabolites extracted for LC-MS analysis. We observed that compared to vehicle treated tumors, combination treated tumors demonstrated forty polar metabolites with |log₂ fold change| > 1 and (negative log₁₀ p-value) > 1; notably this was more than were observed compared to either Pro-905 or JHU395 single agent treatments (Supplemental Fig 6A and 6B). Analysis of these metabolites based on pathways demonstrated that purine and pyrimidine metabolism pathways were the most significantly impacted by combination treatment (Fig 6A). Metabolites in purine pathways were decreased upon combination treatment compared to control (Fig 6B). Of note, guanosine nucleotides dGMP and GDP were among the purine metabolites decreased by the combination treatment.

In a two-week efficacy study, the combination of Pro-905 with JHU395 prevented growth of established tumors to a greater extent than either single agent (Day 12 mean tumor volume: Vehicle 2225 +/− 941 mm³; Pro-905 1018 +/− 287 mm³; JHU395 1423 +/− 1038 mm³; Combo 319 +/− 243 mm³.) (Fig 6C). Tumors from combination treated mice weighed significantly less than those from vehicle treated mice at tissue harvest (Combination 194.9 ± 162.2 mg versus Vehicle 1356.0 ± 570.0 mg; p = 0.004 by one-way ANOVA) (Fig 6D) and had fewer apparent mitoses (Fig 6E). Ki67 staining as a marker of proliferation was significantly reduced in the combination group compared to vehicle treated tumors or single agent Pro-905, and trended towards decreased when compared to JHU395 (mean percent proliferation Vehicle 1.63%, JHU395 0.82%, Pro-905 1.25%, Combination 0.18%) (Fig 6F).