CAN-13-1934 1..12 Therapeutics, Targets, and Chemical Biology Preclinical Therapeutic Efficacy of a Novel Pharmacologic Inducer of Apoptosis in Malignant Peripheral Nerve Sheath Tumors Vincent Chau1,2, S. Kyun Lim2, Wei Mo2, Chiachi Liu1, Amish J. Patel1, Ren�ee M. McKay2, Shuguang Wei3, Bruce A. Posner3, Jef K. De Brabander3, Noelle S. Williams3, Luis F. Parada2,4, and Lu Q. Le1,4 Abstract Neurofibromatosis type I (NF1) is an autosomal disorder that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. Patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (MPNST), a main cause of mortality. Currently, surgery is the primary course of treatment for MPNST, but with the limitation that these tumors are highly invasive. Radiotherapy is another treatment option, but is undesirable because it can induce additional mutations. Patients with MPNST may also receive doxorubicin as therapy, but this DNA-intercalating agent has relatively low tumor specificity and limited efficacy. In this study, we exploited a robust genetically engineered mouse model of MPNST that recapitulates human NF1-associated MPNST to identify a novel small chemical compound that inhibits tumor cell growth. Compound 21 (Cpd21) inhibits growth of all available in vitro models of MPNST and human MPNST cell lines, while remaining nontoxic to normally dividing Schwann cells or mouse embryonic fibroblasts. We show that this compound delays the cell cycle and leads to cellular apoptosis. Moreover, Cpd21 can reduce MPNST burden in a mouse allograft model, underscoring the compound's potential as a novel chemotherapeutic agent. Cancer Res; 74(2); 1–12. �2013 AACR. Introduction Neurofibromatosis type I (NF1) is an autosomal dominant genetic disorder that affects 1 in 3,500 people (1). Patients typically present with a constellation of signs, including caf�e- au-lait pigmentation spots, benign neurofibromas, axillary or inguinal freckling, optic gliomas, Lisch nodules in the eye, bone abnormalities, and multiple additional less penetrant mani- festations (2). Furthermore, NF1 patients are predisposed to malignant peripheral nerve sheath tumors (MPNST; ref. 3), which are a significant source of mortality. Neurofibromas are benign tumors that grow on the skin (dermal) or inside the body (plexiform; ref. 4). Neurofibromas have complex cellularity, including Schwann lineage cells, fibro- blasts, perineurial cells, mast cells, collagen deposits, and phys- ical proximity to peripheral nerves. Plexiform neurofibromas appear in 30% of NF1 patients (5), and although they retain the capacity to enlarge throughout life, many lines of evidence point to an embryonic origin (4). Dermal neurofibromas can arise from a type of adult stem cell called skin-derived precursors (SKP; ref. 4), whereas plexiform neurofibromas arise from embryonic neural crest lineage Schwann cell progenitors (6). Whether the two cell types are related or the same remains unclear (7). NF1 patients can also develop malignant tumors, including leukemia, rhabdomyosarcoma, and neuroblastoma in children (2). The most common malignancy, however, is MPNST, which arises from and appears in, approximately 9% to 21% of patients with plexiform neurofibromas (8). MPNSTs are ini- tially difficult to detect, metastasize widely, and have a poor prognosis (9). Mutation of Nf1, which encodes a RAS-GTPase activating enzyme (10), is known to activate RAS downstream effectors, including the ERK, PI3K, and mTOR pathways (11– 15). The two most common cancer-associated mutations present in MPNSTs in addition to Nf1 mutation are the tumor suppressors p53 (16–18) and CDKN2 (16, 19–23). Mice harbor- ing mutated Nf1 and p53 or Nf1 and Cdkn2 spontaneously develop MPNST (24, 25). In addition to Nf1 and its downstream effector Ras, the conditional deletion of Pten also leads to MPNST in mice (26). In this study, we exploit a robust model of MPNST that histologically and molecularly recapitulates human MPNST. We used primary cells from these MPNSTs to screen for compounds that specifically arrest tumor cell growth. Such compounds would not only have potential as a platform for novel therapeutic development but also for probing the biology of MPNST. Elucidating the mechanism of action of such compounds could identify MPNST specific pathways and point to therapeutic opportunities. We report a novel small Authors' Affiliations: Departments of 1Dermatology, 2Developmental Biology, and 3Biochemistry, and 4Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas Corresponding Authors: Lu Q. Le or Luis F. Parada, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9069. Phone: 214-633-1846, Fax: 214-648-5559; E-mail: lu.le@utsouthwestern.edu or luis.parada@utsouthwestern.edu doi: 10.1158/0008-5472.CAN-13-1934 �2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ molecule, compound 21 (Cpd21 or identification number SW106065), which induces apoptosis in all models of MPNST tested but spares normal cells and tissues. Moreover, we demonstrate that this compound can decrease the tumor burden of MPNST in a mouse allograft model. Materials and Methods Cell and tissue samples S462 and SNF96.2 cells were a gift from Karen Cichowski. Schwann cells (ScienCell) were cultured in Schwann Cell Medi- um (ScienCell) andwere immortalized with Myc-retrovirus(27). High-throughput screening sMPNST and Schwann cells (400 cells/well) were seeded in 384-well plates and grown overnight. Compounds (final con- centration of 2.5 mmol/L) were added and incubated for 96 hours. CellTiter-Glo (Promega) was added and luminescence was quantified using Envision 2102 Multilabel Reader (Perkin Elmer). Soft agar assay Acellular layer of 0.6% Bacto-agar in MPNST media was plated. Cells were trypsinized, pelleted, resuspended, mixed in 1:1 ratio with 0.6% agar to a final density of 1 � 104 cells/mL, and plated on top. After incubation for 10 days, colonies were stained with 0.5 mL 0.005% crystal violet. Cell-cycle analysis BrdUrd (10 mmol/L) was added to cells for 30 minutes. Cells werecollected, resuspendedin100mL PBS, fixed in5 mL ice-cold ethanol, and then stored overnight at 4�C. 2N HCl/Triton X-100 was added to denature DNA. After neutralization, samples were incubated with 50 mL 0.5% Tween 20/1%BSA/PBS, 20 mL anti- BrdU-FITC (BD Biosciences), and 5 mL of 10 mg/mL RNAse overnight. Samples were pelleted, resuspended in 1 mL 5 mg/mL propidium iodide (Sigma), and analyzed by flow cytometry. Western blotting Cells were collected, trypsinized, and lysed with 600 mL radioimmunoprecipitation assay buffer. Samples were run on 5%–20% SDS-PAGE gel (Bio-Rad). The following primary anti- bodies were used: Cyclin D1, H3 (Millipore), Caspase-3, pERK, ERK, pAKT, AKT (Cell Signaling Technology), PARP (Novus Biologicals and Millipore), a-Tubulin (Sigma), and GAPDH (Santa Cruz). Annexin V staining Floating and adherent cells were resuspended in binding buffer (0.01 mol/L Hepes/NaOH (pH 7.4), 0.14 mol/L NaCl, 2.5 mmol/L CaCl2), and processed according to manufacturer (MACS Miltenyi Biotec). Three hundred microliters of suspen- sion was added to 5 mL Annexin V antibody (MACS Miltenyi Biotec) and 2.5 mL propidium iodide, and analyzed by flow cytometry. Metabolic stability studies Cpd21 levels for metabolic stability and pharmaco- kinetic studies were monitored by liquid chromatography (LC/MS-MS) with AB/Sciex (Applied Biosystems) 3200 Qtrap mass spectrometer coupled to Shimadzu Prominence LC. Cpd21 was detected with the mass spectrometer in positive MRM (multiple reaction monitoring) mode by following the precursor to fragment ion transition 205.0 to 111.1. An Agilent C18 XDB 5 mm packing column (50 � 4.6 mm) was used for chromatography. For S9 studies, Cpd21 (2 mmol/L) was incubated in a 1 mL incubation volume with 1 mg of murine CD-1 S9 (combined cytosol and microsome) fractions (Celsis/In Vitro Technologies) and phase I (the NADPH regenerating system) cofactors (Sigma) for 0 to 240 minutes. Reactions were quenched by mixing the incuba- tion mixture with 1 mL of methanol/0.2 ng/mL n-benzyl- benzamide/0.2% formic acid. The quenched mixture was vortexed for 15 seconds, incubated at room temperature for 10 minutes, and spun for 5 minutes at 986 � g. Super- natants were spun in a refrigerated microcentrifuge for 5 minutes at 16,100 � g. The second supernatant was trans- ferred to an HPLC vial and analyzed by LC/MS-MS. Pharmacokinetic studies Six- to 7-week-old NCR-nu/nu female mice were implanted with 2.5 � 105 MPNST cells in the left flank, subcutaneously. When tumors reached 800 mm3, animals were injected with Cpd21 at20 mg/kg in0.2 mL10% ethanol, 10% PEG400, 80% of a solution of 5% dextrose, pH 7.4. Blood was drawn at 0, 10, 30, 60, 180, 360, and 1,440 minutes using the anticoagulant, ACD (acidified citrate dextrose) and plasma isolated by centrifuga- tion. One hundred microliters of plasma was mixed with 200 mL acetonitrile containing 0.15% formic acid and 37.5 ng/mL n- benzylbenzamide IS. Samples were vortexed 15 seconds, incu- bated at room temperature for 10 minutes, and spun twice at 16,100 � g at 4�C. Amount of Cpd21 present in plasma was quantified by LC/MS-MS to determine the rate of clearance from mouse blood. A Cpd21 standard curve was generated using blank plasma spiked with known Cpd21 concentrations and processed as described above. Concentrations of Cpd21in each time-point sample were quantified using Analyst 1.4.2. The limit of detection was defined as 3-fold above the signal obtained from blank plasma. The limit of quantitation (LOQ) was defined as the lowest concentration at which back calcu- lation yielded a concentration within 20% of theoretical. The LOQ for plasma was 5 ng/mL and 1 ng/mL for tumor. Back calculation of points on the standard curve yielded values within 15% of theoretical over 4 orders of magnitude (5,000 to 5 or 1 ng/mL). Pharmacokinetic parameters were calculated using the noncompartmental analysis tool of WinNonLin (Pharsight). Bioluminescent imaging sMPNST-lucþ cells (2 � 505 cells/200 mL) were injected in both flanks of nude mice. Mice were injected with 40 mg/kg Cpd21 or vehicle (10% ethanol, 10% PEG400, and 80% of 5% dextrose in water) intraperitoneally twice per day for 4 weeks. Mice were injected with 160 mL 20 mg/mL D-Luciferin Potas- sium Salt (Perkin Elmer) in 0.9% sterile NaCl, and total flux was quantified using IVIS Lumina II (Caliper Life Sciences) weekly. Chau et al. Cancer Res; 74(2) January 15, 2014 Cancer ResearchOF2 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ Immunohistochemistry Tissues were fixed in 10% formalin and paraffin embedded. After sectioning, immunohistochemical studies were per- formed as previously described (29). The following primary antibodies were used:S100 (28), GAP43 (Abcam), Ki67 (Thermo Scientific), Caspase-3 (Cell Signaling Technology), and PARP (Novus Biologicals). Results Nf1 and p53-deficient SKPs form MPNST NF1-associated dermal and plexiform neurofibromas are histologically compatible despite having significantly different natural history and tumor progression properties. Recently, we reported that SKPs are the cell-of-origin of dermal neurofibro- mas (4). Given the potential in mice for neural crest progenitors to give rise to MPNST after additional loss of the p53 tumor suppressor, we tested the consequences of dual Nf1 and p53 loss of function (NP) in SKPs. SKPs from mice with Nf1f/f;p53f/f genotype were cultured and infected with adenovirus contain- ing Cre-GFP (Ad-Cre) to induce tumor suppressor recombina- tion (Fig. 1A, a; ref. 30). The majority of infected cells displayed green fluorescence, indicating effective viral entry and expres- sion (Fig. 1A, b). We confirmed effective Cre-mediated recom- bination of all floxed alleles by PCR analysis (Fig. 1Ab). The Nf1�/�;p53�/� cells (NP SKPs) were reimplanted into the same mouse from which they were derived (autograft) and highly aggressive, malignant tumors developed in all cases (Fig. 1A, c). These tumors could be successfully cultured as either spheres or adherent cells (data not shown) or allografted into nude mice(Fig. 1A,d). Thesetumors(termedsMPNST, for SKP- derived MPNST) were assayed for human MPNST-associated genes, including embryonic Schwann cell markers, S100 (24, 25), Krox20 (6), PLP (31), and GAP43 (32, 33); developing glia marker, Dhh (34); and neural-crest-associated marker, snail (35). The quantitative reverse transcriptase-PCR (qRT-PCR) Figure 1. MPNSTs can be generated from SKPs that are deficient in Nf1 and p53. A, SKPs were isolated from the skin of cis-Nf1f/f;p53f/f mice, cultured, and infected with Ad-Cre-GFP (a). Infected SKPs were screened for expression of GFP and genotyped for the deletion of Nf1(b). Lane 1, Nf1 f/f control. Lane 2, wild-type Nf1 þ/þ control. Lanes 3 and 4, Nf1 �/� SKPs that were infected with Ad-Cre-GFP. These Nf1 �/� ;p53 �/� SKPs were then autologously transplanted back into the original mouse (c). Resultant tumors were transplanted into nude mice via xenotransplantation (d). qRT-PCR results (e). Lanes 1–4, MPNSTs derived from cis-Nf1þ/�;p53þ/� mice. Lanes 5–7, MPNSTs derived from PLP-CreERT-Nf1f/f;p53f/f mice that had been injected with tamoxifen to induce recombination. Lanes 8–11, sMPNSTs. B, a, hematoxylin and eosin staining of sMPNST. Immunohistochemical staining for S100B in sMPNST (b) and skin (c), and for GAP43 in sMPNST (d) and skin (e). Scale bars, 100 mm. Small-Molecule Inhibitor of MPNST www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 OF3 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ Figure 2. Identification of Cpd21 by high-throughput, small-molecule screening and validation. A, summary of screening procedure. B, high-throughput screening dataforsMPNST cells(top) and Schwann cells (bottom).Intensity of blueor redsignal correlates withdegreeof inhibitionor inductionof ATPlevels by small molecules, respectively. C, structure of Cpd21. D, dose-response curves of Cpd21 (0.125, 0.25, 0.5, 1, 2.5, 5, 10, and 20 mmol/L) in sMPNST cells, MEF, Schwann cells, and MPNST cells from multiple murine models. E and F, dose-response curves of Cpd21 treatment on human MPNST cell lines, S462 and SNF96.2, respectively, compared with mouse NIH3T3 cells and sMPNST cells. G, dose-response curves of Cpd21 treatment on wild-type Schwann cells, compared with sMPNST and MPNST from cis-Nf1f/f;p53f/f mice. H, soft agar assay of Cpd21-treated (0.25, 0.5, 1, 2.5, or 5 mmol/L) sMPNST cells compared with DMSO. Arrows indicate colonies, which may be out of plane in agar. Scale bars, 100 mm. I, quantification of H. J, soft agar assay of MPNST cells from cis-Nf1 þ/� ;p53 þ/� mice and that were treated with DMSO or Cpd21 at 0.25, 0.5, 1, 2.5, or 5 mmol/L. Arrows indicate colonies, which may be out of plane in agar. Scale bars, 100 mm. K, quantification of J. All values, mean � SD. The Student t test used for significance testing (�, P < 0.05; ��, P < 0.01; ���, P < 0.001). Chau et al. Cancer Res; 74(2) January 15, 2014 Cancer ResearchOF4 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ data demonstrated expression of all markers in all sMPNSTs analyzed (Fig. 1Ae). Thus, sMPNSTs exhibit molecular char- acteristics of human NF1 associated MPNST. Hematoxylin and eosin (H&E) staining of sMPNSTs showed presence of spindle-shaped cells that interweave in a poorly differentiated, wavy appearance, reminiscent of human MPNST histology (Fig. 1B, a). In addition, the com- monly used MPNST markers, S100 and GAP43, were present (Fig. 1B—, b–e). Therefore, our results indicate that SKPs, cells that give rise to neurofibromas upon loss of Nf1, can also give rise to MPNST with additional loss of p53. The facility of generating and explanting sMPNSTs made this a favorable system for performing chemical compound screens. Novel MPNST inhibitory small molecule We hold the concern that over time in culture, tumor cells will drift from their original intrinsic cellular and molecular programs in unpredictable ways. These uncontrollable and unmeasurable changes may inadvertently alter fundamental properties of the original tumor cells. To mitigate these con- cerns, the relative facility of generating primary cultures from sMPNSTs afforded the advantage of propagating sufficient numbers of cells for a high-throughput screening while keeping the cell passage number low. We performed a limited high- throughput, small-molecule screen using sMPNST cells pro- duced above in an extension of a related ongoing large-scale screen in our research group. We carried out a 200,000 com- pound screen on primary mouse glioblastoma multiforme cells that are deficient for Nf1, p53, and Pten (S. Kyun Lim and Luis F. Parada, unpublished data). This original glioblastoma multi- forme screen yielded 4,480 compounds, identified as inhibitory to glioblastoma multiforme cell growth. We reasoned that since sMPNST cells are deficient in two of these same tumor suppressors, some compounds might have conserved sMPNST cell growth inhibitory properties. A well-established luminescence assay for measuring ATP levels (an indication of cellular metabolic activity) was adopted (CellTiter-Glo, Promega), and performed 96 hours after com- pound exposure. A total of 1,515 compounds exhibited more than 20% decrease in ATP levels at 2.5 mmol/L (Fig. 2A and B). To exclude compounds that exert general cell toxicity, or perturb the generic cell cycle or mitotic machinery, we count- er-screened the 1,515 compounds against Myc-immortalized Schwann cells (27, 36) and eliminated compounds that showed more than 20% lower ATP levels. The resultant 119 compounds were rescreened on sMPNSTs with a cutoff at 70% inhibition. Twenty-eight compounds passed this test and were further tested in dose response studies ranging from 125 nmol/L to 20 mmol/L. The twenty-first compound (Fig. 2C; Cpd21 or SW106065) inhibited ATP consumption of sMPNST and all other models of MPNST tested (Fig. 2D) with an EC50 of 1 mmol/L. We extended the functional analysis of Cpd21 to additional cell lines. Human MPNST cell lines, S462 (Fig. 2E) and SNF96.2 (Fig. 2F), were assayed for dose-dependent growth, and EC50 concentrations of 439.0 and 753.6 nmol/L, respective- ly, were determined. In contrast, NIH3T3 cells and wild-type Figure 3. Effect of Cpd21 on cell-cycle machinery. A, effect of Cpd21 on cell-cycle distribution. sMPNST cells were treated with DMSO, D, or Cpd21 (0.25, 0.5, 1, 2.5, or 5 mmol/L) for 24 hours, pulsed with BrdUrd for 30 minutes, and analyzed by flow cytometry. Percentage of cells in each phase is shown. B–D, sMPNST cells were treated with DMSO, D, or with Cpd21 (0.25, 0.5, 1, 2.5, or 5 mmol/L) for 24 hours and RNA harvested for qRT-PCR for cyclins (B), cdks (C), or CDKIs (D). E, Western blot analysis for cyclin D1 in sMPNST cells that were treated with DMSO, D, or Cpd21 (0.25, 0.5, 1, and 2.5 mmol/L). All values, mean � SD. The Student t test used for significance testing (�, P < 0.05; ��, P < 0.01; ���, P < 0.001). Small-Molecule Inhibitor of MPNST www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 OF5 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ Schwann cells showed a considerably higher dose resistance (Fig. 2G). We also tested sMPNST response to Cpd21 in anchor- age-independent soft agar growth assays and found that Cpd21 inhibited colony formation (Fig. 2H and I). This effect was also observed in MPNST cells derived from- cis-Nf1þ/�;p53þ/� mice (Fig. 2J andK). Thus, Cpd21 hasgeneral toxic activity on Nf1 and p53-deficient MPNST cells but not on diverse nontumorigenic, mitotically active fibroblasts or Schwann cells. Cpd21 delays the cell cycle in sMPNST To address the mechanism of action of Cpd21, we treated sMPNST cells at 0.25, 0.5, 1, 2.5, and 5 mmol/L for 24 hours, and pulsed with BrdUrd for 30 minutes. Subsequent flow cytometry analysis revealed a decreased percentage of cells in S-phase, and a corresponding increased percentage in G1–G0 and G2–M, compared with treatment with DMSO alone (Fig. 3A). Several cell-cycle regulatory genes exhibited decreased mRNA in the presence of Cpd21, including cyclin A2, cyclin B1, cyclin D1, cyclin E, cdk4, and cdk6 (Fig. 3B and C). Increased levels of cdkn1a and cdkn2a mRNA were observed in a dose- dependent manner (Fig. 3D). Also, levels of cyclin D1 protein, which is known to interact with cdk4/6 in regulating G1–S progression, were decreased (Fig. 3E; refs. 37, 38). These results indicate that following Cpd21 exposure, cells in S-phase are underrepresented, consistent with cell-cycle delay and an arrest of the mitotic machinery. We examined Cpd21-treated sMPNST cells for apoptosis by Annexin V-FITC–conjugated flow cytometry. Tumor cells treated with Cpd21 concentrations at or above 0.5 mmol/L showed a statistically significant increase in the percentage of apoptotic cells (Fig. 4A). At the EC50 concentration of 1 mmol/L, 11.23 � 1.56% of the cells were Annexin V-positive, compared with 5.02% � 0.83% in DMSO-treated control cells. We also performed Western blotting of Cpd21-treated sMPNST cells and observed caspase-3 (Fig. 4B) and PARP (Fig. 4C) cleavage, consistent with induced apoptosis. We further validated this mechanism in MPNST cells cultured from spon- taneous MPNSTs from our cis-Nf1þ/�;p53þ/� mouse model (Fig. 4D), and in the human MPNST cell line, S462 (Fig. 4E). Other cell types, including Myc-immortalized Schwann cells (Fig. 4F) and MEF cells (Fig. 4G), did not undergo caspase-3 cleavage. Together, these data indicate that, in vitro, Cpd21 Figure 4. Cpd21 induces apoptosis in vitro. A, Annexin V staining as detected by flow cytometry. sMPNST cells were treated with DMSO, D, or Cpd21 (0.25, 0.5, 1, and 2.5 mmol/L). sMPNST cells were treated with staurosporine (St.) as positive control. B–G, Western blotting for caspase-3 cleavage in sMPNST cells (B), PARP cleavage in sMPNST cells (C), caspase-3 cleavage in MPNSTs from cis-Nf1þ/�;p53þ/� mice (D), caspase-3 cleavage in human MPNST cell line, S462 (E), caspase-3 cleavage in Myc-SC (F), and caspase-3 cleavage in MEF cells (G). Each cell type was treated with staurosporine (St.) as a positive control. Total caspase-3 (T. casp.-3) and cleaved caspase-3 (Cl. casp.-3) were assayed in each case. All values, mean � SD. The Student t test used for significance testing (�, P < 0.05; ��, P < 0.01; ���, P < 0.001). Chau et al. Cancer Res; 74(2) January 15, 2014 Cancer ResearchOF6 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ Small-Molecule Inhibitor of MPNST www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 OF7 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ induced mitotic arrest of MPNST cells that ultimately results in cellular apoptosis, and that this phenotype was not observed in untransformed dividing cells. Cpd21 decreases tumor burden and induces apoptosis in vivo To determine whether Cpd21 could be directly tested in vivo, we sought to define the metabolic properties in vitro according to previously published methods (39, 40). To examine stability, Cpd21 was incubated with purified, hepatic, enzymatic S9 fraction over a period of 240 minutes, and measured over time (Fig.5A). Thehalf-life intheS9fraction is22.65 minutes. We also incubated Cpd21 with cultured hepatocytes and found that the half-life is 121.6 minutes (Fig. 5B). These assays demonstrate that Cpd21 would have reasonable stability in vivo. We next sought to define the pharmacokinetic properties of Cpd21 according to previously published methods (40). sMPNST (2.5 � 105) cells were injected subcutaneously into nude mice, and when tumors reached 800 mm3, a single dose of Cpd21 was injected intraperitoneally at 20 mg/kg. Plasma and tumor samples were collected after 10, 30, 90, 180, 360, and 960 minutes and assayed by mass spectrometry (Fig. 5C). We observed that Cpd21 rapidly distributes from the plasma into sMPNST tissues and that the half-life in sMPNST tissues is 6 to 8 hours once the compound is completely distributed. Impor- tantly, Cpd21 concentration remains above the EC50 concen- tration of 1 mmol/L (204 ng/mL) in the sMPNSTs for more than 6 hours. These results indicate that Cpd21 is successfully delivered to the tumor tissue in adequate concentrations and with an adequate half-life for in vivo studies. We next determined a dose tolerable for chronic in vivo administration of Cpd21. Cpd21 dissolved in a mixture of ethanol, PEG400, and dextrose was delivered intraperitoneally at 20 or 40 mg/kg twice per day, into nude mice over the course of 2 weeks for comparison with vehicle administration. These concentrations were selected because 40 mg/kg is the maxi- mum solubility in the aforementioned formulation. Over this period, the mice were weighed daily and observed for signs of toxicity. Neither concentration of Cpd21 produced measurable weight loss or distress in the mice (data not shown). Therefore, we selected 40 mg/kg twice per day intraperitoneally as the dosing regimen for efficacy studies. We then tested the effects of Cpd21 administration on tumor burden in vivo. Nude mice were injected with sMPNST cells carrying a luciferase gene (MPNST-lucþ) to permit noninva- sive in vivo tumor growth assessment by luminescence. One week after allograft, recipient mice were intraperitoneally injected with either 40 mg/kg of Cpd21 or vehicle twice daily over 4 weeks, and mice were imaged weekly. We observed that treatment with Cpd21 reduced the tumor burden as measured by total flux normalized to baseline flux levels (Fig. 5D and E). BrdUrd was injected into mice before necropsy. Both BrdUrd and Ki67 evaluation indicated a significant decrease in tumor cell proliferation following Cpd21 treatment (Fig. 5F–I). These results are consistent with our observations that Cpd21 inhi- bits the cell cycle in vitro, resulting in a decrease in the proliferative capacity of sMPNSTs. Consistent with in vitro data, Cpd21-treated sMPNSTs exhibited elevated activated caspase-3 and PARP (Fig. 5J– M). These results were confirmed by Western blotting to detect cleaved caspase-3 and PARP in tumors from our in vivo model (Fig. 5N). Taken together, our results demonstrate that Cpd21 can be delivered to mice in concentrations to sufficiently penetrate sMPNST tissue, and inhibit tumor development. To extend the previous observation that chronic Cpd21 exposure did not appear to cause general toxicity, we examined various tissues following necropsy for histologic evidence of Cpd21-induced toxicity. In addition to tumor tissue (Fig. 6A), we examined liver (Fig. 6B), kidney (Fig. 6C), brain (Fig. 6D), skin (Fig. 6E), and sciatic nerve (Fig. 6F), and found no evidence of abnormal histology. Activated caspase-3 could only be detected in tumor samples (Fig. 6G) and not in other tissues examined (Fig. 6H–L). In contrast to other chemotherapeutic agents such as doxorubicin that induce toxicity in a variety of tissues (41), Cpd21 appears to act most notably on malignant tumor tissue. Interaction with other anticancer agents In addition to doxorubicin, MPNSTs have been shown to be sensitive to pathway-specific inhibitors of MAPK/ERK (42), PI3K/AKT (43), mTOR (43–45), and CXCR4 (7). We therefore examined whether Cpd21 in conjunction with doxorubicin or specific inhibitors, including U0126, a MEK inhibitor (42); LY294002, an AKT inhibitor (43); rapamycin, an mTOR inhib- itor (43–45); and AMD3100, a CXCR4 inhibitor (7), might better inhibit sMPNST cell growth as assessed by ATP consumption (46). We found the inhibition of the PI3K pathway combined with Cpd21 treatment resulted in a decrease in cellular growth and survival that is greater than either inhibitor alone (Fig. 7A and B). Phospho-AKT Western blots of sMPNST cells treated with LY294002 at 20 mmol/L for 30 or 60 minutes verified effective pAKT inhibition in sMPNST cells (Fig. 7C). Inhibition of the MEK/ERK pathway combined with Cpd21 treatment did not produce detectable additive or synergistic effects (Fig. 7D). Phospho-ERK inhibition by U0126 was verified by Western blotting (Fig. 7E). Inhibition of the mTOR pathway combined with Cpd21 treatment did not produce detectable additive or synergistic effects (Fig. 7F). In fact, sMPNST cellular growth Figure 5. Effect of Cpd21 in vivo. A and B, Cpd21 was incubated with murine, hepatic S9 fractions (A) or murine hepatocyte (B) for indicated times. Amount of Cpd21 remaining was quantified by mass spectrometry. C, Cpd21 was injected at 20 mg/kg i.p. and quantified in plasma or tumor over time. Pharmacokinetic parameters were quantified using the noncompartmental analysis tool in WinNonlin (Pharsight). D, sMPNST-lucþ cells were injected subcutaneously in nude mice. Cpd21 (40 mg/kg) or vehicle was injected intraperitoneally twice per day for 4 weeks. Mice were imaged every 7 days. Images shown are from week 4. E, quantification of tumor images in D by normalizing total flux at week 4 with baseline flux (n ¼ 10 for vehicle, n ¼ 8 for Cpd21). F, BrdUrd staining of Cpd21- or vehicle-treated tumors. G, quantification of F. H, immunohistochemistry of Ki67. I, quantification of H. J, immunohistochemistry of cleaved caspase-3. K, quantification of J. L, immunohistochemistry of cleaved PARP. M, quantification of L. N, Western blotting for caspase-3 and PARP cleavage in tumor lysates. All microscopic images taken at �20. Scale bars, 100 mm. All values, mean � SD. The Student t test was used for significance testing (�, P < 0.05; ��, P < 0.01; ���, P < 0.001). Chau et al. Cancer Res; 74(2) January 15, 2014 Cancer ResearchOF8 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ seemed relatively resistant to rapamycin, despite exhibiting decreased pS6 levels after rapamycin treatment (Fig. 7G). None of the other tested reagents produced measurable additive or synergistic effects (data not shown). We extended the above studies of dual LY294002 and Cpd21 treatment to MPNST cells derived from cis-Nf1þ/�;p53þ/� mice, and again, the combined effect of both inhibitors is greater than that of each individual inhibitor (Fig. 7H). We tested the effects of both inhibitors on phosphorylation of AKT and found that pAKT was inhibited only when LY294002 is present (Fig. 7C and I). Thus, in contrast to published reports of successful application of MEK inhibi- tors to suppress NF1-based solid tumors and leukemias, in the context of Cp21, coinhibition of the PI3K pathway was most effective in impeding tumor cell growth and survival. Discussion Malignant peripheral nerve tumors are the major source of mortality in NF1. Like most malignant sarcomas, these tumors are resistant to all current chemotherapeutic and radiother- apeutic strategies. It has long been recognized that MPNSTs harbor tumor suppressor mutations in addition to Nf1 loss. Notably, loss-of-function mutations of the p53 tumor suppres- sor have been reported (16–18). This has led to the successful generation of spontaneous mouse models of MPNST by muta- tion of both Nf1 and p53 tumor suppressor genes (24, 25). A further refinement of MPNST modeling came about by iden- tifying SKPs as a source for dermal neurofibromas in the skin and by the capacity of SKPs to engender plexiform neurofi- bromas when placed in the sciatic nerve (4). Additive mutation of p53 together with Nf1 turned SKPs into progenitors of MPNST, thus facilitating high-throughput screens for chemical compounds that might inhibit early passage cell growth. We identified Cpd21 as a small molecule with inhibitory properties on cultured mouse and human MPNSTcells. It was a surprise that this compound originating from a 200,000 chem- ical compound library, had, without chemical modification, a repertoire of desirable in vivo properties, including low micromolar EC50, adequate stability, manageable toxicity, and adequate accumulation in solid tumor tissue. These serendip- itous favorable properties allowed allograft analysis of Figure 6. Apoptotic effect of Cpd21 is specific to tumor tissue. A–F, H&E staining of tumors (A), liver (B), kidney (C), brain (D), skin (E), and sciatic nerve (F) from mice treated with either Cpd21 or vehicle. G–L, immunohistochemistry for cleaved caspase-3 was only detected in tumor tissue (G), but not in liver (H), kidney (I), brain (J), skin (K), or sciatic nerve (L). All images taken at �20. Scale bars, 100 mm. Small-Molecule Inhibitor of MPNST www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 OF9 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ antitumor efficacy and demonstrated a significant reduction in tumor burden by this compound. Future efforts to assess the effectiveness of Cp21 on human MPNST xenografts will pro- vide additional insight into the potential for this compound as a therapeutic agent. At present we cannot identify the specific mechanism whereby Cpd21 exerts its inhibitory action on MPNST cells. Compound exposure to tumor cells results in a cell-cycle delay manifested by depletion of certain cell-cycle–related molecules and eventually leading to cellular apoptosis. A similar course of events is seen when tumor-bearing mice are treated with compound. However, the precise molecular target and mech- anistic mode of compound action remains a topic of investi- gation. Empirical chemical compound screens to identify cancer cell inhibitory compounds have been applied for several decades. A significant proportion of such empirically derived cancer cell inhibitory small molecules that have reached the clinic have turned out to mechanistically impinge on various aspects of cell division, including mitotic spindle machinery, cell-cycle machinery, and DNA replication and repair machin- ery. These cellular properties are not unique to cancer cells and are shared by many normally dividing cells in the body that are Cpd21 only Cpd21 only U0126 only Cpd21 + LY294002 R L U sa m p le /R L U D M S O LY294002 only * * * * * * * * * * 1.0 0.5 0.0 R L U sa m p le /R L U D M S O 1.0 0.5 0.0 R L U sa m p le /R L U D M S O 1.5 1.0 0.5 0.0 R L U sa m p le /R L U D M S O 0.8 0.6 0.4 0.2 0.0 0.5 1 5 0.5 1 5 0.5 1 55 10 20 Cpd21 + LY294002 0.5 1 5 0.5 1 5 0.5 1 55 10 20 Cpd21 + U0126 0.5 1 5 0.5 1 0.5 1 0.5 15 – – 10 20 10 10 20 20–– – 0.5 1 5 0.5 1 55 10 20 Cpd21 + Rapamycin 0.5 1 5 0.005 0.01 0.05 0.5 1 5 Combined (10 µmol/L LY294002) Combined (5 µmol/L U0126) Cpd21 (500 nmol/L) Cpd21 (500 nmol/L) + LY (10 µmol/L)LY294002 (10 µmol/L) DMSOA C F H I G D E B Combined (10 µmol/L U0126) Combined (10 µmol/L LY294002) Combined (20 µmol/L LY294002) Cpd21 only Cpd21 only LY294002 only Cpd21 only Cpd21 (µmol/L) LY294004 (µmol/L) Cpd21 + LY294002 Rapamycin only R a p a m yc in - 3 0 m in R a p a m yc in - 6 0 m in D M S O Combined (0.01 µmol/L Rapamycin) LY294002 (min) LY294002 only pAKT AKT GAPDH pAKT AKT GAPDH pERK D M S O U 0 1 2 6 ERK H3 pS6 D M S O S6 Tubulin D 30 60 Combined (20 µmol/L LY294002) Figure 7. Cpd21 Interacts with PI3K inhibitor. A, ATP assay following combined treatment of Cpd21 and LY294002 in sMPNST cells. Synergism determined by Loewe method. B, phase contrast images of sMPNST cells treated with DMSO, Cpd21 alone, LY294002 (LY) alone, or a combination of both inhibitors. Images taken at �20. Scale bars, 100 mm. C, Western blotting for pAKT in sMPNST cells treated with 20 mmol/L LY294002 for 30 or 60 minutes. D, ATP assay following combined treatment of Cpd21 and U0126 in sMPNST cells. E, Western blotting for pERK in sMPNST cells treated with 10 mmol/L U0126 for 30 minutes. F, ATP assay following combined treatment of Cpd21 and LY294002 in sMPNST cells. G, Western blotting for pS6 in sMPNST cells treated with 0.05 mmol/L (50 nmol/L) rapamycin for 30 or 60 minutes. H, ATP assay following combined treatment of MPNST cells from cis-Nf1 þ/� ;p53 þ/� mice with Cpd21 and LY294002. Synergism determined by Loewe method. I, Western blotting for pAKT and AKT in sMPNST cells treated with Cpd21 alone, LY294002 alone, or with both inhibitors. All graphs plotted in micromolar concentrations. Chau et al. Cancer Res; 74(2) January 15, 2014 Cancer ResearchOF10 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ essential for organ health. Chemotherapeutic toxicity to nor- mal cells represents a major impediment to effective cancer treatment. In this regard, our screen departs from traditional high-throughput screens in that we limited our cells assayed to early passage cells rather than to established cell lines. It is also noteworthy that the cellular specificity of Cpd21 renders it relatively innocuous to the normally dividing cells we tested (MEFs, Myc-Schwann Cells, and wild-type SKPs). This feature is consistent with the idea that the compound target(s) relates to the "cancer state" of the MPNST cells rather than to more general properties of mitotically active cells. Although we currently do not know whether Cpd21 is also effective against the benign plexiform neurofibromas, it is entirely possible that these slowly dividing tumor cells may also be susceptible to Cpd21 activity if they share common pathways with MPNSTs that are potential target(s) of Cpd21. Recent advances in MPNST research have pointed to addi- tional potential therapeutic targets, including the CXCR4 cyto- kine axis (7), the mTOR pathways (44), and the RAS/MEK/ERK pathways(13,47–50).Inthese studies,AMD3100andrapamycin have been shown to inhibit MPNSTs from the cis-Nf1þ/�;p53þ/� -mouse model, and ERK inhibitors, such as U0126, have been verified in a variety of human and mouse derived MPNSTs. Interestingly,whensuchcompoundsweretestedinconjunction with Cp21, we found no additive value. Instead, blockade of the PI3K pathway did cooperate with Cpd21 action to inhibit tumor growth.Thisintriguingresultpoints tothe potentialplasticity of cancer cells and to the complexity of tumor pathway interac- tions. For example, it may be that Cpd21 activity somehow unveils a synthetic dependence on PI3K signaling that is not present in the absence of compound. Continued studies of Cpd21, its specific mode of action, and the mechanistic basis for its activity on MPNST cells but not on nontumorigenic cells may shed light on novel anticancer pathways. Disclosure of Potential Conflicts of Interest V. Chau has ownership interest in patent pending for Cpd21. No potential conflicts of interest were disclosed by the other authors. Authors' Contributions Conception and design: V. Chau, S.K. Lim, W. Mo, L.F. Parada, L.Q. Le Development of methodology: V. Chau, S.K. Lim, W. Mo, L.F. Parada, L.Q. Le Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): V. Chau, S.K. Lim, W. Mo, C. Liu, A.J. Patel, S. Wei, B.A. Posner, N.S. Williams, L.F. Parada, L.Q. Le Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V. Chau, A.J. Patel, R.M. McKay, S. Wei, B.A. Posner, N.S. Williams, L.F. Parada, L.Q. Le Writing, review, and/or revision of the manuscript: V. Chau, S.K. Lim, R.M. McKay, S. Wei, B.A. Posner, L.F. Parada, L.Q. Le Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): V. Chau, S.K. Lim, W. Mo, S. Wei, B.A. Posner, L.Q. Le Study supervision: V. Chau, W. Mo, L.F. Parada, L.Q. Le Input on chemistry, selection of chemical compounds from library screen, and proofreading manuscript: J.K. De Brabander Acknowledgments The authors thank Michael White for helpful discussions throughout this project and Karen Cichowski for sharing the human MPNST cell lines. The authors also thank all members in Luis Parada's laboratory and in Lu Le's laboratory for helpful discussions. Grant Support This work was partially supported by funding from Texas Neurofibromatosis Foundation, the National Cancer Institute (Grant# R01 CA166593; L.Q. Le), the NIH (Grant# P50NS052606; L.F. Parada), CPRIT (RP 100782; L.F. Parada), and the Simmons Cancer Center Support Grant# 5P30 CA 142543-03. L.Q. Le is a Disease- Oriented Clinical Scholar and holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund. L.F. Parada is an American Cancer Society Research Professor. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received July 11, 2013; revised September 30, 2013; accepted October 28, 2013; published OnlineFirst November 27, 2013. References 1. Pulst SM. Prenatal diagnosis of the neurofibromatoses. Clin Perinatol 1990;17:829–44. 2. Brems H, Beert E, de Ravel T, Legius E. Mechanisms in the patho- genesis of malignant tumours in neurofibromatosis type 1. Lancet Oncol 2009;10:508–15. 3. Riccardi VM. Neurofibromatosis: past, present, and future. N Engl J Med 1991;324:1283–5. 4. Le LQ, Shipman T, Burns DK, Parada LF. Cell of origin and microen- vironment contribution for NF1-associated dermal neurofibromas. Cell Stem Cell 2009;4:453–63. 5. Huson SM, Harper PS, Compston DA. Von Recklinghausen neurofi- bromatosis. A clinical and population study in south-east Wales. Brain 1988;111:1355–81. 6. Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF. Neurofibromas in NF1: Schwann cell origin and role of tumor environment. Science 2002;296:920–2. 7. Mo W, Chen J, Patel A, et al. CXCR4/CXCL12 mediate autocrine cell- cycle progression in NF1-associated malignant peripheral nerve sheath tumors. Cell 2013;152:1077–90. 8. Ferner RE, Gutmann DH. International consensus statement on malig- nant peripheral nerve sheath tumors in neurofibromatosis. Cancer Res 2002;62:1573–7. 9. Ducatman BS, Scheithauer BW, Piepgras DG, Reiman HM, Ilstrup DM. Malignant peripheral nerve sheath tumors. A clinicopathologic study of 120 cases. Cancer 1986;57:2006–21. 10. Ballester R, Marchuk D, Boguski M, et al. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell 1990;63:851–9. 11. Le LQ, Parada LF. Tumor microenvironment and neurofibromatosis type I: connecting the GAPs. Oncogene 2007;26:4609–16. 12. DeClue JE, Papageorge AG, Fletcher JA, et al. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell 1992;69: 265–73. 13. Basu TN, Gutmann DH, Fletcher JA, Glover TW, Collins FS, Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature 1992;356:713–5. 14. Dasgupta B, Yi Y, Chen DY, Weber JD, Gutmann DH. Proteomic analysis reveals hyperactivation of the mammalian target of rapamycin pathway in neurofibromatosis 1-associated human and mouse brain tumors. Cancer Res 2005;65:2755–60. 15. Weiss B, Bollag G, Shannon K. Hyperactive Ras as a therapeutic target in neurofibromatosis type 1. Am J Med Genet 1999;89:14–22. 16. Birindelli S, Perrone F, Oggionni M, et al. Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors. Lab Invest 2001;81:833–44. 17. Menon AG, Anderson KM, Riccardi VM, et al. Chromosome 17p deletions and p53 gene mutations associated with the formation of malignant neurofibrosarcomas in von Recklinghausen neurofibroma- tosis. Proc Natl Acad Sci U S A 1990;87:5435–9. Small-Molecule Inhibitor of MPNST www.aacrjournals.org Cancer Res; 74(2) January 15, 2014 OF11 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ 18. Jhanwar SC, Chen Q, Li FP, Brennan MF, Woodruff JM. Cytogenetic analysis of soft tissue sarcomas. Recurrent chromosome abnormal- ities in malignant peripheral nerve sheath tumors (MPNST). Cancer Genet Cytogenet 1994;78:138–44. 19. Nielsen GP, Stemmer-Rachamimov AO, Ino Y, Møller MB, Rosenberg AE, Louis DN. Malignant Transformation of Neurofibromas in Neuro- fibromatosis 1 Is Associated with CDKN2A/p16 Inactivation. Am J Pathol 1999;155:1879–84. 20. Berner JM, Sorlie T, Mertens F, et al. Chromosome band 9p21 is frequently altered in malignant peripheral nerve sheath tumors: studies of CDKN2A and other genes of the pRB pathway. Genes Chromo- somes Cancer 1999;26:151–60. 21. Kourea HP, Orlow I, Scheithauer BW, Cordon-Cardo C, Woodruff JM. Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neurofibromas. Am J Pathol 1999;155: 1855–60. 22. Buchstaller J, McKeever Paul E, Morrison Sean J. Tumorigenic cells are common in mouse MPNSTs but their frequency depends upon tumor genotype and assay conditions. Cancer Cell 2012;21: 240–52. 23. Perrone F, Tabano S, Colombo F, et al. p15INK4b, p14ARF, and p16INK4a inactivation in sporadic and neurofibromatosis type 1-relat- ed malignant peripheral nerve sheath tumors. Clin Cancer Res 2003;9:4132–8. 24. Vogel KS, Klesse LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF. Mouse tumor model for neurofibromatosis type 1. Science 1999; 286:2176–9. 25. Cichowski K, Shih TS, Schmitt E, et al. Mouse models of tumor development in neurofibromatosis type 1. Science 1999;286:2172–6. 26. Gregorian C, Nakashima J, Dry SM, et al. PTEN dosage is essential for neurofibroma development and malignant transformation. Proc Natl Acad Sci 2009;106:19479–84. 27. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–76. 28. Rallis E, Ragiadakou D. Giant plexiform neurofibroma in a patient with neurofibromatosis type I. Dermatol Online J 2009;15:7. 29. Alcantara Llaguno S, Chen J, Kwon CH, et al. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor sup- pressor mouse model. Cancer Cell 2009;15:45–56. 30. Biernaskie JA, McKenzie IA, Toma JG, Miller FD. Isolation of skin- derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nat Protoc 2006;1:2803–12. 31. Mayes DA, Rizvi TA, Cancelas JA, et al. Perinatal or Adult Nf1 Inac- tivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation. Cancer Res 2011;71:4675–85. 32. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell 1992;71:973–85. 33. Kioussi C, Gruss P. Making of a Schwann. Trends in Genetics 1996; 12:84–6. 34. Wu J, Williams JP, Rizvi TA, et al. Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert hedgehog-expres- sing cells. Cancer Cell 2008;13:105–16. 35. Fernandes KJ, McKenzie IA, Mill P, et al. A dermal niche for multipotent adult skin-derived precursor cells. Nat Cell Biol 2004;6:1082–93. 36. Ridley AJ, Paterson HF, Noble M, Land H. Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell trans- formation. EMBO J 1988;7:1635–45. 37. Bates S, Bonetta L, MacAllan D, et al. CDK6 (PLSTIRE) and CDK4 (PSK-J3) are a distinct subset of the cyclin-dependent kinases that associate with cyclin D1. Oncogene 1994;9:71–9. 38. Tam SW, Theodoras AM, Shay JW, Draetta GF, Pagano M. Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression. Oncogene 1994;9:2663–74. 39. Lu J, Ma Z, Hsieh J-C, et al. Structure–activity relationship studies of small-molecule inhibitors of Wnt response. Bioorganic & Med Chem Lett 2009;19:3825–7. 40. Tso SC, Qi X, Gui WJ, etal. Structure-based designand mechanisms of allosteric inhibitors for mitochondrial branched-chain alpha-ketoacid dehydrogenase kinase. Proc Natl Acad Sci U S A 2013;110:9728–33. 41. Brenner DE, Wiernik PH, Wesley M, Bachur NR. Acute doxorubicin toxicity relationship to pretreatment liver function, response, and pharmacokinetics in patients with acute nonlymphocytic leukemia. Cancer 1984;53:1042–8. 42. DeClue JE, Heffelfinger S, Benvenuto G, et al. Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models. J Clin Invest 2000;105:1233–41. 43. ZouCY,Smith KD,Zhu Q-S,etal.Dual targetingof AKT and mammalian target of rapamycin: A potential therapeutic approach for malignant peripheral nerve sheath tumor. Mol Cancer Ther 2009;8:1157–68. 44. De Raedt T, Walton Z, Yecies Jessica L, et al. Exploiting Cancer Cell Vulnerabilities to Develop a Combination Therapy for Ras-Driven Tumors. Cancer Cell 2011;20:400–13. 45. Johansson G, Mahller YY, Collins MH, et al. Effective in vivo targeting of the mammalian target of rapamycin pathway in malignant peripheral nerve sheath tumors. Mol Cancer Ther 2008;7:1237–45. 46. Straetemans R, O'Brien T, Wouters L, et al. Design and analysis of drug combination experiments. Biom J 2005;47:299–308. 47. Katz D, Lazar A, Lev D. Malignant peripheral nerve sheath tumour (MPNST): the clinical implications of cellular signalling pathways. Expert Rev Mol Med 2009;11:e30. 48. Cichowski K, Jacks T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 2001;104:593–604. 49. Rahrmann EP, Watson AL, Keng VW, et al. Forward genetic screen for malignant peripheral nerve sheath tumor formation identifies new genes and pathways driving tumorigenesis. Nat Genet 2013. 50. Jessen WJ, Miller SJ, Jousma E, et al. MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors. J Clin Invest 2013;123:340–7. Chau et al. Cancer Res; 74(2) January 15, 2014 Cancer ResearchOF12 Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ Published OnlineFirst November 27, 2013.Cancer Res Vincent Chau, S. Kyun Lim, Wei Mo, et al. Tumors Inducer of Apoptosis in Malignant Peripheral Nerve Sheath Preclinical Therapeutic Efficacy of a Novel Pharmacologic Updated version 10.1158/0008-5472.CAN-13-1934doi: Access the most recent version of this article at: E-mail alerts related to this article or journal.Sign up to receive free email-alerts Subscriptions Reprints and .pubs@aacr.orgDepartment at To order reprints of this article or to subscribe to the journal, contact the AACR Publications Permissions Rightslink site. (CCC) Click on "Request Permissions" which will take you to the Copyright Clearance Center's .http://cancerres.aacrjournals.org/content/early/2014/01/09/0008-5472.CAN-13-1934 To request permission to re-use all or part of this article, use this link Research. on April 5, 2021. © 2013 American Association for Cancercancerres.aacrjournals.org Downloaded from Published OnlineFirst November 27, 2013; DOI: 10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/cgi/alerts mailto:pubs@aacr.org http://cancerres.aacrjournals.org/content/early/2014/01/09/0008-5472.CAN-13-1934 http://cancerres.aacrjournals.org/ << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Gray Gamma 2.2) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.3 /CompressObjects /Off /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings true /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 0 /Optimize false /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage false /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness false /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Remove /UCRandBGInfo /Remove /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages false /ColorImageMinResolution 200 /ColorImageMinResolutionPolicy /Warning /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 150 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ENU ([Based on '[High Quality Print]'] Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames false /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides true /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks true /AddPageInfo false /AddRegMarks false /BleedOffset [ 18 18 18 18 ] /ConvertColors /NoConversion /DestinationProfileName () /DestinationProfileSelector /NA /Downsample16BitImages true /FlattenerPreset << /ClipComplexRegions true /ConvertStrokesToOutlines false /ConvertTextToOutlines false /GradientResolution 300 /LineArtTextResolution 1200 /PresetName ([High Resolution]) /PresetSelector /HighResolution /RasterVectorBalance 1 >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> << /AllowImageBreaks true /AllowTableBreaks true /ExpandPage false /HonorBaseURL true /HonorRolloverEffect false /IgnoreHTMLPageBreaks false /IncludeHeaderFooter false /MarginOffset [ 0 0 0 0 ] /MetadataAuthor () /MetadataKeywords () /MetadataSubject () /MetadataTitle () /MetricPageSize [ 0 0 ] /MetricUnit /inch /MobileCompatible 0 /Namespace [ (Adobe) (GoLive) (8.0) ] /OpenZoomToHTMLFontSize false /PageOrientation /Portrait /RemoveBackground false /ShrinkContent true /TreatColorsAs /MainMonitorColors /UseEmbeddedProfiles false /UseHTMLTitleAsMetadata true >> ] >> setdistillerparams << /HWResolution [600 600] /PageSize [792.000 1224.000] >> setpagedevice