nf1 deletion generates multiple subtypes of soft-tissue … · soft-tissue sarcomas are a...

Tools and Technologies

NF1 Deletion Generates Multiple Subtypes of Soft-TissueSarcoma That Respond to MEK Inhibition

Rebecca D. Dodd1, Jeffrey K. Mito2, William C. Eward3, Rhea Chitalia1, Mohit Sachdeva1, Yan Ma1,Jordi Barretina5,6,7, Leslie Dodd4, and David G. Kirsch1,2

AbstractSoft-tissue sarcomas are a heterogeneous group of tumors arising from connective tissue. Recently,

mutations in the neurofibromin 1 (NF1) tumor suppressor gene were identified in multiple subtypes of

human soft-tissue sarcomas. To study the effect of NF1 inactivation in the initiation and progression of

distinct sarcoma subtypes, we have developed a novel mouse model of temporally and spatially restricted

NF1-deleted sarcoma. To generate primary sarcomas, we inject adenovirus containing Cre recombinase

into NF1flox/flox; Ink4a/Arfflox/flox mice at two distinct orthotopic sites: intramuscularly or in the sciatic

nerve. The mice develop either high-grade myogenic sarcomas or malignant peripheral nerve sheath

tumor (MPNST)-like tumors, respectively. These tumors reflect the histologic properties and spectrum of

sarcomas found in patients. To explore the use of this model for preclinical studies, we conducted a study

of mitogen-activated protein kinase (MAPK) pathway inhibition with the MEK inhibitor PD325901.

Treatment with PD325901 delays tumor growth through decreased cyclin D1 mRNA and cell proliferation.

We also examined the effects of MEK inhibition on the native tumor stroma and find that PD325901

decreases VEGFa expression in tumor cells with a corresponding decrease in microvessel density. Taken

together, our results use a primary tumor model to show that sarcomas can be generated by loss of NF1

and Ink4a/Arf, and that these tumors are sensitive to MEK inhibition by direct effects on tumor cells

and the surrounding microenvironment. These studies suggest that MEK inhibitors should be further

explored as potential sarcoma therapies in patients with tumors containing NF1 deletion. Mol Cancer Ther;

12(9); 1906–17. �2013 AACR.

IntroductionSoft-tissue sarcomas are an aggressive and heteroge-

neous group of tumors that arise from connective tissue,including the muscle, nerve sheath, blood vessel, fat, andfibrous tissue. These tumors are fatal in 30%ofpatients (1),and the standard treatment of wide resection and radia-tion carries considerable morbidity among survivors.

Recent genomic studies have identified novel mutationsin a diverse spectrum of human soft tissue sarcomas thatmay reveal new molecular targets for therapy (2). One ofthe most frequently mutated gene described is neurofi-bromin 1 (NF1), a tumor suppressor that functions as anegative regulator of the Ras pathway. Although the lossofNF1 iswell characterized inmalignant peripheral nervesheath tumors (MPNST), NF1 mutations in other soft-tissue sarcomas have only been recently identified. Usinggenomic sequencing and copy number analysis, NF1mutations were identified in myxofibrosarcoma (10.5%;n¼ 4 of 38) and pleomorphic liposarcoma (8%; n¼ 2 of 24)(3). Similar studies have identifiedgenomic loss of theNF1locus in 15% of pediatric embryonal rhabdomyosarcomas(n ¼ 4 of 26; ref. 4). This deletion was mutually exclusivewith Ras gene mutations. The identification of novelmutations in sarcomaswith complex karyotypes providesnew opportunities to model human sarcomas and toinvestigate new therapies for this difficult to treat disease.

In addition to identifying NF1 mutations in spontane-ous sarcomas in the general population, patients withneurofibromatosis type 1 (NF1) are at increased risk fordeveloping aggressive soft-tissue sarcomas. NF1 affects 1in 3,000 live births and is caused by germline inactivationof a single allele of the NF1 gene (5). The most common

Authors' Affiliations: Departments of 1Radiation Oncology, 2Pharmacol-ogy and Cancer Biology, 3Orthopaedic Surgery, and 4Pathology, DukeUniversity Medical Center, Durham, North Carolina; 5The Broad Institute ofHarvard and MIT, Cambridge; 6Department of Medical Oncology, and7Center for Cancer Genome Discovery, Dana-Farber Cancer Institute,Harvard Medical School, Boston, Massachusetts

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for J. Barretina: Novartis Institutes for BiomedicalResearch, Cambridge,MA02139; current address for L. Dodd, Departmentof Pathology and Laboratory Medicine, University of North Carolina atChapel Hill, Chapel Hill, NC 27599.

Corresponding Author: David G. Kirsch, Duke University Medical Center,Box 91006, Durham, NC 27708. Phone: 919-681-8605; Fax: 919-681-1867; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0189

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 12(9) September 20131906

on October 7, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 15, 2013; DOI: 10.1158/1535-7163.MCT-13-0189

http://mct.aacrjournals.org/

sarcoma afflicting patients with NF1 is MPNST, with alifetime risk of 8% to 13% (5). Patients withNF1 are also atrisk for developing non-neurogenic sarcomas, includingrhabdomyosarcoma and undifferentiated pleomorphicsarcoma (UPS), with a 3% to 6% lifetime risk (6–8). Mostmalignant NF1-deleted tumors have inactivation of eitherp53 or Ink4a/Arf tumor suppressor genes, with morethan50%ofMPNSTs expressingwild-typep53andhomo-zygous deletion of Ink4a/Arf (9). Deletion of NF1 aloneis not sufficient to cause high-grade sarcomas, as tissue-specific deletion of NF1 with either Krox20-Cre orDhh-Cre results in neurofibromas (10, 11). Similarly, dele-tion of either Ink4a or Arf alone is not sufficient forformation of neurofibromas or MPNSTs on an NF1 het-erozygous background from either NF1þ/�; Ink4a�/�

mice (12) or NF1þ/�; Arf�/� mice (13). In addition, wehave previously reported that conditional deletion ofInk4a/Arf alone by adenovirus-expressing Cre recombi-nase (Ad-Cre) injection is insufficient for sarcoma initia-tion (14). Deletion of both Ink4a and Arf in an NF1heterozygous background (NF1þ/�; Ink4a/Arf�/�) gen-erated sporadic MPNSTs in 26% of mice (12). The mostwidely usedNF1-dependent mouse sarcomamodel is theNF1þ/�; p53þ/� cismouse,whichdevelopsNF1- andp53-null sarcomas throughout the body following LOH (15,16). Despite the prevalence ofNF1mutations in soft-tissuesarcomas, the role of NF1 in the development of soft-tissue sarcomas, particularly in the background of wild-type p53 alleles, has not been fully explored in a primarymouse model of soft-tissue sarcoma. Therefore, we de-signed a study to test whether deletion of both NF1 andInk4a/Arf could generate high-grade soft-tissue sarcomasin an NF1 wild-type background.Genetically engineered mouse models have greatly

advanced our understanding of sarcomas (14, 15, 17–19)and have served as a preclinical platform for therapies(20–25). However, for many models, mice develop sar-comas at multiple sites, which can make tumor detec-tion and assessment of treatment response challenging.Here, we developed a novel mouse model of temporallyand spatially restricted NF1-deleted sarcoma to increaseour understanding of NF1-mutant tumors. Using Cre-loxP technology, tumors are initiated through Cre-mediated deletion of conditional NF1 and Ink4a/Arffloxed alleles (NF1flox/flox; Ink4a/Arfflox/flox). These micedevelop both high-grade myogenic sarcomas andMPNST-like tumors, based on the site of Cre injection.These tumors reflect the histologic properties and spec-trum of sarcomas found in patients. Because sarcomasin this model develop in a spatially and temporallyrestricted manner, this model is particularly useful fortesting therapeutic drug responses, and we have usedthis model as a preclinical platform to assess newtherapies for patients with NF1-deleted sarcomas. Wehave identified mitogen-activated protein kinase(MAPK) inhibition as a promising therapy that slowsthe growth of primary mouse sarcomas through effectson tumor cell growth and the surrounding vasculature.

Importantly, this model is able to directly assess theeffects of MEK inhibition on native tumor stroma, asthese primary tumors develop in immunocompetentmice with native vasculature. By showing the abilityof these tumors to respond to MEK inhibition, we lay afoundation for future studies using this model to test theefficacy of other drugs for NF1-deficient sarcomas in thepreclinical setting.

Materials and MethodsMouse sarcoma model

All mouse work was conducted in accordance withDuke University Institutional Animal Care and UseCommittee approved protocols. The NF1flox/flox mice(11) and Ink4a/Arfflox/flox mice (26) have been previouslyreported. Primers sequences used to show recombinationof the appropriate alleles are found in SupplementaryTable S1 or have been previously reported (11). Tumorswere generated by injection of Ad-Cre (University ofIowa, Iowa City, IA; Vector core) into the lower left leg ofNF1flox/flox; Ink4a/Arfflox/flox compound mutant mice aspreviously described (14). For sciatic nerve injections, thenerve was exposed by an incision in the surroundingmuscle, followed by injection of 25 mL Ad-Cre into thenerve. Tumor volumes were calculated using the formulaV ¼ (p � L � W � H)/6, with L, W, and H representingthe length, width, and height of the tumor in mm,respectively.

Histology and immunostainingAntibodies for immunohistochemistry included: S100

(DakoCytomation), MyoD1 (DakoCytomation), pERK(Cell Signaling Technology), pS6 (Cell Signaling Technol-ogy), Ki67 (BD Pharmagen), CD31 (BD Pharmagen),pVEGFR-2 (Cell Signaling Technology), and MECA-32(BD Pharmagen). All immunostaining was conductedwith citrate-based antigen retrieval, with the exceptionof MECA-32 and pVEGFR2, which were conducted withEDTA-based retrieval. Terminal deoxynucleotidyl trans-ferase–mediated dUTP nick end labeling (TUNEL) stain-ing used the In Situ Cell Death Detection Kit, TMR Red(Sigma), as per the manufacturer’s instructions. To visu-alize mast cells, slides were stained with toluidine bluesolution (0.02% toluidine blue in 1% NaCl, pH 2.2) for 2minutes, followed by two washes in distilled water andthree washes in 100% ethanol.

PD325901 inhibitor treatmentTreatment started when tumors were between 200 and

300 mm3. Mice receiving vehicle alone were treated withNMP 10% (1-methyl-2-pyrrolidone)/PEG300 90% (Fluka)orally 5 days per week. PD325901 (ref. 27; Sigma) wasdissolved in NMP 10%/PEG300 90%. Mice were treatedwith 5mg/kgofPD325901orally 5daysperweek from thefirst dayof the treatment. Treatment for allmice continueduntil tumors regressed or it was necessary to sacrifice themouse for animal welfare concerns (i.e., animal was mor-ibund; tumor volume reached 2,000 mm3).

A Novel Mouse Model of NF1-Deleted Soft-Tissue Sarcoma

www.aacrjournals.org Mol Cancer Ther; 12(9) September 2013 1907




Tumor growth analysisCriteria for defining treatment response include both

volumetric analysis and a delay in tumor growth kine-tics. A partial response (PR) is classified as a tumor that:(i) shows a decrease in tumor volume from baselinemeasurements and (ii) shows delayed growth kinetics(defined as requiring more than 10 days to increase involume 150%). For waterfall plot analyses, percentage ofmaximal change is reported as the greatest percentage ofloss in tumor volume from baseline for tumors respond-ing to treatment. Tumors that did not respond to treat-ment are shown as greatest percentage of gain in tumorvolume from baseline over the course of the treatment. Inthis presentation of the data, change in tumor volume iscapped at more than 100%.

Statistical analysisGraphs and statistics were conducted in GraphPad 4.0.

A nonparametric Student t test was conducted to deter-mine differences between treatment groups. A P value ofless than 0.05 was considered statistically significant.

In vitro studiesThe mouse sarcoma cell lines 1863 and 3017 were

derived from primary sarcomas in NF1flox/flox; Ink4a/Arfflox/flox mice that developed after intramuscular Ad-Cre injection. These cell lines were authenticated byPCR genotyping. The cells were cultured in Dulbecco’smodified Eagle medium (DMEM) þ 10% FBS. For col-ony formation studies, cells were seeded at 500/well ina 6-well dish and treated with dimethyl sulfoxide(DMSO) or 50 nmol/L PD325901 (Sigma) for 3 to 7days. The number of existing colonies was determinedby crystal violet staining. Each experiment was carriedout in triplicate, and the experiment was repeated threetimes. Data shown are representative of one indepen-dent experiment. For colony formation studies withprior PD325901 treatment, cells were cultured overnightin 50 nmol/L PD325901, followed by splitting the cellsinto DMSO control or PD325901 for colony formation.Experiments were analyzed as described previously.Cell proliferation assays were conducted with cellscultured in either DMSO or 50 nmol/L PD325901, withthe number of cell doublings calculated daily. Forquantitative real-time PCR (qRT-PCR) analyses, cellswere treated with 50 nmol/L PD325901 for 4 hours,followed by RNA harvest into TRIzol. Data shownrepresent the average of four independent experiments.

Western blot analysis1863 cells were treated with 50 nmol/L PD325901 for

either 1 or 4 hours before harvest. Cells were washedonce with cold PBS (Sigma) and lysed for 10 minuteson ice with radioimmunoprecipitation assay (RIPA)buffer (Sigma), supplemented with phosphatase inhi-bitors (Sigma; P5726 and P0044). Protein concentrationwas conducted with BCA Protein Concentration Assay(Thermo Scientific). MiniProtean TGX gels (Bio-Rad)

were transferred to polyvinylidene difluoride (PVDF)by wet transfer.

Quantitative RT-PCRRNA was isolated from tumors and cells by TRIzol.

cDNA synthesis was conducted with iScript (Bio-Rad).qRT-PCR was conducted on an iQ5 instrument (Bio-Rad)using the DDCt method. Primer sequences can be found inSupplementary Table S2.

ResultsGenerating inducible NF1-deleted sarcomas in themouse

As NF1 mutations have been recently detected in avariety of human soft-tissue sarcoma subtypes (3, 4), wesought to determine whether loss of NF1 and the tumorsuppressor Ink4a/Arf was sufficient to generate soft-tis-sue sarcomas in a p53- and NF1-wild-type background.Furthermore, we wished to generate an inducible mousemodel of NF1-deleted sarcoma that could reflect thediverse spectrum of NF1-associated sarcomas found inpatients. Therefore, we generated mice with conditionalmutations in both NF1 and Ink4a/Arf (NF1flox/flox; Ink4a/Arfflox/flox). These mice were injected with Ad-Cre at oneof two sites: intramuscular injections to model tumorsarising in the skeletal muscle, and sciatic nerve injectionsto model tumors arising in the nerve sheath. Mice wereinjected into either the hind limb muscle (intramuscular;n ¼ 10) or sciatic nerve (n ¼ 12) of the lower left leg.Following injection, Cre recombinase can delete bothalleles of NF1 and Ink4a/Arf (Fig. 1A). Mice developedtumors 3 to 12 months after Cre exposure at the site ofinjection. We successfully derived two cell lines (1863and 3017) from sarcomas generated after intramuscularinjection of Ad-Cre. Using these sarcoma cell lines, wehave confirmed recombination of both alleles of NF1 andInk4a/Arf by PCR (Fig. 1B), in addition to loss of bothmRNAandprotein for p16, p19, andNF1 (SupplementaryFig. S1A–S1D). For clarity, we refer to sarcomas and celllines that are deleted for NF1, p16, and p19 as "NF1-deleted." These cell lines maintained an intact p53-depen-dent DNA damage response, as shown by elevation ofp21 in response to doxorubicin treatment (SupplementaryFig. S1E). We observed that the site of injection influencestumor initiation because sciatic nerve–derived tumorsdeveloped more quickly than intramuscularly derivedtumors (mean time to tumor, 4.1 vs. 6.2 months; P <0.0001; Fig. 1C). Injection of Ad-Cre at both sites resultedin large, ovoid tumors that appear well vascularized(Fig. 1D).

Histopathologic analysis determined that these tumorsreflect the histologic properties and spectrum of diseaseseen in human NF1-associated sarcomas (Fig. 1E). Sciaticnerve injections result in tumors with a fascicular patternof tightly packed spindle cells surrounding the nerve(Supplementary Fig. S2A). As seen in othermousemodelsof nerve sheath tumors (10, 22), these sarcomas show focalpositivity for S100b, but do not show nuclear expression

Dodd et al.

Mol Cancer Ther; 12(9) September 2013 Molecular Cancer Therapeutics1908




for the myogenic-lineage marker MyoD1. In contrast,intramuscular injections generate a spectrum of high-grade spindle cell sarcomas, including rhabdomyosarco-ma (n ¼ 5 of 10) and UPS (n ¼ 5 of 10), the most commonand aggressive form of soft tissue sarcoma in adults(Supplementary Fig. S2B). Both rhabdomyosarcoma andUPS occur in patients with NF1 (6–8), and NF1 mutationshave been identified in these sarcoma subtypes. Similar toother mouse models of myogenic sarcoma and theirhuman tumor subtypes (14, 28), the intramuscular tumorsexpress the myogenic marker MyoD1, but do not stainfocally for S100b. NF1-associated sarcomas in patientshave active RAS/MAPK/ERK and PI3K/AKT/mTORsignaling (29, 30). Therefore, we hypothesized that theMEK target extracellular signal–regulated kinase (ERK)and themTOR target S6wouldbephosphorylated in theseprimary NF1-deleted mouse sarcomas. Immunohisto-chemical analysis for pERK and pS6 reveals that MAPKand mTOR pathways are active in both sciatic nerveand intramuscular primary tumors from NF1flox/flox;Ink4a/Arfflox/floxmice (Fig. 1E). Taken together, these datasuggest that this mouse model reflects the spectrum ofNF1-deleted sarcomas found in the human population,and that distinct sarcoma subtypes can be generated fromthe same mouse strain by Ad-Cre injection into differentorthotopic sites.

Importantly, these primary tumors develop from asmall population of initiating tumor cells within thenative tumor stroma, facilitating the study of endogenousimmune cells, cellular growth, and native vasculature in aprimary tumor context. Thus, we further characterizedthe similarities and differences between the sarcoma sub-types based on the site of Ad-Cre injection using histo-chemical analyses to examine mast cell infiltration, cellproliferation, and vascularization. First, we examinedmast cells, granule-rich inflammatory cells that residewithin the connective tissue and are enriched in NF1-deleted neurofibromas and MPNSTs in patients (31).Infiltrating mast cells are important stromal factors thatcontribute to tumor formation in mouse models of NF1-deleted neurofibromas (22). In sarcomas fromNF1flox/flox;Ink4a/Arfflox/flox mice, toluidine blue staining shows sig-nificant infiltration ofmast cells into both the sciatic nerveand intramuscular tumors. Mast cells were present atsimilar levels in both orthotopic injection models (Fig.2A). Ki67 staining shows a high level of proliferation inboth intramuscular and sciatic nerve tumors. Quantifica-tion of Ki67-positive (Ki67þ) nuclei shows that intramus-cular tumors have a higher proliferative index than sciaticnerve tumors (P¼ 0.04), although there is a wide range ofproliferative index between individual tumors withineach subtype (Fig. 2B). In both tumor types, average

A B

C DN

erv

e

S100 MyoDE pERK pS6

Tail Cells

SN IM

Muscle

H&E

NF1 floxNF1 active

Ink4a/Arf activeInk4a/ArfloxP loxP loxP loxP

loxPloxP

NF1

Inject CreNF1 lost

Ink4a/Arf lost

NF1 Δ

P < 0.0001

Ink4a/Arf flox

0 100

SN (n = 12)IM (n = 10)

100

50

0200 300

Days postinjection

Tum

or-

free s

urv

ival

Ink4a/Arf Δ

Tail Cells

1863 3017Figure 1. Characterization of amouse model of NF1-deleted soft-tissue sarcoma. A, schematic ofNF1 and Ink4a/Arf deletionfollowing injection of Ad-Cre. B,PCR showing recombination ofboth NF1 and Ink4a/Arf floxedalleles in samples from paired tailsand NF1-deleted sarcoma celllines, derived from tumorsgenerated by intramuscular (IM)injection of Ad-Cre. C, Kaplan–Meier curve of tumor developmentbased on site of orthotopicinjection. The average time fortumor development is 4.1 monthsformice injected in the sciatic nerve(SN) and 6.2 months for miceinjected intramuscularly. D,photographs of NF1-deletedtumors generated from injectionsinto the sciatic nerve (left) ormuscle(right). E, histopathology ofintramuscular and sciatic nervetumors. Intramuscular tumorsresemble high-grade myogenicsarcomas and stain focally forMyoD1, but not S100. Sciatic nervetumors resemble MPNSTs andstain focally for S100, but not forMyoD1. Both tumors are positivefor pERK and pS6, indicatingactivity of the MAPK and mTORpathways, respectively. H&E,hematoxylin and eosin.






CD31-positive (CD31þ) microvessel density is similar,although vessel distribution between individual tumorsvaries considerably (Fig. 2C). Taken together, these datasuggest that while this mouse model consistently devel-ops tumors with distinct histologic properties, there isindividual tumor-to-tumor heterogeneity in phenotypessuch as mast cell infiltration, proliferation, and vascular-ity, with intramuscular tumors showing higher Ki67levels. As human NF1-associated sarcomas also displayheterogeneity by histologic analysis (7), we hypothesizethat the heterogeneity observed in this mouse model willbe useful for testing therapies directed against NF1-delet-ed sarcoma in the preclinical setting.

Efficacy of MEK inhibition in NF1-deleted sarcomasThe identification of NF1 mutations in myogenic sar-

coma (3, 4) provides a new foundation for preclinicalstudies examining targeted therapies for soft-tissue sar-comas. To determine the use of this novel mousemodel inexamining therapeutic response, we hypothesized that

NF1-deleted myogenic sarcomas would respond toMAPK inhibition based on two lines of evidence: (i) therole of NF1 as a negative regulator of Ras/MAPK/ERKsignaling, and (ii) the elevation of pERK in myogenic sar-comas generated from the NF1flox/flox; Ink4a/Arfflox/flox

mice. To determine the effect of MEK inhibition on cellproliferation in vitro, we generated cell lines (1863 and3017) from NF1-deleted sarcomas that developed follow-ing intramuscular injection of Ad-Cre into NF1flox/flox;Ink4a/Arfflox/flox mice. We treated the cells with the MEKinhibitor PD325901 and observed decreases in colony-forming units and cell proliferation following exposureto the drug (Fig. 3A and Supplementary Fig. S3A). Similarresults were seen when cells were treated with PD325901before seeding for colony formation to account for cell–cell interaction signals and growth factors that are usuallypresent in subconfluent conditions and in the tumormicroenvironment (Supplementary Fig. S3B). Westernblot analysis of 1863 cells treated with PD325901 for 4hours showed a decrease in phosphorylation of ERK

B

C

A

IM SN0

10

20

30

IM SN

IM SN

IM SN

Mast cells

Ki67+ cells

CD31+ cells

IM SN

IM SN

0

10

800

600

400

200

0

20

30

40

50

P < 0.05

Pe

rce

nta

ge

of

Ki6

7+ n

ucle

iC

D3

1+ e

ve

nts

To

luid

ine

blu

e c

ells

pe

r H

PF

Figure 2. Comparison of NF1-deleted sarcomas initiated byintramuscular (IM) or intrasciaticnerve (SN) injection of Ad-Cre. A,mast cells, visualized by toluidineblue, infiltrate both intramuscularand sciatic nerve tumors at similarlevels. Quantification representsthe average number of cells per20� field, with 10 fields countedper tumor. B, Ki67þ proliferativeindex is higher in intramusculartumors compared with sciaticnerve tumors (P < 0.05).Quantification represents thepercentage of Ki67þ cellsnormalized to total nuclei per 40�field, with 6 fields counted pertumor. C, microvessel density,visualizedbyCD31þ cells, is similarbetween intramuscular and sciaticnerve tumors. Quantificationrepresents the average number ofcells per 20� field, with 6 fieldscounted per tumor. HPF, high-power field.

Dodd et al.





(Fig. 3B), with corresponding changes in phospho-S6 andphospho-AKT levels (Supplementary Fig. S4).To extend these studies to the primary mouse model of

NF1-deleted myogenic sarcoma, we compared micereceiving PD325901 treatment (n ¼ 8) to mice receivingvehicle alone (n ¼ 5). Treatment began when tumorsreached 200 to 300 mm3, with mice receiving treatmentfive times per week. Data are presented as both absolutetumorvolumeand inawaterfall plot that displaymaximalpercentage of change in volume for each tumor followingtreatment (Fig. 3C and D). For the waterfall plots, tumorsthat partially responded to treatment are shown as great-est percentage of loss in tumor volume. Tumors that didnot respond to treatment are shownas greatest percentageof gain in tumor volume during treatment. As predicted,treatmentwith PD325901delayed tumor growth inmajor-

ity of the mice, with a PR rate of 75% (Fig. 3C). Among thetumors that responded to treatment, the degree ofresponse varied, which has been observed in other pri-mary sarcoma models (20, 32). The range of treatmentresponse for individual tumors during PD325901 expo-sure is similar to the individual tumor-to-tumor hetero-geneity observed in Ki67 and CD31 staining (Fig. 2).Immunohistochemical analysis reveals that tumorsresponding to treatment have lower levels of MAPK andmTOR signaling compared with vehicle-treated tumors,as determined by a decrease in pERK and pS6 levels (Fig.3E). In contrast, tumors that did not respond to treatmentshowed hyperelevated levels of both pERK and pS6,suggesting that these tumors escaped from treatment byalternative activation of these pathways. These resultsshow that MEK inhibition slows growth of NF1-deleted

pERK

pS6

Vehicle Responder Nonresponder

A B

E

DC

0 5 10 15 20 25 300

1

2

3

4

Fold

ch

ange in v

olu

me

Co

lon

yfo

rmin

g u

nits

400

300

200

100

0

DMSO DMSO PD325901PD325901

1863 3017

P = 0.019 P = 0.0124

PD325901Vehicle

p-ERK

Total ERK

100

80

60

40

20

0

–20

–40

–60

–80

–100

Perc

enta

ge

maxim

al change

0 1 4

Hours after PD325901

Days of treatment Vehicle PD325901

Figure 3. Activity of a MEK inhibitorin NF1-deleted sarcomas. A, NF1-deleted cell lines (1863 and 3017)were exposed to either DMSO-alone or the MEK inhibitorPD325901 (50 nmol/L) for 7 days inculture. The number of coloniesformed after 7 days wasdetermined by crystal violetstaining. B, Western blot analysisshowing inhibition of pERK levelsfollowing 4-hour exposure of 1863cells to MEK inhibitor PD325901(50 nmol/L). C, primary high-grademyogenic sarcomas generatedfrom intramuscular injection of Ad-Cre into NF1flox/flox; Ink4a/Arfflox/flox

mice were treated with vehicle-alone or PD325901. Exposure toPD325901 slowed tumor growth inmajority of tumors, as shown bythe fold change in tumor volumefollowing initiation of treatment. D,a waterfall plot shows the absolutechange in tumor volume for primaryNF1-deleted myogenic sarcomasfollowing vehicle or PD325901treatment. For tumors that showeda PR to treatment, the maximalpercentage of loss in tumor volumeis reported. For tumors that did notrespond to treatment, themaximal percentage of gain intumor volume is reported. E,immunohistochemistry of pERKandpS6 levels in primarymyogenicsarcomas that received vehicle-alone or PD325901. Tumors thatresponded to treatment show adecrease in pERK and pS6 levelscompared with vehicle-alonetumors, whereas tumors that didnot respond to treatment show acorresponding upregulation inboth pERK and pS6 levels.






rhabdomyosarcoma and UPS, as the histologies for eachof these high-grade sarcoma subtype were evenly distrib-uted between vehicle-treated, responders, and nonre-sponding tumors.

To determine the mechanism of tumor control pro-duced by MEK inhibition, we examined the Ki67 indexbetween vehicle and PD325901-treated tumors (Fig. 4A).Tumors that responded to treatment show a decrease inproliferative index determined byKi67 staining (P < 0.05).Of note, the two nonresponding tumors have Ki67 levelssimilar to vehicle-treated tumors, despite exposure to theinhibitor. TUNEL staining shows that PD325901 treat-ment did not significantly increase apoptosis, althoughnonresponders paradoxically show a high number ofTUNEL-positive (TUNELþ) cells (Fig. 4B). Tumors thatresponded to PD325901 treatment exhibit a significantdecrease in levels of cyclin D1 mRNA (P < 0.05), support-ing previous reports that suggest inhibition of the cellcycle contributes to the decrease in tumor cell prolifera-tion observed with this agent (ref. 33; Fig. 4C). Similardownregulation of cyclinD1mRNA is observed during invitro exposure of NF1-deleted sarcoma cells to PD325901,

showing that the cell-cycle effects are cell autonomous(Fig. 4D and Supplementary Fig. S5). Taken together,these data suggest that PD325901 inhibits NF1-deletedsarcomas through cytostatic, but not cytotoxic, mechan-isms. Very recently, others have used PD325901 to treatneurofibromatosis-associated diseases (34, 35), and treat-ment of a primary neurofibroma model with PD325901resulted in decreased proliferation as measured by Ki67staining, but not an increase in apoptosis (36). However, itis important to note that both the cellular context of thetreated cancer and the specific target that is inhibiteddetermine the effect of an agent to work in cytotoxic orcytostatic contexts.

MEK inhibition alters the tumor microenvironmentThe effect of targeted therapies on the surrounding

tumor stroma is an important component of therapeuticefficacy. Recently, the MEK inhibitor PD325901 wasshown to inhibit angiogenesis and VEGF signaling inxenografted tumor models of renal cell cancer (RCC) andlung cancer (37, 38). Although these xenograft modelsprovide valuable information about tumor response, we

A

B

Vehicle PD325901

Vehicle PD325901

1863 3017

Vehicle VehiclePD325901 PD3259010.0 0.0

0.5

0.51.0

1.01.5

2.0

CTumors

*

* *

Vehicle PD325901 NR0

10

20

30

40

50*

Vehicle PD325901 NR0

10

20

30

40

Cyclin

D1 m

RN

A

Cyclin

D1 m

RN

A

TUNEL+ cells

Ki67+ cells

Nonresponder

Vehicle PD325901 Nonresponder

D Cell lines

Pe

rce

nta

ge

of

Ki6

7+ n

ucl

ei

Perc

en

tag

e o

f TU

NE

L+ n

ucl

ei

Figure 4. NF1-deficient sarcomasrespond to MEK inhibition bycytostatic mechanisms. A, Ki67þ

proliferative index is lower intumors responding to PD325901treatment than in vehicle-alone–treated tumors (�,P <0.05). Tumorsthat did not respond to treatment(nonresponders, NR) show Ki67þ

levels similar to vehicle-alone–treated tumors. Quantificationrepresents the percentage of Ki67þ

cells normalized to total nuclei per40� field, with 6 fields counted pertumor. B, TUNEL stainingdetermined that the number ofapoptotic nuclei is not statisticallydifferent between vehicle-aloneand PD325901-treated tumors.Tumors that did not respond totreatment (nonresponders) showan upregulation in TUNELþ cells.Quantification represents thepercentage of TUNELþ cellsnormalized to total nuclei per40� field, with 6 fields countedper tumor. C, qRT-PCR showsdownregulation of cyclin D1mRNA in NF1-deleted myogenicsarcomas responding toPD325901 treatment (n ¼ 4) incomparison with vehicle-alone–treated tumors (n ¼ 4; �, P < 0.05).D, qRT-PCR showsdownregulation of cyclin D1mRNAin sarcoma cell lines following a4-hour treatment with 50 nmol/Lof PD325901 (�, P < 0.05). Datarepresent an average of fourindependent experiments.

Dodd et al.





sought to extend these findings to our NF1-deleted sar-coma model to determine the effect of MEK inhibition onthe tumor stroma of primary immunocompetent tumorswith native vasculature. First, we examined the effect ofMEK inhibition onmast cell infiltration. Elevated levels ofmast cells correlate with tumor aggressiveness in NF1-mutant neurofibromas (22). The role of mast cells follow-ing targeted therapy has not been examined in a primarysarcoma model, and reports of their prognostic or sup-portive role in other cancer types are conflicting (39–42). Inour NF1-deleted sarcomas, toluidine blue staining showsthat tumors treated with either vehicle alone or PD325901have similar levels of mast cell infiltration (Fig. 5A). Thus,mast cell levels were not altered byMEK inhibition in thismodel.In contrast, MEK inhibition results in a significant

decrease in CD31þ microvessel density (P ¼ 0.010; Fig.5B). As similar effects on the endothelium have beenobserved with MEK inhibition in a primary model ofneurofibroma (36),we further characterized themolecularreprogramming responsible for vascular changes in ourprimary high-grade sarcomas. To determine the changesin several angiogenic markers that may correlate with thedecreased endothelial cell content in the tumors, we con-ducted qRT-PCR on vehicle- and PD325901-treatedtumors. Expression of themRNA for the VEGFa isoformsVEGF 120 andVEGF 165was decreased in treated tumors,whereas message levels for the VEGF 188 isoform werenot significantly altered (Fig. 5C). Levels ofAngiopoietin 2(Angpt 2) mRNAwere lower in treated tumors, but levelsof other endothelial cell markers were unaffected, includ-ing Angiopoietin 1. In addition, mRNA levels of otherangiogenesis-related genes were unchanged, includingTie2, VegfR-1, VegfR-2, Pdgf-b, PdgfR, Neuropilin, Hif1a,and Hif2a (Supplementary Fig. S6). Angpt 2 can haveproangiogenic properties when combined with increasedVEGFa expression (43). Therefore, decreased levels ofAngpt2, VEGF 120, and VEGF 165 mRNA suggest apotential mechanism for the decreased vascularity seenwith PD325901 exposure. To determine whether thechange in angiogenic genes following PD325901 treat-ment was primarily through action on the tumor cells oron tumor stroma, we treated NF1-deficient sarcoma cellswith PD325901. Following a 4-hour treatment with theinhibitor, both cell lines exhibited decreased levels ofVEGF 120 and VEGF 165 mRNA, similar to the primarytumors (Fig. 5C). However, levels of Angpt 2 remainunchanged in the PD325901-treated tumor cells. Thesedata suggest that some of the decrease in VEGF mRNAlevels following PD325901 treatment occurs in the sarco-ma cells, but the changes in Angpt 2 levels observed inintact tumors reflect changes in the surrounding tumorstroma, perhaps in response to altered VEGF levels in thetumor cells (44).To determine whether the decreased levels in VEGF

120, VEGF 165, andAngpt 2mRNA in the primary tumorscorrespond to decreased VEGFa signaling in vivo, weconducted immunofluoresence for pVEGFR-2 in the pri-

mary tumors treated with PD325901 (Fig. 5D). UponVEGF ligand binding, the VEGFR-2 receptor on endothe-lial cells is phosphorylated, activating the VEGF angio-genesis signaling cascade (45). In vehicle-treated tumors,pVEGFR-2 foci are abundant and colocalize with endo-thelial cells, visualized by MECA-32 staining. In contrast,PD325901-treated tumors show marked decrease inpVEGFR-2 foci, alongwith decay of endothelial cell archi-tecture. These data suggest that in addition to decreasingproliferation of tumor cells,MEK inhibition has importanteffects on the surrounding tumor microenvironment byblocking angiogenesis and decreasing tumor microvesseldensity. Taken together, these preclinical data suggestthat theMEK inhibitor PD325901may be a useful therapyfor patients with NF1-deficient sarcomas.

DiscussionWe have described a novel genetically engineered

inducible mouse model of NF1-deficient sarcomas. Thisprimary mouse model can be used to study the tumorbiology and therapeutic response of sarcomas with NF1mutation. Using temporally and spatially controlled con-ditional gene deletion, we have determined that inactiva-tion ofNF1 and Ink4a/Arf in the tumor cells is sufficient toinitiate both myogenic andMPNST-like sarcomas in micewith a wild-type stroma. High-grade myogenic sarcomasdevelop after intramuscular injection of Ad-Cre, andMPNST-like sarcomas develop after sciatic nerve injec-tion. Histopathologic analysis shows that the spectrum ofsarcomas that develop in this model are similar to sarco-mas with NF1 deletion in the patient population. Byinitiating sarcomas through deletion of NF1 and Ink4a/Arf, these mice model the approximately 50% of NF1-deleted sarcomaswithwild-type p53 and complement theexisting models of p53-deleted NF1-mutant sarcomas fortherapeutic studies (21). In this model, tumor growth isdelayed by theMEK inhibitor PD325901, which is accom-panied by a decrease in tumor cell proliferation but doesnot increase cellular apoptosis. Importantly, in this modeltumors develop within their native microenvironment,facilitating the study of stromal contribution to therapeu-tic response, which may be more challenging in celltransplant or xenograft studies. PD325901 treatmentdecreases microvessel density, at least partially througha decrease in expression of proangiogenic genes, such asVEGF 120, VEGF 165, and Angpt 2. Therefore, this studyshows the ability of this system to model NF1-deletedsarcomas and shows its use in characterizing targetedtherapies forNF1-deficient sarcomas. Several recent stud-ies have also reported encouraging results using MEKinhibition to treat other neurofibromin-deficient disordersin mice (34–36).

To our knowledge, this is the first study to examine thebiology of NF1-deleted high-grademyogenic sarcomas ingenetically engineered mouse models, despite the pre-valence of these tumors in the general population andin patients with NF1. Histologic analysis shows thatneither tumor location nor sarcoma subtype influences






the average level of mast cell infiltration or the extent ofvascularization. However, these properties are fairly het-erogeneous between individual tumors, despite the fact

that they all have identical initiating mutations and rep-resent similar sarcoma subtypes. We observed a differ-ence in Ki67 proliferative index between intramuscular

Vehicle

Vehicle

PD325901

600

400

200

1.5

1.0

0.5

0.0

3

2

1

0

0

PD325901 NR

Vehicle

VEGF isoformsVEGF isoforms

Nuclei pVEGFR-2 MergeMECA-32

Cell lines

**

** **

*

*

Tumors

120 165 188 Angpt 1 Angpt 2120 165 188 Angpt 1 Angpt 2

PD325901

Vehic

leP

D325901

NR

50

40

30

20

10

0Tolu

idin

e b

lue c

ells

per

HP

FN

um

ber

of ve

ssels

Rela

tive m

RN

A

Rela

tive m

RN

A

Vehicle PD325901P < 0.01

Mast cellsA

B

C

E

D

CD31+ cells

Figure5. PD325901alters vessel number andexpressionof angiogenicgenes. A,mast cells, visualizedby toluidine blue, infiltrate both vehicle- andPD325901-treated NF1-deleted sarcomas at similar levels. Quantification represents the average number of cells per 20� field, with 10 fields counted per tumor.B, microvessel density, visualized by CD31þ cells, is significantly decreased in tumors responding to PD325901 treatment (P < 0.010). Quantificationrepresents the average number of cells per 20� field, with 6 fields counted per tumor. C, qRT-PCR for angiogenic genes in primary NF1-deleted myogenicsarcomas treated with vehicle alone (black bars) or PD325901 (shaded bars). Data represent an average of four tumors per group. Levels of VEGF 120,VEGF 165, and Angpt2 mRNA were significantly decreased in tumors responding to PD325901 treatment. D, qRT-PCR for angiogenic genes in sarcoma celllines (1863-black, 3017-red) following a 4-hour treatment with either vehicle alone (solid bars) or 50 nmol/L of PD325901 (shaded bars). Data represent anaverage of four independent experiments. Levels of VEGF isoforms [120 and 165 (1863 and 3017), and 188 (3017 only)], but not Angpt2 mRNA, weresignificantly decreased in cells exposed to PD325901. E, coimmunofluoresence for pVEGFR-2–positive endothelial cells in NF1-deletedmyogenic sarcomas.In vehicle-treated tumors, pVEGFR-2 foci (green) are abundant and found in association with multiple endothelial cells (red; MECA-32). However, inPD325901-treated tumors, pVEGFR-2 foci are less abundant and the endothelial cell network is less extensive. NR, nonresponders. �, P < 0.05.

Dodd et al.





and sciatic nerve subtypes, although individual tumorsdisplay abroad range of proliferative capacitywithin eachgroup. This heterogeneity is a salient feature of primarytumor systems (46, 47) that permit modeling of diversebiology andmay reflect differences in additional acquiredsomatic mutations and/or the influence of the complexmicroenvironment.Indeed, the heterogeneity observed across primary

tumors make them excellent models for examining dif-fering tumor responses to therapy, and this model facil-itated examination of tumors that were resistant toPD325901 treatment. Alternatively, these results can beviewed from the perspective that tumors may have suf-ficient phenotypic variation to account for the failure oftreatment in nonresponders. Although this diversity ofresponse could be viewed as a limitation, we view it as astrength of the model. Although we are not able to deter-mine whether these tumors possessed a primary resis-tance to MEK inhibition or acquired resistance rapidlyfollowing the initial treatment, examining tumors that didnot respond to PD325901 provides potential mechanismsto circumvent MEK inhibition. First, the resistant tumorshave hyperelevated Ki67 staining, suggesting they wereable to grow through the treatmentwith increased cellularproliferation—even when accompanied by increased cel-lular apoptosis—resulting in a net gain in tumor growth.Alternatively, the increased apoptotic rate in nonrespond-ing tumorsmaybea reflection of a relatively lowapoptoticrate of the responding tumors at this time point assayed. Itis conceivable that tumors that initially responded totherapies have elevated apoptosis during tumor responsebut have decreased apoptosis during tumor progression.Secondly, although all six tumors that responded to treat-ment showed low pERK and pS6 levels, the two tumorsthat did not respond to PD325901 treatment showed highlevels of pERK and pS6, even above levels observed invehicle-treated tumors. Thus, treatment may have beenineffective due to re-activation of these pathways. Theseresults imply that blocking mTOR in addition to ERKwould be a promising therapeutic strategy for futurestudies. Indeed, others have shown that mTOR inhibitionis effective in slowing growth ofNF1-deficientMPNSTs inmouse models (21, 25, 48). However, we recognize that itcan be challenging to define therapeutic windows forcombination treatment of molecularly targeted inhibitorsin clinical trials. For example, recent reports have shownthat combination of the MEK inhibitor selumetinib withthe AKT inhibitor MK-2206 results in increased toxicitywhen used at single-agent doses (49). Finally, the effect ofPD325901 on the tumor stroma of nonresponders was notwell defined, as changes in the levels of mast cells andmicrovessel density were varied. Although future studieswill be needed to fully explore the mechanisms of resis-tance discussed here, this study shows the use of thismouse model to study the diversity of therapeuticresponses found in NF1-deleted myogenic sarcoma.This study examined the effects of MEK inhibition on

the tumor cells and native surrounding microenviron-

ment in a primary tumor model. Our results support amodel in which PD325901 targets both the tumor cellitself and results in secondary effects on the tumorvasculature. Following MEK inhibition, the sarcomacell decreases both cyclin D1 and VEGFa mRNA levels.Lower cyclin D1 mRNA levels decrease cell prolifera-tion, as shown by lower Ki67 levels. In parallel, lowerVEGFa mRNA levels prevent activation of pVEGFR-2on surrounding endothelial cells, ultimately loweringAngpt 2 mRNA levels in the vasculature. Together withdecreased VEGFa, this results in decreased microvesseldensity. This model is consistent with data from previ-ous in vitro and xenograft studies using MEK inhibitorsin other tumors types (33, 37, 38). Alternatively, it is alsopossible that PD325901 could have direct effects on thetumor endothelial cells, which has been reported in vitro(50, 51).

In summary, we have developed a novel mouse modelof NF1-deleted sarcoma with wild-type p53 status. Thetumor spectrum observed after intramuscular and sciaticnerve injections of Ad-Cre is similar to the clinical spec-trumofNF1-deleted sarcoma.Wehave used thismodel toassess the efficacy of PD325901 and to investigate itsmechanism of action in a primary tumor model. Weanticipate that this model will be useful as a preclinicalplatform for other novel therapies and for further studiesto examine the interplay between the microenvironmentand NF1-deficent sarcoma cells during therapeuticresponse.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R.D. Dodd, J. Barretina, D.G. KirschDevelopment of methodology: R.D. Dodd, J.K. Mito, W.C. EwardAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.D. Dodd, J.K. Mito, W.C. Eward, M. Sachdeva,L. DoddAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): R.D. Dodd, J.K. Mito, R. Chitalia, L. Dodd,D.G. KirschWriting, review, and/or revision of themanuscript:R.D. Dodd, J.K. Mito,W.C. Eward, J. Barretina, L. Dodd, D.G. KirschAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W.C. Eward, R. Chitalia, Y. Ma

AcknowledgmentsThe authors thank Ron Dephino (MD Anderson Cancer Center, Hous-

ton, TX) for providing the Ink4a/Arf flox mice. The authors also thankLaura Jeffords, Rafaela Rodrigues, and Loretta Woodleif for assistancewith mouse work.

Grant SupportThis work was supported by a Children’s Tumor Foundation Young

Investigator Award (to R.D. Dodd), a Liddy Shriver Sarcoma InitiativeGrant (to D.G. Kirsch and R.D. Dodd), and K02-AI093866 (to D.G.Kirsch).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 12, 2013; revised June 27, 2013; accepted July 2, 2013;published OnlineFirst July 15, 2013.






References1. Borden EC, Baker LH, Bell RS, Bramwell V, Demetri GD, Eisenberg BL,

et al. Soft tissue sarcomas of adults: state of the translational science.Clin Cancer Res 2003;9:1941–56.

2. Taylor BS, Barretina J, Maki RG, Antonescu CR, Singer S, Ladanyi M.Advances in sarcoma genomics and new therapeutic targets. Nat RevCancer 2011;11:541–57.

3. Barretina J, Taylor BS, Banerji S, Ramos AH, Lagos-Quintana M,Decarolis PL, et al. Subtype-specific genomic alterations define newtargets for soft-tissue sarcoma therapy. Nat Genet 2010;42:715–21.

4. PaulsonV,ChandlerG, RakhejaD,GalindoRL,WilsonK, Amatruda JF,et al. High-resolution array CGH identifies common mechanisms thatdrive embryonal rhabdomyosarcoma pathogenesis. Genes Chromo-somes Cancer 2011;50:397–408.

5. Evans DG, Baser ME, McGaughran J, Sharif S, Howard E, Moran A.Malignant peripheral nerve sheath tumours in neurofibromatosis 1. JMed Genet 2002;39:311–4.

6. Ferrari A, BisognoG, Macaluso A, CasanovaM, D'Angelo P, Pierani P,et al. Soft-tissue sarcomas in children and adolescents with neurofi-bromatosis type 1. Cancer 2007;109:1406–12.

7. Sorensen SA, Mulvihill JJ, Nielsen A. Long-term follow-up of vonRecklinghausen neurofibromatosis. Survival and malignant neo-plasms. N Engl J Med 1986;314:1010–5.

8. McKeen EA, Bodurtha J, Meadows AT, Douglass EC, Mulvihill JJ.Rhabdomyosarcoma complicating multiple neurofibromatosis. JPediatr 1978;93:992–3.

9. Birindelli S, Perrone F, Oggionni M, Lavarino C, Pasini B, Vergani B,et al. Rb and TP53 pathway alterations in sporadic and NF1-relatedmalignant peripheral nerve sheath tumors. Lab Invest 2001;81:833–44.

10. Wu J, Williams JP, Rizvi TA, Kordich JJ, Witte D, Meijer D, et al.Plexiform and dermal neurofibromas and pigmentation are caused byNf1 loss in desert hedgehog-expressing cells. Cancer Cell2008;13:105–16.

11. Zhu Y, Romero MI, Ghosh P, Ye Z, Charnay P, Rushing EJ, et al.Ablation of NF1 function in neurons induces abnormal development ofcerebral cortex and reactive gliosis in the brain. Genes Dev 2001;15:859–76.

12. Joseph NM, Mosher JT, Buchstaller J, Snider P, McKeever PE, LimM,et al. The loss of Nf1 transiently promotes self-renewal but nottumorigenesis by neural crest stem cells. Cancer Cell 2008;13:129–40.

13. King D, Yang G, Thompson MA, Hiebert SW. Loss of neurofibroma-tosis-1 and p19(ARF) cooperate to induce amultiple tumor phenotype.Oncogene 2002;21:4978–82.

14. KirschDG,DinulescuDM,Miller JB,GrimmJ, SantiagoPM,YoungNP,et al. A spatially and temporally restricted mouse model of soft tissuesarcoma. Nat Med 2007;13:992–7.

15. Cichowski K, Shih TS, Schmitt E, Santiago S, Reilly K,McLaughlinME,et al. Mouse models of tumor development in neurofibromatosis type1. Science 1999;286:2172–6.

16. Vogel KS, Klesse LJ, Velasco-Miguel S, Meyers K, Rushing EJ, ParadaLF. Mouse tumor model for neurofibromatosis type 1. Science1999;286:2176–9.

17. Nishijo K, ChenQR, Zhang L,McCleish AT, Rodriguez A, ChoMJ, et al.Credentialing a preclinical mouse model of alveolar rhabdomyosar-coma. Cancer Res 2009;69:2902–11.

18. Haldar M, Hancock JD, Coffin CM, Lessnick SL, Capecchi MR. Aconditional mouse model of synovial sarcoma: insights into a myo-genic origin. Cancer Cell 2007;11:375–88.

19. DoddRD,Mito J,KirschDG.Animalmodels of soft-tissue sarcoma.DisModel Mech 2010;3:557–66.

20. Kim S, Dodd RD, Mito JK, Ma Y, Kim Y, Riedel RF, et al. Efficacy ofphosphatidylinositol-3 kinase (PI3K) inhibitors in a primary mousemodel of undifferentiated pleomorphic sarcoma (UPS). Sarcoma2012;2012:680708.

21. Johannessen CM, Johnson BW, Williams SM, Chan AW, Reczek EE,Lynch RC, et al. TORC1 is essential for NF1-associated malignancies.Curr Biol 2008;18:56–62.

22. Yang FC, IngramDA, Chen S, Zhu Y, Yuan J, Li X, et al. Nf1-dependenttumors require a microenvironment containing Nf1þ/� and c-kit-dependent bone marrow. Cell 2008;135:437–48.

23. Abraham J, Nelon LD, Kubicek CB, Kilcoyne A, Hampton ST, ZarzabalLA, et al. Preclinical testing of erlotinib in a transgenic alveolar rhab-domyosarcoma mouse model. Sarcoma 2011;2011:130484.

24. WuJ,Dombi E, JousmaE,Scott DunnR, Lindquist D, Schnell BM, et al.Preclincial testing of sorafenib and RAD001 in the Nf(flox/flox);DhhCremouse model of plexiform neurofibroma using magnetic resonanceimaging. Pediatr Blood Cancer 2012;58:173–80.

25. Ghadimi MP, Lopez G, Torres KE, Belousov R, Young ED, Liu J, et al.Targeting the PI3K/mTOR axis, alone and in combination with autop-hagy blockade, for the treatment of malignant peripheral nerve sheathtumors. Mol Cancer Ther 2012;11:1758–69.

26. Aguirre AJ, BardeesyN, SinhaM, Lopez L, TuvesonDA, Horner J, et al.Activated Kras and Ink4a/Arf deficiency cooperate to produce meta-static pancreatic ductal adenocarcinoma. Genes Dev 2003;17:3112–26.

27. Brown AP, Carlson TC, Loi CM, Graziano MJ. Pharmacodynamic andtoxicokinetic evaluation of the novel MEK inhibitor, PD0325901, in therat following oral and intravenous administration. Cancer ChemotherPharmacol 2007;59:671–9.

28. Rubin BP, Nishijo K, Chen HI, Yi X, Schuetze DP, Pal R, et al. Evidencefor an unanticipated relationship between undifferentiated pleomor-phic sarcoma and embryonal rhabdomyosarcoma. Cancer Cell2011;19:177–91.

29. Lau N, FeldkampMM, Roncari L, Loehr AH, Shannon P, Gutmann DH,et al. Loss of neurofibromin is associatedwith activation of RAS/MAPKand PI3-K/AKT signaling in a neurofibromatosis 1 astrocytoma. JNeuropathol Exp Neurol 2000;59:759–67.

30. Gregorian C, Nakashima J, Dry SM, Nghiemphu PL, Smith KB, Ao Y,et al. PTEN dosage is essential for neurofibroma development andmalignant transformation. Proc Natl Acad Sci U S A 2009;106:19479–84.

31. Donhuijsen K, SastryM, Volker B, Leder LD.Mast cell frequency in softtissue tumors. Relation to type and grade of malignancy. Pathol ResPract 1992;188:61–6.

32. De Raedt T, Walton Z, Yecies JL, Li D, Chen Y, Malone CF, et al.Exploiting cancer cell vulnerabilities to develop a combination therapyfor ras-driven tumors. Cancer Cell 2011;20:400–13.

33. Halilovic E, She QB, Ye Q, Pagliarini R, Sellers WR, Solit DB, et al.PIK3CA mutation uncouples tumor growth and cyclin D1 regulationfrom MEK/ERK and mutant KRAS signaling. Cancer Res 2010;70:6804–14.

34. Staser K, Park SJ, Rhodes SD, Zeng Y, He YZ, ShewMA, et al. Normalhematopoiesis and neurofibromin-deficientmyeloproliferative diseaserequire Erk. J Clin Invest 2013;123:329–34.

35. Chang T, Krisman K, Theobald EH, Xu J, Akutagawa J, Lauchle JO,et al. SustainedMEK inhibition abrogatesmyeloproliferative disease inNf1 mutant mice. J Clin Invest 2013;123:335–9.

36. Jessen WJ, Miller SJ, Jousma E, Wu J, Rizvi TA, Brundage ME, et al.MEK inhibition exhibits efficacy in human and mouse neurofibroma-tosis tumors. J Clin Invest 2013;123:340–7.

37. Takahashi O, Komaki R, Smith PD, Jurgensmeier JM, Ryan A,Bekele BN, et al. Combined MEK and VEGFR inhibition in orthotopichuman lung cancer models results in enhanced inhibition of tumorangiogenesis, growth, and metastasis. Clin Cancer Res 2012;18:1641–54.

38. HuangD,DingY, LuoWM,BenderS,QianCN,Kort E, et al. InhibitionofMAPK kinase signaling pathways suppressed renal cell carcinomagrowth and angiogenesis in vivo. Cancer Res 2008;68:81–8.

39. Tanaka T, Ishikawa H. Mast cells and inflammation-associated colo-rectal carcinogenesis. Semin Immunopathol 2013;35:245–54.

40. Xia Q, Wu XJ, Zhou Q, Jing Z, Hou JH, Pan ZZ, et al. No relationshipbetween the distribution of mast cells and the survival of stage IIIBcolon cancer patients. J Transl Med 2011;9:88.

41. Chichlowski M, Westwood GS, Abraham SN, Hale LP. Role of mastcells in inflammatory bowel disease and inflammation-associatedcolorectal neoplasia in IL-10–deficient mice. PLoS ONE 2010;5:e12220.

42. Gulubova M, Vlaykova T. Prognostic significance of mast cellnumber and microvascular density for the survival of patients

Dodd et al.





with primary colorectal cancer. J Gastroenterol Hepatol 2009;24:1265–75.

43. Lobov IB, Brooks PC, Lang RA. Angiopoietin-2 displays VEGF-depen-dent modulation of capillary structure and endothelial cell survival invivo. Proc Natl Acad Sci U S A 2002;99:11205–10.

44. Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J, et al.Angiopoietin-2 differentially regulates angiogenesis through TIE2 andintegrin signaling. J Clin Invest 2012;122:1991–2005.

45. Carmeliet P, Jain RK. Molecular mechanisms and clinical applicationsof angiogenesis. Nature 2011;473:298–307.

46. Chen Z, Cheng K, Walton Z, Wang Y, Ebi H, Shimamura T, et al. Amurine lung cancer co-clinical trial identifies genetic modifiers oftherapeutic response. Nature 2012;483:613–7.

47. Singh M, Lima A, Molina R, Hamilton P, Clermont AC, Devasthali V,et al. Assessing therapeutic responses in Kras mutant cancers usinggenetically engineered mouse models. Nat Biotechnol 2010;28:585–93.

48. Johansson G, Mahller YY, Collins MH, Kim MO, Nobukuni T, Perent-esis J, et al. Effective in vivo targeting of the mammalian target ofrapamycin pathway in malignant peripheral nerve sheath tumors. MolCancer Ther 2008;7:1237–45.

49. Speranza RJK, Khin S, Weil MK, Do KT, Horneffer Y, Juwara L, et al.Pharmacodynamic biomarker-driven trial of MK-2206, and AKT inhib-itor, with AZD6244 (selumetinib), a MEK inhibitor, in patients withadvanced colorectal carcinoma [abstract]. J Clin Oncol 30, 2012(suppl; abstr 3529).

50. Vart RJ, Nikitenko LL, Lagos D, Trotter MW, Cannon M, Bourboulia D,et al. Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6 and G-protein–coupled receptor regulate angiopoietin-2 expressionin lymphatic endothelial cells. Cancer Res 2007;67:4042–51.

51. Mavria G, Vercoulen Y, Yeo M, Paterson H, Karasarides M, Marais R,et al. ERK–MAPK signaling opposes Rho-kinase to promote endothe-lial cell survival and sprouting during angiogenesis. Cancer Cell 2006;9:33–44.






2013;12:1906-1917. Published OnlineFirst July 15, 2013.Mol Cancer Ther Rebecca D. Dodd, Jeffrey K. Mito, William C. Eward, et al. That Respond to MEK InhibitionNF1 Deletion Generates Multiple Subtypes of Soft-Tissue Sarcoma

Updated version

10.1158/1535-7163.MCT-13-0189doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2013/07/15/1535-7163.MCT-13-0189.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/12/9/1906.full#ref-list-1

This article cites 50 articles, 16 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/12/9/1906.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/12/9/1906To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-13-0189

http://mct.aacrjournals.org/content/suppl/2013/07/15/1535-7163.MCT-13-0189.DC1

http://mct.aacrjournals.org/content/12/9/1906.full#ref-list-1

http://mct.aacrjournals.org/content/12/9/1906.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/12/9/1906


nf1 deletion generates multiple subtypes of soft-tissue … · soft-tissue sarcomas are a...

Documents