Ras-Driven Transcriptome Analysis Identiﬁes Aurora Kinase ......Ras-Driven Transcriptome Analysis...

Cancer Therapy: Preclinical

Ras-Driven Transcriptome Analysis Identifies Aurora KinaseA as a Potential Malignant Peripheral Nerve Sheath TumorTherapeutic Target

Ami V. Patel1, David Eaves2, Walter J. Jessen3, Tilat A. Rizvi1, Jeffrey A. Ecsedy5, Mark G. Qian5,Bruce J. Aronow3, John P. Perentesis2, Eduard Serra6, Timothy P. Cripe2,4,Shyra J. Miller1, and Nancy Ratner1

AbstractPurpose: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath

tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or

radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel

therapeutic targets.

Experimental Design: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in

MPNST because of the loss of theNF1 gene, which encodes the RasGAP protein neurofibromin. Therefore,

we created a transgenicmousemodel, CNP-HRas12V, expressing constitutively activeHRas in Schwann cells

and defined a Ras-induced gene expression signature to drive a Bayesian factor regressionmodel analysis of

differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional

significance of Aurora kinase overexpression inMPNST in vitro and in vivo using Aurora kinase short hairpin

RNAs (shRNA) and compounds that inhibit Aurora kinase.

Results:We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were

significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal

nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically

amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked

MPNST cell growth in vitro. Furthermore, anAURKA selective inhibitor,MLN8237, stabilized tumor volume

and significantly increased survival of mice with MPNST xenografts.

Conclusion: Integrative cross-species transcriptome analyses combined with preclinical testing has pro-

vided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora

kinases may be a viable treatment platform for MPNST. Clin Cancer Res; 18(18); 1–11. �2012 AACR.

IntroductionMalignant peripheral nerve sheath tumors (MPNST) are

aggressive soft tissue sarcomas that frequently metastasize

and rarely respond to chemotherapy. Patients with Neuro-fibromatosis type 1 (NF1) inherit mutations in the NF1gene and are predisposed to developing MPNST, identifiedin approximately 10% of NF1 patients. MPNSTs, having a20% to 50% 5-year survival rate, are the major cause ofmortality in adult NF1 patients (1). Approximately 50% ofMPNST cases are sporadic, and some sporadicMPNSTshavemutations in the NF1 gene (2). The most common man-ifestation of NF1 is the development of benign peripheralnerve sheath tumors. Approximately 95% of NF1 patientsharbor smaller benign dermal neurofibromas, and at least30% of NF1 patients develop larger benign plexiform neu-rofibromas, typically associated with deeper nerve trunks. Itis believed that plexiform neurofibromas can transform toMPNSTs (1).

Theprotein encodedby theNF1 gene, neurofibromin, is aRas GTPase activating protein (RasGAP) for all Ras iso-forms, negatively regulating the Ras signal transductionpathway. Ras GAPs accelerate the hydrolysis of activeRas-GTP to inactive Ras-GDP (1). Therefore, having loss offunction mutations in NF1 in NF1-derived MPNSTs (3, 4)

Authors' Affiliations: Divisions of 1Experimental Hematology and CancerBiology, 2Oncology, and 3Biomedical Informatics, Cincinnati Children'sHospital Medical Center, Cincinnati; 4Nationwide Children's Hospital andThe Ohio State University, Columbus, Ohio; 5Millennium Pharmaceuticals,Inc., Cambridge, Massachusetts; and 6Institute of Predictive and Person-alized Medicine of Cancer (IMPPC), Badalona (Barcelona), Spain

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A.V. Patel and D. Eaves contributed equally to this article.

Current address for W.J. Jessen: Biomarker Center of Excellence, Cov-ance, Greenfield, IN 46140.

Corresponding Author: Nancy Ratner, Division of Experimental Hema-tology and Cancer Biology, Children's Hospital Medical Center, 3333Burnet Avenue, M.L.C. 7013, Cincinnati, OH 45229. Phone: 513-636-9469; Fax: 513-636-3549; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1072

�2012 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org OF1

Research. on June 4, 2020. © 2012 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 18, 2012; DOI: 10.1158/1078-0432.CCR-12-1072

http://clincancerres.aacrjournals.org/

have elevated levels of Ras-GTP. Although in benigntumors, prolonged Ras activation can be associated withoncogene-induced senescence, inmalignancy aberrant acti-vation of Ras signaling generally leads to promotion oftumor cell proliferation and/or survival (5). It is unclearwhich Ras isoforms are essential in NF1 tumorigenesis, andthe effects of specific isoforms seem to be cell-type depen-dent (6).

Interestingly, NF1-associated MPNSTs and sporadicMPNSTs show similar pathology and share similar geneexpression signatures, possibly indicating a convergence tosimilar pathways downstream of Ras signaling (7, 8). Mul-tiple effector pathways lie downstream of Ras, many ofwhich have been implicated in NF1 tumorigenesis (1).Neurofibromas andMPNSTs fromNF1 patients andmousemodels with Nf1 mutations have elevated levels of phos-phorylated ERK, the downstream effector of the canonicalRas/Raf/MEK/ERK pathway, and a MEK inhibitor provideda modest reduction in human MPNST cell survival inxenografts (Jessen and colleagues; unpublished data). ThemTOR signaling pathway is also activated in MPNST, butblocking mTOR signaling with rapamycin or its analogsonly transiently delayed tumor growth (9, 10).

MPNSTs have a complex karyotype with amplificationsand deletions ofmany alleles, some of whichmay representtherapeutic targets. Multiple studies have identified indi-vidualmolecular alterations in addition toNF1mutation inMPNST (1). Early alterations in premalignant tumors haveincluded loss of the CDKN2A/B locus, which normallyencodes proteins that negatively regulate the cell cycle(11), and loss of functionmutations in the common tumorsuppressor gene TP53 have been frequently observed (1).

Somatic inactivation of additional tumor suppressor geneshas been used tomodelMPNSTs inmice. Nullmutations inNf1 and p53 in cis produced tumors inmice characteristic ofhuman MPNST (GEM-PNST; refs. 12 and 13). Amplifica-tion and/or overexpression of potential oncogenes, espe-cially those encoding receptor tyrosine kinases, has alsobeen implicated in NF1 tumorigenesis, including PDGFR,CKIT, EGFR (1), and IGF1R (14).

Despite these significant contributions to understand-ing the molecular etiology of NF1, thus far, no chemo-therapeutic approach blocking any molecular target,including growth factor receptors upstream of Ras, Rasitself, Ras downstream effectors, or combinations of tar-gets, has prevented or arrested neurofibroma formationor more than transiently delayed MPNST growth (15).However, a recent combinatorial study including rapa-mycin, an inhibitor of mTOR downstream of Ras, and anHSP90 inhibitor, enhancing proteotoxic stress, showedsynergistic efficacy in the Nf1;p53 cis MPNST mousemodel (16). The results of this study suggest that com-bining a Ras pathway inhibitor with a cytotoxic agent maybe an effective treatment strategy for MPNST, an idea notyet tested in human clinical trials.

As additional candidates for MPNST chemotherapies areneeded, we focused on molecular alterations downstreamof H-Ras activation in Schwann cells, utilizing a Schwanncell–specificH-Ras gene expression signature derived fromanovel transgenic mouse model to identify mechanismscontributing to tumorigenesis and potential therapeutictargets in NF1 tumors. We identify overexpression andamplification of a Ras target gene, AURKA, in MPNSTs.Furthermore, we show that blocking AURKA diminishesMPNST cell growth in vitro and in vivo.

Materials and MethodsCNP-HRas12V expression analysis

The CNP-HRas12V data set consisted of 8 controlnerves encompassing 2 genotypes 4 Nf1 flox/flox; 4P0CreB (controls) and 6 CNP-HRas12V nerves. All sta-tistical comparisons and data visualization were per-formed using GeneSpring GX v7.3.1 (Agilent Technolo-gies). All statistical tests were corrected for multiple test-ing effects by applying the Benjamini and Hochberg falsediscovery rate correction. We used a t test and FDR � 0.01to compare groups.

Bayesian sparse latent class factor modelingGene orthologs of transcripts differentially expressed in

CNP-HRas12V peripheral nerves were used as seeds in aBayesian factor regression modeling analysis. The modelincludes 6 categorical responses: 4 species-specific tumorstates (mouse neurofibroma, mouse MPNST, human neu-rofibroma, human MPNST) and 2 species-independenttumor states (neurofibroma, MPNST). By design, the 6categorical factors are associated with the phenotypic out-come of NF1 tumor progression. Using a threshold of 0.85for factor inclusion probability and 0.95 for variable inclu-sion probability, and constraining such that a minimum of

Translational RelevanceMalignant peripheral nerve sheath tumors (MPNST)

are aggressive sarcomas that arise sporadically or in thecontext of neurofibromatosis type 1 (NF1), especially inNF1 patients with benign plexiform neurofibromas.MPNSTs respond poorly to chemotherapy or radiationand are a major source of mortality in NF1 patients. Noeffective treatments for MPNST are proven, and molec-ular events contributing to malignant transformation ofNF1 tumors are incompletely understood. However,identification of tumorigenic signaling pathways mayfacilitate the development of effective treatments. Usinga combined gene expression database of mouse andhuman neurofibromas andMPNSTs, we identified over-expression of Aurora kinases in MPNSTs. Furthermore,we provide in vitro and in vivo evidence supporting theuse of Aurora kinase inhibitors in MPNST treatmentstrategies. The combination of cross-species transcrip-tome analyses with in vitro and in vivoMPNSTmodels forpreclinical testing provides a powerful translationalpipeline for candidate molecular-targeted therapeuticsinto human clinical trials.

Patel et al.

Clin Cancer Res; 18(18) September 15, 2012 Clinical Cancer ResearchOF2




10 genes are required to exceed the factor threshold toinclude that factor in an expanded model, the analysisterminated with a model on 2,000 gene orthologs distrib-uted across k¼ 100 latent factors and 6 categorical responsefactors.

Gene interaction networkA total of 339 gene orthologs from the 2,000 genes linked

to Ras signaling as determined by Bayesian factor regressionmodeling analysiswere also identified as similarly regulatedtranscripts using frequentist statistics (Supplementary Fig.S1). We used GeneGo’s MetaCore to construct a geneinteraction network using the direct interactions networkbuilding algorithm, which resulted in a single multinodeconnected network of 85 gene orthologs (SupplementaryFig. S2).

AURKA locus copy number analysisFor SNP-array analysis, raw data from Illumina’s

cnv370-duo and h660w-quad SNP array chips were pro-cessed using GenomeStudio (Illumina). B-allele fre-quency and log ratio were calculated using the referencecluster file provided by Illumina. Quantitative PCR anal-ysis of DNA, AURKA gene amplification was determinedby quantitative PCR carried out on a LightCycler 480Instrument (Roche Applied Science), using UniversalProbe Library Technology. A total number of 37 sampleswere analyzed: 13 MPNSTs, 5 MPNST-derived cell lines,8 neurofibromas, and 11 normal samples (which pre-sented a diploid status of AURKA and were used as con-trols). For additional details, see SupplementaryMaterialsand Methods.

Lentiviral short hairpin RNA infectionMPNST cell lines were cultured as described (7, 17). Cells

were evaluated by STR DNA profile analysis of 15 loci plusAmelogenin to exclude cross contamination. For lentiviralshort hairpin RNA (shRNA) infection, MPNST cells at 50%to 60% confluence were infected with lentiviral particlescontaining shRNAs targeting AURKA, AURKB, or shNon-targeting (Sigma-Aldrich; TRC library). The CCHMC ViralVector Core produced virus using a 4-plasmid packagingsystem (http://www.cincinnatichildrens.org/research/div/exp-hematology/translational/vpf/vvc/default.htm). Lenti-viral particles were incubated withMPNST cells (MOI�10)in the presence of polybrene (8 mg/mL; Sigma) for 24 hoursfollowed by selection in 2 mg/mL puromycin.

Quantitative real-time PCR-PCRTotal RNA was isolated from cells using the RNeasy kit

(Qiagen) and used as a template for cDNA synthesis (high-capacity cDNA archive kit; Applied Biosystems) and quan-titative real-time PCR (qRT-PCR; ABI 7500 Sequence Detec-tion System) as described (7). Primers for AurkA wereforward-TAGCCTTCAAGTGCCAGAGTGTGT and reverse-ACAAGACCCTGAATGGGTGTCACT and for AurkB for-ward-TGAGGAGGAAGACAATGTGTGGCA and reverse-AGGTCTCGTTGTGTGATGCACTCT.

Western analysisCell lysates were created andWestern blotting conducted

as described (7), membranes were probed with severaldifferent antibodies including anti-AurkA antibodies (CellSignalling Technology Inc., #3092), anti-AurkB antibody(Cell Signalling Technology, Inc., #3094), anti-histone 3(Cell Signalling Technology, Inc., #9715), anti-p-histone 3(Cell Signalling Technology, Inc., #9701), anti-PARP (CellSignalling Technology Inc., #9542) and then, stripped andreprobed with anti-b-actin antibodies (Cell Signaling Tech-nology, Inc., #4967) as a loading control. Signals weredetected using horseradish peroxidase-conjugated second-ary antibodies (BioRad) and the ECL Plus developing sys-tem (Amersham Biosciences).

In vitro cytotoxicity assayMPNST cell lines as described in ref. 8 were plated in

quadruplicate for each dose of Aurora kinase inhibitor on96-well plates at 500 cells per well in serum-containinggrowth medium. Plates were incubated at 37�C and 5%CO2. Twenty-four hours after plating, cells were treatedwith carrier alone [10% FBS (Hyclone) in DMEM] orinhibitors (LC laboratories). MLN8237 compound wasprovided by Millennium Pharmaceuticals, Inc. Theamount of proliferation was quantified at 2 and 4 daysafter addition of drug by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-razolium, inner salt (MTS) assay using Cell Titer 96Proliferation Kit (Promega). Absorbance was read at490 nm in a Spectramax M2 plate reader (MolecularDevices).

b-Galactosidase stainingCells were washed in PBS, fixed for 3 minutes (room

temperature) in 2% formaldehyde/0.2%glutaraldehyde (or3% formaldehyde), washed, and incubated at 37�C (noCO2) with fresh senescence associated 3-Gal (SA-,3-Gal)stain solution: 1 mg of 5-bromo-4-chloro-3-indolyl P3-D-galactoside (X-Gal) per mL (stock ¼ 20 mg of dimethylfor-mamide permL)/40mmol/L citric acid/sodiumphosphate,pH 6.0/5mmol/L potassium ferrocyanide/5mmol/L potas-sium ferricyanide/150 mmol/L NaCl/2 mmol/L MgCl2.Staining was evident in 12 to 16 hours.

MPNST xenograftThe subcutaneous NF1�/� S462TY human MPNST

xenograft model has been previously described (17).S462TY is a derivative of S462MPNST cells. S462TYMPNSTcells (1.8� 106) were injected into 4- to 5-week-old femalenudemice (Harlan). Administration and dose ofMLN8237was determined by previous animal studies (18). Oralgavage of vehicle constituted of 10% 2-hydroxypropyl-b-cyclodextrin (Fisher Scientific)/1% sodium bicarbonate(Sigma-Aldrich) in sterile water or 20 mg/kg MLN8237 invehicle was given twice daily Monday through Fridayand once daily Saturday and Sunday beginning whentumors reached 250 mm3. We measured tumors andweighed mice twice weekly. Tumor volume was calculated

AURKA Is a Potential Therapeutic Target for MPNST

www.aacrjournals.org Clin Cancer Res; 18(18) September 15, 2012 OF3




by: L � W2(p/6), where L is the longest diameter and W isthe width. Mice were treated until tumors reached 2,500mm3 or a maximum of 66 days. Control tumors (9/11)reached2,500mm3byday 66of treatment. The remaining 2mice had tumors that were >1,800 mm3 and actively grow-ing. Survival was analyzed by log-rank test using GraphPadPrism.

PharmacokineticsBlood was collected by cardiac puncture in nontumor

bearing mice. Blood was drawn at 2, 4, and 16 hours afterdosing from 3 to 5 mice per time point. Plasma wasseparated by centrifugation at 2,200 � g for 10 minutesand MLN8237 concentration quantified by LC/MS/MS(18, 19).

ImmunohistochemistryTeased sciatic nerves from adult wild-type or CNP-

HRAS12v mice were prepared for immune labeling asdescribed (20). Schwann cells were labeled with anti-S100b antibodies (Dako) followed by FITC-conjugatedsecondary antibodies (green); expression of the HA-taggedCNP-HRAS12V transgene was detected with anti-HA anti-bodies (Santa Cruz Biotechnology, Inc.) followed byTRITC-conjugated secondary antibodies (red; Fig. 1A).

Paraffin sections were deparaffinized, hydrated, andtransferred to 0.1 M citrate buffer (pH 6.0) for antigenretrieval. Slideswere boiled for 10�C in citrate buffer, cooledat room temperature for 30 minutes, rinsed in water twice

and in PBS 3 times. Sections were quenched with 3%hydrogen peroxide for 10 minutes, rinsed in PBS, andblocked in 10% normal goat serum with 0.3% Triton-X-100. Sections were incubated overnight in primary anti-body; rabbit cleaved caspase-3 (1:8,000, Cell Signaling,#9661), rabbit p-histone 3 (1:800, Cell Signaling,#9701), rabbit Ki-67 (1:5,000 NCL-Ki67-P; Novocastra),rabbit cyclinB1 (1:500, Cell Signaling, #4138). Sectionswere then incubated in biotinylated goat anti-rabbit sec-ondary antibodies (Vector, at a dilution of 1:200). Sectionswere counterstained with Harris hemotoxylin and coverglassed in histo-mount. For nuclear staining, sections wereincubated with propidium iodide/RNase staining solution(Cell Signaling, #4087) for 15minutes at room temperaturein the dark, rinsed in PBS 3 times and then cover glassedwith Prolong Gold Antifade Reagent (Life Technologies,#P36930). Sections were photographed on the Zeiss LSM510 scanning confocalmicroscope equippedwith an argon-ion multiline, HeNe 543, and HeNe 633 lasers. For nuclearstaining with DAPI (Sigma), sections were incubated inDAPI (1:10,000) for 5 minutes, rinsed in PBS 3 times, andcover glassed in Flouromount G (EM Sciences, #17984-25)mounting medium.

ResultsBayesian factor model analysis and latent structurelinked to Ras signaling

It seemed plausible that specific Ras targets would bereexpressed in the setting of MPNST to overcome

Figure 1. Schwann cell–specific Ras-driven gene expression. A, immunolabeling adult teased nerve fibers detected HRas (as detected by anti-HA, red) at themembrane of myelinated fibers (bottom, white arrow) and smaller unmyelinated fibers (top, white arrow). Schwann cell cytoplasm is counterstainedwith anti-S100 (green),whichpreferentially labelsmyelinatingSchwanncells. B, electronmicrographsof a cross-section throughsaphenousnerves fromadultwild-type or CNP-HRAS12V mice are shown. At high magnification, the unmyelinated axons with accompanying Schwann cell processes in the HRAS12Vmutant showed normal ensheathment but few fibers per bundle. Groups of unmyelinated axons in sapphenous nerve contained diminished numbersof axons in theCNP-HRAS12Vmutant. C, At 1-year old, 13.0�2.2 axonswere groupedby a single nonmyelin formingSchwann cell inwild-typemice, but only6.4� 0.9 in CNP-V12Ras mutants (P� 0.004). Black arrows point to small groups of unmyelinated axons in mutant. The phenotype did not worsen with age(not shown). D, Schwann cell–specific Ras signature in CNP-HRAS12V transgenic mouse model.

Patel et al.





suppressive signaling characteristic of senescence in benigntumors (5). Ras signaling can be cell-type dependent (6),and global transcriptional changes directly downstream ofRas activation specifically in Schwann cells or their precur-sors, the pathogenic cell type(s) in NF1 (21, 22), had notbeen reported previously. Therefore, to focus our analysis,we first identified genes in peripheral nerves associated withRas-GTP expression in Schwann cells.The starting point for our analysis was to generate

a mouse expressing a constitutively active, oncogenicallele of Ras (HRAS12V) in Schwann cells, driven by theCNP promoter (23). The CNP promoter drives expres-sion early in Schwann cell progenitors, and expressionis maintained in mature Schwann cells (23). We choseto express H-Ras (in contrast to K- or N-Ras) as all 3 Rasproteins are expressed by mouse Schwann cells (24)yet the proliferation of Nf1 mutant Schwann cellsin vitro is blocked by farnesyl protein transferase inhibi-tors, which affect H- but not N- or K-Ras (25). Theexpression of the HA-tagged HRAS12V transgene wasvalidated in myelinating and nonmyelinating Schwanncells using an anti-HA antibody by immunolabelingteased sciatic nerve preparations (Fig. 1A). These micewere viable and fertile, lived a normal lifespan, and didnot form neurofibromas or MPNSTs. It remains possiblethat expression of Ras from an endogenous locus wouldcause nerve tumors. No peripheral nerve defects werenoted on gross or pathologic examination. Analysis ofCNP-HRAS12V expressing peripheral nerve by electronmicroscopy shows unmyelinated fiber bundles that areslightly disorganized (Fig. 1B) with a minor decrease inthe number of axons per Remak bundle (Fig. 1C). Thephenotype resembles a milder version of the nerve dis-ruption observed in CNP-EGFR mice (26), and the phe-notype was not enhanced in the Nf1þ/� background(data not shown).We identified 308 genes (154 gene orthologs) differ-

entially expressed between mouse nerves from the CNP-HRas12V and normal control mouse nerves (FDR �0.01; Fig. 1D). We did not observe significant functionalenrichment of genes involved in Ras-MAPK (Raf/MEK/ERK) signaling or senescence after querying the 154orthologs in the DAVID database or the GATACAknowledgebase.Transcriptional changes in CNP-HRas12V nerves com-

pared with wild-type nerves downstream should result,directly or indirectly, from altered Ras signaling in Schwanncells. These Schwann cell–specific Ras pathway gene ortho-logs were used as seeds in a Bayesian factor regressionmodeling analysis of an orthologous transcriptome dataset including mouse and human benign neurofibromasand malignant peripheral nerve sheath tumors (MPNST;ref. 27). The model identified 2,000 genes with probabilityof linkage to Ras signaling (Fig. 2A).To determine which, if any, of the 2,000 gene orthologs

show significant deregulated expression inNF1 samples, wefiltered the 2,000 gene orthologs linked to Ras signalingacross genes upregulated or downregulated in mouse or

human neurofibroma or MPNST relative to respective nor-mal nerve controls. A total of 339 gene orthologs showedincreased or decreased expression in mouse model and

Figure 2. AURKA is overexpressed and amplified in MPNST. A, Bayesianlatent factor regression modeling of microarray gene expression dataidentified 2,000 gene orthologs linked to the Ras pathway in mouse andhumanNF1 tumors. B,AURKA andAURKBwere expressed at high levelsin MPNST cell lines and primary tumors relative to neurofibromas andnormal nerves. Each sample is representedbyavertical line and isdirectlyunder the sample type categories represented in A. Sample typescategories are separated by an open space. The vertical dashed linedividesmouse samples (left) and human samples (right). Expression levelof AURKA (top) or AURKB (bottom) was normalized to mouse or humannormal nerve, respectively, and represented as 1.00 on a log scale. Thered line represents the average expression level among samples withineach sample type (C). Example of SNP-array analysis of an MPNST. Thisfigure shows the logR ratio (LRR) of chromosome 20 for tumor MPNST K(see Supplementary Table S1). A blue line denotes the running average ofLRR values. A value of 0 indicates the presence of 2 copies of each locusanalyzed. A mean value above 0 indicates a copy number gain of thelocus interrogated. A vertical yellow line localizes the AURKA locus.






human neurofibromas or mouse model and humanMPNSTs (Supplementary Fig. S1).

We used the GeneGo MetaCore Systems Biology Ana-lysis Platform MetaCore to explore the connectionsbetween the 339 genes and identified a major network(Supplementary Fig. S2). This network contained the over-expressed AURKA gene and its overexpressed downstreamtargets PLK1, cyclin B1, TPX2, HEC, and TACC3. Upstreamactivators of AURKA, E2F3, and FOXM1, as well as securin(PTTG1), which regulates AURKA phosphorylation ofHistone H3 within pericentromeric heterochromatinearly in the G2 phase (28) and interacts with AURKA toregulate cellular responses to antineoplastic drugs (29),were also overexpressed genes within the network. Anotherprominent theme was decreased expression of PPAR-gand its downstream targets leptin, CD36, cytochrome B,RBP7, MGST, PREG3, and SCD. A decrease in gene copynumber at the PPAR-g locus was not detected by SNP-arrayanalysis (data not shown).

Notably, dramatic overexpression of AURKA (upregu-lated 7.9-fold) was observed in the filtered mouse andhuman MPNST sample data as compared with normalnerve (Fig. 2B) as well as within the Ras-driven latent factorsearch results (Fig. 2A) and K-means clustering of the cross-species NF1 data set (9). Expression of AURKB was mod-erately increased in human MPNST (1.9-fold; Fig. 2B), andAURKC was not differentially expressed in either mouse orhuman tumor samples.

To assess the mechanism underlying high expression ofAURKA, we determined copy number of the AURKA locususing SNP-array and qPCR analyses on DNA from dermalneurofibromas, MPNSTs, and MPNST cell lines (Fig. 2C;Supplementary Fig S3 and Tables S1 and S2). MPNSTsand MPNST cell lines but not neurofibromas showedcopy number gains in the AURKA locus. All (5/5) MPNSTcell lines tested, each previously analyzed by gene expres-sion microarrays, and 3 of 14 (21%) primary MPNSTs(one analyzed by gene expression microarray, MPNST#19) exhibited �3n copies of the AURKA locus. TheSNP-array technique can underestimate gene amplifica-tion in tumor cells (30) because of the presence of normalcells. Therefore, we used qPCR-DNA analysis to detectcopy number gains with more sensitivity. Using thistechnique we confirmed copy number gains in 5/5MPNST cell lines and detected copy number gains in 8of 13 (62%) primary MPNSTs (data not shown). Increasesin copy number were also identified in 1 of 8 neurofi-broma samples, consistent with the low number of struc-tural alterations in the AURKA locus in 19 dermal neu-rofibromas (31). These data suggest that amplification ofAURKA could be an early event in progression to malig-nancy. Indeed, AURKA gene expression was elevated insome human neurofibroma samples (Fig. 2B).

Copy number alteration at the AURKA locus correlatedwith variations in mRNA expression levels across tumorsamples (Fig. 2B and C), indicating that gene amplificationmay contribute to AURKA overexpression in MPNST. Thissupports the idea that increased AURKA expression relative

to AURKB and the other mitotically-regulated genes listedearlier is not simply because of comparing proliferating cellsto nonproliferating normal nerve cells leading to a prolif-erative signature, but that AURKA is distinctly upregulated.

Reducing Aurora kinase gene expression inhibitsMPNST cell survival in vitro

To test whether Aurora kinases play a functional rolein MPNST cells, AURKA or AURKB expression was blockedwith lentiviral shRNAs. Three days after lentiviral infectionof MPNST cells, shAURKA reduced AURKA mRNA expres-sion 3.7-fold relative to a nontargeting shRNA (shNT);shAURKB reduced AURKB mRNA expression 5.8-fold(Fig. 3A). Downregulated expression of each gene wasspecific with respect to the other. Expression of Aurorakinases A and B were confirmed at the protein level byWestern blotting in MPNST cells 3 days after lentiviralshRNA infection (Fig. 3B). Inhibiting either AURKA orAURKB expression with shRNA diminished expression ofthe specific aurora kinase (A or B) proteins, respectively.Knocking down expression of either AURKA or AURKBreduced MPNST cell accumulation relative to an shNTcontrol as analyzed at 5 days after lentiviral infection in 4MPNST cell lines using phase contrast microscopy (Fig. 3C)or an MTS assay (Fig. 3D and Supplementary Fig. S4A).

MLN8237 reduces AURKA activity and MPNST cellsurvival in vitro

These AURK gene expression knock down data suggesteda role for Aurora kinases in MPNST cell survival. To verifythis finding, human MPNST cells were separately treatedwith 3 different Aurora kinase inhibitors. VX-680 andZM447439 have been shown to block Aurora kinases A,B, and C at nanomolar concentrations (32). At concentra-tionsdefined for each inhibitor, bothcompoundseffectivelydecreased cell survival (Supplementary Fig. S4B). In a 4-dayassay, the IC50 values for VX-680 ranged from 40 nmol/L(T265) to 110 nmol/L (26T) (Supplementary Fig. S4B); theIC50 values for ZM447439 ranged from 450 nmol/L (T265)to 2000 nmol/L (S462TY) (Supplementary Fig. S4B). Acompound selectively targeting Aurora kinase A, MLN8237(18), also significantly reduced MPNST cell survival withIC50 values ranging from100 nmol/L (S462) to 210 nmol/L(ST8814) and averaging 164 nmol/L (Fig. 3E). MPNST cellsstained positive for a marker of senescence (b-galactosidasestaining) and displayed enlargement in cell size and flat-tening of the cell body characteristic of senescence at 48hours posttreatment with 100 nmol/L MLN8237, as com-pared with vehicle-treated cells (Fig. 3F). Previous studiesreported an average IC50 of 32 nmol/L for a panel of Ewing’ssarcoma andneuroblastoma cell lines (19). These values arewell below the average trough concentration of 1 mmol/Lreported in human phase I studies using the recommendedphase 2 dose of 50 mg twice daily (33).

Inhibiting Aurora kinase A results in mitotic spindledefects,mitotic delay, and eventually apoptosis (34). There-fore, pharmacodynamic analysis of AurkA inhibitors oftenincludes measuring markers of proliferation and apoptosis,

Patel et al.





including p-histone 3 and PARP, respectively (35). Becausephosphorylation of the serine 10 residue of the N-terminaltail of histone H3 is crucial for chromosome condensationand cell-cycle progression (36), protein levels for p-histone-3 were measured at 24 and 48 hours posttreatment with

MLN8237, in vitro. Although there was no change in totalHistone 3, there was significant reduction in p-histone 3levels at 300 nmol/L and 1 mmol/L treated MPNSTs at bothtime points (Fig. 3G). Similar reduction in p-histone 3 wasobserved for cells treated with lentiviral shRNA against

Figure 3. Blocking AURK geneexpression reduces MPNST cellsurvival in vitro MLN8237 reducesAURKA activity and MPNST cellsurvival in vitro. A, AURKAexpression was diminished byAURKA shRNA; AURKB expressionwas diminished by AURKB shRNA.Blocking each AURK expression atthe RNA level was specific asdetermined by qRT-PCR. B, blockingexpression of either AURK reducedprotein levels of both as detected byWestern blot. C, phase contrastimages of MPNST cells showed adramatic reduction in cell numberafter treating with shAURKA orshAURKB. D, quantification of cellaccumulation by MTS assay showeddecreased survival in response toshAURKA and shAURKB. E,MLN8237 Aurora kinase A inhibitorreduced survival of all 5 MPNST celllines tested at an average IC50 of164 nm. F, MPNST cells becamesenescent 48 hours posttreatmentwith MLN8237 as compared withvehicle-treated cells as seen in thesephase contract images of cellsstained for b-galactosidase (b-gal)staining. G, protein levels for AurkA,p-histone 3, total Histone 3, PARP,and loading control b-actin forMPNST cells (S462TY) treated with100 nmol/L, 300 nmol/L, and1 mmol/L of MLN8237 collected at24 and 48 hours posttreatment withdrug. Untreated and vehicle treatedwere used as a negative control andshAURKA-treated MPNST cellscollected at 4 days post virustreatment serve as a positive control.






AURKA (Fig. 3G), indicating a decrease in mitotic cells. Incontrast, there was no measurable increase in cell death ateither time point, as indicated by an absence of cleavedPARP, whichwas clearly visible after staurosporin exposure.

Cleaved caspase-3 was also not detected (data not shown).Posttreatment with MLN8237, AurkA protein levelsincreased as compared with vehicle-treated MPNST celldoses over 300 nmol/L at 24 hours posttreatment and forall the doses at the 48 hours posttreatment time point.Increase in AurkA protein is likely because of the accumu-lation of cells in the G2–M phase posttreatment with thedrug (Fig. 3G).

MLN8237 inhibits MPNST cell growth in vivoTo test the effect of Aurora kinase inhibition on MPNST

cells in vivo, S462TY MPNST cells derived from an NF1patient were implanted into nu/nu mice and treated withMLN8237 (n¼ 11) or vehicle control (n¼11), at 20mg/kg/dose, a dose 50% of the MTD in most mouse strains.Inhibiting Aurora kinase withMLN8237 diminished tumorvolume (Fig. 4A–C) and increased mouse survival (P ¼0.0005; Fig. 4D). Comparing vehicle treated (Fig. 4A) versusMLN8237 (Fig. 4B) shows that MLN8237 had a profoundeffect on 10/11 tumors. Notably only one of 11mice treatedwith MLN8237 required sacrifice because of tumor burdenwithin a prolonged (66 day) treatment period (Fig. 4B). Onaverage, MLN8237 showed effects starting at 15 to 20 daysafter drug administration (Fig. 4C).

To confirm MLN8237 concentrations that inhibitedMPNST xenograft growth were clinically relevant, pharma-cokinetic analysis of MLN8237 was conducted (Fig. 4E).Mice were administered MLN8237 at 20 mg/kg twice dailyfor 5 days and blood plasma was collected at 2, 4, and 16hours after dosing.Cmax was reached by 4 hours andmeasur-ed 2.9 mmol/L; blood plasma levels reached 2.77 by 2 hours.The blood plasma levels measured at 2 and 4 hours are wellabove the 1 mmol/L dosage necessary to successfully inhibitAurora kinase A in vivo, as previously reported (18, 19).

MLN8237 induces cytomegaly in MPNST xenograftsTo define cellular processes that may contribute to

MLN8237-induced tumor stasis, we examined cell-cyclemarkers using immunohistochemistry on paraffin sectionsfromMPNST xenografts. Therewas amarked decrease in thenumber of cells positively labeled for Ki67 in theMLN8237-treated tissue as compared with vehicle-treated tissue sam-ples (Fig. 5A), indicating a reduction in actively proliferatingcells. No difference was observed in the number of cellslabeled for cleaved caspase-3 in vehicle versus MLN8237-treated xenografts (Fig. 5A), indicating no effect on apo-ptosis because of MLN8237. In spite of a decrease in Ki67,there was no change in percent of cells immunolabeled forphospho-histone-3 in the vehicle versus MLN8237-treatedsamples, indicating possible arrest in the G2–Mphase of thecell cycle.CyclinB1wasused as amarker ofG2–M. Therewasan increase in cyclinB1 in the nucleus and cytoplasmof cellsfrom MLN8237-treated xenograft samples as comparedwith vehicle, supporting the idea that the AURKA inhibitorcaused tumor cells to stall in G2–M. Consistent with G2–Marrest, xenografts treated with MLN8327 contained numer-ous enlarged cells with multiple nuclei, as observed withpropidium iodide (Fig. 5B). Quantification of this effect

Figure 4. MLN8237 reduces tumor volume and increases survival of micewith MPNST xenografts. A–C, inhibiting Aurora kinase A diminishedtumor volume. D, Kaplan–Meier curve shows increased survival of micewith MPNST xenografts in response to MLN8237 (P ¼ 0.0005); mediansurvival for vehicle-treated mice was 41 days; median survival forMLN8237-treated mice was undefined. E, pharmacokinetic analysis ofMLN8237 in MPNST xenografts. Average blood plasma concentrationsof MLN8237 (nmol/L) in 3 control nude mice at 2, 4, and 16 hours afterdosing are plotted on a log scale. Dashed line represents dose (1 mmol/L)required for Aurora kinase inhibition in vivo.

Patel et al.





indicated that 4 or 16 hours after the last dose ofMLN8237,thepopulationof xenograft tumor cellswithmultiple nucleiwas significantly increased (Fig. 5B). As Aurora kinase A is amitotic kinase that regulates mitotic spindle formation andsegregation, the enlarged multinucleated cells indicatearrest in G2–M phase.

DiscussionThe purpose of this study was to identify molecular

alterations downstream of Ras signaling in Schwann cells

relevant to NF1 tumorogenesis. We used a Schwann cell–specific Ras signature to drive Bayesian factor analysis, andgene expression data derived from human NF1 tumors andmouse Nf1 GEM models to identify Ras target genes thatmay lead to tumor progression. Gene amplification of thedownstream MAPK1/ERK2 target AURKA resulted inAURKA overexpression in MPNST. MPNST cells weredependent on AURKA overexpression for survival, as inhi-bition of AURKA reduced cell survival in vitro and causedtumor stasis in vivo. The cumulative results of these experi-ments suggest thatAURKA amplification contributes toNF1mutant Schwann cell progression to malignancy. Our pre-clinical data support advancement of the AURKA-specificinhibitor, MLN8237, as a molecular-targeted clinical can-didate for the treatment of MPNST. Finally, our approachsupports investigation of additional overexpressed Ras tar-gets in NF1 tumorigenesis.

We identified 339 potential Ras target genes differentiallyexpressed in mouse and human neurofibromas and/orMPNSTs relative to normal nerve. We hypothesized thatexpression of some or all of these specific Ras target genesmight enhance the effects of the already activated Raspathway, promoting tumorigenesis. Validating this idea,AURKA, a known transcriptional target of Ras-MAPK sig-naling (37), was identified by Bayesian analysis as aSchwann cell H-Ras target overexpressed in mouse andhuman MPNSTs but to a much lesser extent inneurofibromas.

Notably, AURKA can potentiate HRAS-mediated trans-formation correlating with increased phosphorylation ofMEK and ERK (38, 39). Although the exact mechanism bywhichAURKAandRas pathways cooperate is unknown, it isintriguing that Aurora kinases can directly interact withRasGAP, which is, like neurofibromin, an off signal for Rasproteins (40, 41). AURK genes encode Aurora kinases thatregulate mitosis and promote cell-cycle progression (42).AURKA, AURKB, and AURKC have each been implicatedas oncogenes in multiple types of cancer (42), and AURKAwas robustly overexpressed in our mouse and humanMPNST data sets.

AURKA gene amplification provides a mechanism toincrease AURKA expression, and AURKA amplification isoften observed in cancer (42). The level of AURKAmRNAoverexpression (10-fold) in MPNST is greater than thecopy number gains (2- to 5-fold) detected at the AURKAlocus, suggesting that additional mechanisms, perhapsincluding Ras/Raf/MEK/ERK signaling and/or the over-expression of AURKA upstream activators, contribute toelevated AURKA expression in MPNST. In some studies,AURKA gene amplification showed an inverse relation-ship with response to MLN8237 (43). In contrast,MLN8237 exhibited significant antitumor activity in ourNF1�/� MPNST model with AURKA amplification. Thismodel differed from those previously reported, whichshowed increased phosphor-histone3 in response toMLN8237 and lacked cytomegaly (43), and is similar tothat of Huck and colleagues, which shows senescence inresponse to MLN8237 (44).

Figure 5. MLN8237 induces cytomegaly inMPNST xenografts. A,MPNSTxenografts treatedwith vehicle control orMLN8237andassayed forKi67,a marker of cell proliferation, p-histone 3, a marker of proliferation,cleaved caspase-3, amarker for apoptosis, or cyclinB1, amarker of cell inthe G2–M phase of the cell cycle. Vehicle and MLN8237-treated miceshowed similar levels of p-histone 3 and cleaved caspase-3. Noticeablyfewer cells were positively labeled for Ki67 in theMLN8237-treated tissuesamples as compared with vehicle. There was a marked increase in thepositive staining for cyclin B1 in the MLN8237-treated tissue samplescompared with vehicle-treated samples. B, many multinucleated cellswere detected in MLN8237-treated xenografts, as visualized withpropidium iodide and quantified by counting DAPI-stained nuclei per cellat 4 and 16 hours.






Aurora kinase inhibitors, like many antimitotic com-pounds, kill a variety of tumor cells, including some typesof sarcomas (45, 46). MLN8237 has successfully treatedleukemias that become resistant to the tyrosine kinaseinhibitor, nilotinib (47) and enhanced cisplatin-inducedcell death in esophageal cancer xenografts (43) andmedulloblastoma cell lines (48). However, cells canbecome resistant to Aurora kinase inhibitors (45). Ourpreclinical studies using Aurora kinase inhibitors todiminish MPNST cell growth in vitro and in vivo provideevidence supporting investigation into effectiveness ofAurora kinase inhibitors in NF1 treatment. However,Aurora kinase inhibitors had a static effect on MPNST invivo, and the addition of cytotoxic agents may be required.Importantly, clinical trials are ongoing using Aurorakinase inhibitors in the treatment of solid tumors (42).As Ras-driven tumors are often nonresponsive to conven-tional chemotherapeutics, the development of combina-torial therapeutic regimens is essential (16). CombiningAurora kinase inhibitors with other therapeutics, includ-ing MEK inhibitors or chemo-sensitizing agents, may beuseful. The predominant dose-limiting toxicities associ-ated with MLN8237 in 2 separate phase 1 studies inpatients with advanced solid cancers were myelosuppres-sion and gastrointestinal-associated toxicities includingstomatitis (46). Therefore, careful consideration of doseand schedule will be needed when combining MLN8237with other therapeutic agents that cause similar adverseevents to avoid overlapping toxicities. Given the cell-cyclearrest in G2–M phase, compounds that target the G2

checkpoint may be attractive candidates.In summary, our results support a molecular model in

which amplification of AURKA drives cell-cycle progressionin NF1 mutant Schwann cells, promoting malignant trans-

formation. Several lines of evidence make AURKA andattractive candidate for molecular-targeted MPNST thera-peutics. AURKA expression is downstream of Ras signalingandAURKA and several of its substrates are overexpressed inMPNST. Furthermore, several Aurora kinase inhibitors haveshown efficacy in preclinical studies and clinical trials for avariety of tumors. The recent development and assessmentof the AURKA-specific inhibitor, MLN8237, is highly effec-tive against leukemia and solid tumors in a panel of pre-clinical models of pediatric cancers and has proceeded toclinical trials (19, 49). In our preclinical model, MLN8237increases survival of mice with MPNST xenografts. Theresults of this study support further investigation of Aurorakinase inhibitors, alone or in combination, in the treatmentof MPNST.

Disclosure of Potential Conflicts of InterestJ.A. Ecsedy andM.G.Qian are employees ofMillenniumPharmaceuticals,

Inc. No potential conflicts of interest were disclosed by the other authors.

AcknowledgmentsThe authors thank Robert Hennigan for assistance with microscopy. The

authors also thank Jianqiang Wu and Edwin Jousma for assisting withadministration of MLN8237 to mice and Michelle Meadows for assistancewith MPNST cell culture.

Grant SupportW. J. Jessen was supported in part by an ARRA supplement to NIH grant

R01-NS28840 (N. Ratner). The authors gratefully acknowledge support fromthe DAMD Program on Neurofibromatosis for the NF1 Microarray Consor-tium DODW81XWH-09-1-0135.

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 April 11, 2012; revised June 7, 2012; accepted June 11, 2012;published OnlineFirst July 18, 2012.

References1. 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.

2. Bottillo I, Ahlquist T, Brekke H, Danielsen SA, van den Berg E, MertensF, et al. Germline and somatic NF1 mutations in sporadic and NF1-associated malignant peripheral nerve sheath tumours. J Pathol2009;217:693–701.

3. Basu TN,GutmannDH, Fletcher JA, Glover TW, Collins FS, DownwardJ. Aberrant regulation of ras proteins in malignant tumour cells fromtype 1 neurofibromatosis patients. Nature 1992;356:663–4.

4. DeClue JE, Papageorge AG, Fletcher JA, Diehl SR, Ratner N, VassWC,et al. Abnormal regulation of mammalian p21ras contributes to malig-nant tumor growth in von Recklinghausen (Type 1) neurofibromatosis.Cell 1992;69:265–73.

5. Courtois-Cox S, Genther Williams SM, Reczek EE, Johnson BW,McGillicuddy LT, Johannessen CM, et al. A negative feedback sig-naling network underlies oncogene-induced senescence. Cancer Cell2006;10:459–72.

6. Carroll SL, Ratner N. How does the Schwann cell lineage form tumorsin NF1? Glia 2008;56:1590–605.

7. Miller SJ, Rangwala F, Williams J, Ackerman P, Kong S, Jegga AG,et al. Large-scale molecular comparison of human schwann cells tomalignant peripheral nerve sheath tumor cell lines and tissues. CancerRes 2006;66:2584–91.

8. WatsonMA,Perry A, TihanT,PraysonRA,GuhaA,Bridge J, et al. Geneexpression profiling reveals unique molecular subtypes of Neurofibro-matosis Type I-associated and sporadic malignant peripheral nervesheath tumors. Brain Pathol 2004;14:297–303.

9. 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.

10. 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.

11. Beert E,BremsH,Daniels B,DeWever I, VanCalenbergh F,SchoenaersJ, et al. Atypical neurofibromas in neurofibromatosis type 1 are prema-lignant tumors. Genes Chromosomes Cancer 2011;50:1021–32.

12. 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.

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

14. YangJ,YlipaaA,SunY,ZhengH,ChenK,NykterM, et al.Genomic andmolecular characterization of malignant peripheral nerve sheath tumoridentifies the IGF1R pathway as a primary target for treatment. ClinCancer Res 2011;17:7563–73.

Patel et al.





15. Kalamarides M, Acosta MT, Babovic-Vuksanovic D, Carpen O,Cichowski K, Gareth Evans D, et al. Neurofibromatosis 2011: a reportof the Children's Tumor Foundation Annual Meeting. Acta Neuro-pathol. 2011;123:369–80.

16. 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.

17. Frahm S, Mautner VF, Brems H, Legius E, Debiec-Rychter M,Friedrich RE, et al. Genetic and phenotypic characterization oftumor cells derived from malignant peripheral nerve sheathtumors of neurofibromatosis type 1 patients. Neurobiol Dis 2004;1:85–91.

18. Manfredi MG, Ecsedy JA, Chakravarty A, Silverman L, Zhang M,Hoar KM, et al. Characterization of Alisertib (MLN8237), an inves-tigational small-molecule inhibitor of aurora A kinase usingnovel in vivo pharmacodynamic assays. Clin Cancer Res 2011;17:7614–24.

19. Carol H, Boehm I, Reynolds CP, Kang MH, Maris JM, Morton CL,et al. Efficacy and pharmacokinetic/pharmacodynamic evalua-tion of the Aurora kinase A inhibitor MLN8237 against preclinicalmodels of pediatric cancer. Cancer Chemother Pharmacol 2011;68:1291–304.

20. Daston MM, Scrable H, Nordlund M, Sturbaum AK, Nissen LM, RatnerN. The protein product of the neurofibromatosis type 1 gene isexpressed at highest abundance in neurons, Schwann cells, andoligodendrocytes. Neuron 1992;8:415–28.

21. 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 Cell 2008;13:105–16.

22. Zheng H, Chang L, Patel N, Yang J, Lowe L, Burns DK, et al. Inductionof abnormal proliferation by nonmyelinating schwann cells triggersneurofibroma formation. Cancer Cell 2008;13:117–28.

23. Chandross KJ, Cohen RI, Paras P, Gravel M, Braun PE, Hudson LD.Identifcation and characterization of early glial progenitors using atransgenic selection strategy. J Neurosci 1999;19:759–74.

24. Huang Y, Rangwala F, Fulkerson PC, Ling B, Reed E, Cox AD, et al.Role of TC21/R-Ras2 in enhanced migration of neurofibromin-defi-cient Schwann cells. Oncogene 2004;23:368–78.

25. Kim HA, Ling B, Ratner N. Nf1-deficient mouse Schwann cells areangiogenic and invasive and can be induced to hyperproliferate:reversion of some phenotypes by an inhibitor of farnesyl proteintransferase. Mol Cell Biol 1997;17:862–72.

26. Ling BC,Wu J,Miller SJ, Monk KR, Shamekh R, Rizvi TA, et al. Role forthe epidermal growth factor receptor in neurofibromatosis-relatedperipheral nerve tumorigenesis. Cancer Cell 2005;7:65–75.

27. Chang JT, Carvalho C, Mori S, Bild AH, Gatza ML, Wang Q, et al. Agenomic strategy to elucidate modules of oncogenic pathway signal-ing networks. Mol Cell 2009;34:104–14.

28. Crosio C, FimiaGM, Loury R, KimuraM,Okano Y, ZhouH, et al. Mitoticphosphorylation of histone H3: spatio-temporal regulation by mam-malian Aurora kinases. Mol Cell Biol 2002;22:874–85.

29. Tong Y, Ben-Shlomo A, Zhou C, Wawrowsky K, Melmed S. Pituitarytumor transforming gene 1 regulates Aurora kinase A activity. Onco-gene 2008;27:6385–95.

30. Assie G, LaFramboise T, Platzer P, Bertherat J, Stratakis CA, Eng C.SNP arrays in heterogeneous tissue: highly accurate collection of bothgermline and somatic genetic information from unpaired single tumorsamples. Am J Hum Genet 2008;82:903–15.

31. Garcia-LinaresC, Fernandez-Rodriguez J, Terribas E,Mercade J, ProsE, Benito L, et al. Dissecting loss of heterozygosity (LOH) in neurofi-bromatosis type 1-associated neurofibromas: Importance of copyneutral LOH. Hum Mutat 2011;32:78–90.

32. AgneseV,BazanV, Fiorentino FP, FanaleD,BadalamentiG,ColucciG,et al. The role of Aurora-A inhibitors in cancer therapy. Ann Oncol2007;18 Suppl 6:vi47–52.

33. Dees EC, Infante JR, Cohen RB, O'Neil BH, Jones S, von Mehren M,et al. Phase1studyofMLN8054, a selective inhibitor ofAuroraAkinasein patients with advanced solid tumors. Cancer Chemother Pharmacol2011;67:945–54.

34. Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang D, Nitta M,et al. Aurora-A and an interacting activator, the LIM protein Ajuba,are required for mitotic commitment in human cells. Cell 2003;114:585–98.

35. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, GallantM, et al. Identification and inhibition of the ICE/CED-3 proteasenecessary for mammalian apoptosis. Nature 1995;376:37–43.

36. Nowak SJ, Corces VG. Phosphorylation of histone H3: a balancing actbetween chromosome condensation and transcriptional activation.Trends Genet 2004;20:214–20.

37. Furukawa T, Kanai N, Shiwaku HO, Soga N, Uehara A, Horii A. AURKAis one of the downstream targets of MAPK1/ERK2 in pancreaticcancer. Oncogene 2006;25:4831–9.

38. Tatsuka M, Sato S, Kitajima S, Suto S, Kawai H, Miyauchi M, et al.Overexpression of Aurora-A potentiates HRAS-mediated oncogenictransformation and is implicated in oral carcinogenesis. Oncogene2005;24:1122–7.

39. Huang HY, Chang HY, Chou CH, Tseng CP, Ho SY, Yang CD, et al.sRNAMap: genomicmaps for small non-coding RNAs, their regulatorsand their targets in microbial genomes. Nucleic Acids Res 2009;37:D150–4.

40. GigouxV, L'HosteS,RaynaudF,Camonis J,GarbayC. IdentificationofAurora kinases asRasGAPSrc homology 3domain-bindingproteins. JBiol Chem 2002;277:23742–6.

41. Pamonsinlapatham P, Hadj-Slimane R, Raynaud F, Bickle M, Cor-neloup C, Barthelaix A, et al. A RasGAP SH3 peptide aptamer inhibitsRasGAP-Aurora interaction and induces caspase-independent tumorcell death. PLoS One 2008;3:e2902.

42. Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PN Jr., GandaraDR. Aurora kinases as anticancer drug targets. Clin Cancer Res2008;14:1639–48.

43. Sehdev V, Peng D, Soutto M, Washington MK, Revetta F, Ecsedy JA,et al. The aurora kinase A inhibitor MLN8237 enhances cisplatin-induced cell death in esophageal adenocarcinoma cells. Mol CancerTher 2012;3:763–74.

44. Huck JJ, ZhangM,McDonald A, BowmanD,Hoar KM, Stringer B, et al.MLN8054, an inhibitor of Aurora A kinase, induces senescence inhuman tumor cells both in vitro and in vivo. Mol Cancer Res2010;8:373–84.

45. Dreier MR, Grabovich AZ, Katusin JD, Taylor WR. Short and long-termtumor cell responses to Aurora kinase inhibitors. Exp Cell Res2009;315:1085–99.

46. E. C. Dees et. al. Phase I study of the investigational drugMLN8237, anAurora A kinase (AAK) inhibitor, in patients (pts) with solid tumors.Abstract 2010 ASCO Annual Meeting

47. Kelly KR, Ecsedy J, Medina E, Mahalingam D, Padmanabhan S,Nawrocki ST, et al. The novel Aurora A kinase inhibitor MLN8237 isactive in resistant chronic myeloid leukemia and significantlyincreases the efficacy of nilotinib. J Cell Mol Med 2010;15:2057–70.

48. El-Sheikh A, Fan R, Birks D, Donson A, Foreman NK, Vibhakar R.Inhibition of aurora kinase A enhances chemosensitivity of medullo-blastoma cell lines. Pediatr Blood Cancer 2010;55:35–41.

49. Maris JM, Morton CL, Gorlick R, Kolb EA, Lock R, Carol H, et al. Initialtesting of the aurora kinase a inhibitor MLN8237 by the PediatricPreclinical Testing Program (PPTP). Pediatr Blood Cancer 2010;55:26–34.






Published OnlineFirst July 18, 2012.Clin Cancer Res Ami V. Patel, David Eaves, Walter J. Jessen, et al. Therapeutic TargetA as a Potential Malignant Peripheral Nerve Sheath Tumor Ras-Driven Transcriptome Analysis Identifies Aurora Kinase

Updated version

10.1158/1078-0432.CCR-12-1072doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2012/07/18/1078-0432.CCR-12-1072.DC1Access the most recent supplemental material at:

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

Subscriptions

Reprints and

[email protected] 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://clincancerres.aacrjournals.org/content/early/2012/08/31/1078-0432.CCR-12-1072To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-12-1072

http://clincancerres.aacrjournals.org/content/suppl/2012/07/18/1078-0432.CCR-12-1072.DC1

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

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2012/08/31/1078-0432.CCR-12-1072


Ras-Driven Transcriptome Analysis Identiﬁes Aurora Kinase ......Ras-Driven Transcriptome Analysis...

Documents

Transcript of Ras-Driven Transcriptome Analysis Identiﬁes Aurora Kinase ......Ras-Driven Transcriptome Analysis...