cancer research synthetic lethality screens reveal rps6 ... · the insulin-like growth factor-1...

Ther

SynModInhi

JennyJoanMicha

Abst

Intro

Theimporhuma(1–3).signalisignal

AuthorResearPatholoLos AnCompaand OGermaQuebec

Note:Resear

CurrentCancer

CorresCentre,V5Z 1Lintercha

doi: 10

©2010

Cance8770

Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-1093

Canceresearch

apeutics, Targets, and Chemical Biology

thetic Lethality Screens Reveal RPS6 and MST1R asifiers of Insulin-like Growth Factor-1 Receptor

R

bitor Activity in Childhood Sarcomas

C. Potratz1,4, Darren N. Saunders1, Daniel H. Wai2, Tony L. Ng1, Steven E. McKinney1,
M. Carboni3, Marco M. Gottardis3, Timothy J. Triche2, Heribert Jürgens4, el N. Pollak5, Samuel A. Aparicio1, and Poul H.B. Sorensen1
ractThe

cancerantiboobservremainbitionin sarcthat msarcomsuch acell linalso kMST1

ng events-regulate

s' Affiliatioch Centre, Vgy and Labgeles, Califony, Princetoncology, Uny; and 5De, Canada

Supplemench Online (h

address foResearch P

ponding A675 West3. Phone:nge.ubc.ca

.1158/0008-

American A

r Res; 70(

Downloa

insulin-like growth factor-1 receptor (IGF1R) is emerging as a promising therapeutic target in humans. In the high-risk childhood sarcomas Ewing family tumor and rhabdomyosarcoma, IGF1R-blockingdies show impressive antitumor activity in some but not all patients, and acquired resistance ised. Because tumor IGF1R mutations are not described, the basis of IGF1R inhibitor resistances unknown. We hypothesized that compensatory signaling cascades bypassing targeted IGF1R inhi-might be involved. To test this systematically, we performed small interfering RNA (siRNA) screensoma cell lines to identify IGF1R pathway components or related protein tyrosine kinase (PTK) networksodulate the antitumor efficacy of the BMS-536924 IGF1R kinase inhibitor. This strategy revealed (a) thata cells are exquisitely sensitive to loss of distal rather than proximal IGF1R signaling components,s ribosomal protein S6 (RPS6); (b) that BMS-536924 fails to block RPS6 activation in resistant sarcomaes; and (c) that siRNA knockdown of the macrophage-stimulating 1 receptor tyrosine kinase (MST1R;nown as RON) restores BMS-536924 efficacy, even in highly drug-resistant cell lines. We confirmedR expression across a broad panel of childhood sarcomas, and found that loss of MST1R by RNArence blocks downstream RPS6 activation when combined with BMS-536924 in vitro. These findings
interfe
underscore the importance of fully understanding PTK networks for successful clinical implementationof kinase inhibitor strategies. Cancer Res; 70(21); 8770–81. ©2010 AACR.

3-kina(mTOprocesdrug rficatiocomm

duction

insulin-like growth factor-1 receptor (IGF1R) is antant target for cancer drug development, with severalnized blocking antibodies currently in clinical trialsBinding of IGF1 or IGF2 to IGF1R initiates a myriad of

, including activation of the Ras–extracellulard kinase (ERK) and phosphatidylinositol

bitorsIn h

tumorintensprogremainsdiseas(6–8).promiETS fuETV6-axis toof alvEWS-and awhereing, isIGF2PAX3

ns: 1Department of Molecular Oncology, BC Cancerancouver, British Columbia, Canada; 2Department oforatory Medicine, Children's Hospital Los Angeles,rnia; 3Oncology Drug Discovery, Bristol-Myers Squibbn, New Jersey; 4Department of Pediatric Hematologyniversity Children's Hospital Münster, Münster,partment of Oncology, McGill University, Montréal,

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

r D.N. Saunders: Garvan Institute of Medical Research,rogram, Darlinghurst, New South Wales, Australia.

uthor: Poul H.B. Sorensen, BC Cancer Research10th Avenue, Vancouver, British Columbia, Canada604-675-8202; Fax: 604-675-8218; E-mail: psor@.

5472.CAN-10-1093

ssociation for Cancer Research.

21) November 1, 2010

Research. on March 23, 202cancerres.aacrjournals.org ded from

se (PI3K)–AKT–mammalian target of rapamycinR) pathways, among others, leading to diverse cellularses such as growth, survival, tumor angiogenesis, andesistance (4, 5). Deregulation of IGF1R via IGF1R ampli-n or enhanced activation by ligand overexpression ison in solid tumors, suggesting broad utility for inhi-of this receptor.igh-risk childhood sarcomas such as Ewing family(EFT) and rhabdomyosarcoma (RMS), IGF1R is undere scrutiny as a therapeutic target. Despite impressivess with conventional chemotherapies, prognosis re-very poor for patients with relapsed and metastatice, underscoring the urgent need for new therapeuticsNumerous preclinical findings highlight IGF1R as asing molecular target in childhood sarcomas: EWS-sion proteins of EFT and the congenital fibrosarcomaNTRK3 chimeric kinase require a functional IGF1Rtransform cells, and the PAX3-FKHR fusion protein

eolar RMS acts synergistically with IGF1R (9–11).ETS fusion proteins induce IGF1 promoter activity,n IGF1/IGF1R autocrine loop exists in EFT (12, 13),as IGFBP3, which negatively regulates IGF1R signal-repressed by EWS-FLI1 (14). RMS is associated with
overexpression and loss of IGF2 imprinting, and-FKHR activates IGF1R transcription in alveolar
0. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

RMS (activacells (the enRMSserinenutriemTORphorylriboso4E-bintion oEar

IGF1REFT asion ament.resistamutat(2). Relevelsbodieslevelsof effetreatmantibomergereactivylationcoactias epiconfercotargactivitimpinpro-onTo a

interfemolecalso bsmallstudyPTK nidentifinhibitmecha

Mate

Adddures

Cell liAll

and Mlucifer(BC C

CanadCultur

CompBM

panycontroSignalwere f

siRNAWe

way anmacontargetwe usnontaDetailfound

Cell sRela

determSciencdeathtrogen

WesteWe

(9). FoSupple

FlowApo

after panalyz

TumoFro

Oncolthe Pbanklishedsampl

Modifiers of IGF1R Inhibitor Activity in Childhood Sarcomas

www.a


15–17). Functionally, IGF1R seems to be the majortor of the PI3K-AKT cascade in childhood sarcoma18). IGF1R links PI3K-AKT signaling to activation ofergy-sensitive mTOR complex 1 (mTORC1) in bothand EFT cells (19, 20). Signaling by the mTORC1-threonine kinase drives cell growth and division whennt conditions are appropriate for proliferation (21).C1 is essential for Cap-dependent translation via phos-ation of S6 kinase, which phosphorylates and activatesmal protein S6 (RPS6), and eukaryotic initiating factording protein 1 (4EBP1), which releases 4EBP1 inhibi-f the eIF4E translation factor (22).ly clinical trials have shown remarkable responses to-blocking antibodies in some patients with advancednd RMS (1–3, 8, 23–25). In spite of this, disease progres-nd resistance is seen in many patients even during treat-A key question therefore remains as to what determinesnce toward these agents, as tumor-associated geneticions within the IGF1R pathway have not been detectedcently, Cao and colleagues (18) showed that IGF1Rcorrelate with in vivo sensitivity to IGF1R-blocking anti-in RMS xenografts. Other potential biomarkers includeof IGF1 and IGF-binding proteins (IGFBP), or activationctors such as AKT (1, 3, 18, 26). Interestingly, althoughent of mice bearing human RMS xenografts with IGF1Rdies initially reduced xenograft growth, tumors ree-d after extended treatment. This correlated withation of AKT in tumor cells, although IGF1R phosphor-remained blocked (18). In human epithelial tumors,

vation of receptor protein tyrosine kinases (PTK) suchdermal growth factor receptor (EGFR) and ERBB2s resistance to IGF1R antagonists and vice versa, andeting these PTKs along with IGF1R enhances antitumory (26–28). This suggests that alternative PTKs mayge on pathways downstream of IGF1R to maintaincogenic signaling in human tumors.ddress this in childhood sarcomas, we performed RNArence screens in EFT cell lines combining a small-ule IGF1R kinase inhibitor, BMS-536924 (29), whichlocks the insulin receptor (INSR), with pathway-specificinterfering RNA (siRNA) sublibraries to systematicallythe IGF1R signaling cascade and its relation to otheretworks. The goal of this functional approach was toy PTKs or other signaling molecules that modify IGF1R
or activity, as a means to elucidate potential resistance Angele
Boneby Dr.MicrowereNationarray.ngeneraized u

ImmuMST

nisms and rational cotargeting strategies.

rials and Methods

itional information and detailed experimental proce-are provided in Supplementary Materials and Methods.

nessarcoma cell lines were previously described. MCF-7DA-MB-231 breast cancer cell lines with stable firefly
ase expression were a kind gift of Dr. Marcel Ballyancer Research Centre, Vancouver, British Columbia,
rays bCruz

acrjournals.org

Research. on March 23, 202cancerres.aacrjournals.org Downloaded from

a). HCT116 and HEK293 were from the American Typee Collection.

ounds and reagentsS-536924 was provided by Bristol-Myers Squibb Com-αIR3 was from Calbiochem, and isotype-matched IgGl (MOPC-21)was fromAbcam. Rapamycinwas fromCelling Technology. Recombinant human IGF1 and MST1rom Sigma-Aldrich and R&D Systems, respectively.

screensscreened 31 proteins of the insulin/IGF1 signaling path-d all 88 known PTKs of the human genome using Dhar-siRNA libraries in pools of four siRNA duplexes pergene (SMARTpool; Dharmacon). For control siRNAs,ed a commercially available and validated, functionalrgeting siRNA (nontargeting siRNA #3; Dharmacon).s on experimental procedures and data analysis arein Supplementary Materials and Methods.

urvival and death assaystive cell viability and number (fractional survival) wereined by bioluminescence (D-luciferin; Caliper Lifees) or WST-1 assays (Roche Applied Science). Cellwas analyzed by ethidium homodimer-1 (EthD-1; Invi-) uptake.

rn blottingstern blotting was performed as previously describedr description and dilutions of all antibodies used, seementary Table S1.

cytometryptotic cells and cell cycle distribution were determinedropidium iodine staining of nuclei using a FACScaner (BD Biosciences; refs. 9, 30).

r samples and gene expression analysiszen tumor samples from patients enrolled in Children'sogy Group D9802 and D9803 trials were obtained fromediatric Cooperative Human Tissue Network tumor(Columbus, OH). RMS samples were previously pub-by Davicioni and colleagues (31); additional tumores were obtained from the Children's Hospital Loss tumor bank. All samples contained >80% tumor cells.marrow mesenchymal stem cells (MSC) were providedDarwin Prockop (Tulane University, New Orleans, LA).array protocols (GeneChip U133A array; Affymetrix)as described (31). Data sets are deposited on theal Cancer Institute Cancer Array Database at https://ci.nih.gov/caarray/project/trich-00099. Box plots wereted with Genomics Suite 6.5 (Partek, Inc.) and normal-sing RobustMultichipAverage (RMA).

nohistochemistry1R protein expression was analyzed in tissue microar-
y probing with a MST1R antibody (Ron β C-20; SantaBiotechnology) as previously described (32). Samples
Cancer Res; 70(21) November 1, 2010 8771



werewith m10% to

Resu

EFT cIGF1RTo

sarcomIGF1RTC71dose 5controcomplat dosED50swere 3tary Fthan twhere(SuppIgG coshownof IGFand 24S473 ptein kisistentanalyscatedfractiodeathriboseTC71BMS-5

siRNAblockTo e

ate susurvivof thestablysiRNAlater fmeasusiRNAor sursurvivshowsto RPCBLBothersviduallines,(see Sheat m

definireducevival iconfirshowneffectsthe pocorrelSuppleMax

RPS6,with(Fig. 2RPS6-ducedcial elcourstainedup tovaluescruciaeleme

Knocin vitBec

and dBMS-5such atransfwith vtarget53692were aproximor PDBMS-5dancydownknockbroadto 10-compTC32ThereBMS-5

DistalBMS-To

resistacompotionalyses (Fwere dthe EF

Potratz et al.

Cance8772


scored as follows: high positive, >50% of tumor cellsembranous/cytoplasmic positivity; medium positive,50% positivity; and negative.

lts

ell lines are highly sensitive to the BMS-536924kinase inhibitor

explore IGF1R inhibitor efficacy in high-risk childhoodas, we first evaluated the activity of the BMS-536924kinase inhibitor in well-established EFT cell lines,

and TC32. Dose-response curves showed an effective0 (ED50; dose reducing viability to 50% of nontreatedls) of 100 and 175 nmol/L, respectively, and almostete loss of cellular luminescence (loss of viable cells)es above 1 μmol/L (Fig. 1A). For comparison, theof breast cancer cell lines MCF-7 and MDA-MB-23130 nmol/L and 3.5 μmol/L, respectively (Supplemen-ig. S1A). In TC32 cells, BMS-536924 was more potenthe monoclonal anti-IGF1R antibody αIR3 (13, 33),as in TC71 cells potency was comparable with αIR3lementary Fig. S1B). DMSO vehicle or isotype-specificntrol antibodies did not affect cell viability (data not). Western blotting confirmed BMS-536924 inhibition1R tyrosine phosphorylation at 100 nmol/L at both 1hours in EFT lines, correlating with inhibition of AKThosphorylation (Fig. 1B). Ras-mitogen-activated pro-nase signaling (ERK1/2 phosphorylation) was not con-ly influenced by BMS-536924. Flow cytometric cell cycleis revealed both cell death and cell cycle arrest, as indi-by increased sub-G1 and G1 and decreased S and G2-Mns, respectively (Fig. 1C). BMS-536924 induced cellwas furthermore confirmed by increased poly(ADP-) polymerase (PARP) cleavage (Fig. 1D). Therefore,and TC32 EFT cell lines are highly sensitive to the36924 IGF1R kinase inhibitor.

s targeting distal IGF1R signaling componentsEFT cell survivalxplore which components of the IGF1R cascade medi-rvival of EFT cells, we conducted an siRNA fractionalal screen using a siRNA library targeting 31 key proteinsIGF1R/INSR signaling axis. Briefly, TC71 and TC32 cellsexpressing firefly luciferase were transfected withpools (4 duplexes/target gene) and analyzed 96 hoursor luciferase substrate-induced bioluminescence tore relative cell viability (fractional survival). Thus,s reducing fractional survival likely target progrowthvival proteins, whereas siRNAs that increase fractionalal potentially target tumor suppressors. Figure 2A (top)that, along with the XPO1 positive control, siRNAs

S6, FRAP1 (mTOR), AKT2, AKT3, FOXO1A, IRS-1, andreduced fractional survival in both cell lines, whereassuch as PDPK1 or IGF1R were only effective in indi-lines. To better compare results between the two cellwe defined screening hits according to specific criteria
upplementary Materials and Methods) and generatedaps for visualization (Fig. 2A, bottom). Using these
and thand 2

r Res; 70(21) November 1, 2010


tions, only siRNAs to mTOR and RPS6 reproduciblyd survival, whereas only TSC1 siRNAs increased sur-n both cell lines. Protein knockdown by siRNAs wasmed by Western blotting; representative results arein Fig. 2B and Supplementary Fig. S2A. Moreover,on cell survival of individual siRNA duplexes fromols were confirmed (Supplementary Fig. S2B), and thisated with protein knockdown (shown for IGF1R inmentary Fig. S2C).imal loss of cell viability was observed for siRNAs tothe most distal element of the IGF1R pathway tested,nearly complete cell death in both EFT cell linesA). This was validated over a time course (Fig. 2C, left):targeting siRNAs severely impaired survival and in-cell death within 96 hours, confirming RPS6 as a cru-ement of IGF1R survival signaling. In contrast, timee experiments with siRNAs to TSC1 confirmed sus-effects of TSC1 knockdown, with survival increasing7-fold compared with controls (Fig. 2C, right; absolutein Supplementary Fig. S2D). These data point to

l roles for distal rather than proximal IGF1R pathwaynts in survival of EFT cells, such as RPS6 and TSC1.

kdown of TSC1/TSC2 tumor suppressors inducesro BMS-536924 resistanceause EFT cells are highly sensitive to both BMS-536924istal IGF1R pathway blockade, we wondered whether36924 efficacy is modulated by levels of distal proteinss TSC1/TSC2, RPS6, or mTOR. To test this, cells wereected with siRNAs to the above genes and then treatedarying doses of BMS-536924. First, RPS6- or mTOR-ing siRNAs did not further sensitize cells to BMS-4 because, as shown in Fig. 2A and C, the vast majoritylready dead from knockdown alone. Second, siRNAs toal pathway elements, such as IGF1R itself, AKT, IRS-1,PK1, failed to consistently sensitize cells to low-dose36924 (data not shown). Possibly, functional redun-or complex feedback loops exist that override knock-of proximal pathway components. Third, TSC1down markedly shifted dose-response curves over aBMS-536924 dose range, increasing ED50s by up

fold (Fig. 2D). Moreover, knockdown of TSC2, whichlexes with TSC1 (34), also dramatically desensitizedand BMS-536924–responsive SK-N-MC cells (Fig. 2D).fore, loss of TSC1/TSC2 induces marked resistance to36924 in EFT cells.

IGF1R pathway responses correlate with536924 sensitivitymore rigorously test the link between BMS-536924nce and activation states of distal IGF1R signalingnents, we assessed BMS-536924 sensitivity in six addi-childhood sarcoma cell lines. From dose-response anal-ig. 3A), EFT cell lines SK-N-MC, TTC633, and TTC466esignated BMS-536924 sensitive (ED50 ∼200 nmol/L),T cell line 5838 as intermediate (ED50 ∼500 nmol/L),
e Rh18 and Rh30 RMS cell lines as resistant (ED50 >10μmol/L, respectively). We then analyzed IGF1R signaling
Cancer Research



molectivity dand BMAKT.molecsitiviticlearlyphosp4EBP1at 1 anover, RBMS-5

Fig. S3gets (2Rh18 atoxicitwere spoten53692resistThereinhibit

Figureand graBMS-5as loadstainingfor clea


www.a


ules in response to IGF1 stimulation. BMS-536924 sensi-id not correlate with total IGF1R or AKT levels (Fig. 3B),S-536924 inhibited phosphorylation of both IGF1R and

Therefore, differential levels or activation of proximalules do not explain the distinct in vitro BMS-536924 sen-es in these lines. In contrast, BMS-536924 sensitivitycorrelated with its ability to block RPS6 and 4EBP1

horylation. In fact, BMS-536924 failed to block RPS6 orphosphorylation in both Rh18 and Rh30 cell lines, evend 10 μmol/L (Fig. 3B; Supplementary Fig. S3A). More-

of nuclei. Graphs show one representative experiment (n = 3). D, cell lysved PARP.

PS6 siRNA knockdown induced marked cell death in36924–resistant Rh18 and Rh30 cells (Supplementary

effectosarcom

acrjournals.org


B). Because RPS6 and 4EBP1 are known mTORC1 tar-1), we tested effects of rapamycin on these cell lines.nd Rh30 cells are highly sensitive to rapamycin in cyto-y assays (19, 35), and we found that TC71 and TC32 cellsimilarly sensitive (Supplementary Fig. S3C). Rapamycintly inhibited phosphorylation of RPS6 both in BMS-4–sensitive TC71 and TC32 cells and in BMS-536924–ant Rh18 and Rh30 cells (Supplementary Fig. S3D).fore, although RPS6 and 4EBP1 phosphorylation areed by BMS-536924 in sensitive sarcoma cells, these distal

m B, analyzed for induction of cell death by immunoblotting

1. BMS-536924 is active in EFT cell lines in vitro. A, dose response to 72-h BMS-536924 treatment. Cell survival was analyzed by luminescencephed relative to nontreated control. Points, mean (n = 3); bars, SD. B, BMS-536924 effects on IGF1R signaling. Cells were treated with36924 or DMSO vehicle, IGF1 (100 ng/mL) was added for 15 min before lysis, and cell lysates were immunoblotted as specified; actin serveding control. C, cell cycle distribution following BMS-536924 or DMSO vehicle treatment was analyzed by flow cytometry after propidium iodine

ates fro

rs are not affected by BMS-536924 treatment in resistanta cells (although they remain mTORC1 dependent).




Figure(Rc/Cc)were tra96 h (TSwas plointensitbars, SCells wlumines

Potratz et al.

Cance8774


2. siRNA screening of the IGF1R signaling pathway. A, top, siRNAs rank ordered by effect on cell survival relative to transfection reagent alone; bottom, screening hits presented as heat map (for definitions, see Supplementary Materials andMethods). B, confirmation of protein knockdown. Cellsnsfected with siRNAs targeting RPS6 (siRPS6) and TSC1 (siTSC1) or nontargeting control siRNAs (siCTRL) and analyzed after 20 h (RPS6) orC1). C, time course analysis. In cells transfected as in B, survival was analyzed by luminescence and cell death by EthD-1 uptake. Top, survivaltted relative to siCTRL; middle, cell death was graphed relative to siCTRL and surviving cell fractions to take into account that EthD-1 fluorescencey is directly linked to cell numbers remaining at each time point (for comparison, see absolute values in Supplementary Fig. S2D). Points, mean (n = 3);D. Bottom, microscopic images of TC71 cells at 96-h time point. Scale bars, 100 μm. D, TSC1/TSC2 knockdown induces BMS-536924 resistance.
ere transfected with siTSC1, siRNAs targeting TSC2 (siTSC2), or siCTRL and, 48 h later, exposed to BMS-536924 for another 48 h. Survival analyzed bycence (TSC1) or WST-1 (TSC2) is graphed relative to non-BMS-536924–treated control. Points, mean (n = 3); bars, SD.
r Res; 70(21) November 1, 2010 Cancer Research

Research. on March 23, 2020. © 2010 American Association for Cancercancerres.aacrjournals.org Downloaded from


Knockto BMBas

mightBMS-5vergecontriaticallall 88 kenhanand T(four sBMS-5other.Fig. S4and ansurvivtary FFig. S4(see Suthosefects oand ce

for senot shseveracell linHCK a(Fig. 4targetcusedspondTho

cludedand Fserved(37). Owith pPTK hwas coally pwheredividu(data

FigureBMS-5signalinwas loa posure


www.a


down of MST1R sensitizes sarcoma cellsS-536924ed on the above, we reasoned that alternative pathwaysregulate RPS6 activation in BMS-536924–sensitive and36924–resistant cell lines. Because numerous PTKs con-on mTORC1 signaling, activation of another PTK couldbute to BMS-536924 resistance. To explore this system-y, we performed screens using an siRNA library targetingnown human PTKs to identify PTKs whose inactivationces BMS-536924 activity. Briefly, parallel sets of TC71C32 cells were transfected with a library of siRNA poolsiRNA duplexes/target gene), followed after 48 hours by36924 treatment of one set and vehicle treatment of theA sublethal dose of BMS-536924 (∼ED10; SupplementaryA) was selected to ensure detection of both sensitizingtagonizing siRNAs. After another 48 hours, fractionalal was determined for siRNAs alone (Fig. 4A; Supplemen-ig. S4B) and siRNAs plus BMS-536924 (SupplementaryB). A sensitivity index was calculated for each cell linepplementary Materials and Methods; ref. 36) to definesiRNA pools with either sensitizing or antagonistic ef-

ding control. All panels are equal exposures. Asterisk indicates shorter ex

n BMS-536924 activity (Fig. 4B). Protein knockdownll survival effects of individual siRNAs were validated

downwith d

acrjournals.org


lected PTKs (Fig. 5A; Supplementary Fig. S2B; dataown). In the nondrug-treated screen, knockdown ofl PTKs significantly impaired fractional survival in bothes, including IGF1R, EPHB2, FES, Src family kinasesnd LCK, LMTK2, NTRK2, MUSK, PDGFRβ, and PTK9LA), highlighting these PTKs as potential therapeutics in EFTs to be further studied elsewhere. Here, we fo-on BMS-536924–sensitizing siRNAs because corre-ing proteins may contribute to drug resistance.se siRNAs sensitizing both lines to BMS-536924 in-MST1R (macrophage-stimulating 1 receptor), LTK,

LT3 (Fig. 4B). The most pronounced effects were ob-for MST1R, a MET-related PTK also known as RONverexpression and activation of MST1R is associatedoor clinical outcome in several cancers (38), but thisas not been studied in sarcomas. MST1R knockdownnfirmed by Western blotting (Fig. 5A), and alone actu-romoted survival of TC71 and TC32 cells slightly,as slightly decreasing Rh30 survival (Fig. 5B). Four in-al siRNAs to MST1R had similar effects on cell survivalnot shown). With BMS-536924, however, MST1R knock-

.

3. Distal IGF1R pathway signaling responses correlate with BMS-536924 sensitivity. A, dose-response series of EFT and RMS cell lines treated with36924 for 72 h. Survival was measured by WST-1 assay and graphed relative to nontreated controls. Points, mean (n ≥ 3); bars, SD. B, IGF1Rg in cells treated with BMS-536924 or DMSO vehicle for 1 h followed by IGF1 (100 ng/mL) for 15 min. Cell lysates were probed as indicated; actin

markedly sensitized all three cell lines to BMS-536924,ramatically increased cell death and a left shift of




dose-rThis otizatioFig. S5resistafrom ∼was sucells. I

tizedTC32dispar

MST1BMS-

Potratz et al.

Cance8776


esponse curves (Fig. 5C; Supplementary Fig. S5A and B).ccurred over a broad dose range with maximum sensi-n observed near the ED50s for each line (SupplementaryC). Maximal sensitization occurred in BMS-536924–nt Rh30 cells, where the ED50 was shifted by 10-fold2 μmol/L to 200 nmol/L. Thus, MST1R knockdown
fficient to restore BMS-536924 sensitivity to resistant Nex


TC71 cells to BMS-536924, the opposite was seen incells (Fig. 4B; Supplementary Fig. S4B). The basis for thisity is unknown and warrants further investigation.

R knockdown blocks distal IGF1R signaling in536924–resistant cells
t, we analyzed how BMS-536924 sensitization by
elates with IGF1R signaling in either

urell hreseSuellpa/Ccctssensitivity index (SI).

nterestingly, whereas INSR knockdown strongly sensi- MST1R knockdown corr

Figof arepseeA, ccom(Rceffeby

0. © 2010 America

4. Synthetic lethal siRNA screensuman PTKs. Screening hits arented as heat maps (for definitions,pplementary Materials and Methods).survival effects of specific siRNAs alonered with transfection reagent only). B, sensitizing and antagonizing siRNAon BMS-536924 activity as determined

Cancer Research

n Association for Cancer


FigureMST1RuptakeColumndeath wto takevalues,comporesistanindicate


www.a


5. MST1R knockdown sensitizes sarcoma cells to BMS-536924. A, MST1R protein knockdown in TC32 cells transfected with siRNAs targeting(siMST1R) or nontargeting siCTRL. Ku70 was loading control. B, effects of MST1R knockdown on cell viability. Luminescence and EthD-1were measured 96 h after transfection. Survival was graphed relative to siCTRL, and cell death relative to siCTRL and surviving cell fraction.s, mean (n = 3); bars, SD. C, dose response in cells transfected 48 h previously with siMST1R or siCTRL and treated with BMS-536924 for 48 h. Cellas analyzed by EthD-1 uptake and graphed relative to non-BMS-536924–treated control and surviving cell fractions (Supplementary Fig. S5A)into account effects of siRNAs alone (see B) and that EthD-1 fluorescence is directly linked to cell numbers remaining at each dose (for absolutesee Supplementary Fig. S5B). Points, mean (n = 3); bars, SD. D, MST1R knockdown enhances BMS-536924 activity on distal IGF1R signalingnents. Seventy-two hours after siRNA transfection, cells were treated for 1 h with BMS-536924 (0.01 and 0.1 μmol/L; 0.01 μmol/L not tested in highly
t Rh18 cells), αIR3 antibody (100 ng/mL), or IgG control (100 ng/mL), followed by IGF1 (100 ng/mL, 15 min). Lysates were immunoblotted asd with actin as loading control.
Cancer Res; 70(21) November 1, 2010acrjournals.org 8777



BMS-5resistaBMS-5SeveradownIGF1RTC71,MST1levels,two Band RinhibieffectsiRNAmost ntory eflines (versusof theresistaaloneminimdid noRh30,knocktary Fby αIRknockwith BsuperiparedscreenMST1this Pwith I

MST1We

ligandexpresbut sigtrol bexpresthan anormasion inlatter,marroconfirEFT c(see FactivaFinallysamplEleven12 ofMST1>10%

althoucate tconsisIGF1R

Discu

Thefor hiMounhave ldiseasantibobecomfor repatienmonitwe uselemesurvivgate PIGF1Rdistaloncogthat Rcells eAKT aBMS-5this copropoitor seto funing mCur

expresof IGFAKT (impordictinBMS-5expresdid noIGF1Rcatingaccuraby thoa closin vivochildhhighligcriticaAs a

ical fosent athat aprotei

Potratz et al.

Cance8778


36924–sensitive TC71 and TC32 versus BMS-536924–nt Rh18 and Rh30 cells. We chose a low 10 to 100 nmol/L36924 dose range to critically assess drug sensitization.l key findings emerged (Fig. 5D). First, MST1R knock-alone did not alter expression or baseline activity ofor AKT in the cell lines (Fig. 5D; lanes 1 versus 7 forTC32, and Rh30, and lanes 1 versus 6 for Rh18). Second,R knockdown alone had little effect on phospho-RPS6other than slightly decreasing baseline levels in theMS-536924–resistant cells, Rh30 (lanes 1 versus 7)h18 (lanes 1 versus 6). Third, 100 nmol/L BMS-536924ted IGF1R and AKT activation in all cell lines, buts on RPS6 activation were not apparent in control–transfected cells, consistent with Fig. 3B. Fourth andotably, MST1R knockdown markedly enhanced inhibi-fects of BMS-536924 on RPS6 phosphorylation in all celllanes 4 versus 10 for TC71, TC32, and Rh30, and lanes 38 for Rh18). Therefore, MST1R contributes to activationessential distal IGF1R effector, RPS6, in BMS-536924–nt cell lines. By comparison, αIR3 anti-IGF1R antibodiesblocked IGF1R activation in all cell lines, but onlyally decreased AKT activation (Fig. 5D). Alone, αIR3t appreciably affect RPS6 phosphorylation in TC32,or Rh18 cell lines, nor did it enhance effects of MST1Rdown. In the αIR3-sensitive cell line TC71 (Supplemen-ig. S1B), RPS6 activation was minimally decreased3 alone and enhanced in combination with MST1Rdown (lanes 2, 5, and 11). The basis for this discordanceMS-536924 is unknown, but is consistent with itsor activity on survival of TC71 and TC32 cell lines com-with αIR3 (Supplementary Fig. S1B). Our PTK siRNAing studies therefore reveal an unexpected role forR in modulating distal IGF1R effectors, and highlightTK as a candidate therapeutic target for combinationGF1R inhibitors.

R is expressed in childhood sarcomasnext analyzed mRNA expression of MST1R and its, MST1, in 109 childhood sarcoma samples by genesion profiling. All sarcoma subtypes showed variablenificantly higher MST1R expression than normal con-one marrow–derived MSCs (Fig. 6A). Similarly, MST1sion was significantly higher in all sarcoma types otherlveolar RMS (Fig. 6B). Although MSCs were used asl tissue controls for MST1R and MST1 mRNA expres-sarcomas, this does not infer overexpression in theas the relationship of EFT and RMS to normal bonew–derived MSCs remains unclear. Western blottingmed variable MST1R protein expression in all fiveell lines tested, and in three of four RMS cell linesig. 6C). A phospho-specific MST1R antibody showedtion in all cell lines except TC71 and RD (Fig. 6C)., MST1R expression was examined in primary sarcomaes by immunohistochemistry on tissue microarrays.of 22 (50%) EFT, 15 of 21 (71%) alveolar RMS, and

25 (48%) embryonal RMS (ERMS) cases stained for
R, defined as membranous/cytoplasmic positivity inof cells (Fig. 6D; Supplementary Table S2). Therefore,
As a ddepen



gh additional studies are required, our findings indi-hat MST1R is expressed in some childhood sarcomas,tent with a role for this PTK in activation of distaleffectors.

ssion

re is an urgent need to develop new therapeuticsgh-risk childhood sarcomas such as EFT and RMS.ting preclinical data and early promise in clinical trialsed to intense evaluation of IGF1R inhibitors in thesees. Although there is clear efficacy of IGF1R-blockingdies in some patients, others remain resistant ore so during therapy. However, the biological basissistance remains unknown, and biomarkers to selectts most likely to benefit from IGF1R blockade and toor treatment response are poorly demarcated. Here,ed an siRNA screening approach to determine whichnts of the IGF1R signaling axis are crucial for its pro-al function in childhood sarcoma cells, and to investi-TK networks that influence activity of the BMS-536924inhibitor. Our findings suggest that activation of thesignaling molecule RPS6 plays a critical role in pro-enic IGF1R signaling in both EFT and RMS cells, andPS6 activation is sustained in BMS-536924–resistantven when drug treatment blocks proximal IGF1R andctivation. Moreover, knockdown of MST1R restores36924 efficacy in highly drug-resistant cell lines, andrrelates with inhibition of RPS6 phosphorylation. Wese that MST1R is a potential modifier of IGF1R inhib-nsitivity in childhood sarcoma cells through its abilityction as an alternative activator of distal IGF1R signal-olecules including RPS6.rent biomarkers for IGF1R inhibitor efficacy focus onsion and/or activity of intrinsic proximal components1R signaling, including IGF1, IGFBPs, IGF1R itself, and1, 3, 18, 26). In contrast, our findings highlight antant role for distal IGF1R signaling elements in pre-g response to IGF1R blockade. When we analyzed36924 efficacy in sensitive versus resistant cell lines,sion levels and activity of IGF1R pathway elementst correlate with response. In fact, BMS-536924 blockedand AKT phosphorylation even in resistant cells, indi-that proximal pathway elements do not necessarilytely represent responses. These findings are supportedse of Kurmasheva and colleagues (39), which showe correlation between RPS6 activity and in vitro andresponse to the anti-IGF1R antibody CP-751,871 in

ood sarcoma cell lines. Taken together, our studiesht RPS6 as a key survival factor and its blockade as al indicator of the cytotoxic activity of IGF1R inhibitors.component of the 40S ribosome subunit, RPS6 is crit-r protein translation (40). RPS6 activation may repre-point of “oncogene addiction” in transformed cells

re undergoing high proliferative rates and require rapidn translation, especially of key stress-adaptive mRNAs.
ownstream effector of PI3K-AKT–mediated mTORC1-dent translational control, one approach to target RPS6
Cancer Research



is viaseemshoodmycinpro-onstudieapproever, p

tweencells bsuggesvatingchildhOur

Figure(ARMSbesideexpresscells stMST1R protein expression in tissue microarrays of childhood sarcomas using antibodies to total MST1R. Shown are representative examplesof high , >50%


www.a


mTORC1 inhibitors such as rapamycin. Because IGF1Rto be the major activator of the latter cascade in child-sarcoma cells, combining IGF1R inhibitors with rapa-is an attractive strategy to avoid stimulating thiscogenic feedback loop, and preclinical and clinicals are under way to test the long-term efficacy of this

ly positive MST1R-expressing EFT, ARMS, and ERMS primary tumors (i.e.

ach (http://www.clinicaltrials.gov; refs. 18, 39). How-revious preclinical studies report an uncoupling be-

of BMdown

acrjournals.org


IGF1R and AKT activation in RMS xenografts onceecame resistant to IGF1R-blocking antibodies (18),ting the presence of alternative mechanisms for acti-the PI3K-AKT cascade and downstream pathways inood sarcoma cells.combinatorial siRNA approach to screen for modifiers

positive cells). Scale bars, 100 μm.

6. MST1R is expressed in childhood sarcomas. MST1R (A) and MST1 (B) mRNA expression in 30 EFTs, alveolar RMS with PAX7-FKHR fusion7) and PAX3-FKHR fusion (ARMS3; 15 each), 19 ERMS, 30 osteosarcomas (OS), and MSC controls as analyzed by U133A arrays. Symbolseach box plot represent individual samples. Three individual MST1 probes tested, probe 216320_x_at shown. C, left and right, MST1R proteinion and activation in sarcoma cell lines. Cell lines grown in serum-containing medium were analyzed for phosphorylated and total MST1R; HCT116imulated with MST1 (5 nmol/L, 20 min; lane 1) served as positive and HEK293 cells as negative controls. D, immunohistochemical analysis of

S-536924 activity in EFT cells identified MST1R knock-as dramatically sensitizing cell lines to BMS-536924




over atotoxiActivasistandescripendecommtanceand ottanceboth PIt is tIGF1Rculesd'origi(37).growthas MEprolifeexpresoutcombeen sing, Mand RmRNAvival iHowemay bexpresproteilines aIntereRMSantiboversusas doeMST1may rsensitIGF1Rin chiland Mment,tionalknownmay rof IGF

SmagainpotenparedIn thekinasein EFTfrequeRMSactivitbody,How Brentlydata pkinaseticularIn s

converesistatorialIGF1Rapprotial coscorefunctiimplem

Discl

No p

Ackn

WeIreland)Britishtumor tMDA-M

Grant

U.S.T.J. TriKrebshiWilhelmnationa

Theof pageaccorda

Refe1. Gu

recdir

2. PoNa

3. Roeloles25

Potratz et al.

Cance8780


broad dose range, potently restoring BMS-536924 cy-c activity, and overcoming primary drug resistance.tion of receptor PTKs as a mechanism to confer re-ce to targeted inhibitors in human cancers is wellbed (41). It is widely known that PTKs do not act inde-ntly, but form redundant cellular networks to activateon pathway elements, and as such can confer resis-to specific kinase inhibitors (41). For example, EGFRher HER family receptors are known to mediate resis-to IGF1R inhibitors and vice versa, and cotargetingTKs results in enhanced antitumor activity (26–28).herefore conceivable that MST1R similarly modulatesinhibitor activity through activation of distal mole-such as RPS6. MST1R, also called RON (recepteurne nantais) or PTK8, is a MET family receptor PTKActivated by its specific ligand MST1 (hepatocytefactor–like), MST1R activates many similar pathways

T, including PI3K-AKT and Ras-ERK, to regulate cellration, adhesion, and motility (38, 42). MST1R over-sion and activation is associated with poor clinicale in various epithelial cancers, but this PTK has not

tudied in childhood tumors. By gene expression profil-ST1R mRNA expression was detected not only in EFTMS but also in osteosarcoma primary tumors. MST1Rlevels alone did not affect event-free or overall sur-n the EFT or RMS cohorts tested (data not shown).ver, analysis in an IGF1R inhibitor–treated cohorte necessary to rigorously assess influences of MST1Rsion on survival. In addition, we confirmed MST1Rn expression by Western blotting in EFT and RMS cells well as in primary tumors by immunohistochemistry.stingly, MST1R was activated in almost all EFT andcell lines tested based on MST1R phosphotyrosinedy reactivity. Whether this represents ligand-dependentconstitutive activation remains to be determined,s MST1R activation in primary sarcoma samples. IfR indeed modifies IGF1R inhibitor responses, then itepresent a tractable biomarker for IGF1R inhibitorivity. Moreover, combination therapy targeting bothand MST1R may provide a viable treatment optiondhood sarcomas, particularly with therapeutic MST1RET inhibitors being in preclinical and clinical develop-and we are currently exploring this possibility. Addi-synthetic lethal siRNA screens in cell lines withprimary or secondary resistance to IGF1R inhibitors

targeting improves activity Rece

pment of inhibitors of the insulin-like growth factor-I pathway:sons from the first clinical trials. Mol Cancer Ther 2008;7:75–88.

4. Pofac

5. SainEn

6. Beof

7. HaCu



all-molecule tyrosine kinase inhibitors with activityst both IGF1R and INSR are emerging as a second,tially more potent class of targeting compounds com-with blocking antibodies that target IGF1R alone (1–3).current study, we used the dual specificity IGF1R/INSRinhibitor BMS-536924 and confirmed in vitro activitycell lines and reports of EFT cell lines being more

ntly sensitive to IGF1R inhibition (six of six) thancell lines (zero of two; refs. 26, 29, 43). BMS-536924y was superior to the αIR3 monoclonal IGF1R anti-which showed only modest inhibition of cell survival.MS-536924 compares with humanized antibodies cur-in clinical development remains to be determined. Ouroint to a potential role for small-molecule IGF1Rinhibitors in the therapy of childhood sarcomas, par-ly EFTs.ummary, our findings indicate that other PTKs mayrge on common distal signaling elements to mediatence to IGF1R inhibitors, suggesting that new combina-targeting strategies are required to effectively blocksignaling in drug-resistant tumors. In the screening

ach presented here, we identified MST1R as one poten-mbinatorial target. More generally, our results under-the need to fully understand how PTK networks mayon together in a particular tumor type to successfullyent PTK inhibitor strategies.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

thank Maureen O'Sullivan (Our Lady's Children's Hospital, Dublin,and Catherine Pallen (Children's and Women's Hospital, Vancouver,Columbia, Canada) for generously contributing the childhood solidissue microarrays and Dr. Marcel Bally for contributing MCF-7 andB-231 cell lines.

Support

Department of Defense grant W81XWH-07-1-0580 (P.H.B. Sorensen andche). J.C. Potratz was supported by fellowships from the Deutschelfe (German Cancer Aid) and the Faculty of Medicine, Westfälisches-Universität Münster, and D.N. Saunders was supported by an Inter-l Research Fellowship from Cancer Institute NSW.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 03/29/2010; revised 08/16/2010; accepted 08/30/2010; published
eveal further PTKs whose co 1R inhibitors in childhood sarcomas. OnlineFirst 10/19/2010.
rencesalberto A, Pollak M. Emerging role of insulin-like growth factoreptor inhibitors in oncology: early clinical trial results and futureections. Oncogene 2009;28:3009–21.llak M. Insulin and insulin-like growth factor signalling in neoplasia.t Rev Cancer 2008;8:915–28.don J, DeSantos V, Ferry RJJ, Kurzrock R. Early drug dev-

llak MN, Schernhammer ES, Hankinson SE. Insulin-like growthtors and neoplasia. Nat Rev Cancer 2004;4:505–18.mani AA, Yakar S, LeRoith D, Brodt P. The role of the IGF systemcancer growth and metastasis: overview and recent insights.docr Rev 2007;28:20–47.rnstein M, Kovar H, Paulussen M, et al. Ewing's sarcoma family
tumors: current management. Oncologist 2006;11:503–19.yes-Jordan A, Andrassy R. Rhabdomyosarcoma in children.rr Opin Pediatr 2009;21:373–8.
Cancer Research



8. SuEwop

9. MareccelCe

10. Toinsfor

11. WaPACa

12. CirEwPL

13. Sctortum

14. Prigeanpro

15. AyrecHo

16. El-L. Iity199

17. Zhofsar

18. CafacresCa

19. DilPJdoins

20. Wamypa

21. GuCa

22. Ricby

23. Kimpe

24. Kosar

25. TonemoOn

26. Husen

53Ca

27. Chrecrectin20

28. Hacotor

29. WizogroJ M

30. Ricsta

31. Daexpsar20

32. O'TofproCa

33. Rofortiss

34. vantwHu

35. Windde

36. Swantax49

37. Rorec11

38. Beinv

39. KuHoangiz20

40. Rupro

41. Jance8:7

42. Wapropro


www.a


bbiah V, Anderson P, Lazar AJ, Burdett E, Raymond K, Ludwig JA.ing's sarcoma: standard treatment and experimental treatmenttions. Curr Treat Options Oncol 2009;10:126–40.rtin MJ, Melnyk N, Pollard M, et al. The insulin-like growth factor Ieptor is required for Akt activation and suppression of anoikis inls transformed by the ETV6-NTRK3 chimeric tyrosine kinase. Molll Biol 2006;26:1754–69.retsky JA, Kalebic T, Blakesley V, LeRoith D, Helman LJ. Theulin-like growth factor-I receptor is required for EWS/FLI-1 trans-mation of fibroblasts. J Biol Chem 1997;272:30822–7.ng W, Kumar P, Wang W, et al. Insulin-like growth factor II andX3-FKHR cooperate in the oncogenesis of rhabdomyosarcoma.ncer Res 1998;58:4426–33.oni L, Riggi N, Provero P, et al. IGF1 is a common target gene ofing's sarcoma fusion proteins in mesenchymal progenitor cells.oS One 2008;3:e2634.otlandi K, Benini S, Sarti M, et al. Insulin-like growth factor I recep--mediated circuit in Ewing's sarcoma/peripheral neuroectodermalor: a possible therapeutic target. Cancer Res 1996;56:4570–4.eur A, Tirode F, Cohen P, Delattre O. EWS/FLI-1 silencing andne profiling of Ewing cells reveal downstream oncogenic pathwaysd a crucial role for repression of insulin-like growth factor bindingtein 3. Mol Cell Biol 2004;24:7275–83.alon D, Glaser T, Werner H. Transcriptional regulation of IGF-Ieptor gene expression by the PAX3-FKHR oncoprotein. Growthrm IGF Res 2001;11:289–97.Badry O, Minniti C, Kohn E, Houghton P, Daughaday W, Helmannsulin-like growth factor II acts as an autocrine growth and motil-factor in human rhabdomyosarcoma tumors. Cell Growth Differ0;1:325–31.an S, Shapiro DN, Helman LJ. Activation of an imprinted allelethe insulin-like growth factor II gene implicated in rhabdomyo-coma. J Clin Invest 1994;94:445–8.o L, Yu Y, Darko I, et al. Addiction to elevated insulin-like growthtor I receptor and initial modulation of the AKT pathway define theponsiveness of rhabdomyosarcoma to the targeting antibody.ncer Res 2008;68:8039–48.ling MB, Dias P, Shapiro DN, Germain GS, Johnson RK, Houghton. Rapamycin selectively inhibits the growth of childhood rhab-myosarcoma cells through inhibition of signaling via the type Iulin-like growth factor receptor. Cancer Res 1994;54:903–7.n X, Helman LJ. Effect of insulin-like growth factor II on protectingoblast cells against cisplatin-induced apoptosis via p70 S6 kinasethway. Neoplasia 2002;4:400–8.ertin DA, Sabatini DM. Defining the role of mTOR in cancer.ncer Cell 2007;12:9–22.hter JD, Sonenberg N. Regulation of cap-dependent translationeIF4E inhibitory proteins. Nature 2005;433:477–80.SY, Toretsky JA, Scher D, Helman LJ. The role of IGF-1R in

diatric malignancies. Oncologist 2009;14:83–91.lb EA, Gorlick R. Development of IGF-IR inhibitors in pediatriccoma. Curr Oncol Rep 2009;11:307–13.lcher AW, Sarantopoulos J, Patnaik A, et al. Phase I, pharmacoki-tic, and pharmacodynamic study of AMG 479, a fully humannoclonal antibody to insulin-like growth factor receptor 1. J Clin
col 2009;27:5800–7.ang F, Greer A, Hurlburt W, et al. The mechanisms of differentialsitivity to an insulin-like growth factor-1 receptor inhibitor (BMS-
43. Scinsmu

acrjournals.org


6924) and rationale for combining with EGFR/HER2 inhibitors.ncer Res 2009;69:161–70.akravarti A, Loeffler JS, Dyson NJ. Insulin-like growth factoreptor I mediates resistance to anti-epidermal growth factoreptor therapy in primary human glioblastoma cells through con-ued activation of phosphoinositide 3-kinase signaling. Cancer Res02;62:200–7.luska P, Carboni JM, TenEyck C, et al. HER receptor signalingnfers resistance to the insulin-like growth factor-I receptor inhibi-, BMS-536924. Mol Cancer Ther 2008;7:2589–98.ttman M, Carboni J, Attar R, et al. Discovery of a 1H-benzoimida-l-2-yl)-1H-pyridin-2-one (BMS-536924) inhibitor of insulin-likewth factor I receptor kinase with in vivo antitumor activity.ed Chem 2005;48:5639–43.cardi C, Nicoletti I. Analysis of apoptosis by propidium iodideining and flow cytometry. Nat Protocols 2006;1:1458–61.vicioni E, Anderson JR, Buckley JD, Meyer WH, Triche TJ. Generession profiling for survival prediction in pediatric rhabdomyo-comas: a report from the Children's Oncology Group. J Clin Oncol10;28:1240–6.oole JM, Rabenau KE, Burns K, et al. Therapeutic implicationsa human neutralizing antibody to the macrophage-stimulatingtein receptor tyrosine kinase (RON), a c-MET family member.ncer Res 2006;66:9162–70.hlik QT, Adams D, Kull FC, Jacobs S. An antibody to the receptorinsulin-like growth factor I inhibits the growth of MCF-7 cells inue culture. Biochem Biophys Res Commun 1987;149:276–81.Slegtenhorst M, Nellist M, Nagelkerken B, et al. Interaction be-

een hamartin and tuberin, the TSC1 and TSC2 gene products.m Mol Genet 1998;7:1053–7.an X, Harkavy B, Shen N, Grohar P, Helman LJ. Rapamycinuces feedback activation of Akt signaling through an IGF-1R-pendent mechanism. Oncogene 2006;26:1932–40.anton C, Marani M, Pardo O, et al. Regulators of mitotic arrestd ceramide metabolism are determinants of sensitivity to pacli-el and other chemotherapeutic drugs. Cancer Cell 2007;11:8–512.nsin C, Muscatelli F, Mattei MG, Breathnach R. A novel putativeeptor protein tyrosine kinase of the met family. Oncogene 1993;8:95–202.nvenuti S, Comoglio PM. The MET receptor tyrosine kinase inasion and metastasis. J Cell Physiol 2007;213:316–25.rmasheva RT, Dudkin L, Billups C, Debelenko LV, Morton CL,ughton PJ. The insulin-like growth factor-1 receptor-targetingtibody, CP-751,871, suppresses tumor-derived VEGF and syner-es with rapamycin in models of childhood sarcoma. Cancer Res09;69:7662–71.vinsky I, Meyuhas O. Ribosomal protein S6 phosphorylation: fromtein synthesis to cell size. Trends Biochem Sci 2006;31:342–8.ne PA, Gray N, Settleman J. Factors underlying sensitivity of can-

rs to small-molecule kinase inhibitors. Nat Rev Drug Discov 2009;09–23.ng M, Ronsin C, Gesnel M, et al. Identification of the ron geneduct as the receptor for the human macrophage stimulatingtein. Science 1994;266:117–9.otlandi K, Manara MC, Nicoletti G, et al. Antitumor activity of the
ulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 insculoskeletal tumors. Cancer Res 2005;65:3868–76.



2010;70:8770-8781. Published OnlineFirst October 19, 2010.Cancer Res Jenny C. Potratz, Darren N. Saunders, Daniel H. Wai, et al. Activity in Childhood SarcomasModifiers of Insulin-like Growth Factor-1 Receptor Inhibitor Synthetic Lethality Screens Reveal RPS6 and MST1R as

Updated version

10.1158/0008-5472.CAN-10-1093doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2010/10/18/0008-5472.CAN-10-1093.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/70/21/8770.full#ref-list-1

This article cites 43 articles, 20 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/70/21/8770.full#related-urls

This article has been cited by 9 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] at

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

Permissions

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

.http://cancerres.aacrjournals.org/content/70/21/8770To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-10-1093

http://cancerres.aacrjournals.org/content/suppl/2010/10/18/0008-5472.CAN-10-1093.DC1

http://cancerres.aacrjournals.org/content/70/21/8770.full#ref-list-1

http://cancerres.aacrjournals.org/content/70/21/8770.full#related-urls

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/70/21/8770


cancer research synthetic lethality screens reveal rps6 ... · the insulin-like growth factor-1...

Documents