Trabectedin Efficacy in Ewing Sarcoma Is GreatlyIncreased by Combinationwith Anti-IGF SignalingAgentsAna Teresa Amaral1, Cecilia Garofalo2, Roberta Frapolli3, Maria Cristina Manara2,Caterina Mancarella2, Sarah Uboldi3, Silvana Di Giandomenico3, Jose Luis Ord�o~nez1,Victoria Sevillano1, Roberta Malaguarnera4, Piero Picci2, A. Bass Hassan5,Enrique De Alava1, Maurizio D'Incalci3, and Katia Scotlandi2
Abstract
Purpose: Goal of this study was to identify mechanisms thatlimit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma(EWS), so as todevelop a clinical applicable combination therapy.
Experimental Design: By chromatin immunoprecipitation, weanalyzed EWS–FLI1 binding to the promoters of several targetgenes, such as TGFbR2, CD99, insulin-like growth factor receptor1 (IGF1R), and IGF1, both in vitro and in xenografts treated withtrabectedin or doxorubicin. Combined therapy with trabectedinand anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested invitro and in xenografts.
Results: We confirm that both trabectedin and doxorubicinwere able to strongly reduce EWS–FLI1 (both type I and type II)binding to two representative target genes (TGFbR2 and CD99),both in vitro and in xenografts. However, trabectedin, but not
doxorubicin, was also able to increase the occupancy of EWS–FLI1to IGF1R promoters, leading to IGF1R upregulation. Inhibition ofIGF1R either by the specific AVE1642 human antibody or by thedual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) great-ly potentiate the efficacy of trabectedin in the 13 EWS cell lineshere considered as well as in TC-71 and 6647 xenografts. Com-bined therapy induced synergistic cytotoxic effects. Trabectedinand OSI-906 deliver complementary messages that likely con-verge on DNA-damage response and repair pathways.
Conclusions:We showed that trabectedinmaynot only inhibitbut also enhance the binding of EWS–FLI1 to certain target genes,leading to upregulation of IGF1R. We here provide the rationalefor combining trabectedin to anti-IGF1R inhibitors. ClinCancer Res;21(6); 1373–82. �2015 AACR.
IntroductionEwing sarcoma (EWS) is the second most common primary
developmental bone and soft tissue tumor. It has a very aggressivephenotype andpreferentially occurs in children and young adults.Despite remarkable progress has been achieved in treatment oflocalized disease, where overall cure is now approximately 70%(1, 2), there is still an unmet need for therapy amelioration inpatients with metastatic disease whose overall survival is lower
than 30%. Reduced side effects and improvements in quality oflife are also desirable goal. Unfortunately, few new drugs areavailable for the treatment of patients with EWS and most of therecent results have been achieved thanks to an intensified use ofthe conventional drugs.
One of the few exceptions in this paucity of new therapeuticalternatives is trabectedin (ET-743, Yondelis), a marine derivatefrom the Caribbean tunicate Ecteinascidia turbinata (3), which hasbeen shown to combinedirect cytotoxic activity toward cancer cellswith the peculiar capacity to favorably modify the tumor micro-environment and give potent immunomodulatory effects (for areview see ref. 4). Trabectedin is a tetrahydroisoquinolinemoleculethat binds to the N2 of guanine in theminor groove, causing DNAdamage and affecting transcription regulation in a promoter- andgene-specific manner. Indeed, the specific capabilities of trabecte-din to cause a detachment of the FUS-CHOP chimera, the aberranttranscriptional factor that specifically characterizes myxoid lipo-sarcoma (5), from its target promoters is thought to be responsiblefor the high sensitivity of myxoid/round cell lyposarcoma totrabectedin either in vitro and in vivo (6–10). Similarly, trabectedinwas found to interfere with the activity of EWS–FLI1, the genetichallmark and primary oncogenic driver of EWS (11), reversing theexpression of the EWS–FLI1–induced gene signature and blockingthe promoter activity and expression of critical EWS–FLI1 down-stream targets (12). EWS is characterized by the presence ofbalanced translocations, in which more than 90% of cases presentEWS–FLI1 fusion (EWS–FLI1 type I, II, or III chimeras depending
1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Vir-gen del Rocio/CSIC/Universidad de Sevilla, Department of Pathology,Seville, Spain. 2Experimental Oncology Lab, CRS Development ofBiomolecular Therapies, Rizzoli Institute, Bologna, Italy. 3Departmentof Oncology IRCCS—Istituto di Ricerche FarmacologicheMario Negri,Milan, Italy. 4Department of Health, University of Catanzaro, Catan-zaro, Italy. 5SirWilliamDunnSchool ofPathology,UniversityofOxford,Oxford, United Kingdom.
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
A.T. Amaral and C. Garofalo share first authorship and have contributed equallyto this article.
Corresponding Author: Katia Scotlandi, CRS Development of BiomolecularTherapies, Experimental Oncology Lab, Rizzoli Institute, Via di Barbiano 1/10,40136 Bologna, Italy. Phone: 39-051-6366760; Fax: 39-051-6366763; E-mail:[email protected]
on theexons involved; ref. 13). This hampering effect onEWS–FLI1transcriptional activity is thought to be part of the antitumorefficacy of trabectedin against EWS cells in vitro (14–16). However,phase II clinical trials with trabectedin in EWS reported only amodest activity as single agent (17). In this article, we analyzed theeffects of trabectedin with respect to the insulin-like growth factor(IGF) system based on the evidence that cells made resistant totrabectedin overexpress the IGF receptor 1 (IGF1R) and insulinreceptor substrate (IRS-1; ref. 18). The IGF system is widely rec-ognized to be very important for EWS cells survival andmalignantbehavior (19). Accordingly, EWS is the most sensitive tumor toanti-IGF1R agents, with 10% to 14% of clinical responses whenanti-IGF1R monoclonal antibodies (HAb) were used as singleagents in phase II studies (20–23). EWS–FLI1 is known to interferewith the IGF system either by blocking IGFBP-3 expression (24), amolecule that inhibits IGF signaling by sequestering IGF1 andpreventing its interaction with IGF1R (25), or by inducing IGF1expression (26). In this study, we show that trabectedin, besidescausing detachment of the EWS–FLI1 chimera from some well-recognized target promoters, such as TGFb receptor 2 (TGFbR2;ref. 12), is also able to induce attachment of the chimera to theIGF1R promoter, inducing activation of the IGF1R signaling path-way. This finding strongly supports a combination therapy oftrabectedin with anti-IGF1R agents.
Materials and MethodsDrugs
Trabectedin was provided as lyophilized formulation and asclinical preparation by PharmaMar S.A., Colmenar Viejo. For invitro experiments, trabectedin was dissolved in DMSO. For the invivo studies, the clinical preparationof Yondeliswas used.OSI-906(Linsitinib; Selleck Chemicals) was dissolved in double-distilledwater at the final concentration of 10 mmol/L (stock solution).AVE1642, a humanized version of the anti-IGF1R antibody,EM164 (27),was kindly providedby ImmunoGen Inc. andSanofi.Doxorubicin was purchased from Sigma-Aldrich. Working dilu-tions of all drugs were prepared immediately before use.
Cell linesA panel of 13 EWS human cell lines was analyzed. SK-ES-1, SK-
N-MC, and RD-ES were provided by the American Type Culture
Collection (ATCC); EWS cell lines TC-71 and 6647 were kindlyprovided by T.J. Triche (Children's Hospital, Los Angeles, CA).The EWS cell lines LAP-35, IOR/CAR, and IOR/BRZ-2010 as wellas the TC-71 variants resistant to trabectedin (TC/ET 12 nmol/L)or to anti-IGF1R agents and referred as TC/AVE (resistant toAVE1642 HAb) were obtained in the Experimental OncologyLab, Rizzoli Institute (Bologna, Italy) and as previously described(18, 28, 29). The EWS human cell lines WE-68, RM-82, Cado-ES,and STAET2.1 were kindly provided by F. van Valen (Institute ofExperimental Musculoskeletal Medicine, University HospitalM€unster, M€unster, Germany). All cell lines were recently authen-ticated by short tandem repeat (STR) analysis using genRESVRMPX-2 and genRESVRMPX-3 kits (Serac). The following loci wereverified: D16S539, D18S51, D19S433, D21S11, D2S1338,D3S1358, D5S818, D8S1179, FGA, SE33, TH01, and TPOXVWA.The last control was performed in November 2012. Cells wereroutinely tested forMycoplasma contamination every 3months byMycoAlertMycoplasmadetection set (Lonza). Cultureswere grownin a humidified incubator at 37�C with 5% CO2 and maintainedin standard medium [Iscove Modified Dulbecco's medium,IMDM (Lonza), or RPMI (Gibco, Life Technologies), plus 10%fetal bovine serum (FBS), or 1% Glutamine (Gibco) and 1%Antibiotics (Gibco)].
In vitro assaysTo assess cell growth, the MTT assay (Roche) was used accord-
ing to the manufacturer's instructions. Cells were seeded into 96well-plates (range, 2,500–10,000 cells/well) in standardmedium.After 24 hours, various concentrations of trabectedin (0.3–3nmol/L) or OSI-906 (0.3–3 mmol/L) were added and cells wereexposed up to 72 hours. In combination experiments, cells weretreated for 72 hours with drugs alone (control) or combined infixed ratio 1:1,000.
In vivo antitumor activityFemale athymic nude mice, 6- to 9-week old obtained from
Harlan Italy were used. They were maintained under specificpathogen-free conditions with constant temperature and humid-ity, according to the institutional [Istituto di Ricerche Farmaco-logicheMarioNegri (IRFMN),Milan, Italy] guidelines. TC-71 cells(5 � 106) or 6647 cells (10 � 106) were inoculated subcutane-ously in the right flank of the mice. The growing tumor masseswere measured with the aid of a Vernier caliper, and tumorweights (1 mm3 ¼ 1 mg) were calculated using the formula:length � (width)2/2. When tumor load reached about 100 mg,mice were randomized into experimental groups and treatmentwas started.
Study groups were listed as follows (at least 8 mice per group):Placebo, 0.9%NaCl; trabectedin, 0.15mg/kg; doxorubicin, 8mg/kg; and AVE1642 HAb, 40 mg/kg. Drugs were administered i.v.:trabectedin every 7 days for three times (q7d � 3); doxorubicinevery 7 days for two times (q7d� 2); AVE1642 HAb every 3 daysfor six times (q3d � 6). Drug efficacy was calculated as T/C%,where T and C are the mean tumor weights of treated and controlgroups, respectively. Treatmentwas considered effectivewhenT/C<42%. Procedures were conducted in conformity with the insti-tutional guidelines that are in compliance with national (Legis-lative Decree 116 of Jan. 27, 1992 Authorisationn.169/94-Aissued Dec. 19, 1994 by Ministry of Health) and internationallaws and policies (EEC Council Directive 86/609, OJ L 358. 1,December 12, 1987; Standards for the Care andUse of Laboratory
Translational Relevance
Trabectedin (ET-743, Yondelis) is one of the fewnovel drugsrecently proposed for treatment of patients with sarcoma.However, in clinical setting, the activity observed in Ewingsarcoma (EWS) was quite modest. This work demonstratesthat trabectedin is not only able to inhibit binding to DNA ofthe transcriptional factor EWS–FLI1, the genetic hallmark ofEWS, but also increases its attachment on preferentialsequences. Herein, we found enhanced binding to the IGF1Rpromoter, which resulted in increased IGF1R expression.Considering the relevance of the IGF system in EWS, thisevidence might very well explain why trabectedin has shownlimited efficacy in monotherapeutic regimens in clinical set-ting and provides the rationale for development of a therapythat combines trabectedin with anti-IGF signaling agents.
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Animals, United States National Research Council, Statement ofCompliance A5023-01, November 6, 1998). Animal experimentswere reviewed and approved by the IRFMN Animal Care and UseCommittee (IACUC) that includes members for ethical issues.
Western blottingCells were treated or not (control) with trabectedin (0.5–2.5
nmol/L) orwithOSI-906 (400nmol/L) up to 48hours or silencedfor EWS–FLI1 (75–100 nmol/L siRNA; ref. 30) and lysed aspreviously described (18). The following primary antibodies (Ab)were used: anti-PARP; cleaved-caspase-3, anti-NF-H 200k (CellSignaling Technology); anti-IGF1Rb, anti-FLI, anti-b-actin, andGAP-DH (Santa Cruz Biotechnology); anti-rabbit or anti-mouseantibodies conjugated to horseradish peroxidase (GEHealthcare)were used as secondary antibodies.
Chromatin immunoprecipitationIn vitro and in vivo chromatin immunoprecipitation (ChIP)
assays were performed as previously described (6, 28) using anti-FLI1 (C-19; SantaCruz Biotechnology) and/or anti-FLAG (Sigma)antibodies. PCR was performed with primers flanking Ets-con-taining target promoters fragment (listed in Supplementary TableS1). Amplification products obtained were observed in 1.5%agarose gel with Gel Red staining. For quantitative PCR (qPCR),data are indicated as fold enrichment respect to untreated cells invitro experiments or to placebo in xenografts and calculated usingfollowing formula: % of recruitment ¼ 2DCt � input chromatinpercentage where DCt¼Ct (input)�Ct (FLI1 IP) in accordance toFrank and colleagues (31, 32).
For TaqMan assay design TFSEARCH—Searching TranscriptionFactor Binding Sites, version 1.3 free website was used for theprediction of ETS binding sites in the promoter of IGF1R gene andthe sequence spanning from 1041 bp to 1051 bpwas identified asthe best. BeaconDesigner 4 softwarewas used for the design of theassay spanning from 1005 bp to 1114 bp. CD99 and TGFbR2promoter fragments containing ETS consensus sequence wereused as EWS-FLI immunoprecipitation controls (28, 30).
Immunofluorescence assaysCells were seeded in single slide covers placed in 96-well plates,
pretreated with gelatin (Sigma), and grown in standard medium.After exposure to drug/DMSO for 24 hours, cells were fixed in ice-cold-methanol, permeabilized with Triton X-100, and processedfor immunofluorescence. Primary antibodies as follows: pH2AX(1:100; Cell Signaling Technology); 53BP1 (1:100; Abcam), anti-tubulin b III (dilution 1:50), or anti-H neurofilament 200 kD(clone NE14; dilution 1:40; Sigma). Cells were then stained withthe secondary antibody Cy5 (Jackson ImmunoResearch) andcounterstained with DAPI 1 mg/mL (DAKO). Slide covers weremounted in covers with Mowiol fixing agent (Sigma) and cellsobserved in a Leica Microscope using software LEICA software.
RNA extraction and low-density microarrays by qRT-PCRRNA extraction was performed using Qiagen RNA extraction
kit, following the manufacturer's instructions, as described else-where (26, 33). Retrotranscriptionwas performed using 500 ng oftotal RNA. Low-density microarray, in the form of qRT-PCR 96-well plates Human DNA Repair PCR Array (Qiagen) were per-formed using the iQ5 thermocycler from Bio-Rad. RT-PCR wasrun in the iQ5 BIO-RAD thermocycler with the following proto-
col: 95�C for 10minutes; 40 cycles of 42�C10minutes and 60�C1minute. Results were evaluated using the iQ5 software from Bio-Rad and the online platform (http://pcrdataanalysis.sabios-ciences.com/pcr/arrayanalysis.php). Pathway analysis was per-formed using the Ingenuity Pathway Analysis (IPA) software(Ingenuity system).
Activity of caspase-3 and caspase-7Caspases-3 and -7 activities were determined in cells exposed to
drug/DMSO for 48 hours using the Glow caspase luminescent kitby Promega according to the manufacturer's instructions. Lumi-nescence was read in a plate reader (Tecan).
ImmunohistochemistrySections (5 mm) from formalin-fixed, paraffin-embedded TC-
71 xenografts were placed on poly-L-lysine–coated slides (Sig-ma). Avidin-biotin-peroxidase procedure was used for immu-nostaining, as previously described (34), and slides werestained with anti-tubulin b III (dilution 1:50), anti-H neuro-filament 200 kD (clone NE14; dilution 1:40; Sigma) andIGF1Rb (Santa Cruz Biotechnology). Detection of Ki-67 wasperformed on sections as previously described (34). The ter-minal deoxynucleotidyl transferase–mediated dUTP nick endlabeling (TUNEL) assay was performed with ApopTag PlusPeroxidase in situ apoptosis kit (Merck Millipore) according tothe manufacturer's instructions.
Statistical analysisCorrelations between two variables were obtained by the
Spearman test. IC50 values were calculated from linear transfor-mation of dose–response curves. To define drug–drug interac-tions (in terms of synergism, additivity, or antagonism), thecombination index (CI) was calculated with the isobologramequation (35) by using the CalcuSyn software (Biososoft). Differ-ences among means were analyzed by the Student t test or theANOVA test.
ResultsTrabectedin disrupts EWS–FLI1 binding to some DNA targetsbut increases recruitment to IGF1R promoter in both in vitroand in vivo models
We used ChIP analysis to monitor the binding of EWS–FLI1chimera to some well-known target genes, such as TGFbR2 andCD99, reported tobemodulated byEWS–FLI1 andproven tohavea major role in EWS aggressiveness (28), as well as to IGF1R andIGF1 promoters. ChIP indicated that the amount of EWS–FLI1chimera bound to the TGFbR2 and CD99 promoters was signif-icantly reduced after 1 hour treatment with trabectedin both inTC-71 cells, displaying EWS–FLI1 type I chimera, and in the 6647cell line, that displays EWS–FLI1 type II hybrid, at pharmacologicconcentrations (IC50 value after 1 hour of treatment; Fig. 1A). Thebinding of the chimera EWS–FLI1 type I (TC-71) and type II(6647) to the CD99 and TGFbR2 promoters was evaluatedalso in mouse xenografts after i.v. administration of trabectedin(0.15 mg/kg, every 7 days for three times, q7d � 3) and doxo-rubicin (8 mg/kg, every 7 days for two times, q7d� 2). As shownin Supplementary Fig. S1, in TC-71 EWS model trabectedinwas more active (best T/C 56.2% at days 20) than doxorubicin(reference compound, best T/C 79.5% at days 22). Insteadin 6647 xenograft model, doxorubicin was extremely effective
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Figure 1.Trabectedin caused a dysregulation in EWS–FLI1 chimera binding to specific promoters. ChIP assays were carried out in vitro on TC-71 and 6647 EWScells, or in xenografts after treatments with trabectedin or doxorubicin. EWS–FLI1 was precipitated by the anti-FLI1 antibody. Decrease in the binding of the chimeraEWS–FLI1 type I (TC-71) and type II (6647) to CD99 and TGFbR2 promoters was observed either in in vitro (A) or in xenografts (B) treated as described in Materialsand Methods. Results obtained by qPCR are reported as fold enrichment over the controls (untreated in vitro cells; placebo-treated mice) according to thefollowing formula:%of recruitment¼ 2DCt� input chromatin percentagewhereDCt¼Ct (input)�Ct (FLI1 IP; refs. 31, 32) �,P<0.05; �� ,P<0.001, Student t test. C, left,increased recruitment of EWS–FLI1 on IGF1R promoter in TC-71 and 6647 EWS cells treated for 1 hour with trabectedin or doxorubicin. A representative experiment isshown. Data represent recovery of each DNA fragment relative to total input DNA, respect to control. � , P < 0.05; Student t test. Right, upregulation ofIGF1R at protein level byWestern blotting after exposure to trabectedin (0.5–1 nmol/L) up to 48 hours. GAPDHwas used as loading control. Blots are representativeof two independent experiments. D, time course of FLI1 association with the IGF1R promoter in TC-71 and 6647 xenografts treated with trabectedin (0.15 mg/kg).� , P < 0.05; Student t test.
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(best T/C 14.3% at days 21)while trabectedinwas less active (bestT/C 48.6% at days 21). In both cases, however, trabectedinand doxorubicin were able to displace EWS–FLI1 chimerafrom CD99 and TGFbR2 promoters, although with differentkinetics (Fig. 1B). Trabectedin was able to cause detachment ofthe EWS–FLI1 chimera from both CD99 and TGFbR2 promotersalready 24 hours after the first dose both in TC-71 and 6647cells. Reattachment was observed starting 7 days from the thirdtreatment.
Besides inhibitory effects, trabectedin but not doxorubicin alsocaused a dose- and time-dependent increase in the binding of thechimera to IGF1R promoter in EWS cells (Fig. 1C and Supple-mentary Fig. S2A), while occupancy of the IGF1 promoterappeared to be only slightly affected (Supplementary Fig. S2B).Upregulation of IGF1Rb was also confirmed at protein level aftertrabectedin treatment (Fig. 1C), in line with our previous datashowing increased transcription and expression of IGF1Rb in cellsmade resistant to trabectedin (18). Consistently, silencing ofEWS–FLI1 in TC-71 cells induced downregulation of IGF1Rbprotein (Supplementary Fig. S3). Enhancement of EWS–FLI1occupancy to IGF1R by trabectedin was also observed in vivo inTC-71 and 6647 xenografts (Fig. 1D), further sustaining therelationship between EWS-FLI and IGF1R. These findings provid-ed the rationale for testing the combination of trabectedin withanti-IGF1R agents.
Antitumor activity of the combination between trabectedin andthe anti-IGF1R HAb AVE1642
Antitumor activity of trabectedin alone or in combination withanti-IGF1R AVE1642 HAb was evaluated in TC-71 xenograftmodel. The combination trabectedin and AVE1642 HAb (bestT/C 27.9 at days 20) showed a greater antitumor activity thantrabectedin (best T/C 40.3 at days 20) or AVE1642 HAb (best T/C48.6 at days 15) used as single agents (Fig. 2A). Studies inmyxoid liposarcoma indicate that trabectedin besides inhibit-ing cell proliferation can also act as a differentiating agent byblocking the transactivating ability of the fusion gene product(8). We confirmed the antiproliferative, proapoptotic, andprodifferentiating activity of trabectedin also in EWS (Fig. 2Band Supplementary Fig. S4). Moreover, in keeping with ChIPfindings, xenografts treated with trabectedin showed increasedexpression of IGF1Rb (Fig. 2B). Combination treatments withAVE1642 HAb further inhibited tumor cell proliferation andIGF1R expression while increased apoptotic rate (Fig. 2B). Thissupports the combination of trabectedin with anti-IGF1R HAbagainst EWS.
Efficacy of the dual inhibitor anti-IGF1R/IR OSI-906 incombination with trabectedin
Considering that the great majority of EWSs express concom-itant high levels of IR, which may overcome the IGF1R blockade
Figure 2.Efficacy of the combination oftrabectedin with anti-IGF1R HAbAVE1642 against TC-71 xenografts.A, drugs were administered i.v. asfollows: trabectedin 0.15 mg/kg, every7 days for three times andAVE1642 40mg/kg, every 3 days for six times.ANOVA test: � , P < 0.05; �� , P < 0.01;��� , P < 0.001 compared with controls(Placebo). Points, tumor weightmeans; bars, SE. B, representativeimmunohistochemical evaluation ofKi-67, apoptotic rate by TUNEL assay,and IGF1Rb (magnification, �200), inuntreated or treated tumors.Percentages � SE of positive cells areindicated. � , P < 0.05; �� , P < 0.001,Student t test.
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(29, 36), efficacy of the dual inhibitor anti-IGF1R/IROSI-906wasevaluated in a panel of 13 EWS cell lines (Table 1), including theTC/ET 12nmol/L cell line, highly resistant to trabectedin (18) andTC/AVE, resistant to anti-IGF1R AVE1642 HAb (29) Most of cell
lines were highly sensitive to the inhibitor with submicromolarIC50 values (Table 1).
The combination of OSI-906 with trabectedin gave synergisticeffects in all EWS cell lines, including cells resistant to AVE1642
Table 1. Efficacy of combined treatments of Trabectedin with OSI-906 in EWS cell lines
Cell lines OSI-906 (mmol/L) � SE Trabectedin (nmol/L) � SE Combination (nmol/L) � SEa CI � SEb Effects
Figure 3.Effects of the dual inhibitor anti-IGF1R/IR OSI-906 in combination with trabectedin. A, caspase cleavage activation (RLU) in TC-71 and WE-68 treated withtrabectedin and/or OSI-906 for 48 hours. All treatments are normalized respect to control. Bars, mean of two independent experiments � SE. �� , P < 0.001,Student t test respect to control. B, network and pathway analysis of genes significantly modulated after TC-71 cell exposure to drug combination. Low-densityexpression array focusing on DNA damage and DNA repair genes was used.
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HAb (TC/AVE) or trabectedin (TC/ET 12 nmol/L; Table 1). WhenOSI-906was combined to trabectedin, we observed advantageouseffects in terms of apoptosis, both in p53wt (WE-68) and p53-mutated cells (TC-71; Fig. 3A). This advantageous proapoptoticcell death may derive from the combination of two differentinputs. Although inhibition of the IGF system may block anti-apoptotic effects of IGF1R/IR-A due to disruption of AKT and/or14.3.3/Raf-1/Nedd4 pathways (37, 38), trabectedin acts as aDNA-damaging agent inducing double-strand breaks (DSB;ref. 3). To further characterize drug effects on DNA repair path-ways, we used DNA damage low-density arrays customized tocover homologous recombination (HR), nucleotide excisionrepair (NER), base excision repair (BER), and non-homologousend joining repair (NHEJR) pathways. Treatment with trabecte-din, but not withOSI-906, induced expression of BRCA1, BRCA2,key proteins in HR pathway (39) as well as XRCC1, member ofsingle-strand break repair (SSR) pathway (Supplementary Fig.S5). The drug combination resulted in upregulation of membersfrom theHRpathway (RAD52, BRCA1andBRCA2),NERproteins(XPA and ERCC1), and SSR pathways (XRCC1; Fig. 3B). Asvalidation, we also studied DNA-damage induction by presenceof pH2AX and 53BP1 intranuclear foci after 24 hours treatment(Fig. 4A), showing that trabectedin was a potent DSB inducer, incontrast toOSI-906, and thatDNAdamagewas presentwhen cellswere treated with the combination of trabectedin and OSI-906.DNA fragmentation assay showed that besides trabectedin alsoOSI-906 alone and particularly the combination of the two drugsleads to DNA fragmentation (Fig. 4B). The presence of DNA
fragments (<500 bp) after cell exposure to OSI-906 is not sur-prising because these fragments are likely to be related to DNAdegradationby apoptosis. Accordingly,OSI-906 favors expressionof apoptotic proteins such as cPARP, fraction 90 kDa (Fig. 4C).PARP cleavagewas also observed in the TC/ET 12nmol/L resistantcell line after treatment with increasing doses of OSI-906, thusconfirming complementary proapoptotic effects of the two drugs(Fig. 4D).
DiscussionTrabectedin is a newly licensed chemotherapeutic agent, with
an acceptable toxicity profile (40). In this article, we show thattrabectedin may be advantageously used in combination withinhibitors of the IGF system. Particularly, we provide a rationalefor the use of this combination, showing that trabectedin is able toincrease IGF1R expression by enhancing EWS–FLI1 occupancy ofthe IGF1R promoter. EWS–FLI1 drives the malignant phenotypein EWS cells (11), acting either as a transcriptional activator or as atranscriptional repressor in EWS. Importantly, both the activatingand repressive functions of EWS–FLI1 are required for its onco-genic functions (41). Trabectedin has been previously found toblock the promoter activity and expression of critical EWS–FLI1downstream targets, and a combination with SN38 (the activemetabolite of irinotecan) has been proposed to augment thesuppression of EWS–FLI1 activity (16). However, in this article,we show that these inhibitory effects, although probably preva-lent, are not exclusive. In fact, while we confirm that both
Figure 4.DNA-damage induction after cellexposure to trabectedin and/orOSI-906. A, induction of H2AXphosphorylation (g-H2AX) and53BP1 expression in TC-71 cells afterexposure to trabectedin, and/or OSI-906 (200–400 nmol/L) for 24 hours(magnification, � 400). B, DNAfragmentation induced in TC-71 cellsafter 12 to 24 hours exposure to drugs.C and D, evaluation of caspase-3 andPARP cleavage by Western blottingafter 24 hours exposure to drugs.Equal loading was monitored withb-actin blotting.
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trabectedin and doxorubicin are able to strongly suppress thebinding of EWS–FLI1 (both type I and type II) to two target genes(TGFbR2 and CD99) both in vitro and in vivo, a significantenhancement of EWS–FLI1 occupancy on the IGF1R promoterwasobserved only after exposure to trabectedin. Prior studies havedemonstrated that other DNA-binding agents, including mithra-mycin, and actinomycin D, reduced expression of EWS–FLI1downstream targets and displayed differential specificity, likelydue to preferential sequence binding affinities (42, 43). Thisdiscovery introduces a certain level of specificity in the action ofconventional agents, which is potentially very interesting, butrequires further studies because the effects may vary in relation todrugs, transcription factors, and cellular context. For example,doxorubicin did not affect the binding of FUS-CHOP to targetpromoters inmyxoid liposarcoma (6), whereas here it was shownto inhibit occupancy of EWS–FLI1 on TGFbR2 and CD99 pro-moters, indicating differences among transcriptional hybrids anddrug action in different cellular contexts. By also reporting anincrease and not just a suppression of EWS–FLI1 binding tospecific target promoters, we introduced another variable thatdeservesmore ample investigation. In the specific context of EWS,the increase in IGF1R expression is absolutely reasonable from abiologic point of view considering the importance that the IGFsystem has in maintenance of EWSmalignancy (19, 44), and wasindeed confirmed also in cells made resistant to trabectedin (18).From a clinical point of view, increased expression of IGF1R inresponse to in vitro and in vivo exposure to trabectedin, providesthe rationale for a combined use of trabectedin with anti-IGF1Ragents. Here, we demonstrate the advantages of this combinationeither using HAb AVE1642, a well-tolerated agent that bindshuman IGF1R specifically and with high affinity (45, 46), or thedual inhibitor IGF1R/IR, OSI-906, a small molecule shown tohave antitumoral activity against several tumors (47, 48), includ-ing osteosarcoma (49). The association of OSI-906 with trabec-
tedin gave synergistic effects in all of the 13 EWS cell lines hereconsidered, including cells resistant to trabectedin (18) or to anti-IGF1R agents (29). This appears to be due mainly to the com-plementary proapoptotic effects of the two drugs that by affectingdifferent pathways give rise to a combination able to deliver celldeath messages in all EWS cells, independently from the p53status. Although treatment with IGF1R antagonists is known tolead to downregulation of the proteins involved in cell survivaland inhibition of cell death (38, 47), thereby recovering cellsensitivity to apoptosis, trabectedin has been previously describedas a potent DNA-damaging agent. Trabectedin binds to guaninesin the minor groove with some degree of sequence specificity,inducing SSB that rapidly turn into DSB, the most lethal form ofDNA damage. Tavecchio and colleagues (50) clearly showed thatDSBs are not directly caused by the drug, but are formed duringthe processing/repair of the drug, requiring a functional HRpathway. In addition, trabectedin also poisons the mechanismsof DNA repair through the formation of ternary NER proteins–DNA–trabectedin complexes (for a review see ref. 51). Our resultsdemonstrated that in EWScells trabectedin increases expressionofBRCA1, BRCA2, key proteins in the HR pathway as well as ofXRCC1, which is involved in the SSR pathway, resulting in DNAdamage as indicated by phosphorylation of histone H2AX andaccumulation of intranuclear foci. The drug combination withOSI-906 maintains and even increases upregulation of membersofHR (RAD52, BRCA1, and BRCA2), NER (XPA and ERCC1), andSSR (XRCC1) pathways but also induces a strong downregulationof XRCC4 and XRCC6 as well as of MSH4 and MSH5, twomolecules involved in the maintenance of genomic stability andmitotic DSB repair (52), indicating general alterations of DNA-damage response and repair pathways. This is in line with recentevidence that IGF1R inhibition induces a direct functional defectin DSB repair by both NHEJ and HR, besides indirectly impairingHR through influences on the expression and/or activation of cell-
Figure 5.Schematic representation oftrabectedin activity in EWS.Trabectedin may not only inhibit (1)but also enhance the binding of EWS–FLI1 to target genes. Specifically, IGF1Rexpression is activated after treatmentwith trabectedin (3), thereforeproviding the rationale for combinedtreatments with anti-IGF1R agents.
Amaral et al.
Clin Cancer Res; 21(6) March 15, 2015 Clinical Cancer Research1380
cycle regulators (53). The importance of trabectedin and IGFsystem inhibitors in DNA-damage response and repair pathways,which have implications on the therapeutic efficacy and potentialtoxicity of this combined therapy in the clinic, deserve furtherresearch to better elucidate the molecular mechanisms and pro-tein interactions.
Overall, we provide the rationale for combining trabectedin toanti-IGF1R inhibitors. We showed that trabectedin may not onlyinhibit but also enhance binding of EWS–FLI1 to target genes (Fig.5). Specifically, IGF1R expression activated after treatment withtrabectedin and anti-IGF1R agents improve efficacy of trabectedinin cell lines and xenografts. We thus propose the use of acombination therapy that by exploiting the complementarymechanisms of action of the two drugs may have therapeuticpotential.
Disclosure of Potential Conflicts of InterestM. D'Incalci is a consultant/advisory board member for PharmaMar. No
potential conflicts of interest were disclosed by the other authors.
Authors' ContributionsConception and design: C. Garofalo, P. Picci, A. Bass Hassan, E. De Alava,M. D'Incalci, K. ScotlandiDevelopment of methodology: A.T. Amaral, C. Garofalo, M.C. Manara,C. Mancarella, S. Uboldi, S. Di Giandomenico, J.L. Ord�o~nez, V. Sevillano,R. Malaguarnera, P. Picci, E. De Alava, M. D'IncalciAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.T. Amaral, C. Garofalo, R. Frapolli, M.C. Manara,V. Sevillano, P. PicciAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.T. Amaral, C. Garofalo, M.C. Manara, C. Mancar-ella, S. Uboldi, J.L. Ord�o~nez, P. Picci, K. Scotlandi
Writing, review, and/or revision of the manuscript: A.T. Amaral, C. Garofalo,S. Uboldi, J.L. Ord�o~nez, P. Picci, A. Bass Hassan, E. De Alava, M. D'Incalci,K. ScotlandiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Garofalo, C. Mancarella, R. MalaguarneraStudy supervision: E. De Alava, M. D'Incalci, K. ScotlandiOther (involved in funding of the project from EuroSarc grant): A. BassHassan
AcknowledgmentsThe authors thank Cristina Ghinelli and Alba Balladelli for article
editing.
Grant SupportThis work was financially supported by the EU project FP7-HEALTH-2011-
two-stage, Project ID 278742 EUROSARC; the Italian Association for CancerResearch (Katia Scotlandi—AIRCProjectN.14049;C.Garofalo—MFAGN.11584and M. D'Incalci—AIRC Project N.14658); The Italian Ministry of Research andEducation (F.I.R.B. project number: RBAP11884M_005); The Italian Ministry of
the Health (67/GR-2010-2319511, CUP G71J12000830001). E. de �Alava'slaboratory is supported by the AECC (Asociaci�on Espa~nola contra el C�ancer),the Ministry of Economy and Competitivity of Spain-FEDER (PI081828, RD06/0020/0059 RD12/0036/0017, PT13/0010/0056, PI110018, ISCIII Sara Borrellpostdoc grant CD06/00001), and Fundaci�on Memoria de D. Manuel SolorzanoBarruso,Fundaci�onCris contra el cancer, andFundaci�onMaríaGarcía Estrada. J.L.Ord�o~nez is sponsored by the CSIC and the European Social Fund (postdoctoralgrant JAE DOC), A.T. Amaral is sponsored by the Fundacao para a Ciencia eTecnologia, Portugal (fellowship SFRH/BD/69318/2010); and S. Uboldi isrecipient of a FIRC Fellowship N.13743.
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 July 1, 2014; revised December 11, 2014; accepted January 1, 2015;published OnlineFirst January 21, 2015.
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2015;21:1373-1382. Published OnlineFirst January 21, 2015.Clin Cancer Res Ana Teresa Amaral, Cecilia Garofalo, Roberta Frapolli, et al. Combination with Anti-IGF Signaling AgentsTrabectedin Efficacy in Ewing Sarcoma Is Greatly Increased by
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