Trabectedin Efficacy in Ewing Sarcoma Is Greatly Increased by Combination with Anti-IGF Signaling Agents

Published OnlineFirst January 21, 2015; DOI: 10.1158/1078-0432.CCR-14-1688

Cancer Therapy: Preclinical Clinical Cancer Research Trabectedin Efﬁcacy in Ewing Sarcoma Is Greatly Increased by Combination with Anti-IGF Signaling Agents Ana Teresa Amaral1, Cecilia Garofalo2, Roberta Frapolli3, Maria Cristina Manara2, Caterina Mancarella2, Sarah Uboldi3, Silvana Di Giandomenico3, Jose Luis Ordonez~ 1, 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 that doxorubicin, was also able to increase the occupancy of EWS–FLI1 limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma to IGF1R promoters, leading to IGF1R upregulation. Inhibition of (EWS), so as to develop a clinical applicable combination therapy. IGF1R either by the specific AVE1642 human antibody or by the Experimental Design: By chromatin immunoprecipitation, we dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) great- analyzed EWS–FLI1 binding to the promoters of several target ly potentiate the efficacy of trabectedin in the 13 EWS cell lines genes, such as TGFbR2, CD99, insulin-like growth factor receptor here considered as well as in TC-71 and 6647 xenografts. Com- 1(IGF1R), and IGF1, both in vitro and in xenografts treated with bined therapy induced synergistic cytotoxic effects. Trabectedin trabectedin or doxorubicin. Combined therapy with trabectedin and OSI-906 deliver complementary messages that likely con- and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in verge on DNA-damage response and repair pathways. vitro and in xenografts. Conclusion: We showed that trabectedin may not only inhibit Results: We confirm that both trabectedin and doxorubicin but also enhance the binding of EWS–FLI1 to certain target genes, were able to strongly reduce EWS–FLI1 (both type I and type II) leading to upregulation of IGF1R. We here provide the rationale binding to two representative target genes (TGFbR2 and CD99), for combining trabectedin to anti-IGF1R inhibitors. Clin Cancer Res; both in vitro and in xenografts. However, trabectedin, but not 21(6); 1–10. 2015 AACR.

Introduction than 30%. Reduced side effects and improvements in quality of life are also desirable goal. Unfortunately, few new drugs are Ewing sarcoma (EWS) is the second most common primary available for the treatment of patients with EWS and most of the developmental bone and soft tissue tumor. It has a very aggressive recent results have been achieved thanks to an intensified use of phenotype and preferentially occurs in children and young adults. the conventional drugs. Despite remarkable progress has been achieved in treatment of One of the few exceptions in this paucity of new therapeutic localized disease, where overall cure is now approximately 70% alternatives is trabectedin (ET-743, Yondelis), a marine derivate (1, 2), there is still an unmet need for therapy amelioration in from the Caribbean tunicate Ecteinascidia turbinata (3), which has patients with metastatic disease whose overall survival is lower been shown to combine direct cytotoxic activity toward cancer cells with the peculiar capacity to favorably modify the tumor micro- 1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Vir- environment and give potent immunomodulatory effects (for a gen del Rocio/CSIC/Universidad de Sevilla, Department of Pathology, review see ref. 4). Trabectedin is a tetrahydroisoquinoline molecule 2 Seville, Spain. Experimental Oncology Lab, CRS Development of that binds to the N2 of guanine in the minor groove, causing DNA Biomolecular Therapies, Rizzoli Institute, Bologna, Italy. 3Department of Oncology IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, damage and affecting transcription regulation in a promoter- and Milan, Italy. 4Department of Health, University of Catanzaro, Catan- gene-specific manner. Indeed, the specific capabilities of trabecte- 5 zaro, Italy. Sir William Dunn School of Pathology, University of Oxford, din to cause a detachment of the FUS-CHOP chimera, the aberrant Oxford, United Kingdom. transcriptional factor that specifically characterizes myxoid lipo- Note: Supplementary data for this article are available at Clinical Cancer sarcoma (5), from its target promoters is thought to be responsible Research Online (http://clincancerres.aacrjournals.org/). for the high sensitivity of myxoid/round cell lyposarcoma to A.T. Amaral and C. Garofalo are the first authors and have contributed equally to trabectedin either in vitro and in vivo (6–10). Similarly, trabectedin this article. was found to interfere with the activity of EWS–FLI1, the genetic Corresponding Author: Katia Scotlandi, CRS Development of Biomolecular hallmark and primary oncogenic driver of EWS (11), reversing the Therapies, Experimental Oncology Lab, Rizzoli Institute, Via di Barbiano 1/10, expression of the EWS–FLI1–induced gene signature and blocking 40136 Bologna, Italy. Phone: 39-051-6366760; Fax: 39-051-6366763; E-mail: the promoter activity and expression of critical EWS–FLI1 down- [email protected] stream targets (12). EWS is characterized by the presence of doi: 10.1158/1078-0432.CCR-14-1688 balanced translocations, in which more than 90% of cases present 2015 American Association for Cancer Research. EWS–FLI1 fusion (EWS–FLI1 type I, II, or III chimeras depending

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Collection (ATCC); EWS cell lines TC-71 and 6647 were kindly Translational Relevance provided by T.J. Triche (Children's Hospital, Los Angeles, CA). Trabectedin (ET-743, Yondelis) is one of the few novel drugs The EWS cell lines LAP-35, IOR/CAR, and IOR/BRZ-2010 as well recently proposed for treatment of patients with sarcoma. as the TC-71 variants resistant to trabectedin (TC/ET 12 nmol/L) However, in clinical setting, the activity observed in Ewing or to anti-IGF1R agents and referred as TC/AVE (resistant to sarcoma (EWS) was quite modest. This work demonstrates AVE1642 HAb) were obtained in the Experimental Oncology that trabectedin is not only able to inhibit binding to DNA of Lab, Rizzoli Institute (Bologna, Italy) and as previously described the transcriptional factor EWS–FLI1, the genetic hallmark of (18, 28, 29). The EWS human cell lines WE-68, RM-82, Cado-ES, EWS, but also increases its attachment on preferential and STAET2.1 were kindly provided by F. van Valen (Institute of sequences. Herein, we found enhanced binding to the IGF1R Experimental Musculoskeletal Medicine, University Hospital promoter, which resulted in increased IGF1R expression. Munster,€ Munster,€ Germany). All cell lines were recently authen- Considering the relevance of the IGF system in EWS, this ticated by short tandem repeat (STR) analysis using genRESVR evidence might very well explain why trabectedin has shown MPX-2 and genRESVR MPX-3 kits (Serac). The following loci were limited efficacy in monotherapeutic regimens in clinical set- verified: D16S539, D18S51, D19S433, D21S11, D2S1338, ting and provides the rationale for development of a therapy D3S1358, D5S818, D8S1179, FGA, SE33, TH01, and TPOX VWA. that combines trabectedin with anti-IGF signaling agents. The last control was performed in November 2012. Cells were routinely tested for Mycoplasma contamination every 3 months by MycoAlert Mycoplasma detection set (Lonza). Cultures were grown in a humidified incubator at 37 C with 5% CO2 and maintained fi on the exons involved; ref. 13). This hampering effect on EWS–FLI1 in standard medium [Iscove Modi ed Dulbecco's medium, transcriptional activity is thought to be part of the antitumor IMDM (Lonza), or RPMI (Gibco, Life Technologies), plus 10% efficacy of trabectedin against EWS cells in vitro (14–16). However, fetal bovine serum (FBS), or 1% Glutamine (Gibco) and 1% phase II clinical trials with trabectedin in EWS reported only a Antibiotics (Gibco)]. modest activity as single agent (17). In this article, we analyzed the In vitro effects of trabectedin with respect to the insulin-like growth factor assays (IGF) system based on the evidence that cells made resistant to To assess cell growth, the MTT assay (Roche) was used accord- trabectedin overexpress the IGF receptor 1 (IGF1R) and insulin ing to the manufacturer's instructions. Cells were seeded into 96 – receptor substrate (IRS-1; ref. 18). The IGF system is widely rec- well-plates (range, 2,500 10,000 cells/well) in standard medium. – ognized to be very important for EWS cells survival and malignant After 24 hours, various concentrations of trabectedin (0.3 3 – behavior (19). Accordingly, EWS is the most sensitive tumor to nmol/L) or OSI-906 (0.3 3 mmol/L) were added and cells were anti-IGF1R agents, with 10% to 14% of clinical responses when exposed up to 72 hours. In combination experiments, cells were anti-IGF1R monoclonal antibodies (HAb) were used as single treated for 72 hours with drugs alone (control) or combined in fi agents in phase II studies (20–23). EWS–FLI1 is known to interfere xed ratio 1:1,000. with the IGF system either by blocking IGFBP-3 expression (24), a In vivo molecule that inhibits IGF signaling by sequestering IGF1 and antitumor activity preventing its interaction with IGF1R (25), or by inducing IGF1 Female athymic nude mice, 6- to 9-week old obtained from fi expression (26). In this study, we show that trabectedin, besides Harlan Italy were used. They were maintained under speci c causing detachment of the EWS–FLI1 chimera from some well- pathogen-free conditions with constant temperature and humid- recognized target promoters, such as TGFb receptor 2 (TGFbR2; ity, according to the institutional [Istituto di Ricerche Farmaco- ref. 12), is also able to induce attachment of the chimera to the logiche Mario Negri (IRFMN), Milan, Italy] guidelines. TC-71 cells 6 6 IGF1R promoter, inducing activation of the IGF1R signaling path- (5 10 ) or 6647 cells (10 10 ) were inoculated subcutane- fl way. This finding strongly supports a combination therapy of ously in the right ank of the mice. The growing tumor masses trabectedin with anti-IGF1R agents. were measured with the aid of a Vernier caliper, and tumor weights (1 mm3 ¼ 1 mg) were calculated using the formula: 2 Materials and Methods length (width) /2. When tumor load reached about 100 mg, mice were randomized into experimental groups and treatment Drugs was started. Trabectedin was provided as lyophilized formulation and as Study groups were listed as follows (at least 8 mice per group): clinical preparation by PharmaMar S.A., Colmenar Viejo. For in Placebo, 0.9% NaCl; trabectedin, 0.15 mg/kg; doxorubicin, 8 mg/ vitro experiments, trabectedin was dissolved in DMSO. For the in kg; and AVE1642 HAb, 40 mg/kg. Drugs were administered i.v.: vivo studies, the clinical preparation of Yondelis was used. OSI-906 trabectedin every 7 days for three times (q7d 3); doxorubicin (Linsitinib; Selleck Chemicals) was dissolved in double-distilled every 7 days for two times (q7d 2); AVE1642 HAb every 3 days water at the final concentration of 10 mmol/L (stock solution). for six times (q3d 6). Drug efficacy was calculated as T/C%, AVE1642, a humanized version of the anti-IGF1R antibody, where T and C are the mean tumor weights of treated and control EM164 (27), was kindly provided by ImmunoGen Inc. and Sanofi. groups, respectively. Treatment was considered effective when T/C Doxorubicin was purchased from Sigma-Aldrich. Working dilu- <42%. Procedures were conducted in conformity with the insti- tions of all drugs were prepared immediately before use. tutional guidelines that are in compliance with national (Legis- lative Decree 116 of Jan. 27, 1992 Authorisationn.169/94-A Cell lines issued Dec. 19, 1994 by Ministry of Health) and international A panel of 13 EWS human cell lines was analyzed. SK-ES-1, SK- laws and policies (EEC Council Directive 86/609, OJ L 358. 1, N-MC, and RD-ES were provided by the American Type Culture December 12, 1987; Standards for the Care and Use of Laboratory

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Trabectedin Affects IGF1R and Synergizes with Anti-IGF1R Drug

Animals, United States National Research Council, Statement of col: 95C for 10 minutes; 40 cycles of 42C 10 minutes and 60C1 Compliance A5023-01, November 6, 1998). Animal experiments minute. Results were evaluated using the iQ5 software from Bio- were reviewed and approved by the IRFMN Animal Care and Use Rad and the online platform (http://pcrdataanalysis.sabios- Committee (IACUC) that includes members for ethical issues. ciences.com/pcr/arrayanalysis.php). Pathway analysis was performed using the Ingenuity Pathway Analysis (IPA) software Western blotting (Ingenuity system). Cells were treated or not (control) with trabectedin (0.5–2.5 nmol/L) or with OSI-906 (400 nmol/L) up to 48 hours or silenced Activity of caspase-3 and caspase-7 for EWS–FLI1 (75–100 nmol/L siRNA; ref. 30) and lysed as Caspases-3 and -7 activities were determined in cells exposed to previously described (18). The following primary antibodies (Ab) drug/DMSO for 48 hours using the Glow caspase luminescent kit were used: anti-PARP; cleaved-caspase-3, anti-NF-H 200k (Cell by Promega according to the manufacturer's instructions. Lumi- Signaling Technology); anti-IGF1Rb, anti-FLI, anti-b-actin, and nescence was read in a plate reader (Tecan). GAP-DH (Santa Cruz Biotechnology); anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase (GE Healthcare) Immunohistochemistry were used as secondary antibodies. Sections (5 mm) from formalin-fixed, paraffin-embedded TC- 71 xenografts were placed on poly-L-lysine–coated slides (Sig- Chromatin immunoprecipitation ma). Avidin-biotin-peroxidase procedure was used for immu- In vitro and in vivo chromatin immunoprecipitation (ChIP) nostaining, as previously described (34), and slides were assays were performed as previously described (6, 28) using anti- stained with anti-tubulin b III (dilution 1:50), anti-H neuro- FLI1 (C-19; Santa Cruz Biotechnology) and/or anti-FLAG (Sigma) filament 200 kD (clone NE14; dilution 1:40; Sigma) and antibodies. PCR was performed with primers flanking Ets-con- IGF1Rb (Santa Cruz Biotechnology). Detection of Ki-67 was taining target promoters fragment (listed in Supplementary Table performed on sections as previously described (34). The ter- S1). Amplification products obtained were observed in 1.5% minal deoxynucleotidyl transferase–mediated dUTP nick end agarose gel with Gel Red staining. For quantitative PCR (qPCR), labeling (TUNEL) assay was performed with ApopTag Plus data are indicated as fold enrichment respect to untreated cells in Peroxidase in situ apoptosis kit (Merck Millipore) according to vitro experiments or to placebo in xenografts and calculated using the manufacturer's instructions. C following formula: % of recruitment ¼ 2D t input chromatin percentage where DCt ¼ Ct (input) Ct (FLI1 IP) in accordance to Statistical analysis Frank and colleagues (31, 32). Correlations between two variables were obtained by the — For TaqMan assay design TFSEARCH Searching Transcription Spearman test. IC50 values were calculated from linear transfor- Factor Binding Sites, version 1.3 free website was used for the mation of dose–response curves. To define drug–drug interac- prediction of ETS binding sites in the promoter of IGF1R gene and tions (in terms of synergism, additivity, or antagonism), the the sequence spanning from 1041 bp to 1051 bp was identified as combination index (CI) was calculated with the isobologram the best. Beacon Designer 4 software was used for the design of the equation (35) by using the CalcuSyn software (Biososoft). Differ- assay spanning from 1005 bp to 1114 bp. CD99 and TGFbR2 ences among means were analyzed by the Student t test or the promoter fragments containing ETS consensus sequence were ANOVA test. used as EWS-FLI immunoprecipitation controls (28, 30). Results fl Immuno uorescence assays Trabectedin disrupts EWS–FLI1 binding to some DNA targets Cells were seeded in single slide covers placed in 96-well plates, but increases recruitment to IGF1R promoter in both in vitro pretreated with gelatin (Sigma), and grown in standard medium. and in vivo models fi After exposure to drug/DMSO for 24 hours, cells were xed in ice- We used ChIP analysis to monitor the binding of EWS–FLI1 cold-methanol, permeabilized with Triton X-100, and processed chimera to some well-known target genes, such as TGFbR2 and fl for immuno uorescence. Primary antibodies as follows: pH2AX CD99, reported to be modulated by EWS–FLI1 and proven to have (1:100; Cell Signaling Technology); 53BP1 (1:100; Abcam), anti- a major role in EWS aggressiveness (28), as well as to IGF1R and fi tubulin b III (dilution 1:50), or anti-H neuro lament 200 kD IGF1 promoters. ChIP indicated that the amount of EWS–FLI1 (clone NE14; dilution 1:40; Sigma). Cells were then stained with chimera bound to the TGFbR2 and CD99 promoters was signif- the secondary antibody Cy5 (Jackson ImmunoResearch) and icantly reduced after 1 hour treatment with trabectedin both in counterstained with DAPI 1 mg/mL (DAKO). Slide covers were TC-71 cells, displaying EWS–FLI1 type I chimera, and in the 6647 fi mounted in covers with Mowiol xing agent (Sigma) and cells cell line, that displays EWS–FLI1 type II hybrid, at pharmacologic observed in a Leica Microscope using software LEICA software. concentrations (IC50 value after 1 hour of treatment; Fig. 1A). The binding of the chimera EWS–FLI1 type I (TC-71) and type II RNA extraction and low-density microarrays by qRT-PCR (6647) to the CD99 and TGFbR2 promoters was evaluated RNA extraction was performed using Qiagen RNA extraction also in mouse xenografts after i.v. administration of trabectedin kit, following the manufacturer's instructions, as described else- (0.15 mg/kg, every 7 days for three times, q7d 3) and doxo- where (26, 33). Retrotranscription was performed using 500 ng of rubicin (8 mg/kg, every 7 days for two times, q7d 2). As shown total RNA. Low-density microarray, in the form of qRT-PCR 96- in Supplementary Fig. S1, in TC-71 EWS model trabectedin well plates Human DNA Repair PCR Array (Qiagen) were per- was more active (best T/C 56.2% at days 20) than doxorubicin formed using the iQ5 thermocycler from Bio-Rad. RT-PCR was (reference compound, best T/C 79.5% at days 22). Instead run in the iQ5 BIO-RAD thermocycler with the following proto- in 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 EWS cells, or in xenografts after treatments with trabectedin or doxorubicin. EWS–FLI1 was precipitated by the anti-FLI1 antibody. Decrease in the binding of the chimera EWS–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 Materials and Methods. Results obtained by qPCR are reported as fold enrichment over the controls (untreated in vitro cells; placebo-treated mice) according to the DCt following formula: % of recruitment ¼ 2 input chromatin percentage where DCt ¼ Ct (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 is shown. Data represent recovery of each DNA fragment relative to total input DNA, respect to control. , P < 0.05; Student t test. Right, upregulation of IGF1R at protein level by Western blotting after exposure to trabectedin (0.5–1 nmol/L) up to 48 hours. GAPDH was used as loading control. Blots are representative of 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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Figure 2. Efﬁcacy of the combination of trabectedin with anti-IGF1R HAb AVE1642 against TC-71 xenografts. A, drugs were administered i.v. as follows: trabectedin 0.15 mg/kg, every 7 days for three times and AVE1642 40 mg/kg, every 3 days for six times. ANOVA test: , P < 0.05; , P < 0.01; , P < 0.001 compared with controls (Placebo). Points, tumor weight means; bars, SE. B, representative immunohistochemical evaluation of Ki-67, apoptotic rate by TUNEL assay, and IGF1Rb (magniﬁcation, 200), in untreated or treated tumors. Percentages SE of positive cells are indicated. , P < 0.05; , P < 0.001, Student t test.

(best T/C 14.3% at days 21) while trabectedin was less active (best Antitumor activity of the combination between trabectedin and T/C 48.6% at days 21). In both cases, however, trabectedin the anti-IGF1R HAb AVE1642 and doxorubicin were able to displace EWS–FLI1 chimera Antitumor activity of trabectedin alone or in combination with from CD99 and TGFbR2 promoters, although with different anti-IGF1R AVE1642 HAb was evaluated in TC-71 xenograft kinetics (Fig. 1B). Trabectedin was able to cause detachment of model. The combination trabectedin and AVE1642 HAb (best the EWS–FLI1 chimera from both CD99 and TGFbR2 promoters T/C 27.9 at days 20) showed a greater antitumor activity than already 24 hours after the first dose both in TC-71 and 6647 trabectedin (best T/C 40.3 at days 20) or AVE1642 HAb (best T/C cells. Reattachment was observed starting 7 days from the third 48.6 at days 15) used as single agents (Fig. 2A). Studies in treatment. myxoid liposarcoma indicate that trabectedin besides inhibit- Besides inhibitory effects, trabectedin but not doxorubicin also ing cell proliferation can also act as a differentiating agent by caused a dose- and time-dependent increase in the binding of the blocking the transactivating ability of the fusion gene product chimera to IGF1R promoter in EWS cells (Fig. 1C and Supple- (8). We confirmed the antiproliferative, proapoptotic, and mentary Fig. S2A), while occupancy of the IGF1 promoter prodifferentiating activity of trabectedin also in EWS (Fig. 2B appeared to be only slightly affected (Supplementary Fig. S2B). and Supplementary Fig. S4). Moreover, in keeping with ChIP Upregulation of IGF1Rb was also confirmed at protein level after findings, xenografts treated with trabectedin showed increased trabectedin treatment (Fig. 1C), in line with our previous data expression of IGF1Rb (Fig. 2B). Combination treatments with showing increased transcription and expression of IGF1Rb in cells AVE1642 HAb further inhibited tumor cell proliferation and made resistant to trabectedin (18). Consistently, silencing of IGF1R expression while increased apoptotic rate (Fig. 2B). This EWS–FLI1 in TC-71 cells induced downregulation of IGF1Rb supports the combination of trabectedin with anti-IGF1R HAb protein (Supplementary Fig. S3). Enhancement of EWS–FLI1 against EWS. occupancy to IGF1R by trabectedin was also observed in vivo in TC-71 and 6647 xenografts (Fig. 1D), further sustaining the Efficacy of the dual inhibitor anti-IGF1R/IR OSI-906 in relationship between EWS-FLI and IGF1R. These findings provid- combination with trabectedin ed the rationale for testing the combination of trabectedin with Considering that the great majority of EWSs express concom- anti-IGF1R agents. itant high levels of IR, which may overcome the IGF1R blockade

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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 TC-71 0.4 0.15 0.206 0.013 0.137 0.01 0.819 0.01 Synergistic WE-68 0.45 0.25 0.140 0.083 0.08 0.04 0.356 0.02 Synergistic SK-N-MC 0.59 0.18 0.135 0.02 0.07 0.005 0.789 0.08 Synergistic LAP-35 0.17 0.07 0,148 0.03 0,06 0.01 0.734 0.08 Synergistic IOR/CAR 1.402 0.66 0.266 0.009 0.173 0.06 0.789 0.03 Synergistic 6647 1.25 0.45 0.224 0.04 0.097 0.04 0.687 0.06 Synergistic RD-ES 0.63 0.14 0.154 0.05 0.07 0.006 0.646 0.06 Synergistic SKES-1 0.76 0.15 0.08 0.004 0.032 0.01 0.784 0.03 Synergistic RM82 0.2 0.14 0.54 0.012 0.05 0.01 0.600 0.09 Synergistic Cado-ES 3.77 0.47 0.578 0.058 0.18 0.03 0.770 0.09 Synergistic IOR/BRZ-2010 0.605 0.13 0.182 0.003 0.137 0.03 0.834 0.07 Synergistic TC/ET 12 nmol/L 0.402 0.12 1,305 263 2.32 0.59 0.411 0.09 Synergistic TC/AVE1642 0.268 0.003 1,982 340 0.178 0.004 0.710 0.02 Synergistic a IC50 values are referred to trabectedin. bSynergism: CI < 0.9; additive: 0.90 CI 1.10; according to ref. 34.

(29, 36), efﬁcacy of the dual inhibitor anti-IGF1R/IR OSI-906 was lines were highly sensitive to the inhibitor with submicromolar evaluated in a panel of 13 EWS cell lines (Table 1), including the IC50 values (Table 1). TC/ET 12 nmol/L cell line, highly resistant to trabectedin (18) and The combination of OSI-906 with trabectedin gave synergistic TC/AVE, resistant to anti-IGF1R AVE1642 HAb (29) Most of cell effects in all EWS cell lines, including cells resistant to AVE1642

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 with trabectedin 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-density expression array focusing on DNA damage and DNA repair genes was used.

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Figure 4. DNA-damage induction after cell exposure to trabectedin and/or OSI-906. A, induction of H2AX phosphorylation (g-H2AX) and 53BP1 expression in TC-71 cells after exposure to trabectedin, and/or OSI- 906 (200–400 nmol/L) for 24 hours (magniﬁcation, 400). B, DNA fragmentation induced in TC-71 cells after 12 to 24 hours exposure to drugs. C and D, evaluation of caspase-3 and PARP cleavage by Western blotting after 24 hours exposure to drugs. Equal loading was monitored with b-actin blotting.

HAb (TC/AVE) or trabectedin (TC/ET 12 nmol/L; Table 1). When fragments (<500 bp) after cell exposure to OSI-906 is not sur- OSI-906 was combined to trabectedin, we observed advantageous prising because these fragments are likely to be related to DNA effects in terms of apoptosis, both in p53wt (WE-68) and p53- degradation by apoptosis. Accordingly, OSI-906 favors expression mutated cells (TC-71; Fig. 3A). This advantageous proapoptotic of apoptotic proteins such as cPARP, fraction 90 kDa (Fig. 4C). cell death may derive from the combination of two different PARP cleavage was also observed in the TC/ET 12 nmol/L resistant inputs. Although inhibition of the IGF system may block anti- cell line after treatment with increasing doses of OSI-906, thus apoptotic effects of IGF1R/IR-A due to disruption of AKT and/or confirming complementary proapoptotic effects of the two drugs 14.3.3/Raf-1/Nedd4 pathways (37, 38), trabectedin acts as a (Fig. 4D). DNA-damaging agent inducing double-strand breaks (DSB; ref. 3). To further characterize drug effects on DNA repair pathways, we used DNA damage low-density arrays customized to Discussion cover homologous recombination (HR), nucleotide excision Trabectedin is a newly licensed chemotherapeutic agent, with repair (NER), base excision repair (BER), and non-homologous an acceptable toxicity profile (40). In this article, we show that end joining repair (NHEJR) pathways. Treatment with trabecte- trabectedin may be advantageously used in combination with din, but not with OSI-906, induced expression of BRCA1, BRCA2, inhibitors of the IGF system. Particularly, we provide a rationale key proteins in HR pathway (39) as well as XRCC1, member of for the use of this combination, showing that trabectedin is able to single-strand break repair (SSR) pathway (Supplementary Fig. increase IGF1R expression by enhancing EWS–FLI1 occupancy of S5). The drug combination resulted in upregulation of members the IGF1R promoter. EWS–FLI1 drives the malignant phenotype from the HR pathway (RAD52, BRCA1 and BRCA2), NER proteins in EWS cells (11), acting either as a transcriptional activator or as a (XPA and ERCC1), and SSR pathways (XRCC1; Fig. 3B). As transcriptional repressor in EWS. Importantly, both the activating validation, we also studied DNA-damage induction by presence and repressive functions of EWS–FLI1 are required for its onco- of pH2AX and 53BP1 intranuclear foci after 24 hours treatment genic functions (41). Trabectedin has been previously found to (Fig. 4A), showing that trabectedin was a potent DSB inducer, in block the promoter activity and expression of critical EWS–FLI1 contrast to OSI-906, and that DNA damage was present when cells downstream targets, and a combination with SN38 (the active were treated with the combination of trabectedin and OSI-906. metabolite of irinotecan) has been proposed to augment the DNA fragmentation assay showed that besides trabectedin also suppression of EWS–FLI1 activity (16). However, in this article, OSI-906 alone and particularly the combination of the two drugs we show that these inhibitory effects, although probably preva- leads to DNA fragmentation (Fig. 4B). The presence of DNA lent, are not exclusive. In fact, while we confirm that both

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Figure 5. Schematic representation of trabectedin activity in EWS. Trabectedin may not only inhibit (1) but also enhance the binding of EWS– FLI1 to target genes. Speciﬁcally, IGF1R expression is activated after treatment with trabectedin (3), therefore providing the rationale for combined treatments with anti-IGF1R agents.

trabectedin and doxorubicin are able to strongly suppress the tedin gave synergistic effects in all of the 13 EWS cell lines here binding of EWS–FLI1 (both type I and type II) to two target genes considered, including cells resistant to trabectedin (18) or to anti- (TGFbR2 and CD99) both in vitro and in vivo, a significant IGF1R agents (29). This appears to be due mainly to the com- enhancement of EWS–FLI1 occupancy on the IGF1R promoter plementary proapoptotic effects of the two drugs that by affecting was observed only after exposure to trabectedin. Prior studies have different pathways give rise to a combination able to deliver cell demonstrated that other DNA-binding agents, including mithra- death messages in all EWS cells, independently from the p53 mycin, and actinomycin D, reduced expression of EWS–FLI1 status. Although treatment with IGF1R antagonists is known to downstream targets and displayed differential specificity, likely lead to downregulation of the proteins involved in cell survival due to preferential sequence binding affinities (42, 43). This and inhibition of cell death (38, 47), thereby recovering cell discovery introduces a certain level of specificity in the action of sensitivity to apoptosis, trabectedin has been previously described conventional agents, which is potentially very interesting, but as a potent DNA-damaging agent. Trabectedin binds to guanines requires further studies because the effects may vary in relation to in the minor groove with some degree of sequence specificity, drugs, transcription factors, and cellular context. For example, inducing SSB that rapidly turn into DSB, the most lethal form of doxorubicin did not affect the binding of FUS-CHOP to target DNA damage. Tavecchio and colleagues (50) clearly showed that promoters in myxoid liposarcoma (6), whereas here it was shown DSBs are not directly caused by the drug, but are formed during to inhibit occupancy of EWS–FLI1 on TGFbR2 and CD99 pro- the processing/repair of the drug, requiring a functional HR moters, indicating differences among transcriptional hybrids and pathway. In addition, trabectedin also poisons the mechanisms drug action in different cellular contexts. By also reporting an of DNA repair through the formation of ternary NER proteins– increase and not just a suppression of EWS–FLI1 binding to DNA–trabectedin complexes (for a review see ref. 51). Our results specific target promoters, we introduced another variable that demonstrated that in EWS cells trabectedin increases expression of deserves more ample investigation. In the specific context of EWS, BRCA1, BRCA2, key proteins in the HR pathway as well as of the increase in IGF1R expression is absolutely reasonable from a XRCC1, which is involved in the SSR pathway, resulting in DNA biologic point of view considering the importance that the IGF damage as indicated by phosphorylation of histone H2AX and system has in maintenance of EWS malignancy (19, 44), and was accumulation of intranuclear foci. The drug combination with indeed confirmed also in cells made resistant to trabectedin (18). OSI-906 maintains and even increases upregulation of members From a clinical point of view, increased expression of IGF1R in of HR (RAD52, BRCA1, and BRCA2), NER (XPA and ERCC1), and response to in vitro and in vivo exposure to trabectedin, provides SSR (XRCC1) pathways but also induces a strong downregulation the rationale for a combined use of trabectedin with anti-IGF1R of XRCC4 and XRCC6 as well as of MSH4 and MSH5, two agents. Here, we demonstrate the advantages of this combination molecules involved in the maintenance of genomic stability and either using HAb AVE1642, a well-tolerated agent that binds mitotic DSB repair (52), indicating general alterations of DNA- human IGF1R specifically and with high affinity (45, 46), or the damage response and repair pathways. This is in line with recent dual inhibitor IGF1R/IR, OSI-906, a small molecule shown to evidence that IGF1R inhibition induces a direct functional defect have antitumoral activity against several tumors (47, 48), includ- in DSB repair by both NHEJ and HR, besides indirectly impairing ing osteosarcoma (49). The association of OSI-906 with trabec- HR through influences on the expression and/or activation of cell-

OF8 Clin Cancer Res; 21(6) March 15, 2015 Clinical Cancer Research

Trabectedin Affects IGF1R and Synergizes with Anti-IGF1R Drug

cycle regulators (53). The importance of trabectedin and IGF Writing, review, and/or revision of the manuscript: A.T. Amaral, C. Garofalo, ~ system inhibitors in DNA-damage response and repair pathways, S. Uboldi, J.L. Ordonez, P. Picci, A. Bass Hassan, E. De Alava, M. D'Incalci, which have implications on the therapeutic efficacy and potential K. Scotlandi Administrative, technical, or material support (i.e., reporting or organizing toxicity of this combined therapy in the clinic, deserve further data, constructing databases): C. Garofalo, C. Mancarella, R. Malaguarnera research to better elucidate the molecular mechanisms and pro- Study supervision: E. De Alava, M. D'Incalci, K. Scotlandi tein interactions. Other (involved in funding of the project from EuroSarc grant): A. Bass Overall, we provide the rationale for combining trabectedin to Hassan anti-IGF1R inhibitors. We showed that trabectedin may not only inhibit but also enhance binding of EWS–FLI1 to target genes (Fig. Acknowledgments 5). Specifically, IGF1R expression activated after treatment with The authors thank Cristina Ghinelli and Alba Balladelli for article editing. trabectedin and anti-IGF1R agents improve efficacy of trabectedin in cell lines and xenografts. We thus propose the use of a Grant Support combination therapy that by exploiting the complementary This work was financially supported by the EU project FP7-HEALTH-2011- mechanisms of action of the two drugs may have therapeutic two-stage, Project ID 278742 EUROSARC; the Italian Association for Cancer potential. Research (Katia Scotlandi—AIRC Project N.14049; C. Garofalo—MFAG N.11584 and M. D'Incalci—AIRC Project N.14658); The Italian Ministry of Research and Education (F.I.R.B. project number: RBAP11884M_005); The Italian Ministry of Disclosure of Potential Conflicts of Interest the Health (67/GR-2010-2319511, CUP G71J12000830001). E. de Alava's M. D'Incalci is a consultant/advisory board member for PharmaMar. No laboratory is supported by the AECC (Asociacion Espanola~ contra el Cancer), potential conflicts of interest were disclosed by the other authors. the Ministry of Economy and Competitivity of Spain-FEDER (PI081828, RD06/ 0020/0059 RD12/0036/0017, PT13/0010/0056, PI110018, ISCIII Sara Borrell postdoc grant CD06/00001), and Fundacion Memoria de D. Manuel Solorzano Authors' Contributions Barruso,Fundacion Cris contra el cancer, and Fundacion María García Estrada. J.L. ~ Conception and design: C. Garofalo, P. Picci, A. Bass Hassan, E. De Alava, Ordonez is sponsored by the CSIC and the European Social Fund (postdoctoral ¸ ^ M. D'Incalci, K. Scotlandi grant JAE DOC), A.T. Amaral is sponsored by the Fundacao para a Ciencia e Development of methodology: A.T. Amaral, C. Garofalo, M.C. Manara, Tecnologia, Portugal (fellowship SFRH/BD/69318/2010); and S. Uboldi is C. Mancarella, S. Uboldi, S. Di Giandomenico, J.L. Ordonez,~ V. Sevillano, recipient of a FIRC Fellowship N.13743. R. Malaguarnera, P. Picci, E. De Alava, M. D'Incalci The costs of publication of this article were defrayed in part by the Acquisition of data (provided animals, acquired and managed patients, payment of page charges. This article must therefore be hereby marked advertisement provided facilities, etc.): A.T. Amaral, C. Garofalo, R. Frapolli, M.C. Manara, in accordance with 18 U.S.C. Section 1734 solely to indicate V. Sevillano, P. Picci this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.T. Amaral, C. Garofalo, M.C. Manara, C. Mancar- Received July 1, 2014; revised December 11, 2014; accepted January 1, 2015; ella, S. Uboldi, J.L. Ordonez,~ P. Picci, K. Scotlandi published OnlineFirst January 21, 2015.

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OF10 Clin Cancer Res; 21(6) March 15, 2015 Clinical Cancer Research

Trabectedin Efficacy in Ewing Sarcoma Is Greatly Increased by Combination with Anti-IGF Signaling Agents

Ana Teresa Amaral, Cecilia Garofalo, Roberta Frapolli, et al.

Clin Cancer Res Published OnlineFirst January 21, 2015.

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