title: Trabectedin efficacy in Ewing sarcoma is greatly increased by combination with anti-IGF signaling agents.
abstract: OBJECTIVE
  Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy.
  
  
  METHODS
  By chromatin immunoprecipitation, we analyzed EWS-FLI1 binding to the promoters of several target genes, such as TGFβR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts.
  
  
  RESULTS
  We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS-FLI1 (both type I and type II) binding to two representative target genes (TGFβR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS-FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways.
  
  
  CONCLUSIONS
  We showed that trabectedin may not only inhibit but also enhance the binding of EWS-FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors.
authors.affiliation: Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Department of Pathology, Seville, Spain.
authors.forename: Ana Teresa
authors.initials: AT
authors.lastname: Amaral
authors.affiliation: Experimental Oncology Lab, CRS Development of Biomolecular Therapies, Rizzoli Institute, Bologna, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Garofalo
authors.affiliation: Department of Oncology IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Frapolli
authors.affiliation: Experimental Oncology Lab, CRS Development of Biomolecular Therapies, Rizzoli Institute, Bologna, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Manara
authors.affiliation: Experimental Oncology Lab, CRS Development of Biomolecular Therapies, Rizzoli Institute, Bologna, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Mancarella
authors.affiliation: Department of Oncology IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
authors.forename: Sarah
authors.initials: S
authors.lastname: Uboldi
authors.affiliation: Department of Oncology IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
authors.forename: Silvana
authors.initials: S
authors.lastname: Di Giandomenico
authors.affiliation: Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Department of Pathology, Seville, Spain.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Ordóñez
authors.affiliation: Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Department of Pathology, Seville, Spain.
authors.forename: Victoria
authors.initials: V
authors.lastname: Sevillano
authors.affiliation: Department of Health, University of Catanzaro, Catanzaro, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Malaguarnera
authors.affiliation: Experimental Oncology Lab, CRS Development of Biomolecular Therapies, Rizzoli Institute, Bologna, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Picci
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: A Bass
authors.initials: AB
authors.lastname: Hassan
authors.affiliation: Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Department of Pathology, Seville, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: De Alava
authors.affiliation: Department of Oncology IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: D'Incalci
authors.affiliation: Experimental Oncology Lab, CRS Development of Biomolecular Therapies, Rizzoli Institute, Bologna, Italy. katia.scotlandi@ior.it.
authors.forename: Katia
authors.initials: K
authors.lastname: Scotlandi
mesh_terms: D000071443:12E7 Antigen; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004149:Dioxoles / Q000494:pharmacology*; D004317:Doxorubicin / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors*; D051379:Mice; D008819:Mice, Nude; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D051789:Proto-Oncogene Protein c-fli-1 / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics; D012512:Sarcoma, Ewing / Q000188:drug therapy*; D044005:Tetrahydroisoquinolines / Q000494:pharmacology*; D000077606:Trabectedin
chemical_list: D000071443:12E7 Antigen; C551528:3-(8-amino-1-(2-phenylquinolin-7-yl)imidazo(1,5-a)pyrazin-3-yl)-1-methylcyclobutanol; D015703:Antigens, CD; D018906:Antineoplastic Agents, Alkylating; C522605:Cd99 protein, mouse; D004268:DNA-Binding Proteins; D004149:Dioxoles; C497426:Fli1 protein, mouse; D007093:Imidazoles; D051789:Proto-Oncogene Protein c-fli-1; D011719:Pyrazines; D018125:Receptors, Transforming Growth Factor beta; D044005:Tetrahydroisoquinolines; C519530:insulin-like growth factor-1, mouse; D007334:Insulin-Like Growth Factor I; D004317:Doxorubicin; D017526:Receptor, IGF Type 1; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D053148:Caspase 3; D053179:Caspase 7; D000077606:Trabectedin