title: Identification of an inhibitor of the EWS-FLI1 oncogenic transcription factor by high-throughput screening.
abstract: BACKGROUND
  Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."
  
  
  METHODS
  We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.
  
  
  RESULTS
  Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P < .001).
  
  
  CONCLUSIONS
  Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.
authors.affiliation: Molecular Oncology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Dr-MSC 1104, 10 CRC 1W-3816, Bethesda, MD 20892-1104, USA. groharp@mail.nih.gov
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Grohar
authors.forename: Girma M
authors.initials: GM
authors.lastname: Woldemichael
authors.forename: Laurie B
authors.initials: LB
authors.lastname: Griffin
authors.forename: Arnulfo
authors.initials: A
authors.lastname: Mendoza
authors.forename: Qing-Rong
authors.initials: QR
authors.lastname: Chen
authors.forename: Choh
authors.initials: C
authors.lastname: Yeung
authors.forename: Duane G
authors.initials: DG
authors.lastname: Currier
authors.forename: Sean
authors.initials: S
authors.lastname: Davis
authors.forename: Chand
authors.initials: C
authors.lastname: Khanna
authors.forename: Javed
authors.initials: J
authors.lastname: Khan
authors.forename: James B
authors.initials: JB
authors.lastname: McMahon
authors.forename: Lee J
authors.initials: LJ
authors.lastname: Helman
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D018613:Microscopy, Confocal; D015514:Oncogene Proteins, Fusion / Q000187:drug effects* / Q000235:genetics*; D008926:Plicamycin / Q000494:pharmacology*; D040081:Protein Array Analysis; D051789:Proto-Oncogene Protein c-fli-1 / Q000187:drug effects* / Q000235:genetics*; D034802:RNA-Binding Protein EWS / Q000187:drug effects* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000188:drug therapy / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014178:Translocation, Genetic / Q000187:drug effects; D014183:Transplantation, Heterologous
chemical_list: D000970:Antineoplastic Agents; C106031:EWS-FLI fusion protein; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors; D008926:Plicamycin