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).
CONCLUSION: Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT 
antitumor activity both in vitro and in vivo.