Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor

Clin Cancer Res. 2016 Aug 15;22(16):4105-18. doi: 10.1158/1078-0432.CCR-15-2624. Epub 2016 Mar 15.


Purpose: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients.

Experimental design: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma.

Results: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo

Conclusions: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105-18. ©2016 AACR.

MeSH terms

  • Animals
  • Antibiotics, Antineoplastic / pharmacology*
  • Cell Line, Tumor
  • Disease Models, Animal
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic / drug effects
  • Humans
  • Mice
  • Molecular Targeted Therapy
  • Oncogene Proteins, Fusion / antagonists & inhibitors*
  • Oncogene Proteins, Fusion / metabolism*
  • Plicamycin / pharmacology*
  • Promoter Regions, Genetic
  • Proto-Oncogene Protein c-fli-1 / antagonists & inhibitors*
  • Proto-Oncogene Protein c-fli-1 / metabolism*
  • RNA-Binding Protein EWS / antagonists & inhibitors*
  • RNA-Binding Protein EWS / metabolism*
  • Sarcoma, Ewing / drug therapy
  • Sarcoma, Ewing / genetics
  • Sarcoma, Ewing / metabolism
  • Sarcoma, Ewing / mortality
  • Transcription Factors
  • Xenograft Model Antitumor Assays


  • Antibiotics, Antineoplastic
  • EWS-FLI fusion protein
  • Oncogene Proteins, Fusion
  • Proto-Oncogene Protein c-fli-1
  • RNA-Binding Protein EWS
  • Transcription Factors
  • Plicamycin