Fusion of the EWS gene to FLI1 produces a fusion oncoprotein that drives an 
aberrant gene expression program responsible for the development of Ewing 
sarcoma. We used a homogenous proximity assay to screen for compounds that 
disrupt the binding of EWS-FLI1 to its cognate DNA targets. A number of 
DNA-binding chemotherapeutic agents were found to non-specifically disrupt 
protein binding to DNA. In contrast, actinomycin D was found to preferentially 
disrupt EWS-FLI1 binding by comparison to p53 binding to their respective 
cognate DNA targets in vitro. In cell-based assays, low concentrations of 
actinomycin D preferentially blocked EWS-FLI1 binding to chromatin, and 
disrupted EWS-FLI1-mediated gene expression. Higher concentrations of 
actinomycin D globally repressed transcription. These results demonstrate that 
actinomycin D preferentially disrupts EWS-FLI1 binding to DNA at selected 
concentrations. Although the window between this preferential effect and global 
suppression is too narrow to exploit in a therapeutic manner, these results 
suggest that base-preferences may be exploited to find DNA-binding compounds 
that preferentially disrupt subclasses of transcription factors.