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E-GEOD-45414 - Low-dose actinomycin D preferentially disrupts EWS-FLI1DNA binding
Released on 26 August 2013, last updated on 2 September 2013
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 preferentially blocked EWS-FLI1 binding to chromatin, and disrupted EWS-FLI1-mediated gene expression. Higher concentrations of actinomycin globally repressed transcription. These results demonstrate that actinomycin 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. Using proximity assays in A673 Ewing Sarcoma cells, we screened 7 bioactive-enriched small molecule libraries, totaling 5,200 compounds to identify compounds that could disrupt the binding of EWS-FLI1 to its cognate DNA binding sequence. We defined a set of EWS-FLI1-regulated genes by shRNA depletion of EWS-FLI1in the same cell line. Duplicate knock down experiments were carried out and compared to duplicate scrambled shRNA controls. This signature was used to interrogate the effects in duplicate experiments of low- and high-dose actinomycin D treatment in A673 cells as compared to DMSO and untreated controls (2 each).
transcription profiling by array
Madeleine E. Lemieux <email@example.com>, Andrew L Kung
Differential Disruption of EWS-FLI1 Binding by DNA-Binding Agents. Chen C, Wonsey DR, Lemieux ME, Kung AL. , PMID:23894528