Differential Disruption of EWS-FLI1 Binding by DNA-Binding Agents

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

Biochemical disruption of EWS-FLI1 and p53 binding to DNA.

<p>Dose response for disruption of recombinant EWS-FLI1 (red lines) and p53 (black lines) binding to DNA in the presence of indicated compounds was measured using AlphaScreen proximity assays. Data plotted as mean +/− SD of triplicate samples and are representative of 2 independent experiments.</p

Effects of actinomycin D on the binding of EWS-FLI1 to the NR0B1 promoter.

<p>EWS-FLI1 was immunoprecipiatated using a FLI1 antibody, and quantitative PCR used to determine binding to NR0B1, RPS26, and p53. Data expressed as fold-enrichment over normal IgG control ChIP. Data plotted as mean +/− SD of duplicates are representative of 3 independent experiments.</p

AlphaScreen Results.

<p>AlphaScreen Results.</p

Effect of actinomycin D on gene expression.

<p>Effects of actinomycin D (<b>A</b>), epirubicin (<b>B</b>) doxorubicin (<b>C</b>), ebselen (<b>D</b>) and Erk inhibitor (<b>E</b>) on NR0B1-Luc (solid line) and UbC-Renilla (dotted line) reporter activity. Data plotted as mean +/− SEM of triplicates. <b>F</b>: Quantitative RT-PCR was used to determine the abundance of NR0B1, TP53 and RPS26 mRNA after overnight treatment of A673 cells with the indicated concentrations of actinomycin D. Results normalized to DMSO control. Data plotted as mean +/− SD of quadruplicates, and are representative of 3 independent experiments.</p

Growth effects of actinomycin D on Ewing sarcoma and other cancer cell lines.

<p><b>Cells were treated with DMSO or actinomycin D for 44 hours.</b> Viable cell number was determined with a MTT assay. Data plotted as mean +/− SD of sextuplets and are representative of 3 independent experiments.</p

Myc Stimulates Nuclearly Encoded Mitochondrial Genes and Mitochondrial Biogenesis

Although several genes involved in mitochondrial function are direct Myc targets, the role of Myc in mitochondrial biogenesis has not been directly established. We determined the effects of ectopic Myc expression or the loss of Myc on mitochondrial biogenesis. Induction of Myc in P493-6 cells resulted in increased oxygen consumption and mitochondrial mass and function. Conversely, compared to wild-type Myc fibroblasts, Myc null rat fibroblasts have diminished mitochondrial mass and decreased number of normal mitochondria. Reconstitution of Myc expression in Myc null fibroblasts partially restored mitochondrial mass and function and normal-appearing mitochondria. Concordantly, we also observed in primary hepatocytes that acute deletion of floxed murine Myc by Cre recombinase resulted in diminished mitochondrial mass in primary hepatocytes. Our microarray analysis of genes responsive to Myc in human P493-6 B lymphocytes supports a role for Myc in mitochondrial biogenesis, since genes involved in mitochondrial structure and function are overrepresented among the Myc-induced genes. In addition to the known direct binding of Myc to many genes involved in mitochondrial structure and function, we found that Myc binds the TFAM gene, which encodes a key transcriptional regulator and mitochondrial DNA replication factor, both in P493-6 lymphocytes with high ectopic MYC expression and in serum-stimulated primary human 2091 fibroblasts with induced endogenous MYC. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis