Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
PLoS One. 2013 Jul 22;8(7):e69714. doi: 10.1371/journal.pone.0069714. Print 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 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.
EWS 基因与 FLI1 融合产生融合癌蛋白，驱动异常基因表达程序，导致尤文肉瘤的发生。我们使用均相接近测定法筛选可破坏 EWS-FLI1 与其同源 DNA 靶标结合的化合物。发现许多 DNA 结合化疗药物非特异性地破坏蛋白质与 DNA 的结合。相比之下，与 p53 与其各自的同源 DNA 靶标在体外结合相比，放线菌素 D 优先优先破坏 EWS-FLI1 的结合。在基于细胞的测定中，低浓度的放线菌素 D 优先阻断 EWS-FLI1 与染色质的结合，并破坏 EWS-FLI1 介导的基因表达。较高浓度的放线菌素 D 则全局抑制转录。这些结果表明，放线菌素 D 在选定浓度下优先破坏 EWS-FLI1 与 DNA 的结合。尽管在这种优先效应和全局抑制之间的窗口太窄，无法以治疗方式利用，但这些结果表明碱基偏好可能被利用来寻找优先破坏特定转录因子亚类的 DNA 结合化合物。
