Abstract 378: Discovering potential epigenetic anti-cancer drugs derived from natural compounds

Introduction and Aims: Epigenetics is the study of heritable changes in gene expression that are independent of changes in DNA sequence. The main biochemical modifications that govern epigenetics are DNA methylation and post-translational histone modifications. Small molecules modulating the activity of the DNA/chromatin-modifying enzymes have been shown to be able to revert malignant cells to a more normal epigenetic state. Thus, the opportunity for developing potential epigenetic drugs is of great interest in the fields of cancer biology and drug discovery. Natural compounds are bioactive, have variable structures, and many known anti-cancer drugs are derived from natural compounds. In addition, there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. Methods: We used the YB5 cell line, a colon cancer cell line generated by our lab by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment with the natural compounds, FACS analysis was used to check the GFP expression levels. After the primary screening, top hits were validated by doing dose curves, fluorescence microscopy and qPCR. In addition, hypermethylated genes were checked by qPCR and methylation levels of the CMV promoter were determined by pyrosequencing. Results: We have screened 3040 natural compounds, with 33 potential positive hits as measured by an increase in GFP expression (more than 2.64% of GFP+) after a 24-hour treatment. Among these, 19 hits have been validated through dose curves, fluorescence microscopy and qPCR. Two hits have synergistic effects with Decitabine (a DNA methyltransferase inhibitor). By using a biochemistry-based assay, we have determined that none of the positive hits are HDAC inhibitors. In addition, none of the hits decreased methylation levels of the CMV promoter after 24hr treatment. These hits can be classified into several different categories based on their structures. Four class representative top hits were selected to do further analysis. These four hits can reactivate two hypermethylated genes (CDH13 and WIF1) and upregulate p21. A combination of Decitabine and the four top hits can upregulate hypermethylated gene expression levels synergistically in different patterns. Conclusions: We have identified 33 natural compounds that have potential epigenetic activity. Based on the structural analysis, gene reactivation patterns, as well as synergistic effects analysis, these top hits may work via different mechanisms. Further experiments are being carried out to identify other potential target genes of the top hits, study the biological effects of the hits and discover their potential mechanisms of action. Citation Format: Hanghang Zhang, Noel J-M RAYNAL, Marlene A. Jacobson, Jean-Pierre Issa. Discovering potential epigenetic anti-cancer drugs derived from natural compounds. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 378. doi:10.1158/1538-7445.AM2014-378