Abstract B50: A phenotypic screen to identify novel potential epigenetic anticancer drugs from natural compounds

Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. We screened 3040 natural compounds and derivatives by measuring GFP expression in the YB5 cell line, a colon cancer cell line generated 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 known 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 (average Z9 factor = 0.6), we set a stringent criterion that GFP induction value should be more than the average of all drugs +3 standard deviations in order to be considered as positive. 33 hits were positive (positive rate = 1.1%) among which 18 hits were validated through 24hr dose curves, fluorescence microscopy and qPCR. We then grouped the positive hits based on chemical structures. In class #1, 3 positive hits were discovered from the primary screening using NDL-3040 library, then HH1 was discovered as a potent top lead from a secondary screening using 93 analogs. The Moulder Center then synthesized 56 new analogs based on the lead9s structure and 14 are positive in the YB5 system. The most potent analog (HH2) can induce 10% GFP+ cells upon 5uM treatment after 24hr. This potency is in the same range as that obtained with decitabine (a DNMT inhibitor). All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, these drugs can also reactivate many hypermethylated genes (CDH13, MGMT, SYNE1, RRAD, PYGM etc) in the YB5 cell line. These compounds also synergize with Decitabine in terms of GFP induction and many endogenous hypermethylated reactivation (RARβ, SYNE1, RRAD, FAM184A etc). For this class, we found no effects on DNA methylation, HDAC activity or effects on known histone methyltransferases/demethylases (HMT/HDM) using biochemistry-based assays. Global histone methylation and acetylation level changes were determined using mass spectrometry and we found upregulation of H3K79me2 levels. Proliferation assays showed differential sensitivity of a panel of colon cancer cell lines compared to normal cells (IMR90). These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Thus, a novel epigenetic drug class derived from natural compounds was identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noel J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic screen to identify novel potential epigenetic anticancer drugs from natural compounds. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B50.