Abstract 5064: Identifying novel potential epigenetic anti-cancer drugs from natural compounds using a phenotypic-based screening
Authors: Zhang Hanghang , Raynal Noël J.-M , Sato Takahiro , Okamoto Yasuyuki , Garriga Judith , Garcia Benjamin , Morton George , Childers Wayne , Jacobson Marlene A. , Baylin Stephen B. , Graña Xavier , Abou-Gharbia Magid , Issa Jean-Pierre J. 
Published: July 2017

Abstract
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 gene reactivation, leading to cancer cell differentiation and cell death. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. To explore this idea, our lab developed a phenotypic-based system (YB5) 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. By screening an NDL-3040 natural compounds library and grouping the compounds based on chemical structures, we identified two main drug classes. We then synthesized 77 new analogs based on class #1’s lead’s structure and 23 were positive in the YB5 system. The most potent analog (HH2) can induce ~60% GFP+ cells upon 500nM treatment after 96hr. All the positive hits can also be validated in two other cancer cell lines (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes (MGMT, RARβ, etc) can also be re-expressed upon drug treatment. We then performed RNA-seq analysis to identify global gene expression changes following drug treatment. We observed that most genes (2964 genes) were upregulated upon HH1 treatment (10uM) and that many of the upregulated genes were expressed in normal tissues but repressed in cancer, indicating that they might be potential tumor suppressor genes (TSGs). Consistent with this, 94 TSGs could be reactivated upon 10uM drug treatment. These drug target upregulated genes were also enriched for hypermethylation. By performing connectivity mapping using RNA-seq, we identified X as the class #1 drug target. The on-target effect could be further validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Additionally, when we overexpressed wild-type X, we saw that GFP induction as well as endogenous gene reactivations can be inhibited. Strikingly, by using GFP induction as readout to optimize drugs, we found that the in vitro IC50 against X for our top lead compound (HH2) is only 5nM and it is at least 22-fold selective for X over other X family members. 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, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judith Garriga, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, Magid Abou-Gharbia, Jean-Pierre J. Issa. Identifying novel potential epigenetic anti-cancer drugs from natural compounds using a phenotypic-based screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5064. doi:10.1158/1538-7445.AM2017-5064