Abstract 2958: Discovering therapeutic epigenetic targets using whole genome siRNA screening

Abstract

Abstract Background and aims: In the past decade, epigenetic mechanisms involved in the regulation of tumor-specific gene expression have been elucidated partially, prompting the development of therapeutic approaches in cancer cells. In this study, we used an unbiased screen to investigate therapeutic targets which might be effective combination with DNMT inhibitors in reactivating hypermethylated genes. Methods: We screened a whole genome siRNA library targeting 21360 genes using a colon cancer cell line that harbors a cytomegalovirus (CMV) promoter driven GFP stably integrated and silenced by hypermethylation. GFP-positive cell percentages were measured using Guava EasyCyte Plus flow analyzer software and normalized by Z-score. The primary siRNA library contained SMART pools (Dharmacon) of 4 siRNA duplexes per gene. To validate hits, transfections using individual siRNA were performed, followed by FACS analysis and qRT-PCR. DNA methylation status was evaluated by pyrosequencing analysis and Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis. Results: The screen was conducted in combination with low dose decitabine. Of the 21,630 genes tested, 37 (0.2%) were positive (Z-score > sample mean + 5SD) and proceeded to validation studies. We successfully confirmed top hits by flow cytometry and qRT-PCR. Among them, some genes are known epigenetic regulators such as chromodomain helicase DNA-binding protein 4 (CHD4), activating transcription factor 7 interacting protein (ATF7IP) and DNMT1 itself. Interestingly well-known epigenetic regulators such as other DNMTs, histone deacetylases and histone methylases were generally absent among the top hits. CHD4 depletion in combination with DNMT inhibition synergistically reactivated expression of endogenously methylated genes and inhibited cell growth of colon cancer cell lines with minimal effects on the cell growth of non-cancerous cell lines. Depletion of CHD4 alone also inhibited cell growth of colon cancer cell line but not of non-cancerous cell lines. ATF7IP was previously reported to synergize with DNA methylation to maintain X chromosome inactivation, and here we show that it also plays a role in autosomal gene silencing. Other genes which were not previously known as epigenetic regulators are now under investigation. Conclusions: A whole genome siRNA screen in combination with the DNMT inhibitor decitabine identified many new target genes that might be epigenetic regulators and potential targets for drug development. Citation Format: Yasuyuki Okamoto, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Jean-Pierre J. Issa. Discovering therapeutic epigenetic targets using whole genome siRNA screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2958. doi:10.1158/1538-7445.AM2015-2958