Abstract 373: An siRNA screen identifies CHD4 as a target for epigenetic therapy

Abstract

Abstract Background and aims: In the past decade, epigenetic mechanisms 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 an siRNA library targeting 188 gene predicted as epigenetic regulators using colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated cytomegalovirus (CMV) promoter. GFP-positive cell percentages were measured using Guava EasyCyte Plus flow analyzer software. For genome wide gene expression analysis, affymetrix microarrays were performed. DNA methylation status was evaluated by pyrosequencing analysis and Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis. To access biological effect of the combination therapy, we evaluated cell growth using several cell lines. Results: We identified the chromodomain helicase DNA-binding protein 4 (CHD4) as a target; CHD4 depletion in combination with DNMT inhibition synergistically reactivated expression of GFP and endogenously methylated genes. Synergy was most pronounced for genes with very low basal expression. DREAM analysis revealed that methylated genes with promoter CpG islands were enriched among reactivated genes compared to background genes (57% vs 27%, P<0.0001). DNA methylation levels of reactivating genes after combination therapy were identical with DNMT inhibitor monotherapy, suggesting that siCHD4 as a supplement to DNMT inhibition works by a parallel, synergistic mechanism. Combination treatment inhibited cell growth of colon cancer cell lines better than treatment with a single DNMT inhibitor with minimal effects on the cell growth of non-cancerous cell lines. Interestingly, depletion of CHD4 alone also inhibited cell growth of colon cancer cell line but not of non-cancerous cell lines. Conclusions: Combination treatment of siCHD4 and DNMT inhibitor strongly reactivated hypermethylated and silenced genes in cancer cell lines. CHD4 inhibition is a promising new approach to epigenetic targeted therapy. Citation Format: Yasuyuki Okamoto, Jumpei Yamazaki, Takahiro Sato, Matteo Cesaroni, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Richard A Katz, Jean-Pierre Issa. An siRNA screen identifies CHD4 as a target for epigenetic therapy. [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 373. doi:10.1158/1538-7445.AM2014-373