Abstract 424: The transcriptomic and methylomic changes caused by subtoxic doses of 5-azacytidine and 5-aza-2’-deoxycytidine
Authors: Smit Evelyn , Krishnan Nithya , Conroy Jeffrey , Wang Jianmin , Lui Song , Woloszynska-Read Anna 
Published: May 2015

Two demethylating agents, 5-azacytydine (Vidaza) and 5-Aza-2’-deoxycytidine (Decitabine), are used in the clinic to treat myelodysplastic syndrome (MDS) and some forms of leukemia. There are also several ongoing clinical trials using these demethylating agents to treat solid tumors. DNA incorporation of these drugs leads to the depletion of DNMTs (DNA methyltransferases) in the cell and passive DNA demethylation. The major difference between Decitabine and Vidaza is their respective level of incorporation into nucleic acid. Decitabine is only incorporated into DNA, while 70-90% of Vidaza incorporates into RNA and only 10-30% into DNA. Due to their mechanisms of action, the effects of these drugs on DNA are S-phase dependent. It has been previously demonstrated that various transcriptional changes are initiated by demethylation of DNA, but the majority of studies have focused on transcriptional activation of tumor suppressor genes. The objective of this study is to use genomics approaches to examine the effects of low doses of Vidaza and Decitabine on bladder cancer cells and normal immortalized urothelium. We hypothesize that Vidaza and Decitabine will have distinct biological effects on the methylome and transcriptome in these cells, including alternative splicing. We have generated DNA and RNA samples from normal (SV-HUC) and cancer bladder (T24 and SCaBer) cell lines treated with low concentrations (100nM, 200nM, and 1µM) of Vidaza and Decitabine. The cells were treated with the different concentration of drugs on day 2 and 4, and then harvested on day 5. Before utilizing these samples for genomic screening, we monitored the methylation status of three CpGs in the promoter region of LINE-1, a surrogate marker of pharmacologically induced DNA demethylation. Furthermore, we examined the mRNA expression of NY-ESO-1 cancer testis gene previously shown to be one of the reactivated genes by demethylating agents through a loss of promoter hypermethylation. T24 cells immediately showed a significant decrease of methylation of LINE-1 when treated with 100nM Decitibine, but there was barely any change in methylation status in the other cells lines at this low dose. Meanwhile, treatment with Vidaza did not change the methylation status of LINE-1 and mRNA expression of NY-ESO-1. However, these experiments were merely designed to monitor the general pharmacological effects of Vidaza and Decitabine. We are currently examining the effects of these drugs focusing on genomic regions (exonic and intronic) outside of gene promoters by utilizing deep-coverage paired-end RNA sequencing and 450K Illumina methylation array. The integration of these large-scale data will enable us to better understand pharmacologically induced demethylation in cancer and utilization of these agents in treatment of solid tumors including bladder cancer.

Citation Format: Evelyn Smit, Nithya Krishnan, Jeffrey Conroy, Jianmin Wang, Song Lui, Anna Woloszynska-Read. The transcriptomic and methylomic changes caused by subtoxic doses of 5-azacytidine and 5-aza-2’-deoxycytidine. [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 424. doi:10.1158/1538-7445.AM2014-424