Abstract 2001: 5-Aza-2’-deoxycytidine and cytarabine mediate distinct effects on clonogenic growth, genome wide methylation and RNA expression in AML
Authors: Heuston Elisabeth F. , Farrar Jason E. , Triche Timothy , Buckley Jonathan , Wai Daniel H. , Ochs Michael , Considine Michael , Reaman Greg , Arceci Robert J. 
Published: July 2012

Abstract
5-Aza-2’-deoxcytidine (DAC) and cytarabine (AraC) are cytosine analogues and S-phase dependent chemotherapeutic agents that have contributed to the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Further understanding the cellular and molecular consequences of treatment with these agents may provide important insights into improving their use in patients with these disorders. We have treated four AML cell lines representing different FAB subtypes (Molm13 – FAB M5, NB4 – FAB M3, Kasumi-1 – FAB M2 and UT-7epo – FAB M7) with single low doses (0.01 µM to 1.0 µM) of DAC or AraC, and quantified clonogenic and tumorigenic growth changes, as well as drug-specific DNA methylation and RNA expression alterations. Our results demonstrate that both DAC and AraC treatment induce similar levels of clonogenic depletion in Molm13, NB4 and Kasumi-1, although DAC treatment more selectively inhibits clonogenic cell growth relative to overall cytotoxicity compared to AraC. These data were confirmed in vivo for the Molm13 and NB4 cell lines assayed. The UT-7epo cell line was relatively insensitive to AraC treatment but showed a strong inhibition of clonogenic proliferation when treated with DAC. Methylation and transcription profiling following exposure to low-dose DAC or AraC shows that treatment with the DNA methyltransferase 1 inhibitor, DAC, results in genomic hypomethylation that titrates in a dose-dependent manner, while AraC induces hypermethylation in a mostly non-overlapping subset of promoter regions (6.7% overlap). Correlation of methylation with expression analyses and assessment for gene set enrichment in significantly up- and down-regulated genes illustrates mechanistically distinct and therapeutically relevant patterns of altered expression. Altered activity of metabolic pathways is frequently observed following AraC treatment, while DAC treatment significantly up-regulates expression of several lineage specific antigens, as well as reproductive and hematopoietic development-related pathways. Both drugs induce DNA replication and repair pathways, as well as cell cycle-related genes. These observations demonstrate overlapping effects of DAC and AraC in terms of clonogenicity and cytotoxicity with the exception of a highly selective effect of DAC on the megakaryoblastic line, UT-7epo. In addition, there are profound molecular differences in RNA expression and genome wide methylation patterns mediated by DAC and AraC. These observations have important implications for understanding the different cellular and molecular responses to low dose DNA demethylating agents and their potential role for use in combination with AraC in AML.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2001. doi:10.1158/1538-7445.AM2011-2001