Abstract 3644: DNA hypomethylation by nucleoside analogs with subsequent hemoglobin gene induction in the human leukemia MOLT-4 cell line

Abstract

Abstract Nelarabine (Atriance®), a prodrug of 9-β-D-arabinofuranosylguanine (AraG), is a cytotoxic nucleoside analog used to treat acute T-cell lymphoblastic leukemia and T-cell lymphoblastic lymphoma. Nelarabine is demethoxylated by adenosine deaminase to AraG. Phosphorylation and activation by deoxycytidine and deoxyguanosine kinase (dCK, dGK) to AraG monophosphate is a rate-limiting step in the further metabolism to cytotoxic AraG triphosphate and these enzymes are often down-regulated in cells resistant to AraG or other nucleoside analogs. A microarray experiment was conducted using the acute T lymphoblastic leukemic cell line MOLT-4 and an AraG resistant MOLT-4 variant. The aim was to identify affected genes in response to the resistance induction. Of the more than 8000 genes investigated, over 1000 were significantly (p≤0.05) and differentially expressed between the parental and the resistant cell line. Nucleoside analog resistance genes were only modestly affected. Of the top ten upregulated genes, five were different hemoglobin genes, mainly fetal ones. This was verified with real-time PCR expression and western blot for fetal hemoglobin gamma. Drugs with the ability to hypomethylate DNA, such as hydroxyurea and 5-azacytidine, have been shown to induce hemoglobine genes. Clofarabine (CAFdA, 2-chloro-2′-arabino-fluoro-2′-deoxyadenosin, Evoltra®) has been shown to hypomethylate DNA and cytarabine (AraC, Cytosar) to induce fetal hemoglobin production in baboons. Therefore it was investigated if AraG and other nucleoside analogs have the ability to induce DNA hypomethylation with subsequent hemoglobin induction in MOLT-4 cells. MOLT-4 cells were incubated with different nucleoside analogs and 6-mercaptopurin (6-MP, Purinethol®), an anti-metabolite known to induce DNA hypomethylation during 24 and 48 hours. DNA pulse labeling experiment measuring the quotient between newly synthesized DNA and the presence of methylated cytosines were used to assess global DNA methylation. At 24 and 48 hours of drug incubation a 20-40% decrease in DNA methylation was observed in cells treated with 6-MP, AraG, fludarabine (FaraA, 2-fluoroarabinosyladenine, Fludara®), gemcitabine (dFdC, Gemzar®), and cytarabine, a slight decrease in cells incubated with clofarabine whereas no changes in methylation were detected in cells treated with cladribine (CdA, Leustatin®). After three weeks of weekly drug treatment, real-time PCR measurements revealed upregulation of the hemoglobin gamma gene in all drug treated cells, and in cells incubated with AraG, FaraA and 6-MP the induction was between 15-to 70-fold. The clinical impact of DNA hypomethylation by 5-azacytidine is currently under investigation by other researchers but the impact of hemoglobin gene induction is unknown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3644.