Dual Epigenetic Control of CCAAT/Enhancer Binding Protein α (C/EBPα) Expression in Acute Myeloid Leukemia.

Abstract

CCAAT/enhancer binding protein α (C/EBPα) is a master regulator for myeloid differentiation. As a potential tumor suppressor, its loss of function through mutations and posttranslational mechanisms has been thoroughly investigated in acute myeloid leukemia (AML). Recently, we have demonstrated the importance of epigenetic alterations in deregulating C/EBPα expression patterns in lung cancer and head and neck squamous cell carcinoma (HNSCC). In this study we investigated the role of DNA methylation and other epigenetic factors in the regulation of C/EBPα in AML using 94 patient samples and 7 leukemia cell lines. A comprehensive and quantitative DNA methylation analysis of C/EBPα's large CpG island using MassARRAY (Sequenom©) technology and BioCOBRA identified a distinct and densely methylated upstream promoter region (−1423 bp to −1121 bp in relation to the transcription start site) in 20% (19 of 94) of AML patient samples and in five of seven leukemia cell lines, while the core promoter remained unmethylated. This aberrant DNA methylation pattern was associated with two generally prognostically favorable cytogenetic subgroups: inv(16)(p13q22) and t(15;17)(q22;q21). While DNA methylation levels in normal bone marrow samples (NBM) were very low (median: 0%, range: 0%–5%) when compared to NBM, we observed significantly higher DNA methylation levels in the inv(16) and t(15;17) cytogenetic subgroups, with median DNA methylation levels of 29% (range: 1% to 81%) and 5% (range: 1% to 94%), respectively (p<0.05). However, using semi-quantitative RT-PCR, we could not detect a significant correlation between DNA methylation of C/EBPα and its expression in the inv(16) subgroup. Interestingly, while epigenetic-targeting compounds induced up-regulation of C/EBPα mRNA in vitro, C/EBPα protein was diminished. This unexpected scenario was postulated to be compatible with the presence of a microRNA targeting C/EBPα mRNA. Using a computational microRNA prediction approach and functional studies, we show that C/EBPα mRNA is a target for microRNA-124a. We additionally demonstrate that microRNA-124a is frequently silenced by epigenetic mechanisms in AML, becomes upregulated following epigenetic treatment and targets the C/EBPα 3′-untranslated region (3′UTR). In this way, C/EBPα protein expression is reduced in a posttranscriptional manner. Our results indicate that epigenetic alterations of C/EBPα are a frequent event in AML and that epigenetic treatment can result in down-regulation of a key hematopoietic transcription factor.