Genome-wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene.

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression.

Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.

Identification of acute myeloid leukaemia associated microRNA expression patterns.

MicroRNAs (miRNAs) play an important role in cellular differentiation and cancer pathogenesis. This study analysed the expression of 154 human miRNAs in acute myeloid leukaemia (AML) and control samples using a stem-loop real-time reverse transcription polymerase chain reaction approach. Global patterns of miRNA expression in AML, normal bone marrow (NBM) and CD34(+) progenitor cells allowed correct class predictions similar to whole genome microarray expression analyses that were performed at the same time. At single miRNA species level, MIRN23B was repressed in AML specimens compared to NBM and purified CD34(+) haematopoietic progenitor cells. In contrast, the MIRN221/MIRN222 cluster and MIRN34A were expressed at significantly higher levels in AML blasts. Patients with high MIRN221/MIRN222 expression showed low levels of KIT RNA and protein expression but the correlation between kit protein and KIT mRNA was significantly stronger than the correlation of either one with MIRN221/MIRN222. A global analysis between miRNA expression levels and mRNA expression of predicted target genes revealed only weak associations in the majority of miRNA species. Nonetheless, the presence of two or more miRNA binding sites within the mRNA was usually associated with a decrease in mRNA levels. Taken together, these findings provide evidence that specific miRNA expression patterns exist in AML.

MYST2 acetyltransferase expression and Histone H4 Lysine acetylation are suppressed in AML.

Chromatin-modifying enzymes are frequently altered in acute myeloid leukemia (AML). In the current study, we identified MYST2, a core histone acetyltransferase, to be suppressed in blast cells from AML patients compared with nonmalignant hematopoietic progenitor cells. Functionally, loss of MYST2 accelerated leukemic growth and colony formation, while forced expression of MYST2 induced H4K5 acetylation (H4K5Ac) and suppressed hematopoietic progenitor cell growth. Consistently, global H4K5Ac levels were frequently decreased in AML blasts. Low levels of H4K5Ac were most prominent in patients with complex karyotype AML and were associated with inferior overall survival in univariate but not multivariate analysis. ChIP-seq experiments in primary AML patients' blasts revealed widespread H4K5Ac deregulation, most prominent at gene promoters. Taken together, MYST2 is a repressed growth suppressor in AML mediating reduced acetylation of histone 4 at residue 5 and is associated with inferior AML patient survival.

Chromatin modifications induced by PML-RARalpha repress critical targets in leukemogenesis as analyzed by ChIP-Chip.

The translocation t(15;17) generates the chimeric PML-RARalpha transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARalpha transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)-chip experiments identified 372 direct genomic PML-RARalpha targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARalpha targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARalpha binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARalpha to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARalpha regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.