Aberrant DNA hypermethylation signature in acute myeloid leukemia directed by EVI1.
ABSTRACT: DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34(+) bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs.
Project description:We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.
Project description:DNA methylation patterns are frequently deregulated in t(8;21) acute myeloid leukaemia (AML), but little is known of the mechanisms by which specific gene sets become aberrantly methylated. Here, we found that the promoter DNA methylation signature of t(8;21)+ AML blasts differs from that of t(8;21)- AMLs. This study demonstrated that a novel hypermethylated zinc finger-containing protein, THAP10, is a target gene and can be epigenetically suppressed by AML1-ETO at the transcriptional level in t(8;21) AML. Our findings also show that THAP10 is a bona fide target of miR-383 that can be epigenetically activated by the AML1-ETO recruiting co-activator p300. In this study, we demonstrated that epigenetic suppression of THAP10 is the mechanistic link between AML1-ETO fusion proteins and tyrosine kinase cascades. In addition, we showed that THAP10 is a nuclear protein that inhibits myeloid proliferation and promotes differentiation both in vitro and in vivo Altogether, our results revealed an unexpected and important epigenetic mini-circuit of AML1-ETO/THAP10/miR-383 in t(8;21) AML, in which epigenetic suppression of THAP10 predicts a poor clinical outcome and represents a novel therapeutic target.
Project description:BACKGROUND: Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown. METHODS: We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors. RESULTS: We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML. CONCLUSIONS: Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.
Project description:The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ? 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.
Project description:DNMT3A mutations occur in ?25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ?80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.
Project description:Trisomy 8 acute myeloid leukemia (AML) is the commonest numerical aberration in AML. Here we present a global analysis of trisomy 8 AML using methylated DNA immunoprecipitation-sequencing (MeDIP-seq). The study is based on three diagnostic trisomy 8 AML and their parallel relapse status in addition to nine non-trisomic AML and four normal bone marrows (NBMs). In contrast to non-trisomic DNA samples, trisomy 8 AML showed a characteristic DNA methylation distribution pattern because an increase in the frequency of the hypermethylation signals in chromosome 8 was associated with an increase in the hypomethylation signals in the rest of the chromosomes. Chromosome 8 hypermethylation signals were found mainly in the CpG island (CGI) shores and interspersed repeats. Validating the most significant differentially methylated CGI (P = 7.88 × 10(-11)) identified in trisomy 8 AML demonstrated a specific core region within the gene body of HHEX, which was significantly correlated with HHEX expression in both diagnostic and relapse trisomy 8 AMLs. Overall, the existence of extra chromosome 8 was associated with a global impact on the DNA methylation distribution with identification of HHEX gene methylation as a potential diagnostic marker for trisomy 8 AML.
Project description:DNA methylation plays an important role in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). The current study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) by a comprehensive bioinformatics analysis. In addition, we screened for DEGs affected by DNA methylation modification and further investigated their prognostic values for HNSCC. We included microarray data of DNA methylation (GSE25093 and GSE33202) and gene expression (GSE23036 and GSE58911) from Gene Expression Omnibus. Aberrantly methylated-DEGs were analyzed with R software. The Cancer Genome Atlas (TCGA) RNA sequencing and DNA methylation (Illumina HumanMethylation450) databases were utilized for validation. In total, 27 aberrantly methylated genes accompanied by altered expression were identified. After confirmation by The Cancer Genome Atlas (TCGA) database, 2 hypermethylated-low-expression genes (FAM135B and ZNF610) and 2 hypomethylated-high-expression genes (HOXA9 and DCC) were identified. A receiver operating characteristic (ROC) curve confirmed the diagnostic value of these four methylated genes for HNSCC. Multivariate Cox proportional hazards analysis showed that FAM135B methylation was a favorable independent prognostic biomarker for overall survival of HNSCC patients.
Project description:The goal of this study was to comprehensively identify CpG island methylation alterations between pancreatic cancers and normal pancreata and their associated gene expression alterations.We employed methylated CpG island amplification followed by CpG island microarray, a method previously validated for its accuracy and reproducibility, to analyze the methylation profile of 27,800 CpG islands covering 21 MB of the human genome in nine pairs of pancreatic cancer versus normal pancreatic epithelial tissues and in three matched pairs of pancreatic cancer versus lymphoid tissues from the same individual.This analysis identified 1,658 known loci that were commonly differentially methylated in pancreatic cancer compared with normal pancreas. By integrating the pancreatic DNA methylation status with the gene expression profiles of the same samples before and after treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, and the histone deacetylase inhibitor, trichostatin A, we identified dozens of aberrantly methylated and differentially expressed genes in pancreatic cancers including a more comprehensive list of hypermethylated and silenced genes that have not been previously described as targets for aberrant methylation in cancer.We expected that the identification of aberrantly hypermethylated and silenced genes will have diagnostic, prognostic, and therapeutic applications.
Project description:AML1-ETO is regarded as an initial event and plays a pivotal role in t(8;21) acute myeloid leukemia (AML) which is a highly heterogeneous disease. DNA methylation patterns are frequently deregulated in t(8;21) AML, though little is known of the mechanisms through which specific gene sets become aberrantly methylated. Here, We found that the promoter DNA methylation signature of t(8;21) AML blasts differs from those of normal CD34+ bone marrow cells and other AMLs. This signature contains 408 differentially methylated genes, many of which are genes involved in cancer pathway and myeloid leukemia. Database systematic survey and differential methylated regions (DMR) analysis were performed. These study demonstrated that a novel hypermethylated zinc finger containing protein-THAP10 is a target gene and can be epigenetic silenced by AML1-ETO at transcriptional level in t(8;21) AML and silencing of THAP10 by AML1-ETO predicts a poor clinical outcome. Our findings also identified that THAP10 is a bona fide target of miR-383 which can be epigenetic activated by AML1-ETO. In this study, we provided evidence that epigenetic silencing of THAP10 is the mechanistic link between AML1-ETO fusion proteins and tyrosine kinase cascades. In addition, we showed that THAP10 is a nuclear protein which inhibits myeloid proliferation inhibition and promotes differentiation both in vitro and in vivo. Altogether, our results revealed an unexpected and important link between fusion protein AML1-ETO, mir-383, THAP10 and tyrosine cascades in t(8;21) AML. Overall design: Bisulphite converted DNA from the 12 samples were hybridised to the Illumina HumanMethylation450 BeadChip, including 5 t(8;21)-positive AML, 5 t(8;21)-negative AML M2 subtype according FAB and 2 normal bone marrow.