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Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation.

ABSTRACT: Nuclear localization of non-phosphorylated, active beta-catenin is a measure of Wnt pathway activation and is associated with adverse outcome in patients with acute myeloid leukemia (AML). While genetic alterations of the Wnt pathway are infrequent in AML, inhibitors of this pathway are silenced by promoter methylation in other malignanices. Leukemia cell lines were examined for Wnt pathway inhibitor methylation and total beta-catenin levels, and had frequent methylation of Wnt inhibitors and upregulated beta-catenin by Western blot and immunofluorescence. One hundred sixty-nine AML samples were examined for methylation of Wnt inhibitor genes. Diagnostic samples from 72 patients with normal cytogenetics who received standard high-dose induction chemotherapy were evaluated for associations between methylation and event-free or overall survival. Extensive methylation of Wnt pathway inhibitor genes was observed in cell lines, and 89% of primary AML samples had at least one methylated gene: DKK1 (16%), DKK3 (8%), RUNX3 (27%), sFRP1 (34%), sFRP2 (66%), sFRP4 (9%), sFRP5 (54%), SOX17 (29%), and WIF1 (32%). In contrast to epithelial tumors, methylation of APC (2%) and RASSF1A (0%) was rare. In patients with AML with normal cytogenetics, sFRP2 and sFRP5 methylation at the time of diagnosis was associated with an increased risk of relapse, and sFRP2 methylation was associated with an increased risk for death. In patients with AML: (a) there is a high frequency of Wnt pathway inhibitor methylation; (b) Wnt pathway inhibitor methylation is distinct from that observed in epithelial malignancies; and (c) methylation of sFRP2 and sFRP5 may predict adverse clinical outcome in patients with normal karyotype AML.

SUBMITTER: Griffiths EA

PROVIDER: S-EPMC4000011 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation.

Griffiths Elizabeth A EA Gore Steven D SD Hooker Craig C McDevitt Michael A MA Karp Judith E JE Smith B Douglas BD Mohammad Helai P HP Ye Ying Y Herman James G JG Carraway Hetty E HE

Leukemia & lymphoma 20100901 9

Nuclear localization of non-phosphorylated, active beta-catenin is a measure of Wnt pathway activation and is associated with adverse outcome in patients with acute myeloid leukemia (AML). While genetic alterations of the Wnt pathway are infrequent in AML, inhibitors of this pathway are silenced by promoter methylation in other malignanices. Leukemia cell lines were examined for Wnt pathway inhibitor methylation and total beta-catenin levels, and had frequent methylation of Wnt inhibitors and up ...[more]

PMID: 20795789

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Project description:Acute myeloid leukemia (AML) is the second-most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature of AML. GATA4 has been suggested to be a tumor suppressor gene regulated by promoter hypermethylation in various types of human cancers although the expression and promoter methylation of GATA4 in pediatric AML is still unclear.Transcriptional expression levels of GATA4 were evaluated by semi-quantitative and real-time PCR. Methylation status was investigated by methylation-specific PCR (MSP) and bisulfate genomic sequencing (BGS). The prognostic significance of GATA4 expression and promoter methylation was assessed in 105 cases of Chinese pediatric acute myeloid leukemia patients with clinical follow-up records.MSP and BGS analysis showed that the GATA4 gene promoter is hypermethylated in AML cells, such as the HL-60 and MV4-11 human myeloid leukemia cell lines. 5-Aza treatment significantly upregulated GATA4 expression in HL-60 and MV4-11 cells. Aberrant methylation of GATA4 was observed in 15.0 % (3/20) of the normal bone marrow control samples compared to 56.2 % (59/105) of the pediatric AML samples. GATA4 transcript levels were significantly decreased in AML patients (33.06?±?70.94; P?=?0.011) compared to normal bone marrow/idiopathic thrombocytopenic purpura controls (116.76?±?105.39). GATA4 promoter methylation was correlated with patient leukocyte counts (WBC, white blood cells) (P?=?0.035) and minimal residual disease MRD (P?=?0.031). Kaplan-Meier survival analysis revealed significantly shorter overall survival time in patients with GATA4 promoter methylation (P?=?0.014).Epigenetic inactivation of GATA4 by promoter hypermethylation was observed in both AML cell lines and pediatric AML samples; our study implicates GATA4 as a putative tumor suppressor gene in pediatric AML. In addition, our findings imply that GATA4 promoter methylation is correlated with WBC and MRD. Kaplan-Meier survival analysis revealed significantly shorter overall survival in pediatric AML with GATA4 promoter methylation but multivariate analysis shows that it is not an independent factor. However, further research focusing on the mechanism of GATA4 in pediatric leukemia is required.

Project description:BackgroundAberrant DNA methylation plays a crucial role in the progression of myeloid neoplasms. Previously, our literature reported that slit guidance ligand 2 (SLIT2) promoter methylation was associated with disease progression and indicated a poor prognosis in patients with myelodysplastic syndrome. Herein, we further investigated the clinical implications and role of SLIT2 promoter methylation in patients with chronic myeloid leukemia (CML).MethodsThe level of SLIT2 promoter methylation was determined in 104 CML patients, and its clinical significance was analyzed. Moreover, demethylation studies were performed in K562 cells to determine the epigenetic mechanism by which SLIT2 promoter methylation is regulated in CML.ResultsThe level of SLIT2 promoter methylation was similar between CML patients and controls. However, deeper analysis revealed that the SLIT2 promoter methylation level in the accelerated phase (AP) and blast crisis (BC) was markedly higher than that in the chronic phase (CP) and controls. Additionally, a marked difference was identified between the SLIT2 promoter hypermethylated and non-hypermethylated groups among CML patients grouped by clinical stage. The frequency of SLIT2 hypermethylation was markedly increased with the progression of clinical stage, that is, it was the lowest in CP samples (12/80, 15%), higher in AP samples (4/8, 50%) and the highest in BC samples (11/16, 69%). Importantly, the level/density of SLIT2 promoter methylation was significantly higher in the advanced stage than in the early stage among the 6 tested paired CML patients. Epigenetically, the expression of the SLIT2-embedded non-coding genes SLIT2-IT1 and miR-218 expression was decreased in patients with CML. SLIT2 promoter hypermethylated cases had a markedly lower SLIT2-IT1 expression level than SLIT2 promoter non-hypermethylated cases. Moreover, SLIT2-IT1 and miR-218 expression was remarkably upregulated in a dose-dependent manner after demethylation treatment of K562 cells.ConclusionsHypermethylation of the SLIT2 promoter is correlated with disease progression in CML. Furthermore, SLIT2 promoter methylation may function by regulating the expression of the SLIT2-embedded non-coding genes SLIT2-IT1 and miR-218 during CML progression.

Project description:BackgroundRecent massive sequencing studies have revealed that SWI/SNF complexes are among the most frequently altered functional entities in solid tumors. However, the role of SWI/SNF in acute myeloid leukemia is poorly understood. To date, SWI/SNF complexes are thought to be oncogenic in AML or, at least, necessary to support leukemogenesis. However, mutation patterns in SWI/SNF genes in AML are consistent with a tumor suppressor role. Here, we study the SWI/SNF subunit BCL7A, which has been found to be recurrently mutated in lymphomas, but whose role in acute myeloid malignancies is currently unknown.MethodsData mining and bioinformatic approaches were used to study the mutational status of BCL7A and the correlation between BCL7A expression and promoter hypermethylation. Methylation-specific PCR, bisulfite sequencing, and 5-aza-2'-deoxycytidine treatment assays were used to determine if BCL7A expression was silenced due to promoter hypermethylation. Cell competition assays after BCL7A expression restoration were used to assess the role of BCL7A in AML cell line models. Differential expression analysis was performed to determine pathways and genes altered after BCL7A expression restoration. To establish the role of BCL7A in tumor development in vivo, tumor growth was compared between BCL7A-expressing and non-expressing mouse xenografts using in vivo fluorescence imaging.ResultsBCL7A expression was inversely correlated with promoter methylation in three external cohorts: TCGA-LAML (N = 160), TARGET-AML (N = 188), and Glass et al. (2017) (N = 111). The AML-derived cell line NB4 silenced the BCL7A expression via promoter hypermethylation. Ectopic BCL7A expression in AML cells decreased their competitive ability compared to control cells. Additionally, restoration of BCL7A expression reduced tumor growth in an NB4 mouse xenograft model. Also, differential expression analysis found that BCL7A restoration altered cell cycle pathways and modified significantly the expression of genes like HMGCS1, H1-0, and IRF7 which can help to explain its tumor suppressor role in AML.ConclusionsBCL7A expression is silenced in AML by promoter methylation. In addition, restoration of BCL7A expression exerts tumor suppressor activity in AML cell lines and xenograft models.

Project description:The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARalpha, or PLZF-RARalpha. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (gamma-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. beta-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

Project description:BackgroundAcute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear.MethodsEleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis.ResultsThe MT3 promoter was hypermethylated in leukemia cell lines. More CpG's methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P?<?0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P?=?0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P?<?0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1.ConclusionMT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.

Project description:ObjectiveThe escape from T cell-mediated immune surveillance is an important cause of death for patients with acute myeloid leukemia (AML). This study aims to identify clonal heterogeneity in leukemia progenitor cells and explore molecular or signaling pathways associated with AML immune escape.MethodsSingle-cell RNA sequencing (scRNA-seq) was performed to identified AML-related cellular subsets, and intercellular communication was analyzed to investigate molecular mechanisms associated with AML immune escape. Bulk RNA sequencing (RNA-seq) was performed to screen differentially expressed genes (DEGs) related to hematopoietic stem cell progenitors (HSC-Prog) in AML, and critical ore signaling pathways and hub genes were found by Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The mRNA level of the hub gene was verified using quantitative real-time PCR (qRT-PCR) and the protein level of human leukocyte antigen A (HLA-A) using enzyme-linked immuno sorbent assay (ELISA).ResultsscRNA-seq analysis revealed a large heterogeneity of HSC-Prog across samples, and the intercellular communication analysis indicated a strong association between HSC-Prog and CD8+-T cells, and HSC-Prog also had an association with HLA-A. Transcriptome analysis identified 1748 DEGs, enrichment analysis results showed that non-classical wnt signaling pathway was associated with AML, and 4 pathway-related genes (RHOA, RYK, CSNK1D, NLK) were obtained. After qRT-PCR and ELISA validation, hub genes and HLA-A were found to be down-regulated in AML and up-regulated after activation of the non-classical Wnt signaling pathway.ConclusionIn this study, clonal heterogeneity of HSC-Prog cells in AML was identified, non-classical wnt signaling pathways associated with AML were identified, and it was verified that HLA-A could be upregulated by activation of non-classical wnt signaling, thereby increasing antigen presentation.

Dataset Information

Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation.

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Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation.

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