Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome

We report the isolation of the 5′ flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive

DNA-Methylation Profiling of Fetal Tissues Reveals Marked Epigenetic Differences between Chorionic and Amniotic Samples

Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS) and 16 amniotic cell (AC) samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG) content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis

Array-based DNA methylation profiling of primary lymphomas of the central nervous system

<p>Abstract</p> <p>Background</p> <p>Although primary lymphomas of the central nervous system (PCNSL) and extracerebral diffuse large B-cell lymphoma (DLBCL) cannot be distinguished histologically, it is still a matter of debate whether PCNSL differ from systemic DLBCL with respect to their molecular features and pathogenesis. Analysis of the DNA methylation pattern might provide further data distinguishing these entities at a molecular level.</p> <p>Methods</p> <p>Using an array-based technology we have assessed the DNA methylation status of 1,505 individual CpG loci in five PCNSL and compared the results to DNA methylation profiles of 49 DLBCL and ten hematopoietic controls.</p> <p>Results</p> <p>We identified 194 genes differentially methylated between PCNSL and normal controls. Interestingly, Polycomb target genes and genes with promoters showing a high CpG content were significantly enriched in the group of genes hypermethylated in PCNSL. However, PCNSL and systemic DLBCL did not differ in their methylation pattern.</p> <p>Conclusions</p> <p>Based on the data presented here, PCNSL and DLBCL do not differ in their DNA methylation pattern. Thus, DNA methylation analysis does not support a separation of PCNSL and DLBCL into individual entities. However, PCNSL and DLBCL differ in their DNA methylation pattern from non- malignant controls.</p

Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B cell lymphomas

Histone methylation-modifiers, like EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%) also identified by prior exome or RNAseq studies, we here unravel KDM4C in chromosome 9p24, encoding a histone demethylase, to be recurrently altered. Focal structural variation was the main mechanism of KDM4C alterations, which was independent from 9p24 amplification. We identified KDM4C alterations also in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNAseq and genome sequencing data we predict KDM4C structural variants to result in loss-of-function. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas

Androgen Receptor Function Links Human Sexual Dimorphism to DNA Methylation

Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS) due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aber