Tissue specific patterns of methylated cytosine residues vary with age, can be altered by environmental factors, and are often abnormal in human disease yet the cellular consequences of DNA ...methylation are incompletely understood. Although the bodies of highly expressed genes are often extensively methylated in plants, the relationship between intragenic methylation and expression is less clear in mammalian cells. We performed genome-wide analyses of DNA methylation and gene expression to determine how the pattern of intragenic methylation correlates with transcription and to assess the relationship between methylation of exonic and intronic portions of the gene body. We found that dense exonic methylation is far more common than previously recognized or expected statistically, yet first exons are relatively spared compared to more downstream exons and introns. Dense methylation surrounding the transcription start site (TSS) is uncoupled from methylation within more downstream regions suggesting that there are at least two classes of intragenic methylation. Whereas methylation surrounding the TSS is tightly linked to transcriptional silencing, methylation of more downstream regions is unassociated with the magnitude of gene expression. Notably, we found that DNA methylation downstream of the TSS, in the region of the first exon, is much more tightly linked to transcriptional silencing than is methylation in the upstream promoter region. These data provide direct evidence that DNA methylation is interpreted dissimilarly in different regions of the gene body and suggest that first exon methylation blocks transcript initiation, or vice versa. Our data also show that once initiated, downstream methylation is not a significant impediment to polymerase extension. Thus, the consequences of most intragenic DNA methylation must extend beyond the modulation of transcription magnitude.Sequencing data and gene expression microarray data have been submitted to the GEO online database (accession number SRA012081.1). Supporting information including expanded methods and ten additional figures in support of the manuscript is provided.
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We explored diverse alterations contributing to liposarcomagenesis by sequencing the genome, exome, transcriptome, and cytosine methylome of a primary and recurrent dedifferentiated liposarcoma ...(DLPS) from distinct chemotherapy/radiotherapy-naïve patients. The liposarcoma genomes had complex structural rearrangements, but in different patterns, and with varied effects on the structure and expression of affected genes. While the point mutation rate was modest, integrative analyses and additional screening identified somatic mutations in HDAC1 in 8.3% of DLPS. Liposarcoma methylomes revealed alterations in differentiation pathway genes, including CEBPA methylation in 24% of DLPS. Treatment with demethylating agents, which restored CEBPA expression in DLPS cells, was anti-proliferative and pro-apoptotic in vitro and reduced tumor growth in vivo. Both genetic and epigenetic abnormalities established a role for small RNAs in liposarcomagenesis, typified by methylation-induced silencing of microRNA-193b in DLPS but not its well-differentiated counterpart. These findings reveal an unanticipated role for epigenetic abnormalities in DLPS tumors and suggest demethylating agents as potential therapeutics.
Multimodality sequence analysis of DLPS revealed recurrent mutations and epigenetic abnormalities critical to liposarcomagenesis and to the suppression of adipocyte differentiation. Pharmacologic inhibition of DNA methylation promoted apoptosis and differentiated DLPS cells in vitro and inhibited tumor growth in vivo, providing a rationale for investigating methylation inhibitors in this disease.
We have utilized retinoic acid receptor γ (gamma) knockout (RARγ(-/-)) embryonic stem (ES) cells as a model system to analyze RARγ mediated transcriptional regulation of stem cell differentiation. ...Most of the transcripts regulated by all-trans retinoic acid (RA) in ES cells are dependent upon functional RARγ signaling. Notably, many of these RA-RARγ target genes are implicated in retinoid uptake and metabolism. For instance, Lrat (lecithin:retinol acyltransferase), Stra6 (stimulated by retinoic acid 6), Crabp2 (cellular retinoic acid binding protein 2), and Cyp26a1 (cytochrome p450 26a1) transcripts are induced in wild type (WT), but not in RARγ(-/-) cells. Transcripts for the transcription factors Pbx1 (pre-B cell leukemia homeobox-1), Wt1 (Wilm's tumor gene-1), and Meis1 (myeloid ecotropic viral integration site-1) increase upon RA treatment of WT, but not RARγ(-/-) cells. In contrast, Stra8, Dleu7, Leftb, Pitx2, and Cdx1 mRNAs are induced by RA even in the absence of RARγ. Mapping of the epigenetic signature of Meis1 revealed that RA induces a rapid increase in the H3K9/K14ac epigenetic mark at the proximal promoter and at two sites downstream of the transcription start site in WT, but not in RARγ(-/-) cells. Thus, RA-associated increases in H3K9/K14ac epigenetic marks require RARγ and are associated with increased Meis1 transcript levels, whereas H3K4me3 is present at the Meis1 proximal promoter even in the absence of RARγ. In contrast, at the Lrat proximal promoter primarily the H3K4me3 mark, and not the H3K9/K14ac mark, increases in response to RA, independently of the presence of RARγ. Our data show major epigenetic changes associated with addition of the RARγ agonist RA in ES cells.
Abstract
Formalin-fixed paraffin-embedded samples (FFPE) are in many case, the only available source of biological materiel available for translational studies. However the quantity and quality of ...nucleic acid extracted from FFPE samples is very often challenging (small amount and high level of degradation), which makes genomic analysis in general, and epigenomics analysis in particular, difficult to impossible. Here we are showing that we developed a robust, unbiased and cost effective method for genome-wide analysis of DNA methylation of FFPE DNA. Our method, named STAMP for Sequence Tag Analysis of Methylation Patterns, uses a recombinant protein containing a methyl-CpG binding domain (MBD) to precipitate methylated DNA fragments that are then identified by massively parallel sequencing using the SOLiD sequencer (ABI). We generate ∼15-million sequence tags per specimen and utilize an analytical platform comprised of custom-written R-language algorithms to study genomic DNA methylation patterns. We used this technology to compare the methylation patterns of matched fresh frozen (FF) and FFPE tumor specimens. Tissues had been stored for as long as 6 years. Because the STAMP method utilizes fragmented DNA as starting material we hypothesized that this method would be well-suited to the study of FFPE DNA methylation. Using this method, we found the methylation patterns of the FFPE and matched FF specimens were highly correlated. We successfully detected previously known methylated patterns and found hundreds of novel methylated regions. DNA methylation patterns were observed at all scales across the genome: from whole chromosomes to individual genes. We found that densely methylated elements are often highly conserved or closely associated with gene coding regions and promoters. We identified distinct patterns of DNA methylation surrounding the transcription start and termination sites of all genes. Genome-wide analyses of DNA methylation patterns of FFPE samples using the STAMP assay opens up the possibility to analyze large repositories of tumor specimens as well as samples that have been collected during routine clinical work and clinical trials. Large scales studies of DNA methylation using existing FFPE specimens linked to treatment outcomes may be rapidly translated to new epigenetic prognostic measures for cancer patients.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2196.
Abstract 591
The human genome is adorned with methylated cytosine residues that function in the epigenetic guidance of cellular differentiation and development. Cellular interpretation of this ...epigenetic mark is incompletely understood and tissue specific patterns of DNA methylation vary with age, can be altered by environmental factors, and are often abnormal in human disease. Aberrant DNA methylation is a common means by which tumor suppressor genes (TSGs) are inactivated during carcinogenesis (Baylin, Herman, Graff, Vertino and Issa 1998; Laird and Jaenisch 1996; Singal and Ginder 1999). Unlike genetic mechanisms of gene inactivation, such as gene deletion and mutation, the epigenetic silencing of TSGs by promoter hypermethylation is potentially reversible. This has led to the broad interest of cancer biologists in the study of DNA methylation.
We developed a method for genome-wide analysis of DNA methylation by using a recombinant protein containing a methyl-CpG binding domain (MBD) to enrich methylated DNA fragments that are then identified by massively parallel sequencing using the SOLiD sequencer (ABI). We generated ∼15-million sequence tags per specimen and wrote custom R-language algorithms to develop an analytical platform with which to study DNA methylation. We used this technology to study the pharmacodynamics of DNA methylation in acute myelogenous leukemia (AML) cells following exposure to the hypomethylating agent, 5-aza-2'-deoxycytidine (decitabine). We compared DNA methylation patterns before and after decitabine treatment with transcriptional activity revealed by microarrays (Illumina) and quantitative PCR. We found that Sequence Tag Analysis of Methylation Profiles (STAMP) permits highly reproducible, genome-wide identification of DNA methylation density at near base-pair resolution. This method is cost effective and can be extended, without modification, to any mapped genome.
STAMP analysis revealed patterned DNA methylation at all scales across the genome: from whole chromosomes to individual genes. We found that densely methylated elements (DMEs) of the human genome are often highly conserved or closely associated with gene coding regions and promoters. We identified distinct patterns of DNA methylation surrounding the transcription start and termination sites of all genes. These methylation patterns are associated with transcriptional activity of neighboring genes. Interestingly, genes with a densely methylated transcription start site (TSS) have little methylation in the surrounding regions whereas genes with little or no methylation at the TSS have disproportionately higher methylation within their gene bodies. In untreated cells, we detected ∼75,000 DMEs (false discovery rate <0.01) with a median length ∼600 bp and with 75% being less than 960bp. The longest DMEs extend up to ∼24000 bp and are composed of microsatellite clusters. The majority of the DMEs are not classic CpG islands (CGI) but are GC-rich regions (median 57% GC) with a greater than expected incidence of CpG dinucleotides (median CpG observed/expected 0.49): results that suggest the definition of a CGI excludes the majority of the methylated human genome. Although the pattern of DNA methylation was qualitatively similar in cells treated with decitabine, we found that the density of methylation was generally lower and fewer DMEs (∼50,000) were identified. Decitabine treatment led to increased expression of ∼800 genes involved in cell cycle control, apoptosis and cellular differentiation whereas the ∼50 genes with downregulated expression were most commonly involved in RNA metabolism. Distinct pre-treatment DNA methylation patterns were associated with, and tended to predict, the transcriptional activity following treatment with decitabine.
We developed and utilized a powerful new technology to uncover the genome-wide effects of decitabine on DNA methylation patterns in AML. We found that although decitabine induces genome-wide DNA hypomethylation, its effect on transcription depends upon the pattern of DNA methylation prior to treatment. The STAMP methodology leverages the power and flexibility of massively parallel sequencing with the high selectivity of the MBD for its natural ligand, methyl-CpG. This assay permits robust, unbiased and highly sensitive whole-genome identification of methylated DNA segments.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Ibrutinib is a first-in-class inhibitor of Bruton tyrosine kinase (BTK) and has shown single-agent activity in recurrent/refractory central nervous system (CNS) lymphoma. Clinical responses are often ...transient or incomplete, suggesting a need for a combination therapy approach. We conducted a phase 1b clinical trial to explore the sequential combination of ibrutinib (560 or 840 mg daily dosing) with high-dose methotrexate (HD-MTX) and rituximab in patients with CNS lymphoma (CNSL). HD-MTX was given at 3.5 g/m2 every 2 weeks for a total of 8 doses (4 cycles; 1 cycle = 28 days). Ibrutinib was held on days of HD-MTX infusion and resumed 5 days after HD-MTX infusion or after HD-MTX clearance. Single-agent daily ibrutinib was administered continuously after completion of induction therapy until disease progression, intolerable toxicity, or death. We also explored next-generation sequencing of circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) before and during treatment. The combination of ibrutinib, HD-MTX, and rituximab was tolerated with an acceptable safety profile (no grade 5 events, 3 grade 4 events). No dose-limiting toxicity was observed. Eleven of 15 patients proceeded to maintenance ibrutinib after completing 4 cycles of the ibrutinib/HD-MTX/rituximab combination. Clinical responses occurred in 12 of 15 patients (80%). Sustained tumor responses were associated with clearance of ctDNA from the CSF. This trial was registered at www.clinicaltrials.gov as #NCT02315326.
•Ibrutinib/methotrexate/rituximab combination treatment is safe and shows promising clinical activity in CNSL.•Analysis of ctDNA in CSF may be useful to monitor disease burden in patients with CNSL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability
patterned by distinct mutational processes
, tumour heterogeneity
and intraperitoneal spread
. Immunotherapies ...have had limited efficacy in HGSOC
, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8
T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFβ signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Bloodstream infection by highly antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus (VRE), is a growing clinical problem that increasingly defies medical intervention. ...Identifying patients at high risk for bacterial sepsis remains an important clinical challenge. Recent studies have shown that antibiotics can alter microbial diversity in the intestine. Here, we characterized these effects using 16s rDNA pyrosequencing and demonstrated that antibiotic treatment of mice enabled exogenously administered VRE to efficiently and nearly completely displace the normal microbiota of the small and large intestine. In the clinical setting, we found that intestinal domination by VRE preceded bloodstream infection in patients undergoing allogeneic hematopoietic stem cell transplantation. Our results demonstrate that antibiotics perturb the normal commensal microbiota and set the stage for intestinal domination by bacteria associated with hospital-acquired infections. Thus, high-throughput DNA sequencing of the intestinal microbiota could identify patients at high risk of developing bacterial sepsis.
Bruton tyrosine kinase (BTK) links the B-cell antigen receptor (BCR) and Toll-like receptors with NF-κB. The role of BTK in primary central nervous system (CNS) lymphoma (PCNSL) is unknown. We ...performed a phase I clinical trial with ibrutinib, the first-in-class BTK inhibitor, for patients with relapsed or refractory CNS lymphoma. Clinical responses to ibrutinib occurred in 10 of 13 (77%) patients with PCNSL, including five complete responses. The only PCNSL with complete ibrutinib resistance harbored a mutation within the coiled-coil domain of CARD11, a known ibrutinib resistance mechanism. Incomplete tumor responses were associated with mutations in the B-cell antigen receptor-associated protein CD79B.
-mutant PCNSLs showed enrichment of mammalian target of rapamycin (mTOR)-related gene sets and increased staining with PI3K/mTOR activation markers. Inhibition of the PI3K isoforms p110α/p110δ or mTOR synergized with ibrutinib to induce cell death in
-mutant PCNSL cells.
Ibrutinib has substantial activity in patients with relapsed or refractory B-cell lymphoma of the CNS. Response rates in PCNSL were considerably higher than reported for diffuse large B-cell lymphoma outside the CNS, suggesting a divergent molecular pathogenesis. Combined inhibition of BTK and PI3K/mTOR may augment the ibrutinib response in
-mutant human PCNSLs.
.
Inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC) that leads to the activation of hypoxia-inducible ...transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response that is specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that loss of Polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven cytohesin 1 interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.
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