DNA Methylation Independent Silencing of the RARα Gene Expression in Acute Myeloid Leukemia.

Abstract All-trans-retinoic acid (ATRA) and the gene encoding retinoic acid receptor-α (RARα) have been implicated in the pathogenesis and treatment of acute promyelocytic leukemia (APL). Nevertheless, the role of these molecules in the pathogenesis and therapy of non-APL acute myeloid leukemias (AMLs) remains unclear. Previously we have shown that expression of the ATRA-inducible RARα2 isoform is downregulated in a variety of AML cell lines and increases with hematopoietic differentiation along the myelomonocytic lineage. Using quantitative real-time PCR we have now investigated expression of the RARα gene in primary AML cells (n=23) and report that as in AML cell lines the levels of RARα2 mRNAs are markedly reduced (by 48 fold in APL, p≤0.05, and by 52 fold in non-APL AML, p≤0.01) relative to cord blood (CB) derived CD33 (or CD34) positive cell population (n=4). However, in contrast to the AML cell lines and normal hematopoietic progenitors, the expression of the RARα1 isoform was also significantly reduced in primary AML samples (up to 26 fold, p≤0.05). Examination of potential mechanisms underlying the silencing of the RARα gene expression in AML revealed that the RARα2 promoter possesses two small CpG islands that are fully methylated in all AML cell lines examined. Consistent with the expression pattern of RARα1 a single CpG island in the RARα1 promoter region was unmethylated in all these samples. As expected from such results expression of RARα2, but not RARα1, could be stimulated with a DNA demethylating agent 5-aza-2′deoxycytidine and synergistic effects between 5-aza-2′deoxycytidine and ATRA were observed on both RARα2 expression and cellular differentiation of APL and non-APL AML cell lines. Extending this analysis to clinical material we have surprisingly discovered that RARα1 and RARα2 CpG islands are unmethylated in all AML patient samples, including 3 APL cases, suggesting that DNA methylation may not play a significant role in silencing of the RARα gene expression in primary AML cells. Chromatin immunoprecipitation of the RARα2 regulatory region with antibodies to specific histone modifications revealed presence of other negatively acting chromatin states in primary AML samples (relative to normal CD33 positive CB cells), including decreased histone H3 acetylation as well as decreased di- and tri-methylation of histone H3 lysine 4. A possibility that microRNAs targeting sequences in the common 3′-UTR regions of the RARα1 and α2 isoforms may also contribute to silencing of both RARα1 and α2 expression in AML cannot be excluded. These results underscore the complexities of mechanisms that are responsible for silencing of gene expression in AML and support the notion that diminished RARα expression contributes to leukemogenesis.

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DNA Methylation Predicts the Response of Triple-Negative Breast Cancers to All-Trans Retinoic Acid

Cancers ◽

10.3390/cancers10110397 ◽

2018 ◽

Vol 10 (11) ◽

pp. 397 ◽

Cited By ~ 11

Author(s):

Krysta Coyle ◽

Cheryl Dean ◽

Margaret Thomas ◽

Dejan Vidovic ◽

Carman Giacomantonio ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Retinoic Acid ◽

Cell Lines ◽

Triple Negative ◽

Atra Treatment ◽

Response Elements ◽

Acid Binding Protein ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid

All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment.

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Pyrosequencing Analysis of the Methylation Status of the CpG Island in the Retinoic Acid Receptor-β2 (RAR-β2) Gene Promoter.

Blood ◽

10.1182/blood.v104.11.4297.4297 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4297-4297

Author(s):

Da-Cheng Zhou ◽

David Reynolds ◽

Robert E. Gallagher

Keyword(s):

Retinoic Acid ◽

Cell Lines ◽

Cpg Island ◽

Retinoic Acid Receptor ◽

Methylation Status ◽

Cpg Islands ◽

Leukemia Cell ◽

Gene Promoter ◽

Cpg Dinucleotides ◽

Retinoic Acid Receptor Β2

Abstract CpG islands are associated with the 5′-ends of most housekeeping genes and many regulated genes. We have hypothesized that the methylation status of CpG islands in the promoter region of all-trans retinoic acid (ATRA) target genes such as retinoic acid receptor-β2 (RAR-β2) may be related to ATRA resistance and relapse of acute promyelocytic leukemia (APL). In the present study, we developed a highly quantitative method to assess the degree of DNA methylation at specific sites using PyrosequencingTM technology (Biotage, Uppsala, Sweden). This method is more quantitative than methylation-specific PCR, and is as accurate as but simpler and more robust than combined bisulfite restriction analysis (COBRA) or direct sequencing of plasmid clones of PCR products. We used this method to study 14 CpG dinucleotides in the CpG island of the RAR-β2 promoter. In reconstruction experiments in which 100% methylated and 100% unmethylated DNAs were admixed in different proportions (100:0; 80:20, 60:40, etc), a straightline graph was obtained over the entire range from 0 – 100% for each of the 14 CpG dinucleotides (r2 > 0.98). The results were highly reproducible and the variation between the results obtained from repetitive pyrosequencing of the same DNA was very low (S.D.<2%). Also the standard deviation between measurements of different PCR-amplified, bisulfite-converted DNAs prepared in separate experiments was <5%. We then used this method to measure the methylation level of the CpG island of the RAR-β2 promoter in several leukemia cell lines. Of 3 APL cell lines, the two with PML-RARα mutations, i.e., UF-1 and AP-1060, had higher overall methylation, compared to the NB4 cell line with non-mutant PML-RARα (mean ± SD = 52 ± 25% and 55 ± 21%, versus 43 ± 20%; p = 0.04 and 0.08, respectively; SD calculated from the variation across the 14 CpG dinucleotides for each source). Two myeloid leukemia cell lines with predominantly erythroid lineage characteristics, K562 and TF-1, had much lower levels of RAR-β2 methylation (2.6 ± 0.9% and 8.9 ± 3.2%, respectively). In the AP-1060 culture system, recently developed in our lab, there was little difference in methylation status between the patient bone marrow source and an intermediate, non-immortalized cell strain AP-1060S (27 ± 13% vs. 31 ± 25%). Further, there was no difference between lower and higher passage generations of AP-1060S (31 ± 25% vs. 30 ± 26%), which had markedly different replicative potential, indicating that replicative senescence at higher AP-1060 passages was not associated with altered methylation of the RAR-β2 gene promoter. However, the established, immortalized AP-1060 cell line had significantly greater methylation (52 ± 25%) than either the bone marrow source or AP-1060S (p <0.0001 and p = 0.0002, respectively), consistent with published reports of increased promoter methylation of cell lines. In conclusion, pyrosequencing is a high throughput method with great quantitative strength, and can be used for accurate and consistent analysis of methylation status in large numbers of samples.

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Genome-Wide DNA Methylation Analysis Shows Enrichment of Differential Methylation in “Open Seas” and Enhancers and Reveals Hypomethylation in DNMT3A Mutated Cytogenetically Normal AML (CN-AML)

Blood ◽

10.1182/blood.v120.21.653.653 ◽

2012 ◽

Vol 120 (21) ◽

pp. 653-653 ◽

Cited By ~ 2

Author(s):

Ying Qu ◽

Andreas Lennartsson ◽

Verena I. Gaidzik ◽

Stefan Deneberg ◽

Sofia Bengtzén ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Differential Methylation ◽

Methylation Analysis ◽

Cpg Sites ◽

Genome Wide ◽

Genomic Regions ◽

Methylation Patterns

Abstract Abstract 653 DNA methylation is involved in multiple biologic processes including normal cell differentiation and tumorigenesis. In AML, methylation patterns have been shown to differ significantly from normal hematopoietic cells. Most studies of DNA methylation in AML have previously focused on CpG islands within the promoter of genes, representing only a very small proportion of the DNA methylome. In this study, we performed genome-wide methylation analysis of 62 AML patients with CN-AML and CD34 positive cells from healthy controls by Illumina HumanMethylation450K Array covering 450.000 CpG sites in CpG islands as well as genomic regions far from CpG islands. Differentially methylated CpG sites (DMS) between CN-AML and normal hematopoietic cells were calculated and the most significant enrichment of DMS was found in regions more than 4kb from CpG Islands, in the so called open sea where hypomethylation was the dominant form of aberrant methylation. In contrast, CpG islands were not enriched for DMS and DMS in CpG islands were dominated by hypermethylation. DMS successively further away from CpG islands in CpG island shores (up to 2kb from CpG Island) and shelves (from 2kb to 4kb from Island) showed increasing degree of hypomethylation in AML cells. Among regions defined by their relation to gene structures, CpG dinucleotide located in theoretic enhancers were found to be the most enriched for DMS (Chi χ2<0.0001) with the majority of DMS showing decreased methylation compared to CD34 normal controls. To address the relation to gene expression, GEP (gene expression profiling) by microarray was carried out on 32 of the CN-AML patients. Totally, 339723 CpG sites covering 18879 genes were addressed on both platforms. CpG methylation in CpG islands showed the most pronounced anti-correlation (spearman ρ =-0.4145) with gene expression level, followed by CpG island shores (mean spearman rho for both sides' shore ρ=-0.2350). As transcription factors (TFs) have shown to be crucial for AML development, we especially studied differential methylation of an unbiased selection of 1638 TFs. The most enriched differential methylation between CN-AML and normal CD34 positive cells were found in TFs known to be involved in hematopoiesis and with Wilms tumor protein-1 (WT1), activator protein 1 (AP-1) and runt-related transcription factor 1 (RUNX1) being the most differentially methylated TFs. The differential methylation in WT 1 and RUNX1 was located in intragenic regions which were confirmed by pyro-sequencing. AML cases were characterized with respect to mutations in FLT3, NPM1, IDH1, IDH2 and DNMT3A. Correlation analysis between genome wide methylation patterns and mutational status showed statistically significant hypomethylation of CpG Island (p<0.0001) and to a lesser extent CpG island shores (p<0.001) and the presence of DNMT3A mutations. This links DNMT3A mutations for the first time to a hypomethylated phenotype. Further analyses correlating methylation patterns to other clinical data such as clinical outcome are ongoing. In conclusion, our study revealed that non-CpG island regions and in particular enhancers are the most aberrantly methylated genomic regions in AML and that WT 1 and RUNX1 are the most differentially methylated TFs. Furthermore, our data suggests a hypomethylated phenotype in DNMT3A mutated AML. Disclosures: No relevant conflicts of interest to declare.

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Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽

10.1182/blood.v110.11.492.492 ◽

2007 ◽

Vol 110 (11) ◽

pp. 492-492

Author(s):

Wei-Gang Tong ◽

William G. Wierda ◽

Neby Bekele ◽

Shao-Qing Kuang ◽

Michael J. Keating ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Cpg Island ◽

Trichostatin A ◽

Methylation Status ◽

Cpg Islands ◽

Human Leukemia ◽

Dna Hypomethylation ◽

Line P ◽

Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

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Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse

Blood ◽

10.1182/blood-2007-11-126227 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1366-1373 ◽

Cited By ~ 103

Author(s):

Heike Kroeger ◽

Jaroslav Jelinek ◽

Marcos R. H. Estécio ◽

Rong He ◽

Kimie Kondo ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Leukemia Cell ◽

Transcription Start Sites ◽

Remission Maintenance ◽

Acute Myeloid

AbstractDNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.

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Genome Wide Study of DNA Methylation In AML

Blood ◽

10.1182/blood.v116.21.3618.3618 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3618-3618

Author(s):

Marwa Saied ◽

Sabah Khaled ◽

Thomas Down ◽

Jacek Marzec ◽

Paul Smith ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Differential Methylation ◽

Methylation Analysis ◽

Promoter Regions ◽

Gene Body Methylation ◽

Normal Bone ◽

Genome Wide

Abstract Abstract 3618 DNA methylation is the most stable epigenetic modification and has a major role in cancer initiation and progression. The two main aims for this research were, firstly, to use the genome wide analysis of DNA methylation to better understand the development of acute myeloid leukemia (AML). The second aim was to detect differentially methylated genes/regions between certain subtypes of AML and normal bone marrow (NBM). We used the methylated DNA immunoprecipitation technique followed by high-throughput sequencing by Illumina Genome Analyser II (MeDIP -seq) for 9 AML samples for which ethical approval has been obtained. The selected leukemias included three with the t(8; 21), three with the t(15; 17) translocations and three with normal karyotypes (NK). The control samples were 3 normal bone marrows (NBMs) from healthy donors. The number of reads generated from Illumina ranged between 18– 20 million paired-end reads/lane with a good base quality from both ends (base quality > 30 represented 75%-85% of reads). The reads were aligned using 2 algorithms (Maq and Bowtie) and the methylation analysis was performed by Batman software (Bayesian Tool for Methylation Analysis). The creation of this genome-wide methylation map for AML permits the examination of the patterns for key genetic elements. Investigation of the 35,072 promoter regions identified 80 genes, which showed a significant differential methylation levels in leukemic cases in comparison to NBM; consistently high methylation levels in leukaemia were detected in the promoters of 70 genes e.g. DPP6, ID4, DCC, whereas high methylation levels in NBM, lost in leukaemia was observed in 10 genes e.g. ATF4. For each AML subtype, we also identified significant differentially methylated promoter regions e.g. PAX1 for t(8; 21), GRM7 for t(15; 17), NPM2 for NK. An analysis of gene body methylation identified 49 genes with significantly higher methylation in AML in comparison to NBM e.g. MYOD1 and 31 genes with a higher methylation in NBMs than AML e.g. GNG8. A similar analysis of 23,600 CpG islands identified 400 CpG islands with significant differential methylation levels between leukaemia and NBMs (212 CpG islands were found to have significantly increased methylation in leukaemia and 188 CpG islands had significantly higher methylation in NBMs). The pattern of methylation in CpG island “shores” (2 KB from either side of each CpG island) has been investigated and 312 CpG island shores showed a higher methylation in leukaemia and 88 CpG shores had a significant increase methylation levels in NBMs. This genome wide methylation map has been validated by using direct bisulfite sequencing of the regions identified above (Spearman r= 0.8, P <0.0001) and also by using Illumina Infinium assay (Spearman r= 0.7 P <0.0001) which interrogates regions at single representative CpGs. Comparison of previous array based gene expression data with this methylation map revealed a significant negative correlation between promoter methylation and gene expression (Pearson r= -0.9, P< 0.0001) while, gene body methylation showed a small negative correlation with gene expression, that was found in genes of CpG density >3% (Pearson r= -0.3, P< 0.0001). Conclusion: we have established a high-resolution (100bp) map of DNA methylation in AML and thus identified a novel list of genes, which have significantly differential methylation levels in AML. Disclosures: No relevant conflicts of interest to declare.

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DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

Blood ◽

10.1182/blood-2007-05-088344 ◽

2008 ◽

Vol 111 (4) ◽

pp. 2374-2377 ◽

Cited By ~ 28

Author(s):

Annegret Glasow ◽

Angela Barrett ◽

Kevin Petrie ◽

Rajeev Gupta ◽

Manuel Boix-Chornet ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Retinoic Acid ◽

Cell Lines ◽

Myeloid Leukemia ◽

Retinoic Acid Receptor ◽

Positive Effects ◽

All Trans Retinoic Acid ◽

H3 Acetylation ◽

Acute Myeloid

The retinoic acid receptor (RAR) α gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARα2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARα2 expression is dramatically reduced in primary AML blasts. Expression of the RARα1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARα1 and RARα2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARα2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.

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DOT1L As a Therapeutic Target for the Treatment of DNMT3A-Mutant Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.614.614 ◽

2014 ◽

Vol 124 (21) ◽

pp. 614-614

Author(s):

Rachel E. Rau ◽

Min Luo ◽

Benjamin Rodriguez ◽

Mira Jeong ◽

Allison Rosen ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Therapeutic Target ◽

Myeloid Leukemia ◽

Hox Genes ◽

Time Dependent ◽

Mutant Cells ◽

Acute Myeloid

Abstract Mutations of the DNA methyltransferase, DNMT3A, occur in approximately 20% of adult patients with acute myeloid leukemia (AML), and portend a poor prognosis. The most common of these mutations results in a dominant negative loss of function. Our lab observed that upon conditional inactivation of Dnmt3a in the murine hematopoietic system, Dnmt3a–/– hematopoietic stem cells (HSCs) expanded dramatically while their differentiation was inhibited, consistent with a pre-leukemic state. The likely mechanism by which Dnmt3a loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes, however our current understanding is incomplete. In analyses of gene expression data, we observed that murine Dnmt3a–/– HSCs markedly overexpress the histone 3, lysine 79 (H3K79) methyltransferase, Dot1l. This is of interest given the known functional interplay between DNA methylation and histone modifications. Additionally, DOT1L plays a critical role in leukemia with MLL-rearrangements, lesions that essentially never occur concomitantly with DNMT3A mutations in AML. The mutual exclusion of these lesions combined with the observed overexpression of Dot1l in our murine model, led us to postulate that MLL-rearrangements and DNMT3A mutations are distinct epigenetic aberrations that converge on a common mechanism resulting in dysregulated gene expression, specifically mediated by H3K79 methylation (H3K79me). Therefore, in the pathogenesis of DNMT3A-mutant AML, like in MLL-rearranged leukemia, DOT1L-induced H3K79me may play a central role, and may represent a viable therapeutic target. Throughout the genome of normal HSCs, expansive regions with low DNA methylation (canyons) exist. These canyons span conserved domains frequently containing transcription factors. In our Dnmt3a-/- model, canyon borders, particularly flanking genes frequently dysregulated in human leukemia such as HOX genes, are highly prone to DNA methylation loss when Dnmt3a is deleted, resulting in canyon expansion. However, not all canyons expand with Dnmt3a loss. We found a close association between canyon behavior and the associated histone marks, with expanding canyons characterized by a lack of the repressive histone mark, H3K27me. To determine if in Dnmt3a-mediated malignant hematopoiesis, H3K79me also correlates with altered DNA methylation, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for H3K79 di-methylation (H3K79me2) and aligned these data with whole genome DNA methylation data. This revealed that H3K79me2 specifically coats canyons that lose methylation with Dnmt3a loss, including the HoxA and HoxB clusters, but is not present at canyons without methylation loss. This strong correlation between H3K79me and DNA hypomethylation with Dnmt3a loss suggests a functional interaction. To examine whether this also occurred in human samples with DNMT3A mutations, we analyzed TCGA data, which confirmed many canyon borders as regions with marked DNA methylation loss. Further, many canyon-associated genes, including HOX genes are significantly changed in human DNMT3A-mutant AML. To explore the role of H3K79me, and specifically of DOT1L in human DNMT3A-mutant AML, we utilized the DNMT3A-mutant human AML cell lines OCIAML2 and OCIAML3. These cell lines were found to have increased total H3K79me compared to DNMT3A-wild type controls, consistent with the increased Dot1l expression in Dnmt3a–/– HSCs. We then tested the in vitro efficacy of two selective DOT1L inhibitors, SYC-522 (Anglin. J Med Chem. 2011) and the Epizyme compound, EPZ004777 (Daigle. Cancer Cell. 2011), against DNMT3A-mutant cells. Both compounds led to a dose- and time-dependent inhibition of proliferation and induction of apoptosis in the DNMT3A-mutant cell lines at concentrations comparable to those used for MLL-rearranged cell lines. With treatment, DNMT3A-mutant cells also had evidence of induction of differentiation with increased expression of the mature monocyte marker, CD14. Importantly, oncogenic HOX genes overexpressed in DNMT3A-mutant AML were repressed in a time-dependent fashion with DOT1L inhibitor treatment. In conclusion, our data suggest that DOT1L may be a novel, immediately actionable therapeutic target for the treatment of DNMT3A-mutant AML. Disclosures Rau: Epizyme: Honoraria.

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Characteristics of DNA methylation and gene expression in regulatory features on the Infinium 450k Beadchip

10.1101/032862 ◽

2015 ◽

Cited By ~ 5

Author(s):

David Martino ◽

Richard Saffery

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Association Studies ◽

Cpg Islands ◽

Transcriptional Unit ◽

Gene Body ◽

Genomic Context ◽

Methylation Array ◽

The Relationship

Understanding the relationship between variations in DNA methylation and gene expression has been challenging. Evidence suggests the function of DNA methylation may vary with genomic context, and few consistent rules linking methylation to expression have been noted. For array-based studies, the content of current DNA methylation array platforms provide broad coverage of the genome but target only a fraction of the potentially methylated CG dinucleotides. A better understanding of the interplay between DNA methylation and gene expression is beneficial for users of these platforms, and may aid with candidate prioritization in epigenome-wide association studies (EWAS). To address this we examined the relationship between DNA methylation levels and gene expression in primary T-lymphocytes at discreet genomic regions around the transcriptional unit (Promoters, gene body, untranslated regions) and at CpG island-associated regions (islands, shores and shelves), stratifying by high and low expressed genes. As anticipated we found evidence that DNA methylation at CpG sites near promoter regions are tightly correlated with gene expression in both the stably expressed and developmentally regulated genes, however this is dependent on CpG density. DNA methylation within the gene body was not consistently associated with changes in gene expression. CpG islands and island shores exhibited strong correlations with gene expression, but this was not true for island shelves. We found these relationships were generally preserved at both dynamic and steady state genes, with some notable exceptions. In combination these insights may be useful for prioritising candidates identified in epigenome-wide association studies for subsequent functional studies.

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The proto CpG island methylator phenotype of sessile serrated adenomas/polyps

10.1101/357178 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hannah R. Parker ◽

Stephany Orjuela ◽

Andreia Martinho Oliveira ◽

Fabrizio Cereatti ◽

Matthias Sauter ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Normal Mucosa ◽

Cpg Island Methylator Phenotype ◽

Molecular Features ◽

Colon Cancers ◽

Methylator Phenotype ◽

Serrated Adenomas

AbstractSessile serrated adenomas/polyps (SSA/Ps) are the putative precursors of the ˜20% of colon cancers with the CpG island methylator phenotype (CIMP), but their molecular features are poorly understood. We used high-throughput analysis of DNA methylation and gene expression to investigate the epigenetic phenotype of SSA/Ps. Fresh-tissue samples of 17 SSA/Ps and (for comparison purposes) 15 conventional adenomas (cADNs)—each with a matched sample of normal mucosa— were prospectively collected during colonoscopy (total no. samples analyzed: 64). DNA and RNA were extracted from each sample. DNA was subjected to bisulfite next-generation sequencing to assess methylation levels at ˜2.7 million CpG sites located predominantly in gene regulatory regions and spanning 80.5Mb (˜2.5% of the genome); RNA was sequenced to define the samples’ transcriptomes. An independent series of 61 archival lesions was used for targeted verification of DNA methylation findings. Compared with normal mucosa samples, SSA/Ps and cADNs exhibited markedly remodeled methylomes. In cADNs, hypomethylated regions were far more numerous (18,417 vs 4288 in SSA/Ps) and rarely affected CpG islands/shores. SSA/Ps seemed to have escaped this wave of demethylation. Cytosine hypermethylation in SSA/Ps was more pervasive (hypermethylated regions: 22,147 vs 15,965 in cADNs; hypermethylated genes: 4938 vs 3443 in cADNs) and more extensive (region for region), and it occurred mainly within CpG islands and shores. Given its resemblance to the CIMP typical of SSA/Ps’ putative descendant colon cancers, we refer to the SSA/P methylation phenotype as proto-CIMP. Verification studies of six hypermethylated regions (3 SSA/P-specific and 3 common) demonstrated the high potential of DNA methylation markers for predicting the diagnosis of SSA/Ps and cADNs. Surprisingly, proto-CIMP in SSA/Ps was associated with upregulated gene expression (n=618 genes vs 349 that were downregulated); downregulation was more common in cADNs (n=712 vs 516 upregulated genes). The epigenetic landscape of SSA/Ps differs markedly from that of cADNs. These differences are a potentially rich source of novel tissue-based and noninvasive biomarkers that can add precision to the clinical management of the two most frequent colon-cancer precursors.

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