Journal articles: 'N-4 Cytosine Methylation'

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Grogan, Dennis W. "Cytosine Methylation by the SuaI Restriction-Modification System: Implications for Genetic Fidelity in a Hyperthermophilic Archaeon." Journal of Bacteriology 185, no. 15 (August 1, 2003): 4657–61. http://dx.doi.org/10.1128/jb.185.15.4657-4661.2003.

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ABSTRACT 5-Methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles. To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated. Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S. acidocaldarius DNA. In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs. The results show that the SuaI system uses N 4 methylation to block cleavage of its recognition site, thereby avoiding the creation of G · T mismatches by spontaneous deamination at extremely high temperature.

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Lister, Rolanda. "Intrauterine Programming of Diabetes Induced Cardiac Embryopathy." Diabetes & Obesity International Journal 4, no. 3 (2019): 1–14. http://dx.doi.org/10.23880/doij-16000202.

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Background: Maternal hyperglycemia is a well-recognized risk factor for fetal congenital heart disease. However, the underlying cellular and molecular mechanisms are not well characterized. We hypothesize that maternal hyperglycemia leading to congenital heart are linked to abnormal DNA methylation and mRNA expression at cardiac specific loci. Methods: Hyperglycemia was induced in normal 8-week old CD-1 female mice with a one-time intraperitoneal injection of 150 mg/kg of streptozotocin (STZ) 2 weeks prior to mating. Histological analysis of fetal cardiac morphology was evaluated for malformations on embryonic day (E) 16.5 of control pups and pups exposed to maternal hyperglycemia. We used a massively-parallel sequencing-based methylation sensitive restriction based assay to examine genome-wide cytosine methylation levels at >1.65 million loci in neonatal hearts on post-natal (P) day 0. Functional validation was performed with real time quantitative polymerase chain reaction (RT-qPCR). Results: Cardiac structural defects occurred in 28% of the pups (n=12/45) of hyperglycemic dams versus 7% (n=4/61) of controls. Notable phenotypes were hypoplastic left or right ventricle, double outlet right ventricle, ventricular septal defect, and left ventricular outflow tract obstruction. A 10-fold increase in DNA methylation of gene promoter regions was seen in many cardiac important genes in the experimental versus control P0 neonates and have corresponding decreases in gene expression in 21/32 genes functionally validated. Conclusion: Maternal hyperglycemia alters DNA methylation and mRNA expression of some cardiac genes during heart development. Quantitative, genome-wide assessment of cytosine methylation can be used as a discovery platform to gain insight into the mechanisms of hyperglycemia-induced cardiac anomalies.

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Hsieh, Pei-chung, Jian-ping Xiao, Diana O'loane, and Shuang-yong Xu. "Cloning, Expression, and Purification of a Thermostable Nonhomodimeric Restriction Enzyme,BslI." Journal of Bacteriology 182, no. 4 (February 15, 2000): 949–55. http://dx.doi.org/10.1128/jb.182.4.949-955.2000.

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ABSTRACT BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system fromBacillus species was cloned and expressed inEscherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), thebslIRα gene, and the bslIRβ gene. The α and β subunits of BslI can be expressed independently inE. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (αβ), heterotetramers (α2β2), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramerBslI complex (α2β2). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is α2β2. M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the β group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 inBslI sites in the first or second cytosine are resistant toBslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the α subunit of BslI endonuclease.

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Feng, Zhaohui, Wenwei Hu, William N. Rom, Frederick A. Beland, and Moon-shong Tang. "N-Hydroxy-4-aminobiphenyl-DNA Binding in Humanp53Gene: Sequence Preference and the Effect of C5 Cytosine Methylation†." Biochemistry 41, no. 20 (May 2002): 6414–21. http://dx.doi.org/10.1021/bi020093s.

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Brandão, Yara de Oliveira, Mariana Busato Toledo, Andressa Chequin, Thierry Grima Cristo, Renato Silva Sousa, Edneia Amancio Souza Ramos, and Giseli Klassen. "DNA Methylation Status of the Estrogen Receptor α Gene in Canine Mammary Tumors." Veterinary Pathology 55, no. 4 (March 22, 2018): 510–16. http://dx.doi.org/10.1177/0300985818763711.

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Estrogen receptor α (ERα) has an important role in mammary carcinogenesis, prognosis, and treatment. In human and canine mammary cancer, the most aggressive tumors show loss of ERα expression, which in human breast cancer has been attributed to methylation of the cytosine followed by guanine (CpG) island within the estrogen receptor α gene ( ESR1) promoter. This study aimed to investigate the role of ESR1 CpG island (CGI) methylation in ERα expression in canine mammary tumors. Twenty-one canine mammary samples were sorted into three groups: malignant tumor (n = 9), benign tumor (n = 8), and normal gland (n = 4). Immunohistochemical analysis and reverse-transcription quantitative real-time PCR were performed to assess ERα expression and ESR1 mRNA levels. The methylation status was determined using sodium-bisulfite-treated DNA sequencing. All normal mammary glands and benign tumors showed high ERα expression (score range, 5–8). Six of the nine malignant tumors did not show ERα expression (score 0), two had score 2, and one had score 4. Lower ERα ( P < .005) and ESR1 mRNA levels ( P < .005) were found in malignant mammary tumors than in the other two groups. Canine ESR1 has an intragenic and non-promoter-associated CGI, different from humans. No significant variation in methylation percentage was observed among the groups, suggesting that ESR1 is not regulated by DNA methylation, unlike that in humans. This difference should be considered in further research using ERα as a biomarker for mammary tumors in canine studies on ERα-targeting therapy.

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Piyathilake, Chandrika J., Walter C. Bell, Jennifer Jones, Olga L. Henao, Douglas C. Heimburger, Alain Niveleau, and William E. Grizzle. "Patterns of Global DNA and Histone Methylation Appear to be Similar in Normal, Dysplastic and Neoplastic Oral Epithelium of Humans." Disease Markers 21, no. 3 (2005): 147–51. http://dx.doi.org/10.1155/2005/285134.

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Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n= 48) with those of normal oral epithelium from subjects without oral cancer (n= 93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5-mc), lysine 4 of histone H3(H3-Lys4), and lysine 9 of histone H3(H3-Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H3-Lys4 and H3-Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non-cancer subjects (n= 0.63,p< 0.001 andr= 0.62,p< 0.001 respectively). Similarly, the positive correlations between H3-Lys4 and H3-Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non-cancer subjects (r= 0.77,p< 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms.

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Hajkova, Hana, Jana Markova, Cedrik Haskovec, Jaroslav Cermak, Radka Petrbokova, Kyra Michalova, and Jiri Schwarz. "DNMT3A Mutations Are Connected with Lower DNA Methylation Levels and Numbers of Concurrently Hypermethylated Genes in AML Patients." Blood 118, no. 21 (November 18, 2011): 4629. http://dx.doi.org/10.1182/blood.v118.21.4629.4629.

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Abstract Abstract 4629 Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found in patients with acute myeloid leukemia (AML) and have been confirmed to be connected with adverse clinical outcome. As DNMT3A plays direct role in the process of DNA methylation by adding methyl group to the cytosine residue of CpG dinucleotides, the question is obvious: What is the impact of DNMT3A mutations on DNA methylation levels? We examined 79 AML patients at diagnosis for the presence of aberrant DNA methylation of 12 tumor suppressor genes (TSG) (CDKN2B, CALCA, CDH1, ESR1, SOCS1, MYOD1, DAPK1, TIMP3, ICAM1, TERT, CTNNA1, EGR1) by methylation specific real-time PCR (MethyLight) and for mutations in the gene DNMT3A by direct sequencing. Next we studied methylation status of 24 HOX genes from all four clusters A-D using methylation-restriction endonucleases followed by RQ-PCR arrays in 10 AML samples compared to 4 healthy donor samples. Sequencing of cDNA between amino acids 300 and 930 revealed that 32 of 79 AML patients had DNMT3A mutation. The reason for higher DNMT3A mutation incidence in our patients’ cohort consists in preferential selection of AML patients with a higher percentage of probability of DNMT3A mutation (it means normal karyotype and mutations in NPM1, FLT3 and/or IDH1/2 genes). MethyLight assessment of 12 TSG showed subsequent frequencies of hypermethylation: CDKN2B (47%), CALCA (43%), CDH1 (22%), SOCS1 (24%), MYOD1 (18%), ESR1 (14%). The remaining 6 genes were weakly methylated in less than 10 % AML patients at diagnosis and were therefore excluded from further analysis. DNA methylation arrays revealed a set of differentially methylated HOX genes (n=12): 11 HOX genes were hypermethylated (HOXA4, HOXA6, HOXB13, HOXB3, HOXB4, HOXB7, HOXB8, HOXC8, HOXD10, HOXD11, HOXD3) and HOXA5 was hypomethylated compared to healthy donor samples. Comparing overall cumulative DNA methylation levels and numbers of simultaneously hypermethylated genes to mutational status of DNMT3A gene, we did observe lower levels of DNA methylation (P<0.0001) as well as displaying lower numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3a mutations. We observed the same trend also in DNA methylation levels of HOX genes when compared mutant (n=4) versus wild-type (n=6) DNMT3A patients. These results clearly show that numbers of simultaneously hypermethylated genes and DNA methylation levels of chosen tumor suppressor genes (TSG) as well as HOX genes differs between AML patients with wild type and mutant DNMT3A. This study is part of the COST Action BM0801 (EuGESMA) and is supported by NS10632-3/2009, OC10042 and IHBT00023736. Disclosures: No relevant conflicts of interest to declare.

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Aurich, C., B. Schreiner, N. Ille, M. Alvarenga, and D. Scarlet. "14 EPIGENETIC CHANGES IN EQUINE SPERMATOZOA DURING CRYOPRESERVATION: A PRELIMINARY STUDY." Reproduction, Fertility and Development 28, no. 2 (2016): 136. http://dx.doi.org/10.1071/rdv28n2ab14.

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The use of cryopreserved semen for insemination of mares facilitates breeding management but often results in reduced conception rates. This has been mainly attributed to changes in sperm membrane function caused by the freezing-thawing procedure. However, semen processing may also contribute to epigenetic changes in spermatozoa. In the present study, we therefore addressed changes in sperm DNA-methylation induced by cryopreservation of stallion semen. We hypothesised that the cryoprotectant may influence the DNA-methylation level of frozen-thawed semen. For this purpose, semen was collected from fertile Shetland pony stallions. Global DNA-methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) with a monoclonal antibody sensitive and specific for 5-methylcytosin after DNA extraction and denaturation (100 ng of DNA per sample). The level of 5-methylcytosin in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). Statistical analysis was done with the SPSS Statistics 21 software. Values are means ± standard error of the mean. In Experiment 1, 1.5 mL of raw semen (n = 6 stallions, 1 ejaculate each) was shock-frozen at –196°C for 15 min and thawed at 38°C for 60 s. Semen motility and membrane integrity were completely absent, while DNA-methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In Experiment 2, 3 ejaculates per stallion (n = 6) were included. Semen quality and DNA-methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (Minitube, Tiefenbach, Germany; cryoprotectant: 5% glycerol) or BotuCrio (Nidacon, Mölndal, Sweden; cryoprotectants: 1% glycerol and 4% methylformamid) extender. Semen was frozen in 0.5-mL straws in a computer-controlled rate freezer (IceCube 14 M; Sylab, Purkersdorf, Austria, cooling rates: 20°C to 5°C: 0.3°C min–1, 5°C to 25°C: 10°C min–1, –25°C to –140°C: 25°C min–1). Semen motility, morphology, and membrane integrity were significantly reduced (e.g. total motility before freezing: 88.8 ± 1.4%) by cryopreservation but not influenced by the extender used (e.g. total motility: Ghent 69.5 ± 2.0, BotuCrio 68.4 ± 2.2%; P < 0.001 v. nonfrozen semen). Cryopreservation significantly (P < 0.01) increased the level of DNA-methylation (before freezing: 0.6 ± 0.1%, Ghent 6.4 ± 3.7, BotuCrio 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA-methylation was not correlated with semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA-methylation level in stallion semen. In the present study, this effect occurred irrespective of the cryoprotectant but was not seen after shock-freezing in the absence of cryoprotectants. The reduced fertility of mares after insemination with frozen-thawed semen may at least in part be explained by methylation of sperm DNA, which occurs in response to the cryopreservation procedure.

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Furmanek-Blaszk, Beata, Robert Boratynski, Natalia Zolcinska, and Marian Sektas. "M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target." Microbiology 155, no. 4 (April 1, 2009): 1111–21. http://dx.doi.org/10.1099/mic.0.025023-0.

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Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5′-GAAGA-3′/3′-CTTCT-5′. M1.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ to N 6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yielding N 4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000±1000 Da under denaturing conditions. Divalent cations (Ca2+, Mg2+, Mn2+ and Zn2+) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.

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Alexeeva, Marina, Prashanna Guragain, Almaz N. Tesfahun, Miglė Tomkuvienė, Aysha Arshad, Rūta Gerasimaitė, Audronė Rukšėnaitė, et al. "Excision of the doubly methylated base N 4 ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1748 (April 23, 2018): 20170337. http://dx.doi.org/10.1098/rstb.2017.0337.

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Cytosine (C) in DNA is often modified to 5-methylcytosine (m 5 C) to execute important cellular functions. Despite the significance of m 5 C for epigenetic regulation in mammals, damage to m 5 C has received little attention. For instance, almost no studies exist on erroneous methylation of m 5 C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m 5 C into N 4 ,5-dimethylcytosine (m N 4,5 C) in DNA, m N 4,5 C is probably present in vivo . We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N 4,5 C residues in vitro . The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m 5 C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N 4,5 C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

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Wang, Feng, Kiyomi Morita, Courtney D. DiNardo, Keyur Patel, Kyle MacBeth, Alessandra Tosolini, Mark G. Frattini, et al. "Comprehensive Genomic Analysis of IDH Inhibitor-Treated AML Samples Delineates Molecular Mechanisms of Differentiation and Heterogeneous Patterns of Acquired Resistance." Blood 132, Supplement 1 (November 29, 2018): 441. http://dx.doi.org/10.1182/blood-2018-99-116383.

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Abstract Allosteric inhibitors of mutant IDH1 or IDH2 induce differentiation of IDH-mutant AML myeloblasts, which in some patients (pts) can lead to a life-threatening differentiation syndrome (DS). The in vivo mechanism of how IDH inhibitors induce differentiation and occasionally DS is not fully understood. Furthermore, responders to the inhibitors often lose the differentiation effect, after median of 7 months. Although a recent study identified secondary mutations in the dimer-interface of IDH1/2 as a mechanism for acquired IDH-inhibitor resistance, given the rarity of this event, it is unlikely to represent the primary mechanism in the majority of pts. We conducted a comprehensive genomic analysis (DNA sequencing, single-cell DNA sequencing, RNA sequencing and cytosine methylation array) on longitudinally collected bone marrow specimens from IDH1/2-mutant AML pts treated with one of the IDH1/2 inhibitors (ivosidenib, enasidenib, IDH305 or AG881). In total, 146 samples from 39 pts collected at different time points (median 4 [IQR 3-5] samples per patient) were analyzed. 24 pts were IDH1-mutated receiving IDH1 inhibitor and 15 were IDH2-mutated receiving IDH2 inhibitor. 29 pts responded (CR, CRi, CRp or MLFS) and 10 had no response to the inhibitors. 8 of 29 responders developed DS. 22 of 29 responders relapsed after the median of 5 months. Cytosine methylation analysis revealed significant decrease in global methylation at response than baseline (mean beta value 0.54 at baseline vs. 0.45 at response, P < 0.001). This corroborated with a decrease in serum 2HG level, supporting the role of IDH inhibitors in reversing CpG island methylator phenotype (CIMP) likely through TET restoration. Motif enrichment analysis of differentially methylated promoter regions showed significant de-methylation of PU.1(SPI1) and C/EBP binding motifs at response. Consistent with this, RNA sequencing revealed that PU.1 and C/EBP target genes such as CSF2RA, CSF3R, IL34, TREM2, IL6R, CEBPE, GFI1, and KLF5 had significant upregulation at response. Expression of PU.1 itself was also significantly up-regulated upon response, along with other key hematopoietic differentiation genes and pathways such as GM-CSF, IL8-CXCR1 and IL3 pathways. Pts who developed DS had significantly higher upregulation of PU.1 and GM-CSF pathway compared to other DS-free responders, implicating the potential activation of these pathways in the mechanism of DS. At relapse, these pathways that mediated differentiation were all down-regulated when compared to that at response. Interestingly, methylation level remained suppressed at relapse (mean beta value 0.45 at response vs. 0.47 at relapse, P = 0.566), suggesting that the down-regulation of pro-differentiation signals at relapse is independent of methylation. Combined whole exome and targeted deep sequencing revealed the heterogeneous patterns of acquired mutations at relapse. Of 22 cases with baseline and relapse pairs, 18 acquired new driver mutations at relapse (Fig 1). The most frequently acquired mutations at relapse were in BCOR/BCORL1 (N = 6), hematopoietic differentiation genes (CEBPA [N=2] and RUNX1[N=4]), and RTK pathways (NRAS/KRAS [N=5] and PTPN11 [N=1]). We did not identify acquisition of dimer-interface IDH1/2 mutations at relapse. However, in 1 case, relapse was associated with acquisition of mutation in reciprocal IDH gene (IDH2-mutated case acquired IDH1 mutation). Singe-cell DNA sequencing of the double-mutant revealed that the reciprocal acquisition occurred in a different clone. Analysis of differentially expressed genes between baseline and relapse showed significant upregulation of genes in DNA damage response (DDR) pathways at relapse. Consistent with this, relapse samples showed significantly higher mutation burden than baseline, suggesting that accumulation of mutations are upregulating DDR pathway at relapse. In summary, IDH inhibitors induce differentiation and DS through upregulation of PU.1 and C/EBP target genes, which is associated with de-methylation of their binding motifs. The mechanisms of acquired resistance to IDH inhibitors are heterogeneous and involves various genes and pathways including BCOR/BCORL1, hematopoietic differentiation transcription factors (CEBPA, RUNX1), and RTK pathways (RAS, PTPN11). In a rare case, acquired mutation in reciprocal IDH gene was detected, occurring in a different clone. Figure. Figure. Disclosures DiNardo: Agios: Consultancy; Abbvie: Honoraria; Medimmune: Honoraria; Bayer: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria. MacBeth:Celgene Corporation: Employment, Equity Ownership. Tosolini:Celgene Corporation: Employment. Frattini:Celgene Corporation: Employment, Equity Ownership. Kadia:Abbvie: Consultancy; Celgene: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Celgene: Research Funding; Takeda: Consultancy; Abbvie: Consultancy. Ravandi:Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Sunesis: Honoraria; Bristol-Myers Squibb: Research Funding; Xencor: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Jazz: Honoraria; Seattle Genetics: Research Funding; Abbvie: Research Funding; Macrogenix: Honoraria, Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria. Konopleva:Stemline Therapeutics: Research Funding.

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Kues, W. A., K. Iqbal, B. Barg-Kues, and H. Niemann. "305 REPROGRAMMING EVENTS IN EARLY BOVINE AND MURINE EMBRYOS ARE MIRRORED BY PLASMID-ENCODED MARKER CONSTRUCTS." Reproduction, Fertility and Development 21, no. 1 (2009): 249. http://dx.doi.org/10.1071/rdv21n1ab305.

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Episomal plasmids have emerged as useful tools to achieve stable transgenesis in mammalian cell cultures. Here, the suitability of scaffold/matrix attachment region (S/MAR) carrying episomal plasmids and conventional plasmids for the generation of transgenic murine and bovine embryos was assessed. Bovine zygotes were produced from slaughterhouse ovaries, and murine zygotes were isolated from superovulated and mated NMRI females. Zygote stages were microinjected with approximately 10 pl of plasmid solution. The S/MAR encoding plasmids pEPI or minicircle preparations (gift of J. Bode, Braunschweig, Germany), devoid of most of the plasmid backbone, were used as episomal plasmids. Both plasmids carry an enhanced green fluorescent protein (EGFP) gene driven by the cytomegalovirus promoter (CMV). The plasmids peGFP (CMV-eGFP), pdsRED encoding red fluorescent protein (CMV-RFP), pOct4-GFP (germ line-specific Oct-4 promoter-EGFP), and pgAChR-GFP (muscle-specific γAChR promoter) were used as conventional plasmids. To study the effects of DNA methylation at cytosine/guanine dinucleotids (CpG), plasmid DNA was treated with CpG-methylase in the presence of S-adenosyl-methionin, and in some experiments, completeness of DNA methylation was verified by methylation-sensitive restriction endonucleases. Embryos were analyzed during in vitro culture up to blastocyst stage by fluorescence microscopy, and selected stages were harvested for RT-PCR analysis or DNA recovery. Microinjection of circular plasmids with ubiquitous CMV (n = 505) or germ line-specific Oct-4 promoter (n = 176) driven transcription in bovine zygotes resulted in 159 and 44 blastocysts, of which 94 and 27 showed expression of EGFP. Microinjection of bovine zyotes (n = 179) with S/MAR plasmids yielded a total of 18 blastocysts of which 12 were green fluorescent protein-positive. On average, >50% of the blastocysts were EGFP-positive, irrespective of whether S/MAR carrying episomal plasmids or conventional plasmids had been injected. In contrast, injection of the γAChR (muscle-specific) driven construct did not give rise to EGFP expression (n = 20), suggesting that promoter specificity was maintained. Injection of murine zygotes (n = 126) with CMV or Oct-4 promoter constructs was less successful, about 10 to 20% of the obtained blastocysts expressed EGFP. In the case of unmethylated pOct4-GFP plasmid, the onset of EGFP expression was found to coincide with the time point of major embryonic genome activation [i.e. late 1-cell stage in murine (n = 25) and 4- to 8-cell stages in bovine (n = 75) embryos]. In contrast, injection of CpG-methylated plasmids (murine n = 33; bovine n = 101) delayed the onset of EGFP expression for a further 30 to 40 h. Recovery of plasmid sequences from blastocyst stages and bisulfite sequencing indicated that the majority of plasmids are maintained in an episomal status. Thus, plasmid-mediated transgenesis is a robust method to express foreign DNA in a promoter-specific manner in mammalian embryos and can be employed to analyze reprogramming events. The excellent technical support by E. Lemme and K. Korsawe is acknowledged. Funded by DFG.

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Huang, Yanfang, Xiaohong Jiang, Miao Yu, Rongfu Huang, Jianfeng Yao, Ming Li, Fangfang Zheng, and Xiaoyu Yang. "Beneficial effects of diazepin-quinazolin-amine derivative (BIX-01294) on preimplantation development and molecular characteristics of cloned mouse embryos." Reproduction, Fertility and Development 29, no. 6 (2017): 1260. http://dx.doi.org/10.1071/rd15463.

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Somatic cell nuclear transfer is frequently associated with abnormal epigenetic modifications that may lead to the developmental failure of cloned embryos. BIX-01294 (a diazepine–quinazoline–amine derivative) is a specific inhibitor of the histone methyltransferase G9a. The aim of the present study was to investigate the effects of BIX-01294 on development, dimethylation of histone H3 at lysine 9 (H3K9), DNA methylation and the expression of imprinted genes in cloned mouse preimplantation embryos. There were no significant differences in blastocyst rates of cloned embryos treated with or without 0.1 μM BIX-01294. Relative to clone embryos treated without 0.1 μM BIX-01294, exposure of embryos to BIX-01294 decreased histone H3K9 dimethylation and DNA methylation in cloned embryos to levels that were similar to those of in vivo-fertilised embryos at the 2-cell and blastocyst stages. Cloned embryos had lower expression of octamer-binding transcription factor 4 (Oct4) and small nuclear ribonucleoprotein N (Snrpn), but higher expression of imprinted maternally expressed transcript (non-protein coding) (H19) and growth factor receptor-bound protein 10 (Grb10) compared with in vivo-fertilised counterparts. The addition of 0.1 μM BIX-01294 to the activation and culture medium resulted in lower H19 expression and higher cyclin dependent kinase inhibitor 1C (Cdkn1c) and delta-like 1 homolog (Dlk1) expression, but had no effect on the expression of Oct4, Snrpn and Grb10. The loss of methylation at the Grb10 cytosine–phosphorous–guanine (CpG) islands in cloned embryos was partially corrected by BIX-01294. These results indicate that BIX-01294 treatment of cloned embryos has beneficial effects in terms of correcting abnormal epigenetic modifications, but not on preimplantation development.

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Liu, Xiaochang, Jiuxia Pang, Christopher Seiler, Ryan Kempen, Hao Liu, Aref Al-Kali, Y. Natalia Tretyakova, Mark Litzow, and Shujun Liu. "DNA Cytosine-Demethylating Agent 5-Aza-2'-Deoxycytidine Targets Leukemia Cells through Reducing DNA N6-Methyladenine." Blood 134, Supplement_1 (November 13, 2019): 2513. http://dx.doi.org/10.1182/blood-2019-130490.

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Introduction: It is known that overexpression of DNA methyltransferases (e.g., DNMT1) is frequent and changes of DNA cytosine methylation (5mC) are a constant feature of cancers. DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (Dec), have been in clinics for patients with leukemia. It is classically believed that promoter hypomethylation coupled by reexpression of epigenetically-suppressed tumor suppressors is a core mechanism behind Dec-impaired leukemia cell growth. However, the fact that global DNA methylation profiling barely predicts Dec-response suggests a demethylation-independent mechanism of Dec-induced cell death. N6-methyladenine (m6A) has been identified recently as an abundant DNA modification in eukaryotes (Wu, Nature 2016;532:329). Importantly, m6A is extensively present in the human genome, and m6A abundance is associated with tumorigenesis (Xie, Cell 2018;71:306). Furthermore, the DNA m6A is dynamically modulated by the methyltransferases (i.e., METTL3, N6AMT1) and demethylases (i.e., ALKBH1), and changes in m6A predict gene expression (Wu, Nature 2016;532:329). Given a potential crosstalk between m6A and distinct epigenetic mechanisms (Yao, Nat. Commun 2017;8:1122), we hypothesized that the anticancer actions of Dec may partially result from changes in DNA m6A in leukemia cells. Methods: Protein expression of target genes was assessed by Western blotting. The levels of DNA cytosine methylation (5mC) and N6-methyladenine (m6A) were measured by dotblotting or liquid chromatography-mass spectrometry (LC-MS/MS). The cell viability and apoptosis were determined by the Cell Counting Kit 8 (CCK8) assays as well as the Annexin V/Propidium Iodide staining and flow cytometry. The peripheral blood mononuclear cells (PBMCs) of leukemia patients from Mayo Clinic were prepared by Ficoll-Hypaque gradient centrifugation. Results: To test our hypothesis, leukemia cells, Kasumi-1, MV4-11, K562 and KU812, were exposed to 2 µM Dec, a clinical achievable concentration, for 72 hours. As expected, Dec treatment led to a downregulation of DNMT1 and DNMT3a, a reduction of 5mC levels by dotblotting using anti-5mC antibody, a blockage of cell proliferation and a promotion of cell apoptosis. When genomic DNA was subjected to dotblotting using anti-m6A antibody, the results revealed a marked decrease of DNA m6A levels in all Dec-treated cells. Then genomic DNA from K562 and MV4-11 cells was enzymatically digested to 2'-deoxynucleosides. dA was quantified by HPLC-UV, while the amount of m6A was measured by isotope dilution HPLC-ESI-MS/MS using 15N labeled internal standard. The standard curves were generated using pure standards, from which the m6A/A ratio was calculated. In agreement with dotblotting results, Dec treatment significantly decreased DNA m6A abundance in both cell lines. Mechanistically, exposure to Dec led to a consistent increase of demethylase fat mass and obesity-associated protein (FTO), but not METTL3 nor ALKBH1 and ALKBH5. Further, knockdown of FTO increased DNA m6A, which was further confirmed by treatment with FTO inhibitors rhein and meclofenamic acid (MA). These data indicate that FTO may be responsible for Dec-induced m6A changes in leukemia cells. To investigate the clinical implications of DNA m6A, we obtained PBMCs from AML patients (n = 10), who received Dec therapy (20 mg/m2 daily for 5 days every 4 weeks) in Mayo Clinic. These PBMCs were further cultured for 48 hours, frozen and stored in 100% ethanol before DNA extraction. The results from dotblotting using anti-5mC or anti-m6A showed that a trend of decrease in both m6A and 5mC abundance is observed, and the pattern of changes in m6A and 5mC displays a positive correlation. Finally, exposure of leukemia cells to the combination of Dec (2 µM) with FTO inhibitor MA (50 µM) induced more cell apoptosis and greater inhibition on cell proliferation as compared to single agent in vitro, supporting FTO inhibitors as new therapeutic agents in leukemia. Conclusion: Our studies suggest that the FTO-DNA m6A axis may partially mediate the therapeutic outcomes of Dec in leukemia. Our findings provide a new mechanistic paradigm for the anticancer activities of Dec, and define the m6A methylation status in leukemia cells as a new pharmacodynamic marker for their response to Dec-based therapy, pointing to a novel treatment strategy for incorporating m6A modulators to enhance the therapeutic index of Dec. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding.

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Gough, Sheryl M., Yang Jo Chung, Maegawa Shinji, Jaroslav Jelinek, Jean-Pierre Issa, and Peter Aplan. "Mice with Myelodysplastic Syndrome Respond to Treatment with Azanucleosides: A Preclinical Model for MDS Therapies." Blood 120, no. 21 (November 16, 2012): 3829. http://dx.doi.org/10.1182/blood.v120.21.3829.3829.

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Abstract Abstract 3829 Therapies for the treatment of myelodysplastic syndromes (MDS) are limited. The only known curative therapy for MDS is allogeneic hematopoietic stem cell transplantation. MDS is frequently associated with epigenetic gene silencing via methylation of cytosine residues in gene regulatory regions. Clinical trials with DNA methyl-transferase inhibitors 5'azacytidine and 5-aza-2'-deoxycytidine (Decitabine, DAC) have shown hematologic responses and a survival benefit. Both azanucleosides have recently been approved for use in the clinic. However, responses are typically not durable, thus further characterization of response to treatment is required, as is the identification of new drugs to better treat MDS. Using bone marrow cells from NUP98-HOXD13 (NHD13) transgenic mice, which have previously been shown to faithfully recapitulate key features of MDS, we co-transplanted MDS and wild type (WT) bone marrow cells into WT irradiated recipient mice. The chimaeric bone marrow transplant (BMT) produces a recipient that mimics the human condition, with patient bone marrow comprised of hematopoietic cells derived from both the MDS clone as well as normal hematopoietic precursors. WT and MDS cells in the mice can be distinguished by differential CD45 alleles (CD45.1 and CD45.2, respectively), which enables analysis and purification of the MDS and normal cells; this feat is not easily achieved with human MDS patient samples, which lack cell surface antigens specific for the MDS clone. We treated mice with 0.018mg DAC (or saline) daily for 5 days, given every 4–5 weeks, to approximate MDS patient dosing schedules. Three trial groups were divided into saline (total n=19) and DAC (total n=24) treatment cohorts. Successful treatment of the chimeric MDS mice resulted in increasing levels of WT cells in the peripheral blood, with a concomitant reduction or eradication of the MDS cells. For instance, in trial #1, DAC treated chimaeric WT/MDS mice showed less severe anemia (2.0 g/dL higher Hgb), normalized neutrophil counts (57– 85% of mice vs 20–60%) and a significant survival benefit (median 27 vs 20 weeks, p=0.004) compared to saline treated mice. The response to treatment varied within individual mice and between trials, analogous to the variability in response seen in human MDS patients. We purified WT and MDS cells from treated mice and evaluated CpG island (CGI) DNA methylation in Chd13, a gene aberrantly methylated in MDS patients. Chd13 was hypermethylated in the murine MDS cells compared to WT cells (42–59% vs 10–16%, respectively). Of note, there was considerable variability between mice in the response to DAC treatment; half of the DAC treated mice showed a normalization of Chd13 methylation (14–20%) and half had a modest decrease in Chd13 methylation (38–40%). We next assayed global CGI methylation using a deep sequencing technique (DREAM, Digital Restriction Enzyme Analysis of Methylation). 8.2% of all CGI sites assayed were hypermethylated in MDS cells compared to WT; this increase in methylation was reduced to only 2.0% following DAC treatment of the MDS cells. This genome wide approach documented extensive hypermethylation in the NHD13 mice, similar to findings in a subset of MDS patients. Chimaeric WT/MDS mice demonstrated variable hematologic outcomes and cytosine demethylation in response to DAC treatment, as seen in human MDS patients. Successful treatment resulted in depletion of MDS cells allowing reconstitution of the bone marrow with WT cells as would happen in the successful treatment of patients. Durable responses were seen in several recipients, including one long-term survivor with no evidence of MDS cells in the bone marrow at 24 months of age (16 months post treatment). The chimaeric WT/MDS mice therefore represent a useful pre-clinical model of human MDS, which can be used to better characterize current treatments and test novel therapies. Disclosures: Issa: Johnson & Johnson: Consultancy; Astex: Consultancy. Aplan:NIH Office of Technology Transfer: Patents & Royalties.

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Baak, Ulrike, Helmut Orawa, Nicola Goekbuget, Olaf Hopfer, Thomas Burmeister, Wolf-Karsten Hofmann, Dieter Hoelzer, and Eckhard Thiel. "Promotor Demethylation as a Mechanism of HOX11 Activation in Adult T-ALL?." Blood 108, no. 11 (November 1, 2006): 2288. http://dx.doi.org/10.1182/blood.v108.11.2288.2288.

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Abstract Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

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Dong, Qian, Sarmistha Das, Cheng Chen, John Easton, Heather L. Mulder, Emily Walker, Geoffrey Neale, et al. "Abstract 4514: Variations of blood DNA methylation associated with cancer treatment exposures among childhood cancer survivors of African ancestry." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4514. http://dx.doi.org/10.1158/1538-7445.am2023-4514.

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Abstract We previously showed that cancer treatment associated DNA methylation (DNAm) signatures were present decades following the cancer diagnosis in childhood cancer survivors (CCS) of European ancestry (EA) and that treatment associated DNAm sites mediated the causal pathway from specific treatment exposures to increased risk of chronic health conditions (CHCs). This new analysis further evaluated and compared the treatment and DNAm associations in CCS of African ancestry (AA) from the St. Jude Lifetime Cohort Study. Cancer treatments were abstracted from medical records. DNAm was measured using MethylationEPIC BeadChip with blood-derived DNA. Among 370 AA CCS (53.2% female, median age at blood draw=31.2 [range=18.4-65.1] years), treatments included alkylating agents (54.6%), anthracyclines (48.6%), epipodophyllotoxins (29.2%), corticosteroids (33.0%), and vincristine (61.6%), and radiation therapy (RT) to brain (26.5%), chest (27.6%), abdomen (22.7%), and pelvis (21.6%). Epigenome-wide association study (EWAS) for each treatment, using multivariable linear regression adjusting for sex, age at blood draw, leukocyte cell subtype proportions, genotype principal components and DNAm principal components, showed little inflation with genomic control (GC) factor between 1.1 (brain-RT) and 1.2 (chest-RT). A total of 93 5′-cytosine-phosphate-guanine-3′ (CpG) was associated with one or more cancer treatments (GC-adjusted P<9 × 10−8), including epipodophyllotoxins (n=46), alkylating agents (n=38), corticosteroids (n=3), anthracyclines (n=3), abdominal-RT (n=3), chest-RT (n=2), and pelvic-RT (n=1). A total of 60 differentially methylated regions (DMRs) was identified using DMRcate R package, including alkylating agents (n=28), epipodophyllotoxins (n=22), corticosteroids (n=3), abdominal-RT (n=4), chest-RT (n=2), and anthracyclines (n=1). A total of 39 CpGs from EWAS were nested within 21 DMRs. 92.1% (650/706) chemo-associated CpGs in EA were replicated in AA CCS, and 98.9% (86/87) chemo-associated CpGs in AA were replicated in EA CCS. In contrast, 66.7% (4/6) RT-associated CpGs in AA were replicated in EA CCS, and 71.7% (638/890) RT-associated CpGs in EA were replicated in AA CCS. Moreover, the four CpGs that partially mediated the effect of abdominal-RT on hypercholesterolemia in EA were not associated with abdominal-RT in AA CCS, further suggesting substantial difference in RT-associated CpGs as compared to chemo-associated CpGs between AA and EA CCS. Future studies by including a larger sample size of AA CCS are warranted to assess the difference in treatment-associated DNAm alterations and subsequent disparity in risk of treatment-related CHCs between AA and EA CCS. The DNAm sites can be used as predictors for risk management (treatment decision-making) and potential mechanistic targets for intervention for those at the greatest risk of CHCs. Citation Format: Qian Dong, Sarmistha Das, Cheng Chen, John Easton, Heather L. Mulder, Emily Walker, Geoffrey Neale, Deo Kumar Srivastava, I-Chan Huang, Jinghui Zhang, Melissa M. Hudson, Leslie L. Robison, Kirsten K. Ness, Nan Song, Zhaoming Wang. Variations of blood DNA methylation associated with cancer treatment exposures among childhood cancer survivors of African ancestry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4514.

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Kostadinov, Rumen, Byunggil Yoo, Midhat S. Farooqi, Shannon Kelley, Erin Guest, Michael J. Burke, Sarah Wheelan, and Patrick Brown. "Whole Genome Bisulfite Sequencing (WGBS) Robustly Measures the Pharmacodynamic Effect of Decitabine/Vorinostat Epigenetic Treatment in Relapsed Pediatric ALL Demonstrating Potent Hypomethylation Associated with Upregulation of PRC2 and TP53 Targets." Blood 132, Supplement 1 (November 29, 2018): 918. http://dx.doi.org/10.1182/blood-2018-99-119206.

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Abstract Introduction Epigenetic alterations are prevalent in ALL and have been implicated as determinants of chemotherapy resistance and relapse. Epigenetics modulating drugs, such as DNA methyltransferase and HDAC inhibitors, are of interest as a potential means of enhancing chemotherapy sensitivity and improving clinical outcomes. Interpretation of clinical responses in trials of epigenetic drugs has been hampered by lack of precision in assessing the pharmacodynamic effects of the drugs. In the context of a clinical trial testing the epigenetic modulators decitabine (10-15 mg/m2) and vorinostat (180 mg/m2) (D/V) given for one week prior to reinduction with multiagent chemotherapy in young (age 1 - 21 years) patients with relapsed ALL, we assessed genome-wide cytosine methylation with single base-pair resolution using WGBS. Methods DNA was isolated from leukemia specimens (marrow or blood) obtained before and after 1 week of treatment with D/V from N=10 patients (20 paired samples). For N=6 of these patients (12 paired samples), RNA was also isolated. WGBS at 30X coverage and RNA-seq were performed on the DNA and RNA samples, respectively. We used a custom WGBS analysis pipeline to trim reads (TrimGalore), align to hg19 genome (Bismark/bowtie2), remove PCR duplicates and repeats, call B allele frequency at SNPs, and call methylation states at CpG sites. We used rsem, STAR and EBSeq for RNA-seq alignment and differential gene expression calls between pre- and post- treatment samples. 16 million CpGs were segmented into 1.3 million segments based on pre/post D/V methylation change (HaarSeg/R). We plotted 5 quintiles of gene expression (N=22,522 genes), from most expressed (Q1) to unexpressed (Q5), and linked to associated promoter CpG segment methylation (deepTools, Fig.1). Gene set enrichment analysis (GSEA/MSigDB) was performed on genes ranked in order by greatest promoter hypomethylation, highest significance of differential gene expression, and highest gene log2 fold overexpression post- treatment to create an "epigenetically activated" ranked gene list. Results WGBS showed case-specific areas of leukemia-associated allelic imbalance in each pair of samples, confirming that pre/post DV methylation differences are specific to leukemic blast population. D/V treatment reduced CpG segment methylation by 12.4% on average (range: 5.1-20.7%). Drug-induced hypomethylation was equally distributed (not specific to exon, intron, intergenic, promoter, or enhancer regions). In the 6 cases with paired RNA-seq data, we found a strong correlation between promoter methylation and gene expression in pre-treatment samples, and this correlation was maintained in the hypomethylated post-treatment samples. One of these cases, with global methylation decreased by 12.9%, is shown in Fig.1. GSEA on these 6 cases combined showed that 75/4169 gene sets (including targets of PRC2 and TP53, H3K27ME3- and H3K4ME3- bound, FDR<25%, p<0.01) were overrepresented in the "epigenetically activated" ranked gene list. In 4/6 cases we detected hypomethylated segments at the CD19 promoter and an associated 1.05- to 5.13- fold CD19 upregulation. Conclusions With high resolution, we observed base-pair specific reduction in CpG methylation after epigenetic modulation with decitabine and vorinostat across the genome in all cases, although the pharmacodynamic effect varied by as much as 15.6% between patients. The drug-induced activation of PRC2 targets suggests that epigenetic treatment of relapsed ALL may induce differentiation, and the activation of TP53 targets suggests that epigenetic treatment can reactivate tumor suppressor genes to suppress cell proliferation. Furthermore, epigenetic activation of CD19 expression suggests that epigenetic pre-treatment can potentially sensitize cells to CD19-targeted immunotherapies. Integrated WGBS/RNA-seq represents a powerful tool for pharmacodynamic evaluation of epigenetic therapy. Figure 1. D/V treatment reduced average CpG segment methylation by 12.9% in case T0300. Methylation of gene promoters, defined as ±3kb from the transcription start site (TSS), showed strong correlation with expression of 22,522 genes ranked and split into quintiles in order of upregulated (Q1) to downregulated (Q4-Q5) (A, B). Hypomethylation was equally distributed across promoter regions and gene bodies (TSS-TES, scaled transcription start-end sites) (C, D). Figure 1. Figure 1. Disclosures Burke: AMGEN: Speakers Bureau; JAZZ: Speakers Bureau; Shire: Speakers Bureau.

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Krombholz, Christopher Felix, Lorena Gallego Villar, Pritam Kumar Panda, Sushree Sangita Sahoo, Marcin W. Wlodarski, Daniel B. Lipka, Christoph Plass, Charlotte Niemeyer, Miriam Erlacher, and Christian Flotho. "5-Azacytidine Is Effective for Targeting Leukemia-Initiating Cells in Juvenile Myelomonocytic Leukemia." Blood 132, Supplement 1 (November 29, 2018): 4342. http://dx.doi.org/10.1182/blood-2018-99-116594.

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Abstract Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of young children that originates from early hematopoietic stem/progenitor cells. We have previously developed an in vivo disease model using xenotransplantation of primary JMML cells into Rag2-/-γc-/- mice (Haematologica 2016;101:597). The model reproduces a characteristic JMML phenotype including myelomonocytic proliferation, hepatosplenomegaly, and lung infiltration. Case-specific driver mutations and DNA methylation patterns are unchanged after xenologous engraftment, indicating their origin in leukemia-initiating cells. We and others recently discovered a tight link between prognosis and differential DNA methylation in JMML (Nat Commun 2017;8:2126; Nat Commun 2017;8:2127; Blood 2018;131:1576). We also reported that the DNA methyltransferase inhibitor 5-azacytidine (azacitidine, 5AC) has unprecedented clinical activity in JMML and induces complete or partial remissions before allogeneic HSCT (Blood 2015;125:2311). Cytosine arabinoside (araC) is a structurally related nucleoside which is commonly used for cytoreduction in JMML but lacks the ability to induce remissions. Here we employed the xenotransplantation model to investigate the antileukemic activity and epigenetic effects of 5AC on JMML in comparison to araC. After eight weeks of leukemic expansion, 15 xenograft mice were treated with two cycles of 5AC (3 mg/kg/d x 5 days every two weeks). Control groups included mice treated with araC 20 mg/kg/d (N=15) or carrier solution (0.9% NaCl, N=20). The experimental animals maintained stable body weight, and no major toxicity on murine hematopoiesis was observed. 5AC and araC exhibited antileukemic activity and substantially reduced the human JMML cell content in bone marrow, spleen, liver, and lung. However, we noted that CD34+ stem/progenitor cells within the human leukemia population were depleted after treatment with 5AC but not after araC (5AC, 20.2% +/- 7.3%; araC, 35.6% +/- 6.1 %; carrier, 39.4% +/- 3.5%; p<0.01). To demonstrate that the selective reduction of CD34+ cells impaired the leukemia-initiating capacity of the xenograft, we treated a subsequent series of mice as above and retransplanted the bone marrow into secondary recipient mice. JMML cells obtained from 5AC-treated primary recipients sustained engraftment in only one of 9 secondary recipients at 30 weeks after retransplantation whereas JMML xenografts treated with araC or carrier engrafted in 8/13 or 4/8 secondary mice, respectively (p=0.03). We then examined the genome-wide DNA methylation in 5AC-treated xenografts (N=5) using Infinium 450K arrays. The JMML genomes exhibited global and profound DNA demethylation with near-complete loss of fully methylated CpG sites. A focused analysis of approximately 5,000 CpG sites with JMML-specific methylation illustrated that the profiles of 5AC-treated JMML cells were more similar to healthy human CD34+ cells than untreated JMML cells. As expected, no change in DNA methylation was observed in xenografts treated with araC. Next we studied the early effects of 5AC on the transcriptome and epigenome of JMML. Xenograft mice were treated as above, and JMML cells were harvested from bone marrow on days 0, 2, 4, and 6. RNA sequencing readily identified non-random changes in gene transcription that progressed over time from days 2 to 6 and were reproducible across replicate mice. Between days 0 and 6 we observed >2fold upregulation of 856 transcripts and downregulation of 958 transcripts (<0.01 false discovery rate, multitest-corrected). CpG-rich 5' regions (putative promoters) of corresponding genes were invariably demethylated. Gene Ontology enrichment analysis linked upregulated transcripts to myeloid differentiation whereas downregulated transcripts were involved in nucleosome assembly/organization and chromatin silencing. In summary, the xenograft experiments highlight the therapeutic potential of 5AC in JMML and thus encourage the further clinical development of epigenetic therapy with hypomethylating agents for this disease. Disclosures Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Claus, Rainer, Dietmar Pfeifer, Maika Almstedt, Björn Hackanson, Manuela Zucknick, Christoph Plass, and Michael Lübbert. "Decitabine (DAC) Treatment Induces Very Early In Vivo DNA Methylation and Transcriptome Changes in Primary AML Peripheral Blood Blasts." Blood 118, no. 21 (November 18, 2011): 1360. http://dx.doi.org/10.1182/blood.v118.21.1360.1360.

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Abstract Abstract 1360 Background: The in vitro effects of the DNA hypomethylating agents 5-aza-C (5-azacytidine) and 5-aza-dC (DAC) upon the epigenome and transcriptome of AML cell lines have been investigated by numerous groups, demonstrating induction of gene-specific and global hypomethylation, as well as up- and downregulation of multiple genes. However, their in vivo mechanisms of action are not well-studied, with only very sparse data on primary blasts from patients (pts) with myeloid neoplasias treated with these compounds (Daskalakis et al., Blood 2002, Yang et al., Cancer Research 2006, Fandy et al., Blood 2009). This is primarily due to the substantial methodological challenges of obtaining sufficient cell numbers of bona fide malignant cells during treatment. To study very early in vivo effects, we isolated and analyzed primary cells from AML pts with high numbers of peripheral blood blasts treated with the hypomethylating agent DAC. Patients, Materials and Methods: Of 48 consecutive pts aged >60 years with newly diagnosed AML treated with DAC as described (Lübbert et al., 2011) at a single centre within a multicenter phase II study (trial 00331), successful sequential sampling of peripheral blood blasts in sufficient numbers and purity was feasible in 8 pts. The median WBC at AML diagnosis was 18,400/μl (range 4,800–241,000), the median age was 80 years, and 6/8 had cytogenetic abnormalities. The methylation state of LINE1 and 35 genes (5' regions, represented by a total of 635 evaluable CpG dinucleotides) was quantified by MALDI-TOF mass spectrometry. Gene selection criteria were based on previous identification as potentially silenced tumor suppressors in genome-wide methylation analyses, and on reports as epigenetic targets in myeloid neoplasia. Transcriptional changes were assessed by array-based transcriptome profiling using the HG-U133plus 2.0 GeneChip array. Results: Isolation and purification of sufficient numbers of blasts was done both immediately before and at day 6 (median, range 4–7) after start of DAC infusion. Methylation quantification of pre-treatment blasts yielded patterns clearly distinguishing them from CD34-positive normal hematopoietic precursors of healthy donors (n=10). Specifically, P73, P15 and CDH1 exhibited the strongest and most consistent methylation gains (9–18%, p<0.015) in the malignant vs. normal cells. Early after DAC treatment, a striking decrease in LINE1 methylation was noted in 7/8 pts (p = 0.02), the most consistent effect among all investigated genomic regions. Overall, DAC-induced methylation changes (hypo- and/or hypermethylation) occurred in all eight pts. Hypomethylation was observed in the CpG islands of PBX1 (median 11%, range 5.5–25%, p<0.069), ESR1 (median 1.5%, range 0–9%, p<0.1) and MPO (median 4.3%, range 1–19%, p<0.1). For P73 and TLX3, substantial hypomethylation was observed in a subset of patients (maximum changes of 36% and 34% respectively), while other pts showed unchanged or increased DNA methylation levels. Unsupervised hierarchical clustering revealed that the highest similarities were intra- but not inter-individual. When interrogating unbiased methylation changes at all 635 CpGs, significant hypomethylation was induced in 4/8 pts, significant hypermethylation in 1/8 pts, and non-significant methylation changes seen in 3/8 pts. DAC-induced mRNA expression changes also occurred in all eight pts, without clear correlations between hypomethylation and restored expression. Conclusions: DAC administered at a clinically effective dose and schedule resulted in a consistent and pronounced in vivo decrease of LINE1 methylation already at early time points after treatment start, with significant hypomethylation events in 50% of the pts This might be indicative of effective, early Dnmt1 depletion. Induction of multiple transcriptional changes (but up- and downregulation) was also noted in all pts. This is, to the best of our knowledge, the first report of an integrated methylation/transcriptome analysis in primary leukemia cells isolated at very early time points during therapy with hypomethylating agents, i.e. before clonal selection or even replacement and the occurrence of secondary events. Our observations are compatible with a mechanism of action of DAC that is distinct from those of a non-hypomethylating cytosine analogues such as cytarabine, in line with in vitro results (Flotho et al, Leukemia 2009). Disclosures: No relevant conflicts of interest to declare.

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Dong, Qian, Nan Song, Cheng Chen, Zhenghong Li, John Easton, Heather Mulder, Jinghui Zhang, et al. "Abstract 3764: Epigenome-wide association study of blood lipids among survivors of childhood cancer in the St. Jude Lifetime Cohort." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3764. http://dx.doi.org/10.1158/1538-7445.am2022-3764.

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Abstract Epigenetic studies of blood lipid traits have identified genes underlying lipid metabolism in the general population. However, investigation of this association has not been conducted in survivors of childhood cancer, a population with a much higher burden of dyslipidemia and other cardiometabolic conditions due to exposures of genotoxic cancer therapies. We performed epigenome-wide association studies (EWAS) to identify blood DNA methylation (DNAm) 5’-cytosine-phosphate-guanine-3′ (CpGs) associated with lipid concentrations, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), and triglycerides (TG), for childhood cancer survivors from the St. Jude Lifetime (SJLIFE) cohort. DNAm was generated with blood derived DNA using Illumina MethylationEPIC BeadChip array. Adjusted M-value of DNAm for each CpG was calculated based on a linear regression of M-value against a set of covariates, including sex, age at DNA sampling, leukocyte subtype proportions, top four significant genetic principal components, and top four methylation principal components. In the exposure EWAS, a linear regression model was used for lipid levels prior to DNA sampling as an exposure variable and DNAm as an outcome variable, adjusting for cancer treatments, age at lipid measurement, BMI, cigarette smoking and lipid lowering medicine use. In the outcome EWAS, a linear regression model was used for DNAm as an exposure variable and lipid levels after DNA sampling as outcome variables, adjusting for cancer treatments, age at lipid measurement, BMI, smoking, lipid lowering medicine use, lipid levels measured at DNA sampling, age at DNA sampling, and polygenic risk score for specific lipid levels. Among survivors of European ancestry (N=2052), we identified 43 significant CpGs (P<9×10-8) associated with HDL (n=7), TC (n=3) and TG (n=33) as exposures; and 106 CpGs associated with HDL (n=5), LDL (n=3), TC (n=4), and TG (n=94) as outcomes. Among survivors of African ancestry (N=370), we identified 3 CpGs associated with TG as an exposure, and 5 CpGs associated with LDL (n=1) and TC (n=4) as outcomes. A comparison of effect sizes of significant CpGs between survivors of European and African ancestry suggests moderate to substantial racial differences in epigenetic associations with lipid exposures (14/46 with I2>50) and lipid outcomes (106/111 with I2>50). Additionally, no overlap between CpGs associated with lipid exposures and lipid outcomes suggests that the DNAm levels of these CpGs could be either the cause or consequence of lipid levels. Examination of the EWAS catalog and recent literature, suggests that most of the lipid associated CpGs identified in our study are novel. Blood lipid associated CpGs may be epigenetic biomarkers for identification of survivors with higher risk of dyslipidemia and may inform potential drug targets for future interventions. Citation Format: Qian Dong, Nan Song, Cheng Chen, Zhenghong Li, John Easton, Heather Mulder, Jinghui Zhang, Geoffrey Neale, Emily Walker, I-Chan Huang, Kirsten K. Ness, Melissa M. Hudson, Leslie L. Robison, Zhaoming Wang. Epigenome-wide association study of blood lipids among survivors of childhood cancer in the St. Jude Lifetime Cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3764.

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Shaknovich, Rita, Huimin Geng, Nathalie Johnson, Leandro Cerchietti, Maria E. Figueroa, Lucas Tsikitas, Olivier Elemento, et al. "ABC and GCB DLBCLs Display Unique Biologically Distinct and Clinically Relevant Epigenetic Signatures." Blood 114, no. 22 (November 20, 2009): 619. http://dx.doi.org/10.1182/blood.v114.22.619.619.

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Abstract Abstract 619 Diffuse Large B-cell Lymphoma (DLBCL) is a complex biological entity with heterogeneous genetic, biological and clinical features. Gene expression profiling studies have attempted to resolve some of this heterogeneity. For example, DLBCL patients harboring gene signatures associated with expression of germinal center B-cell genes (GCB) or activated B-cell genes (ABC) vary in their response rate to standard chemo-immunotherapy regimens. Since epigenetic gene regulation can play a fundamental role in determining the phenotype of normal and malignant tissues we asked whether ABC and GCB DLBCLs also display unique epigenetic signatures that might be clinically useful and further explain the biology of these tumors. For this purpose we examined the DNA methylation level of 50,000 cytosine residues distributed among 14,000 gene promoters in a cohort of 159 patients with DLBCL, all of whom were uniformly treated with R-CHOP, using the HELP assay and high-density oligonucleotide microarrays. For a subset of these patients (n=69), we also performed Affymetrix gene expression profiling. First, a Bayesian predictor of ABC/GCB subtypes was trained from a published expression profiling study of 203 DLBCL patients. The predictor was then applied to our cohort of 69 patients. At a probability cutoff of 0.9, 20 patients were classified as ABC, 40 were classified as GCB and 9 could not be classified (i.e. “type III DLBCL”). As expected from published studies, the differences in progression free survival (PFS) and overall survival (OS) of these ABC vs. GCB patients thus determined was highly significant, with p=0.0026 (log rank) and p=0.043 (log rank) respectively, with a much worse prognosis for ABC patients compared to GCB ones. We then asked whether the ABC and GCB subtypes could be predicted from the DNA methylation profiles of the same 69 patients. By performing a t-test we found that 239 genes were differentially methylated between ABC and GCB (p<0.0001) and also displayed >30% difference in methylation level. This DNA methylation signature was incorporated into a new Bayesian predictor, which we showed to predict ABC and GCB DLBCL subtypes from DNA methylation profiles with a 91% accuracy. Using a cross-validation procedure, we estimated that the classification performance on independent cases to be ∼87%. The predicted ABC and GCB cases retained the clinical predictive power of the gene expression profile when applied to the remaining 90 patients that did not have gene expression profiles, confirming its clinical relevance (difference in PFS p=0.0148, log rank). Gene set enrichment analysis showed that the ABC DNA methylation signature was enriched in genes involved in antigen dependent B and T-cell responses and in TNF inflammatory responses (p<1.01E-4 and <6.01E-4 respectively). A computational analysis of promoter DNA sequences of the genes involved in this signature revealed over-representation of binding sites for transcription factors including MYB, STAT5A, MAZ, and JUN and Sp1; many of these factors have a known role in B cell development and function. The role of Sp1 in these tumors is under further examination. Among the 239 genes that were differentially methylated and the 411 genes that were differentially expressed between ABC and GCB there was an overlap of 16 genes (greater than expected by chance Fisher Exact p=0.005). A predictor based on the methylation profiles of these 16 genes was on its own 92% accurate in identifying ABC vs. GCB cases among our cohort of DLBCLs. Although there was a general trend for inverse correlation in expression between the 239 differentially methylated genes, these 16 overlapping genes displayed marked and extreme inverse correlation. This was validated by single locus quantitative methylation sequencing (MassArray) and QPCR. The 16 genes included genes known to play critical roles in B-cell differentiation, proliferation and metabolism but not previously implicated in DLBCL. Gain and loss of function assays of a subset of these genes in ABC and GCB DLBCL cell lines show that they have tumor suppressor functions in DLBCL. Our results demonstrate for the first time that ABC and GCB DLBCLs are epigenetically distinct diseases; they identify new biological differences and candidate tumor suppressor genes between these tumors and demonstrate that a DNA methylation classifier can be used to distinguish GCB and ABC DLBCL subtypes. Disclosures: No relevant conflicts of interest to declare.

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Jankowska, Anna, Myunggon Ko, Yun Huang (equal contribution), Utz J. Pape, Hadrian Szpurka, Mamta Tahiliani, Hozefa S. Bandukwala, et al. "Impaired Hydroxylation of 5-Methylcytosine In TET2 mutated Patients with Myeloid Malignancies." Blood 116, no. 21 (November 19, 2010): 1. http://dx.doi.org/10.1182/blood.v116.21.1.1.

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Abstract Abstract 1 TET2 mutations are frequently found across broad spectrum of myeloid malignancies but how these mutations contribute to diseases is still unknown. Preliminary results from our laboratory have suggested that TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and consequently, the levels of 5-hmC may be lower in genomes of mutant bone marrow cells. To facilitate study of TET2 function we developed a blot assay to detect 5-hmC in genomic DNA with a specific antiserum to 5-hmC. In a second improved assay with increased sensitivity and precision, we treated genomic DNA with bisulfite in order to convert 5-hmC to cytosine 5-methylenesulfonate (CMS) and measured 5-hmC levels indirectly using a specific anti-CMS serum. Based on the results of this technique we demonstrate here for the first time that indeed TET2 mutations in predicted catalytic residues and other positions compromised TET2 function. We studied 102 patients with various myeloid malignancies (4/28 MDS, 14%, 26/48 MDS/MPN, 54% and 1/4 MPN, 2% and primary 2/11 AML 18% and 3/11 sAML, 27% TET2 mutants, respectively) and compared to wt cases or controls (N=17). Mutations were found throughout the entire coding region and were mostly inactivating (33/45 TET2 mutations). The levels of 5-hmC in genomic DNA from TET2 mutants were significantly decreased in comparison to wt cases and controls (p=4.5e-08 and p=1.8e-09, respectively). Particularly low levels of 5-hmC were found in patients with homozygous (UPD)/hemizygous (deletion) TET2 mutations and those with biallelic mutations. Surprisingly, 18% of all TET2 WT patients also showed low levels of genomic 5-hmC (despite normal TET2 mRNA expression), suggesting that these patients may carry not yet identified variants/lesions in TET2 or other partner proteins involved in TET2-mediated catalysis. To further investigate the impact of TET2 mutations associated with myeloid malignancies we also introduced 9 different missense mutations corresponding to those found in patients into murine Tet2 cells; severe loss of enzymatic activity was observed in 7/9 cases as measured by greatly diminished 5-hmC levels. To study the role of Tet2 in normal hematopoiesis we depleted Tet2 in C57BL/6 mice by retrovirus-mediated transduction of shRNA against Tet2. Tet2 depletion is associated with skewing of hematopoietic differentiation towards the monocyte/macrophage lineage. To further investigate the function of TET2 we transduced the myeloid THP-1 cell line with lentiviral vector containing TET2 cDNA (TET2+) or an empty vector. This manipulation allowed us to select clones showing 19-fold increase in TET2 mRNA expression without significantly alterations of proliferation kinetics. Using this model we studied the impact of TET2 overexpression on resultant methylation pattern of CpG sites. We have applied Illumina Infinium HumanMethylation27 arrays (27,5K CpG sites/14.4K genes). Overexpression of TET2 resulted in a distinct promoter methylation patterns with 169 altered CpG sites with difference of averaged β>0.5 (considered significant as compared to control). Among these differentially methylated loci, 27 promoters were significantly hypomethylated while 42 were hypermethylated as compared to control cells. Change in methylation pattern observed through overexpression of TET2 in vitro prompted us to analyze methylation patterns in patients with and without TET2 mutations or those with decreased 5-hmC levels. Using methylation arrays a total of 62 cases were analyzed. When patients were grouped based on the levels of 5hmC, an associated methylation signature can be clearly discerned with 2512 differentially methylated loci and distinct skewing towards hypomethylation (2510 sites; e.g., TMEM102, ABCC11) vs. hypermethylation (2 sites, AIM2 and SP140), consistent with the observation made in the TET2+ cells line. In sum, our results provide strong evidence for TET2 as the first mutated gene in myeloid malignancies that is involved in conversion of 5-mC to 5-hmC in DNA, indicating the novel role of TET2 in a substantial component of epigenetic deregulation in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

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Lavelle, Donald, Kestis Vaitkus, Virryan Banzon, Maria Hankewych, Pasit Phiasivongsva, Sanjeev Redkar, Sarath Kanekal, et al. "Fetal Hemoglobin Induction in Baboons (P. Anubis) Following Admistration of a Novel Decitabine Dinucleotide (S110) Compound." Blood 110, no. 11 (November 16, 2007): 571. http://dx.doi.org/10.1182/blood.v110.11.571.571.

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Abstract Increased fetal hemoglobin (HbF) levels can ameliorate the symptoms of patients with sickle cell disease and increase their life span. The DNA methyltransferase (DNMT) inhibitor decitabine increases HbF in non-human primates (baboons) and sickle cell patients and offers great promise as an effective therapeutic agent for the treatment of this disease. The goal of this investigation was to develop and test new compounds that inhibit DNMT, increase HbF, and offer significant advantages compared to decitabine such as increased stablity, reduced cytotoxicity, and a lower potential for mutagenicity. S110 is decitabine-guanine dinucleotide developed by Supergen, Inc., that is cytosine deaminase resistant and more stable in plasma than decitabine. Experiments were performed to compare the effect of S110 and decitabine on γ-globin expression in human erythroid progenitor cell cultures derived from CD34+ peripheral blood cells. Cultures were treated with decitabine (1 X 10−6 M) or S110 (1 and 5 X 10−6 M) on day 8. Forty eight hours later, RNA was purified for real time PCR analysis of ε- and γ-globin gene expression by the ΔΔCT method using α-globin as a control. Effects on globin polypeptide chain expression was determined by HPLC analysis of freeze-thaw lysates prepared 72 hours following drug addition. Both S110 and decitabine increased expression of ε-globin and γ-globin mRNA and the γ/γ + β polypeptide chain ratio (see Table). Effect of S110 on ε-and γ-globin mRNA and the γ / γ + β chain ratio in Human Erythroid Progenitor Cultures (n=4) DRUG CONCENTRATION ε-globin mRNA (Fold increase) γ-globin mRNA (Fold increase) Globin chain ratio (γ/γ + β) Untreated 3 +/− 3.3 Decitabine 1 X 10-6 M 3 +/− 24.8 2 +/− 2.8 8 +/− 3.2 S110 1 x 10-6 M 5 +/− 4.2 4 +/− 1.1 8 +/− 1.9 S110 5 X 10−6 9 +/− 34.5 35 +/− 1.5 2 +/− 2.9 The effect of S110 on HbF levels in vivo was then tested in non-human primates. Two baboons (PA 7256; PA 7470, P. anubis) were phlebotomized for eight days to achieve a HCT of 20 followed by treatment with S110 (1mg/kg/d; sc) for ten days. Pretreatment HbF levels were 3.1% (PA 7256) and 3.5% (PA 7470). Following S110 treatment, peak HbF levels of 46.1% (PA 7256) and 75.5% (PA 7470) were attained that were similar to levels observed in animals treated with equivalent molar doses of decitabine. Bisulfite sequence analysis showed that the level of DNA methylation of 5 CpG sites within the γ-globin promoter in purified BM erythroblasts following S110 administration was significantly lower (41.5%, PA 7256; 16.3%, PA 7470) compared to erythroblasts from bled, untreated baboons (70.8%; n=4). Pharmacokinetics was also evaluated in parallel to assess systemic exposure. These data demonstrate that S110, a newly developed decitabine-guanine dinucleotide, effectively increased HbF and reduced DNA methylation in cultured human erythroid progenitor cells and in experimental non-human primates.

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Chen, Qingxiao, Jingsong He, Xing Guo, Jing Chen, Xuanru Lin, Xi Huang, Haimeng Yan, et al. "TET2 Downregulation Promotes AML Cell Proliferation Via Pim-1 Expression." Blood 128, no. 22 (December 2, 2016): 5085. http://dx.doi.org/10.1182/blood.v128.22.5085.5085.

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Abstract Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults which is still incurable although novel drugs and new combination of chemotherapies are used . With the development of genetic and molecular biology technologies, more and more genes are found to be related to leukemogenesis and drug resistance of AML. TET2, a member of the ten-eleven-translocation gene family which can modify DNA by catalyzing the conversion of 5-mehtyl-cytosine to 5-hydroxymethyl-cytosine , is often inactivated through mutation or deletion in myeloid malignancies. Recent research reported that TET2 knock-down can promote proliferation of hematopoietic stem cells and leukemic cells. Also, several clinical studies showed that patients with TET2 mutation or low levels of TET2 expression have more aggressive disease courses than those with normal levels of TET2. However, the mechanism of the phenomenon is unknown. Our aim is to uncover how TET2 protein level is negatively correlated with AML cell proliferation and to provide a better view of target therapy in AML. Methods: We determined the expression levels of TET2 and other target genes in acute leukemia cell lines, bone marrow AML specimens, and peripheral blood mononuclear cells from healthy donors by qRT-PCR and Western blot. We also determined the mutation status of TET2 in AML cell lines. CCK8 and flow cytometry were used to determine cell proliferation, cell apoptosis, and cell cycle profile. Methylation-specific PCR were used to examine the methylation status in gene promoter regions. Also, we developed TET2 knock-down lentivirus to transfect AML cell lines to examine the effect of TET2 depletion. Last, RNA-seq was used to compare gene expression level changes between TET2 knock-down cell lines and the control cell lines. Results: AML cells from AML cell lines (KG-1,U937, Kasumi, HL-60, THP-1, and MV4-11) and AML patients' specimens expressed lower levels of TET2 than those of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expressed TET2, while KG-1 expressed TET2 at a relatively higher level than those of other AML cell lines. We constructed a TET2 shRNA to transfect KG-1,THP-1,MV-4-11,Kasumi,and HL-60, and used qRT-PCR and western blot to verify the knock-down efficiency. CCK8 confirmed that knocking down TET2 could increase leukemia cell proliferation (P<0.05). Flow cytometry showed that cell cycle profile was altered in TET2 knock-down cells compared to the negative control cells. In order to identify target genes, we performed RNA-seq on wildtype and TET2 knockdown KG-1 cells and found that the expression of cell cycle related genes, DNA replication related genes, and some oncogenes were changed. We focused on Pim-1, an oncogene related to leukemogenesis, which was significantly up-regulated in the RNA-seq profile. Western blot and qPCR verified the RNA-seq results of Pim-1 expression in the transfected cells . Also, AML patients' bone marrow samples (n=35) were tested by qPCR and 28 of them were found to express low TET2 but high Pim-1 with the other 7 being opposite. For detailed exploration in expression regulation of Pim-1 via TET2, we screened genes affecting Pim-1 expression and found SHP-1, a tumor suppress gene which is often silenced by promoter methylation in AML. Western blot band of SHP-1 was attenuated in TET2 knockdown KG-1 cells. Moreover, methylation-specific PCR showed that after knocking down TET2 in KG-1 cell line, the promoter regions were methylated much more than the control cells. These results indicated that the function of TET2 in epigenetic modulation plays an important role in regulating Pim-1 expression. Finally, using flow cytometry and CCK8 we surprisingly found that knocking down TET2 expression could lead leukemic cells (KG-1, THP-1 and MV-4-11) more sensitive to Pim-1 inhibitor (SGI-1776 free base) and decitabine (a demethylation agent treating MDS and AML) (P<0.05). Conclusion: Our study showed that knocking down TET2 promoted leukemic cell proliferation. This phenomenon may correlate to Pim-1 up-regulation. Our clinical data also showed that the expression of TET2 and Pim-1 have an inverse relationship. The mechanism of TET2 regulating Pim-1 expression may be related to the epigenetic modulation function of TET2. Finally, we found TET2 downregulation could increase leukemia vulnerability to Pim-1 inhibitor and decitbine, and provide a novel view of target therapy in AML. Disclosures No relevant conflicts of interest to declare.

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Gaidzik, Verena I., Richard F. Schlenk, Peter Paschka, Anja Stölzle, Andrea Corbacioglu, David Nachbaur, Thomas Kindler, et al. "DNMT3A mutations Predict for Inferior Outcome in NPM1-Wildtype and Molecular Unfavorable Cytogenetically-Normal Acute Myeloid Leukemia: A Study of the German-Austrian AMLSG." Blood 118, no. 21 (November 18, 2011): 415. http://dx.doi.org/10.1182/blood.v118.21.415.415.

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Abstract Abstract 415 Background: Alteration of DNA methylation, a hallmark of epigenetic modification, is currently discussed as one important pathomechanism in leukemogenesis. Using a next-generation sequencing approach, a frameshift mutation of the gene encoding the DNA methyltransferase (DNMT3A) in an acute myeloid leukemia (AML) case was identified. DNMT3A catalyses the addition of a methyl group to the cytosine residue of CpG dinucleotides, thereby affecting promoter methylation status and gene expression. Subsequent sequencing analysis in an independent cohort of 288 AML patients (pts) revealed DNMT3A mutations (DNMT3Amut) in 22% of the pts; mutations were associated with intermediate-risk cytogenetics and poor outcome. Aims: To evaluate frequency and clinical impact of DNMT3Amut in pts with AML aged 18 to 61 years who were treated within AMLSG treatment trials AML HD98A (Schlenk et al., J Clin Oncol 2010;28:4642–8) and AMLSG 07–04 (NCT00151242). Methods: DNMT3A mutation analysis was performed in 1218 AML (HD98A, n=685; AMLSG 07–04, n=533; de novo AML, n=1102; s-AML, n=45; t-AML, n=69) using a DNA-based PCR assay for all coding exons (1 to 23) followed by direct sequencing. The median follow-up was 5.06 years. Results: DNMT3A mut were found with an overall frequency of 19.6% (239/1218); 189 mutations were located in the MTase domain clustering at amino acid R882 (79%). All but one mutation were heterozygous; only 4 cases had two mutations. DNMT3A sequence alterations included 17 frameshift, 4 nonsense, and 222 missense mutations. DNMT3A mut pts were significantly older (P=.01), more frequently females (P=.001), had higher white blood cell and platelet counts (both P<.0001), and higher bone marrow blasts percentage (P=.001). DNMT3Amut were associated with cytogenetically-normal AML (CN-AML, P<.0001), while DNMT3Amut were rare in favorable and adverse-risk karyotypes (P<.0001). Correlations with other molecular markers (NPM1, CEBPA, FLT3, IDH1/2, TET2, ASXL1) revealed a significant association with NPM1 (P<.0001), FLT3-ITD (P<.0001), and IDH1/2 (IDH1R132, P<.0001; IDH2R140, P=.0003; IDH2R172, P=.03) mutations, while co-occurrence of CEBPA (P=.02) and ASXL1 (P=.02) mutations was less frequent. DNMT3A mutational status did not impact complete remission (CR) rate, event-free (EFS) and relapse-free survival (RFS), neither in the whole cohort (P=.09, P=.98, P=.11; respectively) nor in the subgroup of CN-AML (P=.39, P=.79, P=.19, respectively). DNMT3Amut had a negative impact on overall survival (OS) in trend in the whole cohort (P=.07) and significantly in CN-AML (P=.02). In multivariable analyses, DNMT3Amut were in trend associated with a negative prognostic impact on OS (hazard ratio, 1.24; P=.06). In addition, we performed subgroup analyses according to (1) the NPM1 mutational status, and (2) the molecular risk groups of CN-AML (as defined by the European LeukemiaNet classification). DNMT3Amut did not impact OS in NPM1-mutated patients in the whole cohort as well as in CN-AML (P=.34; P=.22; respectively), while in NPM1-wildtype patients DNMT3Amut were associated with inferior OS in both, the whole cohort and in CN-AML (P=.001; P=.005; respectively). In molecular unfavorable CN-AML (NPM1-wildtype with or without FLT3-ITD, NPM1-mutated with FLT3-ITD, CEBPA-wildtype), DNMT3Amut were significantly associated with worse OS (P=.002) compared with DNMT3A-wildtype pts, even outweighing FLT3-ITD as an unfavorable prognostic marker. There was no effect of DNMT3Amut in molecular favorable-risk CN-AML. Conclusions: DNMT3A mutations are confirmed as frequent genetic aberrations in AML, associated with normal karyotype, NPM1, FLT3-ITD, and IDH1/2 mutations. DNMT3Amut predicts for inferior outcome in molecularly-defined subsets of AML, that is, NPM1-wildtype AML and molecular unfavorable CN-AML. As a single marker, DNMT3Amut only had a moderate effect on outcome. Disclosures: No relevant conflicts of interest to declare.

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Saba, Hussain I., Michael Lübbert, and P. W. Wijermans. "Response Rates of Phase 2 and Phase 3 Trials of Decitabine (DAC) in Patients with Myelodysplastic Syndromes (MDS)." Blood 106, no. 11 (November 16, 2005): 2515. http://dx.doi.org/10.1182/blood.v106.11.2515.2515.

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Abstract Background: MDS is characterized by ineffective hematopoiesis, resulting in cytopenias with dysplastic morphology of peripheral blood cells and bone marrow. Decitabine (Dacogen™ DAC) is a cytosine analog that reverses aberrant DNA methylation, leading to re-expression of silenced tumor suppressor genes. Due to the requirement for DNA synthesis and subsequent demethylation, decitabine may require prolonged administration to achieve maximum benefit. Overall response rates (ORR) (CR+PR) from 1 pivotal Phase 3 (D-0007) and 3 supportive Phase 2 trials (91–01, 95–11 and 97–19) in patients with intermediate and high risk MDS receiving DAC are being presented. Methods: The Phase 2 trials were open-label and single-arm, with a minimum of 4 treatment cycles and a maximum of 8 cycles, while the D-0007 was a 1:1 randomized comparison of DAC plus supportive care (SC) vs. SC alone, with a maximum of 10 cycles of therapy. The D-0007 study design dictated that patients be removed from therapy following 8 cycles of decitabine if CR was not achieved, and 6 cycles in the absence of PR. Patients who maintained a CR for 2 cycles were removed from therapy. Results: A total of 271 unique patients were exposed to DAC in the studies (n= 89 in D-0007, n=29 in 91–01, n = 66 in 95–11, n = 87 in 97–19). Patients receiving DAC had similar demographics and disease characteristics in all trials. Responses were observed in all IPSS and FAB subgroups. The percent of patients classified as intermediate-2 and high risk (according to the International Prognostic Scoring System) in the Phase 3 trial was 69% vs. 72% in the Phase 2 trials. By intent-to-treat analysis, the ORRs were 45%, 26%, and 26% respectively, for the Phase 2 trials. These results were corroborated in the Phase 3 trial, where the response rates were evaluated according to the more robust International Working Group MDS criteria, following a blinded, centralized bone marrow review. The D-0007 overall response rate was 17% for DAC (9% CR, 8% PR) vs. 0% for SC (p<0.001). Responses were durable, lasting a median of 266 days. The 95–11 and 97–19 response rates were also centrally reviewed, while 91–01 responses were investigator-assessed. In the 91–01 trial, the ORR was 45% (28% CR, 17% PR) with a median duration of response of 217 days, the 95–11 ORR was 26% (21% CR, 5% PR) with a median duration of 250 days, and the 97–19 ORR was 26% (22% CR, 5% PR) with a median duration of 146 days. Hematologic improvement (HI) was also evaluated according to varied criteria in conjunction with the response rates in all 4 studies; 12 patients (13%) had HI in D-0007, 2 patients (7%) in 91–01, 8 patients (12%) in 95–11, and 13 patients (15%) in 97–19. The D-0007 trial design dictated that patients who maintained a CR for 2 cycles be removed from therapy. As a result, the median number of cycles delivered was 3, with only 48% of patients receiving ≥4 cycles. In the Phase 2 studies, the median number of cycles is clearly higher (median 4), with the majority of patients receiving at least 4 cycles and approximately one-third of patients receiving ≥6 cycles. Conclusion: While response rates of ≥17% were demonstrated in these trials, the optimization of hypomethylating agents for maximum efficacy is very likely to include prolonged therapy, which may correlate with increases in response rate and duration.

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Saba, Hussain, Craig Rosenfeld, Jean-Pierre Issa, John DiPersio, Azra Raza, Virginia Klimek, James Slack, Carlos de Castro, Karl Mettinger, and Hagop Kantarjian. "First Report of the Phase III North American Trial of Decitabine in Advanced Myelodysplastic Syndrome (MDS)." Blood 104, no. 11 (November 16, 2004): 67. http://dx.doi.org/10.1182/blood.v104.11.67.67.

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Abstract MDS is a disease of the elderly marked by dysplasia of hematopoetic cell lines resulting in cytopenias and AML progression. There is currently no treatment approved to alter the natural history of the disease. Aberrant methylation associated with cancer is a potential target for pharmacologic therapy, and DacogenTM (decitabine) for injection (SuperGen, Inc.), a cytosine analogue, indirectly depletes methylcytosine after incorporation into DNA with subsequent inactivation of DNA methyltransferases. We report the results of a Phase III trial of decitabine (DAC) vs. supportive care (Supp.Care) in adult MDS patients with IPSS Intermediate (Int)-1 (31%), Int-2 (44%) and high risk disease (26%). Secondary MDS (14%) and previously treated (27%) MDS patients were not excluded. Bone marrows were assessed by a blinded, independent pathologist. 170 patients (accrued from July 2001 through April 2003 at 23 centers) were randomized 1:1 to either Supp.Care or DAC (a 3 hr infusion of 15mg/m2/hr every 8 hrs on 3 consecutive days every 6 wks for up to 10 cycles. The groups were comparable for numerous risk factors, including time from diagnosis (median 29 weeks for DAC and 35 weeks for Supp.Care). Kaplan Meier (KM) curves for time to AML progression or death showed early and clinically meaningful separation (consistent with clinical benefit) in favor of DAC in all patients (intent to treat; ITT), Int-2/High Risk, and High Risk patients. Using the Cox proportional hazards model for the ITT population, the probability of progression to AML or death was 1.68-fold greater for Sup.Care than for DAC (p=0.023). Median Time to AML or Death (Days) MDS group (n) DAC Supp.Care p-value 1Protocol specified test. 2Preferred test for analysis of early separation of KM curves. n=89 n=81 Wilcoxon1,2 Log rank1 All Patients (170) 338 263 0.046 0.204 Int-2/High Risk (118) 334 189 0.005 0.040 High Risk (44) 260 79 0.001 0.006 Treatment naïve (124) 354 189 0.005 0.034 Figure Figure Investigator reported response rate by International Working Group criteria was 25% (10% CR, 15% PR) for DAC vs. 0% for Supp.Care (p< 0.001), with responses equally distributed across baseline subgroups. Time to response was 100 days and median duration was estimated at >9 months. Responders (CR and PR) vs. non-responders had a median survival of 678 days vs. 406 days (p= 0.038 Wilcoxon). There were no treatment related deaths. As expected, grade 3–4 toxicity (including hematologic toxicity, febrile neutropenia) occurred in more DAC patients than in Supp.Care patients. Most patients tolerated treatment well. DAC appears a promising therapy for MDS with manageable toxicity. Final results of independently reviewed response rates and clinical benefit (transfusion independence; Quality of Life) will be presented.

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Li, Jia, Yue Wei, Minjung Lee, Caleb Class, Hui Yang, Deqiang Sun, Guillermo Garcia-Manero, and Yun Huang. "Comprehensive DNA 5-Hydroxymethylation Landscapes in Myelodysplastic Syndromes (MDS)." Blood 134, Supplement_1 (November 13, 2019): 2996. http://dx.doi.org/10.1182/blood-2019-128599.

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Background: Epigenetic regulators as well as epigenetic marks play an essential role in regulating normal hematopoiesis. Epigenetic alterations are one of the early events reflecting changes in cell identify during the transition of a pre-leukemic condition into a malignant phenotype. Capturing epigenetic alterations at early stages of pre-leukemic conditions could benefit the diagnosis and understanding the disease progression during hematopoietic malignant transformation. DNA 5-hydroxymethylation (5hmC) is the one of the major oxidation products of 5-methycytosine (5mC) mediated by the TET protein family dioxygenase. 5hmC has been reported to be involve in the regulation of chromatin accessibility and gene transcription. The dynamic changes of 5hmC have been reported as a hallmark in myeloid malignancies. In our study, we applied an improved 5hmC profiling method (sCMSIP) developed in our lab to systematically profile 5hmC and evaluate the 5hmC dynamics in patients with myeloid malignancies. Methods: In order to profile 5hmC using low amount of input DNA isolated from patient bone-marrow aspirates, we improved our previously developed anti-CMSIP method to capture 5hmC enriched regions using ultra-low amount of genomic DNA (10-20 ng gDNA). Cytosine-5-methylenesulfonatem (CMS) is the 5hmC derivatives upon sodium bisulfite treatment. A home-made anti-CMS antibody was developed to specifically recognize CMS genome-wide after sodium bisulfite treatment. The traditional anti-CMSIP has been widely used to study the genome-wide 5hmC distribution in various biological systems, including mESC, HSC and immune cells. We further optimized the immunoprecipitation procedure to profile genome-wide 5hmC with ultra-low amount of genomic DNA (sensitive CMSIP, sCMSIP). Using this improved CMS-IP method, we performed genome-wide 5hmC analysis in ~100 individuals including healthy donors (n = 10), patients with MDS (n = 62), AML (n = 4) and CMML (n = 13). In parallel, we performed RRBS (DNA methylation) and RNA-seq (transcriptome) analysis on matched samples. We then performed integrative bioinformatics analyses to unveil the potential diagnostic and prognostic values of 5hmC in myeloid neoplasms. Results and Conclusions: Based on our analysis, we found distinct epigenetic landscapes and transcriptomes between healthy donor and patients with myeloid malignancies. Interestingly, we could further separate patients with MDS into three clusters by comparing their 5hmC landscapes with normal, AML and CMML individuals, suggesting that 5hmC might be a sensitive epigenetic mark to reflect the disease status of MDS. Furthermore, we identified a positive correlation between 5hmC distribution and the blast ratio in paired patients before and after hypomethylating agent (HMA) treatment. These findings suggest that 5hmC might be used as a prognostic marker for HMA treatment. Detailed analyses further elucidate the impaired 5hmC enrichment in various key transcription factors that are essential for HSC function and myeloid lineage specification. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding.

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Sahin, Deniz, Shiva Bamezai, Fabio Ciccarone, Elena Fischbein, Fabian Mohr, Medhanie A. Mulaw, Alex Pulikkottil Jose, et al. "TET1 Promotes Leukemic Growth in T-ALL Via Maintenance of 5-Hydroxymethylation Marks and Can be Antagonized By the PARP Inhibitor Olaparib." Blood 128, no. 22 (December 2, 2016): 737. http://dx.doi.org/10.1182/blood.v128.22.737.737.

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Abstract T-cell acute lymphoid leukemia (T-ALL) is an aggressive hematological cancer predominantly occurring in children and adolescents. T-ALL is characterized by aberrant expression of genes involved in T-cell differentiation and self-renewal accompanied by aberrant DNA cytosine methylation patterns. The mechanisms underlying these aberrations are not well understood. The Ten-eleven translocation 1 (TET1) enzyme is an iron-dependent dioxygenase that catalyzes the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation and regulation of gene expression. Using quantitative real time (qRT)-PCR we now demonstrate that T-ALL is characterized by high and aberrant expression of TET1: TET1 was significantly and >20-fold higher expressed in human T-ALL cell lines compared to other B-lymphoid or myeloid leukemia cell lines (p<0.001, n=3). This was confirmed in primary patient samples, in which TET1 was more than 10-fold higher expressed in 95% of T-ALL cases (n=10) compared to other leukemia subtypes (B-ALL, n=29; AML, n=39; CLL, n=23, p<0.001). Furthermore, expression levels of TET1 in T-ALL patient samples were over 50-fold higher than in normal PB and BM derived healthy CD3+ T cells (n=6, p<0.001) in line with published cDNA microarray data from a large patient cohort of 174 T-ALL cases (Haferlach et al., JCO 2010). Knockdown (KD) of TET1 mRNA using two independent shRNAs in T-ALL cell lines adversely affected their cell growth (>70% reduction compared to scrambled, p<0.001, n=4) clonogenicity (>70% reduction compared to scrambled, p<0.001, n=4) and blocked cells in the G2/M phase of the cell cycle in vitro (n=3). Furthermore, TET1 KD significantly reduced the leukemic engraftment potential of T-ALL cell line Jurkat in xenografts (shRNA, n=16; scrambled, n=8). Global analysis of 5hmC levels using hMeDIP-seq performed on TET1 depleted Jurkat cells revealed lower global 5hmC levels as compared to cells transduced with scrambled control (n=2). Promoters (-5kb TSS) of 5,181 genes showed reduction of 5hmC marks (log2 fold diff.; p-value < 1e-10). Global 5mC analysis (MeDIP-seq) revealed 12,007 genes that gained 5mC marks at their promoters as a result of TET1 KD (log2 fold diff. > 1.5; p-value < 1-10). Of note, genes demonstrating a loss of 5hmC marks and corresponding a gain in 5mC marks on their promoters belonged to gene families involved innucleosome organization (HIST family), DNA damage response (DDR)(RAD, PARP), T-cell development (NOTCH3, RUNX1, LEF1) and homeobox genes (q-value < 1e-5). These changes were paralleled by changes in expression after TET1 depletion as measured by RNA-Seq in Jurkat cells: GO TERM analysis (log2 fold diff. > 1.5; p<0.05) revealed deregulation of genes belonging to nucleosome assembly, DNA damage, nucleosome organization and transcriptional deregulation in cancer. In line with these observations these pathways showed differential expression in T-ALL patients divided into the categories high vs. low TET1 expression based on previously published microarray data (Haferlach et al., JCO 2010) (median as cut-off; n=174)(log2 fold diff. >1.5; p<0.05).The aberrantly high TET1 expression in T-ALL cell lines shares a potential causal relationship with the hypomethylated status of the TET1 promoter (determined by LC/MS), and the enrichment of euchromatic marks such as H3K4me3 and acetylated H3 as determined by ChIP-qPCR. Since it was shown that PARP activity positively influences TET1 expression by regulating its promoter epigenetically, we tested the impact of the PARP inhibitor olaparib on TET1 activity in our model: of note, aberrant expression of TET1 could be reverted by the PARP inhibitor olaparib, which induced reduction of euchromatic marks on the TET1 promoter, in parallel with decreased TET1 expression and protein levels as well as 5hmC levels in Jurkat cells without affecting expression of other TET family members. Olaparib treatment caused a >75% reduction in cell growth and clonogenicity of T-ALL cell lines in vitro (n=3) and delayed leukemic engraftment in NSG mice (42%±8 in scrambled vs 9%±7 for the shRNAs, n=4 per arm; p<0.001). In conclusion, these data demonstrate for the first time that TET1 plays an important oncogenic role in T-ALL. We also identify PARPs as upstream regulators of TET1 in T-ALL, opening the way to antagonize TET1 pharmacologically in this poor prognosis leukemia. Disclosures Mulaw: NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

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Haque, Shabirul, Jennifer Nieto, Hyunjoo Lee, Nicholas Chiorazzi, and Patricia Mongini. "Evidence for Allelic Exclusion of p53 within Single Sorted Human B Cells." Blood 118, no. 21 (November 18, 2011): 1122. http://dx.doi.org/10.1182/blood.v118.21.1122.1122.

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Abstract Abstract 1122 The survival of replicating B cells, with DNA damage arising from oxidative stress and/or activation-induced cytosine deaminase (AID), appears in part to be under p53 control. Importantly, a common C>G single nucleotide polymorphism (SNP) within codon 72 of p53 influences p53 function. Among other differences, p53-72R (CGC=Arginine) is notably more effective than p53-72P (CCC=Proline) at inducing apoptosis. The SNP has been linked to clinical outcome in multiple settings, including malignancy. Most individuals in the US population display heterozygosity. In this study, we have examined whether B cells, whose genomic DNA is heterozygous for the codon 72 SNP, exhibit allelic exclusion at the level of expressed RNA. This was suggested by reported evidence that p53 expression is strongly regulated by gene methylation status; mRNA of peripheral blood cells from p53-72P/R heterozygous individuals is skewed toward the representation of only one SNP, depending upon ethnic status; and a p53 intron 2 SNP, representing a potential methylation site, is in strong linkage disequilibrium with the codon 72 SNP (PLoS ONE 6:e15320, 2011). Evidence for allelic exclusion of this functionally relevant p53 SNP would suggest that not all identically-stimulated B cells have equal likelihoods of survival within p53-72P/R heterozygous individuals. To investigate this issue, we first confirmed that p53 was expressed in non-transformed human B cells replicating in response to surrogate C3d-bound antigen, IL4 and BAFF. These physiologically-relevant stimuli synergize to induce a burst of T cell-independent clonal expansion, followed by apoptosis of many of the divided progeny (J. Immunol. 175:6143, 2005). Expression of p53 was monitored by immunoblotting and flow cytometry. Consistent with a role in regulating clonal burst size, p53 protein and mRNA/protein of p53-regulated pro-apoptotic genes were significantly elevated in blasts, prior to apoptosis. This contrasted with undectable p53 protein in non-stimulated B cells. To assess whether expressed p53 within a single lymphoblast derives from one allele, i.e. demonstrates allelic exclusion, we first identified tonsil donors heterozygous for the codon 72 polymorphism. This involved PCR-restriction fragment length polymorphism (RFLP) analysis of genomic DNA, as described by others (Leukemia Research 30:1113, 2006). Subsequently, purified resting B2 cells from cryopreserved tonsil cell suspensions, determined to be heterozygous for p53-72P/R, were labeled with CFSE and stimulated as above. At d5, single B lymphoblasts were sorted into 96 well PCR plates containing lysis buffer and cDNA prepared using random hexamer primers. A p53 sequence comprising exons 2a, 3, and 4 was subjected to two rounds of PCR amplification with the following primers, Forward: 5-cagccagactgccttccg-3 & Reverse: 5-gcaagtcacagacttggctg-3. Nucleotide sequencing of PCR-amplified p53 was performed commercially (Applied Biosystems Big Dye Terminator v3.1 cycle sequencing) and analyzed by chromasPro software. In two experiments, only 39 cells of a total of 236 assayed were positive for p53. By contrast, β-actin cDNA from two rounds of PCR amplification yielded 88% of the single cell-containing wells positive for actin. These yields indicate that p53 mRNA levels within a single cell are significantly more limiting than those for actin and are consistent with quantitative PCR of cDNA obtained from activated cell pools. Interestingly, analysis of the cDNA p53 chromatogram sequence of the p53-positive single cells (n=39), whose genomic DNA was heterozygous for the p53 codon 72 SNP, showed only a single homogenous sequence: either P (n=21) or R (n=18), but never both. This was in marked contrast to the chromatogram of cDNA derived from a pool of activated B cells within each experiment. In the latter cases, two overlapping peaks indicating co-expression of p72-P and p72-R were noted. Taken together, our findings suggest that p53 mRNA expression from a single non-transformed human B lymphocyte arises from the transcriptional activation of a single allele, i.e. shows allelic exclusion. Although the mechanism for this phenomenon requires further investigation, these results imply that B cells within individuals heterozygous for the functionally important p53-p72 polymorphism might vary considerably in their resistance to apoptosis. Disclosures: No relevant conflicts of interest to declare.

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Duan, J. E., Z. Jiang, F. Alqahtani, I. Mandoiu, H. Dong, X. Zheng, S. L. Marjani, J. Chen, and X. C. Tian. "1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos." Reproduction, Fertility and Development 31, no. 1 (2019): 126. http://dx.doi.org/10.1071/rdv31n1ab1.

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Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

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Adelman, Emmalee R., Jian Shi, and Maria E. Figueroa. "Aging Human Hematopoietic Stem Cells Manifest Massive Epigenetic Reprogramming and Altered Gene Splicing of Key Hematopoietic Gene Sets." Blood 128, no. 22 (December 2, 2016): 885. http://dx.doi.org/10.1182/blood.v128.22.885.885.

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Abstract Aging leads to impairment of hematopoietic stem cell (HSC) function with decreased self-renewal, imbalanced differentiation potential and an increased risk to develop myeloid malignancies. These malignancies are associated with epigenetic deregulation, which contributes to pathogenesis. Notably, studies in murine models have revealed epigenetic changes in aged HSC. However, it is unknown if this occurs in normal human HSC aging and whether it may contribute to HSC dysfunction. Therefore, we performed comprehensive epigenomic and transcriptional profiling in primary human HSC (Lin-, CD34+, CD38-) isolated from young (18-30 yo), mid (45-55 yo) and old (65-75 yo) healthy donors. Using a micro-ChIP-seq protocol we profiled H3K4me1, H3K4me3, H3K27me3 and H3K27ac in 4-7 donors per age group, as well as genome-wide DNA methylation (5mC), hydroxymethylation (5hmC) and RNA-seq. Analysis of enhancer-associated marks revealed that with age there is marked reduction in both H3K4me1 and H3K27ac (20,783 and 15,625 peaks lost, respectively; log10likelihood ratio >3). Gene ontology analysis of these lost peaks revealed their association with genes involved in hematopoiesis and, RNA splicing and chromatin organization, respectively (ChIPenrich, FDR<0.05). In addition, regions depleted in H3K4me1 are enriched for PU.1, FLI1, ETS, and CTCF binding sites (Homer, q<0.00001). We next asked if aging results in specific remodeling of poised (H3K4me1>H3K4me3, H3K27ac-) and active (H3K4me1>H3K4me3, H3K27ac+) enhancers. We found age-related loss of H3K4me1 enrichment at 10,696 poised enhancers, which are associated with hematopoiesis and T- and B-cell receptor signaling (FDR<0.05). We also identified 17,242 active enhancers in young HSC, 7,057 of which are depleted in old HSC. This loss of active enhancers targets genes associated with hematopoiesis, immune signaling and myeloid malignancies (FDR<0.05). Next we analyzed the impact of aging on promoter-associated marks, H3K4me3 and H3K27me3. Remarkably, while aging leads to loss of 22,689 H3K4me3 peaks, only 1,339 H3K27me3 peaks are lost. Loss of H3K4me3 targets genes involved in inflammatory response, development and WNT signaling (FDR<0.05). Given this uneven change in H3K4me3/H3K27me3 with aging, we hypothesized this may correlate with changes in bivalently marked promoters, which regulate key developmental genes. Out of 3,947 bivalent promoters in young HSC, 842 are lost in aged HSC. This loss of bivalency affects genes involved in WNT, Cadherin and Hedgehog signaling pathways (FDR<0.05). Next we analyzed changes in cytosine modifications. We observe widespread gain of 5hmC (n=14,554 differentially hydroxymethylated regions [DHMR]; FDR <0.005), with specific enrichment at introns and exons (p<2.2e-16), as well as enrichment for GATA and KLF binding sites (Homer, q<0.00001). These DHMR target genes involved in hematopoiesis, proteins regulated by alternative splicing, and pathways associated with cancer (FDR<0.05). In contrast, much more subtle changes are found in 5mC with HSC aging, with only 529 differentially methylated regions (q-value <0.05, meth.diff >20%). However, these subtle changes also target genes associated with cadherin and WNT signaling. Finally, RNA-seq analysis revealed that this age-associated epigenetic reprogramming is accompanied by an overall trend to gene downregulation. Amongst the genes most affected are the nuclear lamin gene LMNA (mutated in progeria syndrome), splicing factors SRSF7 and U2AF1 and, the transcription factors KLF3/6 and HIF1α (FDR <0.05, fold change >1.5). Notably, changes in expression also include significant differential exon usage, which may be mediated by DHMR at intron-exon boundaries: 575 genes show altered exon usage (FDR <0.05, fold change>1.5) including LMNA and the epigenetic modifiers BRD9, CITED2, KDM6A and SETD6. In summary, we have completed the first comprehensive epigenomic profiling of aging in human HSC. Our findings show massive epigenetic remodeling in aged HSC, consisting of loss of activating histone marks primarily targeting enhancers and bivalent promoters at genes involved in hematopoiesis and developmental pathways. Cytosine modifications show widespread changes in 5hmC, targeting intron-exon boundaries. Globally, this epigenetic reprogramming results in overall gene downregulation and altered splicing of genes important for HSC regulation. Disclosures No relevant conflicts of interest to declare.

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Sun, Jie, Yinghui Zhu, Zonghui Ding, Haojie Dong, Yimei Feng, Qiang Chen, Xiaochun Yu, et al. "TET2 Activity Is Modulated By SIRT1-Mediated Protein Deacetylation: A Potential Therapeutic Target in Myelodysplastic Syndrome." Blood 128, no. 22 (December 2, 2016): 1053. http://dx.doi.org/10.1182/blood.v128.22.1053.1053.

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Abstract DNA methylation at cytosine position 5 (5mC) is a genetic modification that regulates gene expression and is critical for cancer initiation and development. The methylcytosine dioxygenase TET2 participates in active DNA demethylation by converting 5mC to 5-hydroxymethylcytosine (5hmC) and its subsequent intermediates thereby regulating gene expression. TET2 gene is frequently mutated in hematological disorders, including 15-25% of myelodysplastic syndrome (MDS) cases. Loss of TET2 function leads to DNA hypermethylation and subsequent dysregulated gene expression in hematopoietic stem cells, and has been considered as an initial step of myeloid malignant transformation including MDS and acute myeloid leukemia (AML). While enzymatic activity of TET2 is well studied, little is known about post-translational modifications (PTMs) that regulate its activity in hematopoietic cells. Lysine acetylation, one of the most crucial PTMs, occurs in a variety of proteins and modulates protein-protein or protein-nucleic acids interactions. Here, we first observed that ectopic expression of histone acetyltransferase EP300 or CREB binding protein (CBP) increased the endogenous TET2 acetylation in 293T cells. Mass spectrometry analysis of TET2 identified several conserved acetylated lysine residues located on the exterior surface of its catalytic domain (CD), which is predicted to be essential for the interaction of TET2 with DNA. Modeling of TET2/DNA interaction using x-ray crystal structure (PDB code: 4NM6) indicates acetylated TET2 interferes with DNA binding thereby impairing the catalytic activity of the enzyme. Like other acetylated proteins, TET2 modification is reversible, since ectopic expression of the protein deacetylase Sirtuin1 (SIRT1) significantly decreases acetylation of TET2. A domain mapping analysis revealed that SIRT1 preferentially interacts with TET2 CD, further supporting the regulatory role of SIRT1 on TET2. To explore the role of SIRT1 mediated deacetylaion of TET2 in more physiological condition, SIRT1 knockdown (KD) MDS-L cells as well as SIRT1 knockout (KO) murine hematopoietic c-kit+ cells were used. In MDS-L cells derived from a MDS patient, SIRT1 KD promoted hyperacetylation of endogenous TET2 which is associated with 50% decrease in 5hmC levels compared to control (p=0.03, n=3). Similarly, SIRT1 KO murine c-Kit+ cells showed TET2 hyperacetylation and 62% decrease in 5hmC compared to wildtype (WT) counterpart (p=0.009, n=5). Phenotypically, the transforming ability of SIRT1 shRNA transduced MDS-L cells was evaluated in vitro through serial replating assay. In contrast to first plating which showed no difference between two groups, the number of colonies derived from SIRT1 KD cells was 2.39 fold higher than that from the control shRNA transduced group after two rounds of replating (p=0.0002, n=3). Importantly, we tested the in-vivo effect of SIRT1 KD on MDS-L cells engrafted in immunodeficient NSGS mice. SIRT1 KD significantly enhanced MDS-L cell engraftment compared to control shRNA (50±5% of human CD45+ cells in SIRT1 KD group vs. 19±4% human CD45+ cells in control group, p=0.003, n=6). Interestingly, increased transformation capacity of SIRT1 KD MDS-L cells was associated with decreased expression of TET2 regulated tumor suppressor genes. For example, expression levels of Mtss1 and Dusp6 were significantly decreased in SIRT1 KD MDS-L cells compared to control shRNA counterpart (Mtss1, 49±5% decrease compared to control, p=0.005; Dusp6, 25±2% decrease compared to control, p=0.01). Similar results were observed in SIRT1 KO cells. The validation of physical binding of TET2 to its targets is ongoing. Moreover, the effect of an SIRT1 allosteric activator-SRT1720 in MDS-L cells is similar to that of Vitamin C, a known TET2 activator. Specifically, SRT1720 significantly increased Mtss1 expression in MDSL cells (p=0.0005, n=3), indicating that SIRT1 agonist may lead to activation of TET2 downstream tumor suppressor genes. In summary, our data demonstrate that TET2 activity can be functionally modified by acetylation and may be enhanced in MDS patients that do not harbor loss-of-function mutations through the SIRT1 mediated deacetylation. These results support further exploration of molecular mechanisms inducing TET2 acetylation, and evaluation of SIRT1 activation as a potential therapeutic approach in MDS. Disclosures No relevant conflicts of interest to declare.

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Carraway, Hetty E., Judith E. Karp, Ivana Gojo, Keith W. Pratz, Nilanjan Ghosh, Steven D. Gore, Margaret M. Showel, et al. "A Phase I Study Of The Histone Deacetylase Inhibitor Entinostat Plus Clofarabine For Philadelphia Chromosome Negative, Poor Risk (Newly Diagnosed Older Adults or Adults with Relapsed and Refractory Disease) Acute Lymphoblastic Leukemia Or Bilineage/Biphenotypic Leukemia." Blood 122, no. 21 (November 15, 2013): 1427. http://dx.doi.org/10.1182/blood.v122.21.1427.1427.

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Abstract Introduction Adult patients (pts) diagnosed with acute lymphocytic leukemia (ALL) are known to have a poor clinical outcome as compared to children. Studies report a 2 year event free survival of 30-40% for Philadelphia chromosome negative (Ph-) patients age >30 yrs and 17% for age >50 yrs. In order to improve outcome for adult ALL, agents that are effective, safe and associated with a low morbidity are needed. Clofarabine, a second generation purine nucleoside analog, has clinical activity as a single agent and in combination with cytosine arabinoside (ara-C) against refractory and relapsed ALL. Clofarabine exerts its cytotoxicity through multiple mechanisms of action, with major effects via inhibition of ribonucleotide reductase (RR) and DNA polymerase-alpha, and incorporation into DNA leading to DNA damage and activation of apoptotic pathways. Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing of tumor suppressor genes. HDAC inhibitors are shown to be apoptogenic in vitro for ALL cell lines and have received FDA approval for the treatment of CTCL and peripheral T cell lymphoma. Pre-treatment with entinostat has been shown to enhance the cytotoxic activity of fludarabine in leukemia cell lines in vitro (Maggio et al, Cancer Research 2004). Given the similarity of clofarabine to fludarabine, and its FDA approval for children with refractory ALL, the combination of entinostat with clofarabine was pursued. Methods A Phase I window of opportunity study using overlapping schedule of entinostat and clofarabine was used in adult pts with ALL (B precursor) or Acute Bilineage Leukemia (ABL). Pts were enrolled onto one of two arms; arm “A” received repeated cycles of entinostat-clofarabine every 21 days as long as there was evidence of response (CR, CRi, or PR following cycle 1) and pts on arm “B” received one cycle of entinostat-clofarabine prior to standard multi-agent chemotherapy. Entinostat was administered orally on day 1 and day 8 (with dose escalation from flat dosing of 4mg to 6mg to 8mg from cohort 1 to 3). Clofarabine was administered intravenously at a fixed dose for all dose cohorts at 10mg/m2 for 5 days (day 3-7). Adults >40 yrs with newly diagnosed, Ph- B-lineage ALL or ABL were eligible. Additionally, adults > 21 yrs with relapsed and refractory, Ph- ALL or ABL were eligible. Eligibility criteria included serum creatinine < 2.0 mg/dl, hepatic enzymes < or = 2.5 ULN and bilirubin <2.0 mg/dl. WBC <150,000/mm3 with no evidence for ongoing or impending leukostasis was required. Results 23 pts from 3 institutions were enrolled on this study (18 at JHH, 3 at UMD, 2 at UColorado). 17 pts were treated on arm A and 6 pts on arm B. 6 pts were treated in each dose level with responses as follows: in dose level one (1 CR, 5 NR) and dose level two (1 CR, 1 CRi, 4NR) and dose level three (3 PR and 3 NR). The dose level 3 cohort was expanded with a total of 5 additional pts to date (1 PR, 1 SD, 3NR). Thus, the overall response rate on dose level 3 was 4PR, 1SD, 6NR. The 4 pts with CR/CRi/PR were all de novo treated elderly pts from Arm A. Notably, one pt on arm A has been in remission for over 1.5 yrs. Toxicities to date included expected but manageable grade (G) 3 and 4 cytopenias. There were G3 elevations of ALT (N=2) and AST (2) and bilirubin (1) and one bacteremia (1) and G4 cellulitis (1). Planned correlative studies are ongoing and include evaluation of acetylation of target proteins using multiparameter flow cytometry and western blot as well as methylation evaluation. Conclusions Combination therapy with entinostat-clofarabine is feasible and is well tolerated with minimal toxicity. Promising durable responses were observed in older pts that were not otherwise able to receive multi-agent induction chemotherapy upfront. This is notable given the low dose of clofarabine used in every cohort. Correlative studies evaluating protein hyperacetylation and DNA methylation in serial samples from treated pts are in progress. Disclosures: Off Label Use: This clinical study is a Phase 1 investigation and it discusses non-FDA approved doses of both clofarabine and entinostat for adults with Acute Lymphocytic Leukemia. The reason for this is that this study examines these agents in combination in a Phase 1 fashion and we started with low doses of each agent. Ordentlich:Syndax: Employment. Trepel:Syndax: Research Funding.

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Grayburn, W. S., and E. U. Selker. "A natural case of RIP: degeneration of the DNA sequence in an ancestral tandem duplication." Molecular and Cellular Biology 9, no. 10 (October 1989): 4416–21. http://dx.doi.org/10.1128/mcb.9.10.4416-4421.1989.

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5S rRNA genes of Neurospora crassa are generally dispersed in the genome and are unmethylated. The xi-eta region of Oak Ridge strains represents an informative exception. Most of the cytosines in this region, which consists of a diverged tandem duplication of a 0.8-kilobase-pair segment including a 5S rRNA gene, appear to be methylated (E. U. Selker and J. N. Stevens, Proc. Natl. Acad. Sci. USA 82:8114-8118, 1985). Previous work demonstrated that the xi-eta region functions as a portable signal for de novo DNA methylation (E. U. Selker and J. N. Stevens, Mol. Cell. Biol. 7:1032-1038, 1987; E. U. Selker, B. C. Jensen, and G. A. Richardson, Science 238:48-53, 1987). To identify the structural basis of this property, we have isolated and characterized an unmethylated allele of the xi-eta region from N. crassa Abbott 4. The Abbott 4 allele includes a single 5S rRNA gene, theta, which is different from all previously identified Neurospora 5S rRNA genes. Sequence analysis suggests that the xi-eta region arose from the theta region by duplication of a 794-base-pair segment followed by 267 G.C to A.T mutations in the duplicated DNA. The distribution of these mutations is not random. We propose that the RIP process of N. crassa (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987; E. U. Selker, and P. W. Garrett, Proc. Natl. Acad. Sci. USA 85:6870-6874, 1988; E. B. Cambareri, B. C. Jensen, E. Schabtach, and E. U. Selker, Science 244:1571-1575, 1989) is responsible for the numerous transition mutations and DNA methylation in the xi-eta region. A long homopurine-homopyrimidine stretch immediately following the duplicated segment is 9 base pairs longer in the Oak Ridge allele than in the Abbott 4 allele. Triplex DNA, known to occur in homopurine-homopyrimidine sequences, may have mediated the tandem duplication.

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Grayburn, W. S., and E. U. Selker. "A natural case of RIP: degeneration of the DNA sequence in an ancestral tandem duplication." Molecular and Cellular Biology 9, no. 10 (October 1989): 4416–21. http://dx.doi.org/10.1128/mcb.9.10.4416.

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5S rRNA genes of Neurospora crassa are generally dispersed in the genome and are unmethylated. The xi-eta region of Oak Ridge strains represents an informative exception. Most of the cytosines in this region, which consists of a diverged tandem duplication of a 0.8-kilobase-pair segment including a 5S rRNA gene, appear to be methylated (E. U. Selker and J. N. Stevens, Proc. Natl. Acad. Sci. USA 82:8114-8118, 1985). Previous work demonstrated that the xi-eta region functions as a portable signal for de novo DNA methylation (E. U. Selker and J. N. Stevens, Mol. Cell. Biol. 7:1032-1038, 1987; E. U. Selker, B. C. Jensen, and G. A. Richardson, Science 238:48-53, 1987). To identify the structural basis of this property, we have isolated and characterized an unmethylated allele of the xi-eta region from N. crassa Abbott 4. The Abbott 4 allele includes a single 5S rRNA gene, theta, which is different from all previously identified Neurospora 5S rRNA genes. Sequence analysis suggests that the xi-eta region arose from the theta region by duplication of a 794-base-pair segment followed by 267 G.C to A.T mutations in the duplicated DNA. The distribution of these mutations is not random. We propose that the RIP process of N. crassa (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987; E. U. Selker, and P. W. Garrett, Proc. Natl. Acad. Sci. USA 85:6870-6874, 1988; E. B. Cambareri, B. C. Jensen, E. Schabtach, and E. U. Selker, Science 244:1571-1575, 1989) is responsible for the numerous transition mutations and DNA methylation in the xi-eta region. A long homopurine-homopyrimidine stretch immediately following the duplicated segment is 9 base pairs longer in the Oak Ridge allele than in the Abbott 4 allele. Triplex DNA, known to occur in homopurine-homopyrimidine sequences, may have mediated the tandem duplication.

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Gupta, Mamta, Gaungzhen Hu, Linda Wellik, Steven Offer, Robert B. Diasio, Ahmet Dogan, and Thomas E. Witzig. "Silencing of SHP1 Is Frequent in Phospho-STAT3 Positive Diffuse Large Cell Lymphoma and This Silencing Is Independent of Methylation of Cpg Islands,." Blood 118, no. 21 (November 18, 2011): 3678. http://dx.doi.org/10.1182/blood.v118.21.3678.3678.

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Abstract Abstract 3678 The JAK/STAT pathway is becoming a relevant target for lymphoma therapy and inhibitors of the pathway are now in clinical trial. It would be ideal to identify patients for trial by assessment of pathway activation. We analyzed the primary tumors from 50 patients with diffuse large B cell lymphoma (DLBCL) using antibody against phosphorylated tyrosine signal transducer and activator of transcription 3 (STAT3) revealed that 52% (21/40) of DLBCL tumors express tyrosine phosphorylated STAT3 (pSTAT3). There are several potential mechanisms of activation of the JAK/STAT pathway. Protein tyrosine phosphatases such as SHP1 are key negative regulators of this pathway. SHP-1 is also considered a tumor suppressor gene because of its ability to antagonize the action of tyrosine kinases. Persistent activation of STAT3 seems to play a critical role in the pathogenesis of several malignancies. In an attempt to determine mechanism of STAT3 phosphorylation, we analyzed SHP1 expression and its correlation to STAT3 phosphorylation in DLBCL tumors. We analyzed SHP1 expression by immunohistochemistry (IHC) in a tissue microarray composed of 27 DLBCL tumor samples. SHP1 was found silenced in 33% (7/27) of DLBCL cases. To confirm our IHC data SHP1 mRNA expression was also analyzed in DLBCL patients (n=7) and CD19+ normal B cells (n=3) using reverse transcriptase polymerase chain reaction (RT-PCR). SHP1 expression was found silenced in 57% (4/7) of cases, however normal B cells expressed a significant amount of SHP1 mRNA transcripts. We next sought to determine the mechanism of SHP1 silencing in these patients. DNA methylation is the most common mechanism of gene silencing and gene inactivation in human cancers and methylation of a cluster of CPG sites near the proximal SHP1 promoter 2 has been shown in myeloma and T cell lymphoma cells. We analyzed the frequency of SHP1 DNA methylation in 40 DLBCL patients and in DLBCL cell lines by analyzing the proximal promoter regions for SHP1 with methylation specific (MSP) and unmethylation specific (UMSP) PCR primers. We found SHP1 methylation in 2.5% (1/40) DLBCL tumors and none of the DLBCL cell lines tested (OCILy3, OCILy10, SUDHL2, SUDHL6, OCILy19 and OCILy1) were positive for methylation. Furthermore, bisulphite sequencing of the MSP product confirmed methylated cytosines in the one MSP-positive DLBCL patient sample and in the positive control (methylated DNA). We next confirmed our MSP methylation results in the 40 DLBCL tumors DNA with a high resolution melting (HRM) methylation array for accurate detection of changes in the CpG methylation. The primers were designed to flank CpG islands, not to specifically amplify (converted) methylated or unmethylated DNA (as in MSP PCR). Data were normalized to an unmethylated control. Based on the melting profile, there was no methylation in the controls but detectable methylation (>5%) in the SHP1 promoter region was found in 10% (4/40) of DLBCL samples. The one sample that was positive with MSP PCR was also >50% methylated by HRM. Next we analyzed whether there is any correlation between SHP1 expression and tyrosine phoshorylated STAT3 (pSTAT3). In the 27 cases of DLBCL studied, 16 were pSTAT3 negative and 11 were pSTAT3 positive by IHC. 50% (8/16) of the pSTAT3 negative cases had very high expression of SHP1. Similarly, 45 % (5/11) of pSTAT3 positive cases were SHP1 negative consistent with loss of SHP1 gene expression. These data suggest that loss of SHP1 expression is one of several potential mechanisms that can contribute to increased tyrosine STAT3 activation in DLBCL. In addition, these data suggest that CPG island promoter hypermethylation in the SHP1 promoter is not associated with SHP1 gene silencing in DLBCL tumors. Disclosures: No relevant conflicts of interest to declare.

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Das, Deepika Sharma, Ze Tian, Arghya Ray, Durgadevi Ravillah, Yan Song, Paul G. Richardson, Bryan Oronsky, Jan Scicinski, Dharminder Chauhan, and Kenneth C. Anderson. "Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001." Blood 124, no. 21 (December 6, 2014): 4712. http://dx.doi.org/10.1182/blood.v124.21.4712.4712.

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Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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Jeong, Mira, Deqiang Sun, Min Luo, Aysegul Ergen, Hongcang Gu, Christoph Bock, Alexander Meissner, Gretchen Darlington, Wei Li, and Margaret A. Goodell. "Genome Wide DNA Methylation and Transcriptome Analysis in HSC Aging." Blood 118, no. 21 (November 18, 2011): 2367. http://dx.doi.org/10.1182/blood.v118.21.2367.2367.

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Abstract Abstract 2367 Hematopoietic stem cell (HSC) Aging is a complex process linked to number of changes in gene expression and functional decline of self-renewal and differentiation potential. While epigenetic changes have been implicated in HSC aging, little direct evidence has been generated. DNA methylation is one of the major underlying mechanisms associated with the regulation of gene expression, but changes in DNA methylation patterns with HSC aging have not been characterized. We hypothesize that revealing the genome-wide DNA methylation and transcriptome signatures will lead to a greater understanding of HSC aging. Here, we report the first genome-scale study of epigenomic dynamics during normal mouse HSC aging. We isolated SP-KSL-CD150+ HSC populations from 4, 12, 24 month-old mouse bone marrow and carried out genome-wide reduced representative bisulfite sequencing (RRBS) and identified aging-associated differentially methylated CpGs. Three biological samples were sequenced from each aging group and we obtained 30–40 million high-quality reads with over 30X total coverage on ∼1.1M CpG sites which gives us adequate statistical power to infer methylation ratios. Bisulfite conversion rate of non-CpG cytosines was >99%. We analyzed a variety of genomic features to find that CpG island promoters, gene bodies, 5'UTRs, and 3'UTRs generally were associated with hypermethylation in aging HSCs. Overall, out of 1,777 differentially methylated CpGs, 92.8% showed age-related hypermethylation and 7.2% showed age-related hypomethylation. Gene ontology analyses have revealed that differentially methylated CpGs were significantly enriched near genes associated with alternative splicing, DNA binding, RNA-binding, transcription regulation, Wnt signaling and pathways in cancer. Most interestingly, over 579 splice variants were detected as candidates for age-related hypermethylation (86%) and hypomethylation (14%) including Dnmt3a, Runx1, Pbx1 and Cdkn2a. To quantify differentially expressed RNA-transcripts across the entire transcriptome, we performed RNA-seq and analyzed exon arrays. The Spearman's correlation between two different methods was good (r=0.80). From exon arrays, we identified 586 genes that were down regulated and 363 gene were up regulated with aging (p<0.001). Most interestingly, overall expression of DNA methyl transferases Dnmt1, Dnmt3a, Dnmt3b were down regulated with aging. We also found that Dnmt3a2, the short isoform of Dnmt3a, which lacks the N-terminal region of Dnmt3a and represents the major isoform in ES cells, is more expressed in young HSC. For the RNA-seq analysis, we focused first on annotated transcripts derived from cloned mRNAs and we found 307 genes were down regulated and 1015 gene were up regulated with aging (p<0.05). Secondly, we sought to identify differentially expressed isoforms and also novel transcribed regions (antisense and novel genes). To characterize the genes showing differential regulation, we analyzed their functional associations and observed that the highest scoring annotation cluster was enriched in genes associated with translation, the immune network and hematopoietic cell lineage. We expect that the results of these experiments will reveal the global effect of DNA methylation on transcript stability and the translational state of target genes. Our findings will lend insight into the molecular mechanisms responsible for the pathologic changes associated with aging in HSCs. Disclosures: No relevant conflicts of interest to declare.

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Ng, Kwok Peng, Soledad Negrotto, Zhenbo Hu, Kevin A. Link, Santosh L. Saraf, Anjali Advani, Mikkael A. Sekeres, Jaroslaw P. Maciejewski, James C. Mulloy, and Yogenthiran Saunthararajah. "Non-p53 Dependent, Leukemia Initiating-Cell Selective, Therapy." Blood 114, no. 22 (November 20, 2009): 2077. http://dx.doi.org/10.1182/blood.v114.22.2077.2077.

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Abstract Abstract 2077 Poster Board II-54 Conventional drug therapy for AML is limited by toxic effects on normal hematopoietic stem cells (nHSC), and dependence on p53/apoptosis pathways that are impaired in malignancy. In hematopoiesis, key transcription factors (TF) determine cell-fate. Here, a difference in nHSC versus leukemia initiating-cell (LIC) TF expression is used to overcome the above limitations. The DNA methylating enzyme DNA methyl-transferase 1 (DNMT1) is also a component of multi-protein histone methyl-transferase complexes. Accordingly, shRNA mediated depletion of DNMT1 in hematopoietic cells hypomethylated DNA and decreased global H3K27 and H3K9 trimethylation (histone marks associated with transcription repression) by >70%. These epigenetic modifications were reproduced using a clinically relevant method: the cytosine analogue decitabine, added to normal human CD34+ hematopoietic precursor cells at 0.2–0.5uM 2–3X/week, depleted DNMT1, H3K27 and H3K9 trimethylation by >70% and significantly hypomethylated DNA (Illumina CpG Microarray). These decitabine levels did not cause measurable DNA damage (H2AX phosphorylation and Fast Micromethod) or apoptosis (Annexin staining and caspase 3 activity). Therefore, at low levels, decitabine can produce broad chromatin changes that increase TF access to target genes, without causing measurable DNA damage or apoptosis. The gene-expression/cell-fate consequences of opening chromatin with decitabine likely depend on the pre-existing TF expression pattern. HOXB4 (stem cell TF), CEBPa (lineage-specifying TF), and CEBPe (late differentiation TF) levels were measured by RQ-PCR in CD34+ cells from AML (n=3) versus normal bone marrow (n=3). AML CD34+ expressed >50-fold higher CEBPa, but HOXB4 and CEBPe levels comparable to normal CD34+ cells, a pattern confirmed in microarray gene expression analysis (CD34+ and myeloblasts, AML n=321, normal n=51 (GEO)). Repression of late differentiation TF likely involves chromatin-modification, regardless of underlying cause. Therefore, depleting DNMT1 to open chromatin in AML cells expressing high lineage-specifying TF could resume differentiation and terminate AML self-renewal, while nHSC, with high stem cell TF and little lineage-specifying TF, should continue to self-renew. nHSC and human MLL-AF9 AML cells were treated identically with decitabine for 7 days, then 300,000 each viable MLL-AF9 and nHSC were combined and transplanted into NSG mice (n = 8). Mice that received PBS treated cells died by week 5 (>90% human myeloblasts in bone marrow). Mice that received decitabine treated cells remained healthy until sacrifice for analysis at week 12 (log-rank p = 0.02, no detectable leukemia, >80% normal human hematopoietic cell marrow engraftment). Direct treatment of mice with established MLL-AF9 leukemia with very low dose decitabine 1mg/m2 3X/week extended survival by >20% (log-rank p = 0.04). Decitabine 0.5uM 2X/week induced morphologic differentiation, but not early apoptosis, in primary patient samples (n=15) and leukemia cell-lines (n=4). Cell-cycle exit by differentiation versus apoptosis may utilize different cyclin dependent kinase inhibitors (CDKN). The THP1 AML cell line contains a homozygous frame-shift mutation in TP53 (p.R174fs*3) and no detectable p53 RNA/protein. THP1 cells were treated with equimolar Ara-C or decitabine. Ara-C weakly upregulated CDKN1A (p21) but not CDKN2B (p15), and produced a transient decrease in cell-counts (D3-5) with recovery and growth similar to control by D7. Decitabine strongly upregulated p15, weakly upregulated p21, and produced gradual but complete and durable abrogation of cell growth by D7. A 66y patient with transfusion dependent RCMD with 5q-, 15q- and severe comorbidities was treated with metronomic (instead of cycled) very low dose SQ decitabine (0.2mg/kg [7.5mg/m2] 2X/week) to avoid cytotoxicity and sustain differentiation modification. Platelets increased by week 4, hematologic remission occurred by week 8 and cytogenetic remission by week 14 (without significant side-effects). Rationalizing dose and schedule of decitabine exploits a difference in nHSC and LIC TF expression to selectively terminate LIC self-renewal by a non-p53 dependent differentiation pathway. This approach, distinct from conventional apoptosis-based therapy, could have a very favorable safety profile, with efficacy in MDS/AML with complex cytogenetic abnormalities. Disclosures: Off Label Use: Decitabine, to treat myelodysplastic syndrome using a novel dose and schedule. Advani:Cephalon: Research Funding. Saunthararajah:HemaQuest: Consultancy.

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Wang, Jiajing, Hongmei Zhang, Faisal I. Rezwan, Caroline Relton, S. Hasan Arshad, and John W. Holloway. "Pre-adolescence DNA methylation is associated with BMI status change from pre- to post-adolescence." Clinical Epigenetics 13, no. 1 (March 25, 2021). http://dx.doi.org/10.1186/s13148-021-01042-4.

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Abstract Background Previous studies have shown that DNA methylation (DNAm) is associated with body mass index (BMI). However, it is unknown whether DNAm at pre-adolescence is associated with BMI status transition from pre- to post-adolescence. In the Isle of Wight (IoW) birth cohort, genome-wide DNA methylation in whole blood was measured using Illumina Infinium Human450 and EPIC BeadChip arrays in n = 325 subjects, and pre- to post-adolescence BMI transition was classified into four groups: (1) normal to normal, (2) normal to overweight or obese, (3) overweight or obese to normal, and (4) persistent overweight or obese. We used recursive random forest to screen genome-wide Cytosine-phosphate-Guanine (CpG) sites with DNAm potentially associated with BMI transition for each gender, and the association of BMI status transition with DNAm at an earlier age was assessed via logistic regressions. To evaluate gender specificity, interactions between DNAm and gender were included in the model. Findings in the IoW cohort were further tested in an independent cohort, the Avon Longitudinal Study of Parents and Children (ALSPAC). Results In total, 174 candidate CpGs were selected including CpGs from screening and CpGs previously associated correctionally with BMI in children and adults. Of these 174 CpGs, pre-adolescent DNAm of 38 CpGs in the IoW cohort was associated with BMI status transition, including 30 CpGs showing gender-specific associations. Thirteen CpGs showed consistent associations between the IoW cohort and the ALSPAC cohort (11 of which were gender-specific). Conclusion Pre-adolescence DNAm is associated with the change in BMI status from pre- to post-adolescence and such associations are likely to be gender-specific.

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Vida, Carmen, Matilde Alique, Patricia De Sequera Ortiz, Guillermo Bodega, Carlos Oliva, Julia Carracedo, and Manuel Ramírez Chamond. "P1052EXPANDED HEMODIALYSIS (HDX) DOES NOT AFFECT EPIGENETIC INTERCELLULAR SIGNALS INVOLVED IN INFLAMMATION AND CARDIOVASCULAR DISEASE." Nephrology Dialysis Transplantation 35, Supplement_3 (June 1, 2020). http://dx.doi.org/10.1093/ndt/gfaa142.p1052.

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Abstract Background and Aims Epigenetic signals play a principal role in homeostasis, but also may promote diseases including cardiovascular diseases (CVDs) when are altered. Extracellular vesicles (EVs) or plasma circulating DNA and RNA may have relevant functions in both physiological and pathophysiological contexts related to the epigenetic intercellular communication. Thus, changes in the endothelial or platelet EVs, or the plasma circulating methylated DNA may contribute to the chronic inflammation and the subsequent CVDs in chronic kidney disease patients, particularly when are in hemodialysis (HD). Dialysis membranes do not usually allow the passage of molecules larger than 30-40 kDa. However, the new system of expanded hemodialysis (HDx) with a medium-cut-off membrane (MCO), due to its characteristics, could alter the plasma content of these EVs and DNA methylation, and thereby, promote the development of CVDs. Therefore, our study evaluates whether global plasma DNA methylation and EVs content are modified during an HDx session. Method For this study, we selected 12 dialysis patients: HDx patients (n=6; dialyzed with MCO) and control group (n=6; dialyzed with other HD membranes). Before and after a dialysis session, plasma samples were obtained. EVs were isolated by ultracentrifugation, and the total number of EVs and platelet and endothelial-derived EVs were characterized and quantified by flow cytometry. RNA and DNA extraction and quantification were carried out using different kits and NanoDrop spectrophotometer. DNA methylation was assessed with a 5-methyl cytosine (5-mC) DNA assay kit. Results As shown in the figure, after a dialysis session with the HDx, no significant differences were observed in the total number of EVs, as well in the number of platelet- and endothelial-derived EVs, in comparison to those observed in HDx patients before the dialysis session. By contrast, patients dialyzed with other HD membranes presented differences in the number of total EVs and platelet and endothelial EVs, which decreased significantly (p<0.05) after the dialysis session. Concerning DNA methylation, no statistically significant changes in total DNA 5-mC (%) were observed in both HDx and control patients after the dialysis session. However, a slight tendency to decrease methylated DNA was observed with the HDx compared to other HD membranes (control). Moreover, no significant changes in DNA and RNA % were observed after dialysis session in both HDx and control group. Conclusion To our knowledge, this is the first study to investigate the influence of the HDx technique on the content of plasma cellular EVs and DNA methylation status. HDx does not affect EVs levels, although it shows a tendency to purify plasma methylated DNA. Although this study was not designed to analyze the comparative effectiveness between different membranes, interestingly this effect in epigenetic signals was not observed with other HD membranes, where patients showed a marked reduction of EVs content. The differential activity of HDx about other HD membranes deserves further investigation. Funding (PI17/01029; PI19/00240; ISCIII-FEDER). Santander/UCM PR41/17-20964. Spanish Society of Nephrology 2018. UAH-GP2018-4

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Gondalia, Rahul, Antoine R. Baldassari, Katelyn M. Holliday, Raúl Méndez-Giráldez, Anne E. Justice, James D. Stewart, Duanping Liao, et al. "Abstract P101: Methylome-wide Association Study Provides Evidence of Particulate Matter Air Pollution-associated Dna Methylation at Cardiovascular Disease-related Genes." Circulation 137, suppl_1 (March 20, 2018). http://dx.doi.org/10.1161/circ.137.suppl_1.p101.

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Background: DNA methylation (DNAm), a heritable but dynamic epigenetic modification that can influence gene expression without altering the genome, may underlie the associations between air pollution and cardiovascular disease risk. Therefore, our objective was to evaluate associations between DNAm and ambient concentrations of particulate matter (PM) ≤ 2.5 and ≤ 10 micrometers in diameter (PM 2.5; PM 10 ). Methods: We conducted a methylome-wide association study among twelve cohort- and race/ethnicity- stratified subpopulations from the Women’s Health Initiative and the Atherosclerosis Risk in Communities study (discovery n = 6,720; replication n = 1,936; mean age: 61.3 yrs; 83% female; 46% African American; 9% Hispanic/Latino American). We averaged geocoded address-specific estimates of daily and monthly mean PM concentrations over 2, 7, 28, and 365 days and 1 and 12 months before exams at which we measured leukocyte DNAm in whole blood. In each subpopulation, we estimated PM-DNAm associations at approximately 485,000 Cytosine-phosphate-Guanine (CpG) sites in multi-level, linear mixed-effects models adjusting for sociodemographic, behavioral, and meteorological characteristics; estimated leukocyte proportions; and technical covariates. We combined subpopulation-specific PM-DNAm associations in fixed-effects, inverse variance-weighted meta-analyses of the discovery, replication, and overall populations. Then we conducted in silico characterization of CpG sites at which PM-DNAm associations exceeded methylome-wide significance and were not heterogeneous ( P < 1.0 x 10 -7 ; P Cochran’s Q > 0.10) to assess their putative function and biological plausibility. Results: Discovery analyses identified significant PM 2.5 - and PM 10 -DNAm associations at four CpG sites, but none survived Bonferroni correction. Overall analyses identified significant associations at two CpG sites. On chromosome 20 near MATN4 , 28-day mean PM 10 was associated with increased DNAm at cg19004594 ( P all = 2.8 x 10 -8 ; P Cochran’s Q = 0.61). MATN4 is expressed in heart and lung tissues. It encodes Matrilin 4, a von Willebrand factor A domain-containing protein linked to cardiac remodeling. On chromosome 10 near ARPP21 , 1-month mean PM 10 was inversely associated with DNAm at cg24102420 ( P all = 4.8 x 10 -8 ; P Cochran’s Q = 0.51). ARPP21 is expressed in the brain/spinal cord and neutrophils. It encodes cAMP-regulated phosphoprotein 21, a regulator of calmodulin/calcium signaling. Conclusions: Findings from this methylome-wide association study suggest that ambient PM 10 concentrations affect DNA methylation at regions of the genome potentially related to cardiovascular disease among racially, ethnically and environmentally diverse populations of U.S. men and women. Further investigation is warranted to uncover epigenetic mechanisms of PM-associated cardiovascular traits.

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Liu, Yanmin, Yanhui Wang, Geng Chen, Chunxu Li, Shoulin Jiang, Megha N. Parajulee, and Fajun Chen. "Elevated CO2 alters transgene methylation not only in promoterregion but also in codingregion of Bt rice under different N-fertilizer levels." Scientific Reports 10, no. 1 (October 23, 2020). http://dx.doi.org/10.1038/s41598-020-75121-6.

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Abstract The earth has been undergoing climate change, especially in recent years, driven by increasing concentration of atmospheric carbon dioxide (CO2) and rising earth-surface temperature, which could reduce N allocation to Bt toxin for transgenic Bt crops (Bt crops), but the N fertilization is considered to be an effective method to enhance the C–N balance in Bt crops in the case of elevated CO2 in future. DNA methylation not only in promoterregion but also in codingregion of transgene plays a critical role in transgene expression regulation and silencing of transgenic crops. Recent research has emphasized the risks of increased transgene silencing of Bacillus thuringiensis (Bt) rice under elevated CO2. In this study, the effects of elevated CO2 (vs. ambient CO2) on exogenous Bt toxins and transgene expression in promoterregion and codingregion of Bt rice during tillering stage (cv. HH1 expressing fused Cry1Ab/Cry1Ac) were evaluated under three nitrogen (N) fertilizer rate (1/4, 1 and 2 N levels). The aboveground and belowground biomass, and foliar Bt protein content of Bt rice were all significantly increased with the augmentation of N-fertilizer. And elevated CO2 significantly increased belowground biomass, total soluble protein content, transgene methylation levels in promoterregion (P1), and in total of promoterregion(P1) and codingregion (P2 + P3) (i.e., P1 + P2 + P3) at 1 N level, and it also increased transgene methylation levels in codingregion (P2), and in total of promoterregion and codingregion (P1 + P2 + P3) at 2 N level. In addition, elevated CO2 decreased foliar Bt protein content at 1 N level. The transgene methylation levels in promoterregion and codingregion were negatively correlated with Bt-transgene expression level. The methylation level of cytosines located at CG sites was higher than those at CHG and CHH sites in P1, P2 and P3 fragments regardless of the CO2 or N-fertilizer level. The correlation of transgene mehtylation in promoterregion with transgene expression is even stronger than that in codingregion. These data indicate that N fertilization supply will increase the Bt toxin content in transgenic Bt rice, especially under elevated CO2.

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Ibeagha-Awemu, Eveline M., Nathalie Bissonnette, Suraj Bhattarai, Mengqi Wang, Pier-Luc Dudemaine, Stephanie McKay, and Xin Zhao. "Whole Genome Methylation Analysis Reveals Role of DNA Methylation in Cow’s Ileal and Ileal Lymph Node Responses to Mycobacterium avium subsp. paratuberculosis Infection." Frontiers in Genetics 12 (December 21, 2021). http://dx.doi.org/10.3389/fgene.2021.797490.

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Johne’s Disease (JD), caused by Mycobacterium avium subsp paratuberculosis (MAP), is an incurable disease of ruminants and other animal species and is characterized by an imbalance of gut immunity. The role of MAP infection on the epigenetic modeling of gut immunity during the progression of JD is still unknown. This study investigated the DNA methylation patterns in ileal (IL) and ileal lymph node (ILLN) tissues from cows diagnosed with persistent subclinical MAP infection over a one to 4 years period. DNA samples from IL and ILLN tissues from cows negative (MAPneg) (n = 3) or positive for MAP infection (MAPinf) (n = 4) were subjected to whole genome bisulfite sequencing. A total of 11,263 and 62,459 differentially methylated cytosines (DMCs), and 1259 and 8086 differentially methylated regions (DMRs) (FDR<0.1) were found between MAPinf and MAPneg IL and ILLN tissues, respectively. The DMRs were found on 394 genes (denoted DMR genes) in the IL and on 1305 genes in the ILLN. DMR genes with hypermethylated promoters/5′UTR [3 (IL) and 88 (ILLN)] or hypomethylated promoters/5′UTR [10 (IL) and 25 (ILLN)] and having multiple functions including response to stimulus/immune response (BLK, BTC, CCL21, AVPR1A, CHRNG, GABRA4, TDGF1), cellular processes (H2AC20, TEX101, GLA, NCKAP5L, RBM27, SLC18A1, H2AC20BARHL2, NLGN3, SUV39H1, GABRA4, PPA1, UBE2D2) and metabolic processes (GSTO2, H2AC20, SUV39H1, PPA1, UBE2D2) are potential DNA methylation candidate genes of MAP infection. The ILLN DMR genes were enriched for more biological process (BP) gene ontology (GO) terms (n = 374), most of which were related to cellular processes (27.6%), biological regulation (16.6%), metabolic processes (15.4%) and response to stimulus/immune response (8.2%) compared to 75 BP GO terms (related to cellular processes, metabolic processes and transport, and system development) enriched for IL DMR genes. ILLN DMR genes were enriched for more pathways (n = 47) including 13 disease pathways compared with 36 enriched pathways, including 7 disease/immune pathways for IL DMR genes. In conclusion, the results show tissue specific responses to MAP infection with more epigenetic changes (DMCs and DMRs) in the ILLN than in the IL tissue, suggesting that the ILLN and immune processes were more responsive to regulation by methylation of DNA relative to IL tissue. Our data is the first to demonstrate a potential role for DNA methylation in the pathogenesis of MAP infection in dairy cattle.

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Han, Luhang, Hongmei Zhang, Akhilesh Kaushal, Faisal I. Rezwan, Latha Kadalayil, Wilfried Karmaus, A. John Henderson, et al. "Changes in DNA methylation from pre- to post-adolescence are associated with pubertal exposures." Clinical Epigenetics 11, no. 1 (December 2019). http://dx.doi.org/10.1186/s13148-019-0780-4.

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Abstract Background Adolescence is a period characterized by major biological development, which may be associated with changes in DNA methylation (DNA-M). However, it is unknown to what extent DNA-M varies from pre- to post-adolescence, whether the pattern of changes is different between females and males, and how adolescence-related factors are associated with changes in DNA-M. Methods Genome-scale DNA-M at ages 10 and 18 years in whole blood of 325 subjects (n = 140 females) in the Isle of Wight (IOW) birth cohort was analyzed using Illumina Infinium arrays (450K and EPIC). Linear mixed models were used to examine DNA-M changes between pre- and post-adolescence and whether the changes were gender-specific. Adolescence-related factors and environmental exposure factors were assessed on their association with DNA-M changes. Replication of findings was attempted in the comparable Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. Results In the IOW cohort, after controlling for technical variation and cell compositions at both pre- and post-adolescence, 15,532 cytosine–phosphate–guanine (CpG) sites (of 400,825 CpGs, 3.88%) showed statistically significant DNA-M changes from pre-adolescence to post-adolescence invariant to gender (false discovery rate (FDR) = 0.05). Of these 15,532 CpGs, 10,212 CpGs (66%) were replicated in the ALSPAC cohort. Pathway analysis using Ingenuity Pathway Analysis (IPA) identified significant biological pathways related to growth and development of the reproductive system, emphasizing the importance of this period of transition on epigenetic state of genes. In addition, in IOW, we identified 1179 CpGs with gender-specific DNA-M changes. In the IOW cohort, body mass index (BMI) at age 10 years, age of growth spurt, nonsteroidal drugs use, and current smoking status showed statistically significant associations with DNA-M changes at 15 CpGs on 14 genes such as the AHRR gene. For BMI at age 10 years, the association was gender-specific. Findings on current smoking status were replicated in the ALSPAC cohort. Conclusion Adolescent transition is associated with changes in DNA-M at more than 15K CpGs. Identified pathways emphasize the importance of this period of transition on epigenetic state of genes relevant to cell growth and immune system development.

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Journal articles on the topic 'N-4 Cytosine Methylation'

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