Global DNA Hypomethylation Research Articles

Chronic Dietary Exposure of Roosters to a Glyphosate-Based Herbicide Increases Seminal Plasma Glyphosate and AMPA Concentrations, Alters Sperm Parameters, and Induces Metabolic Disorders in the Progeny.

The effects of chronic dietary Roundup (RU) exposure on rooster sperm parameters, fertility, and offspring are unknown. We investigated the effects of chronic RU dietary exposure (46.8 mg kg−1 day−1 glyphosate) for 5 weeks in 32-week-old roosters (n = 5 RU-exposed and n = 5 control (CT)). Although the concentrations of glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) increased in blood plasma and seminal fluid during exposure, no significant differences in testis weight and sperm concentrations were observed between RU and CT roosters. However, sperm motility was significantly reduced, associated with decreased calcium and ATP concentrations in RU spermatozoa. Plasma testosterone and oestradiol concentrations increased in RU roosters. These negative effects ceased 14 days after RU removal from the diet. Epigenetic analysis showed a global DNA hypomethylation in RU roosters. After artificial insemination of hens (n = 40) with sperm from CT or RU roosters, eggs were collected and artificially incubated. Embryo viability did not differ, but chicks from RU roosters (n = 118) had a higher food consumption, body weight and subcutaneous adipose tissue content. Chronic dietary RU exposure in roosters reduces sperm motility and increases plasma testosterone levels, growth performance, and fattening in offspring.

Low Exposure Extended Dosing Mimicking Clinical Exposures of the Oral Formulation of Azacitidine Results in a Sustained Hypomethylation and Targets Leukemic Stem Cells

Acute Myeloid Leukemia (AML) is characterized by uncontrolled proliferation of incompletely differentiated myeloid stem/ progenitor cells. Despite recent advances in therapy, high rates of clinical relapse, even in patients who achieve complete remission, remains a critically unmet need. One of the strategies to prolong remission post-induction in AML is to employ effective maintenance therapies. Oral Azacitidine (Oral-AZA; CC-486) is the first and only currently approved maintenance therapy in AML. However, the mechanism of action of Oral-AZA and whether it is differentiated from the Injectable-AZA used in AML induction therapy, remains unclear.In this work, we explore the mechanism of Oral-AZA by in vitro modelling of the relative clinical exposures of Oral-AZA vs Injectable-AZA in AML cell line models. Following Vu et al (Nat. Commun. 2020), we used the Injectable-AZA concentration (1 μM aZA) as a single dose (HELD - High Exposure Limited Duration). A fractionated dose of 0.2 μM each day over 5 days (LEED - Low Exposure Extended Duration) was used to model Oral-AZA.Azacitidine incorporates into RNA and DNA of AML cells leading to hypomethylation driven gene expression changes. We found that HELD but not LEED dosing produced an acute anti-proliferative effect in sensitive AML cell lines suggesting potential for a non-hypomethylation mediated/integrated stress response (ISR) driven mechanism. This ISR effect was rescued by co-treatment with ISRIB, an ISR inhibitor. With HELD, we observed robust ATF4 activation as early as 6 hours that was sustained up to 24 hours. In contrast, LEED induced modest and transient ATF4 activation. Thus, an Injectable-AZA-like regimen activated the ISR pathway more robustly than an Oral-AZA-like regimen. Interestingly, decitabine, a DNA-only incorporating cytidine analog did not activate ISR.In AML cell lines, the target of Azacitidine, DNMT1 was depleted (about 90% compared to control) within 24 hours in both HELD and LEED regimens. However, LEED produced a more sustained DNMT1 loss, up to 7 days whereas in HELD, DNMT1 protein levels recovered 96 hours post-dosing. Given the relative level and duration of DNMT1 loss, we hypothesized that LEED would lead to a more durable hypomethylation. We performed whole genome bisulfite sequencing (WGBS) in 3 AML cell lines (OCI-AML2, MV-4-11 and SKM1) at 48- and 96-hours post-initiation of HELD and LEED dosing. Consistent with the DNMT1 depletion kinetics, at 48 hours we observed almost 75% global DNA hypomethylation with both HELD and LEED treatments. At 96 hours, HELD demonstrated a recovery effect and reverted to 50% hypomethylation. In contrast, LEED showed up to 85% hypomethylated sites, demonstrating the durability of the hypomethylation mediated by an Oral-AZA-like (LEED) regimen.Next, we explored the effect of Oral-AZA-like dosing in the differentiation of leukemic stem cells (LSCs) towards a more mature phenotype. In an in vitro LSC model (OCI-AML-20) with flow cytometry analysis, LEED dosing resulted in a greater depletion (2-fold more) of LSCs (CD34+/38- or 38 low) and concomitant enrichment of cells with more differentiated phenotype (CD34+/38+) compared to HELD dosing. To further validate these observations at the transcriptomic level, we performed single cell RNAseq in OCI-AML-20 cells at different timepoints (3, 5 and 7 days). Data were analyzed using a classifier to identify leukemic myeloid cell lineages (Van Galen et al., Cell 2019). Compared to untreated cells, at day 7, both LEED and HELD dosing resulted in an increase of GMP and promonocytes and those differences were more pronounced with Oral-AZA-like (50% GMP and 20% promonocytic) than the Injectable-AZA-like regimen (28% GMP and 12.5% promonocytic).Taken together, our in vitro modeling of the clinically relevant exposures of Oral-AZA vs Injectable-AZA by using HELD and LEED dosing strategies show a shift of the molecular mechanism between the clinical entities. Our work demonstrates that an Injectable-AZA-like regimen mediates cytotoxicity through an early ISR driven effect. Oral-AZA mechanism leads to a more sustained pharmacodynamic DNA hypomethylation effect that in turn could be linked to a differentiation inducing effect on the LSC population. DisclosuresJeyaraju: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Alapa: Bristol Myers Squibb: Current Employment. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Risueño: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Wang: Bristol Myers Squibb: Current Employment. Subramanyam: Bristol Myers Squibb: Current Employment. Sriganesh: Bristol Myers Squibb: Current Employment. Anand: Bristol Myers Squibb: Current Employment. Jain: Bristol Myers Squibb: Current Employment. Reddy: Bristol Myers Squibb: Current Employment. Ghosh: Bristol Myers Squibb: Current Employment. Kyriakopoulos: Bristol Myers Squibb: Current Employment. Lailler: Rancho Biosciences: Current Employment. Hartl: Rancho Biosciences: Current Employment. Lopes de Menezes: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Evaluation of DNA Methylation at Birth in Monozygotic Twin Pairs Discordant for Acute Lymphoblastic Leukemia

Background: Aberrant patterns of DNA methylation constitute a key feature of pediatric acute lymphoblastic leukemia (ALL) at diagnosis, however its role as a predisposing or early contributor to the development of ALL remains unknown. We employed a discordant monozygotic twin model to identify epigenetic variation associated with future development of pediatric ALL through evaluation of DNA methylation at birth.Methods: Twin pairs discordant for the development of pediatric ALL were identified using linked data from the California Cancer Registry and California Birth Statistical Master File spanning from 1989 to 2015. Archived dried neonatal blood spots were obtained from 86 same-gender twin pairs with available materials from the California Biobank Program. Following isolation of genomic DNA from DBS samples, monozygosity was confirmed in 43 of 86 twin pairs through an identity-by-descent analysis from a genome-wide SNP-array. Epigenome-wide DNA methylation assessment of the 43 discordant monozygotic twin pairs was conducted using the Illumina Infinium MethylationEPIC BeadChip kit (Illumina, San Diego, CA, USA). Data preprocessing and quality control measures were conducted in R, using SeSAMe for data normalization. Two twin pairs were omitted due to failure to pass quality control measures. Within-pair analysis was conducted through identification of array probes with absolute differences in methylation beta values greater than 15% between case and control siblings of a twin pair unit. Differentially methylated probes (DMP) were identified using a conditional logistic regression model accounting for array-specific variation, nucleated cell proportions, and appropriate control for the paired nature of the dataset. Differentially methylated regions (DMR) were defined by regional correlation of p-values from the conditional logistic regression model. Gene set enrichment analysis was conducted on significant probes identified through the within pair and regression analysis.Results: The discordant twin cohort (n = 41 pairs) included 24 female and 17 male pairs. Median gestational age was 258 days, ranging from 184 to 306 days. Age of diagnosis in the case twin ranged from <1 to 23 years (median = 5). There was no significant association between birthweight and case status (paired Wilcoxon signed rank test p = 0.22). No significant differences in nucleated cell proportions were identified in deconvolution analysis. Within-pair analysis identified a total of 18,001 probes with absolute methylation variation greater than 15% across the 41 twin pairs, with 3,984 recurrently variable across more than one pair. Gene ontology analysis of these recurrently variable sites revealed an enrichment of immune-related processes in 7 of the top 15 terms with nominal p-value <0.05, though no terms were significant after correction for multiple comparisons. Conditional logistic regression was conducted on 37 twin pairs, with T-cell cases (n=4) omitted to improve data resolution. This resulted in 240 significant DMPs with p-values below an FDR threshold of 0.05. Of these significant probes, 20 associate with genes previously reported to have altered DNA methylation in ALL at diagnosis. Regional analysis identified 10 significant DMRs with adjusted p-values below 0.05, with the top association encompassing a 454bp region on chromosome 6 located near TRIM39-RPP21 (adjusted p-value 2.39e-05). Notably, conditional regression analysis revealed a significant negative bias in coefficients (409,812 of 710,010 probes, binomial exact test p <2.2e-16), indicating a global tendency toward hypomethylation in cases compared to unaffected siblings (Figure 1). The strength of this bias was greater in probes associated with open sea regions compared to those in island regions, as well as promoter-associated probes.Conclusions: This novel analysis of DNA methylation at birth in ALL-discordant monozygotic twins identified sites of differential methylation associated with immune regulation. In addition, these results provide evidence of an association between global DNA hypomethylation and future development of ALL in one member of a genetically identical twin pair. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epigenetic-based targeted therapies for well-differentiated pancreatic neuroendocrine tumors: recent advances and future perspectives

ABSTRACT Introduction Well-differentiated pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of primary tumors of the endocrine pancreas. Dysregulation of chromatin remodeling, gene expression alteration, global DNA hypomethylation of non-coding regions, DNA hypermethylation and silencing of tumor suppressor gene promoters are frequent epigenetic changes in PanNETs. These changes exert a role in neoplastic transformation and progression. As epigenetic mechanisms, converse to genetic mutations, are potentially reversible, they are an interesting and promising therapeutic target for the treatment of PanNETs. Areas covered We reviewed main epigenetic alterations associated with the development, biological and clinical features and progression of PanNETs, as well as emerging therapies targeting epigenetic changes, which may prove effective for the treatment of human PanNETs. Expert opinion Constant advances in the PanNET medical approach, as reported in the clinical and therapeutic recommendations of ESMO, improved the overall survival of patients over the years. However, over 60% of the patients with metastatic disease still have poor prognosis. Epigenetic regulator drugs, currently approved to treat some human malignancies, that showed anti-tumoral activity also on PanNETs, in pre-clinical and clinical studies, could concur to ameliorate the prognosis and OS of advanced and metastatic PanNET, in combination with surgery and currently employed medical therapies.

An Exploratory Pilot Study of Changes in Global DNA Methylation in Patients Undergoing Major Breast Surgery Under Opioid-Based General Anesthesia.

This study aimed to investigate DNA methylation levels in patients undergoing major breast surgery under opioid-based general anesthesia. Blood samples were collected from eleven enrolled patients, before, during and after anesthesia. PBMC were isolated and global DNA methylation levels as well as DNA methyltransferase (DNMT) and cytokine gene expression were assessed. DNA methylation levels significantly declined by 26%, reversing the direction after the end of surgery. Likewise, DNMT1a mRNA expression was significantly reduced at all time points, with lowest level of −68%. DNMT3a and DNMT3b decreased by 65 and 71%, respectively. Inflammatory cytokines IL6 and TNFα mRNA levels showed a trend for increased expression at early time-points to end with a significant decrease at 48 h after surgery. This exploratory study revealed for the first time intraoperative global DNA hypomethylation in patients undergoing major breast surgery under general anesthesia with fentanyl. The alterations of global DNA methylation here observed seem to be in agreement with DNMTs gene expression changes. Furthermore, based on perioperative variations of IL6 and TNFα gene expression, we hypothesize that DNA hypomethylation may occur as a response to surgical stress rather than to opiate exposure.

Quantification of Global DNA Methylation in Canine Melanotic and Amelanotic Oral Mucosal Melanomas and Peripheral Blood Leukocytes From the Same Patients With OMM: First Study.

Oral mucosal melanomas (OMMs) are aggressive and resistant cancers of high importance in veterinary oncology. Amelanotic OMM produces comparatively less melanin and is considered to be more aggressive than melanotic OMM. Global DNA methylation profiles with hypomethylated or hypermethylated patterns have both been associated with aggressive neoplasms; however, global DNA hypomethylation seems to correlate to higher aggressiveness. Accordingly, global DNA methylation in peripheral blood leukocytes has been investigated to understand the role of systemic or environmental factors in cancer development. This study aimed to quantify global DNA methylation in canine melanotic and amelanotic OMM samples and in the peripheral blood leukocytes of the same dogs. Tumor tissue samples were collected from 38 dogs, of which 19 were melanotic and 19 were amelanotic OMM. These were submitted to immunohistochemistry (IHC) with anti-5-methylcytosine (5mC) and anti-Ki67 primary antibodies. Ki67- and 5mC-positive nuclei were manually scored with the help of an image analysis system. Peripheral blood samples were collected from 18 among the 38 OMM-bearing dogs and from 7 additional healthy control dogs. Peripheral blood leukocytes were isolated from the 25 dogs, and DNA was extracted and analyzed by high-performance liquid chromatography (HPLC) for global DNA methylation. The pattern of global DNA methylation in both canine melanotic and amelanotic OMM indicated higher percentages of weakly or negatively stained nuclei in most of the OMM cells, presuming predominant global DNA hypomethylation. In addition, Ki67 counts in amelanotic OMM were significantly higher than those in melanotic OMM (p < 0.001). Global DNA methylation different immunostaining patterns (strong, weak or negative) correlated with Ki67 scores. Global DNA methylation in circulating leukocytes did not differ between the 9 melanotic and 9 amelanotic OMM or between the 18 OMM-bearing dogs and the 7 healthy dogs. This study provides new information on canine melanotic and amelanotic OMM based on global DNA methylation and cell proliferation.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present studyaimedto identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

Effects of low lead exposure on sperm quality and sperm DNA methylation in adult men

InstructionLead (Pb) exposure is a risk factor for male infertility, but the epigenetic changes in sperm DNAattributable to lead exposure is poorly defined.MethodsIn this study, we investigated whether low Pb exposure (< 10 µg/dL) affects the sperm quality. Blood, urine, and semen samples of 297 men of childbearing age were analyzed for all relevant parameters. Based on the blood Pb level (BLL), participants were allocated to RL (0–2.5 µg/dL), RM (2.5–5 µg/dL), and RH (5–10 µg/dL) groups. The 5-methylcytosine and 5-hydroxymethylcytosine patterns in the sperm DNA were identified using methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation sequencing.ResultsThe non-progressive motility (NP) was significantly increased and associated with global hypomethylation of sperm DNA in the RH group compared with the RL group, indicating that aberrant sperm methylation due to low Pb exposure is possibly associated with reduced sperm motility. The hypomethylated promoter regions were primarily enriched in the calcium (Ca) homeostasis pathway. Further, the interaction between Ca and Pb was associated with sperm rapid progressive motility and asthenospermia risk, although no significant methylation abnormality was observed in those with BLL < 5 µg/dL. When BLL was > 5 µg/dL or when predicting NP, no significant Pb–Ca interaction was observed.DiscussionOverall, our results indicate that aberrant DNA methylation of the Ca homeostasis pathway, induced by low Pb exposure, is the potential cause for reduced sperm velocity.

Epigenetic Reprogramming of Tumor-Associated Fibroblasts in Lung Cancer: Therapeutic Opportunities.

Simple SummaryLung cancer is the leading cause of cancer death among both men and women, partly due to limited therapy responses. New avenues of knowledge are indicating that lung cancer cells do not form a tumor in isolation but rather obtain essential support from their surrounding host tissue rich in altered fibroblasts. Notably, there is growing evidence that tumor progression and even the current limited responses to therapies could be prevented by rescuing the normal behavior of fibroblasts, which are critical housekeepers of normal tissue function. For this purpose, it is key to improve our understanding of the molecular mechanisms driving the pathologic alterations of fibroblasts in cancer. This work provides a comprehensive review of the main molecular mechanisms involved in fibroblast transformation based on epigenetic reprogramming, and summarizes emerging therapeutic approaches to prevent or overcome the pathologic effects of tumor-associated fibroblasts. Lung cancer is the leading cause of cancer-related death worldwide. The desmoplastic stroma of lung cancer and other solid tumors is rich in tumor-associated fibroblasts (TAFs) exhibiting an activated/myofibroblast-like phenotype. There is growing awareness that TAFs support key steps of tumor progression and are epigenetically reprogrammed compared to healthy fibroblasts. Although the mechanisms underlying such epigenetic reprogramming are incompletely understood, there is increasing evidence that they involve interactions with either cancer cells, pro-fibrotic cytokines such as TGF-β, the stiffening of the surrounding extracellular matrix, smoking cigarette particles and other environmental cues. These aberrant interactions elicit a global DNA hypomethylation and a selective transcriptional repression through hypermethylation of the TGF-β transcription factor SMAD3 in lung TAFs. Likewise, similar DNA methylation changes have been reported in TAFs from other cancer types, as well as histone core modifications and altered microRNA expression. In this review we summarize the evidence of the epigenetic reprogramming of TAFs, how this reprogramming contributes to the acquisition and maintenance of a tumor-promoting phenotype, and how it provides novel venues for therapeutic intervention, with a special focus on lung TAFs.

The Co-occurrence of Chronic Hepatitis B and Fibrosis Is Associated With a Decrease in Hepatic Global DNA Methylation Levels in Patients With Non-alcoholic Fatty Liver Disease.

Global DNA hypomethylation has been reported in patients with chronic hepatitis B (CHB) and non-alcoholic fatty-liver disease (NAFLD). However, the global DNA methylation profile of patients with concurrent NAFLD and CHB (NAFLD + CHB) is still unclear. We aimed to detect the hepatic global DNA methylation levels of NAFLD + CHB patients and assess the associated risk factors. Liver biopsies were collected from 55 NAFLD patients with or without CHB. The histological characteristics of the biopsy were then assessed. Hepatic global DNA methylation levels were quantified by fluorometric method. The hepatic global DNA methylation levels in NAFLD + CHB group were significantly lower than that in NAFLD group. Participants with fibrosis showed lower levels of hepatic global DNA methylation than those without fibrosis. Participants with both CHB and fibrosis had lower levels of hepatic global DNA methylation than those without either CHB or fibrosis. The co-occurrence of CHB and fibrosis was significantly associated with a reduction in global DNA methylation levels compared to the absence of both CHB and fibrosis. Our study suggests that patients with NAFLD + CHB exhibited lower levels of global DNA methylation than patients who had NAFLD alone. The co-occurrence of CHB and liver fibrosis in NAFLD patients was associated with a decrease in global DNA methylation levels.

Comprehensive analysis of partial methylation domains in colorectal cancer based on single-cell methylation profiles.

Epigenetic aberrations have played a significant role in affecting the pathophysiological state of colorectal cancer, and global DNA hypomethylation mainly occurs in partial methylation domains (PMDs). However, the distribution of PMDs in individual cells and the heterogeneity between cells are still unclear. In this study, the DNA methylation profiles of colorectal cancer detected by WGBS and scBS-seq were used to depict PMDs in individual cells for the first time. We found that more than half of the entire genome is covered by PMDs. Three subclasses of PMDS have distinct characteristics, and Gain-PMDs cover a higher proportion of protein coding genes. Gain-PMDs have extensive epigenetic heterogeneity between different cells of the same tumor, and the DNA methylation in cells is affected by the tumor microenvironment. In addition, abnormally elevated promoter methylation in Gain-PMDs may further promote the growth, proliferation and metastasis of tumor cells through silent transcription. The PMDs detected in this study have the potential as epigenetic biomarkers and provide a new insight for colorectal cancer research based on single-cell methylation data.

Pilot Use of a Novel DNA Methytransferase -1 (DNMT1) Flow Cytometric Pharmacodynamic Assay to Evaluate Response to Low-Dose Azacitidine Therapy for Acute Myeloid Leukemia or Myelodysplastic Syndrome Relapse after Allogeneic Hematopoietic Cell Transplantation

Background Azacitadine (AZA) is a cytidine analogue that incorporates into replicating DNA or RNA and binds to DNA methyltransferase (DNMT1) leading to its degradation and global DNA hypomethylation. The optimal dosing of AZA for DNA hypomethylation without overt cytotoxicity is not well understood. We used a novel flow cytometry-based assay to measure DNMT1 in leukemia cells as a pharmacodynamic marker of intracellular drug targeting in patients receiving low dose AZA for relapsed AML/MDS post hematopoietic cell transplantation (HCT).Disease relapse is the most common cause of treatment failure post-HCT with no established standard of care. Time to relapse is the major factor impacting outcome and survival in those with relapsed disease within 6 months is estimated to be less <5% (Bejanyan N etal BBMT 2015;21(3):454-9).Methods We retrospectively reviewed 29 AML and MDS patients (median age 63.5 years, range 28-74) who received low dose intravenous AZA for treatment of disease relapse at a median of 105 days (range 23-1477) after allogeneic HCT from 2009 to 2016. All were considered ineligible for second transplant due to early relapse or poor functional status. Median AZA dose was 40 mg/m2/day x5 days (range 16-100), determined per clinician preference. We assessed DNMT1 in CD34+/CD117+ blasts in blood or marrow in 8 patients with available samples. We previously reported a novel S-phase specific flow cytometric DNMT1 assay to measure the direct pharmacodynamic effects of AZA (William B etal BBMT, Volume 21, Issue 2, S318 - S319). Cryopreserved mononuclear cells from patient blood or marrow were thawed and fixed using formaldehyde (1%) and methanol (90%). Fixed cells were stained for DNMT1 and cell surface antigens. Cell cycle analysis was performed using DAPI staining. The expression of DNMT1 in leukemia cells in S-phase was measured as mean fluorescent intensity (MFI) normalized to a control.Results Patient characteristics are summarized in Table 1. Disease relapse occurred at a median of 105 days (range 23-1477) after HCT. Median starting dose of AZA was 40mg/m2 for 5 or7 days of every 28 day cycle. Median number of AZA cycles administered was 3 (range 1-19). Ten patients received at least one DLI (median DLI per patient=2, range 1-5). With a median follow up of 6.3 months (range 1.3-41.1 months), 7 patients (27%) responded to AZA with complete remission (CR), hematologic improvement, or improvement in donor chimerism. Additional six (21%) patients exhibited stable disease. One patient achieved a sustained CR >2.5 years despite continued immunosuppression due to ongoing chronic graft versus host disease.Median survival was 6.8 months (95% CI 3.8-11.1). Any response to AZA was associated with a higher survival (7.6 vs 3.2 months, p=0.026) (Figure 1). Survival was not significantly different between the two AZA dose groups (<40 or >40mg/m2/day, OS 7.6 vs 4.9 months, p=0.5).Eight patients (3 MDS, 5 AML) had available samples (marrow or blood) at baseline and after initiation of AZA. Four patients with AML had a subsequent decrease in DNMT1.Two patients with MDS had in increase in DNMT1 after initiation of AZA, however the samples were early in the treatment course (C1D3 and C1D15). Given the heterogeneity in sampled time points in relation to therapy, correlation to clinical response is unable to be determined. Within these limitations, a higher pre-treatment DNMT1 level and notable decrease was observed in the AML patient (Subject 2) who achieved CR from hematological relapse sustained for 3 years. In the remaining 7 patients who did not respond or had a short duration of response, the variation in DNMT1 level pre and post-treatment was minimal.Conclusion Within the limitations of a small retrospective study, our data suggest that low dose AZA is comparable to high dose AZA for treatment of AML /MDS patients who relapse post-HCT. A significant proportion of this poor- risk population, with median time to relapse of 105 days, responded to low-dose AZA. Furthermore, we obtained serial quantification of DNMT1 levels throughout AZA therapy using a novel flow cytometry assay. One patient with sustained response exhibited high baseline DNMT1 level and decrease after therapy. These results suggest that lower doses of AZA may achieve similar therapeutic efficacy with less myelosuppression; however, this hypothesis should be further evaluated in the standard dose setting. [Display omitted] DisclosuresCaimi:Seattle Genetics: Equity Ownership; Incyte: Equity Ownership; Abbvie: Equity Ownership; Celgene: Speakers Bureau. Cooper:Novartis: Research Funding. Little:Hemex Health: Equity Ownership. Malek:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees. De Lima:Celgene Corporation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Tet1 regulates epigenetic remodeling of the pericentromeric heterochromatin and chromocenter organization in DNA hypomethylated cells.

Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.

PCB cause global DNA hypomethylation of human peripheral blood monocytes in vitro

We have recently reported significant associations between exposure to polychlorinated biphenyls (PCB) and alterations on genome-wide methylation of leukocyte DNA of healthy volunteers and provided evidence in support of an etiological link between the observed CpG methylation variations and chronic lymphocytic leukemia. The present study aimed to elucidate the effects of PCB in human lymphocytes’ methylome in vitro. Therefore, U937 cells and human peripheral blood monocytes (PBMC) were exposed in vitro to the dioxin-like PCB-118, the non-dioxin-like PCB-153, and hexachlorobenzene (HCB) and thorough cytotoxicity, genotoxicity and global CpG methylation analyses were performed. All compounds currently tested did not show any consistent significant genotoxicity at all exposure periods and concentrations used. On the contrary, extensive dose-dependent hypomethylation was observed, even at low concentrations, in stimulated PBMC treated with PCB-118 and PCB-153 as well as a small but statistically significant hypomethylation in HCB-treated stimulated cells.

Tannic acid-crosslinked chitosan matrices enhance osteogenic differentiation and modulate epigenetic status of cultured cells over glutaraldehyde crosslinking

ABSTRACT Tannic acid (TA) is a polyphenol with potential to crosslink biomacromolecules. Though, feasibility of TA crosslinking is known, the more toxic glutaraladehyde (GA) continue to be used extensively for developing novel chitosan-based scaffolds, hindering in vivo translation. Thus, a direct comparison between TA- and GA-crosslinked chitosan membranes with respect to physico-chemical, biocomptability and biofunctional properties is required. Role of TA to modulate epigenetic changes in cultured cells is also investigated. TA-crosslinked scaffolds differed from GA in contact angle and swelling by 20–25% while mechanical properties and degradation differed by 50%. Cell viability on TA-crosslinked scaffolds was ~1.4 times higher compared to GA-crosslinked samples, osteocalcin expression was two-fold higher and ROS and cox-2 expression was reduced by ~1.4 times (p < .01). TA crosslinking modulated epigenetic changes in cultured cells by evidencing global DNA hypomethylation. It is concluded that TA can provide a flexible scaffold with ability to modulate epigenetics and osteogeneticity.

Contiguous erosion of the inactive X in human pluripotency concludes with global DNA hypomethylation.

SUMMARYFemale human pluripotent stem cells (hPSCs) routinely undergo inactive X (Xi) erosion. This progressive loss of key repressive features follows the loss of XIST expression, the long non-coding RNA driving X inactivation, and causes reactivation of silenced genes across the eroding X (Xe). To date, the sporadic and progressive nature of erosion has obscured its scale, dynamics, and key transition events. To address this problem, we perform an integrated analysis of DNA methylation (DNAme), chromatin accessibility, and gene expression across hundreds of hPSC samples. Differential DNAme orders female hPSCs across a trajectory from initiation to terminal Xi erosion. Our results identify a cis-regulatory element crucial for XIST expression, trace contiguously growing reactivated domains to a few euchromatic origins, and indicate that the late-stage Xe impairs DNAme genome-wide. Surprisingly, from this altered regulatory landscape emerge select features of naive pluripotency, suggesting that its link to X dosage may be partially conserved in human embryonic development.

STK11/LKB1 Loss of Function Is Associated with Global DNA Hypomethylation and S-Adenosyl-Methionine Depletion in Human Lung Adenocarcinoma.

STK11 (liver kinase B1, LKB1) is the fourth most frequently mutated gene in lung adenocarcinoma, with loss of function observed in up to 30% of all cases. Our previous work identified a 16-gene signature for LKB1 loss of function through mutational and nonmutational mechanisms. In this study, we applied this genetic signature to The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples and discovered a novel association between LKB1 loss and widespread DNA demethylation. LKB1-deficient tumors showed depletion of S-adenosyl-methionine (SAM-e), which is the primary substrate for DNMT1 activity. Lower methylation following LKB1 loss involved repetitive elements (RE) and altered RE transcription, as well as decreased sensitivity to azacytidine. Demethylated CpGs were enriched for FOXA family consensus binding sites, and nuclear expression, localization, and turnover of FOXA was dependent upon LKB1. Overall, these findings demonstrate that a large number of lung adenocarcinomas exhibit global hypomethylation driven by LKB1 loss, which has implications for both epigenetic therapy and immunotherapy in these cancers. SIGNIFICANCE: Lung adenocarcinomas with LKB1 loss demonstrate global genomic hypomethylation associated with depletion of SAM-e, reduced expression of DNMT1, and increased transcription of repetitive elements.

Moderate DNA hypomethylation suppresses intestinal tumorigenesis by promoting caspase-3 expression and apoptosis

Global DNA hypomethylation is a most common epigenetic alteration in human neoplasia. However, accumulative evidence shows that global DNA hypomethylation impacts tumorigenesis in a tissue-specific manner, promoting tumorigenesis in some but suppressing tumorigenesis in others including colorectal cancer. The underlying mechanisms, especially how DNA hypomethylation suppresses tumorigenesis, remain largely unknown. Here, we investigate how DNA hypomethylation affects intestinal tumorigenesis by using an Uhrf1 tandem tudor domain knockin mutant mouse model (Uhrf1ki/ki) that exhibits a moderate ~10% reduction of global DNA methylation. We found that both chemical-induced colorectal carcinogenesis and Apc loss of heterozygosity (LOH)-induced intestinal tumorigenesis are substantially suppressed in the Uhrf1 mutant mice. Furthermore, unlike Dnmt1 hypomorphic mice in which DNA hypomethylation suppresses the incidence of macroscopic intestinal tumors but promotes the formation of microadenoma in ApcMin/+ background, Uhrf1ki/ki/ApcMin/+ mice have markedly reduced incidence of both microadenoma and macroadenoma. DNA hypomethylation does not appear to affect Apc LOH, activation of the Wnt or Hippo pathway, or tumor cell proliferation, but acts cooperatively with activated Wnt pathway to enhance the caspase-3 gene expression, activation, and apoptosis. Furthermore, increased caspase-3 expression correlates with DNA hypomethylation within the caspase-3 enhancer regions. Taken together, we present a new mouse model for investigating the role of and the molecular mechanisms by which DNA hypomethylation suppresses intestinal tumorigenesis. Our finding that a moderate DNA hypomethylation is sufficient to suppress intestinal tumorigenesis by promoting caspase-3 expression and apoptosis sheds new light on DNA-methylation inhibitor-based colorectal cancer therapeutics.

Principles of signaling pathway modulation for enhancing human naive pluripotency induction.

SummaryIsolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Homocysteine-induced neural tube defects in chick embryos via oxidative stress and DNA methylation associated transcriptional down-regulation of miR-124.

Although moderate homocysteine (HCY) elevation is associated with neural tube defects (NTDs), the underlying mechanisms have not been elucidated. In this study, we aimed to investigate that whether HCY-induced NTDs were associated with oxidative stress and methyl metabolism in chick embryos. The potential role of miR-124 in neurogenesis was also investigated. In this study, increased intracellular oxidative species and alterations in DNA methylation were observed following HCY treatment. This alteration coincided with decreases of Mn superoxide dismutase and glutathione peroxidase activities, as well as the expression of anti-rabbit DNA methyltransferase (DNMT) 1 and 3a. In addition, HCY induced significant decreases of S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) (P<0.05). N-acetyl-L-cysteine and choline ameliorated global DNA hypomethylation induced by HCY. MiR-124 levels were significantly suppressed by HCY (P<0.05), while elevated by 5-aza-2'-deoxycytidine (5-aza-dC). MiR-124 knockdown resulted in spina bifida occulta. Our research suggests that HCY-induced NTDs were associated with oxidative stress and methyl metabolism in chick embryos. MiR-124 down-regulation may occur via epigenetic mechanisms and contribute to HCY-induced NTDs in chick embryo models.