Global DNA Methylation Patterns Research Articles

Dynamics of proteins, carbohydrates and global DNA methylation patterns during induction of nodular cluster cultures from seeds of Vriesea reitzii

Tissue culture techniques have been employed for bromeliad mass propagation by means of the morphogenetic route of nodular cluster cultures (NCs). This study aimed to assess protein, carbohydrate and global DNA methylation (GDM) level dynamics during NCs induction from Vriesea reitzii seeds. Seeds were inoculated into Murashige and Skoog (MS) liquid medium supplemented with 4 µM α-naphthaleneacetic acid (NAA) to induce NCs and in culture medium without plant growth regulators to form normal seedlings. Samples collected at 0, 3, 7, 10, 14, and 21 days of culture were analyzed. All parameters assessed showed the same variation pattern. However, seeds inducing NCs showed significantly lower starch (6.0 mg g-1 FM), carbohydrate (10.7 mg g-1 FM) and GDM (11.0%) levels than seeds forming normal seedlings after 21 days in culture. On the other hand, the protein content (9.1 mg g-1 FM) was significantly higher during induction. NCs induction process through seeds is the result of gene reprogramming in the explant, which leads to morphological, biochemical and metabolic alterations. This involves dedifferentiation, high cell proliferation, high energy demand and protein synthesis, which is related to elevated metabolic activity.

Stearoyl‐CoA desaturase 1 determines pancreatic β‐cell fate through regulation of DNA methylation pattern

Study objectiveStearoyl CoA desaturase 1 (SCD1) is a pivotal enzyme involved in saturated fatty acids metabolism that exhibits a protective role against lipotoxicity in pancreatic β‐cells. It has been shown that SCD1 deficiency contributes to impaired insulin secretion, β‐cell failure and development of type 2 diabetes (T2D). In the present study we investigated the involvement of SCD1 in regulation of DNA methylation and expression of transcription factors (TFs) responsible for maintenance of mature β‐cells function and identity.MethodsThe experiments were carried out ex vivo on pancreatic islets isolated from wild type (WT) and SCD1 knock‐out (SCD1 KO) mice, and in vitro on INS‐1E pancreatic β‐cell line, where lipotoxicity was induced by palmitic acid treatment. Morphological and functional characteristics of islets was evaluated by ELISA assays, immunofluorescence labeling and transmission electron microscopy techniques. DNA methylation and gene expression levels were assessed by bisulfite sequencing and real‐time quantitative PCR analyses, respectively.ResultsOur data demonstrated that pancreatic islets of SCD1 KO mice are characterized by different microarchitecture, secretory granules content and functional abnormalities manifested by diminished insulin secretion and greater proinsulin secretion in comparison to WT islets. On both ex vivo and in vitro models we observed significantly reduced expression of TFs crucial to maintenance of β‐cell identity such as Pdx1, MafA, Nkx6.1 and Neurod1. Interestingly, transcriptional repression of Pdx1 and MafA was related to excessive methylation of promoter regions of those TFs. In addition, we showed that downregulation of SCD1 activity/gene expression affects global DNA methylation pattern and changes methyl groups distribution within chromosomes in pancreatic β‐cells. DNA hypomethylation which occurred in SCD1‐deficient β‐cells was followed by decreased expression of DNA methyltransferase 1 (Dnmt1). We indicated that activation of AMP‐activated protein kinase (AMPK) axis influences post‐translational regulation of Dnmt1, thereby mediating the effect of SCD1 inhibition on global DNA methylation profile in β‐cells.ConclusionsTogether, our findings suggest that SCD1 is a gatekeeper that preserves epigenetic inheritance of DNA methylation pattern in β‐cells. Thus, SCD1 protects β‐cells from lipid‐derived loss of identity in methylation‐dependent manner through regulation of expression of TFs responsible for maintenance of β‐cell identity. Our results provide additional mechanistic insights toward understanding the role of SCD1 in T2D pathogenesis.Support or Funding InformationThis work was supported by grants from the National Science Center, Poland, grants no UMO‐2017/27/N/NZ3/01987 (AMD) and UMO‐2015/19/D/NZ4/03705 (JJ), and by The National Centre for Research and Development, grant no STRATEGMED 3/305813/2/NCBR/2017 (AD).

Global RNA Expression and DNA Methylation Patterns in Primary Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is one of the most malignant tumors, with a median survival of only a few months. The tumorigenic processes of this disease have not yet been completely unraveled. Here, we report an mRNA expression and DNA methylation analysis of fourteen primary ATCs. ATCs clustered separately from normal thyroid tissue in unsupervised analyses, both by RNA expression and by DNA methylation. In expression analysis, enrichment of cell-cycle-related genes as well as downregulation of genes related to thyroid function were seen. Furthermore, ATC displayed a global hypomethylation of the genome but with hypermethylation of CpG islands. Notably, several cancer-related genes displayed a correlation between RNA expression and DNA methylation status, including MTOR, NOTCH1, and MAGI1. Furthermore, TSHR and SLC26A7, encoding the thyroid-stimulating hormone receptor and an iodine receptor highly expressed in normal thyroid, respectively, displayed low expression as well as aberrant gene body DNA methylation. This study is the largest investigation of global DNA methylation in ATC to date. It shows that aberrant DNA methylation is common in ATC and likely contributes to tumorigenesis in this disease. Future explorations of novel treatments should take this into consideration.

Shading Effects on Leaf Color Conversion and Biosynthesis of the Major Secondary Metabolites in the Albino Tea Cultivar "Yujinxiang".

Albino became a novel kind of tea cultivar in China recently. In this study, transcriptome and whole-genome bisulfite sequencing (WGBS) were employed to investigate the shading effects on leaf color conversion and biosynthesis of three major secondary metabolites in the albino tea cultivar "Yujinxiang". The increased leaf chlorophyll level was likely the major cause for shaded leaf greening from young pale or yellow leaf. In comparison with the control, the total catechin level of the shading group was significantly decreased and the abundance of caffeine was markedly increased, while the theanine level was nearly not influenced. Meanwhile, differentially expressed genes (DEGs) enriched in some biological processes and pathways were identified by transcriptome analysis. Furthermore, whole-genome DNA methylation analysis revealed that the global genomic DNA methylation patterns of the shading period were remarkably altered in comparison with the control. In addition, differentially methylated regions (DMRs) and the DMR-related DEG analysis indicated that the DMR-related DEGs were the critical participants in biosynthesis of the major secondary metabolites. These findings suggest that DNA methylation is probably responsible for changes in the contents of the major secondary metabolites in Yujinxiang.

Dynamics of proteins, carbohydrates and global DNA methylation patterns during induction of nodular cluster cultures from seeds of Vriesea reitzii

ABSTRACT. Tissue culture techniques have been employed for bromeliad mass propagation by means of the morphogenetic route of nodular cluster cultures (NCs). This study aimed to assess protein, carbohydrate and global DNA methylation (GDM) level dynamics during NCs induction from Vriesea reitzii seeds. Seeds were inoculated into Murashige and Skoog (MS) liquid medium supplemented with 4 µM α-naphthaleneacetic acid (NAA) to induce NCs and in culture medium without plant growth regulators to form normal seedlings. Samples collected at 0, 3, 7, 10, 14, and 21 days of culture were analyzed. All parameters assessed showed the same variation pattern. However, seeds inducing NCs showed significantly lower starch (6.0 mg g-1 FM), carbohydrate (10.7 mg g-1 FM) and GDM (11.0%) levels than seeds forming normal seedlings after 21 days in culture. On the other hand, the protein content (9.1 mg g-1 FM) was significantly higher during induction. NCs induction process through seeds is the result of gene reprogramming in the explant, which leads to morphological, biochemical and metabolic alterations. This involves dedifferentiation, high cell proliferation, high energy demand and protein synthesis, which is related to elevated metabolic activity.

Influence of quercetin on the global DNA methylation pattern in pigs.

The plant flavonol quercetin causes multiple health-promoting effects in human and animals. In this study, we investigated the possible effects of quercetin on global DNA methylation in growing pigs after 7 weeks of feeding a quercetin-enriched diet. The results indicated that a trend for an improved feed conversion was observed in the quercetin fed group compared to the control group. Furthermore, quercetin influenced global DNA methylation by upregulating DNA methyltransferase 1 (DNMT1) in both mRNA and protein expressions in a tissue specific manner. The observed bioactivity of quercetin against the global methylation marker in pigs indicates that quercetin could be a potential inducer of DNA methylation which might be of economic significance for effective nutritional interventions affecting the health and productivity of animals.

Temporal DNA methylation pattern and targeted therapy in colitis-associated cancer.

DNA methylation plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). However, the global and temporal DNA methylation pattern during initiation and progression of colitis-associated cancer (CAC) are still unknown, including the potential therapeutic strategy of targeting methylation for CAC. In the present study, the global DNA methylation pattern was determined at different time points during CAC using DNA methylation sequencing, followed by the Starburst plot integrating alterations and potential functional prediction analysis. After demonstrating the regulatory role of DNA methyltransferases (DNMTs) on the expression of hub-genes in CRC cells, DNMT inhibitors were administered to treat CAC mice. Our results indicated that 811 genes were hypermethylated at different time points during initiation and progression of CAC. Genes that were downregulated and hypermethylated during CAC, including hub-genes BAD and inositol polyphosphate phosphatase-like 1 (INPPL1), were involved in MAPK signaling pathways, kit receptor signaling pathways, apoptosis and EGF/EGFR signaling pathways. Upregulated DNMTs (DNMT1, DNMT3A and DNMT3B) mediated downregulation and hypermethylation of BAD and INPPL1 in CAC and CRC cells. Low doses of DNMT inhibitors (decitabine (DAC) and azacitidine (AZA)) exerted efficient antitumor effects in CAC, accompanied with upregulation of BAD and INPPL1 expression, and apoptosis induction. In summary, the present study demonstrates the temporal DNA methylation pattern during CAC and provides a novel therapeutic strategy for treating this disease.

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

PATH-16. HISTOPATHOLOGICAL EPENDYMOMA VARIANTS ARE ASSOCIATED WITH DISTINCT CLINICAL PARAMETERS AND DNA METHYLATION PATTERNS

Abstract According to the current WHO classification, ependymal tumors are classified as subependymomas, myxopapillary ependymomas, classic ependymomas, anaplastic ependymomas and RELA-fusion positive ependymoma (RELA-EPN). Among classic ependymomas, the WHO defines rare histological variants, i.e. the clear-cell, papillary, and tanycytic ependymoma. In parallel to this WHO classification scheme, DNA methylation patterns can distinguish nine distinct molecular ependymoma subgroups, some of which tightly overlap with certain histopathological subgroups, e.g. subependyomas or myxopapillary ependymomas. Since very little is known about the molecular background of histological classic ependymoma variants, we analyzed histomorphology, clinical parameters and global DNA methylation patterns of diagnosed tanycytic ependymomas (n=12), clear-cell ependymomas (n=14) and papillary ependymomas (n=19). Surprisingly, up to 42% of these variants did not match to ependymomas using a previously published DNA methylation-based classifier for brain tumors. Among the tumors with a match to one of the nine known ependymoma methylation classes, tanycytic ependymomas were predominantly located in the spine, but showed diverse molecular methylation patterns. Most clear-cell ependymomas showed a common histomorphology, were found supratentorially and fell into the methylation class of RELA-EPN. Papillary ependymomas showed a “papillary”, “trabecular” or “pseudo-papillary” growth pattern. Interestingly, a true papillary growth pattern was strongly associated with the molecular class B of posterior fossa ependymoma (PFB), but tumors displayed DNA methylation sites that were significantly different when compared to PFB ependymomas without papillary growth. Our results show that the diagnosis of classic histological ependymoma variants can be challenging. While clear-cell and papillary ependymomas harbor common molecular features, tanycytic ependymoma may not represent a molecularly distinct subgroup.

Molecular characterization of histopathological ependymoma variants.

According to the WHO classification, ependymal tumors are classified as subependymomas, myxopapillary ependymomas, classic ependymomas, anaplastic ependymomas, and RELA-fusion-positive ependymomas (RELA-EPN). Among classic ependymomas, the WHO defines rare histological variants, i.e., the clear cell, papillary, and tanycytic ependymoma. In parallel, global DNA methylation patterns distinguish nine molecular groups, some of which tightly overlap with histopathological subgroups. However, the match of the aforementioned histological variants to DNA methylation classes remains unclear. We analyzed histomorphology, clinical parameters, and global DNA methylation of tumors with the initial histological diagnoses of tanycytic (n = 12), clear cell (n = 14), or papillary ependymoma (n = 19). Forty percent of these tumors did not match to the epigenetic profile of ependymomas, using a previously published DNA methylation-based classifier for brain tumors. Instead, they were classified as low-grade glioma (n = 3), plexus tumor (n = 2), CNS high-grade neuroepithelial tumor with MN1 alteration (n = 2), papillary tumor of the pineal region (n = 2), neurocytoma (n = 1), or did not match to any known brain tumor methylation class (n = 8). Overall, integrated diagnosis had to be changed in 35.6% of cases as compared to the initial diagnosis. Among the tumors molecularly classified as ependymoma (27/45 cases), tanycytic ependymomas were mostly located in the spine (5/7 cases) and matched to spinal or myxopapillary ependymoma. 6/8 clear cell ependymomas were found supratentorially and fell into the methylation class of RELA-EPN. Papillary ependymomas with a positive ependymoma match (12/19 cases) showed either a "papillary" (n = 5), a "trabecular" (n = 1), or a "pseudo-papillary" (n = 6) growth pattern. The papillary growth pattern was strongly associated with the methylation class B of posterior fossa ependymoma (PFB, 5/5 cases) and tumors displayed DNA methylation sites that were significantly different when compared to PFB ependymomas without papillary growth. Tumors with pseudo-papillary histology matched to the methylation class of myxopapillary ependymoma (4/6 cases), whereas the trabecular case was anatomically and molecularly a spinal ependymoma. Our results show that the diagnosis of histological ependymoma variants is challenging and epigenetic profiles may improve diagnostic accuracy of these cases. Whereas clear cell and papillary ependymomas display correlations between localization, histology, and methylation, tanycytic ependymoma does not represent a molecularly distinct subgroup.

Epigenetic patterns associated with an ascidian invasion: a comparison of closely related clades in their native and introduced ranges

Environmentally induced epigenetic modifications have been proposed as one mechanism underlying rapid adaptive evolution of invasive species. Didemnum vexillum is an invasive colonial ascidian that has established in many coastal waters worldwide. Phylogenetic analyses have revealed that D. vexillum populations consist of two distinct clades; clade B appears to be restricted to the native range (Japan), whereas clade A is found in many regions throughout the world, including New Zealand. The spread of D. vexillum clade A suggests that it might be intrinsically more invasive than clade B, despite low levels of genetic diversity compared to populations from the native region. This study investigated whether D. vexillum clade A exhibits epigenetic signatures (specifically differences in DNA methylation) associated with invasiveness. Global DNA methylation patterns were significantly different between introduced clade A colonies, and both clades A and B in the native range. Introduced colonies also showed a significant reduction in DNA methylation levels, which could be a mechanism for increasing phenotypic plasticity. High levels of DNA methylation diversity were maintained in the introduced population, despite reduced levels of genetic diversity, which may allow invasive populations to respond quickly to changes in new environments. Epigenetic changes induced during the invasion process could provide a means for rapid adaptation despite low levels of genetic variation in introduced populations.

Developmental subtypes assessed by DNA methylation-iPLEX forecast the natural history of chronic lymphocytic leukemia

AbstractAlterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

MiR-424 prevents astrogliosis after cerebral ischemia/reperfusion in elderly mice by enhancing repressive H3K27me3 via NFIA/DNMT1 signaling.

Global DNA and histone methylation patterns in astrocytes following ischemia are influenced by age; however, it is unknown whether aberrant methylation can induce reactive astrogliosis after ischemic stroke in elderly rodents. Here we showed that phosphorylated signal transducer and activator of transcription 3 (STAT3) level increased along with that of the astrogliosis marker glial fibrillary acidic protein (GFAP) on days 1, 3, and 14 post-reperfusion in 9-month-old male mice with middle cerebral artery occlusion (MCAO). Methylation of the STAT3 binding site in the GFAP gene promoter was increased in these mice on days 3 and 14 postreperfusion. The repressive modification histone 3 lysine 27 trimethylation (H3K27me3) was decreased, whereas the permissive modification histone 3 lysine 4 trimethylation was increased in GFAP-positive cells in the ipsilateral cortex. Furthermore, DNA methyltransferase 1 (DNMT1) expression in astrocytes was upregulated in the ischemic brain. In primary astrocyte cultures, the microRNA miR-424 was found to target nuclear factor IA (NFIA); miR-424 agomir increased DNMT1 and H3K27me3 levels in U87 cells subjected to oxygen and glucose deprivation and induced cell cycle arrest in primary astrocytes while suppressing reactive astrocytosis, thereby preserving the structure of neurons and their axons in MCAO mice. These results demonstrate that miR-424 prevents astrogliosis following cerebral ischemia/reperfusion in elderly mice by enhancing H3K27me3 via NFIA/DNMT1 signaling.

Ovine oocytes display a similar germinal vesicle configuration and global DNA methylation at prepubertal and adult ages

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p<0.0001). In conclusion, our results indicate that adult-size oocytes derived from 3 to 4 month old prepubertal ewes show similar GV morphology and DNA methylation staining patterns to those obtained from adult animals, despite exhibiting a lower developmental competence.

Global DNA Methylation Patterns in Human Gliomas and Their Interplay with Other Epigenetic Modifications.

During the last two decades, several international consortia have been established to unveil the molecular background of human cancers including gliomas. As a result, a huge outbreak of new genetic and epigenetic data appeared. It was not only shown that gliomas share some specific DNA sequence aberrations, but they also present common alterations of chromatin. Many researchers have reported specific epigenetic features, such as DNA methylation and histone modifications being involved in tumor pathobiology. Unlike mutations in DNA, epigenetic changes are more global in nature. Moreover, many studies have shown an interplay between different types of epigenetic changes. Alterations in DNA methylation in gliomas are one of the best described epigenetic changes underlying human pathology. In the following work, we present the state of knowledge about global DNA methylation patterns in gliomas and their interplay with histone modifications that may affect transcription factor binding, global gene expression and chromatin conformation. Apart from summarizing the impact of global DNA methylation on glioma pathobiology, we provide an extract of key mechanisms of DNA methylation machinery.

Global DNA Methylation Pattern of Fibroblasts in Idiopathic Pulmonary Fibrosis.

Our previous transcriptome study of cultured fibroblasts identified 178 genes that were differentially expressed by 8 idiopathic pulmonary fibrosis (IPF) fibroblasts compared with 4 controls. Here, we performed genome-wide DNA methylation analysis to evaluate the relationship of CpG methylation to differential gene expression. Among 485,577 loci, 5850 loci on 2282 genes showed significant differences between the 2 groups (delta-beta >10.21 and p-value <0.05). Among these, beta values of 80 CpGs (30 hypermethylated and 50 hypomethylated) were significantly correlated with mRNA expression of 34 genes (19.1%) of the 178 differentially expressed genes between the 2 groups (13 downregulated and 21 upregulated). Gene ontology enrichment of these genes included cell adhesion, molecule binding, chemical homeostasis, surfactant homeostasis, and receptor binding. One-third of them are involved in the known process of fibrosis; the others are novel genes with respect to pulmonary fibrosis. We identified relationships between the altered DNA methylation levels and about one-fifth of the corresponding changes in gene expression by lung tissue fibroblasts. Findings from this study provide new information on novel genes responsible for the pathogenesis of IPF under the control of CpG methylation changes in IPF lungs.

Gut Microbiota Composition Is Associated With the Global DNA Methylation Pattern in Obesity.

Objective: Obesity and obesity-related metabolic diseases are characterized by gut microbiota and epigenetic alterations. Recent insight has suggested the existence of a crosstalk between the gut microbiome and the epigenome. However, the possible link between alterations in gut microbiome composition and epigenetic marks in obesity has been not explored yet. The aim of this work is to establish a link between the gut microbiota and the global DNA methylation profile in a group of obese subjects and to report potential candidate genes that could be epigenetically regulated by gut microbiota in adipose tissue. Methods: Gut microbiota composition was analyzed in DNA stool samples from 45 obese subjects by 16S ribosomal RNA (rRNA) gene sequencing. Twenty patients were selected based on their Bacteroidetes-to-Firmicutes ratio (BFR): HighBFR group (BFR > 2.5, n = 10) and LowBFR group (BFR < 1.2, n = 10). Genome-wide analysis of DNA methylation pattern in both whole blood and visceral adipose tissue of these selected patients was performed with an Infinium EPIC BeadChip array-based platform. Gene expression analysis of candidate genes was done in adipose tissue by real-time quantitative PCR. Results: Genome-wide analysis of DNA methylation revealed a completely different DNA methylome pattern in both blood and adipose tissue in the low BFR group vs. the high BFR group. Two hundred fifty-eight genes were differentially methylated in both blood and adipose tissue, of which several potential candidates were selected for gene expression analysis. We found that in adipose tissue, both HDAC7 and IGF2BP2 were hypomethylated and overexpressed in the low BFR group compared with the high BFR group. β values of both genes significantly correlated with the BFR ratio and the relative abundance of Bacteroidetes and/or Firmicutes. Conclusions: In this study, we demonstrate that the DNA methylation status is associated with gut microbiota composition in obese subjects and that the expression levels of candidate genes implicated in glucose and energy homeostasis (e.g., HDAC7 and IGF2BP2) could be epigenetically regulated by gut bacterial populations in adipose tissue.

Abstract 833: Integrative analysis of TCGA pancreatic ductal adenocarcinoma data

Abstract Pancreatic cancer is a heterogeneous type of cancer that exhibits different histopathological, molecular and genomic phenomena. Pancreatic ductal adenocarcinoma (PDAC) is the deadliest type of pancreatic cancer with a five-year survival of only 8%. Prognostic biomarkers for early detection of PDAC are not available. In this study, we used clinical, DNA methylation, and gene expression profiles of 146 TCGA PDAC patients. We analyzed the global patterns of DNA methylation and correlated the promoter DNA methylation with corresponding gene expression. CpG sites with a difference of 0.2 in the mean β value of the primary tumor and normal samples were considered as differentially methylated. We observed differential methylation of about 12-thousand CpG sites between tumor and normal samples, the majority of which are hypermethylated. Chromosome 1, 20, 18 and 19 are predominately hypermethylated and chromosome 9 is hypomethylated. CpG islands and regions that are in close proximity to CpG islands (shores) have more hypermethylated sites compared to the far away regions (shelf). Pathway enrichment analysis of differentially methylated genes enabled us to understand how changes in methylation affect biological pathways involved in the progression of PDAC. Our analysis reveals enrichment of MAPK signaling, Rap1 signaling, calcium signaling pathway, etc. Promoter DNA methylation is highly associated with its corresponding gene expression. A significant correlation between methylated CpG sites within 1.5kb from TSS and corresponding gene expression was observed between 4,971 CpGs and 1,744 corresponding genes, out of these about 92% of the genes showed a negative correlation. Survival analysis showed a statistically significant correlation between the expression levels of genes, NEK2, and ASPM with overall survival of the patients. In the future, we will further analyze the long non-coding RNA (lncRNA) promoter methylation, and role of lncRNA and miRNA in PDAC patients’ survival. Citation Format: Nitish K. Mishra, Siddesh Southekal, Chittibabu Guda. Integrative analysis of TCGA pancreatic ductal adenocarcinoma data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 833.

Abstract 3646: (Epi-)genomic homogeneity in radiation-induced glioblastoma with recurrent PDGFRA amplification and loss of CDKN2A/B following primary acute lymphatic leukemia and medulloblastoma

Abstract As an essential pillar of today’s cancer treatment, radiation therapy has led to improved survival rates of patients with childhood malignancies including leukemia and central nervous system (CNS) tumors. However, long-term complications such as radiation-induced malignancies occur in a subset of patients following radiation therapy, especially observed in pediatric patients due to their long follow-up period in case of survival. Radiation-induced glioblastomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). Histopathologically, most RIGs are best described as high-grade gliomas resembling de novo glioblastoma, and histopathological features to distinguish RIGs from their sporadic counterparts are lacking. Recent large-scale genomic and epigenomic analyses have revealed key genetic alterations in various types of CNS tumors. Here, we performed comprehensive (epi-)genomic and gene expression profiling of RIGs following radiation therapy for primary MB (n=24) and ALL (n=8). DNA methylation profiling demonstrates a high similarity of global DNA methylation patterns among RIGs, regardless of the primary malignancy. Known genetic alterations in high-grade gliomas such as PDGFRA amplification (53%, 17/32) and loss of CDKN2A/B (63%, 20/32) occur in RIGs. None of the RIGs harbored somatic hotspot mutations in genes encoding histone variants H3.3 and H3.1/H3.2 or IDH1/2, which are frequently observed in high-grade glioma subtypes of children and young adults. Germline alterations causing cancer predisposition syndromes were not found more frequently in RIG patients than in patients suffering from high-grade gliomas without previous irradiation treatment. The genetic homogeneity of RIGs with the absence of histone 3 and IDH1/2 mutations suggests that RIGs share a common cell of origin, which might be particularly vulnerable to radiation. We performed in vitro drug screens on patient-derived RIG tumor spheres, exhibiting amplified and overexpressed PDGFRA, alongside with non-PDGFRA-amplified GBM cell lines to assess the potential efficacy of RTK inhibitors. The median latency time between cranial irradiation and RIG occurrence was 4.5 years (range: 2.5-15). Patients treated for ALL were diagnosed with RIG at age 9-14 years, exposing a particularly vulnerable but narrow time frame for RIG occurrence in ALL patients, in contrast to RIG patients treated for MB (range: 3-39 years). In summary, our study uncovers diagnostic biomarkers and potential targetable alterations in RIG, which could become relevant for the stratification into future clinical trials with e.g. specific RTK inhibitors, with the objective of improving the outcome of survivors of childhood cancer. Citation Format: Maximilian Y. Deng, Dominik Sturm, Elke Pfaff, Gnana P. Balasubramanian, Jens Schittenhelm, Martin Ebinger, Martin U. Schuhmann, Andrey Korshunov, Andreas von Deimling, Olaf Witt, Stefan M. Pfister, David T. Jones. (Epi-)genomic homogeneity in radiation-induced glioblastoma with recurrent PDGFRA amplification and loss of CDKN2A/B following primary acute lymphatic leukemia and medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3646.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the differentiation of embryonic stem cells towards pancreatic lineage and pancreatic beta cell function

Animal and epidemiological studies demonstrated association of persistent exposure of TCDD, an endocrine disrupting chemical, to susceptibility of type 2 diabetes (T2D). High doses of TCDD were commonly employed in experimental animals to illustrate its diabetogenic effects. Data linking the epigenetic effects of low doses of TCDD on embryonic cells to T2D susceptibility risks is very limited. To address whether low dose exposure to TCDD would affect pancreatic development, hESCs pretreated with TCDD at concentrations similar to human exposure were differentiated towards pancreatic lineage cells, and their global DNA methylation patterns were determined. Our results showed that TCDD-treated hESCs had impaired pancreatic lineage differentiation potentials and altered global DNA methylation patterns. Four of the hypermethylated genes (PRKAG1, CAPN10, HNF-1B and MAFA) were validated by DNA bisulfite sequencing. PRKAG1, a regulator in the AMPK signaling pathway critical for insulin secretion, was selected for further functional study in the rat insulinoma cell line, INS-1E cells. TCDD treatment induced PRKAG1 hypermethylation in hESCs, and the hypermethylation was maintained after pancreatic progenitor cells differentiation. Transient Prkag1 knockdown in the INS-1E cells elevated glucose stimulated insulin secretions (GSIS), possibly through mTOR signaling pathway. The current study suggested that early embryonic exposure to TCDD might alter pancreatogenesis, increasing the risk of T2D.