Hypermethylation Patterns Research Articles

EPCO-21. STING PROMOTER EPIGENETIC SILENCING IN GLIOBLASTOMA

Abstract The Stimulator of interferon genes (STING) protein is a critical component of the innate immune response to pathogenic and cytosolic self DNA. The importance of STING signalling has recently been demonstrated in a number of tumor types, including glioblastoma (GBM), and its activation through STING agonism is a promising antitumor therapeutic avenue. Recent studies have shown a loss of STING expression in cancer and an overall dysregulation of the CGAS/STING pathway. A potential mechanism of STING silencing is the methylation of its promoter. Using bulk methylation array data from patient samples, we explore epigenetic control of the TMEM173 gene that encodes STING. We find that the STING promoter is highly methylated in glioblastoma and that STING RNA expression is correspondingly low. Methylation data from normal brain and non-cancer cerebral diseases reveal a consistent pattern of STING promoter hypermethylation in these diverse states. The degree of bulk STING methylation in GBM samples inversely correlates with tumor purity and markers of immune infiltration. Single-cell transcriptome sequencing reveals that STING is expressed specifically in immune cells and endothelial cells, but not glioma cells. Collectively these results suggest that STING signalling is disrupted in GBM, that STING is epigenetically silenced by DNA methylation in the tumor, and that STING expression in bulk GBM tumor samples arises from infiltrating immune cells in the tumor microenvironment. Therapeutic approaches stimulating the innate STING signalling pathway are therefore likely to be effective on infiltrating immune cells rather than tumor cells themselves. This work motivates further study into mechanisms of STING activation in the broader GBM tumor microenvironment.

Genome-wide methylation analysis in Silver\u2013Russell syndrome, Temple syndrome, and Prader\u2013Willi syndrome

BackgroundImprinting disorders (IDs) show overlapping phenotypes, particularly in Silver–Russell syndrome (SRS), Temple syndrome (TS14), and Prader–Willi syndrome (PWS). These three IDs include fetal and postnatal growth failure, feeding difficulty, and muscular hypotonia as major clinical features. However, the mechanism that causes overlapping phenotypes has not been clarified. To investigate the presence or absence of methylation signatures associated with overlapping phenotypes, we performed genome-wide methylation analysis (GWMA).ResultsGWMA was carried out on 36 patients with three IDs (SRS [n = 16], TS14 [n = 7], PWS [n = 13]) and 11 child controls using HumanMethylation450 BeadChip including 475,000 CpG sites across the human genome. To reveal an aberrantly methylated region shared by SRS, TS14, and PWS groups, we compared genome-wide methylation data of the three groups with those of control subjects. All the identified regions were known as SRS-, TS14-, and PWS-related imprinting-associated differentially methylated regions (iDMRs), and there was no hypermethylated or hypomethylated region shared by different ID groups. To examine the methylation pattern shared by SRS, TS14, and PWS groups, we performed clustering analysis based on GWMA data. The result focusing on 620 probes at the 62 known iDMRs (except for SRS-, TS14-, and PWS-related iDMRs) classified patients into two categories: (1) category A, grossly normal methylation patterns mainly consisting of SRS group patients; and (2) category B, broad and mild hypermethylation patterns mainly consisting of TS14 and PWS group patients. However, we found no obvious relationship between these methylation patterns and phenotypes of patients.ConclusionsGWMA in three IDs found no methylation signatures shared by SRS, TS14, and PWS groups. Although clustering analysis showed similar mild hypermethylation patterns in TS14 and PWS groups, further study is needed to clarify the effect of methylation patterns on the overlapping phenotypes.

Differential epigenetic regulation between the alternative promoters, PRDM1\u03b1 and PRDM1\u03b2, of the tumour suppressor gene PRDM1 in human multiple myeloma cells

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. The transcription factor PRDM1 is a master regulator of plasma cell development and is considered to be an oncosuppressor in several lymphoid neoplasms. The PRDM1β isoform is an alternative promoter of the PRDM1 gene that may interfere with the normal role of the PRDM1α isoform. To explain the induction of the PRDM1β isoform in MM and to offer potential therapeutic strategies to modulate its expression, we characterized the cis regulatory elements and epigenetic status of its promoter. We observed unexpected patterns of hypermethylation and hypomethylation at the PRDM1α and PRDM1β promoters, respectively, and prominent H3K4me1 and H3K9me2 enrichment at the PRDM1β promoter in non-expressing cell lines compared to PRDM1β-expressing cell lines. After treatment with drugs that inhibit DNA methylation, we were able to modify the activity of the PRDM1β promoter but not that of the PRDM1α promoter. Epigenetic drugs may offer the ability to control the expression of the PRDM1α/PRDM1β promoters as components of novel therapeutic approaches.

Histone H3K9 and H3K14 acetylation at the promoter of the LGALS9 gene is associated with mRNA levels in cervical cancer cells

Galectin-9 levels have been reported to be altered in several cancer types, but the mechanism that regulates the expression of Galectin-9 has not been clarified. Galectin-9 is encoded by the LGALS9 gene, which gives rise to eight mRNA variants. The aims of this study were: (a) to identify the mRNA variants of LGALS9, (b) to characterize CpG methylation and H3K9 and H3K14 histone acetylation at the promoter of the LGALS9 gene, and (c) to characterize the relationship between these modifications and LGALS9 expression level in cervical cancer cells. All mRNA variants were detected in HaCaT (nontumoural keratinocytes) and SiHa cells, and seven were observed in HeLa cells. The promoter region of LGALS9 contains eight CpG dinucleotides. No hypermethylation pattern related to low LGALS9 expression was identified in tumour cells. Chromatin immunoprecipitation analysis demonstrated higher acetylation of H3K9ac and H3K14ac in HaCaT cells, which was related to higher mRNA levels. The presence of the mRNA variants suggests that alternative splicing may regulate the expression of galectin-9 isoforms. The results of this study suggest that histone acetylation, but not promoter CpG methylation, may be involved in the transcriptional regulation of the LGALS9 gene.

An Evolutionary Cancer Epigenetic Approach Revealed DNA Hypermethylation of Ultra-Conserved Non-Coding Elements in Squamous Cell Carcinoma of Different Mammalian Species.

Background: Ultra-conserved non-coding elements (UCNEs) are genomic sequences that exhibit > 95% sequence identity between humans, mammals, birds, reptiles, and fish. Recent findings reported their functional role in cancer. The aim of this study was to evaluate the DNA methylation modifications of UNCEs in squamous cell carcinoma (SCC) from different mammal species. Methods: Fifty SCCs from 26 humans, 17 cats, 3 dogs, 1 horse, 1 bovine, 1 badger, and 1 porcupine were investigated. Fourteen feline stomatitis and normal samples from 36 healthy human donors, 7 cats, 5 dogs, 5 horses, 2 bovines and 1 badger were collected as normal controls. Bisulfite next generation sequencing evaluated the DNA methylation level from seven UCNEs (uc.160, uc.283, uc.416, uc.339, uc.270, uc.299, and uc.328). Results: 57/59 CpGs were significantly different according to the Kruskal–Wallis test (p < 0.05) comparing normal samples with SCC. A common DNA hypermethylation pattern was observed in SCCs from all the species evaluated in this study, with an increasing trend of hypermethylation starting from normal mucosa, through stomatitis to SCC. Conclusions: Our findings indicate that UCNEs are hypermethylated in human SCC, and this behavior is also conserved among different species of mammals.

Abstract 1248: DNA methylation biomarkers for MPNST detection in patients with neurofibromatosis type 1

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are a highly aggressive soft tissue sarcoma affecting approximately 10-15% of individuals with neurofibromatosis type 1 (NF1). MPNST development is characterized by an altered DNA methylation landscape and presents a promising area for developing MPNST-specific biomarkers for screening NF1 patients in the clinic. Here, using genome-wide DNA methylation profiling of a cohort of 13 matched pairs of MPNSTs and adjacent neurofibroma tissues, along with MPNST cell lines and control samples, we conducted intensive DNA methylation analysis towards identification and validation of candidate MPNST specific CpG sites. We utilized a logistic regression model with LASSO regularization to identify the best candidate biomarkers for MPNST detection in patient tissue and/or blood samples. For model fitting, we used TCGA data along with an independent, publicly available dataset. The selected candidate biomarkers were validated on an independent cohort of 22 tissue samples using bisulfite sequencing as well as optimized droplet digital PCR (ddPCR). Our comprehensive analysis revealed the presence of a unique hypermethylation pattern during MPNST development that is also recapitulated within the in vitro MPNST model. Further, based on application of stringent feature selection criteria among an MPNST and NF comparison, together with building a prediction model, we provide a list of candidate MPNST-specific CpGs. Moreover, we successfully applied a ddPCR method to measure the DNA level of five selected candidate biomarkers, all of which showed high performance for MPNST detection (sensitivity: &amp;gt;88%, specificity: &amp;gt;91%) in tissue samples. Overall, our findings confirmed the appearance of a unique hypermethylation pattern during malignant transformation that is also maintained across an in vitro MPNST model. Importantly, we provide a panel of candidate CpGs that have great potential to be further applied as biomarkers in clinical settings for MPNST detection in NF1 patients' tissue and/or liquid biopsy samples. Citation Format: Katarzyna J. Tomczak, Christine B. Peterson, S Carson Callahan, Sharon M. Landers, Christopher J. Terranova, Gabryella S. Serrao, Angela D. Bhalla, Jace P. Landry, Zachary A. Mulder, Michelle G. Yeagley, Ayush T. Raman, Erica L. Gardenhire, Davis R. Ingram, Alexander Lazar, Wei-Lien Wang, Ian E. McCutcheon, John M. Slopis, Kunal Rai, Keila E. Torres. DNA methylation biomarkers for MPNST detection in patients with neurofibromatosis type 1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1248.

Abstract 1251: Epigenetics in early onset colorectal cancer (EOCRC)

Abstract Colorectal cancer (CRC) incidence rates have increased among young adults while the rates have decreased among older adults over 50 years of age. While the etiology of the growing prevalence of young adults with newly diagnosed CRC is unknown, there has been growing interest in the roles implicated for diabetes, obesity, environmental changes, and epigenetics. Among the epigenetic marks implicated in EOCRC, the most widely studied are the global DNA hypomethylation. Using mouse studies, our group has demonstrated that DNA methylation as an epigenetic mechanism to control gene expression is essential to postnatal development. We have discovered a key set of genes that undergo epigenetic changes in colonic epithelial stem cells, and these changes are stable over a lifetime. We have shown that the gut microbiome influences this process. Importantly, perturbation of established intestinal DNA methylation leads to accelerated intestinal tumor development. To translate our mechanistic studies in the animal model to humans, we have confirmed these CpG loci that are concordant between the two species (i.e. homologous regions and/or conserved CpG islands). Here, we hypothesize that the dysregulation of developmentally programmed intestinal DNA methylation is associated with enhanced susceptibility to colon cancer in young adult. We have identified a population of patients within the Harris Health and Baylor College of Medicine system under the age of 50 with mismatch repair stable colorectal adenocarcinoma. Patient characteristics including age, sex, race, and primary tumor profile (stage at diagnosis, histology, and RAS status) have been defined. Using a panel of 6 genes and bisulfite pyrosequencing for quantitative assessment of DNA methylation, we compared the level of methylation in EOCRC tumors to paired normal adjacent tissues as well as normal adult tissue (age 50 or greater) to traditional CRC tumors. Our preliminary data suggest hypermethylation patterns are distinct in patients of EOCRC relative to traditional CRC. Our ultimate goal is to better understand the etiology of EOCRC in order to devise better prevention and treatment strategies for EOCRC. Citation Format: Karen Riggins, Benjamin Musher, Michael Scheurer, Li Yang, Patricia Castro, Neda Zarrin-Khameh, Lanlan Shen. Epigenetics in early onset colorectal cancer (EOCRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1251.

Selenium accumulation characteristics of Cyphomandra betacea (Solanum betaceum) seedlings.

A pot experiment was conducted to study the selenium (Se) accumulation characteristics and the tolerance of Cyphomandra betacea (Solanum betaceum) seedlings under different soil Se concentrations. The 5mg/kg soil Se concentration increased the C. betacea seedling biomass and photosynthetic pigment contents (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid), whereas the other soil Se concentrations (10, 25, and 50mg/kg) inhibited seedling growth. Increases in the soil Se concentrations tended to decrease the superoxide dismutase activity and soluble protein content, but had the opposite effect on the peroxidase and catalase activities. The 5, 10, and 25mg/kg soil Se concentrations decreased the DNA methylation levels of C. betacea seedlings because of an increase in demethylation patterns (versus 0mg/kg), whereas the 50mg/kg soil Se concentration increased the DNA methylation levels because of an increase in hypermethylation patterns (versus 0mg/kg). Increases in the soil Se concentrations were accompanied by an increasing trend in the Se content of C. betacea seedlings. Moreover, the amount of Se extracted by the shoots was highest for the 25mg/kg soil Se concentration. Therefore, C. betacea may be able to accumulate relatively large amounts of Se and its growth may be promoted in 5mg/kg soil Se.

Characterization and comparative DNA methylation profiling of four adipogenic genes in adipose-derived stem cells and dedifferentiated fat cells from aging subjects

Adipose-derived stem cells (ASCs) and dedifferentiated fat (DFAT) cells are alternative cell sources in tissue engineering and regeneration because they are easily obtained and exhibit multilineage differentiation. However, aging may attenuate their regenerative potential and metabolic functions. Reports characterizing DFAT cells derived from aging donors are rare, and comparisons of DNA methylation profiles between aging ASCs and DFAT cells are poorly understood. Therefore, this study aimed to characterize DFAT cells relative to ASCs derived from aging subjects and compare the DNA methylation profiles of four adipogenic genes in these cells. ASCs and DFAT cells from aging donors exhibited characteristics similar to those of stem cells, including colony formation, proliferation, and multilineage differentiation abilities. However, compared with ASCs, DFAT cells exhibited increased proliferation, smooth muscle actin alpha (SMA-α) expression and decreased cellular senescence. DNA methylation profiling of ASCs and DFAT cells by combined bisulfite restriction analysis (COBRA) demonstrated hypermethylation patterns in three potent adipogenic genes—peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid-binding protein 4 (FABP4), and lipoprotein lipase (LPL)—but hypomethylation of CCAAT/enhancer binding protein alpha (C/EBPα) in the aging group. Statistically significant differences were observed between the aging group and the young group. Epigenetic regulation maintains the stability of ASCs and DFAT cells in an age-dependent manner. Our findings suggested that although the DNA methylation patterns of three adipogenic genes correlated with hypermethylation and aging, ASCs and DFAT cells exhibited cellular stability and several stem cell characteristics, offering further opportunities for personalized regeneration and energy maintenance by adipogenesis during aging.

The role of NAT2 polymorphism and methylation in anti-tuberculosis drug-induced liver injury in Mongolian tuberculosis patients.

Anti-tuberculosis drug-induced liver injury (ATLI) is one of the most significant adverse reactions for this line of therapy. N-acetyltransferase 2 (NAT2) is an important metabolic enzyme involved in drug metabolism and detoxification. Genetic polymorphism and DNA methylation have been proven to be key factors that affect the expression of NAT2. Therefore, the objective of the study was to investigate the relationship between NAT2 gene polymorphism and DNA methylation in the promoter region with ATLI risk in Mongolian tuberculosis patients. Our study is a case-control design. Chi-square test, Mann-Whitney U non-parametric test and Pearson test were all used to analyse existing relationships. The association between NAT2 gene acetylation phenotype and the total methylation of the NAT2 promoter region was analysed by means of binary logistic regression analysis. The general situation of the patients was evaluated by questionnaire, and the NAT2 genotyping of the three major polymorphism loci of gene coding was carried out by a gene sequencing technique. The methylation status of the NAT2 gene promoter region was detected by bisulphite sequencing and mass spectrometry. Our study found that the detection rate of ATLI in Mongolian tuberculosis patients was 27.6%. There were no significant differences in demographic characteristics and living habits amongst the two groups, while significant differences were observed in the polymorphism of the NAT2 genes 481 (rs1799929) and 590 (rs1799930) and the acetylation phenotype. Moreover, the composition and distribution of the NAT2*4/4 and NAT2*4/5 genotypes were found in the two groups. The risk of ATLI in the slow acetylation type was 3.56 times higher than that of the fast acetylation type. Compared with the control group, the CpG5, CpG10, CpG11.12 and total methylation of the NAT2 promoter region in the ATLI group showed a hypermethylated pattern (P<.05). However, on performing binary logistic regression, neither the slow acetylation, intermediate acetylation nor rapid acetylation were found to be associated with ATLI (P>.05). It was found that the total methylation of NAT2 gene promoter region was an independent influencing factor of ATLI in Mongolian tuberculosis patients. With the increase of the total methylation level of NAT2 gene promoter region, the risk of ATLI increased gradually. (OR=8.371, 95% CI: 2.391~29.315). CpG1, CpG4, CpG9, CpG10 and CpG11.12 were positively correlated with a total methylation level in the ATLI group. The detection rate of ATLI in Mongolian tuberculosis patients was 27.6%, and there were differences in the NAT2 genotypes and acetylated phenotypes. The slow acetylated type was the risk factor for ATLI. Methylation in the promoter region of the NAT2 gene has an effect on the risk of ATLI. After adjusting for the interference of three acetylation types, it was found that the total methylation of the promoter region of NAT2 gene in Mongolian tuberculosis patients is an independent influencing factor of ATLI. Furthermore, there is a moderate to high correlation between some sites and the overall level of methylation.

Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation.

As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.

DNA hypermethylation is associated with invasive phenotype of malignant melanoma.

Tumor cell invasion is one of the key processes during cancer progression, leading to life-threatening metastatic lesions in melanoma. As methylation of cancer-related genes plays a fundamental role during tumorigenesis and may lead to cellular plasticity which promotes invasion, our aim was to identify novel epigenetic markers on selected invasive melanoma cells. Using Illumina BeadChip assays and Affymetrix Human Gene 1.0 microarrays, we explored the DNA methylation landscape of selected invasive melanoma cells and examined the impact of DNA methylation on gene expression patterns. Our data revealed predominantly hypermethylated genes in the invasive cells affecting the neural crest differentiation pathway and regulation of the actin cytoskeleton. Integrative analysis of the methylation and gene expression profiles resulted in a cohort of hypermethylated genes (IL12RB2, LYPD6B, CHL1, SLC9A3, BAALC, FAM213A, SORCS1, GPR158, FBN1 and ADORA2B) with decreased expression. On the other hand, hypermethylation in the gene body of the EGFR and RBP4 genes was positively correlated with overexpression of the genes. We identified several methylation changes that can have role during melanoma progression, including hypermethylation of the promoter regions of the ARHGAP22 and NAV2 genes that are commonly altered in locally invasive primary melanomas as well as during metastasis. Interestingly, the down-regulation of the methylcytosine dioxygenase TET2 gene, which regulates DNA methylation, was associated with hypermethylated promoter region of the gene. This can probably lead to the observed global hypermethylation pattern of invasive cells and might be one of the key changes during the development of malignant melanoma cells.

Aberrant Promoter Hypermethylation of RASSF1a and BRCA1 in Circulating Cell-Free Tumor DNA Serves as a Biomarker of Ovarian Carcinoma.

Objective:Ovarian cancer is one of the leading causes of cancer deaths in women. Ovarian cancer is diagnosed at the late stages and generally relapses within 12-14 months of cytoreductive surgery. This is attributed to lack of precise molecular detection methodologies to detect and track the disease. Epigenetic alteration such as aberrant promoter hypermethylation is an important early event that occurs during cancer development and progression. This study focuses on development of a minimally invasive methylation marker that could be used for detection and prognosis of ovarian cancer patients. Methods:Aberrant promoter hypermethylation of RASSF1a and BRCA1 was assessed in circulating DNA of 72 EOC patients using methylation-specific PCR. The findings were correlated with various clinicopathological parameters. Statistical analysis was done using the Fisher exact test and chi-square test. Results:The aberrant methylation patterns of RASSF1a and BRCA1 was identified to be present in the cancerous samples. A total of 31.9 % and 56.9% methylation was observed for RASSF1a and BRCA1 respectively. A striking 50% methylation of BRCA1 was identified in the benign sample cohort, which marks the significance of assessing the hypermethylation pattern to detect cancer at its early stages. Methylation of the two tumor suppressor genes was evident across various stages and grades of ovarian tumors suggesting that this could also help as a prognostic marker. Conclusion:The results of the current study hold significance since the hypermethylation patterns can be identified in the cell-free circulating tumor DNA from a small volume of blood plasma and is a simple and minimally-invasive method. Assessment of hypermethylation patterns of a panel of TSG along with the existing screening markers could aid in better diagnosis and management of the disease. It could also aid in designing specifically tailored treatment strategies to fight the disease.

Estrogen suppresses bcl2 expression through dnmt1 in Japanese eel ovarian follicle.

Japanese eels are commercially valuable species in Asian aquaculture. This study evaluated whether salmon pituitary extract (SPE) or 17β-estradiol (E2) treatment can induce cytotoxic activity in eel ovarian follicles. Follicular cells died after exposure SPE for 24-h culture in an in vitro culture. Moreover, the E2 treatment also significantly reduced follicular cell counts. These results reveal that the inhibition of follicular cell numbers by SPE may occur through SPE-induced steroidogenesis. Results of a quantitative PCR analysis indicated that adding E2 to the culture decreased bcl2 and increased dnmt1 mRNA expression in Japanese eel follicular cells after 24 h. The results of a promoter assay revealed that E2 significantly increase dnmt1 promoter activity through estrogen receptor-binding site. An in silico analysis predicted several putative transcription factors targeting the bcl2 gene promoter region. Methylation of the bcl2 promoter accounted for the downregulation of bcl2 by E2-mediated dnmt1. The DNA methylation level of the bcl2 gene was significantly higher in E2-treated follicular cells than that in the control group. Finally, the E2-induced hypermethylation pattern of the bcl2 promoter and the reduction in follicular cell numbers were suppressed by adding an MTase inhibitor. Our findings demonstrate that estrogen has a negative effect on the reproductive system of female eels by regulating an epigenetic mechanism during artificial maturation.

Genes Controlled by DNA Methylation Are Involved in Wilms Tumor Progression.

To identify underlying mechanisms involved with metastasis formation in Wilms tumors (WTs), we performed comprehensive DNA methylation and gene expression analyses of matched normal kidney (NK), WT blastemal component, and metastatic tissues (MT) from patients treated under SIOP 2001 protocol. A linear Bayesian framework model identified 497 differentially methylated positions (DMPs) between groups that discriminated NK from WT, but MT samples were divided in two groups. Accordingly, methylation variance grouped NK and three MT samples tightly together and all WT with four MT samples that showed high variability. WT were hypomethylated compared to NK, and MT had a hypermethylated pattern compared to both groups. The methylation patterns were in agreement with methylases and demethylases expression. Methylation data pointed to the existence of two groups of metastases. While hierarchical clustering analysis based on the expression of all 2569 differentially expressed genes (DEGs) discriminated WT and MT from all NK samples, the hierarchical clustering based on the expression of 44 genes with a differentially methylated region (DMR) located in their promoter region revealed two groups: one containing all NKs and three MTs and one containing all WT and four MTs. Methylation changes might be controlling expression of genes associated with WT progression. The 44 genes are candidates to be further explored as a signature for metastasis formation in WT.

Abstract LB-301: Integrative genomic analysis identifies distinct mutational, epigenetic and immunological patterns among triple-negative breast cancer subtypes

Abstract Triple-negative breast cancer is an inherently heterogeneous disease defined by the absence of estrogen receptor, progesterone receptor and human epithelial growth factor receptor amplifications. The lack of therapeutically targetable, high-frequency “driver” alterations has impeded development of targeted therapies for TNBC. TNBCs display transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Each subtype displays unique biology and differentially responds to standard-of-care chemotherapy. Therefore, to better understand TNBC genomic alterations within molecular subtypes we have performed an integrative analysis of mutation, copy-number, gene expression, miRNA expression, reverse-phase protein array and methylation of 192 TNBC patients in The Cancer Genome Atlas and drug sensitivity of TNBC cell lines. Gene expression analysis confirmed our previous subtype-specific findings with enrichment of cell cycle, DNA replication response in BL1, Wnt and KRAS signaling BL2, androgen response in LAR and TGF-beta and mesenchymal differentiation in M. Despite having higher mutational burdens and similar copy number alterations, the BL1 and M subtypes display very different patterns of gene expression. These differences likely originate from epigenetic changes, as global methylation analysis demonstrated large hypomethylation across the genomes of M subtype. In contrast, the highly differentiated LAR subtype displayed global patterns of hypermethylation. Mutation analysis revealed enrichment of PIK3CA mutations in LAR tumors, NOTCH1, NOTCH2, NOTCH3 and FBXW7 mutations in M and BL1 tumors and activating MAPK pathway mutations (KRAS, NRAS, BRAF, MAP2K1, MAPK9 and MAP3K13) exclusive to BL2 tumors. Differential copy number analysis identified amplifications specific to the BL1 subtype (AKT3, KDR and KIT), BL2 subtype (TERT, EGFR and KRAS), M subtype (MYCN and WNT5B) and NOTCH pathway (BL1 and M). Reverse-phase protein array analysis confirmed activated MAPK signaling unique to the BL2 subtype and PI3K signaling in LAR. Cell line subtype correlations and published drug sensitivity screens verified LAR sensitivity to AR antagonists and PI3K/AKT/mTORC inhibitors, while BL2 cell models displayed sensitivity to MEK inhibitors. Subtype-specific differences in lymphocyte composition were inferred from gene expression with BL1 subtype displaying the highest CD8 levels. Despite having the greatest level of macrophages, the M subtype had the lowest ratio of M1/M2 polarized macrophages. These data suggest immune checkpoint therapies may be more effective in BL1 subtype and additional strategies targeting the immune suppressive macrophage population necessary for M tumors. Using a comprehensive multi-omics approach we defined the multi-platform genomic landscape of TNBC, identify potential therapeutic strategies and shed light on the evolutionary paths of TNBC. Citation Format: Brian D. Lehmann, Antonio Colaprico, Jianjiao Chen, Lily Wang, Jennifer Pietenpol, Xi Chen. Integrative genomic analysis identifies distinct mutational, epigenetic and immunological patterns among triple-negative breast cancer subtypes [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 LB-301.

Peroxisomal β-oxidation regulates histone acetylation and DNA methylation in Arabidopsis

Epigenetic markers, such as histone acetylation and DNA methylation, determine chromatin organization. In eukaryotic cells, metabolites from organelles or the cytosol affect epigenetic modifications. However, the relationships between metabolites and epigenetic modifications are not well understood in plants. We found that peroxisomal acyl-CoA oxidase 4 (ACX4), an enzyme in the fatty acid β-oxidation pathway, is required for suppressing the silencing of some endogenous loci, as well as Pro35S:NPTII in the ProRD29A:LUC/C24 transgenic line. The acx4 mutation reduces nuclear histone acetylation and increases DNA methylation at the NOS terminator of Pro35S:NPTII and at some endogenous genomic loci, which are also targeted by the demethylation enzyme REPRESSOR OF SILENCING 1 (ROS1). Furthermore, mutations in multifunctional protein 2 (MFP2) and 3-ketoacyl-CoA thiolase-2 (KAT2/PED1/PKT3), two enzymes in the last two steps of the β-oxidation pathway, lead to similar patterns of DNA hypermethylation as in acx4 Thus, metabolites from fatty acid β-oxidation in peroxisomes are closely linked to nuclear epigenetic modifications, which may affect diverse cellular processes in plants.

A distinct epigenetic program underlies the 1;7 translocation in myelodysplastic syndromes

The unbalanced translocation dic(1;7)(q10;p10) in myelodysplastic syndromes (MDS) is originated by centromeric juxtaposition resulting into 1q trisomy and 7q monosomy. More than half of cases arise after chemo/radio-therapy. To date, given the absence of genes within the centromeric regions, no specific molecular events have been identified in this cytogenetic subgroup. We performed the first comprehensive genetic and epigenetic analysis of MDS with dic(1;7)(q10;p10) compared to normal controls and therapy-related myeloid neoplasms (t-MNs). RNA-seq showed a unique downregulated signature in dic(1;7) cases, affecting more than 80% of differentially expressed genes. As revealed by pathway and gene ontology analyses, downregulation of ATP-binding cassette (ABC) transporters and lipid-related genes and upregulation of p53 signaling were the most relevant biological features of dic(1;7). Epigenetic supervised analysis revealed hypermethylation at intronic enhancers in the dicentric subgroup, in which low expression levels of enhancer putative target genes accounted for around 35% of the downregulated signature. Enrichment of Krüppel-like transcription factor binding sites emerged at enhancers. Furthermore, a specific hypermethylated pattern on 1q was found to underlie the hypo-expression of more than 50% of 1q-deregulated genes, despite trisomy. In summary, dic(1;7) in MDS establishes a specific transcriptional program driven by a unique epigenomic signature.

Parallel profiling of DNA methylation and hydroxymethylation highlights neuropathology-associated epigenetic variation in Alzheimer\u2019s disease

BackgroundAlzheimer’s disease is a progressive neurodegenerative disorder that is hypothesized to involve epigenetic dysfunction. Previous studies of DNA modifications in Alzheimer’s disease have been unable to distinguish between DNA methylation and DNA hydroxymethylation. DNA hydroxymethylation has been shown to be enriched in the human brain, although its role in Alzheimer’s disease has not yet been fully explored. Here, we utilize oxidative bisulfite conversion, in conjunction with the Illumina Infinium Human Methylation 450K microarray, to identify neuropathology-associated differential DNA methylation and DNA hydroxymethylation in the entorhinal cortex.ResultsWe identified one experiment-wide significant differentially methylated position residing in the WNT5B gene. Next, we investigated pathology-associated regions consisting of multiple adjacent loci. We identified one significant differentially hydroxymethylated region consisting of four probes spanning 104 bases in the FBXL16 gene. We also identified two significant differentially methylated regions: one consisting of two probes in a 93 base-pair region in the ANK1 gene and the other consisting of six probes in a 99-base pair region in the ARID5B gene. We also highlighted three regions that show alterations in unmodified cytosine: two probes in a 39-base pair region of ALLC, two probes in a 69-base pair region in JAG2, and the same six probes in ARID5B that were differentially methylated. Finally, we replicated significant ANK1 disease-associated hypermethylation and hypohydroxymethylation patterns across eight CpG sites in an extended 118-base pair region in an independent cohort using oxidative-bisulfite pyrosequencing.ConclusionsOur study represents the first epigenome-wide association study of both DNA methylation and hydroxymethylation in Alzheimer’s disease entorhinal cortex. We demonstrate that previous estimates of DNA hypermethylation in ANK1 in Alzheimer’s disease were underestimates as it is confounded by hypohydroxymethylation.

Age-dependent DNA methylation patterns on the Y chromosome in elderly males.

The Y chromosome, a sex chromosome that only exists in males, has been ignored in traditional epigenetic association studies for multiple reasons. However, sex differences in aging‐related phenotypes and mortality could suggest a critical role of the sex chromosomes in the aging process. We obtained blood‐based DNA methylation data on the Y chromosome for 624 men from four cohorts and performed a chromosome‐wide epigenetic association analysis to detect Y‐linked CpGs differentially methylated over age and cross‐validated the significant CpGs in the four cohorts. We identified 40–219 significant CpG sites (false discovery rate <0.05) with >82% of them hypermethylated with increasing age, which is in strong contrast to the patterns reported on the autosomal chromosomes. Comparing the rate of change in the Y‐linked DNA methylation across cohorts that represent different age intervals revealed a trend of acceleration in DNA methylation with increasing age. The age‐dependent DNA methylation patterns on the Y chromosome were further examined for their association with all‐cause mortality with results suggesting that the predominant pattern of age‐related hypermethylation on the Y chromosome is associated with reduced risk of death.