Promoter CpGs Research Articles

Epigenetic Modifications of White Blood Cell DNA Caused by Transient Fetal Infection with Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5-2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle.

PB1776: ABC TRANSPORTERS AFFECT THE SENSITIVITY OF ACUTE MYELOID LEUKEMIA BLASTS TO CHEMOTHERAPY

Background: Approximately 40 % of acute myeloid leukemia (AML) patients does not achieve a complete hematologic remission (CR) after the first cycle of 3 + 7 induction therapy and thus face an adverse prognosis. Mechanisms of chemoresistance are still poorly understood, but drug export from cytosol plays a crucial role. 7 ABC transporters (ABCA2, ABCA5, ABCB1, ABCB6, ABCC1, ABCC3, ABCG2) were selected from literature for expression, methylation, genotype and efflux activity analysis. Aims: The aim of this study is to clarify the role of selected ABC transporters in reduced sensitivity to chemotherapy in AML. Methods: Peripheral blood was collected at diagnosis (day 0) and at the third day of 3 + 7 therapy (day 3). Leukemic blasts were separated on MACS (CD34+ or CD117+, Miltenyi Biotec). Gene expression was measured on StepOne Plus (Applied Biosystems) using TaqMan assays, B2M and YWHAZ were used as reference genes (n=85). Promotor, exons and splicing regions were sequenced on MiSeq (V2, Illumina) using Roche reagents (n=109). Methylation analysis was performed on Pyromark Q24 (Qiagen) for 85 CpGs in promoters of ABCA2, ABCC1 and ABCG2 (n=51). Efflux activity was assessed with Efflux-ID kit (Enzo) (n=12). For data analysis, we divided patients by primary response status into responding and refractory group based on a CR achievement after the first cycle of 3 + 7 therapy. Data analysis was performed in GraphPad Prism 9.1.2 and R software 4.0.0. Results: Higher expression of ABCA2 (p=0.005), ABCA5 (p=0.016), ABCC1 (p<0.0001) and ABCG2 (p=0.001) at day 0 was associated with primary treatment failure. ABCA2 (p=0.018) and ABCC1 (p=0.008) remained significant in the Cox regression analysis adjusted to age, leukocyte count at diagnosis and ELN risk stratification. Moreover, higher number of concurrently overexpressed ABC transporters (threshold defined by Cutoffinder) was predictive of failure in CR achievement (p=0.006) by Cox regression. We detected a significant rise of expression at day 3 in ABCA5 (p=0.003), ABCB6 (p=0.001) and ABCC3 (p<0.0001). Significantly higher rise of ABCC3 (p=0.029) expression was detected in responders compared to refractory patients. In terms of a CR achievement, we identified 6 unfavorable DNA variants (rs2301837, rs8077268, rs17563146, rs1373970189, rs113369901, 9:137022522-137022522), and 3 favorable DNA variants (rs556735807, rs2235048, rs1407267536). The risk variant rs113369901 correlated with higher expression of ABCC1 (p=0.039), which was also associated with chemoresistance. Cox regression analysis confirmed prognostic relevance for four variants: rs2301837 (p=0.032), rs8077268 (p=0.033), 9:137022522-137022522 (p=0.003) and rs1373970189 (p=0.039). Higher methylation of ABCG2 promoter correlated with its lower expression (p=0.0001). Promoters of ABCA2 and ABCC1 were basically unmethylated, especially ABCC1, which had the highest expression among the studied genes. The greatest contribution to cell efflux had transporters inhibited by MK-571 inhibitor, following by Verapamil. These influence activity of ABCC1 and ABCB1. Summary/Conclusion: We described differences in expression levels and genetic variants of selected ABC transporters in association with treatment response in AML. ABCC1 seems to contribute substantially as illustrated by expression, genotyping, DNA methylation as well as activity data. With such detailed knowledge of chemoresistance mechanisms in leukemic cells and an emergence of new drugs, a different therapeutic strategy for chemoresistant patients may be considered in the future. Supported by MHCR (00023736, IHBT).

Abstract 3632: Differential DNA methylation in the benign and cancerous prostate tissue of African American and European American men

Abstract In this study, we compare patterns of differential DNA methylation (DNAm) distinguishing tumor and benign prostate tissue in European American (EA) and African American (AA) men to improve our understanding of biological mechanisms that may underlie prostate cancer (PCa) disparities. In the United States, AA men are more likely to be diagnosed with and to die of PCa compared with EA men. Changes in DNAm accompany tumor development and progression, and differences in DNAm across ancestry groups may be an important lens through which to study PCa disparities. We collected paired tumor and histologically benign formalin-fixed paraffin-embedded tissue blocks from 151 (76 AA, 75 EA) PCa patients undergoing prostatectomy at the University of Chicago. DNA was extracted and genome-wide DNAm was measured at ~850,000 CpG sites using the Illumina MethylationEpic Array. After quality control, 682,694 CpGs remained for analysis. We conducted differential methylation analyses, adjusting for batch, age, and individual and identified 29,236 and 38,035 tumor-associated CpGs in AA and EA respectively (p&amp;lt;5x10-8 and Δβ&amp;gt;0.3) with 25,263 CpGs common to both sets. There was a depletion of differentially methylated CpGs in CpG islands and promoters in both groups, but depletion was stronger in AA. Tumor-associated CpGs within islands and in promoters were more likely to be hypermethylated for both groups, though this was stronger in EA. We identified 2,392 genes with differentially methylated promoters common to both ancestry groups; 223 and 1,045 were unique to AA and EA respectively. Shared gene regions included GSTP1, APC, RARB, and GRASP, which have been previously reported. We assigned CpG sites to genes and used Gene Set Enrichment Analysis to identify differentially methylated pathways. The identified pathways include several previously associated with cancer development including the epithelial mesenchymal transition. We also identified ancestry-specific pathways including the early and late estrogen response pathways in AA. Examining the predictive ability of our differentially methylated CpGs, we found that relatively few CpGs (~10) were sufficient to distinguish tumor and benign tissue in AA and EA men (AUC&amp;gt;0.9). Scores constructed from differentially methylated CpGs in EA were able to predict tumor vs benign in AA (and vice versa) with high accuracy. Finally, we examined the association between ancestry and DNAm in both tumor and benign tissue. Here, 89 and 423 ancestry-associated CpGs were identified in tumor and benign respectively. Overall, we find that differential methylation patterns distinguishing tumor and benign tissue are generally similar for EA and AA men. However, differenced in tumor-associated DNAm as well as the presence of ancestry-associated CpGs provide a rich area for future studies exploring the impact of these differences on cancer development in AA and EA men. Citation Format: Meytal B. Chernoff, Marc Gillard, Kathryn Demanelis, Dayana Delgado, Anthony Williams, Donald Vander Griend, Brandon L. Pierce. Differential DNA methylation in the benign and cancerous prostate tissue of African American and European American men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3632.

Abstract 3737: DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia

Abstract Infants and young children (&amp;gt; 3 yrs.) harboring ETO2-GLIS2 have the worst outcomes amongst all pediatric acute myeloid leukemia (AML) subtypes, with poor responses to induction therapy, high incidence of relapse, and dismal 5-year survival rates. Previous studies demonstrated that the ETO2-GLIS2 fusion leads to imbalance in the expression of ETS-related transcription factor ERG and GATA1 with neo-super enhancer (SE) activities in a single oncogenic hit (Thirant et al. 2017). ERG was found to co-localize with ETO2-GLIS2 fusion at 90% (372/419) of H3K27ac marked SE-regions and was strongly upregulated (9.44-fold). Herein, we aimed to investigate cooperativity of DNA methylation and chromatin marks on ERG overexpression in this AML subtype. We analyzed transcriptomic data from pediatric ETO2-GLIS2 AML patients (n=40) from dbGAP (phs000465.v19. p8) in comparison to normal bone marrow (NBM) (n=71) samples, confirming upregulation (median log FC: 2.4-fold) in ERG expression in the ETO2-GLIS2 subgroup compared to NBM. Next, we performed an unbiased genome-wide methylation array using Infinium MethylationEPIC 850K-arrays (Illumina). Differences in mean methylation (β-value) at individual CpG sites between NBM and AML samples were considered significant at differences greater than ±10%. We observed reduced methylation at 72% (5/7) CpGs at the ERG-promoter (1500bp upstream of transcription start site) in ETO2-GLIS2 subgroup compared to NBM. Overall median methylation across the promoter was reduced by 12% in the AML subgroup. In comparison, 22 differentially methylated CpGs were observed in the body, of which methylation increased at 68% (15/22) of the CpGs, with median methylation similarly increased by 15% in the AML patients. This represents a classic example of methylation-expression relation, having a promoter demethylation and body-hypermethylation, linked to upregulation of the gene-expression. Next, we investigated the overlaps between CpGs and the distribution of DNAse clusters and H3K27ac marks based on ENCODE data on myeloid leukemia cell line K562. We observed frequent overlaps between demethylated CpGs and DNAse clusters, while H3K27ac broad peaks were coincident with both demethylated CpGs at promoter or hypermethylated CpGs at body. Based on our findings, we thus conclude that DNA-methylation both at the ERG promoter and body is tightly linked to the overlapping chromatin marks of the gene. DNA-methylation might aid in opening of adjacent chromatin on ERG to possibly facilitate SE enrichment and over-expression the gene. Future studies will investigate targeted alterations of DNA-methylation on the gene to test the impact on ERG expression and leukemic growth. Citation Format: Samrat Roy Choudhury, Jordan T. Bird, Stephanie Byrum, Jason E. Farrar. DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3737.

Abstract 3750: Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology

Abstract Background: DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA hypermethylation events co-occur in groups of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Here we designed and performed a first step of validation of a novel next-generation sequencing (NGS) targeted methylation panel that can simultaneously evaluate the methylation levels of multiple cancer-related genes. Methods: The WG00446_MET_A AmpliSeq™ Methylation Panel for Cancer Research was designed using the Ion AmpliSeq technology to amplify 152 regions with an amplicon size range of 125-175bp, that covers a total of 1107 promoter and intragenic CpGs of 18 cancer-related genes. The performance of the panel was assessed by running in duplicate commercially available fully methylated and unmethylated control human gDNA samples and a variable mixture of them to simulate 10%, 25%, 75%, and 90% of global methylation. The libraries (manual preparation using the Ion AmpliSeq Plus Library Kit) were loaded on 530 chips and run on the GeneStudio S5 Sequencer (Thermo Fisher Scientific). The sequencing output was analyzed using the methylation_analysis plugin, available on Ion Torrent Suite v5.16 (Thermo Fisher Scientific). Results: The WG00446_MET_A panel allowed to run 6 samples per 530 chips to reach an observed mean target depth &amp;gt;2,000X (observed reads on-targets: &amp;gt;80%; average number of mapped reads: &amp;gt;450,000/sample). The conversion efficiency, determined by spiking-in Unmethylated Lambda DNA into each sample before the bisulfite conversion process, was &amp;gt;97% for all samples. The observed % of global methylation for all CpGs were &amp;gt;98% and &amp;lt;2% for fully methylated and unmethylated gDNA samples, respectively. The observed results for the variable mixtures were in agreement with what expected. Conclusions: The methylation-specific NGS analysis using the WG00446_MET_A panel represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. It will offer the opportunity to design a more robust algorithm for disease prediction for those cancer patients with a low quantity of biological material available. The orthogonal testing on DNA extracted by FFPE and frozen samples is ongoing with a subset of amplicons from the WG00446_MET_A panel. Citation Format: Federico Pio Fabrizio, Stefano Castellana, Angelo Sparaneo, Tommaso Mazza, Flavia Centra, Domenico Trombetta, Vito Michele Fazio, Lucia Anna Muscarella. Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3750.

Nucleus Type-Specific DNA Methylomics Reveals Epigenetic "Memory" of Prior Adaptation in Skeletal Muscle.

Using a mouse model of conditional and inducible in vivo fluorescent myonuclear labeling (HSA-GFP), sorting purification of nuclei, low-input reduced representation bisulfite sequencing (RRBS), and a translatable and reversible model of exercise (progressive weighted wheel running, PoWeR), we provide the first nucleus type-specific epigenetic information on skeletal muscle adaptation and detraining. Adult (>4 mo) HSA-GFP mice performed PoWeR for 8 wk then detrained for 12 wk; age-matched untrained mice were used to control for the long duration of the study. Myonuclei and interstitial nuclei from plantaris muscles were isolated for RRBS. Relative to untrained, PoWeR caused similar myonuclear CpG hypo- and hyper-methylation of promoter regions and substantial hypomethylation in interstitial nuclear promoters. Over-representation analysis of promoters revealed a larger number of hyper- versus hypo-methylated pathways in both nuclear populations after training and evidence for reciprocal regulation of methylation between nucleus types, with hypomethylation of promoter regions in Wnt signaling-related genes in myonuclei and hypermethylation in interstitial nuclei. After 12 wk of detraining, promoter CpGs in documented muscle remodeling-associated genes and pathways that were differentially methylated immediately after PoWeR were persistently differentially methylated in myonuclei, along with long-term promoter hypomethylation in interstitial nuclei. No enduring gene expression changes in muscle tissue were observed using RNA-sequencing. Upon 4 wk of retraining, mice that trained previously grew more at the whole muscle and fiber type-specific cellular level than training naïve mice, with no difference in myonuclear number. Muscle nuclei have a methylation epi-memory of prior training that may augment muscle adaptability to retraining.

DNA methylomic homogeneity and heterogeneity in muscles and testes throughout pig adulthood.

DNA methylome pattern is significantly different among tissues, ages, breeds, and genders. We assessed 20 methylome and transcriptome data in longissimus dorsi (LD) or testicles from Bamaxiang (BMX) and Large White pigs (LW) by deep sequencing technology. We identified ~55.7M CpGs and 5.30M, 0.20M, 1.20M, and 0.16M differential CpGs (P<0.01) between tissues, ages, breeds, and genders, respectively. Interestingly, 7.54% of differentially methylated regions (DMRs) are co-localized with promoters, which potentially regulate gene expression. RNA-seq analysis revealed that 23.42% CpGs are significantly correlated with gene expression (mean |r|=0.58, P<0.01), most of which are enriched in tissue-specific functions. Specially, we also found that the methylation levels in promoters of 655 genes were strongly associated with their expression levels (mean |r|=0.66, P<0.01). In addition, differentially methylated CpGs (DMCpGs) between breeds in HOXC gene cluster imply important regulatory roles in myocytes hypertrophy and intermuscular fat (IMF) deposition. Dramatically, higher similarity of methylation pattern was observed within pedigree than across pedigrees, which indicates the existence of heritable methylation regions. In summary, a part of CpGs in promoter can change its methylation pattern and play a marked regulatory function in different physiological or natural environments.

Aging of Preleukemic Thymocytes Drives CpG Island Hypermethylation in T-Cell Acute Lymphoblastic Leukemia

During the last decade, aberrant DNA methylation has been identified as a hallmark of human cancer and several studies have highlighted the potential of DNA methylation as a clinically or diagnostically relevant biomarker. In comparison to their normal healthy counterparts, cancer cells generally display DNA hypermethylation at specific CpG islands, but the actual mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. To profile the DNA methylation landscape of human T-cell acute lymphoblastic leukemia (T-ALL), we analyzed 109 T-ALLs together with 10 stages of normal T cell development, which are considered the normal human counterparts of this disease, by 850 EPIC arrays. Here, we show that CpG islands are hypermethylated in all human T- ALLs compared to their normal counterparts. We designed a DNA methylation signature that can distinguish two types of T-ALL, with low or high levels of CpG island hypermethylation. This profile is dominated by CpGs in promoters of PRC2 target genes. T-ALLs with high levels of CpG island hypermethylation show low levels of H3K27me3 and vice versa, resulting in gene repression in both subtypes of T-ALL by different mechanisms. Furthermore, we found that aberrant CpG island hypermethylation shows a strong correlation with the epigenetic age of the leukemic T cells. By investigating the DNA methylation profile of two distinct mouse T-ALL models, the Lck-Cre Ptenfl/fland the CD2-Lmo2 transgenic mouse model, by Reduced Representation Bisulfite Sequencing, we could indeed recapitulate the DNA methylation features of the two human T-ALL subtypes in mice (Fig.1). The aggressive, fast-transforming Ptenfl/flmouse model displays low levels of CpG island hypermethylation, which correlated with human T-ALLs that have a shorter proliferative history and a worse prognosis. In contrast, murine CD2-Lmo2 T-ALLs have a longer disease latency and display a CpG island hypermethylation phenotype that is similar to human T-ALLs with a longer proliferative history. In CD2-Lmo2 mice, a pre-leukemic phase is present with self-renewing thymocytes. We elucidate that the CpG island methylation signature is gradually established in aging pre-leukemic thymocytes of 8, 16 and 24 weeks old CD2-Lmo2 mice (Fig.1). Of note, this hypermethylation phenotype is completely absent in age matched Lck-Cre Ptenfl/flmice that did not yet develop leukemia, suggesting that the proliferative history is responsible for aberrant CpG island DNA methylation observed in human T-ALL. Notably, this provides the first evidence that a pre-leukemic phase might be present in a large subset of human T-ALLs, and that epigenetic aberrations, either in the DNA methylation or histone methylation machinery are one of the first detectable alterations during T-ALL development. Finally, using patient derived xenografts (PDX), we show that DNA hypomethylation by the FDA-approved hypomethylating agent Decitabine is very effective in treating T-ALL. Gene expression profiling revealed that the anti-leukemic effect is exerted by down-regulation of the oncogenic MYC pathway. However, by profiling these PDX T-ALLs by EPIC arrays, we unexpectedly uncover that the age-related CpG island hypermethylation signature is completely resistant to Decitabine treatment. Altogether, our work demonstrates that DNA methylation reflects the epigenetic history of leukemic T cells and suggests that methylation-based subtypes of human T-ALL have followed a different trajectory towards T-cell transformation, possibly mediated by differences in the self-renewing capacity of the putative T-ALL cell-of-origin. Given that the concept of preleukemic thymocytes has only been reported in T-ALL mouse models so far, we here provide, for the first time, conceptual evidence that a pre-leukemic phase might also be involved in the pathogenesis of the human disease. Disclosures No relevant conflicts of interest to declare.

Epigenome-wide association study of DNA methylation and microRNA expression highlights novel pathways for human complex traits

ABSTRACTDNA methylation (DNAm) and microRNAs (miRNAs) have been implicated in a wide-range of human diseases. While often studied in isolation, DNAm and miRNAs are not independent. We analyzed associations of expression of 283 miRNAs with DNAm at >400K CpG sites in whole blood obtained from 3565 individuals and identified 227 CpGs at which differential methylation was associated with the expression of 40 nearby miRNAs (cis-miR-eQTMs) at FDR<0.01, including 91 independent CpG sites at r2 < 0.2. cis-miR-eQTMs were enriched for CpGs in promoter and polycomb-repressed state regions, and 60% were inversely associated with miRNA expression. Bidirectional Mendelian randomization (MR) analysis further identified 58 cis-miR-eQTMCpG-miRNA pairs where DNAm changes appeared to drive miRNA expression changes and opposite directional effects were unlikely. Integration of genetic variants in joint analyses revealed an average partial between cis-miR-eQTM CpGs and miRNAs of 2% after conditioning on site-specific genetic variation, suggesting that DNAm is an important epigenetic regulator of miRNA expression. Finally, two-step MR analysis was performed to identify putatively causal CpGs driving miRNA expression in relation to human complex traits. We found that an imprinted region on 14q32 that was previously identified in relation to age at menarche is enriched with cis-miR-eQTMs. Nine CpGs and three miRNAs at this locus tested causal for age at menarche, reflecting novel epigenetic-driven molecular pathways underlying this complex trait. Our study sheds light on the joint genetic and epigenetic regulation of miRNA expression and provides insights into the relations of miRNAs to their targets and to complex phenotypes.

Abstract 4102: DNA methylation and repressive histones in the promoters of immune checkpoints in tumor tissues and peripheral blood of breast and colorectal cancer patients

Abstract Higher expression of immune checkpoints in tumor microenvironment plays significant roles in inhibiting anti-tumor immunity, which is associated with poor prognosis and progression of cancer patients. Major epigenetic modifications in both DNA and histone could be involved in upregulation of immune checkpoints (ICs) in cancer. We investigated transcriptomic expression of different ICs and some ligands including PD-L1 and galectin-9 in the tumor microenvironment (TME) and in circulation of primary breast cancer (PBC) and colorectal cancer (CRC) patients. We found that transcriptomic expressions of PD-1, CTLA-4, TIM-3 and LAG-3 in PBC and PD-1, CTLA-4, TIM-3, TIGIT, PD-L1 and galectin-9 in CRC were significantly upregulated in tumor tissues (TT), compared with normal tissues (NT). Additionally, expressions of TIM-3, TIGIT, and PD-L1 were significantly upregulated, while LAG-3 was downregulated in peripheral blood of both PBC and CRC patients. We then investigated the epigenetic modifications beyond this upregulation of immune checkpoint genes. Interestingly, we found that CpG islands in the promoters of PD-1, CTLA-4 and TIM-3 in PBC, and CTLA-4 and TIGIT in CRC patients were significantly hypomethylated in tumor compared with normal tissues. Notably, CpGs in PD-L1 promoter were completely hypomethylated in PBC and CRC in both TT and NT. In addition, PD-L1 was hypomethylated in in peripheral blood of both PBC and CRC patients, while TIGIT was hypomethylated only in circulation of CRC patients, compared with healthy donors. Next, we checked the abundance of repressive histones (H3K9me3 and H3K27me3) in the promoter regions of ICs/ligands. We found that in PBC TT, distribution of both H3K9me3 and H3K27me3 was lower in the promoters of PD-1, CTLA-4 and LAG-3, while distribution of only H3K27me3 was lower in TIM-3 promoter, compared with NT. Furthermore, in CRC TT, the distribution of H3K9me3 was lower in PD-1 and TIGIT promoters, and H3K27me3 was lower in CTLA-4 promoter, while both H3K9me3 and H3K27me3 were lower in TIM-3 promoter, compared with NT. Our data demonstrate that both DNA methylation and repressive histones are involved in upregulation of ICs and their ligands in the tumor microenvironment and peripheral blood of breast and colorectal cancer patients. These epigenetic modifications could be useful as diagnostic/prognostic biomarkers and/or therapeutic targets in breast and colorectal cancers. Citation Format: Eyad Elkord, Varun Sasidharan Nair, Salman M. Toor. DNA methylation and repressive histones in the promoters of immune checkpoints in tumor tissues and peripheral blood of breast and colorectal cancer patients [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 4102.

3-Oxoacid CoA transferase 1 as a therapeutic target gene for cisplatin-resistant ovarian cancer.

Ovarian cancer (OC) is the second leading cause of mortality from gynecological malignancies and has the highest mortality rate worldwide. As it is commonly asymptomatic during the early stages of the disease, >70% of patients with OC are diagnosed at advanced stages with metastasis. Despite treatment methods, including optimal debulking surgery and chemotherapy with the platinum-based drug cisplatin, OC recurrence is often inevitable, with an overall 5-year survival rate of 45%, mostly due to the steady development of cisplatin resistance. To identify genes involved in cisplatin resistance, the present study determined the half-maximal inhibitory concentrations of eight different OC cell lines and classified them into two groups (sensitive and resistant). mRNA expression was analyzed with GeneChip Human Gene 1.0 ST Arrays, and DNA methylation profiles were evaluated with the HumanMethylation450 BeadChip. Using an integrated approach of analyzing gene expression levels and DNA methylation profiles simultaneously, 26 genes were selected that were differentially expressed and methylated between the resistant and sensitive groups. Among these 26 genes, 3-oxoacid CoA transferase 1 (OXCT1), which was demonstrated to be downregulated and hypermethylated at promoter CpGs in the cisplatin-resistant group compared with the cisplatin-sensitive group, was selected for further investigation. Treatment with a DNA methyltransferase inhibitor restored hypermethylation-mediated gene silencing of OXCT1 in the cisplatin-resistant group, but not in the cisplatin-sensitive group. Furthermore, overexpression of OXCT1 conferred sensitivity to cisplatin in OC cells. The results of the present study suggest that OXCT1 serves an important role in conferring cisplatin sensitivity, and may provide a potential therapeutic target for cisplatin chemotherapy in patients with recurrent OC.

TGFβ promotes mesenchymal phenotype of pancreatic cancer cells, in part, through epigenetic activation of VAV1.

The highly homeostasis-resistant nature of cancer cells leads to their escape from treatment and to liver metastasis, which in turn makes pancreatic ductal adenocarcinoma (PDAC) difficult to treat, especially the squamous/epithelial-to-mesenchymal transition (EMT)-like subtype. As the molecular mechanisms underlying tumour heterogeneity remain elusive, we investigated whether epigenetic regulation might explain inter-individual differences in the progression of specific subtypes. DNA methylation profiling performed on cancer tissues prior to chemo/radiotherapy identified one hypermethylated CpG site (CpG6882469) in the VAV1 gene body that was correlated with demethylation of two promoter CpGs (CpG6772370/CpG6772811) in both PDAC and peripheral blood. Transforming growth factor β treatment induced gene-body hypermethylation, dissociation of DNMT1 from the promoter, and VAV1 expression via SMAD4 and mutant KrasG12D. Pharmacological inhibition of TGFβ-VAV1 signalling decreased the squamous/EMT-like cancer cells, promoted nuclear VAV1 localization, and enhanced the efficacy of gemcitabine in prolonging the survival of KPfl/flC mice. Together, the three VAV1 CpGs serve as biomarkers for prognosis and early detection, and the TGFβ-VAV1 axis represents a therapeutic target.

Genome-wide DNA methylation profiling of peripheral blood mononuclear cells in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a stress-sensitive disorder. Environmental factors including stress can trigger epigenetic changes, which have not been well-studied in IBS. We performed a pilot study investigating genome-wide DNA methylation of IBS patients and healthy controls (HCs) to identify potential epigenetic markers and associated pathways. Additionally, we investigated relationships of epigenetic changes in selected genes with clinical traits. Twenty-seven IBS patients (59% women; 10 IBS-diarrhea, 8 IBS-constipation, 9 IBS-mixed) and 23 age- and sex-matched HCs were examined. DNA methylation from peripheral blood mononuclear cells (PBMCs) was measured using HM450 BeadChip, and representative methylation differences were confirmed by bisulphite sequencing. Gene expression was measured using quantitative PCR. Gastrointestinal (GI) and non-GI symptoms were measured using validated questionnaires. Associations were tested using non-parametric methods. Genome-wide DNA methylation profiling of IBS patients compared with HCs identified 133 differentially methylated positions (DMPs) (mean difference ≥10%; p < 0.05). These genes were associated with gene ontology terms including glutathione metabolism related to oxidative stress and neuropeptide hormone activity. Validation by sequencing confirmed differential methylation of subcommissural organ (SCO)-Spondin (SSPO), glutathione-S-transferases mu 5 (GSTM5), and tubulin polymerization promoting protein genes. Methylation of two promoter CpGs in GSTM5 was associated with epigenetic silencing. Epigenetic changes in SSPO gene were positively correlated with hospital anxiety and depression scores in IBS patients (r > 0.4 and false discovery rate <0.05). This study is the first to comprehensively explore the methylome of IBS patients. We identified DMPs in novel candidate genes which could provide new insights into disease mechanisms; however, these preliminary findings warrant confirmation in larger, independent studies.

Abstract 1105: Clinical associations between altered bladder cancer genes

Abstract Next generation sequencing (NGS) has identified frequently altered cancer genes in urologic cancers including bladder cancer (BCa). We examined BCa tumors from 54 U.S. patients using exome NGS and targeted sequencing, and compared these to 99 extensively characterized Chinese patient tumors (Guo et al., 2013). We are first to show that telomerase (TERT) is the gene most frequently altered by somatic sequence changes in 37/54 (69%) of tumors and that TERT is also altered by rare and common germline variants in 30/54 (56%) tumors. Most TERT germline variants were novel (19/20) and three variants were confirmed as both somatic and germline in distinct tumors, including c. - 245 T&amp;gt;C (rs2853669), the most recurrent variant. A subset of variants created or altered transcription factor binding sites and promoter CpGs. Telomere length was significantly shorter in tumor versus adjacent normal tissue from the same patient (p = 0.004) indicating TERT minimally lengthens telomeres in rapidly proliferating BCa cells. Somatic TERT alterations were not associated with stage, grade, or alterations in other BCa genes identified by exome sequencing. STAG2 is altered by somatic mutations in 20/153 (13%) tumors and by X chromosome deletions and promoter hypermethylation. STAG2 mutant tumors had higher levels of aneuploidy (p = 0.01) and Kaplan-Meier analysis indicated a poor prognosis due to significantly shorter survival times (p &amp;lt; 0.001). Somatic BAP1 alterations were associated with papillary histologic features in 15% of Caucasian patient tumors but were infrequently observed in Asian patients (p = 0.003), which remained marginally significant after accounting for differences in tumor grade and stage (p = 0.037). BAP1 mutations co-occurred with histone 3 lysine 27-specific demethylase (KDM6A) mutations (p = 0.017), indicating the cancer genes might provide complementary survival advantages to cancer cells. We find that cancer gene alterations are highly predictive of canonical features of the BCa phenotype and ∼4-5 tumor suppressors are altered for each oncogene. X chromosome cancer genes such as STAG2 and KDM6A have a single copy in men and their lethality (as shown for STAG2) likely contributes to the gender bias in cancer. We speculate that TERT alterations are early events in BCa and identification of somatic alterations in urine may provide a biomarker for early BCa diagnosis. BAP1 mutations contribute to frequent BRCA pathway alterations in BCa and indicate treatment with poly(ADP ribose) polymerase inhibitors would be effective. Citation Format: Michael L. Nickerson, Kate M. Im, Sevilay Turan, Lee E. Moore, Michael Dean, Dan Theodorescu. Clinical associations between altered bladder cancer genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1105. doi:10.1158/1538-7445.AM2015-1105

Abstract 1066: Racial disparities of genome-wide DNA methylation profiles and association with breast tissues biomarkers in healthy women

Abstract Breast cancer is the most common cancer in women, with more aggressive disease observed among African Americans (AA) compared to European Americans (EA). It is a complex and heterogeneous disease, characterized by genetic and epigenetic alterations. Racial differences in DNA methylation have been shown in breast cancers, but it is not clear if these differences exist in healthy women who have not had breast cancer. We identified genome-wide differentially methylated (DM) CpGs by race, in order to: 1) understand methylation patterns of promoter and non-promoter related DM CpGs; 2) study their association with tissue biomarkers of proliferation and differentiation; and 3) examine if these correlations were similar for AA and EA. Normal breast tissues were collected from healthy women undergoing reduction mammoplasty (N = 83). Tissue DNA was analyzed using the Infinium HumanMethylation450 BeadChip for differential genome-wide methylation between AA (N = 22) and EA (N = 61). Analysis methods included age and BMI-adjusted Generalized Linear Regression models, hierarchical clustering and Principal Component Analysis (PCA). Correction for multiple comparisons was performed. We further evaluated the performance of the threshold for classification between EA and AA using the area under the curve (AUC) and by 10-fold cross validation. Biological functions of the DM genes were assigned using the Ingenuity Pathway Analysis (IPA). Partial correlation analysis was performed between the DM CpGs and proliferation (Ki-67, Leptin, IGF1, and IGFBP3) or differentiation (Adiponectin)-related tissue biomarkers. 485 or 23 DM CpGs were identified after FDR or Bonferroni correction at p&amp;lt;0.05, respectively. The AUC was 0.8 and 1 and% correct rates were 78% and 100%, respectively. A heat map and PCA showed distinct methylation patterns between EA and AA. A larger variation was observed in promoters while relatively consistent methylation levels were found in intergenic regions. IPA among 177 DM CpGs in promoter regions showed 136 genes associated with cancers that play roles in cell death and survival, cellular development, and cell-to-cell signaling. Of the 23 DM CpGs, 9 CpGs are known oncogenes or tumor suppressors in breast or other cancers. 8 CpGs were significantly correlated with at least one biomarker in EA (p&amp;lt;0.05), but not in AA. One CpG site was significantly correlated in AA only (p&amp;lt;0.05). 4 DM CpGs were differentially correlated between EA and AA at P-interaction&amp;lt;0.05. This is the first genome-wide study to show differences in methylation between AA and EA in normal breast tissues. A subset of the CpGs was correlated with breast tissue biomarkers of proliferation and differentiation and these correlation patterns were different by race. These findings may provide further insights on the contribution of DNA methylation differences as determinants of racial disparities in breast cancer. Citation Format: Min-Ae Song, Catalin Marian, Theodore M. Brasky, Daniel Weng, Cenny Taslim, Jo L. Freudenheim, Peter G. Shields. Racial disparities of genome-wide DNA methylation profiles and association with breast tissues biomarkers in healthy women. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1066. doi:10.1158/1538-7445.AM2015-1066

Genome-Wide Scan for Methylation Profiles in Keloids.

Keloids are benign fibroproliferative tumors of the skin which commonly occur after injury mainly in darker skinned patients. Medical treatment is fraught with high recurrence rates mainly because of an incomplete understanding of the biological mechanisms that lead to keloids. The purpose of this project was to examine keloid pathogenesis from the epigenome perspective of DNA methylation. Genome-wide profiling used the Infinium HumanMethylation450 BeadChip to interrogate DNA from 6 fresh keloid and 6 normal skin samples from 12 anonymous donors. A 3-tiered approach was used to call out genes most differentially methylated between keloid and normal. When compared to normal, of the 685 differentially methylated CpGs at Tier 3, 510 were hypomethylated and 175 were hypermethylated with 190 CpGs in promoter and 495 in nonpromoter regions. The 190 promoter region CpGs corresponded to 152 genes: 96 (63%) were hypomethylated and 56 (37%) hypermethylated. This exploratory genome-wide scan of the keloid methylome highlights a predominance of hypomethylated genomic landscapes, favoring nonpromoter regions. DNA methylation, as an additional mechanism for gene regulation in keloid pathogenesis, holds potential for novel treatments that reverse deleterious epigenetic changes. As an alternative mechanism for regulating genes, epigenetics may explain why gene mutations alone do not provide definitive mechanisms for keloid formation.

Abstract 565: Telomerase reverse transcriptase promoter alterations in human bladder cancer

Abstract The telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of the enzyme telomerase, (aka telomere terminal transferase), which contains both protein and RNA subunits. TERT is a RNA-dependent polymerase which increases the length of telomeres at the ends of each chromosome by adding TTAGGG repeats. TERT polymerase uses RNA as a complementary primer to elongate telomeric G-rich repeat sequences in the 5’ to 3’ direction. TERT is essential and active in germ cells, stem cells, primary tumors, and immortalized cells and acts to maintain adequate telomeres. However, in somatic cells, TERT expression is reduced or absent and telomeres gradually shorten during the S phase of each cell cycle due to replication-dependent loss of telomere terminal sequences. Adequate telomere length is important for preserving genome stability, regulating cell replication, and preventing cell death. Somatic TERT promoter alterations were recently reported in melanoma, glioma, and in small sample sets from other cancers including bladder cancer. These are thought to drive re-expression of TERT in cancer cells. In this study, given recent reports of somatic TERT promoter alterations in bladder cancer, polymerase chain reaction and Sanger sequencing were performed to examine a region of the TERT promoter in 58 bladder tumors. The TERT promoter was altered in 90% of 58 tumors by 44 germline (21 unique, 20 novel) and 53 somatic variants (11 unique, 4 novel). Somatic alterations were observed in 67% of tumors. The majority of variants were C&amp;gt;T DNA changes, three variants occurred as both germline and somatic, and a subset of variants created or altered transcription factor binding sites and promoter CpGs. Analyses of telomere length showed significantly shorter telomeres in tumor tissue versus adjacent normal tissue from the same patient (p=0.004) indicating TERT may not sufficiently lengthen telomeres in rapidly proliferating cancer cells. Interestingly, somatic TERT promoter alterations were not associated with stage, grade, or alterations in other bladder cancer genes identified by exome sequencing. We speculate that TERT promoter alterations are frequent and early events in bladder cancer and identification of TERT alterations in urine may provide a biomarker for early diagnosis and monitoring of bladder cancer. Note: This abstract was not presented at the meeting. Citation Format: Sevilay Turan. Telomerase reverse transcriptase promoter alterations in human bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2014-565

Identification of methylation quantitative trait loci (mQTLs) influencing promoter DNA methylation of alcohol dependence risk genes.

Interaction of DNA methylation and sequence variants that are methylation quantitative trait loci (mQTLs) may influence susceptibility to diseases such as alcohol dependence (AD). We used genome-wide genotype data from 268 African Americans (AAs: 129 AD cases and 139 controls) and 143 European Americans (EAs: 129 AD cases and 14 controls) to identify mQTLs that were associated with promoter CpGs in 82 AD risk genes. 282 significant mQTL-CpG pairs (9.9 × 10(-100) ≤ P(nominal) ≤ 7.7 × 10(-8)) in AAs and 313 significant mQTL-CpG pairs (2.7 × 10(-53) ≤ P(nominal) ≤ 9.9 × 10(-8)) in EAs were identified [i.e., mQTL-CpG associations survived multiple-testing correction, q values (false discovery rate) ≤ 0.05]. The most significant mQTL was rs1800759, which was strongly associated with CpG cg12011299 in both AAs (P(nominal) = 9.9 × 10(-100); q = 6.7 × 10(-91)) and EAs (P(nominal) = 2.7 × 10(-53); q = 1.4 × 10(-44)). Rs1800759 (previously known to be associated to AD) and CpG cg12011299 (distance: 37 bp) are both located in alcohol dehydrogenase (ADH) 4 gene (ADH4) promoter region. In general, the strength of association between mQTLs and CpGs was inversely correlated with the distance between them. Association was also influenced by race and AD. Additionally, 48.3 % of the mQTLs identified in AAs and 65.6 % of the mQTLs identified in EAs were predicted to be expression QTLs. Three mQTLs (rs2173201, rs4147542, and rs4147541 in ADH1B-AHD1C gene cluster region) found in AAs were previously identified by our genome-wide association studies as being significantly associated with AD in AAs. Thus, DNA methylation, which can be influenced by sequence variants and is implicated in gene expression regulation, appears to at least partially underlie the association of genetic variation with AD.

Methylation Regulation of NANOGP8 Expression in Lung Cancer Cell Line

The nearly identified retrogene NANOGP8 is from a stem cell transcription factor NANOG gene, which expressed in multiple cancers,but generally not in normal tissues and its function is related to cancer development and progression. To investigate the relationship between NANOGP8 expression level and methylation sites at the gene promoters. Bisulphate Sequencing in lung cancer cell line A549 as well as the normal fibroblast cells were performed. Genomic DNA was extracted from these cells and treated with bisulphate,then PCR was performed in the NANOG promoter,encompassing a total of 10 CpGs within nucleotides-1449 to-1295 and-1168 to-952 relative to the transcription start site( TSS) reported by Park. Two regions including 9 CpGs in the potential NANOGP8 promoter,which located within nucleotides-1529 to-1401 and-690 to-587 relative to the TSS of NANOGP8 predicted by Methprimer program were also examined. PCR products were then ligated to pGEM-T vector. The recombinant plasmid was transformed into E. coli DH5α and eight clones for each region were sequenced and blasted. There are no significant changes of the percentage of clones with methylation in NANOG promoter in A549 cell line compared to in the fibroblast cells( 65% vs. 67. 5%,P = 0. 62). However, the percentage at the two regions of NANOGP8 shows a significant increase from A549 cell line to the fibroblast cells( 12. 5% vs. 59. 7%,P 0. 001). NANOGP8 transcriptional level was detected by RT-PCR in both cell lines and found NANOGP8 is highly expressed in A549 cell line but not in the fibroblast cells. The methylation status of 5'-flanking regions of human NANOGP8 is associated with the NANOGP8 expression in lung cell line and may relate to lung cancer development and progression.

Quantitative leukocyte BDNF promoter methylation analysis in bipolar disorder.

BackgroundBipolar disorder (BD) is a complex psychiatric phenotype with a high heritability and a multifactorial etiology. Multisite collaborative efforts using genome-wide association studies (GWAS) have identified only a portion of DNA sequence-based risk factors in BD. In addition to predisposing DNA sequence variants, epigenetic misregulation may play an etiological role in BD and account for monozygotic twin discordance, parental origin effects, and fluctuating course of BD. In this study, we investigated DNA methylation of the brain-derived neurotrophic factor (BDNF) gene in BD.MethodsFifty participants with BD were compared to the same number of age- and sex-matched controls for DNA methylation differences at BDNF promoters 3 and 5. DNA methylation reads were obtained using a mass spectrophotometer for 64 cytosine-guanine (CpG) sites in 36 CpG ‘units’ across three amplicons of BDNF promoters 3 and 5.Results and DiscussionMethylation fractions differed between BD participants and controls for 11 of 36 CpG units. Five CpG units, mostly in promoter 5, remained significant after false discovery rate correction (FDR) (p values ≤ 0.004) with medium to large effect sizes (Cohen's d ≥ 0.61). Several of the significant CpGs overlapped with or were immediately adjacent to transcription factor binding sites (TFBSs) - including two of the FDR-significant CpG units in promoter 5. For the CpGs in promoter 3, there was a positive and significant correlation between age at sample collection and DNA methylation fraction (rho = 0.56, p = 2.8 ×10−5) in BD cases, but not in controls. Statistically significant differences in mean methylation fraction at 5/36 CpG units (after FDR), some at or immediately adjacent to TFBSs, suggest possible relevance for the current findings to BD etiopathogenesis. The positive correlation between age and methylation seen in promoter 3 is consistent with age-related decline in BDNF expression previously reported. Future studies should provide more exhaustive epigenetic study of the BDNF locus to better characterize the relationship between BDNF methylation differences and BD.Electronic supplementary materialThe online version of this article (doi:10.1186/2194-7511-1-28) contains supplementary material, which is available to authorized users.