Comprehensive DNA Methylation Analysis Research Articles

Comprehensive review and updated analysis of DNA methylation in hepatocellular carcinoma: From basic research to clinical application.

Hepatocellular carcinoma (HCC) is a primary malignant tumour, ranking second in global mortality rates and posing significant health threats. Epigenetic alterations, particularly DNA methylation, have emerged as pivotal factors associated with HCC diagnosis, therapy, prognosis and malignant progression. However, a comprehensive analysis of the DNA methylation mechanism driving HCC progression and its potential as a therapeutic biomarker remains lacking. This review attempts to comprehensively summarise various aspects of DNA methylation, such as its mechanism, detection methods and biomarkers aiding in HCC diagnosis, treatment and prognostic assessment of HCC. It also explores the role of DNA methylation in regulating HCC's malignant progression and sorafenib resistance, alongside elaborating the therapeutic effects of DNA methyltransferase inhibitors on HCC. A detailed examination of these aspects underscores the significant research on DNA methylation in tumour cells to elucidate malignant progression mechanisms, identify diagnostic markers and develop new tumour-specific inhibitors for HCC. KEY POINTS: A comprehensive summary of various aspects of DNA methylation, such as its mechanism, detection methods and biomarkers aiding in diagnosis and treatment. The role of DNA methylation in regulating hepatocellular carcinoma's (HCC) malignant progression and sorafenib resistance, alongside elaborating therapeutic effects of DNA methyltransferase inhibitors. Deep research on DNA methylation is critical for discovering novel tumour-specific inhibitors for HCC.

Genome-wide methylation profiling reveals extracellular vesicle DNA as an ex vivo surrogate of cancer cell-derived DNA

Extracellular vesicle-derived DNA (evDNA) encapsulates the complete genome and mutational status of cells; however, whether cancer cell-derived evDNA mirrors the epigenetic features of parental genomic DNA remains uncertain. This study aimed to assess and compare the DNA methylation patterns of evDNA from cancer cell lines and primary cancer tissues with those of the nuclear genomic DNA. We isolated evDNA secreted by two cancer cell lines (HCT116 and MDA-MB-231) from various subcellular compartments, including the nucleus and cytoplasm. Additionally, we obtained evDNA and nuclear DNA (nDNA) from the primary cancer tissues of colon cancer patients. We conducted a comprehensive genome-wide DNA methylation analysis using the Infinium Methylation EPIC BeadChip, examining > 850,000 CpG sites. Remarkable similarities were observed between evDNA and nDNA methylation patterns in cancer cell lines and patients. This concordance extended to clinical cancer tissue samples, showcasing the potential utility of evDNA methylation patterns in deducing cellular origin within heterogeneous populations through methylation-based deconvolution. The observed concordance underscores the potential of evDNA as a noninvasive surrogate marker for discerning tissue origin, particularly in cancer tissues, offering a promising future for cancer diagnostics. This finding enhances our understanding of cellular origins and would help develop innovative diagnostic and therapeutic strategies for cancer.

Integrated analysis of DNA methylome and transcriptome revealing epigenetic regulation of CRIR1-promoted cold tolerance

BackgroundDNA methylation contributes to the epigenetic regulation of nuclear gene expression, and is associated with plant growth, development, and stress responses. Compelling evidence has emerged that long non-coding RNA (lncRNA) regulates DNA methylation. Previous genetic and physiological evidence indicates that lncRNA-CRIR1 plays a positive role in the responses of cassava plants to cold stress. However, it is unclear whether global DNA methylation changes with CRIR1-promoted cold tolerance.ResultsIn this study, a comprehensive comparative analysis of DNA methylation and transcriptome profiles was performed to reveal the gene expression and epigenetic dynamics after CRIR1 overexpression. Compared with the wild-type plants, CRIR1-overexpressing plants present gained DNA methylation in over 37,000 genomic regions and lost DNA methylation in about 16,000 genomic regions, indicating a global decrease in DNA methylation after CRIR1 overexpression. Declining DNA methylation is not correlated with decreased/increased expression of the DNA methylase/demethylase genes, but is associated with increased transcripts of a few transcription factors, chlorophyll metabolism and photosynthesis-related genes, which could contribute to the CRIR1-promoted cold tolerance.ConclusionsIn summary, a first set of transcriptome and epigenome data was integrated in this study to reveal the gene expression and epigenetic dynamics after CRIR1 overexpression, with the identification of several TFs, chlorophyll metabolism and photosynthesis-related genes that may be involved in CRIR1-promoted cold tolerance. Therefore, our study has provided valuable data for the systematic study of molecular insights for plant cold stress response.

Reduced CHH methylation levels reveal a critical role of aging pathway genes in Moso bamboo flowering

Moso bamboo holds significant economic importance in China, serving various purposes, such as food, material, ornamentation, and greenery. Despite its versatility, the occurrence of flowering in Moso bamboo poses a threat to bamboo forests, resulting in substantial losses. The underlying cause of bamboo flowering remains elusive. Dynamic fluctuations in DNA methylation govern the transcriptional levels of crucial genes pivotal for plant growth and development. In this study, we conducted comprehensive DNA methylation (by whole-genome bisulfite sequencing) and transcriptome (by RNA-seq) analyses on non-flowering leaves, flowering leaves, and spikelets of Moso bamboo. Our findings revealed a notable reduction in the overall DNA methylation level, particularly CHH methylation, from leaves to spikelets, influencing the expression of differentially regulated genes. Notably, we identified DNA methylation as a regulatory mechanism for numerous flowering-related genes, including SPLs, FT, and SOC1. Specifically, the SPL3f gene, a key regulator of the aging pathway, exhibited hypomethylation and a high expression level in spikelets. Conversely, SOC1c displayed transcriptional silencing attributed to hypermethylation in the CHH context in the leaves of non-flowering plants. DNA methylation may affect the flowering mechanism of Moso bamboo by regulating the expression of key genes. In summary, our results shed light on the dynamic changes in DNA methylation between leaves and spikelets, unraveling an important epigenetic modification mechanism for flowering in Moso bamboo.

Dapagliflozin administration to a mouse model of type 2 diabetes induces DNA methylation and gene expression changes in pancreatic islets

Decreased pancreatic β-cell volume is a serious problem in patients with type 2 diabetes mellitus, and there is a need to establish appropriate treatments. Increasingly, sodium/glucose cotransporter 2 (SGLT2) inhibitors, which have a protective effect on pancreatic β-cells, are being prescribed to treat diabetes; however, the underlying mechanism is not well understood. We previously administered SGLT2 inhibitor dapagliflozin to a mouse model of type 2 diabetes and found significant changes in gene expression in the early-treated group, which led us to hypothesize that epigenetic regulation was a possible mechanism of these changes. Therefore, we performed comprehensive DNA methylation analysis by methylated DNA immunoprecipitation using isolated pancreatic islets after dapagliflozin administration to diabetic model mice. As a result, we identified 31 genes with changes in expression due to DNA methylation changes. Upon immunostaining, cystic fibrosis transmembrane conductance regulator and cadherin 24 were found to be upregulated in islets in the dapagliflozin-treated group. These molecules may contribute to the maintenance of islet morphology and insulin secretory capacity, suggesting that SGLT2 inhibitors’ protective effect on pancreatic β-cells is accompanied by DNA methylation changes, and that the effect is long-term and not temporary. In future diabetes care, SGLT2 inhibitors may be expected to have positive therapeutic effects, including pancreatic β-cell protection.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

Abstract 5106: Pharmacological targeting of LSD1 prolongs animal survival times in patient derived orthotopic xenograft models of posterior fossa ependymoma in combination with fractionated radiation

Abstract Background: Posterior Fossa Group A Ependymoma (PFA EPN) constitute one of the most aggressive and highly recurrent molecular subtypes of childhood Ependymomas. We recently performed a comprehensive genome-wide DNA methylation analysis in recurrent pediatric EPN tumors from patients that were followed up for over 13 years. Integrative analysis between tumor DNA methylation and histone modification identified LSD1 as a druggable driver of PFA EPN recurrence. In this study, we examined the therapeutic efficacy of our newly developed LSD1 inhibitor SYC-836 in PDOX models of recurrent PFA EPN. Methodology: To determine the in vitro anti-tumor activities of SYC-836, paired PFA EPN xenograft cell cultures (monolayer and 3D neurospheres) were treated with SYC-836 at 8 concentrations (0.02-25 µM) and examined for changes of cell proliferation every 3-4 days through day 14. To validate the drug’s in vivo efficacy, two established posterior fossa EPN PDOX models, ICb-4423EPN and ICb-2002EPN, were utilized. Forty 8-week old SCID mice of both genders received intra-cerebellar tumor cell implantation and were divided into 4 treatment groups (n=10 per group): 1) control, 2) radiation (2 Gy/day x 5 days), 3) SYC-836 (15 mg/kg, daily intra-peritoneal injection for 28 days), and 4) combination (radiation + SYC-836). Animal survival times were analyzed using log-rank analysis. Changes in LSD1 protein expression and histone lysine methylation were examined through western hybridization. Results: SYC-836 strongly suppressed PFA EPN cell growth in vitro, in both, a time- and dose-dependent manner. While there was no survival benefit with either SYC-836 only (P=0.186) or XRT only (P=0.205); combining SYC-836 with XRT significantly (P=0.04) prolonged the median survival time of ICb-4423EPN, from 46 days in the untreated group to 59 days; and in ICb-2002EPN, even more prominent, from 136 days to 180 days (P=0.007). SYC-836 was well tolerated in mice with no loss of body weight or other toxicities. Changes in LSD1 protein expression and H3K4me2 were detected through western hybridization during and after SYC-836 treatment. Conclusion: Our data support LSD1 as a druggable driver of PFA recurrence and highlights strong anti-tumor activities of SYC-836 in combination with radiation, a standard therapy of pediatric EPN. SYC-836 may have a role in the clinical setting by either reducing radiation dosages or be an adjuvant agent to other chemotherapy drugs in our treatment approach for ependymoma. Citation Format: Milagros M. Suarez Palacios, Sibo Zhao, Huiyuan Zhang, Lin Qi, Holly Lindsay, Mari Kogiso, Frank Braun, Adekunle M. Adesina, Yuchen Du, Yongcheng Song, Xiao-Nan Li. Pharmacological targeting of LSD1 prolongs animal survival times in patient derived orthotopic xenograft models of posterior fossa ependymoma in combination with fractionated radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5106.

Abstract 155: Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered

Abstract Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumor type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumors. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogenous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large pediatric and adult cohort of 29 tumors H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbor Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbors atypical radiological and histopathological profiles with calcification and/or a solid tumor component both for BRAFV600E and FGFR1MUT cases. Contrary to other DMG, these tumors occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. The analyses of a H3.3-K27M BRAFV600E tumor at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis, indicating a distinct oncogenesis from classical DMG despite an identical founder H3K27 alteration, and possible subclonal evolution.In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype-phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management. Our work also lays the foundations for therapeutic development highly-needed against this deadly disease. Citation Format: Lucie Auffret, Yassine Ajlil, Arnault Tauziede-Espariat, Thomas Kergrohen, Chloé Puiseux, Laurent Riffaud, Pascale Blouin, Anne-Isabelle Bertozzi, Pierre Leblond, Klas Blomgren, Sebastien Froelich, Alberto Picca, Mehdi Touat, Marc Sanson, Kevin Beccaria, Thomas Blauwblomme, Volodia Dangouloff-Ros, Nathalie Boddaert, Pascale Varlet, Marie-Anne Debily, Jacques Grill, David Castel. Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 155.

Comparative and functional analysis unveils the contribution of photoperiod to DNA methylation, sRNA accumulation, and gene expression variations in short-day and long-day grasses.

Photoperiod employs complicated networks to regulate various developmental processes in plants, including flowering transition. However, the specific mechanisms by which photoperiod affects epigenetic modifications and gene expression variations in plants remain elusive. In this study, we conducted a comprehensive analysis of DNA methylation, small RNA (sRNA) accumulation, and gene expressions under different daylengths in facultative long-day (LD) grass Brachypodium distachyon and short-day (SD) grass rice. Our results showed that while overall DNA methylation levels were minimally affected by different photoperiods, CHH methylation levels were repressed under their favorable light conditions, particularly in rice. We identified numerous differentially methylated regions (DMRs) that were influenced by photoperiod in both plant species. Apart from differential sRNA clusters, we observed alterations in the expression of key components of the RNA-directed DNA methylation pathway, DNA methyltransferases, and demethylases, which may contribute to the identified photoperiod-influenced CHH DMRs. Furthermore, we identified many differentially expressed genes in response to different daylengths, some of which were associated with the DMRs. Notably, we discovered a photoperiod-responsive gene MYB11 in the transcriptome of B.distachyon, and further demonstrated its role as a flowering inhibitor by repressing FT1 transcription. Together, our comparative and functional analysis sheds light on the effects of daylength on DNA methylation, sRNA accumulation, and gene expression variations in LD and SD plants, thereby facilitating better designing breeding programs aimed at developing high-yield crops that can adapt to local growing seasons.

Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.

A new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered: a clinico-radiological and histomolecular characterisation

Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumour type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumours. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogeneous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large paediatric and adult cohort of 29 tumours H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbour Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbours atypical radiological and histopathological profiles with calcification and/or a solid tumour component both for BRAFV600E and FGFR1MUT cases. The analyses of a H3.3-K27M BRAFV600E tumour at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis. Contrary to other DMG, these tumours occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype/phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management.

The Effect of Nanomaterials on DNA Methylation: A Review.

DNA methylation is an epigenetic mechanism that involves the addition of a methyl group to a cytosine residue in CpG dinucleotides, which are particularly abundant in gene promoter regions. Several studies have highlighted the role that modifications of DNA methylation may have on the adverse health effects caused by exposure to environmental toxicants. One group of xenobiotics that is increasingly present in our daily lives are nanomaterials, whose unique physicochemical properties make them interesting for a large number of industrial and biomedical applications. Their widespread use has raised concerns about human exposure, and several toxicological studies have been performed, although the studies focusing on nanomaterials' effect on DNA methylation are still limited. The aim of this review is to investigate the possible impact of nanomaterials on DNA methylation. From the 70 studies found eligible for data analysis, the majority were in vitro, with about half using cell models related to the lungs. Among the in vivo studies, several animal models were used, but most were mice models. Only two studies were performed on human exposed populations. Global DNA methylation analyses was the most frequently applied approach. Although no trend towards hypo- or hyper-methylation could be observed, the importance of this epigenetic mechanism in the molecular response to nanomaterials is evident. Furthermore, methylation analysis of target genes and, particularly, the application of comprehensive DNA methylation analysis techniques, such as genome-wide sequencing, allowed identifying differentially methylated genes after nanomaterial exposure and affected molecular pathways, contributing to the understanding of their possible adverse health effects.

Abstract 6016: A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells

Abstract Background: Gut microbial colonization, represents a critical time period for establishing the overall health of an individual. The intestine is a highly dynamic tissue with high turnover of intestinal epithelial cells throughout the life span of an individual making intestinal stem cells (ISCs) critical for normal gut homeostasis. Currently, there exists a lack of knowledge in how the gut microbiota regulates DNA methylation to influence the behavior of these ISCs. Aberrant DNA methylation is now documented to be critical in the disease pathogenesis of intestinal disorders such as colorectal cancer, thus understanding how the gut microbiome influences DNA methylation and ISCs is fundamental for improving human health. Methods: We utilized the Lgr5-GFP mouse model in which endogenous GFP expression is driven by the bonafide intestinal stem cell marker Lgr5. These mice were maintained under two different housing conditions, conventional and germ free. We collected mice under the two conditions and performed dissociation of the colon and small intestine epithelial cells using EDTA dissociation. We then analyzed cell populations using flow cytometry, and collected two cell populations: the differentiated cell population (EpiCam +, GFP -) and intestinal stem cell population (EpiCam +, GFP +). To perform a genome wide comprehensive DNA methylation analysis we performed Whole Genome Bisulfite Sequencing (WGBS) which included both stem cell and differentiated cell populations of females and males at the two age groups points (postnatal day 21 (p21) and p100). Results: There was no significant difference in CpG methylation at the global level between the two housing conditions. Next, we analyzed age matched samples from germ free and conventional mice to determine if there are differentially methylated regions (DMRs) associated with the presence of intestinal microbiota. We have identified DMRs that are maintained in germ free stem cells from p21 to p100 compared to conventional mice, as well as DMRs in the differentiated cell population at both ages. Interestingly in our analysis several genes displayed robust hypomethylation of control mice versus germ free mice at the p100 time point in both the stem cell and differentiated cell populations. One such gene, Ifitm3, has been heavily associated with chronic inflammation in the colonic epithelium and a poor risk factor for colonic cancer (Li D et. Al, Clin Cancer Res. 2011). We validated Ifitm3 methylation and expression changes between cohorts using pyrosequencing and RT-PCR respectively. Conclusions: This comprehensive analysis has yielded top candidate genes for studying functional consequences of DNA methylation changes that have been previously linked to severe patient diseases including cancer. Citation Format: Robert Craig Peery, Jiejun Shi, Leah Farmer, Li Yang, Emily Lawrence, Yumei Li, Wei Li, Lanlan Shen. A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6016.

Abstract 761: Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric cancer

Abstract Aberrant DNA methylation accumulates in non-malignant gastric mucosa after exposure to environmental pathogens such as H. pylori (HP). To understand how environmental factors and DNA methylation interplay to influence primary gastric cancer (GC) risk, we performed an integrated analysis of clinical factors and DNA methylation data of gastric tissues from a longitudinally monitored cohort in Japan, with validation in a separate cohort from Singapore. The Japanese check-up cohort included 4,234 healthy subjects who underwent gastric mucosal biopsy. The median observation period was 4.2 years, and 77 subjects developed GC. GC incidence correlated with age, drinking, smoking, GC family history, and HP status in the multivariable Cox model. Next, we conducted comprehensive DNA methylation analysis using Infinium MethylationEPIC arrays on gastric tissues (n=164), including (1) mucosal biopsies from subjects who later developed GC (“future GC patients”), (2) mucosal biopsies from HP(+) subjects who did not later develop GC (“future non-GC subjects”), (3) mucosal biopsies from future non-GC HP(−) subjects (“control mucosae”), and (4) GCs and surrounding mucosae. Infinium data of gastric mucosae (n=137) collected in the GCEP cohort (Singapore) were also analyzed. DNA methylation of promoter region observed in GCs, accumulated not only in mucosae adjacent to GCs but also in the biopsy mucosae of future GC patients. Mucosae of future non-GC subjects were more methylated than control mucosae but less methylated than mucosae of future GC patients. Similar findings were observed in the GCEP cohort. DNA methylation levels were associated with clinical factors and histopathological alterations - however, in multivariable analyses, DNA methylation remained an independent GC risk factor. Methylation levels were predictive of not only higher GC risk but also a shorter period to GC incidence. We then focused on the associations between environmental factors and methylation. Heavy drinking and smoking were associated with the accumulation of DNA methylation only in HP(+) subjects. The increases in methylation over time in subjects who quit smoking were significantly attenuated compared to continuous smokers. These results suggest that pro-carcinogenic epigenetic alterations initiated by HP exposure are amplified by unfavorable but modifiable lifestyle choices. Furthermore, target genes methylated by each environmental factor may overlap in part; however, they were not necessarily methylated similarly, suggesting that the best markers for stratifying GC risk may differ for each subgroup classified by exposure to environmental factors. Indeed, candidate markers for stratifying GC risk overlapped among subgroups, whereas markers unique to each subgroup were also identified, highlighting the potential of integrating environmental, lifestyle, and epigenetic information to inform GC precision prevention. Citation Format: Genki Usui, Keisuke Matsusaka, Kie K. Huang, Feng Zhu, Tomohiro Shinozaki, Masaki Fukuyo, Bahityar Rahmutulla, Norikazu Yogi, Tomoka Okada, Motoaki Seki, Eiji Sakai, Kazutoshi Fujibayashi, Stephen K. Tsao, Christopher Khor, Tiing L. Ang, Hiroyuki Abe, Hisahiro Matsubara, Masashi Fukayama, Toshiaki Gunji, Nobuyuki Matsuhashi, Teppei Morikawa, Tetsuo Ushiku, Khay G. Yeoh, Patrick Tan, Atsushi Kaneda. Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 761.

Clinical characteristics and prognostic significance of DNA methylation regulatory gene mutations in acute myeloid leukemia

BackgroundDNA methylation is a form of epigenetic modification that regulates gene expression. However, there are limited data on the comprehensive analysis of DNA methylation regulated gene mutations (DMRGM) in acute myeloid leukemia (AML) mainly referring to DNA methyltransferase 3α (DNMT3A), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2), and Tet methylcytidine dioxygenase 2 (TET2).ResultsA retrospective study of the clinical characteristics and gene mutations in 843 newly diagnosed non-M3 AML patients was conducted between January 2016 and August 2019. 29.7% (250/843) of patients presented with DMRGM. It was characterized by older age, higher white blood cell count, and higher platelet count (P < 0.05). DMRGM frequently coexisted with FLT3-ITD, NPM1, FLT3-TKD, and RUNX1 mutations (P < 0.05). The CR/CRi rate was only 60.3% in DMRGM patients, significantly lower than in non-DMRGM patients (71.0%, P = 0.014). In addition to being associated with poor overall survival (OS), DMRGM was also an independent risk factor for relapse-free survival (RFS) (HR: 1.467, 95% CI: 1.030–2.090, P = 0.034). Furthermore, OS worsened with an increasing burden of DMRGM. Patients with DMRGM may be benefit from hypomethylating drugs, and the unfavorable prognosis of DMRGM can be overcome by hematopoietic stem cell transplantation (HSCT). For external validation, the BeatAML database was downloaded, and a significant association between DMRGM and OS was confirmed (P < 0.05).ConclusionOur study provides an overview of DMRGM in AML patients, which was identified as a risk factor for poor prognosis.

Comprehensive DNA Methylation Analysis Indicates That Pancreatic Intraepithelial Neoplasia Lesions Are Acinar-Derived and Epigenetically Primed for Carcinogenesis.

Analysis of DNA methylation landscapes provides insights into the cell-of-origin of PanIN lesions, clarifies the role of PanIN lesions as metaplastic precursors to human PDAC, and suggests potential targets for chemoprevention.

Clinicopathological and prognostic value of lysyl oxidase expression in gastric cancer: a systematic review, meta-analysis and bioinformatic analysis

The association between the expression of Lysyl oxidase (LOX) and its clinicopathological parameters and prognosis in patients with gastric cancer (GC) is still disputed. We performed this meta-analysis and bioinformatics analysis to clarify the relationship between the expression and methylation level of LOX with its clinicopathological parameters and prognostic value. We applied odds ratios with a 95% confidence interval to study the associations between LOX expression and clinicopathological parameters and overall survival (OS) in GC patients. In addition, association analysis of promoter methylation levels and expression of LOX with its prognostic value was performed using the Cancer Genome Atlas (TCGA) and four Gene Expression Omnibus (GEO) datasets. The PRISMA 2020 checklist was used to guide the data extraction and analysis. This meta-analysis includes seven clinical studies with a total of 1435 GC patients. LOX expression was related to lymph node metastasis and tumor distant metastasis in GC patients, but not to gender, tumor differentiation, Lauren classification, or tumor depth of invasion. Patients with GC grouped in high-expression of LOX had a much worse OS than those in low-expression. In addition, TCGA and four GEO datasets with 1279 samples were included in the bioinformatics analysis. The bioinformatics analysis showed that patients with high LOX levels had poor OS; low levels of methylation at some cg sites in the LOX gene were strongly related to poor OS and PFS; and methylation levels of LOX are negatively correlated with advanced tumor stage. The conclusion from comprehensive DNA methylation and gene expression analysis supports LOX as a specific diagnostic and prognosis biomarker in GC. LOX expression was related to lymph node metastasis, tumor distant metastasis and poor prognosis in GC. Low methylation levels were related to advanced tumor stage and poor prognosis in GC. Integrative analysis supports LOX as a specific diagnostic and prognosis biomarker in GC.

Comprehensive Analysis of DNA Methylation and Transcriptome to Identify PD-1-Negative Prognostic Methylated Signature in Endometrial Carcinoma.

Background Epigenetic mechanism plays an important role in endometrial carcinoma (EC). This study was designed to analyze the epigenetic mechanism between DNA methylation-driven genes (DEDGs) and drugs targeting DEDGs and to develop a DEDG score model for predicting the prognosis of EC. Methods Expression profile and methylation profile data of PD-1-negative EC samples were obtained from TCGA. To obtain intersected DEDGs, differentially expressed genes (DEGs) and differentially methylated genes from tumor tissues and normal tissues were analyzed by limma. A linear discriminant classification model was constructed using the gene expression profile of DMDGs, methylation profile of TSS1500, TSS200, and gene body regions. Principal component analysis (PCA) and ROC analysis were conducted. The protein-drug interactions analysis of DMDGs was performed using Network Analyst 3.0 tool. Lasso Cox regression analysis was used to screen prognostic DNA methylation driving gene and to build a risk score model. The ROC curve and Kaplan-Meier survival curve were plotted to evaluate the model prediction capability. Results A total of 96 DMDGs were screened from the three regions, distributed on 22 chromosomes, with consistent methylation patterns in different gene regions. Both the expression profile and methylation profile of the three regions can neatly distinguish tumor samples from normal ones, with a high classifying performance. A gene signature, which consisted of ELFN1-AS1 and ZNF132, could classify EC patients into a high-risk group and low-risk group. Prognosis of the high-risk group was significantly worse than that of the low-risk group. The risk model showed a high performance in predicting the prognosis of EC. Conclusion We successfully established a risk score system with two DMDGs, which showed a high prediction accuracy of EC prognosis.

Comprehensive analysis of DNA methylation for periodontitis

BackgroundPeriodontitis is an infectious disease, and a risk factor for peri-implantitis that could result in the implant loss. DNA methylation has an essential role in the etiology and pathogenesis of inflammatory disease. However, there is lack of study on methylation status of genes in periodontitis. This study sought to explore the gene methylation profiling microarray in periodontitis.MethodsThrough searching in the Gene Expression Omnibus database, a gene methylation profiling data set GSE173081 was identified, which included 12 periodontitis samples and 12 normal samples, respectively. Thereafter, the data of GSE173081 was downloaded and analyzed to determined differentially methylated genes (DMGs), which then were used to perform Gene Ontology analysis and pathway enrichment analyses through online database. In addition, the DMGs were applied to construct the protein–protein interaction (PPI) network information, predict the hub genes in pathology of periodontitis.ResultsIn total 668 DMGs were sorted and identified from the data set, which included 621 hypo-methylated genes and 47 hyper-methylated genes. Through the function and ontology analysis, these 668 genes are mainly classified into intracellular signaling pathway, cell components, cell–cell interaction, and cellular behaviors. The pathway analysis showed that the hypo-methylated genes were mostly enriched in the pathway of cGMP–PKG signaling pathway; RAF/MAP kinase; PI3K–Akt signaling pathway, while hyper-methylated genes were mostly enriched in the pathway of bacterial invasion of epithelial cells; sphingolipid signaling pathway and DCC mediated attractive signaling. The PPI network contained 630 nodes and 1790 interactions. Moreover, further analysis identified top 10 hub genes (APP; PAX6; LPAR1; WNT3A; BMP2; PI3KR2; GATA4; PLCB1; GATA6; CXCL12) as central nodes that are involved in the immune system and the inflammatory response.ConclusionsThis study provides comprehensive information of methylation status of genes to the revelation of periodontitis pathogenesis that may contribute to future research on periodontitis.

Epigenetic remodelling of Fxyd1 promoters in developing heart and brain tissues

FXYD1 is a key protein controlling ion channel transport. FXYD1 exerts its function by regulating Na+/K+-ATPase activity, mainly in brain and cardiac tissues. Alterations of the expression level of the FXYD1 protein cause diastolic dysfunction and arrhythmias in heart and decreased neuronal dendritic tree and spine formation in brain. Moreover, FXYD1, a target of MeCP2, plays a crucial role in the pathogenesis of the Rett syndrome, a neurodevelopmental disorder. Thus, the amount of FXYD1 must be strictly controlled in a tissue specific manner and, likely, during development. Epigenetic modifications, particularly DNA methylation, represent the major candidate mechanism that may regulate Fxyd1 expression. In the present study, we performed a comprehensive DNA methylation analysis and mRNA expression level measurement of the two Fxyd1 transcripts, Fxyd1a and Fxyd1b, in brain and heart tissues during mouse development. We found that DNA methylation at Fxyd1a increased during brain development and decreased during heart development along with coherent changes in mRNA expression levels. We also applied ultra-deep methylation analysis to detect cell to cell methylation differences and to identify possible distinct methylation profile (epialleles) distribution between heart and brain and in different developmental stages. Our data indicate that the expression of Fxyd1 transcript isoforms inversely correlates with DNA methylation in developing brain and cardiac tissues suggesting the existence of a temporal-specific epigenetic program. Moreover, we identified a clear remodeling of epiallele profiles which were distinctive for single developmental stage both in brain and heart tissues.