Genome-wide DNA Methylation Analysis Research Articles

Genome-Wide DNA methylation profile analysis identifies differentially methylated loci associated with personal PM2.5 exposure in adults with asthma

Particulate matter with aerodynamic diameters ≤2.5 µm (PM2.5) is a major environmental risk factor for acute asthma exacerbation, and the underlying mechanism is not completely understood. Studies have indicated that DNA methylation is a potential mechanism linking PM2.5 to its health effects. We conducted a panel study involving 24 adult patients with asthma in Beijing,China between 2017 and 2019. PM2.5 and other atmospheric pollutant exposure data were repeatedly measured. Blood samples were collected for genome-wide DNA methylation analysis. A linear mixed-effects (LME) model was conducted to identify differentially methylated probes (DMPs) associated with PM2.5 exposure. After filtering out probes that did not meet the criteria through quality control, 811,001 CpG sites were included in the LME model, and 36 DMPs were strongly associated with personal PM2.5 exposure at false discovery rate (FDR) < 0.05, of which 22 and 14 DMPs negatively and positively correlated with personal PM2.5 exposure, respectively. Functional analysis revealed that DMPs affected smooth muscle cell contraction and development, extracellular matrix synthesis and secretion, T cell activation and differentiation, and inflammatory factor production. This study provides evidence linking personal PM2.5 exposure to genome-wide DNA methylation in adult patients with asthma. Identifying enrichment pathways can provide biological insights into the acute health effects of PM2.5.

Methylome-wide analysis in systemic microbial-induced experimental periodontal disease in mice with different susceptibility

ObjectiveThe study delved into the epigenetic factors associated with periodontal disease in two lineages of mice, namely C57bl/6 and Balb/c. Its primary objective was to elucidate alterations in the methylome of mice with distinct genetic backgrounds following systemic microbial challenge, employing high-throughput DNA methylation analysis as the investigative tool.MethodsPorphyromonas gingivalis (Pg)was orally administered to induce periodontitis in both Balb/c and C57bl/6 lineage. After euthanasia, genomic DNA from both maxilla and blood were subjected to bisulfite conversion, PCR amplification and genome-wide DNA methylation analysis using the Ovation RRBS Methyl-Seq System coupled with the Illumina Infinium Mouse Methylation BeadChip.ResultsOf particular significance was the distinct methylation profile observed within the Pg-induced group of the Balb/c lineage, contrasting with both the control and Pg-induced groups of the C57bl/6 lineage. Utilizing rigorous filtering criteria, we successfully identified a substantial number of differentially methylated regions (DMRs) across various tissues and comparison groups, shedding light on the prevailing hypermethylation in non-induced cohorts and hypomethylation in induced groups. The comparison between blood and maxilla samples underscored the unique methylation patterns specific to the jaw tissue. Our comprehensive methylome analysis further unveiled statistically significant disparities, particularly within promoter regions, in several comparison groups.ConclusionThe differential DNA methylation patterns observed between C57bl/6 and Balb/c mouse lines suggest that epigenetic factors contribute to the variations in disease susceptibility. The identified differentially methylated regions associated with immune regulation and inflammatory response provide potential targets for further investigation. These findings emphasize the importance of considering epigenetic mechanisms in the development and progression of periodontitis.

Multiomics analysis in IgM monoclonal gammopathies reveals epigenetic influence on oncogenesis via DNA methylation

AbstractCurrently, the role of DNA methylation in the immunoglobulin M (IgM)–monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenstrom macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

Respiratory infection- and asthma-prone, low vaccine responder children demonstrate distinct mononuclear cell DNA methylation pathways

BackgroundInfants with frequent viral and bacterial respiratory infections exhibit compromised immunity to routine immunizations. They are also more likely to develop chronic respiratory diseases in later childhood. This study investigated the feasibility of epigenetic profiling to reveal endotype-specific molecular pathways with potential for early identification and immuno-modulation. Peripheral blood mononuclear cells from respiratory infection allergy/asthma-prone (IAP) infants and non-infection allergy/asthma prone (NIAP) were retrospectively selected for genome-wide DNA methylation and single nucleotide polymorphism analysis. The IAP infants were enriched for the low vaccine responsiveness (LVR) phenotype (Fisher's exact p-value = 0.02).ResultsAn endotype signature of 813 differentially methylated regions (DMRs) comprising 238 lead CpG associations (FDR < 0.05) emerged, implicating pathways related to asthma, mucin production, antigen presentation and inflammasome activation. Allelic variation explained only a minor portion of this signature. Stimulation of mononuclear cells with monophosphoryl lipid A (MPL), a TLR agonist, partially reversed this signature at a subset of CpGs, suggesting the potential for epigenetic remodeling.ConclusionsThis proof-of-concept study establishes a foundation for precision endotyping of IAP children and highlights the potential for immune modulation strategies using adjuvants for future investigation.

Genome-wide DNA methylation status is a predictor of the efficacy of anti-EGFR antibodies in the second-line treatment of metastatic colorectal cancer: Translational research of the EPIC trial

PurposeThe genome-wide DNA methylation status (GWMS) predicts of therapeutic response to anti-epidermal growth factor receptor (EGFR) antibodies in treating metastatic colorectal cancer. We verified the significance of GWMS as a predictive factor for the efficacy of anti-EGFR antibodies in the second-line treatment of metastatic colorectal cancer.MethodsClinical data were obtained from a prospective trial database, and a genome-wide DNA methylation analysis was performed. GWMS was classified into high-methylated colorectal cancer (HMCC) and low-methylated colorectal cancer (LMCC). The patients were divided into subgroups according to the treatment arm (cetuximab plus irinotecan or irinotecan alone) and GWMS, and the clinical outcomes were compared between the subgroups.ResultsOf the 112 patients, 58 (51.8%) were in the cetuximab plus irinotecan arm, and 54 (48.2%) were in the irinotecan arm; 47 (42.0%) were in the HMCC, and 65 (58.0%) were in the LMCC group regarding GWMS. Compared with the LMCC group, the progression-free survival (PFS) was significantly shortened in the HMCC group in the cetuximab plus irinotecan arm (median 1.4 vs. 4.1 months, p = 0.001, hazard ratio = 2.56), whereas no significant differences were observed in the irinotecan arm. A multivariate analysis showed that GWMS was an independent predictor of PFS and overall survival (OS) in the cetuximab plus irinotecan arm (p = 0.002, p = 0.005, respectively), whereas GWMS did not contribute to either PFS or OS in the irinotecan arm.ConclusionsGWMS was a predictive factor for the efficacy of anti-EGFR antibodies in the second-line treatment of metastatic colorectal cancer.

Aberrant methylation and expression of TNXB promote chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling.

Recurrent joint bleeding in hemophilia patients frequently causes hemophilic arthropathy (HA). Drastic degradation of cartilage is a major characteristic of HA, but its pathological mechanisms has not yet been clarified. In HA cartilages, we found server matrix degradation and increased expression of DNA methyltransferase proteins. We thus performed genome-wide DNA methylation analysis on human HA (N=5) and osteoarthritis (OA) (N=5) articular cartilages, and identified 1228 differentially methylated regions (DMRs) associated with HA. Functional enrichment analyses revealed the association between DMR genes (DMGs) and extracellular matrix (ECM) organization. Among these DMGs, Tenascin XB (TNXB) expression was down-regulated in human and mouse HA cartilages. The loss of Tnxb in F8-/- mouse cartilage provided a disease-promoting role in HA by augmenting cartilage degeneration and subchondral bone loss. Tnxb knockdown also promoted chondrocyte apoptosis and inhibited phosphorylation of AKT. Importantly, AKT agonist showed chondroprotective effects following Tnxb knockdown. Together, our findings indicate that exposure of cartilage to blood leads to alterations in DNA methylation, which is functionally related to ECM homeostasis, and further demonstrate a critical role of TNXB in HA cartilage degeneration by activating AKT signaling. These mechanistic insights allow development of potentially new strategies for HA cartilage protection.

The survival, gene expression, and DNA methylation of Paralichthys olivaceus impacted by the decay of green tide and bacterial infection in both laboratory and field simulation experiments

The recurring appearance of Ulva prolifera green tides has become a pressing environmental issue, especially for marine transportation, tourism, and aquaculture in the stage of decomposition. An abundance of decaying U. prolifera leads to water acidification, hypoxia and pathogenic microorganism proliferation, threatening marine germplasm resources, particularly benthic organisms with weak escape ability. Epigenetic modification is considered to be one of the molecular mechanisms involved in the plastic adaptive response to environmental changes. However, few studies concerning the specific impact of decaying green tide on benthic animals at the epigenetic level. In this study, decomposing algal effluents of U. prolifera, sediments containing uncorrupted U. prolifera, pathogenic microorganism were considered as impact factors, to reveal the effect of decaying U. prolifera on marine economic benthic species, Paralichthys olivaceus, using both field and laboratory simulation experiments. Field simulation experiment showed higher mortality rates and serious histopathological damage than the laboratory simulation experiment. And both the decaying U. prolifera and the sediment containing U. prolifera were harmful to P. olivaceus. Genome-wide DNA methylation and transcription correlation analyses showed that the response of P. olivaceus to green tide stress and bacterial infection was mainly mediated by immune signaling pathways such as PI3K-Akt signaling pathway. DNA methylation regulates the expression of immune-related genes involved in the PI3K-Akt signaling pathway, which enables P. olivaceus to adapt to the adverse environmental stresses by resisting apoptosis. In summary, this research analyzed the potential role of P. olivaceus in decaying U. prolifera, which is of great significance for understanding the impact of decaying green tide on marine commercial fish and also provides some theoretical guidance for the proliferation and release of fish seedlings.

Genome-Wide DNA Methylation in Early-Onset-Dementia Patients Brain Tissue and Lymphoblastoid Cell Lines.

Epigenetics, a potential underlying pathogenic mechanism of neurodegenerative diseases, has been in the scope of several studies performed so far. However, there is a gap in regard to analyzing different forms of early-onset dementia and the use of Lymphoblastoid cell lines (LCLs). We performed a genome-wide DNA methylation analysis on sixty-four samples (from the prefrontal cortex and LCLs) including those taken from patients with early-onset forms of Alzheimer's disease (AD) and frontotemporal dementia (FTD) and healthy controls. A beta regression model and adjusted p-values were used to obtain differentially methylated positions (DMPs) via pairwise comparisons. A correlation analysis of DMP levels with Clariom D array gene expression data from the same cohort was also performed. The results showed hypermethylation as the most frequent finding in both tissues studied in the patient groups. Biological significance analysis revealed common pathways altered in AD and FTD patients, affecting neuron development, metabolism, signal transduction, and immune system pathways. These alterations were also found in LCL samples, suggesting the epigenetic changes might not be limited to the central nervous system. In the brain, CpG methylation presented an inverse correlation with gene expression, while in LCLs, we observed mainly a positive correlation. This study enhances our understanding of the biological pathways that are associated with neurodegeneration, describes differential methylation patterns, and suggests LCLs are a potential cell model for studying neurodegenerative diseases in earlier clinical phases than brain tissue.

Genome-wide DNA methylation analysis in blood identifies differentially methylated regions related to polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is considered a multifactorial disorder caused due to predisposing alleles of various genes and environmental factors which include lifestyle, dietary habits, and exposure to toxicants. Recently the emphasis has shifted to understanding epigenetic alterations due to the poor correlation between genotypes and the clinical profile of women with PCOS. Investigators have been focusing on differential methylation signatures associated with PCOS, therefore, a pilot study was carried out to investigate PCOS-associated differential DNA methylation regions in the population of north India. A cross-sectional study with sixteen participants comprising eight women with PCOS diagnosed under the Rotterdam criterion and eight age and BMI-matched healthy controls. Subjects were classified into four groups based on BMI and two different groups were compared each time making four comparison types in total. Genome-wide DNA methylation in whole blood was evaluated using an Infinium Human Methylation 850k array which identified PCOS-associated differential sites and differential methylation regions (DMRs) with promoter region, and CpG islands considering (p ≤ 0.01) as statistically significant. Multiple significantly associated hypomethylated and hypermethylated loci of genes were identified. Thirty-four hypomethylated and one hundred twenty-six hypermethylated sites of genes were suggested when all the subjects were of low BMI. But when the groups comprised with high BMI, twenty-seven hypomethylated genes and fifty-nine hypermethylated genes were identified. In silico analysis suggests that most of these genes are involved in estrogen biosynthesis, lipid biosynthetic process, and activation of matrix metalloproteinases (MMP). Seven genes that are found common in both of these comparisons are mostly involved in the inflammasome pathway indicated by BioPlanet and Reactome databases. The study provides information about DMRs in the blood tissue which may be investigated further to understand underlying pathogenesis and as therapeutic targets in the future.

DNA methylation by DNMT3a drives endothelial inflammation and death during acute lung injury

Vascular endothelium, the inner most lining of all blood vessels, controls the transport of nutrients, protein, water, and leukocytes across the vessel wall and thereby maintains lung tissue and fluid homeostasis. Damage to endothelial integrity caused by mediagenic, inflammatory, or toxic agents results in pulmonary edema, inflammation, organ failure, and high lethality. A key pathogenic characteristic of vascular injury involves an excessive loss of endothelial cells (EC) accompanied by the generation of inflammatory cytokines. Genome-wide analyses of DNA methylation, the most enduring epigenetic modification of DNA, and its impact on EC transcriptomes and cellular functions, still lack comprehensive understanding. We demonstrate that lipopolysaccharide (LPS) elevated DNA methylation in EC within the lungs, primarily through a mechanism dependent on DNA methyltransferase 3a (DNMT 3a). Mice with endothelial cell-specific deletion of DNMT3a ( EC-DNMT3a−/−) promptly recovered from lung injury, attributed to a reduction of EC death. Furthermore, we observed that the deletion of DNMT3a in EC led to an increase in the expression of the apoptosis inhibitor B-cell lymphoma 2 (BCL2). Collectively, the data implies that inhibiting DNMT3a activity plays a crucial role in reshaping the EC methylome and promoting BCL-2 activity. This, in turn, facilitates the generation of reparative EC and contributes to the resolution of acute lung injury (ALI). These findings offer insights into potential targets for preventing ALI. American Heart Association (AHA) Postdoctoral Fellowship (19POST34450241). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genome-wide epigenetic signatures facilitated the variant classification of the PURA gene and uncovered the pathomechanism of PURA-related neurodevelopmental disorders

PurposeRare genetic variants in the PURA gene cause the PURA-related neurodevelopmental disorder (PURA-NDD), characterized by neonatal abnormalities and developmental delay. Using genome-wide DNA methylation analysis on patients with PURA variants, we aim to establish a PURA-NDD-specific methylation profile and provide further insights on the molecular basis of the PURA-NDD. MethodsTwenty three individuals (including 12 unpublished) carrying PURA variants were enrolled. We conducted the Illumina Infinium EPIC microarray analysis in 17 PURA-NDD individuals. In vitro experiments were performed to examine how PURA variants affect Pur-a expression. ResultsAdditional phenotypes in 12 newly identified patients were described in this study. Genome-wide DNA methylation analysis unveiled distinctive methylation profiles to PURA-NDD, and the established classifier can reclassify PURA variants of uncertain significance. Patients bearing PURA hapoloinsufficient and missense variants have comparable DNA methylation profiles, and cells expressing these PURA variants showed consistent Pur-a downregulation, suggesting a haploinsufficiency mechanism. ConclusionPatients with PURA-NDD exhibit a specific episignature, which has potential to aid identification and diagnosis of PURA-NDD patients and offer implications for further functional investigations.

Genome-wide DNA methylation and transcriptomic analysis of liver tissues subjected to early ischemia/reperfusion injury upon human liver transplantation

Introduction and ObjectivesEpigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. The present study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors. Patients and Methods12 liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq. ResultsSignificant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-α, NFKB, and interleukin signaling. Cell enrichment analysis individuates macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response. ConclusionsIn the process of liver graft procurement-transplantation, IRI induces significant epigenetic changes that primarily act on the signaling pathways of inflammatory responses dependent on TNF-α, NFKB, and interleukins. Our DNAm datasets are the early IRI methylome literature and will serve as a launch point for studying the impact of epigenetic modification in IRI.

The association analysis of DNA methylation and transcriptomics identified BpCYCD3;2 as a participant in influencing cell division in autotetraploid birch (Betula pendula) leaves

Polyploidization plays a crucial role in plant breeding and genetic improvement. Although the phenomenon of polyploidization affecting the area and number of plant epidermal pavement cells is well described, the underlying mechanism behind this phenomenon is still largely unknown. In this study, we found that the leaves of autotetraploid birch (Betula pendula) stopped cell division earlier and had a larger cell area. In addition, compared to diploids, tetraploids have a smaller stomatal density and fewer stomatal numbers. Genome-wide DNA methylation analysis revealed no significant difference in global DNA methylation levels between diploids and tetraploids. A total of 9154 differential methylation regions (DMRs) were identified between diploids and tetraploids, with CHH-type DMRs accounting for 91.73% of all types of DMRs. Further research has found that there are a total of 2105 differentially methylated genes (DMEGs) with CHH-type DMRs in birch. The GO functional enrichment results of DMEGs showed that differentially methylated genes were mainly involved in terms such as cellular process and metabolic process. The analysis of differentially methylated genes and differentially expressed genes suggests that hyper-methylation in the promoter region may inhibit the gene expression level of BpCYCD3;2 in tetraploids. To investigate the function of BpCYCD3;2 in birch, we obtained overexpression and repressed expression lines of BpCYCD3;2 through genetic transformation. The morphogenesis of both BpCYCD3;2-OE and BpCYCD3;2-RE lines was not affected. However, low expression of BpCYCD3;2 can lead to inhibition of cell division in leaves, and this inhibition of cell proliferation can be compensated for by an increase in cell size. Additionally, we found that the number and density of stomata in the BpCYCD3;2-RE lines were significantly reduced, consistent with the tetraploid. These data indicate that changes in cell division ability and stomatal changes in tetraploid birch can be partially attributed to low expression of the BpCYCD3;2 gene, which may be related to hyper-methylation in its promoter region. These results will provide new insights into the mechanism by which polyploidization affects plant development.

Differential DNA methylation in iPSC-derived dopaminergic neurons: a step forward on the role of SNORD116 microdeletion in the pathophysiology of addictive behavior in Prader-Willi syndrome.

A microdeletion including the SNORD116 gene (SNORD116 MD) has been shown to drive the Prader-Willi syndrome (PWS) features. PWS is a neurodevelopmental disorder clinically characterized by endocrine impairment, intellectual disability and psychiatric symptoms such as a lack of emotional regulation, impulsivity, and intense temper tantrums with outbursts. In addition, this syndrome is associated with a nutritional trajectory characterized by addiction-like behavior around food in adulthood. PWS is related to the genetic loss of expression of a minimal region that plays a potential role in epigenetic regulation. Nevertheless, the role of the SNORD116 MD in DNA methylation, as well as the impact of the oxytocin (OXT) on it, have never been investigated in human neurons. We studied the methylation marks in induced pluripotent stem-derived dopaminergic neurons carrying a SNORD116 MD in comparison with those from an age-matched adult healthy control. We also performed identical neuron differentiation in the presence of OXT. We performed a genome-wide DNA methylation analysis from the iPSC-derived dopaminergic neurons by reduced-representation bisulfite sequencing. In addition, we performed RNA sequencing analysis in these iPSC-derived dopaminergic neurons differentiated with or without OXT. The analysis revealed that 153,826 cytosines were differentially methylated between SNORD116 MD neurons and control neurons. Among the differentially methylated genes, we determined a list of genes also differentially expressed. Enrichment analysis of this list encompassed the dopaminergic system with COMT and SLC6A3. COMT displayed hypermethylation and under-expression in SNORD116 MD, and SLC6A3 displayed hypomethylation and over-expression in SNORD116 MD. RT-qPCR confirmed significant over-expression of SLC6A3 in SNORD116 MD neurons. Moreover, the expression of this gene was significantly decreased in the case of OXT adjunction during the differentiation. SNORD116 MD dopaminergic neurons displayed differential methylation and expression in the COMT and SLC6A3 genes, which are related to dopaminergic clearance.

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model.

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Abstract 1463: Characterizing inflammation-induced epigenetic and transcriptomic alterations driving NSCLC using lung organoid models

Abstract Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic alterations. These, in turn, modulate the microenvironment to facilitate emergence of tumor promoting immune evasive mechanisms. The evolution of epigenetic events and their role, both alone, and in association with lung cancer specific genetic drivers in initiation of lung cancer is not well delineated. Specific pathological subtypes of non-small cell lung cancer (NSCLC) have distinct cells of origin, suggestive of cell type-specific changes in driving their development. We employed lung organoid models with chronic exposure to cigarette smoke condensate (CSC) to elucidate sequential, cell type-specific epigenetic and transcriptomic alterations and their role in co-operating with genetic events to drive development of NSCLC. We examined epigenomic and transcriptomic alterations over a 6-month exposure period by DNA methylation arrays, ATAC-seq, and RNA-seq. We observe that CSC exposure causes distinct morphological changes in organoid structure and composition, accompanied by an increase in proliferative potential. Using flow cytometry and immunofluorescent staining approaches, we demonstrate a chronic inflammation-driven shift in stem cell populations accompanied by reduction in differentiated cell types. Bulk and single-cell RNA sequencing approaches identified distinct temporal changes in transcriptional signatures in CSC exposed organoids suggestive of evolution of an immune evasive and pro-tumorigenic phenotype. Gene set enrichment analysis revealed a downregulation of interferon signaling and upregulation of Myc targets and oxidative phosphorylation pathways in CSC treated organoids. Notably, co-culturing organoids with key immune cell populations, demonstrate the ability of the CSC exposed organoids to modulate shifts in immune cells from a pro- to anti- inflammatory state. In accordance with this shift in immune cell phenotype, CSC treated organoids secreted decreased levels of pro-inflammatory cytokines as measured by multiplex ELISA assays. Genome wide DNA methylation analysis reveal that CSC methylated genes were associated with differentiation and cell fate commitment as well as transcription factor activity. Finally, we demonstrate that introduction of key NSCLC-specific driver events at the 6-month time point sensitize only CSC treated organoids to tumor formation in vivo, leading to the development of distinct genetic driver-specific subtypes of NSCLC. Altogether, our results demonstrate the ability of chronic inflammation to drive key cell type-specific transcriptomic and epigenetic changes leading to the development of distinct subtypes of NSCLC. Our findings will help identify molecular correlates for early detection and aid development of novel therapeutic strategies for disease interception. Citation Format: Na Wang, Kara Lombardo, Raksha Padaki, Ray-Whay Chiu Yen, Malcolm V. Brock, Leslie Cope, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing inflammation-induced epigenetic and transcriptomic alterations driving NSCLC using lung organoid models [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 1463.

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.

Genome-Wide DNA Methylation Analysis and Functional Validation of Litter Size Traits in Jining Grey Goats.

DNA methylation (DNAm) is associated with the reproductive system. However, the genetic mechanism through which DNAm regulates gene expression and thus affects litter size in goats is unclear. Therefore, in the present work, genome-wide DNAm profiles of HP and LP Jining Grey goat ovary tissues were comprehensively analyzed via WGBS, and RNA-Seq data were combined to identify candidate genes associated with litter size traits in the Jining Grey goat. Finally, BSP and RT-qPCR were used to verify the sequencing results of the key genes. Notably, the DNMT genes were downregulated at the expression level in the HP group. Both groups exhibited comparable levels of methylation. A total of 976 differentially methylated regions (DMRs) (973 DMRs for CG and 3 DMRs for CHG) and 310 differentially methylated genes (DMGs) were identified in this study. Through integration of WGBS and RNA-Seq data, we identified 59 differentially methylated and differentially expressed genes (DEGs) and ultimately screened 8 key DMGs (9 DMRS) associated with litter size traits in Jining Grey goats (SERPINB2: chr24_62258801_62259000, NDRG4: chr18_27599201_27599400, CFAP43: chr26_27046601_27046800, LRP1B. chr2_79720201_79720400, EPHA6: chr1_40088601_40088800, TTC29: chr17_59385801_59386000, PDE11A: chr2_117418601_117418800 and PGF: chr10_ 16913801_16914000 and chr10_16916401_16916600). In summary, our research comprehensively analyzed the genome-wide DNAm profiles of HP and LP Jining Grey goat ovary tissues. The data findings suggest that DNAm in goat ovaries may play an important role in determining litter size.

Genome-wide DNA methylation analysis related to ALS patient progression and survival.

Epigenetics contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS). We aimed to characterize the DNA methylation profiles associated with clinical heterogeneity in disease progression and survival among patients. We included a cohort of 41 patients with sporadic ALS, with a median follow-up of 86.9months, and 27 rigorously matched healthy controls. Blood-based genome-wide DNA methylation analysis was conducted. A total of 948 progression rate-associated differentially methylated positions, 298 progression rate-associated differentially methylated regions (R-DMRs), 590 survival time-associated DMPs, and 197 survival time-associated DMRs (S-DMRs) were identified, using complementary grouping strategies. Enrichment analysis of differentially methylated genes highlighted the involvement of synapses and axons in ALS progression and survival. Clinical analysis revealed a positive correlation between the average methylation levels of the R-DMR in PRDM8 and disease progression rate (r = 0.479, p = 0.002). Conversely, there was an inverse correlation between the average methylation levels of the R-DMR in ANKRD33 and disease progression rate (r = -0.476, p = 0.002). In addition, patients with higher methylation levels within the S-DMR of ZNF696 experienced longer survival (p = 0.016), while those with elevated methylation levels in the S-DMR of RAI1 had shorter survival (p = 0.006). DNA methylation holds promise as a potential biomarker for tracking disease progression and predicting survival outcome and also offers targets for precision medicine.

Comparative epigenomics indicate a common origin of ectopic and intrasellar corticotroph pituitary neuroendocrine tumors/adenomas: a case report.

Ectopic pituitary neuroendocrine tumors (PitNET)/adenomas are rare and diagnostically challenging extra-sellar tumors. Previous studies have demonstrated the impact of epigenomic analyses in the diagnostics of sellar neoplasms and characterized the close relationship of epigenomic signatures and cellular origins of PitNET/adenomas. As of today, little is known about the pathogenesis of ectopic PitNET/adenomas, and epigenomic analyses have not been performed in these rare tumors. We report on the clinical course of an 81-year-old patient with sphenoid ectopic sparsely granulated corticotroph PitNET/adenoma and deploy genome-wide DNA methylation analysis to compare its methylation profile to a reference cohort of sellar neoplasms. Genome-wide methylation analysis revealed an epigenomic profile analogous to reference sellar corticotroph PitNET/adenomas, and the copy number variation profile showed loss of chromosomes 18 and 22. The methylation profile shows concordance with sellar corticotroph PitNET/adenomas suggesting a common cellular origin and confirming the reliability of methylation analyses as a diagnostic method in these rare tumors. This is the first data suggesting that epigenetic profiles of ectopic PitNET/adenoma do not differ from their sellar counterparts.