Methylation Landscape Research Articles

Estrogen receptor signaling alters sperm DNA methylation landscape in adult male rats.

Estrogen through its receptors, ERα and ERβ, regulate various aspects of spermatogenesis and male fertility. Since the sperm epigenome is an important contributing factor to male fertility, we evaluated the effects of estrogen signaling activation through the ERs on sperm DNA methylome in adult rats. Whole genome-bisulfite sequencing (WGBS) in caudal sperm DNA was performed. The differentially methylated CpG sites (DMCs) were validated by pyrosequencing, and the expression of differentially methylated genes (DMGs) was evaluated in testis by q-RT-PCR. Activation of ERα signaling brought about large-scale changes in the sperm DNA methylome as compared to ERβ. There were 28074 DMCs and 5189 DMGs obtained after ERα agonist PPT treatment, whereas 1492 DMCs and 336 DMGs for ERβ agonist DPN. In genic regions, most of the DMCs were intronic, followed by promoter and upstream region. DMCs were distributed around the TSS and in transcription factor binding regions, implicating their plausible role in gene expression regulation. Genes important for spermatogenesis were identified and validated which showed a similar trend of differential methylation as obtained by WGBS. The expression of the DMGs was also found to be altered in the testis. There was a considerable overlap (14 to 50%) of PPT DMGs with the DMGs reported to be affected in clinical conditions of male infertility. This study highlights the role of ERs in shaping the sperm epigenome and that aberrant estrogen signaling could be a contributing factor in clinical conditions of male infertility.

Non-invasive diagnosis for urothelial carcinoma using a dual-target DNA methylation biomarker panel.

Urothelial carcinoma (UC) is a common malignancy worldwide. Aberrant DNA methylation is implicated in UC carcinogenesis. This study sought to delineate the DNA methylation landscape in UC and identify DNA methylation-based biomarkers for early detection of UC. Whole genome bisulfite sequencing (WGBS) was conducted on bladder cancer tissues and paired normal tissues. By integrating WGBS data with The Cancer Genome Atlas (TCGA) UBC data, a DNA methylation-based biomarker was identified. When combined with a known UC biomarker AL021918.2, the performance of the dual-target test was evaluated in voided urine samples from 224 UC patients and 419 controls. Notable hypomethylation was observed in UC samples compared to normal samples. Through differential methylation analysis, differential methylation CpG sites, regions, and genes were identified. Of these, Transmembrane protein 106A gene (TMEM106A) was screened as a new UC biomarker. In a dual-target test, using triplex quantitative methylation-specific PCR (qMSP) to examine TMEM106A and AL021918.2 methylation levels, the training set showed a sensitivity of 89.0%, a specificity of 92.9%, and an area under the curve (AUC) value of 0.941 (95% confidence interval [CI]: 0.913-0.969). Similarly, the validation set showed a sensitivity of 90.0%, a specificity of 91.1%, and an AUC value of 0.922 (95% CI: 0.881-0.962). Thus, our dual-target test demonstrated outstanding detection rates for low-grade or early-stage tumors. We provide a comprehensive analysis of DNA methylation profiles in UC, and highlight the promising clinical potential of dual-target urine tests for UC detection.

Comprehensive investigation of matrix metalloproteinases in skin cutaneous melanoma: diagnostic, prognostic, and therapeutic insights

The dysregulation of matrix metalloproteinases (MMPs) in skin cutaneous melanoma (SKCM) represents a critical aspect of tumorigenesis. In this study, we investigated the diagnostic, prognostic, and therapeutic aspects of the MMPs in SKCM. Thirteen SKCM cell lines and seven normal skin cell lines were cultured under standard conditions for experimental analyses. RNA and DNA were extracted, followed by RT-qPCR to assess MMP expression and promoter methylation analysis to determine methylation levels. Functional assays, including cell proliferation, colony formation, and wound healing, were conducted post-MMP7 knockdown using siRNA in A375 cells. Databases like GEPIA2, HPA, MEXPRESS, and miRNet were employed for expression, survival, methylation, and miRNA-mRNA network analyses. We investigated the expression and promoter methylation landscape of MMPs in SKCM cell lines, revealing significant (p-value < 0.05) up-regulation of MMP1, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, and MMP25, alongside down-regulation of MMP2, MMP3, and MMP21. Furthermore, our analysis demonstrated a significant (p-value < 0.05) inverse correlation between MMP expression levels and promoter methylation status, suggesting a potential regulatory role of DNA methylation in MMP dysregulation. Notably, MMP7, MMP11, and MMP14 exhibited significant (p-value < 0.05) associations with the overall survival of SKCM patients, emphasizing their prognostic significance. Additionally, Receiver operating characteristic (ROC) curve analysis highlighted the significant (p-value < 0.05) diagnostic potential of MMP7, MMP11, and MMP14 in distinguishing SKCM from normal individuals. Subsequent validation across multiple cohorts confirmed significant (p-value < 0.05) elevated MMP expression levels in SKCM tissues, particularly in advanced disease stages, further emphasizing their role in tumor progression. Furthermore, we elucidated potential regulatory pathways involving miR-22-3p, which targets MMP7, MMP11, and MMP14 genes in SKCM. Our findings also revealed associations between MMP expression and immune modulation, drug sensitivity, and functional states of SKCM cells. Lastly, MMP7 knockdown in A375 cells significantly significant (p-value < 0.05) impacted several characteristics, including cell proliferation, colony formation, and wound healing. Our findings highlight the diagnostic, prognostic, and therapeutic potential of MMP7, MMP11, and MMP14 in SKCM. These MMPs could serve as biomarkers for early detection and targets for therapy. Future efforts should focus on preclinical and clinical validation to translate these insights into personalized diagnostic and therapeutic strategies.

Cross-tissue comparison of epigenetic aging clocks in humans.

Epigenetic clocks are a common group of tools used to measure biological aging-the progressive deterioration of cells, tissues, and organs. Epigenetic clocks have been trained almost exclusively using blood-based tissues, but there is growing interest in estimating epigenetic age using less-invasive oral-based tissues (i.e., buccal or saliva) in both research and commercial settings. However, differentiated cell types across body tissues exhibit unique DNA methylation landscapes and age-related alterations to the DNA methylome. Applying epigenetic clocks derived from blood-based tissues to estimate epigenetic age of oral-based tissues may introduce biases. We tested the within-person comparability of common epigenetic clocks across five tissue types: buccal epithelial, saliva, dry blood spots, buffy coat (i.e., leukocytes), and peripheral blood mononuclear cells. We tested 284 distinct tissue samples from 83 individuals aged 9-70 years. Overall, there were significant within-person differences in epigenetic clock estimates from oral-based versus blood-based tissues, with average differences of almost 30 years observed in some age clocks. In addition, most epigenetic clock estimates of blood-based tissues exhibited low correlation with estimates from oral-based tissues despite controlling for cellular proportions and other technical factors. Notably, the Skin and Blood clock exhibited the greatest concordance across all tissue types, indicating its unique ability to estimate chronological age in oral- and blood-based tissues. Our findings indicate that application of blood-derived epigenetic clocks in oral-based tissues may not yield comparable estimates of epigenetic age, highlighting the need for careful consideration of tissue type when estimating epigenetic age.

DNA methylation landscapes before and after Pacific Oyster Mortality Syndrome are different within and between resistant and susceptible Magallana gigas.

Pacific oysters face recurring outbreaks of Pacific Oyster Mortality Syndrome (POMS), a polymicrobial multifactorial disease. Although this interaction is increasingly understood, the role of epigenetics (e.g., DNA methylation) appears to be of fundamental importance because of its ability to shape oyster resistance/susceptibility and respond to environmental triggers, including infections. In this context, we comprehensively characterized basal (no infection) and POMS-induced changes in the methylome of resistant and susceptible oysters, focusing on the gills and mantle. Our analysis identified differentially methylated regions (DMRs) that revealed distinct methylation patterns uniquely associated with the susceptible or resistant phenotypes in each tissue. Enrichment analysis of genes bearing DMRs highlighted that these epigenetic changes were specifically linked to immunity, signaling, metabolism, and transport. Notably, 31 genes with well-known immune functions were differentially methylated after POMS, with contrasting methylation patterns between the phenotypes. Based on the methylome differences between phenotypes, we identified a set of candidate epibiomarkers that could characterize whether an oyster is resistant or susceptible (1998 candidates) and whether a site has been exposed to POMS (164 candidates). Overall, the findings provide a deeper understanding of the molecular interactions between oysters and POMS infection, opening new questions about the broader implications of epigenetic mechanisms in host-pathogen dynamics and offering promising strategies for mitigating the impacts of this devastating disease. Beyond its biological aspects, this study provides insights into potential epigenetic biomarkers for POMS disease management and targets for enhancing oyster health and productivity.

Somatic mutations and DNA methylation identify a subgroup of poor prognosis within lower-risk myelodysplastic syndromes.

Lower risk (LR) myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem and progenitor disorders caused by the accumulation of somatic mutations in various genes including epigenetic regulators that may produce convergent DNA methylation patterns driving specific gene expression profiles. The integration of genomic, epigenomic, and transcriptomic profiling has the potential to spotlight distinct LR-MDS categories on the basis of pathophysiological mechanisms. We performed a comprehensive study of somatic mutations and DNA methylation in a large and clinically well-annotated cohort of treatment-naive patients with LR-MDS at diagnosis from the EUMDS registry (ClinicalTrials.gov.NCT00600860). Unsupervised clustering analyses identified six clusters based on genetic profiling that concentrate into four clusters on the basis of genome-wide methylation profiling with significant overlap between the two clustering modes. The four methylation clusters showed distinct clinical and genetic features and distinct methylation landscape. All clusters shared hypermethylated enhancers enriched in binding motifs for ETS and bZIP (C/EBP) transcription factor families, involved in the regulation of myeloid cell differentiation. By contrast, one cluster gathering patients with early leukemic evolution exhibited a specific pattern of hypermethylated promoters and, distinctly from other clusters, the upregulation of AP-1 complex members FOS/FOSL2 together with the absence of hypermethylation of their binding motif at target gene enhancers, which is of relevance for leukemic initiation. Among MDS patients with lower-risk IPSS-M, this cluster displayed a significantly inferior overall survival (p < 0.0001). Our study showed that genetic and DNA methylation features of LR-MDS at early stages may refine risk stratification, therefore offering the frame for a precocious therapeutic intervention.

Ectopic expression of DNMT3L in human trophoblast stem cells restores features of the placental methylome.

The placental DNA methylation landscape is unique, with widespread partially methylated domains (PMDs). The placental "methylome" is conserved across mammals, a shared feature of many cancers, and extensively studied for links with pregnancy complications. Human trophoblast stem cells (hTSCs) offer exciting potential for functional studies to better understand this epigenetic feature; however, whether the hTSC epigenome recapitulates primary trophoblast remains unclear. We find that hTSCs exhibit an atypical methylome compared with trophectoderm and 1st trimester cytotrophoblast. Regardless of cell origin, oxygen levels, or culture conditions, hTSCs show localized DNA methylation within transcribed gene bodies and a complete loss of PMDs. Unlike early human trophoblasts, hTSCs display a notable absence of DNMT3L expression, which is necessary for PMD establishment in mouse trophoblasts. Remarkably, we demonstrate that ectopic expression of DNMT3L in hTSCs restores placental PMDs, supporting a conserved role for DNMT3L in de novo methylation in trophoblast development in human embryogenesis.

DEMETER DNA demethylase reshapes the global DNA methylation landscape and controls cell identity transition during plant regeneration

Abstract Background Plants possess a high potential for somatic cell reprogramming, enabling the transition from differentiated tissue to pluripotent callus, followed by the formation of de novo shoots during plant regeneration. Despite extensive studies on the molecular network and key genetic factors involved in this process, the underlying epigenetic landscape remains incompletely understood. Results Here, we explored the dynamics of the methylome and transcriptome during the two-step plant regeneration process. During the leaf-to-callus transition in Arabidopsis Ler, CG methylation shifted across genic regions, exhibiting a similar number of differentially methylated regions (DMRs) for both hypo- and hypermethylation. Pericentromeric regions underwent substantial CG and extensive CHH hypomethylation, alongside some CHG hypermethylation. Upon shoot regeneration from callus, genic regions displayed extensive reconfiguration of CG methylation, while pericentromeric methylation levels highly increased across all cytosine contexts, coinciding with the activation of the RNA-directed DNA methylation (RdDM) pathway. However, mutation in DEMETER (DME) DNA demethylase gene resulted in significant genic CG redistribution and global non-CG hypomethylation in pericentromeric regions, particularly during shoot regeneration. This non-CG hypomethylation observed in dme-2 mutants was, at least partly, due to RdDM downregulation. The dme-2 mutants affected gene expression involved in pluripotency and shoot meristem development, resulting in enhanced shoot regeneration through a reprogrammed state established by pericentromeric hypomethylation compared to wild type. Conclusion Our study demonstrates epigenetic changes, accompanied by transcriptome alterations, during pluripotency acquisition (leaf-to-callus) and regeneration (callus-to-de novo shoot). Additionally, it highlights the functions of the DME demethylase, particularly its close association with the RdDM pathway, which underlies pericentromeric non-CG methylation maintenance. These results provide important insights into the epigenetic reconfiguration associated with cell identity transition during somatic cell reprogramming.

Integration of multi-omics layers empowers precision diagnosis through unveiling pathogenic mechanisms on maple syrup urine disease.

Maple syrup urine disease (MSUD) is a rare inherited metabolic disorder characterized by deficient activity of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex, required to metabolize the amino acids leucine, isoleucine, and valine. Despite its profound metabolic implications, the molecular alterations underlying this metabolic impairment had not yet been completely elucidated. We performed a comprehensive multi-omics integration analysis, including genomic, epigenomic, and transcriptomic data from fibroblasts derived from a cohort of MSUD patients and unaffected controls to genetically characterize an MSUD case and to unravel the MSUD pathophysiology. MSUD patients exhibit a defined episignature that reshapes the global DNA methylation landscape, resulting in the stimulation of HOX cluster genes and the restriction of cell cycle gene-related signatures. Subsequent data integration revealed the impact of AP1-related and CEBPB transcription factors on the observed molecular reorganization, with MEIS1 emerging as a potential downstream candidate affected by robust epigenetic repression in MSUD patients. Furthermore, the integration of multi-omics layers facilitated the identification of a strong epigenetic repression in the DBT promoter in a patient wherein no BCKDH pathogenic variants had been detected. A Circular Chromatin Conformation Capture assay indicated a disturbance of the interactions of DBT promoter, thereby unveiling alternative modes of disease inheritance. Integration of multi-omics data unveiled underlying molecular networks rewired in MSUD patients and represents a powerful approach with diagnostic potential for rare genetic disorders with unknown genetic bases.

Unraveling the DNA methylation landscape in dog blood across breeds

BackgroundDNA methylation is a covalent bond modification that is observed mainly at cytosine bases in the context of CG pairs. DNA methylation patterns reflect the status of individual tissues, such as cell composition, age, and the local environment, in mammals. Genetic factors also impact DNA methylation, and the genetic diversity among various dog breeds provides a valuable platform for exploring this topic. Compared to those in the human genome, studies on the profiling of methylation in the dog genome have been less comprehensive.ResultsOur study provides extensive profiling of DNA methylation in the whole blood of three dog breeds using whole-genome bisulfite sequencing. The difference in DNA methylation between breeds was moderate after removing CpGs overlapping with potential genetic variation. However, variance in methylation between individuals was common and often occurred in promoters and CpG islands (CGIs). Moreover, we adopted contextual awareness methodology to characterize DNA primary sequences using natural language processing (NLP). This method could be used to effectively separate unmethylated CGIs from highly methylated CGIs in the sequences that are identified by the conventional criteria.ConclusionsThis study presents a comprehensive DNA methylation landscape in the dog blood. Our observations reveal the similar methylation patterns across dog breeds, while CGI regions showed high variations in DNA methylation level between individuals. Our study also highlights the potential of NLP approach for analyzing low-complexity DNA sequences, such as CGIs.

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

FoxA1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma.

The ability of cancer cells to undergo identity changes (i.e., lineage plasticity) plays a key role in tumor progression and response to therapy. Loss of the pulmonary lineage specifier NKX2-1 in KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and causes a FoxA1/2-dependent pulmonary-to-gastric lineage switch. However, the mechanisms by which FoxA1/2 activate a latent gastric identity in the lung remain largely unknown. Here, we show that FoxA1/2 reprogram the epigenetic landscape of gastric-specific genes after NKX2-1 loss in mouse models by facilitating ten-eleven translocation (TET)2/3 recruitment, DNA demethylation, histone 3 lysine 27 acetylation (H3K27ac) deposition, and three-dimensional (3D) chromatin interactions. FoxA1/2-mediated DNA methylation changes are highly conserved in human endodermal development and in progression of human lung and pancreatic neoplasia. Furthermore, oncogenic signaling is required for specific elements of FoxA1/2-dependent epigenetic reprogramming. This work demonstrates the role of FoxA1/2 in rewiring the DNA methylation and 3D chromatin landscape of NKX2-1-negative LUAD to drive cancer cell lineage switching.

Single-cell DNA methylome and 3D genome atlas of the human subcutaneous adipose tissue.

Human subcutaneous adipose tissue (SAT) contains a diverse array of cell-types; however, the epigenomic landscape among the SAT cell-types has remained elusive. Our integrative analysis of single-cell resolution DNA methylation and chromatin conformation profiles (snm3C-seq), coupled with matching RNA expression (snRNA-seq), systematically cataloged the epigenomic, 3D topology, and transcriptomic dynamics across the SAT cell-types. We discovered that the SAT CG methylation (mCG) landscape is characterized by pronounced hyper-methylation in myeloid cells and hypo-methylation in adipocytes and adipose stem and progenitor cells (ASPCs), driving nearly half of the 705,063 detected differentially methylated regions (DMRs). In addition to the enriched cell-type-specific transcription factor binding motifs, we identified TET1 and DNMT3A as plausible candidates for regulating cell-type level mCG profiles. Furthermore, we observed that global mCG profiles closely correspond to SAT lineage, which is also reflected in cell-type-specific chromosome compartmentalization. Adipocytes, in particular, display significantly more short-range chromosomal interactions, facilitating the formation of complex local 3D genomic structures that regulate downstream transcriptomic activity, including those associated with adipogenesis. Finally, we discovered that variants in cell-type level DMRs and A compartments significantly predict and are enriched for variance explained in abdominal obesity. Together, our multimodal study characterizes human SAT epigenomic landscape at the cell-type resolution and links partitioned polygenic risk of abdominal obesity to SAT epigenome.

Epigenetic Landscape of DNA Methylation in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, characterized by its aggressive progression and dismal prognosis. Advances in epigenetic profiling, specifically DNA methylation analysis, have significantly deepened our understanding of PDAC pathogenesis. This review synthesizes findings from recent genome-wide DNA methylation studies, which have delineated a complex DNA methylation landscape differentiating between normal and cancerous pancreatic tissues, as well as across various stages and molecular subtypes of PDAC. These studies identified specific differentially methylated regions (DMRs) that not only enhance our grasp of the epigenetic drivers of PDAC but also offer potential biomarkers for early diagnosis and prognosis, enabling the customization of therapeutic approaches. The review further explores how DNA methylation profiling could facilitate the development of subtype-tailored therapies, potentially improving treatment outcomes based on precise molecular characterizations. Overall, leveraging DNA methylation alterations as functional biomarkers holds promise for advancing our understanding of disease progression and refining PDAC management strategies, which could lead to improved patient outcomes and a deeper comprehension of the disease's underlying biological mechanisms.

DNMT3AR882H Is Not Required for Disease Maintenance in Primary Human AML, but Is Associated With Increased Leukemia Stem Cell Frequency.

Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy.

MRNA-seq-based analysis predicts: AEG-1 is a therapeutic target and immunotherapy biomarker for pan-cancer, including OSCC.

The aberrant expression of AEG-1 is significantly correlated with tumorigenesis, development, neurodegeneration and inflammation. However, the relationship between AEG-1 expression and immune infiltration in OSCC, as well as other tumor types, has yet to be comprehensively analyzed. The expression levels, prognostic and clinicopathological characteristics, mutation patterns and methylation landscapes of AEG-1 in various tumors were obtained from multiple databases, including TIMER, GEPIA, HPA, TCGA, UALCAN, cBioPortal, SMART and TISIDB, in addition to single-cell RNA-seq data. The integration of these datasets facilitated the elucidation of the relationships among pan-cancer cellular heterogeneity, immune infiltration and AEG-1 expression levels. In vitro experiments created AEG-1 overexpressing cell lines, and mRNA-seq analyzed AEG-1-related differential genes in OSCC. RT-PCR validated these findings in vivo using xenograft tumors. Tumor cell lines were developed to study AEG-1's effects through H&E, Masson, and PAS staining. Immunohistochemistry examined AEG-1-related gene expression patterns. Our analysis demonstrated that AEG-1 is highly expressed across various cancer types and is associated with tumor grade and patient prognosis. Additionally, AEG-1 amplification was observed in multiple cancers. Notably, we identified a significant elevation of AEG-1 expression in OSCC, which strongly correlated with patient prognosis and immune infiltration. Through mRNA-seq analysis of differentially expressed genes and immune-related gene sets, we identified a strong correlation between AEG-1 and immune infiltration markers such as LCP2, CD247, HLA-DPA1, HLA-DRA, HLA-DRB1, CIITA and CD74 in OSCC. Additionally, AEG-1 was found to regulate Th1/Th2 immune homeostasis, promote glycogen accumulation, and contribute to tumor fibrosis. In conclusion, AEG-1 significantly correlates with prognosis and immune infiltration across various cancer types and holds potential as a novel prognostic immune biomarker for OSCC. This finding may facilitate the identification of patients who are most likely to benefit from adjuvant immunotherapy.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer

BackgroundMuscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes.MethodsPaired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC.ResultsThe prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3’ untranslated region (3’UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3’UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub (https://github.com/lelelililele/Nanopore-m6A-analysis).ConclusionsThe state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.

DNA Methylation Profiling of Salivary Gland Tumors Supports and Expands Conventional Classification

Tumors of the major and minor salivary glands histologically encompass a diverse and partly overlapping spectrum of frequent diagnostically challenging neoplasms. Despite recent advances in molecular testing and the identification of tumor-specific mutations or gene fusions, there is an unmet need to identify additional diagnostic biomarkers for entities lacking specific alterations. In this study, we collected a comprehensive cohort of 363 cases encompassing 20 different salivary gland tumor entities and explored the potential of DNA methylation to classify these tumors. We were able to show that most entities show specific epigenetic signatures and present a machine learning algorithm that achieved a mean balanced accuracy of 0.991. Of note, we showed that cribriform adenocarcinoma is epigenetically distinct from classical polymorphous adenocarcinoma, which could support risk stratification of these tumors. Myoepithelioma and pleomorphic adenoma form a uniform epigenetic class, supporting the theory of a single entity with a broad but continuous morphologic spectrum. Furthermore, we identified a histomorphologically heterogeneous but epigenetically distinct class that could represent a novel tumor entity. In conclusion, our study provides a comprehensive resource of the DNA methylation landscape of salivary gland tumors. Our data provide novel insight into disputed entities and show the potential of DNA methylation to identify new tumor classes. Furthermore, in future, our machine learning classifier could support the histopathologic diagnosis of salivary gland tumors.

Influence of RNA Methylation on Cancerous Cells: A Prospective Approach for Alteration of In Vivo Cellular Composition.

RNA methylation is a dynamic and ubiquitous post-transcriptional modification that plays a pivotal role in regulating gene expression in various conditions like cancer, neurological disorders, cardiovascular diseases, viral infections, metabolic disorders, and autoimmune diseases. RNA methylation manifests across diverse RNA species including messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA), exerting pivotal roles in gene expression regulation and various biological phenomena. Aberrant activity of writer, eraser, and reader proteins enables dysregulated methylation landscape across diverse malignancy transcriptomes, frequently promoting cancer pathogenesis. Numerous oncogenic drivers, tumour suppressors, invasion/metastasis factors, and signalling cascade components undergo methylation changes that modulate respective mRNA stability, translation, splicing, transport, and protein-RNA interactions accordingly. Functional studies confirm methylation-dependent alterations drive proliferation, survival, motility, angiogenesis, stemness, metabolism, and therapeutic evasion programs systemically. Methyltransferase overexpression typifies certain breast, liver, gastric, and other carcinomas correlating with adverse clinical outcomes like diminished overall survival. Mapping efforts uncover nodal transcripts for targeted drug development against hyperactivated regulators including METTL3. Some erasers and readers also suitable lead candidates based on apparent synthetic lethality. Proteomic screens additionally highlight relevant methylation-sensitive effector pathways amenable to combinatorial blockade, reversing compensatory signalling mechanisms that facilitate solid tumour progression. Quantifying global methylation burdens and responsible enzymes clinically predicts patient prognosis, risk stratification for adjuvant therapy, and overall therapeutic responsiveness.