Methylome Analysis Research Articles

Physiological aging and inflammation-induced cellular senescence may contribute to oligodendroglial dysfunction in MS

Aging affects all cell types in the CNS and plays an important role in CNS diseases. However, the underlying molecular mechanisms driving these age-associated changes and their contribution to diseases are only poorly understood. The white matter in the aging brain as well as in diseases, such as Multiple sclerosis is characterized by subtle abnormalities in myelin sheaths and paranodes, suggesting that oligodendrocytes, the myelin-maintaining cells of the CNS, lose the capacity to preserve a proper myelin structure and potentially function in age and certain diseases. Here, we made use of directly converted oligodendrocytes (dchiOL) from young, adult and old human donors to study age-associated changes. dchiOL from all three age groups differentiated in an comparable manner into O4 + immature oligodendrocytes, but the proportion of MBP + mature dchiOL decreased with increasing donor age. This was associated with an increased ROS production and upregulation of cellular senescence markers such as CDKN1A, CDKN2A in old dchiOL. Comparison of the transcriptomic profiles of dchiOL from adult and old donors revealed 1324 differentially regulated genes with limited overlap with transcriptomic profiles of the donors’ fibroblasts or published data sets from directly converted human neurons or primary rodent oligodendroglial lineage cells. Methylome analyses of dchiOL and human white matter tissue samples demonstrate that chronological and epigenetic age correlate in CNS white matter as well as in dchiOL and resulted in the identification of an age-specific epigenetic signature. Furthermore, we observed an accelerated epigenetic aging of the myelinated, normal appearing white matter of multiple sclerosis (MS) patients compared to healthy individuals. Impaired differentiation and upregulation of cellular senescence markers could be induced in young dchiOL in vitro using supernatants from pro-inflammatory microglia. In summary, our data suggest that physiological aging as well as inflammation-induced cellular senescence contribute to oligodendroglial pathology in inflammatory demyelinating diseases such as MS.

Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

Multimodal analysis of cfDNA methylomes for early detecting esophageal squamous cell carcinoma and precancerous lesions

Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.

489. Deciphering the Molecular Basis of Aging in Substance Use Disorder: Integrative Transcriptomic and Methylomic Analysis

489. Deciphering the Molecular Basis of Aging in Substance Use Disorder: Integrative Transcriptomic and Methylomic Analysis

Coordinated Regulation of Myonuclear DNA Methylation, mRNA, and miRNA Levels Associates with Mitochondrial Function During an Acute Skeletal Muscle Growth Stimulus

The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature miRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth. Y.W. was supported by the National Institutes of Health (K99 AR081367) K.A.M. was supported by the National Institutes of Health (R00 AG063994 and R01 AG080047). F.v.W. was supported by the Swedish research council (2022-01392), AFM-Telethon (23137), SMDF, Åke Wiberg, Swedish Medical Association, and the Swedish Research Council for Sport Science (2022/10). 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.

Neuronal-Specific Methylome and Hydroxymethylome Analysis Reveal Replicated and Novel Loci Associated With Alcohol Use Disorder

Neuronal-Specific Methylome and Hydroxymethylome Analysis Reveal Replicated and Novel Loci Associated With Alcohol Use Disorder

Analysis of Methylome of Different Forms of Basal Cell Hyperplasia and Squamous Cell Metaplasia of Bronchial Epithelium.

Squamous cell lung cancer (SCLC) occurs as a result of dysregenerative changes in the bronchial epithelium: basal cell hyperplasia (BCH), squamous cell metaplasia (SM), and dysplasia. We previously suggested that combinations of precancerous changes detected in the small bronchi of patients with SCLC may reflect various "scenarios" of the precancerous process: isolated BCH→stopping at the stage of hyperplasia, BCH+SM→progression of hyperplasia into metaplasia, SM+dysplasia→progression of metaplasia into dysplasia. In this study, DNA methylome of various forms of precancerous changes in the bronchial epithelium of SCLC patients was analyzed using the genome-wide bisulfite sequencing. In BCH combined with SM, in contrast to isolated BCH, differentially methylated regions were identified in genes of the pathogenetically significant MET signaling pathway (RNMT, HPN). Differentially methylated regions affecting genes involved in inflammation regulation (IL-23, IL-23R, IL12B, IL12RB1, and FIS1) were detected in SM combined with dysplasia in comparison with SM combined with BCH. The revealed changes in DNA methylation may underlie various "scenarios" of the precancerous process in the bronchial epithelium.

Transcriptome and methylome analyses unveil the effects of low and high temperatures on the postharvest senescence of plum fruit

Temperature strongly affects plum fruit senescence, but the epigenetic mechanism is unclear. Analysis of data on firmness, weigh loss rate, and decay rate showed that high temperature (HT) accelerates postharvest senescence, while low temperature (LT) delays it. Transcriptome analysis suggests that genes related to cell wall metabolism are associated with fruit softening. DNA methylation analysis shows that HT promotes fruit senescence accompanied by an increase in DNA methylation level, while LT delays senescence accompanied by a decrease in DNA methylation level. Further analysis suggests that PsDRM2 and PsDML1 may be responsible for regulating changes in methylation level. Association analysis showed that HT accelerates senescence through DNA methylation-mediated regulation of flavonoid metabolism, whereas LT delays senescence through DNA methylation-mediated regulation of lipid metabolism. These findings highlight the importance of temperature and DNA methylation in controlling postharvest senescence and provide valuable insights for developing effective strategies to control fruit senescence.

Integrative analysis in head and neck cancer reveals distinct role of miRNome and methylome as tumour epigenetic drivers

Head and neck cancer is the sixth most common malignancy worldwide, with the relatively low 5-year survival rate, mainly because it is diagnosed at a late stage. Infection with HPV is a well known aetiology, which affects the nature of these cancers and patients’ survival. Besides, it is considered that the main driving force for this type of cancer could be epigenetics. In this study we aimed to find potential epigenetic biomarkers, by integrating miRNome, methylome, and transcriptome analyses. From the fresh head and neck cancer tissue samples, we chose a group for miRNome, methylome and transcriptome profiling, in comparison to adequate control samples. Bioinformatics analyses are performed in R v4.2.2. Count normalisation and group differential expression for mRNA and the previously obtained miRNA count data was performed with DESeq2 v1.36. Gene set enrichment analysis was performed and visualised using gProfiler2 v0.2.1 Identification of miRNA targets was performed by querying in miRTarBase using multiMiR v1.18.0. Annotation of CpG sites merging into islands was obtained from RnBeads.hg19 v1.28.0. package. For the integrative analysis we performed kmeans clustering using stats v4.2.2 package, using 8–12 clusters and nstart 100. We found that transcriptome analysis divides samples into cancers and controls clusters, with no relation to HPV status or cancer anatomical location. Differentially expressed genes (n = 2781) were predominantly associated with signalling pathways of tumour progression. We identified a cluster of genes under the control of the transcription factor E2F that are significantly underexpressed in cancer tissue, as well as T cell immunity genes and genes related to regulation of transcription. Among overexpressed genes in tumours we found those that belong to cell cycle regulation and vasculature. A small number of genes were found significantly differentially expressed in HPV-positive versus HPV-negative tumours (for example NEFH, ZFR2, TAF7L, ZNF541, and TYMS). In this comprehensive study on an overlapping set of samples where the integration of miRNome, methylome and transcriptome analysis were performed for head and neck cancer, we demonstrated that the majority of genes were associated exclusively with miRNome or methylome and, to a lesser extent, under the control of both epigenetic mechanisms.

Integrated analysis of the gonadal methylome and transcriptome provides new insights into the expression regulation of sex determination and differentiation genes in spotted scat (Scatophagus argus)

The spotted scat (Scatophagus argus) is an economically important mariculture fish. There have been no studies of gonadal DNA methylation at the genomic level in S. argus. Here, the gonadal methylome profiles of male and female S. argus were constructed and a link between DNA methylation and transcriptional regulation was identified. The gonads at stage IV of three male and female S. argus individuals were subjected to whole-genome bisulfite sequencing (WGBS), generating an average of 17.76 Gb of clean data for each sample. Analysis of methylation levels in different genomic regions revealed that both male and female S. argus gonads exhibited low methylation levels in the promoters and first exons, but high levels in the inner exons and introns. In general, DNA methylation levels were higher in the testes than in the ovaries. A total of 234,482 differentially methylated regions (DMRs) were identified between testes and ovaries, including 234,245, 26 and 211 DMRs in the CpG, CHG, and CHH backgrounds, respectively. Combined methylation and transcriptome analysis revealed that the relationship between methylation patterns and gene expression was generally negatively correlated. The male-specific Dmrt1Y, which is the candidate sex-determining gene of S. argus, was lowly modified by DNA methylation at its promoter region in the testis. While its allele on the X chromosome, Dmrt1ΔX, was highly modified by DNA methylation in both the testis and ovary. The expression levels of some other genes involved in sex differentiation and sex steroid synthesis were also regulated by DNA methylation. This study sheds light on the divergent methylation patterns in the gonadal genomes of male and female S. argus. These findings laid the foundation for subsequent analysis of differentially expressed sex determination and differentiation genes between male and female S. argus, providing a crucial theoretical basis for sex-control breeding efforts in S. argus.

Genome-wide DNA methylation-analysis of blastic plasmacytoid dendritic cell neoplasm identifies distinct molecular features

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid dendritic cells (pDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES and RNA-seq as well as ATAC-seq on selected cases. We determined the BPDCN DNA methylation profile and observed a dramatic loss of DNA methylation during malignant transformation from early and mature DCs towards BPDCN. DNA methylation profiles further differentiate between BPDCN, AML, CMML, and T-ALL exhibiting the most striking global demethylation, mitotic stress, and merely localized DNA hypermethylation in BPDCN resulting in pronounced inactivation of tumor suppressor genes by comparison. DNA methylation-based analysis of the tumor microenvironment by MethylCIBERSORT yielded two, prognostically relevant clusters (IC1 and IC2) with specific cellular composition and mutational spectra. Further, the transcriptional subgroups of BPDCN (C1 and C2) differ by DNA methylation signatures in interleukin/inflammatory signaling genes but also by higher transcription factor activity of JAK-STAT and NFkB signaling in C2 in contrast to an EZH2 dependence in C1-BPDCN. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

Abstract LB193: Enhanced single cell DNA methylation analysis using combinatorial indexing

Abstract The epigenetic landscape of the human brain undergoes a plethora of changes during malignant transformation. In recent years, DNA methylation-based epigenetic modifications have been widely studied using traditional techniques like bisulfite sequencing and enzymatic methyl sequencing (EM-seq). However, these methods analyze bulk cell populations and lack the granularity of single-cell analysis. Although advances in single-cell analysis have revolutionized our understanding of cellular heterogeneity and functional diversity within complex biological systems, they are still costly, low throughput, and laborious. To address these challenges, ScaleBio has pioneered combinatorial indexing technology, enabling a significant increase in cell throughput. This method utilizes the cell itself as a compartment to perform 2-3 rounds of sequential barcoding in a plate-based workflow, eliminating the need for complex instrumentation. This technology has been successfully adapted to assess DNA methylation at the single-cell level offering a robust, affordable, high-throughput protocol that enhances yield, diversity, and coverage. In this study we used ScaleBio's single-cell methylation kit to investigate DNA methylation patterns during oncogenesis using cancer cells with widespread DNA methylation changes, such as human isocitrate dehydrogenase (IDH) mutant glioma cells at the single-cell level. The IDH gene family, comprising of IDH1, IDH2, and IDH3, encodes enzymes involved in the tricarboxylic acid (TCA) cycle. IDH1/2 mutations are present in over 80% of low-grade gliomas while IDH-mutant gliomas constitute about 1/5th of all adult diffuse gliomas, thus making them one of the most common brain tumor subtypes. As such, IDH mutations have emerged as attractive therapeutic targets for glioma treatment. By uncovering DNA methylation patterns at the single cell level, we provide here an epigenetic map to better understand this complex disease and better inform clinical discoveries. We achieved high cell recovery and robust cytosine coverage throughout our analysis of single cell methylomes isolated from human glioma tumor tissue. Using this data we generated a ranked list of the top hypo- and hypermethylated genomic regions and identified cell type specific clusters seen in different pathological states by looking at Differentially Methylated Regions (DMR) uncovering unique single-cell methylation profiles that may be obscured by bulk or pseudo-bulk analysis. These data show that the ScaleBio single-cell methylation workflow offers increased sensitivity, specificity, and accuracy in identifying DNA methylation sites when compared to other techniques while offering a comprehensive view of methylomes and providing insights into cellular heterogeneity and trajectories. Citation Format: Sanika Khare, Dominic Skinner, Maggie Nakamoto, Hosu Sin, Ashley Woodfin, Eric Pu, Lin Lin, Jason Koth, Beth Walczak. Enhanced single cell DNA methylation analysis using combinatorial indexing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB193.

Neuronal-specific methylome and hydroxymethylome analysis reveal significant loci associated with alcohol use disorder.

Background: Alcohol use disorder (AUD) is a complex condition associated with adverse health consequences that affect millions of individuals worldwide. Epigenetic modifications, including DNA methylation (5mC), have been associated with AUD and other alcohol-related traits. Epigenome-wide association studies (EWAS) have identified differentially methylated genes associated with AUD in human peripheral and brain tissue. More recently, epigenetic studies of AUD have also evaluated DNA hydroxymethylation (5hmC) in the human brain. However, most of the epigenetic work in postmortem brain tissue has examined bulk tissue. In this study, we investigated neuronal-specific 5mC and 5hmC alterations at CpG sites associated with AUD in the human orbitofrontal cortex (OFC). Methods: Neuronal nuclei from the OFC were evaluated in 34 human postmortem brain samples (10 AUD, 24 non-AUD). Reduced representation oxidative bisulfite sequencing was used to assess 5mC and 5hmC at the genome-wide level. Differential 5mC and 5hmC were evaluated using the methylKit R package and significance was set at false discovery rate < 0.05 and differential methylation > 2. Functional enrichment analyses were performed, and gene-level convergence was evaluated in an independent dataset that assessed 5mC and 5hmC of AUD in bulk cortical tissue. Results: We identified 417 5mC and 3635hmC significant differential CpG sites associated with AUD, with 59% in gene promoters. Some of the identified genes have been previously implicated in alcohol consumption, including SYK, DNMT3A for 5mC, GAD1, DLX1, DLX2, for 5hmC and GATA4 in both. Convergence with a previous AUD 5mC and 5hmC study was observed for 28 genes. We also identified 5 and 35 differential regions for 5mC and 5hmC, respectively. Lastly, GWAS enrichment analysis showed an association with AUD for differential 5mC genes. Discussion: This study reveals neuronal-specific methylome and hydroxymethylome dysregulation associated with AUD, identifying both previously reported and potentially novel gene associations with AUD. Our findings provide new insights into the epigenomic dysregulation of AUD in the human brain.

Integrative methylome and transcriptome analysis reveals epigenetic regulation of Fusobacterium nucleatum in laryngeal cancer.

The aetiological mechanisms of Fusobacterium nucleatum in laryngeal cancer remain unclear. This study aimed to reveal the epigenetic signature induced by F. nucleatum in laryngeal squamous cell carcinoma (LSCC). Combined analysis of methylome and transcriptome data was performed to address the functional role of F. nucleatum in laryngeal cancer. Twenty-nine differentially expressed methylation-driven genes were identified by mapping the methylation levels of significant differential methylation sites to the expression levels of related genes. The combined analysis revealed that F. nucleatum promoted Janus kinase 3 (JAK3) gene expression in LSCC. Further validation found decreased methylation and elevated expression of JAK3 in the F. nucleatum-treated LSCC cell group; F. nucleatum abundance and JAK3 gene expression had a positive correlation in tumour tissues. This analysis provides a novel understanding of the impact of F. nucleatum in the methylome and transcriptome of laryngeal cancer. Identification of these epigenetic regulatory mechanisms opens up new avenues for mechanistic studies to explore novel therapeutic strategies.

Abstract 7544: Bromodomain and extra-terminal protein inhibitors deplete nuclear factor 1C and reduce stemness and growth of human small cell lung cancer cells in vitro and in vivo

Abstract Objectives: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to examine complex epigenetic mechanisms underpinning the aggressiveness of SCLC, with a focus on understanding those mediating stemness in these highly lethal cancers. Methods: Normal small airway epithelial cells (SAECs) were reprogrammed into iPSCs, validated by various assays, such as spectral karyotyping and teratoma formation. Genome-wide chromatin landscapes of lung iPSCs (Lu-iPSC) and SAECs, as well as NSCLC and SCLC lines were characterized using RNA-seq, DNA methylome, and DNase-hypersensitivity sequencing (DHS). Gene knock-downs were established using siRNA/shRNA techniques, along with qRT-PCR, immunoblotting, proliferation, and tumorigenicity assays. Glycolysis was analyzed using Seahorse assays. In-silico analysis of the NFIC promoter was performed using TFmapper. NFIC and related gene expression in human SCLC cells following NFIC or Bromodomain-containing protein (BRD) knockdown or exposure to BET inhibitors (BETi) were evaluated. Stemness scores were calculated using established RNA-seq criteria, and gene set enrichment analyses were conducted. Proliferation assays and xenograft models were used for in-vitro and in-vivo SCLC growth assessment. Results: RNA-seq,DNA methylome and DHS analyses revealed shared characteristics among SAEC, Lu-iPSC, SCLC, and NSCLC, whereas SOXs and POUs showed strong activity in Lu-iPSC but not SCLC. NFIC, with elevated chromatin occupancy in SCLC, exhibited increased expression compared to SAEC. NFIC KD in SCLC significantly curtailed cell growth in-vitro and in-vivo, affecting multiple pathways, including glycolysis and stem cell signaling etc, confirmed by Seahorse assays. Furthermore, we identified occupancy sites for BRD4 in the NFIC promoter, and knockdown of BRD4, but not BRD2 or BRD3, reduced NFIC expression in SCLC cells. BET inhibitor (BETi) treatment dose-dependently decreased NFIC expression in SCLC lines and human SCLC PDXs, irrespective of subtype. BETi treatment also reduced SCLC stemness and showed a significant overlap in gene regulation with NFIC knockdown, affecting pathways related to gluconeogenesis, neuronal differentiation, and embryonic organ morphogenesis. Importantly, BETi treatment effectively reduced SCLC cell growth in-vitro and in-vivo, suggesting the potential therapeutic value of targeting NFIC via BET inhibition in SCLC. Conclusions: NFIC, a unique transcription factor in SCLC, emerges as a key downstream target of BRD4. These findings underscore the potential druggability of NFIC in clinical settings, provide compelling rationale for evaluating BETi for SCLC therapy. Citation Format: Haitao Wang, Ruihong Wang, Frank Villa-Hernandez, Vivek Shukla, Lyuba Varticovski, Songjoon Baek, Frank Echtenkamp, Sudheer Kumar Gara, Mary R. Zhang, Stephanie Shiffka, Razi Raziuddin, Leonard M. Neckers, W. Marston Linehan, Gordon L. Hager, Shamus R. Carr, Chuong D. Hoang, Haobin Chen, David S. Schrump. Bromodomain and extra-terminal protein inhibitors deplete nuclear factor 1C and reduce stemness and growth of human small cell lung cancer cells in vitro and in vivo [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 7544.

Abstract 280: Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors

Abstract Our purpose is to identify stromal-derived signals required for IDH1/2-mut hematopoietic stem cells (HSCs) to become leukemogenic. Mutations in IDH genes are found in 20% of acute myeloid leukemia (AML) patients and are not transformative. They produce the oncometabolite 2HG which inhibits key epigenetic regulators. We explored whether epigenetic changes in the bone marrow (BM) niche could provide cooperative signals to IDH-mut HSCs leading to their transformation and disease development. We found that 2HG is elevated in the BM plasma of MDS/AML patients with IDH1/2 mutations as compared to IDH-wt patients. Interestingly, 2HG is taken up by BM stromal cells (BMSCs) suggesting it may induce aberrant methylation patterns in them. Indeed, methylome analysis showed hypermethylation in BMSCs of AML patients with IDH1/2 mutations compared to wt. Among the most hypermethylated genes in the IDH-mut stroma are AXIN1, AXIN2 and NDK2, all negative regulators of β-catenin; this pathway activation was previously shown to cause MDS/AML in mice and present in 38% of patients. In agreement with these findings βcat activation in the stroma of IDH-mut vs IDH-wt AML patients showed a strong positive correlation as 50% of IDH-mut patients had activated βcat in their BMSCs. Inquiring whether stroma methylation precedes HSC transformation, we analyzed samples from healthy individuals with IDH-mut clonal hematopoiesis. They exhibited increased global and AXIN1 promoter methylation compared to samples without such clones, but not active βcat in osteoblasts, indicating this might be a later step along with disease development. In fact, transplantation of HSCs from Idh2-mut mice to recipients expressing active βcat in osteoblasts led to increase in blasts and shortened survival compared to wt mice transplanted with Idh2-mut HSCs. As many patients relapse following treatment with IDH inhibitors, we investigated a potential role for βcat signaling in treatment outcome. IDH-mut AML patients who do not respond to treatment with the IDHi present more frequently with activated βcat in osteoblasts compared to responders. Analyzing samples from the same patient pre- and post-treatment with IDHi, we found before treatment most patients were positive for βcat stromal activation, whereas after treatment, all patients with complete response were negative, while patients with progressive disease remained positive. Our model suggests IDH-mut HSCs secrete 2HG inducing epigenetic alterations in the niche, some of which cause activation of the βcat pathway. In turn, this additional signal leads to the transformation of HSCs to dysplastic cells and MDS/AML development. This work uncovers a new mechanism for transformation to MDS/AML and resistance to IDHi treatment. Thus, targeting βcat activation in the stroma, in combination with IDHi, could improve treatment outcome. Citation Format: Paraskevi Vgenopoulou, Luis Flores, Ioanna Mosialou, Junfei Zhao, X. Shawn Liu, Pamela Sung, Virginie Penard-Lacronique, Martin Carroll, Stavroula Kousteni. Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors [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 280.

Abstract 7022: Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. Traditionally, sodium bisulfite conversion was used to distinguish 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) from cytosines. However, this method damages DNA and introduces significant sequencing bias. NEBNext® Enzymatic Methyl-seq (EM-seq™) is an enzymatic conversion approach that minimizes DNA damage therefore enabling longer insert sizes, lower duplication rates and a more accurate quantification of methylation in DNA samples with inputs ranging from 10 ng to 200 ng. An improved enzymatic conversion method, EM-seq v2, has a more streamlined workflow and an expanded DNA input range of 100 pg to 200 ng. Libraries made using NA12878 genomic DNA show improved yields, consistent global methylation and an even GC bias representation. The increased library yields subsequently reduces sequencing duplications and costs. Sequencing costs are an important consideration when performing whole genome methylome analysis as this often needs high sequencing depth to attain meaningful coverage to accurately call methylation. Reduced representation libraries, a commonly used method to investigate DNA methylation, relies on enrichment of CpGs within libraries digested using MspI. These CpGs are commonly found in regulatory regions. Although lacking comprehensive CpG coverage, for many researchers this method provides enough cost effective CpG methylation coverage for their studies. Reduced Representation EM-seq libraries (RREM-seq) and Reduced Representation Bisulfite libraries (RRBS) were made using 200 ng to 100 pg of NA12878 DNA. EM-seq adaptors were ligated to MspI digested DNA followed by conversion using either EM-seq v2 or sodium bisulfite. Libraries were PCR amplified and sequenced on an Illumina NovaSeq platform. RREM-seq resulted in higher yields and reproducible results regardless of input amounts compared to RRBS libraries. RREM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. RREM-seq libraries have superior sequencing metrics resulting in robust methylation profiling. Focusing on a subset of the genome generates higher coverage DNA methylation data at a lower DNA sequencing cost. Citation Format: Vaishnavi Panchapakesa, Chaithanya Ponnaluri, Daniel Evanich, Ariel Erijman, Bradley Langhorst, Louise Williams. Whole genome and reduced representation enzymatic methyl-seq enable cost effective methylomes [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 7022.

Unveiling the Impact of ApoF Deficiency on Liver and Lipid Metabolism: Insights from Transcriptome-Wide m6A Methylome Analysis in Mice.

Lipid metabolism participates in various physiological processes and has been shown to be connected to the development and progression of multiple diseases, especially metabolic hepatopathy. Apolipoproteins (Apos) act as vectors that combine with lipids, such as cholesterol and triglycerides (TGs). Despite being involved in lipid transportation and metabolism, the critical role of Apos in the maintenance of lipid metabolism has still not been fully revealed. This study sought to clarify variations related to m6A methylome in ApoF gene knockout mice with disordered lipid metabolism based on the bioinformatics method of transcriptome-wide m6A methylome epitranscriptomics. High-throughput methylated RNA immunoprecipitation sequencing (MeRIP-seq) was conducted in both wild-type (WT) and ApoF knockout (KO) mice. As a result, the liver histopathology presented vacuolization and steatosis, and the serum biochemical assays reported abnormal lipid content in KO mice. The m6A-modified mRNAs were conformed consensus sequenced in eukaryotes, and the distribution was enriched within the coding sequences and 3' non-coding regions. In KO mice, the functional annotation terms of the differentially expressed genes (DEGs) included cholesterol, steroid and lipid metabolism, and lipid storage. In the differentially m6A-methylated mRNAs, the functional annotation terms included cholesterol, TG, and long-chain fatty acid metabolic processes; lipid transport; and liver development. The overlapping DEGs and differential m6A-modified mRNAs were also enriched in terms of lipid metabolism disorder. In conclusion, transcriptome-wide MeRIP sequencing in ApoF KO mice demonstrated the role of this crucial apolipoprotein in liver health and lipid metabolism.

Biological Insights from Cell-Free DNA Methylome Analysis in Preeclampsia.

Biological Insights from Cell-Free DNA Methylome Analysis in Preeclampsia.

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed.DesignTo assess circulating marker...