Methylated DNA Immunoprecipitation Sequencing Research Articles

P.102 Integration of Ultrasensitive electroluminescent immunoassay and cell-free DNA methylation analysis for the non-invasive discrimination of adult diffuse gliomas

Background: Gliomas are highly aggressive brain tumors with nearly universal recurrence rate. Despite this, the ability to accurately predict tumor recurrence relies solely on serial MRI imaging, highlighting the need for prognostic biomarkers. Due to the low accuracies of individual serum markers, we have proposed the use of an integrated, multi-platform approach to biomarker discovery. Methods: A cohort of 107 glioma plasma samples, including 30 pairs, underwent plasma proteomic, consisting of a panel of serum proteins (FABP4, GFAP, NFL, Tau and MMP3,4 &7) quantified through ultrasensitive electrochemiluminescence multiplexed immunoassays, and plasma DNA methylation analysis, captured through cell-free methylated DNA immunoprecipitation and high-throughput sequencing. Results: Unsupervised hierarchal clustering revealed robust separation of primary and recurrent tumors through plasma proteomics, associated with a distinct plasma methylation signature. NFL, Tau and MMP3 levels differed between primary and recurrent samples; pair-wise analysis revealed increased in NFL and Tau concentrations upon recurrence. Tau levels predicted outcome independent of WHO Grade and IDH status. A predictive model created through the integration of the proteomic and methylation signatures revealed an AUC of 0.83. Conclusions: The combination of DNA methylation and plasma proteomics showcases that an integrative approach may improve the ability of these techniques for the serial monitoring of gliomas patients.

Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy.

DNA methylation alterations are widespread in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. Although the presence of somatic mutations in peripheral blood can predict the risk of development of AML and MDS, its accuracy remains unsatisfactory. We performed global DNA methylation profiling in a case control study nested within the Singapore Chinese Health Study to evaluate whether DNA methylation alterations were associated with AML/MDS development. Targeted deep sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-seq) were performed on peripheral blood collected a median of 9.9 years before diagnosis of AML or MDS, together with age-matched still-healthy individuals as controls. Sixty-six individuals who developed AML or MDS displayed significant DNA methylation changes in the peripheral blood compared with 167 age- and gender-matched controls who did not develop AML/MDS during the follow-up period. Alterations in methylation in the differentially methylation regions were associated with increased odds of developing AML/MDS. The epigenetic changes may be acquired independently and before somatic mutations that are relevant for AML/MDS development. The association between methylation changes and the risk of pre-AML/MDS in these individuals was considerably stronger than somatic mutations, suggesting that methylation changes could be used as biomarkers for pre-AML/MDS screening.

Abstract 2427: The development of a tissue-agnostic genome-wide methylome enrichment MRD assay for applications across the cancer care continuum for head and neck malignancies

Abstract Background: Plasma-based tests to quantify circulating cell-free DNA cancer signal have emerged as viable applications across the cancer continuum, from early detection to optimal disease management. Here we demonstrate the feasibility of a tissue-agnostic, genome-wide methylome enrichment platform based on cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMEDIP-seq) for cancer detection, cancer signal quantification, and prognostication in head and neck cancer (HNC). Methods: Pre-treatment plasma samples from individuals with newly diagnosed stage I-IV HPV+ or HPV- HNC were analyzed with a bisulfite-free, non-degradative, genome-wide methylome enrichment platform using 5-10 ng of cell-free DNA. For cancer detection, a machine learning classifier used differentially methylated regions to distinguish cancers from non-cancer controls. The area under the receiver operating characteristic curve (AUC) and 95% confidence intervals were calculated. Cancer signals were quantified from average normalized counts across informative methylated regions and a 95% specificity threshold. For prognostication, events were defined as recurrence, progression, or death due to HNC, whichever occurred earliest. Time to event was compared for samples with cancer signal quantities above versus below the threshold. Post-treatment and longitudinal plasma samples from individuals with Stage I-IVB HNC (HPV+ and HPV- included) will be analyzed for recurrence prediction and detection of relapse. More than 100 patients and 300 samples will be analyzed. Results: For cancer detection, 92 pre-treatment plasma samples from HNC cases were distinguished from 674 controls with an AUC of 0.96 (0.94, 0.98). The AUC was 0.93 (0.86, 1.0) for Stage I, 0.93 (0.83, 1.0) for Stage II, 0.96 (0.94, 0.99) for Stage III, and 0.97 (0.96, 0.99) for Stage IV. For prognostication, 91 pre-treatment samples were included (7 stage I, 16 stage II, 23 stage III, 45 stage IV). Median follow-up time was 50.6 months with 27 events. Likelihood of recurrence or progression was significantly higher in samples with cancer signal above the threshold [hazard ratio 5.4 (95% CI 2.25, 12.95), log-rank P<0.001]. In the upcoming analysis, data will be reported on the ability to predict recurrence and relapse in post-treatment samples. Conclusions: The cfMeDIP-seq approach demonstrated robust detection of HNC, across all stages and subtypes, and the ability to predict recurrence and progression from pre-treatment samples. We will report training data with cross validation to predict recurrence and relapse using post-treatment and longitudinal sampling. Collectively, data from these studies indicate that genome wide methylome enrichment has multiple use cases across the care continuum for patients with HNC. Citation Format: Geoffrey Liu, Jun Min, Yarong Wang, Justin Burgener, Ben Brown, Karen Budhraja, Junjun Zhang, Owen Hall, Shu Yi Shen, Martha Pienkowski, Shao Hui Huang, Laurie Ailles, Katrina Rey-McIntyre, Jeremy B. Provance, Eduardo Sosa, Cynthia Frye, Scott Bratman, Brian Allen, Joshua T. Jones, Abel Licon, Jing Zhang, Anne-Renee Hartman, Daniel D. De Carvalho. The development of a tissue-agnostic genome-wide methylome enrichment MRD assay for applications across the cancer care continuum for head and neck malignancies [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 2427.

Abstract 5024: Analytical performance of a genome-wide methylome enrichment platform to detect minimal residual disease from plasma-derived cell-free DNA

Abstract Introduction: Plasma-derived cell-free DNA (cfDNA) can be used to identify cancer signals, including minimal residual disease (MRD), in patients who have undergone curative cancer treatments. The cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) methodology is combined with custom algorithms that leverage differentially methylated regions (DMRs) found in cfDNA to distinguish between cancer and non-cancer signals. This novel non-degradative, tissue-agnostic approach was developed to bypass the limitations of bisulfite-sequencing and tissue-informed methods used in other liquid biopsy technologies. Here we present preliminary analytical performance metrics of an algorithm in development for detecting MRD. Methods: In the genome-wide methylome enrichment platform, plasma-derived cfDNA is subjected to standard library preparation, combined with DNA filler, denatured, and immunoprecipitated using an anti-5-mC antibody. Captured DNA is amplified and sequenced. For this study, a candidate algorithm comprised of differentially methylated regions (DMRs) and 12 cancer types was used to quantify cancer-specific methylation. Control samples from 12 non-cancer donors were used to establish a selected 95% true negative rate threshold. This threshold was used to determine assay sensitivity, which was evaluated using contrived cancer samples intended to mimic low-level circulating tumor DNA (ctDNA) representative of MRD. Samples were contrived by titrating fragmented DNA from three immortalized tumor-derived cell-lines into plasma-derived cfDNA in a titration series targeting <1% ctDNA levels. Cell-lines were of non-small cell lung cancer and head and neck squamous cell carcinoma origin; titration series were replicated for a total of 65 tests. Results: All non-cancer and contrived cancer samples met in-process quality control metrics. This included ≥98.5% methylation specificity and ≥80 million unique molecules. Limit of detection calculations at 95% sensitivity (LoD95) were <0.1%. Conclusions: Tissue-agnostic approaches for detecting cancer signals from plasma have significant benefit, especially in settings where tissue is not accessible for evaluation. However, these tests must have highly sensitive methods of cancer signal detection for clinical applications. Our preliminary analytical data demonstrates the use of a blood-based, tissue- agnostic genome-wide methylome enrichment platform utilizing non-degradative methodology combined with specific algorithms and DMRs for MRD quantification and prognostic prediction, using treatment-naïve plasma samples. Future studies in post-treatment and longitudinal samples are ongoing to evaluate the utility of this genome-wide methylome enrichment platform for cancer management. Citation Format: Shu Yi Shen, Iulia Cirlan, Felicia Vincelli, Ben Brown, Jun Min, Justin Burgener, Junjun Zhang, Yulia Newton, Margaret Gruca, Abel Licon, Jing Zhang, Anne-Renee Hartman, Alan Williams, Hestia Mellert, Daniel D. De Carvalho. Analytical performance of a genome-wide methylome enrichment platform to detect minimal residual disease from plasma-derived cell-free DNA [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 5024.

Abstract 3454: Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk

Abstract Cell-free DNA (cfDNA) epigenetic and fragmentomic profiling has emerged as prominent non-invasive approaches for early cancer detection and subtyping. However, owing to difficulties in observing the early development of human malignancies, most cancer biomarker and evolution studies to date have primarily examined biologics following a diagnosis. Utilizing cfDNA as a screening tool for early disease detection requires assessment of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers. Here, we leverage the Ontario Health Study (OHS), a prospective longitudinal population cohort, to assess cfDNA profiles in blood plasma collected from participants that developed breast (n=476), prostate (n=342) and pancreatic (n=32) cancers throughout the study follow up time between 2009 and 2019. We performed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) on baseline plasma samples from incident cancer cases up to eight years prior to diagnosis, in addition to matched controls (n=404) with no history of cancer through follow-up. Genome-wide plasma cfDNA differential methylation analysis revealed significantly hypermethylated regions, particularly among CpG island and shore regions, were predictive of early breast cancers, and overlapped hypermethylated regions in solid cancer tissues relative to adjacent normal tissues and peripheral blood leukocytes. Using a repeated cross-validation strategy we found that as few as 200 hypermethylated regions can identify individuals with breast cancer in blood up to seven years prior to diagnosis among discovery set samples (AUC=0.725) and are highly generalizable to samples processed in separate batches (AUC = 0.650). Remarkably, integrating biomarker hypermethylated regions with fragmentomic features including fragment length and 5’ tetranucleotide motif proportions further increased accuracy for detecting individuals with early prostate cancers (AUC=0.804). In addition, we observed shared genome-wide cfDNA methylome changes associated with aging and heavy alcohol consumption in cfDNA. We identified increased shedding of tissue-specific methylation markers from liver and pancreatic tissue among individuals consuming more than three drinks a week relative to non-drinkers, reflective of potential signatures of tissue damage. Citation Format: Nicholas Cheng, Kimberly Skead, Tom Oullette, Scott Bratman, Daniel De Carvalho, David Soave, Philip Awadalla. Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk [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 3454.

KIDNEY-PAGER: analysis of circulating tumor DNA as a biomarker in renal cancer - an observational trial. Study protocol.

Management of localized renal cell carcinoma (RCC) is challenged by inaccurate methods to assess the risk of recurrence and deferred detection of relapse and residual disease after radical or partial nephrectomy. Circulating tumor DNA (ctDNA) has been proposed as a potential biomarker in RCC. Conduction of an observational study to evaluate the validity of ctDNA as a biomarker of the risk of recurrence and subclinical residual disease to improve postoperative surveillance. Urine and blood will be prospectively collected before and after surgery of the primary tumor from up to 500 patients until 5 years of follow-up. ctDNA analysis will be performed using shallow whole genome sequencing and cell-free methylated DNA immunoprecipitation sequencing. ctDNA levels in plasma and urine will be correlated to oncological outcomes. Residual blood and urine as well as tissue biopsies will be biobanked for future research. Results will pave the way for future ctDNA-guided clinical trials aiming to improve RCC management.

The Hypothalamic Epigenetic Landscape in Dietary Obesity.

The hypothalamus in the brain plays a pivotal role in controlling energy balance in vertebrates. Nutritional excess through high-fat diet (HFD) feeding can dysregulate hypothalamic signaling at multiple levels. Yet, it remains largely unknown in what magnitude HFD feeding may impact epigenetics in this brain region. Here, it is shown that HFD feeding can significantly alter hypothalamic epigenetic events, including posttranslational histone modifications, DNA methylation, and chromatin accessibility. The authors comprehensively analyze the chromatin immunoprecipitation-sequencing (ChIP-seq), methylated DNA immunoprecipitation-sequencing (MeDIP-seq), single nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), and RNA-seq data of the hypothalamus of C57 BL/6 mice fed with a chow or HFD for 1 to 6 months. The chromatins are categorized into 6 states using the obtained ChIP-seq data for H3K4me3, H3K27ac, H3K9me3, H3K27me3, and H3K36me3. A 1-month HFD feeding dysregulates histone modifications and DNA methylation more pronouncedly than that of 3- or 6-month. Besides, HFD feeding differentially impacts chromatin accessibility in hypothalamic cells. Thus, the epigenetic landscape is dysregulated in the hypothalamus of dietary obesity mice.

Early Cancer Detection in Li-Fraumeni Syndrome with Cell-Free DNA.

By utilizing an integrated cell-free DNA approach, liquid biopsy shows earlier detection of cancer in patients with LFS compared with current clinical surveillance methods such as imaging. Liquid biopsy provides improved accessibility and sensitivity, complementing current clinical surveillance methods to provide better care for these patients. See related commentary by Latham et al., p. 23. This article is featured in Selected Articles from This Issue, p. 5.

Hypermethylation of the ADIRF promoter regulates its expression level and is involved in NNK-induced malignant transformation of lung bronchial epithelial cells.

The carcinogenic mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a well-known tobacco carcinogen, has not been fully elucidated in epigenetic studies. 5-Methylcytosine (5mC) modification plays a major role in epigenetic regulation. In this study, the 5mC level increased in both BEAS-2B human bronchial epithelium cells treated with 100mg/L NNK for 24h and NNK-induced malignant-transformed BEAS-2B cells (2B-NNK cells), suggesting that 5mC modification is associated with the malignant transformation mechanism of NNK. Using a combination of Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq), RNA sequencing (RNA-seq), and bioinformatics analysis of data from the Genomic Data Commons database, we found that the Adipogenesis regulatory factor (ADIRF) promoter region was abnormally hypermethylated, yielding low ADIRF mRNA expression, and that ADIRF overexpression could inhibit the proliferation, migration, and invasion of 2B-NNK cells. This finding suggests that ADIRF plays a tumor suppressor role in the NNK-induced malignant transformation of cells. Subsequently, using 5-Aza-2'-deoxycytidine (5-Aza-2'-dC) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Catalytically Dead Cas9 (dCas9 system), we verified that the demethylation of the ADIRF promoter region in 2B-NNK cells inhibited the proliferation, migration, and invasion ability of the cells and increased their apoptosis ability. These results suggest that abnormal 5mC modification of the ADIRF promoter plays a positive regulatory role in the pathogenesis of NNK-induced lung cancer. This study offers a new experimental basis for the epigenetic mechanism of NNK-induced lung cancer.

DNMT3A mutation promotes leukemia development through NAM-NAD metabolic reprogramming

BackgroundDNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia (AML) with Arg882His (R882H) as the hotspot mutation. It has been reported that DNMT3A mutation plays a key role in leukemogenesis through hypomethylation of some target genes associated with cell growth and differentiation. In this study, we investigated the function of DNMT3A R882H in the malignant progression of AML by regulating metabolic reprogramming.MethodsUltra-High Performance Liquid Chromatography–High Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) was used to detect metabolites in the serum of mice harboring Dnmt3a R878H mutation and the wild-type Dnmt3a. Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) were used to analyze the levels of DNA methylation and mRNA expression of genes in mouse Gr1+ bone marrow cells respectively. The TCGA and GO databases were used to analyze the differential genes between human samples carrying the DNMT3A R882 mutation and the wild-type DNMT3A. Co-immunoprecipitation and immunoblotting were used to illustrate the binding levels of Cyclins-CDKs and CDK inhibitors including CDKN1A and CDKN1B. Flow cytometry was used to analyze the cell differentiation, division, apoptosis and cell cycle. The effect of NAMPT inhibition on leukemia was evaluated by using in vivo fluorescence imaging in NOG mouse model bearing OCI-AML3 cells.ResultsDNMT3A mutation caused high expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage synthetic pathway, through DNA hypomethylation, and finally led to abnormal nicotinamide (NAM) metabolism and NAD synthesis. The NAM-NAD metabolic abnormalities caused accelerated cell cycle progression. Inhibition of NAMPT can reduce the binding degree between Cyclins-CDKs, and increase the binding interaction of the CDK inhibitors with Cyclins-CDKs complexes. Moreover, cells with high expression of NAMPT were more sensitive to the NAMPT inhibitor FK866 with a lower IC50. The inhibition of NAMPT can remarkably extend the survival time of tumor-bearing mice and reduce the infiltration of tumor cells.ConclusionsTaken together, our data showed that DNMT3A mutation caused NAMPT overexpression to induce the reprogramming of NAM-NAD metabolism and contribute to abnormal proliferation, which provided a potential direction for targeted therapy at the metabolic level in AML with DNMT3A mutation.

MEDIPIPE: an automated and comprehensive pipeline for cfMeDIP-seq data quality control and analysis.

Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) has emerged as a promising liquid biopsy technology to detect cancers and monitor treatments. While several bioinformatics tools for DNA methylation analysis have been adapted for cfMeDIP-seq data, an end-to-end pipeline and quality control framework specifically for this data type is still lacking. Here, we present the MEDIPIPE, which provides a one-stop solution for cfMeDIP-seq data quality control, methylation quantification, and sample aggregation. The major advantages of MEDIPIPE are: (i) ease of implementation and reproducibility with Snakemake containerized execution environments that will be automatically deployed via Conda; (ii) flexibility to handle different experimental settings with a single configuration file; and (iii) computationally efficiency for large-scale cfMeDIP-seq profiling data analysis and aggregation. This pipeline is an open-source software under the MIT license and it is freely available at https://github.com/pughlab/MEDIPIPE.

Global circulating free DNA methylation and fragmentome deconvolution in patients with metastatic renal cell carcinoma treated with immunotherapy (GOLDEN).

4544 Background: Metastatic renal cell carcinoma (mRCC) lacks molecular biomarkers. Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) is a new, non-invasive approach to the detection of RCC-derived DNA in the circulation. In the GOLDEN study (NCT03702309), we explored the role of cfMeDIP-seq and fragmentomic dynamics as a biomarker, during immune checkpoint inhibition (ICI)-based treatment in mRCC patients (pts). Methods: Blood samples from mRCC pts were collected before and during treatment. Response to treatment was defined by the treating oncologist as: response, any degree of radiological tumor regression; stable disease (SD), no radiological change; or progressive disease (PD), radiological/clinical progression. More than 10ng of plasma cfDNA was subject to cfMeDIP-seq. A prognostic pan-cancer methylation signature previously generated using TCGA tissue methylation arrays was applied to cfMeDIP-seq output and cancer-signal methylation (CSM) score was calculated. An RCC-specific methylation signature, previously generated by identifying differentially methylated regions between RCC pts and controls, was applied ( Nuzzo et al, Nat Med 2020). Genome-wide fragmentation profiles were generated using DELFI. Using medians as cut off, log-rank test was performed to assess overall survival (OS) and progression-free survival (PFS). Results: Thirty-five patients were enrolled and n=33 received systemic treatment. 31/35 pts (89%) had clear cell and 4/35 (11%) non-clear cell histology. Median age was 61 (30-81) years and the M:F ratio was 6:1. Patients’ IMDC prognostic risk score was favorable n=6/35 (17%), intermediate n=23/35 (65%) and poor n=6/35 (17%). 20/33 (61%) patients received ipilimumab/nivolumab, 10/33 (30%) nivolumab and n=3/33 (9%) pembrolizumab/axitinib. There were n=14/33 (42%) responders, n=4/33 (12%) pts with SD and 15/33 (45%) pts with PD. Patients with high pan-cancer CSM score at baseline trended towards worse OS (HR 3.86, p = 0.079). Fragmentomics signature, consisting of increased proportion of short cfDNA fragments, showed a trend for worse PFS (HR = 3.04, p = 0.075) in on-treatment samples. Using the RCC-specific methylation signature, we found that those with an increase in signature score from baseline to on-treatment had better PFS (HR=2.4x10-10, p=0.002, n=10) and a trend towards better OS (HR=0.17, p=0.074, n=10). We hypothesize that RCC tissue-specific cell death early during ICI treatment could underpin this increase in the methylation score. Conclusions: This small study is the first report on the role of cfMeDIPseq-derived methylation and fragmentomic signatures in patients with mRCC undergoing ICI-based treatment. We found several trends and one significant association between these signatures and pt outcomes, which support further validation in larger cohorts. Clinical trial information: NCT03702309 .

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Promoting GSDME expression through DNA demethylation to increase chemosensitivity of breast cancer MCF-7 / Taxol cells.

Breast cancer is the most common and high-incidence cancer in women. It is mainly treated by surgery combined with chemoradiation. The main challenge in treating breast cancer patients is developing resistance to chemotherapeutics, so it is urgent to find potential strategies that can improve the chemotherapy effect of patients. In this study, we aimed to explore the role of GSDME methylation in the sensitivity of chemotherapy for breast cancer. Here, we identified breast cancer MCF-7 / Taxol cells models using quantitative real-time PCR (qRT-PCR), Western blotting (WB), and cell counting kit-8 (CCK-8) analyses. Epigenetic changes in it were detected by Methylated DNA immunoprecipitation-sequencing and methylation-specific PCR. The expression level of GSDME in breast cancer cells was observed by qPCR and WB analyses. CCK-8 and colony formation assay were used to detect cell proliferation. Finally, pyroptosis was detected by LDH assay, flow cytometry, and WB analyses. Our results indicate that ABCB1 mRNA and p-GP expression are significantly increased in breast cancer MCF-7 / Taxol cells. GSDME enhancer methylation was found in drug-resistant cells and was associated with the down-regulation of GSDME expression. After treatment with decitabine (5-Aza-2'-deoxycytidine), the demethylation of GSDME induced the occurrence of pyroptosis and thereby inhibited the proliferation of MCF-7 / Taxol cells. We found that the upregulation of GSDME enhances the chemosensitivity of MCF-7 / Taxol cells to paclitaxel by inducing pyroptosis. Taken together, we identified decitabine increases GSDME expression through DNA demethylation and induces pyroptosis, thus increasing the chemosensitivity of MCF-7 / Taxol cells to Taxol. Use of decitabine / GSDME / pyroptosis-based treatment strategies may be a new way to overcome the resistance of breast cancer to paclitaxel chemotherapy.

Abstract OT1-23-01: Development of a hoRizontal data intEgration classifier for NOn-invasive early diAgnosis of breasT cancEr: the RENOVATE trial

Abstract Background: The detection of breast lesions through self-examination or during screening tests is a frequent finding. Breast biopsy is required in case of radiologically suspect lesions, bestowing a high burden on both patients and national healthcare system, since only one every four biopsies is breast cancer (BC). To date, the assessment of circulating biomarkers failed to demonstrate clinical utility in the early diagnosis of BC, for its suboptimal accuracy and difficult transferability to clinical practice. The combination of novel cutting-edge methods for the assessment of circulating analytes in an integrated multiomic classifier may overcome such limitations, possibly allowing liquid biopsy to become a novel noninvasive procedure for the differential diagnosis of BC. Design: In the RENOVATE trial (NCT04781062), women with suspect (BI-RADS-4/5) breast lesions ≤ 2 cm (cT1) are asked, before biopsy, to donate ~ 35 mL of blood collected in four dedicated tubes and ~ 50 mL of urine at the Diagnostic Senology Unit of Ospedale Policlinico San Martino (Genoa, IT). Plasma cell-free DNA methylation and copy number alterations are assessed in a cohort of patients diagnosed with early BC and a matched set of patients with histologically proven benign lesions through cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMeDIPseq), as well as ultra-low pass whole genome sequencing (ULP-WGS). Thanks to the volume and quality of our sample set, other experimental techniques will be tested as well. Results from cfMeDIP-seq and ULP-WGS, possibly in combination with other findings, will be integrated in a unique classifier for the noninvasive differential diagnosis of suspect breast lesions. Eligibility criteria: Patients with radiologically suspect breast lesions ≤ 2 cm (i.e. BIRADS 4/5) are eligible. Patients with previous history of cancer, or diagnosed with autoimmune or active allergic diseases, acute or chronic hepatic, renal, or cardiac diseases, or acute or chronic infectious diseases are excluded from the present trial. Specific aims: The primary aim of the present trial is to develop a noninvasive classifier for the differential diagnosis of suspect breast lesions detected through mammography and/or ultrasound. For such purpose we will assess the performance of plasma cfMeDIPseq, ULP-WGS, and other promising techniques for the differential diagnosis of BC. Such techniques will be integrated in a unique classifier in order to reach the maximum possible accuracy. Statistical methods: Sample size was calculated with a semi-parametric simulation-based approach from beta-distributions of PBMC datasets: assuming to test 20,000 CpG regions, with 300 differentially methylated target CpGs, a target maximal difference in DNA methylation of 0.2 between groups and an FDR of 0.05, 1 – beta ~ 0.90 would be achieved with an overall sample size of 150 samples split in a 1:2 ratio. Target accrual and present accrual: Minimum target accrual is set at 49 patients with BC and 98 patients with benign lesions. To date, we have collected plasma samples from 74 eligible patients with BC and 115 eligible patients with benign lesions. A validation cohort accounting for ~30% of our sample set will be recruited at Istituto Nazionale dei Tumori (Milan, IT). Contact information: For further information, please contact Gabriele Zoppoli at gabriele.zoppoli@unige.it. Citation Format: Francesco Ravera, Martina Dameri, Isabella Lombardo, Mario Stabile, Alberto Tagliafico, Massimo Calabrese, Alberto Ballestrero, Lorenzo Ferrando, Gabriele Zoppoli. Development of a hoRizontal data intEgration classifier for NOn-invasive early diAgnosis of breasT cancEr: the RENOVATE trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-23-01.

Integrated, Longitudinal Analysis of Cell-free DNA in Uveal Melanoma.

Here, we demonstrate integrated, longitudinal cfDNA sequencing using multi-omic approaches is more effective than unimodal analysis. This approach supports the use of frequent blood testing using comprehensive genomic, fragmentomic, and epigenomic techniques.

Genome-wide characterization of mitochondrial DNA methylation in human brain.

There is growing interest in the role of DNA methylation in regulating the transcription of mitochondrial genes, particularly in brain disorders characterized by mitochondrial dysfunction. Here, we present a novel approach to interrogate the mitochondrial DNA methylome at single base resolution using targeted bisulfite sequencing. We applied this method to investigate mitochondrial DNA methylation patterns in post-mortem superior temporal gyrus and cerebellum brain tissue from seven human donors. We show that mitochondrial DNA methylation patterns are relatively low but conserved, with peaks in DNA methylation at several sites, such as within the D-LOOP and the genes MT-ND2, MT-ATP6, MT-ND4, MT-ND5 and MT-ND6, predominantly in a non-CpG context. The elevated DNA methylation we observe in the D-LOOP we validate using pyrosequencing. We identify loci that show differential DNA methylation patterns associated with age, sex and brain region. Finally, we replicate previously reported differentially methylated regions between brain regions from a methylated DNA immunoprecipitation sequencing study. We have annotated patterns of DNA methylation at single base resolution across the mitochondrial genome in human brain samples. Looking to the future this approach could be utilized to investigate the role of mitochondrial epigenetic mechanisms in disorders that display mitochondrial dysfunction.

Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls

SummaryCell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) identifies genomic regions with DNA methylation, using a protocol adapted to work with low-input DNA samples and with cell-free DNA (cfDNA). We developed a set of synthetic spike-in DNA controls for cfMeDIP-seq to provide a simple and inexpensive reference for quantitative normalization. We designed 54 DNA fragments with combinations of methylation status (methylated and unmethylated), fragment length (80 bp, 160 bp, 320 bp), G + C content (35%, 50%, 65%), and fraction of CpG dinucleotides within the fragment (1/80 bp, 1/40 bp, 1/20 bp). Using 0.01 ng of spike-in controls enables training a generalized linear model that absolutely quantifies methylated cfDNA in MeDIP-seq experiments. It mitigates batch effects and corrects for biases in enrichment due to known biophysical properties of DNA fragments and other technical biases.

MeDIP-seq and RNA-seq analysis during porcine testis development reveals functional DMR at the promoter of LDHC

Testis development requires tight regulation of gene expression programmed by epigenetic modifiers. However, their mechanism remains to be elucidated. Here, we investigated the genome-wide DNA methylation landscape in the Duroc and Meishan boar testes using methylated DNA immunoprecipitation sequencing (MeDIP-seq). We identified over 1100 promoter differential methylation genes (DMGs) before and after puberty, most of which are associated with testis development. Furthermore, we discovered that the expression of lactate dehydrogenase C (LDHC) gene during testis development is regulated by DNA methylation. The promoter of LDHC in pre-pubertal testes is substantially methylated, whereas considerably demethylated in post-pubertal testes. Artificial demethylation with the demethylating agent 5-Aza-CdR induced LDHC expression in immature Sertoli cells (SCs). Mechanistically, we confirmed the transcription factor SP1 was recruited to bind in hypomethylated differentially methylated regions (DMRs) in LDHC promoter, which upregulated the expression of LDHC. Functionally, we demonstrated that LDHC was activated in mature SCs (mSCs) and its overexpression significantly increases lactate secretion in SCs. In conclusion, our results highlight the function and regulation of dynamic DNA methylation in testis development.