Methylated DNA Immunoprecipitation Research Articles

A phase II trial of olaparib and durvalumab in patients with recurrent IDH-mutated gliomas.

2013 Background: Isocitrate dehydrogenase mutations (IDHmt) define astrocytomas and oligodendrogliomas. IDHmt results in accumulation of R-2-hydroxyglutarate (2HG), leading to epigenetic dysregulation and defective homologous recombination repair, providing a rationale for poly (adenosine 5’-diphophate-ribose) polymerase (PARP) inhibitors. PARP inhibition upregulates PD-L1 so the combination with immune checkpoint inhibition is potentially synergistic. Methods: Patients (pts) with recurrent high-grade IDHmt gliomas were enrolled in this phase II open-label study (NCT03991832). Eligibility included progressive disease with up to 2 prior lines of systemic therapies and ECOG 0–1. Pts received olaparib 300 mg twice daily continuously and durvalumab 1500 mg IV every 4 weeks. Simon’s optimal two-stage design was used. The primary objective was overall response rate (ORR) and disease control rate (DCR) by RANO criteria. Secondary objectives included overall survival (OS), progression free survival (PFS) and safety.Exploratory biomarkers of response and resistance were assessed with serial blood samples using cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as well as multiplex-immunohistochemistry. Results: In the 29 pts enrolled between January 2020–February 2023, median age was 40.5 (range 23–66) and 41% were female. Initial tumor grade was 2 in 9, 3 in 8 and 4 in 12 pts, respectively. Median time to enrollment from tumour diagnosis was 5.9 years. All had prior resection and median number of prior systemic therapies was 2. One patient clinically deteriorated before starting treatment. ORR was 10%, 95% CI 2.2–27%, with responses in 3 pts. One pt with grade 4 astrocytoma had a complete response and remains on treatment after 33 months. The other 2 responders with grade 4 astrocytoma had response durations of 4.1 and 9.8 months. DCR was 28% (95% CI 12.7–47.2%) with 5 additional pts demonstrating stable disease. With a median follow-up of 33 months, mOS was 9.5 months (95% CI 4.3–19.3) and mPFS was 1.9 months (95% CI 1.8–3.0). There was no treatment-related grade 3–4 toxicities. Any grade toxicities included fatigue (48%), nausea (17%), diarrhea (10%), and cytopenias (3%). Using serial cfMeDIP-seq, cell free DNA methylomes comparing responders versus progressors using the top differentially methylated regions can predict treatment response with high accuracy (AUC 0.833). Post progression, differentially methylated genes converge on TGF-β and signal transduction pathways, suggesting possible mechanisms of resistance. Multi-modal analysis of long-term responders will additionally be presented. Conclusions: Combination treatment with olaparib and durvalumab for pts with IDHmt glioma is well tolerated but has limited efficacy in unselected pts. cfMeDIP-seq can reliably predict tumour progression providing a blood-based biomarker of treatment response. Clinical trial information: NCT03991832 .

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.

Prenatal nicotine exposure leads to epigenetic alterations in peripheral nervous system signaling genes in the testis of the rat

BackgroundPrenatal nicotine exposure (PNE) has been documented to cause numerous deleterious effects on fetal development. However, the epigenetic changes promoted by nicotine exposure on germ cells are still not well understood.ObjectivesIn this study, we focused on elucidating the impact of prenatal nicotine exposure on regulatory epigenetic mechanisms important for germ cell development.MethodsSprague-Dawley rats were exposed to nicotine during pregnancy and male progeny was analyzed at 11 weeks of age. Testis morphology was analyzed using frozen testis sections and expression of germ cell markers was examined by RT-qPCR; histone modifications were assessed by Western Blot (WB). DNA methylation analysis was performed by methylation-specific PCR of bisulfite converted DNA. Genome-wide DNA methylation was analyzed using Methylated DNA immunoprecipitation (MeDIP)-seq. We also carried out transcriptomics analysis of pituitary glands by RNA-seq.ResultsWe show that gestational exposure to nicotine reduces germ cell numbers, perturbs meiosis, affects the expression of germ line reprogramming responsive genes, and impacts the DNA methylation of nervous system genes in the testis. PNE also causes perturbation of gene expression in the pituitary gland of the brain.ConclusionsOur data demonstrate that PNE leads to perturbation of male spermatogenesis, and the observed effects are associated with changes of peripheral nervous system signaling pathways. Alterations in the expression of genes associated with diverse biological activities such as cell migration, cell adhesion and GABA signaling in the pituitary gland underscore the complexity of the effects of nicotine exposure during pregnancy.

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.

Comprehensive DNA methylation profiling by MeDIP-NGS identifies potential genes and pathways for epithelial ovarian cancer.

Ovarian cancer, among all gynecologic malignancies, exhibits the highest incidence and mortality rate, primarily because it is often presents with non-specific or no symptoms during its early stages. For the advancement of Ovarian Cancer Diagnosis, it is crucial to identify the potential molecular signatures that could significantly differentiate between healthy and ovarian cancerous tissues and can be used further as a diagnostic biomarker for detecting ovarian cancer. In this study, we investigated the genome-wide methylation patterns in ovarian cancer patients using Methylated DNA Immunoprecipitation (MeDIP-Seq) followed by NGS. Identified differentially methylated regions (DMRs) were further validated by targeted bisulfite sequencing for CpG site-specific methylation profiles. Furthermore, expression validation of six genes by Quantitative Reverse Transcriptase-PCR was also performed. Out of total 120 differentially methylated genes (DMGs), 68 genes were hypermethylated, and 52 were hypomethylated in their promoter region. After analysis, we identified the top 6 hub genes, namely POLR3B, PLXND1, GIGYF2, STK4, BMP2 and CRKL. Interestingly we observed Non-CpG site methylation in the case of POLR3B and CRKL which was statistically significant in discriminating ovarian cancer samples from normal controls. The most significant pathways identified were focal adhesion, the MAPK signaling pathway, and the Ras signaling pathway. Expression analysis of hypermethylated genes was correlated with the downregulation of the genes. POLR3B and GIGYF2 turned out to be the novel genes associated with the carcinogenesis of EOC. Our study demonstrated that methylation profiling through MeDIP-sequencing has effectively identified six potential hub genes and pathways that might exacerbate our understanding of underlying molecular mechanisms of ovarian carcinogenesis.

Coordinated activation of DNMT3a and TET2 in cancer stem cell-like cells initiates and sustains drug resistance in hepatocellular carcinoma

BackgroundResistance to targeted therapies represents a significant hurdle to successfully treating hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical determinants of HCC relapse and therapeutic resistance, the underlying mechanisms are poorly understood. We aimed to address whether and how dysregulated epigenetic regulators have regulatory and functional communications in establishing and maintaining drug resistance.MethodsHCC-resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction and promoter binding of DNMT3a-TET2 were investigated by co-immunoprecipitation, ChIP-qPCR. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.ResultsWe identified the coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with a less favorable response to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased promoter DNA methylation.ConclusionsWe demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and thus are promising therapeutic targets for refractory HCC.

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.

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.

TET1 inhibits the migration and invasion of cervical cancer cells by regulating autophagy

ABSTRACT Methylation modifications play pertinent roles in regulating gene expression and various biological processes. The silencing of the demethylase enzyme TET1 can affect the expressions of key oncogenes or tumour suppressor genes, thus contributing to tumour formation. Nonetheless, how TET1 affects the progression of cervical cancer is yet to be elucidated. In this study, we found that the expression of TET1 was significantly downregulated in cervical cancer tissues. Functionally, TET1 knockdown in cervical cancer cells can promote cell proliferation, migration, invasion, cervical xenograft tumour formation and EMT. On the contrary, its overexpression can reverse the aforementioned processes. Moreover, the autophagy level of cervical cancer cells can be enhanced after TET1 knockdown. Mechanistically, methylated DNA immunoprecipitation (MeDIP)-sequencing and MeDIP quantitative real-time PCR revealed that TET1 mediates the methylation of autophagy promoter regions. These findings suggest that TET1 affects the autophagy of cervical cancer cells by altering the methylation levels of NKRF or HIST1H2AK, but the specific mechanism needs to be investigated further.

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.

DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development

Increased expression of the angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl− cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to the development of hypertension. However, little is known about transcriptional control of Agtr1a and Slc12a2 in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wister-Kyoto rats (WKY). Chromatin immunoprecipitation-qPCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and MeCP2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

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.

Klotho Overexpression Is Frequently Associated With Upstream Rearrangements in Fusion-Negative Phosphaturic Mesenchymal Tumors of Bone and Sinonasal Tract

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered “fusion-negative” (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.

Accumulation of endogenous adenosine improves cardiomyocyte metabolism via epigenetic reprogramming in an ischemia-reperfusion model

Adenosine kinase (ADK) plays the major role in cardiac adenosine metabolism, so that inhibition of ADK increases myocardial adenosine levels. While the cardioprotective actions of extracellular adenosine against ischemia/reperfusion (I/R) are well-established, the role of cellular adenosine in protection against I/R remains unknown. Here we investigated the role of cellular adenosine in epigenetic regulation on cardiomyocyte gene expression, glucose metabolism and tolerance to I/R. Evans blue/TTC staining and echocardiography were used to assess the extent of I/R injury in mice. Glucose metabolism was evaluated by positron emission tomography and computed tomography (PET/CT). Methylated DNA immunoprecipitation (MeDIP) and bisulfite sequencing PCR (BSP) were used to evaluate DNA methylation. Lentiviral/adenovirus transduction was used to overexpress DNMT1, and the OSI-906 was administered to inhibit IGF-1. Cardiomyocyte-specific ADK/IGF-1-knockout mice were used for mechanistic experiments.Cardiomyocyte-specific ADK knockout enhanced glucose metabolism and ameliorated myocardial I/R injury in vivo. Mechanistically, ADK deletion caused cellular adenosine accumulation, decreased DNA methyltransferase 1 (DNMT1) expression and caused hypomethylation of multiple metabolic genes, including insulin growth factor 1 (IGF-1). DNMT1 overexpression abrogated these beneficial effects by enhancing apoptosis and decreasing IGF-1 expression. Inhibition of IGF-1 signaling with OSI-906 or genetic knocking down of IGF-1 also abrogated the cardioprotective effects of ADK knockout, revealing the therapeutic potential of increasing IGF-1 expression in attenuating myocardial I/R injury. In conclusion, the present study demonstrated that cardiomyocyte ADK deletion ameliorates myocardial I/R injury via epigenetic upregulation of IGF-1 expression via the cardiomyocyte adenosine/DNMT1/IGF-1 axis.

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.

Genome-wide DNA methylation analysis in schizophrenia with tardive dyskinesia: a preliminary study.

Tardive dyskinesia (TD) develops in 20-30% of schizophreniapatients and up to 50% in patients > 50years old. DNA methylation may play an important role in the development of TD. DNA methylation analyses in schizophrenia with TD. We conducted a genome-wide DNA methylation analysis in schizophrenia with TD using methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-Seq) in a Chinese sample including five schizophrenia patients with TD and five without TD (NTD), and five healthy controls. The results were expressed as the log2FC, fold change of normalized tags between two groups within the differentially methylated region (DMR). For validation, the pyrosequencing was used to quantify DNA methylation levels of several methylated genes in an independent sample (n = 30). Through genome-wide MeDIP-Seq analysis, we identified 116 genes that were significantly differentially methylated in promotor regions in comparison of TD group with NTD group including 66 hypermethylated genes (top 4 genes are GABRR1, VANGL2, ZNF534, and ZNF746) and 50 hypomethylated genes (top 4 genes are DERL3, GSTA4, KNCN, and LRRK1). Part of these genes (such as DERL3, DLGAP2, GABRR1, KLRG2, LRRK1, VANGL2, and ZP3) were previously reported to be associated with methylation in schizophrenia. Gene Ontology enrichment and KEGG pathway analyses identified several pathways. So far, we have confirmed the methylation of 3 genes (ARMC6, WDR75, and ZP3) in schizophrenia with TD using pyrosequencing. This study identified number of methylated genes and pathways for TD and will provide potential biomarkers for TD and serve as a resource for replication in other populations.

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.