Crosstalk of DNA Methylation Triggered by Pathogen in Poplars With Different Resistances

DNA methylation plays crucial roles in responses to environmental stimuli. Modification of DNA methylation during development and abiotic stress responses has been confirmed in increasing numbers of plants, mainly annual plants. However, the epigenetic regulation mechanism underlying the immune response to pathogens remains largely unknown in plants, especially trees. To investigate whether DNA methylation is involved in the response to infection process or is related to the resistance differences among poplars, we performed comprehensive whole-genome bisulfite sequencing of the infected stem of the susceptible type Populus × euramerican ‘74/76’ and resistant type Populus tomentosa ‘henan’ upon Lonsdalea populi infection. The results revealed that DNA methylation changed dynamically in poplars during the infection process with a remarkable decrease seen in the DNA methylation ratio. Intriguingly, the resistant P. tomentosa ‘henan’ had a much lower basal DNA methylation ratio than the susceptible P. × euramerican ‘74/76’. Compared to mock-inoculation, both poplar types underwent post-inoculation CHH hypomethylation; however, significant decreases in mC and mCHH proportions were found in resistant poplar. In addition, most differentially CHH-hypomethylated regions were distributed in repeat and promoter regions. Based on comparison of DNA methylation modification with the expression profiles of genes, DNA methylation occurred in resistance genes, pathogenesis-related genes, and phytohormone genes in poplars during pathogen infection. Additionally, transcript levels of genes encoding methylation-related enzymes changed during pathogen infection. Interestingly, small-regulator miRNAs were subject to DNA methylation in poplars experiencing pathogen infection. This investigation highlights the critical role of DNA methylation in the poplar immune response to pathogen infection and provides new insights into epigenetic regulation in perennial plants in response to biotic stress.

Circulating tumor DNA methylation marker MYO1-G for diagnosis and monitoring of colorectal cancer

Background Circulating tumor DNA (ctDNA) is a promising diagnostic and prognostic marker for many cancers and has been actively investigated in recent years. Previous studies have already demonstrated the potential use of ctDNA methylation markers in the diagnosis and prognostication of colorectal cancer (CRC). This retrospective study validated the value of methylation biomarker MYO1-G (cg10673833) in CRC diagnosis and disease monitoring using digital droplet PCR (ddPCR), a biomarker selected from our previous study due to its highest diagnostic efficiency. Methods Blood samples of CRC and control samples from tumor-free individuals at two institutions were collected to quantify the methylation ratio using ddPCR. Area under curve (AUC) was calculated after constructing receiver operating characteristic curve (ROC) for CRC diagnosis. Sensitivity and specificity were estimated and comparisons of methylation ratio in different groups were performed. Results We collected 673 blood samples from 272 patients diagnosed with stage I-IV CRC and 402 normal control samples. The methylation biomarker discriminated patients with CRC from normal controls with high accuracy (area under curve [AUC] = 0.94) and yielded a sensitivity of 84.3% and specificity of 94.5%. Besides, methylation ratio of MYO1-G was associated with tumor burden and treatment response. The methylation ratio was significantly lower in patients after their radical operation than when compared with those before surgeries (P < 0.001). Methylation ratio was significantly higher in patients with disease progression than those with stable disease (P = 0.002) and those with complete response or partial response (P = 0.009). Conclusions Together, our study indicated that this methylation marker can serve as a potential biomarker for diagnosing and monitoring CRC.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

Hypomethylation of the DAZ3 promoter in idiopathic asthenospermia: a screening tool for liquid biopsy

Given the role of the deleted in azoospermia gene in male infertility, whether the somatic deleted in azoospermia methylation status is associated with idiopathic asthenospermia should be determined. To investigate the methylation levels of the deleted in azoospermia promoter in peripheral white blood cells from idiopathic asthenospermia patients relative to those in normozoospermia controls, 61 ethylene diamine tetraacetic acid anticoagulant blood samples were drawn from all participants for DNA isolation. The deleted in azoospermia promoter methylation ratio was detected by MassARRAY-based methylation quantification and confirmed by quantitative methylation-specific polymerase chain reaction. A MassARRAY-based methylation analysis showed that the deleted in azoospermia 3 promoter (0 to − 2 kbp) was significantly hypomethylated in peripheral white blood cells from idiopathic asthenospermia males, specifically one CpG site (− 246 to − 247). Quantitative methylation-specific polymerase chain reaction data further confirmed that the methylation level of the deleted in azoospermia 3 promoter region in idiopathic asthenospermia patients was significantly lower than that in normozoospermia males. The area under the receiver operating characteristic curve determined by quantitative methylation-specific polymerase chain reaction was 0.737 (95% confidence interval: 0.552 to 0.924), with a sensitivity of 53.9% and a specificity of 88.2% at a cut-off level of 74.7%. Therefore, our results suggested that methylation ratio detection of the deleted in azoospermia 3 promoter region by real-time polymerase chain reaction assay is a promising and feasible tool for liquid biopsy in the clinical laboratories. The methylation status of other reported infertility-related genes should also be investigated in peripheral white blood cells.

SAT-552 Epigenetic Regulation of 11beta-Hydroxysteroid Dehydrogenase 1 and 2 Gene in Salt-Sensitive Hypertensive Rats

ENDO 2020 Epigenetic regulation of 11beta-hydroxysteroid dehydrogenase 1 and 2 gene in salt-sensitive hypertensive rats [Objective]11Beta-hydroxysteroid dehydrogenase type1 (11-HSD1) is the modulator of glucocorticoid hormone and type2 (11-HSD2) is the modulator of mineralocorticoid hormone. We investigated the effect of high salt diet on the methylation of both enzyme gene in salt-sensitive hypertensive (SSH) rats [Methods]SSH rats were fed a high (7% NaCl) or normal (0.45%) salt chow for 4 weeks. Body weight, blood pressure, plasma and urinary aldosterone concentration and PRA were measured. DNA was extracted from kidneys and visceral fats. Bisulfite sequencing and Pyrosequencing were done for the analysis of methylation status of 11-HSD1 and 2 gene. [Results] High salt diet significantly decreased methylation ratio of 11-HSD1 gene in the visceral fats of SSH rats compared with controls (p&lt;0.05). The methylation ratio of 11-HSD2 gene in the kidney of SSH rats was not influenced by high salt diet. [Discussion and Conclusion]11-HSD1 overexpression in visceral fats in mice was reported to show SSH. We reported decreased 11-HSD2 activity in the artery in SSH rats. In this study high salt diet affected methylation status of 11-HSD1 in the adipose tissue but not 11-HSD2 gene in the kidney in SSH. Food intake such as salt may influence the epigenesis of 11-HSD and induce hypertension.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer

Background: More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance. Purpose: We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy. Methods: We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward. Results: Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] ( P = .013), chromosome 3 [140200000-140399999] ( P = .037), and chromosome 12 [101200000-101399999] ( P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival ( P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively. Conclusion: Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Photoperiodic stress induces genotype-specific shift in DNA methylation in Tartary buckwheat

Tartary buckwheat, known for its rich source of health beneficial secondary metabolites, is cultivated in many areas of the world. Among different environmental factors, photoperiod strongly influence its growth, flowering time, and ultimately the yield. In this context, epigenetics could contribute significantly in the regulation of plant response against changing environment. Therefore, with the aim to study the involvement of DNA methylation in photoperiod mediated plant response, genome-wide DNA methylation analysis was performed in two accessions (A1 and A2) of Tartary buckwheat using three photoperiodic treatments, i.e., 10-hr light/day (T1), 12-hr light/day (T2), and 14-hr light/day (T3). Flowering time and plant fresh weight data revealed that accessions A1 and A2 prefer T1 and T2 treatments, respectively. Total DNA methylation ratio increased with the increase in photoperiod in accession A1 but decreased under same conditions in accession A2. Full methylation increased significantly while intensive decrease in hemimethylation was noted from T2 to T3 in A1, whereas full methylation strongly increased and hemimethylation strongly decreased from T1 to T2 in A2. Overall, the DNA methylation events appeared more frequently than demethylation events. This study reports for the first time an accession-/genotype specific pattern of shift in the DNA methylation under different photoperiodic treatments that will pave the way toward identification of specific genes involved in the regulation of plant response against photoperiodic stress.

ACTR-53. MGMT PROMOTER METHYLATION ANALYSIS FOR ALLOCATING COMBINED CCNU/TMZ CHEMOTHERAPY: LESSONS LEARNED FROM THE CeTeG/NOA-09 TRIAL

BACKGROUND The CeTeG/NOA-09 trial recently showed a survival benefit for combination chemotherapy with CCNU/TMZ in glioblastoma patients with a methylated MGMT promoter as determined by quantitative Methylation-Specific PCR (qMSP). Identifying patient subgroups with a pronounced benefit from this novel treatment is crucial. Here, we report on the prognostic and predictive value of MGMT promoter methylation ratio determined by qMSP and investigate the concordance of pyrosequencing (PSQ) and qMSP for patients in this trial. METHODS qMSP and PSQ were used for MGMT promoter methylation analysis. The mITT population of the CeTeG/NOA-09 trial was used for multivariate analysis including the parameters MGMT promoter methylation ratio, RPA class and study center. RESULTS Patients of the mITT population of the CeTeG/NOA-09 trial (n=129) with MGMT promoter methylation ratio greater than 4 (qMSP) showed a superior overall survival compared to patients with borderline methylation ratio of 2–4 (p=0.0251). In the latter patients, treatment with CCNU/TMZ did not show a survival benefit (p=0.924). Multivariate analysis with treatment arm, RPA class and study center as covariates did not confirm a prognostic or predictive value of MGMT promoter methylation ratio (qMSP) for patients of the mITT population (n=129, HR=0.88; 95% CI: 0.72 – 1.08) or patients with a ratio greater than 4 (n=117, HR =0.86; 95% CI: 0.69 – 1.07). In a subset of 49 trial patients, qMSP and PSQ showed not only a high qualitative (45/49; 91.8%), but also a high quantitative concordance rate (Spearman correlation, r=0.83, p< 0.0001). CONCLUSION Glioblastoma patients with borderline MGMT promoter methylation (qMSP ratio 2–4) do not seem to benefit from combination treatment with CCNU/TMZ. Thus, we propose a qMSP cut-off of 4 as a novel decision tool for clinicians. qMSP and PSQ show a high concordance rate indicating that a decision for combination therapy can also be based on PSQ results.

Stratified monotherapy approach according to MGMT methylation status in elderly patients with glioblastoma.

2050 Background: The elderly patients with glioblastoma have an extremely poor prognosis. As they often have some degree of age-related vulnerability, it is especially important to minimize a risk of treatment-related adverse events by optimizing treatment intensity for this population. We conducted phaseⅡ clinical trial to investigate the efficacy of stratified monotherapy approach according to O6-methylguanine-DNA methyltransferase (MGMT) methylation status in elderly patients with glioblastoma. Methods: Patients aged 70 years or older with Karnofsky performance status (KPS) of at least 60 were eligible for this study. MGMT methylation status was quantitatively assessed by pyrosequencing based on the average methylation ratio of 16 CpG sites in the MGMT gene promoter. The patients with highly methylated MGMT promoter defined as an average methylation ratio with 30% or higher were treated with temozolomide (TMZ) monotherapy (standard 5/28 regimen), while the others with low or intermediate levels of MGMT promoter methylation were treated with radiation therapy (40Gy/15fr) alone. Results: Between April 2013 and December 2017, 70 patients were enrolled in this study. Median age was 78 years (70-91) and median KPS was 60 (60-100). Of 70 patients, 19 patients with highly methylated MGMT promoter received TMZ monotherapy, while the remaining 51 patients were treated with radiation therapy. Median progression-free survival (PFS) and median overall survival (OS) were 7.5 and 17.4 months in the TMZ group, respectively. Median PFS and median OS were 4.6 and 10.4 months in the radiotherapy group, respectively. Conclusions: For elderly glioblastoma patients with highly methylated MGMT promoter, TMZ monotherapy could be a treatment option. Clinical trial information: UMIN000012172.

miR-10b Downregulated by DNA Methylation Acts as a Tumor Suppressor in HPV-Positive Cervical Cancer via Targeting Tiam1

Background/Aims: microRNAs (miRNAs) are known to act as oncogenes or tumor suppressors in diverse cancers. Although miR-10b is an oncogene implicated in many tumors, its role in cervical cancer (CC) remains largely unclear. Here, we investigated the function and underlying mechanisms of miR-10b in human CC. Methods: Quantitative RT-PCR was used to measure miR-10b expression in CC and normal tissues, and its association with clinicopathologic features was analyzed. Methylation of CpG sites in the miR-10b promoter was analyzed by methylation sequencing. Cell proliferation, apoptosis, migration, and invasion assays were used to elucidate the biological effects of miR-10b and expression of the target gene was assayed with Western blot. Results: miR-10b was downregulated in CC tissues compared with normal tissues, and less miR-10b expression was associated with larger tumors, vascular invasion and HPV-type 16 positivity. miR-10b expression decreased in HeLa (HPV18-positive) and SiHa (HPV16-positive) cells compared with C-33A (HPV-negative), but increased after treatment with 5-Aza-CdR. Methylation ratio of site -797 in the miR-10b promoter in C-33A was lower than that in HeLa and SiHa. Further analysis indicates that site -797 is located within a transcription factor AP-2A (TFAP2A) binding element. Functionally, overexpression of miR-10b in HeLa and SiHa suppressed cell proliferation, migration and invasion, and induced apoptosis and miR-10b downregulation had opposite effects. Mechanistically, T-cell lymphoma invasion and metastasis 1 (Tiam1) was identified as a direct and functional target of miR-10b. Conclusion: miR-10b acts as a tumor suppressor in CC by suppressing oncogenic Tiam1, and its expression may be downregulated through methylation of TFAP2A binding element by HPV.