Epigenome-wide association study of lung function in Latino children and youth with asthma

Introduction DNA methylation studies have associated methylation levels at different CpG sites or genomic regions with lung function. Moreover, genetic ancestry has been associated with lung function in Latinos. However, no epigenome-wide association study (EWAS) of lung function has been performed in this population. Here, we aimed to identify DNA methylation patterns associated with lung function in pediatric asthma among Latinos. Results We conducted an EWAS in whole blood from 250 Puerto Rican and 148 Mexican American children and young adults with asthma. A total of five CpGs exceeded the genome-wide significance threshold of p = 1.17 × 10−7 in the combined analyses from Puerto Ricans and Mexican Americans: cg06035600 (MAP3K6, p = 6.13 × 10−8) showed significant association with pre-bronchodilator Tiffeneau–Pinelli index, the probes cg00914963 (TBC1D16, p = 1.04 × 10−7), cg16405908 (MRGPRE, p = 2.05 × 10−8), and cg07428101 (MUC2, p = 5.02 × 10−9) were associated with post-bronchodilator forced vital capacity (FVC), and cg20515679 (KCNJ6) with post-bronchodilator Tiffeneau–Pinelli index (p = 1.13 × 10−8). However, these markers did not show significant associations in publicly available data from Europeans (p > 0.05). A methylation quantitative trait loci analysis revealed that methylation levels at these CpG sites were regulated by genetic variation in Latinos and the Biobank-based Integrative Omics Studies (BIOS) consortium. Additionally, two differentially methylated regions in REXOC and AURKC were associated with pre-bronchodilator Tiffeneau–Pinelli index (adjusted p < 0.05) in Puerto Ricans and Mexican Americans. Moreover, we replicated some of the previous differentially methylated signals associated with lung function in non-Latino populations. Conclusions We replicated previous associations of epigenetic markers with lung function in whole blood and identified novel population-specific associations shared among Latino subgroups.

Epigenome-wide epidemiologic studies of human immunodeficiency virus infection, treatment, and disease progression

Despite significant advances in the treatment and care of people with HIV (PWH), several challenges remain in our understanding of disease pathogenesis to improve patient care. HIV infection can modify the host epigenome and as such can impact disease progression, as well as the molecular processes driving non-AIDS comorbidities in PWH. Epigenetic epidemiologic studies including epigenome-wide association studies (EWAS) offer a unique set of tools to expand our understanding of HIV disease and to identify novel strategies applicable to treatment and diagnosis in this patient population. In this review, we summarize the current state of knowledge from epigenetic epidemiologic studies of PWH, identify the main challenges of this approach, and highlight future directions for the field. Emerging epigenetic epidemiologic studies of PWH can expand our understanding of HIV infection and health outcomes, improve scientific validity through collaboration and replication, and increase the coverage of diverse populations affected by the global HIV pandemic. Through this review, we hope to highlight the potential of EWAS as a tool for HIV research and to engage more investigators to explore its application to important research questions.

HDAC8 suppresses the epithelial phenotype and promotes EMT in chemotherapy-treated basal-like breast cancer

Background Basal-like breast cancer (BLBC) is one of the most aggressive malignant diseases in women with an increased metastatic behavior and poor prognosis compared to other molecular subtypes of breast cancer. Resistance to chemotherapy is the main cause of treatment failure in BLBC. Therefore, novel therapeutic strategies counteracting the gain of aggressiveness underlying therapy resistance are urgently needed. The epithelial-to-mesenchymal transition (EMT) has been established as one central process stimulating cancer cell migratory capacity but also acquisition of chemotherapy-resistant properties. In this study, we aimed to uncover epigenetic factors involved in the EMT-transcriptional program occurring in BLBC cells surviving conventional chemotherapy. Results Using whole transcriptome data from a murine mammary carcinoma cell line (pG-2), we identified upregulation of Hdac4, 7 and 8 in tumor cells surviving conventional chemotherapy. Subsequent analyses of human BLBC patient datasets and cell lines established HDAC8 as the most promising factor sustaining tumor cell viability. ChIP-sequencing data analysis identified a pronounced loss of H3K27ac at regulatory regions of master transcription factors (TFs) of epithelial phenotype like Gata3, Elf5, Rora and Grhl2 upon chemotherapy. Interestingly, impairment of HDAC8 activity reverted epithelial-TFs levels. Furthermore, loss of HDAC8 activity sensitized tumor cells to chemotherapeutic treatments, even at low doses. Conclusion The current study reveals a previously unknown transcriptional repressive function of HDAC8 exerted on a panel of transcription factors involved in the maintenance of epithelial cell phenotype, thereby supporting BLBC cell survival to conventional chemotherapy. Our data establish HDAC8 as an attractive therapeutically targetable epigenetic factor to increase the efficiency of chemotherapeutics. Graphical abstract

DNA methylation signatures associated with cardiometabolic risk factors in children from India and The Gambia: results from the EMPHASIS study

Background The prevalence of cardiometabolic disease (CMD) is rising globally, with environmentally induced epigenetic changes suggested to play a role. Few studies have investigated epigenetic associations with CMD risk factors in children from low- and middle-income countries. We sought to identify associations between DNA methylation (DNAm) and CMD risk factors in children from India and The Gambia. Results Using the Illumina Infinium HumanMethylation 850 K Beadchip array, we interrogated DNAm in 293 Gambian (7–9 years) and 698 Indian (5–7 years) children. We identified differentially methylated CpGs (dmCpGs) associated with systolic blood pressure, fasting insulin, triglycerides and LDL-Cholesterol in the Gambian children; and with insulin sensitivity, insulinogenic index and HDL-Cholesterol in the Indian children. There was no overlap of the dmCpGs between the cohorts. Meta-analysis identified dmCpGs associated with insulin secretion and pulse pressure that were different from cohort-specific dmCpGs. Several differentially methylated regions were associated with diastolic blood pressure, insulin sensitivity and fasting glucose, but these did not overlap with the dmCpGs. We identified significant cis-methQTLs at three LDL-Cholesterol-associated dmCpGs in Gambians; however, methylation did not mediate genotype effects on the CMD outcomes. Conclusion This study identified cardiometabolic biomarkers associated with differential DNAm in Indian and Gambian children. Most associations were cohort specific, potentially reflecting environmental and ethnic differences.

Therapeutical interference with the epigenetic landscape of germ cell tumors: a comparative drug study and new mechanistical insights

Background Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs. Results We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers (MZ-1, LP99) and polycomb-repressive complexes (PRT4165, GSK343). Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT. Conclusion Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs.

The CpG island methylator phenotype increases the risk of high-grade squamous intraepithelial lesions and cervical cancer

Background High-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer, but additional alterations are necessary for its development. Abnormal DNA methylation has an important role in the origin and dissemination of cervical cancer and other human tumors. In this work, we analyzed the methylation of eight genes (AJAP1, CDH1, CDH13, MAGI2, MGMT, MYOD1, RASSF1A and SOX17) that participate in several biological processes for the maintenance of cell normality. We analyzed DNA methylation by methylation-specific PCR (MSP) and HPV infection using the INNO‑LiPA genotyping kit in 59 samples diagnostic of normal cervical tissue (non-SIL), 107 low-grade squamous intraepithelial lesions (LSILs), 29 high-grade squamous intraepithelial lesions (HSILs) and 51 cervical cancers (CCs). Results We found that all samples of LSIL, HSIL, and CC were HPV-positive, and the genotypes with higher frequencies were 16, 18, 51 and 56. In general, the genes analyzed displayed a significant tendency toward an increase in methylation levels according to increasing cervical lesion severity, except for the CDH13 gene. High CpG island methylator phenotype (CIMP) was associated with a 50.6-fold (95% CI 4.72–2267.3)-increased risk of HSIL and a 122-fold risk of CC (95% CI 10.04–5349.7). Conclusions We found that CIMP high was significantly associated with HSIL and CC risk. These results could indicate that CIMP together with HR-HPV infection and other factors participates in the development of HSIL and CC.

Pembrolizumab plus azacitidine in patients with chemotherapy refractory metastatic colorectal cancer: a single-arm phase 2 trial and correlative biomarker analysis

Background DNA mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) is not responsive to pembrolizumab monotherapy. DNA methyltransferase inhibitors can promote antitumor immune responses. This clinical trial investigated whether concurrent treatment with azacitidine enhances the antitumor activity of pembrolizumab in mCRC. Methods We conducted a phase 2 single-arm trial evaluating activity and tolerability of pembrolizumab plus azacitidine in patients with chemotherapy-refractory mCRC (NCT02260440). Patients received pembrolizumab 200 mg IV on day 1 and azacitidine 100 mg SQ on days 1–5, every 3 weeks. A low fixed dose of azacitidine was chosen in order to reduce the possibility of a direct cytotoxic effect of the drug, since the main focus of this study was to investigate its potential immunomodulatory effect. The primary endpoint of this study was overall response rate (ORR) using RECIST v1.1., and secondary endpoints were progression-free survival (PFS) and overall survival (OS). Tumor tissue was collected pre- and on-treatment for correlative studies. Results Thirty chemotherapy-refractory patients received a median of three cycles of therapy. One patient achieved partial response (PR), and one patient had stable disease (SD) as best confirmed response. The ORR was 3%, median PFS was 1.9 months, and median OS was 6.3 months. The combination regimen was well-tolerated, and 96% of treatment-related adverse events (TRAEs) were grade 1/2. This trial was terminated prior to the accrual target of 40 patients due to lack of clinical efficacy. DNA methylation on-treatment as compared to pre-treatment decreased genome wide in 10 of 15 patients with paired biopsies and was significantly lower in gene promoter regions after treatment. These promoter demethylated genes represented a higher proportion of upregulated genes, including several immune gene sets, endogenous retroviral elements, and cancer-testis antigens. CD8+ TIL density trended higher on-treatment compared to pre-treatment. Higher CD8+ TIL density at baseline was associated with greater likelihood of benefit from treatment. On-treatment tumor demethylation correlated with the increases in tumor CD8+ TIL density. Conclusions The combination of pembrolizumab and azacitidine is safe and tolerable with modest clinical activity in the treatment for chemotherapy-refractory mCRC. Correlative studies suggest that tumor DNA demethylation and immunomodulation occurs. An association between tumor DNA demethylation and tumor-immune modulation suggests immune modulation and may result from treatment with azacitidine. Trial registration ClinicalTrials.gov, NCT02260440. Registered 9 October 2014, https://clinicaltrials.gov/ct2/show/NCT02260440.

Decreased mitochondrial D-loop region methylation mediates an increase in mitochondrial DNA copy number in CADASIL

Background Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a typical neurodegenerative disease associated with mitochondrial dysfunction. Methylation of the D-loop region and mitochondrial DNA copy number (mtDNAcn) play a critical role in the maintenance of mitochondrial function. However, the association between D-loop region methylation, mtDNAcn and CADASIL remains unclear. Methods Overall, 162 individuals were recruited, including 66 CADASIL patients and 96 age- and sex-matched controls. After extracting genomic DNA from the peripheral white blood cells, levels of D-loop methylation and mtDNAcn were assessed using MethylTarget sequencing and real-time PCR, respectively. Results We observed increased mtDNAcn and decreased D-loop methylation levels in CADASIL patients compared to the control group, regardless of gender stratification. Besides, we found a negative correlation between D-loop methylation levels and mtDNAcn. Mediation effect analysis shows that the proportion of the association between mtDNAcn and CADASIL that is mediated by D-loop methylation is 11.6% (95% CI 5.6, 22.6). After gender stratification, the proportions of such associations that are mediated by D-loop methylation in males and females were 7.2% (95% CI 2.4, 19.8) and 22.0% (95% CI 7.4, 50.1), respectively. Conclusion Decreased methylation of the D-loop region mediates increased mtDNAcn in CADASIL, which may be caused by a compensatory mechanism of mitochondrial dysfunction in patients with CADASIL.

Epigenetic biomarkers of ageing are predictive of mortality risk in a longitudinal clinical cohort of individuals diagnosed with oropharyngeal cancer

Background Epigenetic clocks are biomarkers of ageing derived from DNA methylation levels at a subset of CpG sites. The difference between age predicted by these clocks and chronological age, termed “epigenetic age acceleration”, has been shown to predict age-related disease and mortality. We aimed to assess the prognostic value of epigenetic age acceleration and a DNA methylation-based mortality risk score with all-cause mortality in a prospective clinical cohort of individuals with head and neck cancer: Head and Neck 5000. We investigated two markers of intrinsic epigenetic age acceleration (IEAAHorvath and IEAAHannum), one marker of extrinsic epigenetic age acceleration (EEAA), one optimised to predict physiological dysregulation (AgeAccelPheno), one optimised to predict lifespan (AgeAccelGrim) and a DNA methylation-based predictor of mortality (ZhangScore). Cox regression models were first used to estimate adjusted hazard ratios (HR) and 95% confidence intervals (CI) for associations of epigenetic age acceleration with all-cause mortality in people with oropharyngeal cancer (n = 408; 105 deaths). The added prognostic value of epigenetic markers compared to a clinical model including age, sex, TNM stage and HPV status was then evaluated. Results IEAAHannum and AgeAccelGrim were associated with mortality risk after adjustment for clinical and lifestyle factors (HRs per standard deviation [SD] increase in age acceleration = 1.30 [95% CI 1.07, 1.57; p = 0.007] and 1.40 [95% CI 1.06, 1.83; p = 0.016], respectively). There was weak evidence that the addition of AgeAccelGrim to the clinical model improved 3-year mortality prediction (area under the receiver operating characteristic curve: 0.80 vs. 0.77; p value for difference = 0.069). Conclusion In the setting of a large, clinical cohort of individuals with head and neck cancer, our study demonstrates the potential of epigenetic markers of ageing to enhance survival prediction in people with oropharyngeal cancer, beyond established prognostic factors. Our findings have potential uses in both clinical and non-clinical contexts: to aid treatment planning and improve patient stratification.

High performance methylated DNA markers for detection of colon adenocarcinoma

Background Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA. Methods Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann–Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy. Results In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue. Conclusions We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.