Mutations in regulators of the epigenome and their effects on the DNA methylome

Genome-wide profiling for genetic alterations in cancer has identified mutations in genes that are associated with epigenetic programming of genomes for DNA methylation patterns, histone modifications patterns and the positioning of nucleosomes. Here a systematic evaluation of the available cancer genome profiling data established by large international consortia, in order to identify recurrently mutated genes or pathways was described. Using curated list of approximately 700 epigenetic regulators and currently available genome-wide datasets on genetic and epigenetic alterations in cancers, the distribution of alterations in epigenetic regulators was described. Epigenetic genes were classified as potential oncogenic or those with tumor-suppressor function based on the location of mutations relative to functional domains and their frequencies. A panel of 50 epigenetic genes, including: DNMTs, histones (H3F3A, HIST1H3B), histone editors (KDM5C, KDM6A) and writers (MLLs, SETD2, EZH2, ATM) that can promote epigenetic changes in cancer was identified. Using correlative analysis of publicly available methylation data with information on deregulated epigenetic driver genes, many identified subtype-specific methylation clusters were correlated with groups of up to 3 epigenetic regulators. This analysis provides a source for the identification and link between methylation groups and deregulated epigenetic genes. Major cancer specific methylation changes have been observed in promoters and gene bodies. Tissue-specific cancer methylation differences have been located in enhancers and regulatory regions of non-coding RNAs. Based on identified results, the major mechanism of non-coding RNA deregulation in cancer has been investigated on independent data cohort. Using integrative analysis of non-coding RNA in early-onset prostate cancer, non-coding RNAs were classified as tumor-suppressive and oncogenic. About 120 novel prostate cancer specific non-coding RNAs that have been epigenetically deregulated have been identified. Our study on the defects in regulators of the epigenome will help to understand mechanisms leading to distinct epigenetic patterns and will allow the molecular validation of defined correlations in experimental settings

DNA methylation at an enhancer of the three prime repair exonuclease 2 gene (TREX2) is linked to gene expression and survival in laryngeal cancer

Background: Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 (TREX2) locus in laryngeal (n = 256) and colorectal cancer cases (n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA). Results: Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts (p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 (p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients (p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression. Conclusions: The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies

Lavandula Angustifolia Mill. українського походження: порівняльне дослідження хімічного складу та антимікробного потенціалу екстрактів з трави

Provide updated data on the antimicrobial activity and chemical composition of original dry extracts from Lavandula angustifolia herb of Ukrainian origin. The aim – an experimental comparative study of the chemical profile and antimicrobial activity of the original dry extracts of Lavandula angustifolia herb and their effect on the ability to destroy biofilms of microbial cultures or prevent their formation in vitro.   Materials and methods. The objects of the study are dry extracts obtained from the lavender herb with purified water and ethanol solutions (40 and 70 %). The main biologically active substances (BAS) of the extracts were determined by the Thin-layer chromatography and Absorption spectrophotometry methods. The microbiological properties of the test samples of the investigated plant extracts were studied in vitro by the two-fold serial dilutions method. The ability of microorganisms to form a biofilm was determined by the method of adhesion to polystyrene in flat-bottomed plastic plates. The optical density of the initial bacterial suspension was measured on the Densi-La-Meter device, and the density of inoculated bacterial cells on the Multiskan EX photometer at a wavelength of 540 nm. The study of the antimicrobial activity of water and ethanol extracts of lavender herb in a wide range of concentrations was carried out by the agar diffusion method in the "wells" modification, which is commonly used in microbiological practice. Results. Water and water-ethanol extracts of lavender of Ukrainian origin were obtained. Terpenoids (linalool, linylyl acetate and traces of 1,8-cineol), flavonoids (hyperoside, isoquercitrin), and hydroxycinnamic acids (rosmarinic, chlorogenic acids) were identified in the extracts. The total content of phenolic compounds is 2.02–2.60 mg/g, flavonoids – 1.46–3.17 mg/g. The largest amount of BAS was extracted with 70 % ethanol. According to the results of experimental studies, the extracts of the lavender herb, obtained by extraction with a water-ethanol solution (40 and 70 % ethanol) at a concentration of 1 mg/ml, have antimicrobial properties against a wide range of infectious agents (S. aureus, E. coli, K. pneumoniae, P. aeruginosa, C. albicans). Studies of the influence of test samples of lavender extracts at a concentration of 1 mg/ml on the ability of microorganisms (S. aureus, E. coli, K. pneumoniae, P. aeruginosa) to form biofilms demonstrated that the highest inhibitory activity against biofilm formation was found in the case of the action of test of a sample of phytoextract obtained by extraction with a water-ethanol solution (40 % ethanol), which accounted for S. aureus ‒ 57.8 %, P. aeruginosa – 66.7 %. A wide spectrum of antimicrobial action was established for the tested lavender phytoextracts under the conditions of application of the concentration range of 10-60 μg/ml. The best spectrum of antimicrobial action and the highest activity corresponds to the lavender extract, obtained by extraction with 70 % ethanol, with the effect depending on the concentration. Conclusion. The lavender herb of Ukrainian origin is a promising and affordable source of potential antimicrobial active pharmaceutical ingredients (API). Water-ethanol lavender extract (70 % ethanol), according to research results, has shown high antimicrobial and antifungal potential. According to preliminary data, antimicrobial activity correlates with the content of phenolic compounds. The obtained results may be useful for the search for original substances for the complex correction of symptoms of neurological deficits of infectious etiologyДоповнені дані про антимікробну активність оригінальних сухих екстрактів трави лаванди вузьколистої українського походження та їх хімічного складу. Мета – експериментальне порівняльне дослідження хімічного профілю, та антимікробної активності оригінальних сухих екстрактів трави лаванди вузьколистої та їх впливу на здатність руйнувати біоплівки мікробних культур або запобігати їх утворенню в умовах in vitro. Матеріали та методи. Об’єкти дослідження – сухі екстракти, які отримали з трави лаванди вузьколистої водою очищеною та розчинами етанолу (40 та 70 %). Основні біологічно активні речовини (БАР) екстрактів визначали методами тонкошарової хроматографії та абсорбційної спектрофотометрії. Мікробіологічні властивості тест-зразків досліджуваних рослинних екстрактів вивчали in vitro методом двократних серійних розведень. Здатність мікроорганізмів до утворення біоплівки визначали методом адгезії до полістиролу в плоскодонних пластикових планшетах. Вимірювання оптичної щільності вихідної бактеріальної суспензії проводили на приладі «Densi-La-Meter», інокульованих бактеріальних клітин ‒ на фотометрі «Multiskan EX» за довжини хвилі 540 нм. Дослідження антимікробної активності водних та етанольних екстрактів трави лаванди в широкому спектрі концентрацій проводили загальноприйнятим у мікробіологічній практиці методом дифузії в агар у модифікації «колодязів». Результати. Одержані водний та водно-етанольні екстракти лаванди вузьколистої українського походження. В екстрактах ідентифіковано терпеноїди (ліналоол, лінілілацетат та сліди 1,8-цинеолу), флавоноїди (гіперозид, ізокверцитрин) та гідроксикоричні кислоти (розмаринова, хлорогенова). Сумарний вміст фенольних речовин становить 2,02-2,60 мг/г, флавоноїдів – 1,46-3,17 мг/г. Найбільша кількість БАР вилучалася етанолом 70%.  Згідно з результатами експериментальних досліджень екстракти трави лаванди вузьколистої, отримані екстракцією водно-етанольним розчином (40  і 70 % етанолом) у концентрації 1 мг/мл, володіють антимікробними властивостями щодо широкого спектра збудників інфекцій (S. aureus, E. coli,  K. рneumoniae, P. aeruginosa, C. albicans). Дослідження впливу тест-зразків екстрактів лаванди в концентрації 1 мг/мл на здатність мікроорганізмів (S. aureus, E. coli,  K. рneumoniae, P. aeruginosa) до формування біоплівок продемонстрували, що найвища інгібіторна активність щодо біоплівкоутворен­ня виявлена у разі дії тест-зразка фітоекстракту, отриманого екстракцією водно-етанольним розчином (40 % етанолом), яка склала відповідно до  S. aureus ‒ 57,8 %, P. aeruginosа – 66,7 %. Установлений широкий спектр антимікробної дії для досліджуваних фітоекстрактів лаванди в умовах застосування діапазону концентрацій 10-60 мкг/мл. Найкращий спектр антимікробної дії та найвища активність відповідає екстракту лаванди вузьколистої, отриманому екстракцією 70 % етанолом, залежність ефекту від концентрації. Висновки. Трава лаванди вузьколистої українського походження є перспективним та доступним джерелом потенційних антимікробних активних фармацевтичних інгредієнтів (АФІ). Екстракт лаванди водно-спиртовий 70% за результатами досліджень виявив високий антимікробний та протигрибковий потенціал. За попередніми даними антимікробна активність корелює із вмістом фенольних речовин. Одержані результати можуть бути корисними для пошуку оригінальних субстанцій для комплексної корекції симптомів неврологічного дефіциту інфекційної етіологі

Identification of NHLRC1 as a Novel AKT Activator from a Lung Cancer Epigenome-Wide Association Study (EWAS)

Changes in DNA methylation identified by epigenome-wide association studies (EWAS) have been recently linked to increased lung cancer risk. However, the cellular effects of these differentially methylated positions (DMPs) are often unclear. Therefore, we investigated top differentially methylated positions identified from an EWAS study. This included a putative regulatory region of NHLRC1. Hypomethylation of this gene was recently linked with decreased survival rates in lung cancer patients. HumanMethylation450 BeadChip array (450K) analysis was performed on 66 lung cancer case-control pairs from the European Prospective Investigation into Cancer and Nutrition Heidelberg lung cancer EWAS (EPIC HD) cohort. DMPs identified in these pre-diagnostic blood samples were then investigated for differential DNA methylation in lung tumor versus adjacent normal lung tissue from The Cancer Genome Atlas (TCGA) and replicated in two independent lung tumor versus adjacent normal tissue replication sets with MassARRAY. The EPIC HD top hypermethylated DMP cg06646708 was found to be a hypomethylated region in multiple data sets of lung tumor versus adjacent normal tissue. Hypomethylation within this region caused increased mRNA transcription of the closest gene NHLRC1 in lung tumors. In functional assays, we demonstrate attenuated proliferation, viability, migration, and invasion upon NHLRC1 knock-down in lung cancer cells. Furthermore, diminished AKT phosphorylation at serine 473 causing expression of pro-apoptotic AKT-repressed genes was detected in these knock-down experiments. In conclusion, this study demonstrates the powerful potential for discovery of novel functional mechanisms in oncogenesis based on EWAS DNA methylation data. NHLRC1 holds promise as a new prognostic biomarker for lung cancer survival and prognosis, as well as a target for novel treatment strategies in lung cancer patients

FGFR1 overexpression in non-small cell lung cancer is mediated by genetic and epigenetic mechanisms and is a determinant of FGFR1 inhibitor response

Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response. In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123. A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123. The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.De tre första författarna delar förstaförfattarskapetDe två sista författarna delar sistaförfattarskapet</p

Differentially methylated regions within lung cancer risk loci are enriched in deregulated enhancers

Genome-wide association studies (GWAS) have identified SNPs linked with lung cancer risk. Our aim was to discover the genes, non-coding RNAs, and regulatory elements within GWAS-identified risk regions that are deregulated in non-small cell lung carcinoma (NSCLC) to identify novel, clinically targetable genes and mechanisms in carcinogenesis. A targeted bisulphite-sequencing approach was used to comprehensively investigate DNA methylation changes occurring within lung cancer risk regions in 17 NSCLC and adjacent normal tissue pairs. We report differences in differentially methylated regions between adenocarcinoma and squamous cell carcinoma. Among the minimal regions found to be differentially methylated in at least 50% of the patients, 7 candidates were replicated in 2 independent cohorts (n = 27 and n = 87) and the potential of 6 as methylation-dependent regulatory elements was confirmed by functional assays. This study contributes to understanding the pathways implicated in lung cancer initiation and progression, and provides new potential targets for cancer treatment

Evolution of DNA Methylation Is Linked to Genetic Aberrations in Chronic Lymphocytic Leukemia

ABSTRACT Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefi ned. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintain-ing low methylation heterogeneity and up to 10 % of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and inde-pendent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL. SIGNIFICANCE: Epigenetic alterations are pervasive in cancer and continually develop during disease progression; however, the mechanisms that promote changes in the tumor epigenome at large are cur-rently undefi ned. The current work provides insight into the coevolution of genetic and epigenetic aber-rations and highlights the infl uential role of genetic aberrations in the selection of novel methylatio

Intratumor DNA Methylation Heterogeneity Reflects Clonal Evolution in Aggressive Prostate Cancer

Despite much evidence on epigenetic abnormalities in cancer, it is currently unclear to what extent epigenetic alterations can be associated with tumors’ clonal genetic origins. Here, we show that the prostate intratumor heterogeneity in DNA methylation and copy-number patterns can be explained by a unified evolutionary process. By assaying multiple topographically distinct tumor sites, premalignant lesions, and lymph node metastases within five cases of prostate cancer, we demonstrate that both DNA methylation and copy-number heterogeneity consistently reflect the life history of the tumors. Furthermore, we show cases of genetic or epigenetic convergent evolution and highlight the diversity in the evolutionary origins and aberration spectrum between tumor and metastatic subclones. Importantly, DNA methylation can complement genetic data by serving as a proxy for activity at regulatory domains, as we show through identification of high epigenetic heterogeneity at androgen-receptor-bound enhancers. Epigenome variation thereby expands on the current genome-centric view on tumor heterogeneity

Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia

Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefined. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintaining low methylation heterogeneity and up to 10% of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and independent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL