Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML.

BACKGROUND: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. RESULTS: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34-3.8). CONCLUSIONS: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action.

"Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer".

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060-109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lncPVT1/ZBTB7A axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes. The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

Developmental Dyslexia: Insights from EEG-Based Findings and Molecular Signatures-A Pilot Study.

Developmental dyslexia (DD) is a learning disorder. Although risk genes have been identified, environmental factors, and particularly stress arising from constant difficulties, have been associated with the occurrence of DD by affecting brain plasticity and function, especially during critical neurodevelopmental stages. In this work, electroencephalogram (EEG) findings were coupled with the genetic and epigenetic molecular signatures of individuals with DD and matched controls. Specifically, we investigated the genetic and epigenetic correlates of key stress-associated genes (NR3C1, NR3C2, FKBP5, GILZ, SLC6A4) with psychological characteristics (depression, anxiety, and stress) often included in DD diagnostic criteria, as well as with brain EEG findings. We paired the observed brain rhythms with the expression levels of stress-related genes, investigated the epigenetic profile of the stress regulator glucocorticoid receptor (GR) and correlated such indices with demographic findings. This study presents a new interdisciplinary approach and findings that support the idea that stress, attributed to the demands of the school environment, may act as a contributing factor in the occurrence of the DD phenotype.

Transcriptional repression of lncRNA and miRNA subsets mediated by LRF during erythropoiesis.

Non-coding RNA (ncRNA) species, mainly long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been currently imputed for lesser or greater involvement in human erythropoiesis. These RNA subsets operate within a complex circuit with other epigenetic components and transcription factors (TF) affecting chromatin remodeling during cell differentiation. Lymphoma/leukemia-related (LRF) TF exerts higher occupancy on DNA CpG rich sites and is implicated in several differentiation cell pathways and erythropoiesis among them and also directs the epigenetic regulation of hemoglobin transversion from fetal (HbF) to adult (HbA) form by intervening in the γ-globin gene repression. We intended to investigate LRF activity in the evolving landscape of cells' commitment to the erythroid lineage and specifically during HbF to HbA transversion, to qualify this TF as potential repressor of lncRNAs and miRNAs. Transgenic human erythroleukemia cells, overexpressing LRF and further induced to erythropoiesis, were subjected to expression analysis in high LRF occupancy genetic loci-producing lncRNAs. LRF abundance in genetic loci transcribing for studied lncRNAs was determined by ChIP-Seq data analysis. qPCRs were performed to examine lncRNA expression status. Differentially expressed miRNA pre- and post-erythropoiesis induction were assessed by next-generation sequencing (NGS), and their promoter regions were charted. Expression levels of lncRNAs were correlated with DNA methylation status of flanked CpG islands, and contingent co-regulation of hosted miRNAs was considered. LRF-binding sites were overrepresented in LRF overexpressing cell clones during erythropoiesis induction and exerted a significant suppressive effect towards lncRNAs and miRNA collections. Based on present data interpretation, LRF's multiplied binding capacity across genome is suggested to be transient and associated with higher levels of DNA methylation. KEY MESSAGES: During erythropoiesis, LRF displays extensive occupancy across genetic loci. LRF significantly represses subsets of lncRNAs and miRNAs during erythropoiesis. Promoter region CpG islands' methylation levels affect lncRNA expression. MiRNAs embedded within lncRNA loci show differential regulation of expression.

Contingent Synergistic Interactions between Non-Coding RNAs and DNA-Modifying Enzymes in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with maturation and differentiation defects exhibiting morphological dysplasia in one or more hematopoietic cell lineages. They are associated with peripheral blood cytopenias and by increased risk for progression into acute myelogenous leukemia. Among their multifactorial pathogenesis, age-related epigenetic instability and the error-rate DNA methylation maintenance have been recognized as critical factors for both the initial steps of their pathogenesis and for disease progression. Although lower-risk MDS is associated with an inflammatory bone marrow microenvironment, higher-risk disease is delineated by immunosuppression and clonal expansion. "Epigenetics" is a multidimensional level of gene regulation that determines the specific gene networks expressed in tissues under physiological conditions and guides appropriate chromatin rearrangements upon influence of environmental stimulation. Regulation of this level consists of biochemical modifications in amino acid residues of the histone proteins' N-terminal tails and their concomitant effects on chromatin structure, DNA methylation patterns in CpG dinucleotides and the tissue-specific non-coding RNAs repertoire, which are directed against various gene targets. The role of epigenetic modifications is widely recognized as pivotal both in gene expression control and differential molecular response to drug therapies in humans. Insights to the potential of synergistic cooperations of epigenetic mechanisms provide new avenues for treatment development to comfort human diseases with a known epigenetic shift, such as MDS. Hypomethylating agents (HMAs), such as epigenetic modulating drugs, have been widely used in the past years as first line treatment for elderly higher-risk MDS patients; however, just half of them respond to therapy and are benefited. Rational outcome predictors following epigenetic therapy in MDS and biomarkers associated with disease relapse are of high importance to improve our efforts in developing patient-tailored clinical approaches.

LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for ß-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF's potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the ß-type globin's expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of ß-type globin's locus, triggering the transcriptional events from γ- to ß-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an "open" chromatin conformation across the γ-δ intergenic region and accomplish ß-globin expression and hemoglobin switch.

Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

BACKGROUND: Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. METHODS: Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. RESULTS: Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. CONCLUSIONS: We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

LRF/ZBTB7A conservation accentuates its potential as a therapeutic target for the hematopoietic disorders.

Conserved sequences across species have always provided valuable insights to improve our understanding on the human genome's entity and the interplay among different loci. Lymphoma/leukemia related factor (LRF) is encoded by ZBTB7A gene and belongs to an evolutionarily conserved family of transcription factors, implicated in vital cellular functions. The present data, demonstrating the wide-spread and the high overlap of the LRF/ZBTB7A recognition sites with genomic segments identified as CpG islands in the human genome, suggest that its binding capacity strongly depends on a specific sequence-encoded feature within CpGs. We have previously shown that de-methylation of the CpG island 326 lying in the ZBTB7A gene promoter is associated with impaired pharmacological induction of fetal hemoglobin in ß-type hemoglobinopathies patients. Within this context we aimed to investigate the extent of the LRF/ZBTB7A conservation among primates and mouse genome, focusing our interest also on the CpG island flanking the gene's promoter region, in an effort to further establish its epigenetic regulatory role in human hematopoiesis and pharmacological involvement in hematopoietic disorders. Comparative analysis of the human ZBTB7A nucleotide and amino acid sequences and orthologous sequences among non-human primates and mouse, exhibited high conservation scores. Pathway analysis, clearly indicated that LRF/ZBTB7A influences conserved cellular processes. These data in conjunction with the high levels of expression foremost in hematopoietic tissues, highlighted LRF/ZBTB7A as an essential factor operating indisputably during hematopoiesis.

HLA Polymorphisms and Food Allergy Predisposition.

Food allergy (FA) is a growing health problem that affects ∼8% of the children worldwide. Although the prevalence of FA is increasing, the underlying genetic mechanisms responsible for the onset of this immune disorder are not yet clarified. Genetic factors seem to play a leading role in the development of FA, though interaction with environmental factors cannot be excluded. The broader network of genetic loci mediating the risk of this complex disorder remains to be identified. The human leucocyte antigen (HLA) has been associated with various immune disorders, including FA. This review aims to unravel the potential associations between HLA gene functions and the manifestation and outcome of FA disorders. Exploring new aspects of FA development with the perspective to improve our understanding of the multifaceted etiology and the complex biological mechanisms involved in FA is essential.

The multi-faceted functioning portrait of LRF/ZBTB7A.

Transcription factors (TFs) consisting of zinc fingers combined with BTB (for broad-complex, tram-track, and bric-a-brac) domain (ZBTB) are a highly conserved protein family that comprises a multifunctional and heterogeneous group of TFs, mainly modulating cell developmental events and cell fate. LRF/ZBTB7A, in particular, is reported to be implicated in a wide variety of physiological and cancer-related cell events. These physiological processes include regulation of erythrocyte maturation, B/T cell differentiation, adipogenesis, and thymic insulin expression affecting consequently insulin self-tolerance. In cancer, LRF/ZBTB7A has been reported to act either as oncogenic or as oncosuppressive factor by affecting specific cell processes (proliferation, apoptosis, invasion, migration, metastasis, etc) in opposed ways, depending on cancer type and molecular interactions. The molecular mechanisms via which LRF/ZBTB7A is known to exert either physiological or cancer-related cellular effects include chromatin organization and remodeling, regulation of the Notch signaling axis, cellular response to DNA damage stimulus, epigenetic-dependent regulation of transcription, regulation of the expression and activity of NF-κB and p53, and regulation of aerobic glycolysis and oxidative phosphorylation (Warburg effect). It is a pleiotropic TF, and thus, alterations to its expression status become detrimental for cell survival. This review summarizes its implication in different cellular activities and the commonly invoked molecular mechanisms triggered by LRF/ZBTB7A's orchestrated action.

Combined study on clastogenic, aneugenic and apoptotic properties of doxorubicin in human cells in vitro.

BACKGROUND: Doxorubicin is a widely used anticancer drug due to its broad spectrum of antitumor activity. Various mechanisms have been proposed for its cytostatic activity, including DNA intercalation, topoisomerase II inhibition, generation of free radicals and apoptosis. The present study aims to further clarify the cytostatic activity of doxorubicin by its specific effect on (a) DNA damage, (b) micronucleation and (c) apoptosis, using a combination of different methods and cell systems such as human lymphocytes and HL-60 human leukemic cells. DNA lesions were analyzed by the alkaline comet assay in combination with formamidopyrimidine (Fpg) and human 8-oxoguanine (hOGG1) repair enzymes. Micronucleation was investigated by the Cytokinesis-Block Micronucleus assay (CBMN) in combination with Fluorescence In Situ Hybridization analysis. Impairment on mitotic apparatus was investigated by double immunofluorescence of ß- and γ-tubulin. Apoptotic cell frequency was determined by the CBMN cytome assay. Complementary to the above, caspase-3 level was investigated by Western blot. RESULTS: It was found that doxorubicin generates DNA breakage induced by oxidative damage in DNA bases, which can be repaired by the Fpg and hOGG1 enzymes. Increased micronucleus frequency was identified mainly through chromosome breakage and, at a lesser extent, through chromosome delay. Analysis of mitotic spindle showed disturbance of chromosome orientation and centrosome duplication and/or separation, leading to aneuploidy. Enhanced frequency of apoptotic leukemic cells was also observed. Caspase-3 seems to be involved in the generation of apoptosis. CONCLUSIONS: The aforementioned findings derived from different treatment schedules, doses and time of exposure on primary versus transformed cells extend our knowledge about doxorubicin genotoxicity and contribute to the better understanding of the mechanisms by which doxorubicin induces genotoxic effects on human cells.

Impact of ZBTB7A hypomethylation and expression patterns on treatment response to hydroxyurea.

BACKGROUND: We aimed to clarify the emerging epigenetic landscape in a group of genes classified as "modifier genes" of the ß-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/ß-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented. RESULTS: We examined the CpG islands' DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients' monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU. CONCLUSIONS: This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5' CpG sequences of all studied human HBB cluster "modifier genes." Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the ß-type hemoglobinopathy patients.

Whole transcriptome analysis of human erythropoietic cells during ontogenesis suggests a role of VEGFA gene as modulator of fetal hemoglobin and pharmacogenomic biomarker of treatment response to hydroxyurea in ß-type hemoglobinopathy patients.

BACKGROUND: Human erythropoiesis is characterized by distinct gene expression profiles at various developmental stages. Previous studies suggest that fetal-to-adult hemoglobin switch is regulated by a complex mechanism, in which many key players still remain unknown. Here, we report our findings from whole transcriptome analysis of erythroid cells, isolated from erythroid tissues at various developmental stages in an effort to identify distinct molecular signatures of each erythroid tissue. RESULTS: From our in-depth data analysis, pathway analysis, and text mining, we opted to focus on the VEGFA gene, given its gene expression characteristics. Selected VEGFA genomic variants, identified through linkage disequilibrium analysis, were explored further for their association with elevated fetal hemoglobin levels in ß-type hemoglobinopathy patients. Our downstream analysis of non-transfusion-dependent ß-thalassemia patients, ß-thalassemia major patients, compound heterozygous sickle cell disease/ß-thalassemia patients receiving hydroxyurea as fetal hemoglobin augmentation treatment, and non-thalassemic individuals indicated that VEGFA genomic variants were associated with disease severity in ß-thalassemia patients and hydroxyurea treatment efficacy in SCD/ß-thalassemia compound heterozygous patients. CONCLUSIONS: Our findings suggest that VEGFA may act as a modifier gene of human globin gene expression and, at the same time, serve as a genomic biomarker in ß-type hemoglobinopathy disease severity and hydroxyurea treatment efficacy.

Correlation of SIN3A genomic variants with ß-hemoglobinopathies disease severity and hydroxyurea treatment efficacy.

AIMS: Hemoglobinopathies, particularly ß-thalassemia and sickle cell disease, are characterized by great phenotypic variability in terms of disease severity, while notable differences have been observed in hydroxyurea treatment efficacy. In both cases, the observed phenotypic diversity is mostly dependent on the elevated fetal hemoglobin levels, resulting from the persistent fetal globin gene expression in the adult erythroid stage orchestrated by intricate mechanisms that still remain only partly understood. We have previously shown that several protein factors act as modifiers of fetal hemoglobin production, exerting their effect via different pathways. MATERIALS & METHODS: Here, we explored whether SIN3A could act as a modifier of fetal globin gene expression, as it interacts with KLF10, a known modifier of fetal hemoglobin production. RESULTS: We show that SIN3A genomic variants are associated both with ß-thalassemia disease severity (rs11072544) as well as hydroxyurea treatment response (rs7166737) in ß-hemoglobinopathies patients. CONCLUSION: Our findings further underline that fetal hemoglobin production is the result of a complex interplay in which several human globin gene cluster variants interact with protein factors encoded by modifier genes to produce the observed clinical outcome.

Individualizing fetal hemoglobin augmenting therapy for ß-type hemoglobinopathies patients.

Individual genetic composition is an important cause of variations in the response and tolerance to drug treatment. Pharmacogenomics is a modern discipline aiming to delineate individual genomic profiles and drug response. To date, there are several medical disciplines where pharmacogenomics is readily applicable, while in others its usefulness is yet to be demonstrated. Recent experimental evidence suggest that besides genomic variation within the human ß-globin gene cluster, other variants in modifier genes residing outside the human ß-globin gene cluster are significantly associated with response to hydroxyurea treatment in ß-type hemoglobinopathies patients, deducted from the increase in fetal hemoglobin levels. This article aims to provide an update and to discuss future challenges on the application of pharmacogenomics for ß-type hemoglobinopathies therapeutics in relation to the current pharmacological treatment modalities for those disorders.