A human genome editing-based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

PCDHGC3 hypermethylation as a potential biomarker of intestinal neuroendocrine carcinomas.

Neuroendocrine neoplasms (NENs) encompass tumors arising from neuroendocrine cells in various organs, including the gastrointestinal tract, pancreas, adrenal gland, and paraganglia. Despite advancements, accurately predicting the aggressiveness of gastroenteropancreatic (GEP) NENs based solely on pathological data remains challenging, thereby limiting optimal clinical management. Our previous research unveiled a crucial link between hypermethylation of the protocadherin PCDHGC3 gene and neuroendocrine tumors originating from the paraganglia and adrenal medulla. This epigenetic alteration was associated with increased metastatic potential and succinate dehydrogenase complex (SDH) dysfunction. Expanding upon this discovery, the current study explored PCDHGC3 gene methylation within the context of GEP-NENs in a cohort comprising 34 cases. We uncovered promoter hypermethylation of PCDHGC3 in 29% of GEP-NENs, with a significantly higher prevalence in gastrointestinal (GI) neuroendocrine carcinomas (NECs) compared with both pancreatic (Pan) NECs and neuroendocrine tumors (NETs) of GI and Pan origin. Importantly, these findings were validated in one of the largest multi-center GEP-NEN cohorts. Mechanistic analysis revealed that PCDHGC3 hypermethylation was not associated with SDH mutations or protein loss, indicating an SDH-independent epigenetic mechanism. Clinically, PCDHGC3 hypermethylation emerged as a significant prognostic factor, correlating with reduced overall survival rates in both patient cohorts. Significantly, whereas PCDHGC3 hypermethylation exhibited a strong correlation with TP53 somatic mutations, a hallmark of NEC, its predictive value surpassed that of TP53 mutations, with an area under the curve (AUC) of 0.95 (95% CI 0.83-1.0) for discriminating GI-NECs from GI-NETs, highlighting its superior predictive performance. In conclusion, our findings position PCDHGC3 methylation status as a promising molecular biomarker for effectively stratifying patients with GI-NENs. This discovery has the potential to advance patient care by enabling more precise risk assessments and tailored treatment strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

Metformin and Glucose Concentration as Limiting Factors in Retinal Pigment Epithelial Cell Viability and Proliferation.

Metformin is a well-established drug for the treatment of type 2 diabetes; however, the mechanism of action has not been well described and many aspects of how it truly acts are still unknown. Moreover, regarding in vitro experiments, the glycaemic status when metformin is used is generally not considered, which, added to the suprapharmacological drug concentrations that are commonly employed in research, has resulted in gaps of its mechanism of action. The aim of this study was to determine how glucose and metformin concentrations influence cell culture. Considering that diabetic retinopathy is one of the most common complications of diabetes, a retinal pigment epithelial cell line was selected, and cell viability and proliferation rates were measured at different glucose and metformin concentrations. As expected, glucose concentration by itself positively influenced cell proliferation rates. When the metformin was considered, results were conditioned, as well, by metformin concentration. This conditioning resulted in cell death when high concentrations of metformin were used under physiological concentrations of glucose, while this did not happen when clinically relevant concentrations of metformin were used independently of glucose status. Our study shows the importance of in vitro cell growth conditions when drug effects such as metformin's are being analysed.

Maternal obesity and gestational diabetes reprogram the methylome of offspring beyond birth by inducing epigenetic signatures in metabolic and developmental pathways.

BACKGROUND: Obesity is a negative chronic metabolic health condition that represents an additional risk for the development of multiple pathologies. Epidemiological studies have shown how maternal obesity or gestational diabetes mellitus during pregnancy constitute serious risk factors in relation to the appearance of cardiometabolic diseases in the offspring. Furthermore, epigenetic remodelling may help explain the molecular mechanisms that underlie these epidemiological findings. Thus, in this study we explored the DNA methylation landscape of children born to mothers with obesity and gestational diabetes during their first year of life. METHODS: We used Illumina Infinium MethylationEPIC BeadChip arrays to profile more than 770,000 genome-wide CpG sites in blood samples from a paediatric longitudinal cohort consisting of 26 children born to mothers who suffered from obesity or obesity with gestational diabetes mellitus during pregnancy and 13 healthy controls (measurements taken at 0, 6 and 12 month; total N = 90). We carried out cross-sectional and longitudinal analyses to derive DNA methylation alterations associated with developmental and pathology-related epigenomics. RESULTS: We identified abundant DNA methylation changes during child development from birth to 6 months and, to a lesser extent, up to 12 months of age. Using cross-sectional analyses, we discovered DNA methylation biomarkers maintained across the first year of life that could discriminate children born to mothers who suffered from obesity or obesity with gestational diabetes. Importantly, enrichment analyses suggested that these alterations constitute epigenetic signatures that affect genes and pathways involved in the metabolism of fatty acids, postnatal developmental processes and mitochondrial bioenergetics, such as CPT1B, SLC38A4, SLC35F3 and FN3K. Finally, we observed evidence of an interaction between developmental DNA methylation changes and maternal metabolic condition alterations. CONCLUSIONS: Our observations highlight the first six months of development as being the most crucial for epigenetic remodelling. Furthermore, our results support the existence of systemic intrauterine foetal programming linked to obesity and gestational diabetes that affects the childhood methylome beyond birth, which involves alterations related to metabolic pathways, and which may interact with ordinary postnatal development programmes.

Conservation of Aging and Cancer Epigenetic Signatures across Human and Mouse.

Aging and cancer are two interrelated processes, with aging being a major risk factor for the development of cancer. Parallel epigenetic alterations have been described for both, although differences, especially within the DNA hypomethylation scenario, have also been recently reported. Although many of these observations arise from the use of mouse models, there is a lack of systematic comparisons of human and mouse epigenetic patterns in the context of disease. However, such comparisons are significant as they allow to establish the extent to which some of the observed similarities or differences arise from pre-existing species-specific epigenetic traits. Here, we have used reduced representation bisulfite sequencing to profile the brain methylomes of young and old, tumoral and nontumoral brain samples from human and mouse. We first characterized the baseline epigenomic patterns of the species and subsequently focused on the DNA methylation alterations associated with cancer and aging. Next, we described the functional genomic and epigenomic context associated with the alterations, and finally, we integrated our data to study interspecies DNA methylation levels at orthologous CpG sites. Globally, we found considerable differences between the characteristics of DNA methylation alterations in cancer and aging in both species. Moreover, we describe robust evidence for the conservation of the specific cancer and aging epigenomic signatures in human and mouse. Our observations point toward the preservation of the functional consequences of these alterations at multiple levels of genomic regulation. Finally, our analyses reveal a role for the genomic context in explaining disease- and species-specific epigenetic traits.

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.

Epigenetic downregulation of TET3 reduces genome-wide 5hmC levels and promotes glioblastoma tumorigenesis.

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Longitudinal genome-wide DNA methylation analysis uncovers persistent early-life DNA methylation changes.

BACKGROUND: Early life is a period of drastic epigenetic remodeling in which the epigenome is especially sensitive to extrinsic and intrinsic influence. However, the epigenome-wide dynamics of the DNA methylation changes that occur during this period have not been sufficiently characterized in longitudinal studies. METHODS: To this end, we studied the DNA methylation status of more than 750,000 CpG sites using Illumina MethylationEPIC arrays on 33 paired blood samples from 11 subjects at birth and at 5 and 10 years of age, then characterized the chromatin context associated with these loci by integrating our data with histone, chromatin-state and enhancer-element external datasets, and, finally, validated our results through bisulfite pyrosequencing in two independent longitudinal cohorts of 18 additional subjects. RESULTS: We found abundant DNA methylation changes (110,726 CpG sites) during the first lustrum of life, while far fewer alterations were observed in the subsequent 5 years (460 CpG sites). However, our analysis revealed persistent DNA methylation changes at 240 CpG sites, indicating that there are genomic locations of considerable epigenetic change beyond immediate birth. The chromatin context of hypermethylation changes was associated with repressive genomic locations and genes with developmental and cell signaling functions, while hypomethylation changes were linked to enhancer regions and genes with immunological and mRNA and protein metabolism functions. Significantly, our results show that genes that suffer simultaneous hyper- and hypomethylation are functionally distinct from exclusively hyper- or hypomethylated genes, and that enhancer-associated methylation is different in hyper- and hypomethylation scenarios, with hypomethylation being more associated to epigenetic changes at blood tissue-specific enhancer elements. CONCLUSIONS: These data show that epigenetic remodeling is dramatically reduced after the first 5 years of life. However, there are certain loci which continue to manifest DNA methylation changes, pointing towards a possible functionality beyond early development. Furthermore, our results deepen the understanding of the genomic context associated to hyper- or hypomethylation alterations during time, suggesting that hypomethylation of blood tissue-specific enhancer elements could be of importance in the establishment of functional states in blood tissue during early-life.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Interindividual epigenetic variability: Sound or noise?

Interindividual variability is an inherent characteristic of biological systems. Whereas the underlying molecular sources of interindividual variability remain poorly understood, recent work by Ecker et al. (Ecker S, Chen L, Pancaldi V, Bagger FO, et al. 2017. Genome Biol 18: 18.) sheds light on the characterization of this phenomenon in a complex biological scenario. By combining data from the BLUEPRINT Epigenome Project with a novel analytical approach, these authors were able to measure the degree of transcriptional and epigenetic variability across a wide panel of samples and types of immune cell. Interestingly, neutrophils displayed increased variability compared to monocytes and T cells, which may be related to the crucial role of the former as an initial mediator of immune responses. Here we review recent literature in this area, and discuss some important issues raised by these innovative analyses. Furthermore, we summarize other potential sources of epigenetic variability, such as epigenetic drift and the epigenetic clock, as well as the current ongoing direction of the field.

Autoregulatory loop of nuclear corepressor 1 expression controls invasion, tumor growth, and metastasis.

Nuclear corepressor 1 (NCoR) associates with nuclear receptors and other transcription factors leading to transcriptional repression. We show here that NCoR depletion enhances cancer cell invasion and increases tumor growth and metastatic potential in nude mice. These changes are related to repressed transcription of genes associated with increased metastasis and poor prognosis in patients. Strikingly, transient NCoR silencing leads to heterochromatinization and stable silencing of the NCoR gene, suggesting that NCoR loss can be propagated, contributing to tumor progression even in the absence of NCoR gene mutations. Down-regulation of the thyroid hormone receptor ß1 (TRß) appears to be associated with cancer onset and progression. We found that expression of TRß increases NCoR levels and that this induction is essential in mediating inhibition of tumor growth and metastasis by this receptor. Moreover, NCoR is down-regulated in human hepatocarcinomas and in the more aggressive breast cancer tumors, and its expression correlates positively with that of TRß. These data provide a molecular basis for the anticancer actions of this corepressor and identify NCoR as a potential molecular target for development of novel cancer therapies.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

Epigenetics and the environment: emerging patterns and implications.

Epigenetic phenomena in animals and plants are mediated by DNA methylation and stable chromatin modifications. There has been considerable interest in whether environmental factors modulate the establishment and maintenance of epigenetic modifications, and could thereby influence gene expression and phenotype. Chemical pollutants, dietary components, temperature changes and other external stresses can indeed have long-lasting effects on development, metabolism and health, sometimes even in subsequent generations. Although the underlying mechanisms remain largely unknown, particularly in humans, mechanistic insights are emerging from experimental model systems. These have implications for structuring future research and understanding disease and development.