ABSTRACT
Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes.
Subject(s)
DNA Methylation/genetics , Inflammation/genetics , Sirtuin 1/genetics , Sirtuin 2/genetics , Acetylation , Cell Differentiation/genetics , Chromatin/genetics , Gene Expression Regulation/genetics , Histones/genetics , Humans , Inflammation/chemically induced , Inflammation/pathology , Lipopolysaccharides/toxicity , Macrophages/metabolism , Promoter Regions, Genetic , Transcriptional Activation/geneticsABSTRACT
OBJECTIVE: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that mainly targets joints. Monocytes and macrophages are critical in RA pathogenesis and contribute to inflammatory lesions. These extremely plastic cells respond to extracellular signals which cause epigenomic changes that define their pathogenic phenotype. Here, we interrogated how DNA methylation alterations in RA monocytes are determined by extracellular signals. METHODS: High-throughput DNA methylation analyses of patients with RA and controls and in vitro cytokine stimulation were used to investigate the underlying mechanisms behind DNA methylation alterations in RA as well as their relationship with clinical parameters, including RA disease activity. RESULTS: The DNA methylomes of peripheral blood monocytes displayed significant changes and increased variability in patients with RA with respect to healthy controls. Changes in the monocyte methylome correlate with DAS28, in which high-activity patients are divergent from healthy controls in contrast to remission patients whose methylome is virtually identical to healthy controls. Indeed, the notion of a changing monocyte methylome is supported after comparing the profiles of same individuals at different stages of activity. We show how these changes are mediated by an increase in disease activity-associated cytokines, such as tumour necrosis factor alpha and interferons, as they recapitulate the DNA methylation changes observed in patients in vitro. CONCLUSION: We demonstrate a direct link between RA disease activity and the monocyte methylome through the action of inflammation-associated cytokines. Finally, we have obtained a DNA methylation-based mathematical formula that predicts inflammation-mediated disease activity for RA and other chronic immune-mediated inflammatory diseases.
Subject(s)
Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/genetics , Cytokines/blood , Epigenome/immunology , Inflammation Mediators/blood , Biomarkers/blood , DNA Methylation/immunology , Humans , Leukocytes, Mononuclear/immunology , Macrophages/immunology , Tumor Necrosis Factor-alpha/bloodABSTRACT
The plasticity of myeloid cells is illustrated by a diversity of functions including their role as effectors of innate immunity as macrophages (MACs) and bone remodelling as osteoclasts (OCs). TET2, a methylcytosine dioxygenase highly expressed in these cells and frequently mutated in myeloid leukemias, may be a key contributor to this plasticity. Through transcriptomic and epigenomic analyses, we investigated 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and gene expression changes in two divergent terminal myeloid differentiation processes, namely MAC and OC differentiation. MACs and OCs undergo highly similar 5hmC and 5mC changes, despite their wide differences in gene expression. Many TET2- and thymine-DNA glycosylase (TDG)-dependent 5mC and 5hmC changes directly activate the common terminal myeloid differentiation programme. However, the acquisition of differential features between MACs and OCs also depends on TET2/TDG. In fact, 5mC oxidation precedes differential histone modification changes between MACs and OCs. TET2 and TDG downregulation impairs the acquisition of such differential histone modification and expression patterns at MAC-/OC-specific genes. We prove that the histone H3K4 methyltransferase SETD1A is differentially recruited between MACs and OCs in a TET2-dependent manner. We demonstrate a novel role of these enzymes in the establishment of specific elements of identity and function in terminal myeloid differentiation.
Subject(s)
Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Macrophages/metabolism , Osteoclasts/metabolism , Proto-Oncogene Proteins/genetics , Thymine DNA Glycosylase/genetics , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Cell Lineage/genetics , DNA-Binding Proteins/metabolism , Dioxygenases , Gene Expression Profiling , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Humans , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/cytology , Macrophages/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , Primary Cell Culture , Proto-Oncogene Proteins/metabolism , RANK Ligand/pharmacology , Thymine DNA Glycosylase/metabolism , TranscriptomeABSTRACT
Compelling evidences highlight the critical role of the tumor microenvironment as mediator of tumor progression and immunosuppression in several types of cancer. The reciprocal interplay between neoplastic and non-tumoral host cells is mediated by direct cell-to-cell contact, soluble factors and exosomes that result in differential gene expression patterns that are driven by epigenetic mechanisms. In this regard, extensive literature has described the abnormalities in the DNA methylation status and histone modification profiles in tumor cells. However, little is known about the mechanisms of epigenetic dysregulation that participate as a consequence of the intricate crosstalk among the cells within the tumor niche. This review summarizes the current knowledge on epigenetic changes that result from the interactions between myeloid, stromal and cancer cells in the tumor microenvironment and its functional impact in both tumorigenesis and tumor progression. We also discuss potential niche-specific epigenetic biomarkers to improve the prognosis and clinical treatment of cancer patients.
Subject(s)
Epigenesis, Genetic , Myeloid Cells/metabolism , Neoplasms/genetics , Stromal Cells/metabolism , Tumor Escape/genetics , Tumor Microenvironment/genetics , Disease Progression , Gene Expression , Humans , Myeloid Cells/immunology , Neoplasms/immunology , Stromal Cells/immunology , Tumor Escape/immunology , Tumor Microenvironment/immunologyABSTRACT
BACKGROUND: Inflammasomes are cytosolic multiprotein complexes in macrophages. They assemble after infection- or stress-associated stimuli, activating both caspase-1-mediated inflammatory cytokine secretion and pyroptosis. Increased inflammasome activity resulting from gene mutations is related to monogenic autoinflammatory syndromes. However, variable penetrance among patients with the same gene mutations suggests involvement of additional mechanisms associated with inflammasome gene regulation. OBJECTIVE: We sought to investigate the role of DNA demethylation in activating inflammasome genes during macrophage differentiation and monocyte activation in healthy control subjects and patients with autoinflammatory syndrome. METHODS: Inflammasome-related genes were tested for DNA methylation and mRNA levels by using bisulfite pyrosequencing and quantitative RT-PCR in monocytes in vitro differentiated to macrophages and exposed to inflammatory conditions. The contribution of Tet methylcytosine dioxygenase 2 (TET2) and nuclear factor κB to DNA demethylation was tested by using chromatin immunoprecipitation, small interfering RNA-mediated downregulation, and pharmacologic inhibition. RESULTS: We observed that inflammasome-related genes are rapidly demethylated in both monocyte-to-macrophage differentiation and on monocyte activation. Demethylation associates with increased gene expression, and both mechanisms are impaired when TET2 and nuclear factor κB are downregulated. We analyzed DNA methylation levels of inflammasome-related genes in patients with cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever, 2 archetypical monogenic autoinflammatory syndromes. Under the above conditions, monocytes from untreated patients with CAPS undergo more efficient DNA demethylation than those of healthy subjects. Interestingly, patients with CAPS treated with anti-IL-1 drugs display methylation levels similar to those of healthy control subjects. CONCLUSION: Our study is the first to demonstrate the involvement of DNA methylation-associated alterations in patients with monogenic autoinflammatory disease and opens up possibilities for novel clinical markers.
Subject(s)
Cryopyrin-Associated Periodic Syndromes/genetics , DNA Methylation/genetics , Inflammasomes/genetics , Cryopyrin-Associated Periodic Syndromes/metabolism , Cytokines/genetics , Cytokines/metabolism , DNA-Binding Proteins/genetics , Dioxygenases , Familial Mediterranean Fever/genetics , Familial Mediterranean Fever/metabolism , Humans , Macrophages/metabolism , Monocytes/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins/geneticsABSTRACT
IL-8 promotes cancer cell growth, survival, angiogenesis, and metastasis in several tumors. Herein, we investigated the sources of IL-8 production in multiple myeloma (MM) and its potential roles in MM pathogenesis. We found that bone marrow cells from patients with MM secreted higher amounts of IL-8 than healthy donors. IL-8 production was detected in cultures of CD138(+) plasma cells and CD138(-) cells isolated from bone marrows of MM patients, and in three of seven human myeloma cell lines (HMCLs) analyzed. Interactions between MM and stromal cells increased IL-8 secretion by stromal cells through cell-cell adhesion and soluble factors. Interestingly, IL8 expression also increased in HMCLs, stromal cells, and osteoclasts after treatment with the antimyeloma drugs melphalan and bortezomib. In fact, the effect of bortezomib on IL-8 production was higher than that exerted by stromal-MM cell interactions. Addition of exogenous IL-8 did not affect growth of HMCLs, although it protected cells from death induced by serum starvation through a caspase-independent mechanism. Furthermore, IL-8 induced by stromal-MM cell interactions strongly contributed to osteoclast formation in vitro, because osteoclastogenesis was markedly reduced by IL-8-specific neutralizing antibodies. In conclusion, our results implicate IL-8 in myeloma bone disease and point to the potential utility of an anti-IL-8 therapy to prevent unwanted effects of IL-8 up-regulation on survival, angiogenesis, and osteolysis in MM.
Subject(s)
Interleukin-8/biosynthesis , Multiple Myeloma/pathology , Osteogenesis/physiology , Cell Separation , Cell Survival , Coculture Techniques , Enzyme-Linked Immunosorbent Assay , Humans , Multiple Myeloma/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Polymerase Chain Reaction , Stromal Cells/metabolism , Up-RegulationABSTRACT
INTRODUCTION: The RAS/RAF/MEK/ERK signaling pathway plays an important role in osteoclast (OC) differentiation and survival mediated by macrophage-colony stimulating factor (M-CSF). Also, vascular endothelial growth factor (VEGF) may greatly influence OC formation and resorption through VEGFR1 and VEGFR2. RAF265 is a novel, orally bioavailable dual inhibitor of RAF kinase and VEGFR2. METHODS: Effect of RAF265 on osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) and OC resorption on calcium-coated wells was assessed by appropriate in vitro assays. Immunoblotting, real-time RT-PCR and flow cytometry were used to evaluate RAF265 mechanism of action. RESULTS: RAF265 significantly impaired in vitro differentiation of PBMCs to OCs induced by receptor activator of NF-kB ligand (RANKL) and M-CSF (IC(50) â 160 nM). In parallel, RAF265 exerted a potent inhibition of OC resorptive capacity (IC(50) â 20 nM). RAF265 treatment led to ERK inhibition and diminished expression of c-fos and NFATc1 (nuclear factor of activated T cells, calcineurin-dependent 1), which would likely account for inhibition of osteoclastogenesis. The reduced gene expression of aVb3 integrin, CCR1, cathepsin K, carbonic anhydrase II, matrix metalloproteinase 9, urokinase and tissue-type plasminogen activators, vacuolar H(+)-ATPase subunit (ATP6V1A) and Rab7 GTPase would probably mediate RAF265 hindered resorption. RAF265 inhibitory effect on VEGFR2 (noticeable at 10-50 nM) was also found to be implicated in the potent inhibition of this agent on OC function. CONCLUSIONS: We have found a new therapeutic application for RAF265 as an inhibitory agent of osteoclastogenesis and OC function, which might be useful for the treatment of skeletal disorders associated with increased bone resorption.
Subject(s)
Imidazoles/pharmacology , Osteoclasts/drug effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyridines/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Bone Resorption/drug therapy , Cells, Cultured , Humans , Leukocytes, Mononuclear/cytology , Osteoclasts/cytologyABSTRACT
Despite the advantage observed with novel drugs such as bortezomib, thalidomide, or lenalidomide, multiple myeloma (MM) remains incurable and there is a clear need for new drugs or combinations based on the pathogenetic mechanism of MM. One of the proposed mechanisms in MM pathogenesis is the involvement of kinase molecules in the growth and survival of myelomatous cells. In this study, we have explored the optimal combination for dasatinib, a tyrosine kinase inhibitor, in MM cells. A clear synergistic effect was observed with the triple combination of dasatinib with bortezomib and dexamethasone which was evident even in the presence of bone marrow microenvironment. Experiments performed on freshly isolated patients' cells also demonstrated potentiation of response in the triple as compared with the agents alone or in double combinations. Gene expression profiling experiments provided some clues on the transcriptional rationale underlying this potentiation, as the triple combination led to significant deregulation of genes involved in cell death, cell growth, proliferation, DNA replication, repair and recombination, and cell-cell signaling. Some of these results were further confirmed by apoptosis and cell cycle experiments and also by Western blot and PCR. These data provide the rationale for the use of this novel combination in MM patients.
Subject(s)
Antineoplastic Agents/therapeutic use , Boronic Acids/therapeutic use , Dexamethasone/therapeutic use , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Pyrazines/therapeutic use , Pyrimidines/therapeutic use , Thiazoles/therapeutic use , Transcriptome/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Boronic Acids/pharmacology , Bortezomib , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dasatinib , Dexamethasone/pharmacology , Drug Synergism , Gene Expression Profiling , Humans , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Multiple Myeloma/physiopathology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Pyrimidines/pharmacology , Thiazoles/pharmacologyABSTRACT
Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.
Subject(s)
Endotoxin Tolerance/genetics , Gram-Negative Bacteria/immunology , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/immunology , Monocytes/immunology , Monocytes/microbiology , Sepsis/genetics , Sepsis/immunology , Case-Control Studies , DNA Methylation/genetics , DNA Methylation/immunology , Endotoxin Tolerance/drug effects , Endotoxin Tolerance/immunology , Endotoxins/toxicity , Epigenesis, Genetic , Female , Gram-Negative Bacterial Infections/microbiology , Humans , In Vitro Techniques , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Janus Kinase 2/immunology , Lipopolysaccharides/toxicity , Male , Monocytes/drug effects , STAT Transcription Factors/genetics , STAT Transcription Factors/immunology , Sepsis/microbiology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/immunologyABSTRACT
Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.
Subject(s)
Antineoplastic Agents/pharmacology , Bone Diseases/drug therapy , DNA Methylation/drug effects , Enzyme Inhibitors/pharmacology , Mesenchymal Stem Cells/drug effects , Multiple Myeloma/drug therapy , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Agents/therapeutic use , Bone Diseases/diagnosis , Bone Diseases/genetics , Bone Diseases/pathology , Bone Marrow/pathology , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA (Cytosine-5-)-Methyltransferases/metabolism , Enzyme Inhibitors/therapeutic use , Epigenesis, Genetic/drug effects , Female , Femur/diagnostic imaging , Femur/pathology , Gene Expression Regulation, Neoplastic/drug effects , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/metabolism , Humans , Male , Mesenchymal Stem Cells/pathology , Mice , Middle Aged , Multiple Myeloma/complications , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Osteogenesis/drug effects , Osteogenesis/genetics , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis. METHODS: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time. RESULTS: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient. CONCLUSION: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases.
Subject(s)
Autoimmune Diseases/classification , Autoimmune Diseases/genetics , Epigenome , Gene Expression Profiling , Adult , Aged , Antiphospholipid Syndrome/genetics , Antiphospholipid Syndrome/immunology , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Autoimmune Diseases/immunology , Case-Control Studies , Cluster Analysis , Cross-Sectional Studies , Epigenomics , Female , Humans , Inflammation/immunology , Interferons/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Male , Middle Aged , Mixed Connective Tissue Disease/genetics , Mixed Connective Tissue Disease/immunology , Scleroderma, Systemic/genetics , Scleroderma, Systemic/immunology , Sjogren's Syndrome/genetics , Sjogren's Syndrome/immunology , Undifferentiated Connective Tissue Diseases/genetics , Undifferentiated Connective Tissue Diseases/immunologyABSTRACT
In the past decade, we have witnessed considerable developments in understanding the roles and functions of miRNAs. In parallel, the identification of alterations in miRNA expression in inflammatory disease indicates their potential as therapeutic targets. Pharmacological treatments targeting abnormally expressed miRNAs for inflammatory diseases are not yet in clinical practice; however, some small compounds and nucleic acids targeting miRNAs have shown promise in preclinical development. Here, we focus on recent advances in understanding miRNA deregulation in inflammatory diseases and provide an overview of the current development of miRNA-based therapeutics in these diseases with an emphasis on newly discovered miRNA therapeutic targets.
Subject(s)
Inflammation/genetics , Inflammation/therapy , MicroRNAs/genetics , Animals , Gene Expression Regulation , Humans , Inflammation/metabolism , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Molecular Targeted TherapyABSTRACT
BACKGROUND: Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. METHODS: Bead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. RESULTS: Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. CONCLUSION: We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction.
Subject(s)
Cytokines/genetics , DNA Methylation , DNA/analysis , Inflammation Mediators/metabolism , Inflammation/complications , Monocytes/pathology , Multiple Organ Failure/complications , Sepsis/diagnosis , Aged , Case-Control Studies , DNA/genetics , Female , Humans , Inflammation/genetics , Male , Middle Aged , Monocytes/immunology , Monocytes/metabolism , Multiple Organ Failure/genetics , Phenotype , Sepsis/etiology , Sepsis/metabolism , Signal TransductionABSTRACT
Multi-omics approaches use a diversity of high-throughput technologies to profile the different molecular layers of living cells. Ideally, the integration of this information should result in comprehensive systems models of cellular physiology and regulation. However, most multi-omics projects still include a limited number of molecular assays and there have been very few multi-omic studies that evaluate dynamic processes such as cellular growth, development and adaptation. Hence, we lack formal analysis methods and comprehensive multi-omics datasets that can be leveraged to develop true multi-layered models for dynamic cellular systems. Here we present the STATegra multi-omics dataset that combines measurements from up to 10 different omics technologies applied to the same biological system, namely the well-studied mouse pre-B-cell differentiation. STATegra includes high-throughput measurements of chromatin structure, gene expression, proteomics and metabolomics, and it is complemented with single-cell data. To our knowledge, the STATegra collection is the most diverse multi-omics dataset describing a dynamic biological system.
Subject(s)
B-Lymphocytes , Cell Differentiation , Animals , B-Lymphocytes/cytology , B-Lymphocytes/physiology , Cell Line , Genomics , Metabolomics , Mice , ProteomicsABSTRACT
Lysyl oxidase-like 3 (LOXL3) is a member of the lysyl oxidase family comprising multifunctional enzymes with depicted roles in extracellular matrix maturation, tumorigenesis, and metastasis. In silico expression analyses followed by experimental validation in a comprehensive cohort of human cell lines revealed a significant upregulation of LOXL3 in human melanoma. We show that LOXL3 silencing impairs cell proliferation and triggers apoptosis in various melanoma cell lines. Further supporting a pro-oncogenic role in melanoma, LOXL3 favors tumor growth in vivo and cooperates with oncogenic BRAF in melanocyte transformation. Upon LOXL3 depletion, melanoma cells display a faulty DNA damage response (DDR), characterized by ATM checkpoint activation and inefficient ATR activation leading to the accumulation of double-strand breaks (DSBs) and aberrant mitosis. Consistent with these findings, LOXL3 binds to proteins involved in the maintenance of genome integrity, in particular BRCA2 and MSH2, whose levels dramatically decrease upon LOXL3 depletion. Moreover, LOXL3 is required for efficient DSB repair in melanoma cells. Our results reveal an unexpected role for LOXL3 in the control of genome stability and melanoma progression, exposing its potential as a novel therapeutic target in malignant melanoma, a very aggressive condition yet in need for more effective treatment options.
Subject(s)
Amino Acid Oxidoreductases/metabolism , DNA Breaks, Double-Stranded , DNA Repair , Genomic Instability , Melanoma/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Amino Acid Oxidoreductases/genetics , Cell Line, Tumor , Cell Survival , Humans , Melanoma/genetics , Melanoma/pathology , Proto-Oncogene Proteins B-raf/geneticsABSTRACT
Myeloid cells are extremely plastic as they respond and terminally differentiate into a plethora of functional types, in the blood or tissues, in response to a variety of growth factors, cytokines and pathogenic molecules. This plasticity is also manifested by the subversion of normal differentiation into the aberrant generation of a variety of tolerogenic myeloid cells in the tumoral microenvironment, where a variety of factors are released. Epigenetic mechanisms are in great part responsible for the plasticity of myeloid cells both under physiological and tumoral conditions. The development of compounds that inhibit epigenetic enzymes provides novel therapeutic opportunities to intercept the crosstalk between cancer cells and host myeloid cells. Here, we summarize our current knowledge on the myeloid cell types generated in the cancer environment, the factors and epigenetic enzymes participating in these processes and propose a number of potential targets for future pharmacological use.
Subject(s)
Cell Differentiation/genetics , Epigenesis, Genetic , Myeloid Cells/cytology , Tumor Microenvironment/genetics , Animals , Dendritic Cells/immunology , Humans , Macrophages/immunology , Osteoclasts/metabolism , Tumor Microenvironment/immunologyABSTRACT
Activation-induced cytidine deaminase (AID) triggers antibody diversification in B cells by catalysing deamination and subsequently mutating immunoglobulin (Ig) genes. Association of AID with RNA Pol II and occurrence of epigenetic changes during Ig gene diversification suggest participation of AID in epigenetic regulation. AID is mutated in hyper-IgM type 2 (HIGM2) syndrome. Here, we investigated the potential role of AID in the acquisition of epigenetic changes. We discovered that AID binding to the IgH locus promotes an increase in H4K20me3. In 293F cells, we demonstrate interaction between co-transfected AID and the three SUV4-20 histone H4K20 methyltransferases, and that SUV4-20H1.2, bound to the IgH switch (S) mu site, is replaced by SUV4-20H2 upon AID binding. Analysis of HIGM2 mutants shows that the AID truncated form W68X is impaired to interact with SUV4-20H1.2 and SUV4-20H2 and is unable to bind and target H4K20me3 to the Smu site. We finally show in mouse primary B cells undergoing class-switch recombination (CSR) that AID deficiency associates with decreased H4K20me3 levels at the Smu site. Our results provide a novel link between SUV4-20 enzymes and CSR and offer a new aspect of the interplay between AID and histone modifications in setting the epigenetic status of CSR sites.
Subject(s)
Cytidine Deaminase/genetics , Epigenesis, Genetic/immunology , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Hyper-IgM Immunodeficiency Syndrome/genetics , Immunoglobulin Class Switching/genetics , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Binding Sites , Cell Line, Tumor , Cytidine Deaminase/immunology , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Histone-Lysine N-Methyltransferase/immunology , Histones/immunology , Humans , Hyper-IgM Immunodeficiency Syndrome/immunology , Hyper-IgM Immunodeficiency Syndrome/pathology , Immunoglobulin G/genetics , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Models, Biological , Mutation , Protein Binding , RNA Polymerase II/genetics , RNA Polymerase II/immunology , Signal TransductionABSTRACT
PURPOSE: PIM kinases are a family of serine/threonine kinases recently proposed as therapeutic targets in oncology. In the present work, we have investigated the effects of the novel pan-PIM kinase inhibitor, PIM447, on myeloma cells and myeloma-associated bone disease using different preclinical models. EXPERIMENTAL DESIGN: In vitro/ex vivo cytotoxicity of PIM447 was evaluated on myeloma cell lines and patient samples. Synergistic combinations with standard treatments were analyzed with Calcusyn Software. PIM447 effects on bone cells were assessed on osteogenic and osteoclastogenic cultures. The mechanisms of PIM447 were explored by immunoblotting, qPCR, and immunofluorescence. A murine model of disseminated multiple myeloma was employed for in vivo studies. RESULTS: PIM447 is cytotoxic for myeloma cells due to cell-cycle disruption and induction of apoptosis mediated by a decrease in phospho-Bad (Ser112) and c-Myc levels and the inhibition of mTORC1 pathway. Importantly, PIM447 demonstrates a very strong synergy with different standard treatments such as bortezomib + dexamethasone (combination index, CI = 0.002), lenalidomide + dexamethasone (CI = 0.065), and pomalidomide + dexamethasone (CI = 0.077). PIM447 also inhibits in vitro osteoclast formation and resorption, downregulates key molecules involved in these processes, and partially disrupts the F-actin ring, while increasing osteoblast activity and mineralization. Finally, PIM447 significantly reduced the tumor burden and prevented tumor-associated bone loss in a disseminated murine model of human myeloma. CONCLUSIONS: Our results demonstrate dual antitumoral and bone-protective effects of PIM447. This fact, together with the very strong synergy exhibited with standard-of-care treatments, supports the future clinical development of this drug in multiple myeloma. Clin Cancer Res; 23(1); 225-38. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , Bone and Bones/drug effects , Bone and Bones/pathology , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Protective Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Bone Resorption/drug therapy , Bone Resorption/genetics , Bone Resorption/metabolism , Bone Resorption/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Female , Gene Expression , Humans , Mice , Multiple Myeloma/drug therapy , Osteoclasts/drug effects , Osteoclasts/metabolism , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-pim-1/genetics , Proto-Oncogene Proteins c-pim-1/metabolism , Standard of Care , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life.
Subject(s)
Chromatin Assembly and Disassembly , DNA Helicases/metabolism , Homeostasis , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Muscle, Striated/metabolism , Aging/pathology , Animals , Cardiomyopathies/metabolism , Cardiomyopathies/pathology , Cell Differentiation/genetics , CpG Islands/genetics , Gene Expression Regulation, Developmental , Heart/embryology , Mice, Transgenic , Mitochondria, Heart/metabolism , Muscle, Striated/embryology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Promoter Regions, Genetic/genetics , Protein BindingABSTRACT
BACKGROUND: Monocyte-to-osteoclast conversion is a unique terminal differentiation process that is exacerbated in rheumatoid arthritis and bone metastasis. The mechanisms implicated in upregulating osteoclast-specific genes involve transcription factors, epigenetic regulators and microRNAs (miRNAs). It is less well known how downregulation of osteoclast-inappropriate genes is achieved. RESULTS: In this study, analysis of miRNA expression changes in osteoclast differentiation from human primary monocytes revealed the rapid upregulation of two miRNA clusters, miR-212/132 and miR-99b/let-7e/125a. We demonstrate that they negatively target monocyte-specific and immunomodulatory genes like TNFAIP3, IGF1R and IL15. Depletion of these miRNAs inhibits osteoclast differentiation and upregulates their targets. These miRNAs are also upregulated in other inflammatory monocytic differentiation processes. Most importantly, we demonstrate for the first time the direct involvement of Nuclear Factor kappa B (NF-κB) in the regulation of these miRNAs, as well as with their targets, whereby NF-κB p65 binds the promoters of these two miRNA clusters and NF-κB inhibition or depletion results in impaired upregulation of their expression. CONCLUSIONS: Our results reveal the direct involvement of NF-κB in shutting down certain monocyte-specific genes, including some anti-inflammatory activities, through a miRNA-dependent mechanism for proper osteoclast differentiation.