OBJECTIVE: We report a combination of BK virus-specific T cells and pembrolizumab as a treatment option in progressive multifocal leukoencephalopathy (PML). RESULTS: A 57-year-old male patient diagnosed with PML presented a fast-progressing right hemiparesis, aphasia, and cognitive deficits. Brain MRI showed a severe leukoencephalopathy with diffusion restriction. The patient was treated with 10 doses of pembrolizumab (2 mg/kg body weight) in differing intervals and 2 partially human leukocyte antigen-matched allogenic BK virus-specific T cell transfusions after the fifth pembrolizumab treatment. Although pembrolizumab alone decreased the viral load but failed to control the virus, BK-specific T cell transfer further enhanced the decline of JC virus copies in the CSF. Moreover, the regression of leukoencephalopathy and disappearance of diffusion restriction in subsequent brain MRI were observed. The combined treatment resulted in a clinical stabilization with improvements of the cognitive and speech deficits. DISCUSSION: This case supports the hypothesis that pembrolizumab is more efficient in the presence of an appropriate number of functional antigen-specific T cells. Thus, the combined treatment of pembrolizumab and virus-specific T cells should be further evaluated as a treatment option for PML in future clinical trials.
Antibodies, Monoclonal, Humanized/therapeutic use , BK Virus/physiology , Leukoencephalopathy, Progressive Multifocal/therapy , T-Lymphocytes/physiology , Humans , Leukoencephalopathy, Progressive Multifocal/cerebrospinal fluid , Male , Middle Aged , Treatment Outcome , Viral Load
BACKGROUND & AIMS: Methyl-CpG binding protein 2, MECP2, which binds to methylated regions of DNA to regulate transcription, is expressed by hepatic stellate cells (HSCs) and is required for development of liver fibrosis in mice. We investigated the effects of MECP2 deletion from HSCs on their transcriptome and of phosphorylation of MECP2 on HSC phenotype and liver fibrosis. METHODS: We isolated HSCs from Mecp2-/y mice and wild-type (control) mice. HSCs were activated in culture and used in array analyses of messenger RNAs and long noncoding RNAs. Kyoto Encyclopedia of Genes and Genomes pathway analyses identified pathways regulated by MECP2. We studied mice that expressed a mutated form of Mecp2 that encodes the S80A substitution, MECP2S80, causing loss of MECP2 phosphorylation at serine 80. Liver fibrosis was induced in these mice by administration of carbon tetrachloride, and liver tissues and HSCs were collected and analyzed. RESULTS: MECP2 deletion altered expression of 284 messenger RNAs and 244 long noncoding RNAs, including those that regulate DNA replication; are members of the minichromosome maintenance protein complex family; or encode CDC7, HAS2, DNA2 (a DNA helicase), or RPA2 (a protein that binds single-stranded DNA). We found that MECP2 regulates the DNA repair Fanconi anemia pathway in HSCs. Phosphorylation of MECP2S80 and its putative kinase, HAS2, were induced during transdifferentiation of HSCs. HSCs from MECP2S80 mice had reduced proliferation, and livers from these mice had reduced fibrosis after carbon tetrachloride administration. CONCLUSIONS: In studies of mice with disruption of Mecp2 or that expressed a form of MECP2 that is not phosphorylated at S80, we found phosphorylation of MECP2 to be required for HSC proliferation and induction of fibrosis. In HSCs, MECP2 regulates expression of genes required for DNA replication and repair. Strategies to inhibit MECP2 phosphorylation at S80 might be developed for treatment of liver fibrosis.
Chemical and Drug Induced Liver Injury/metabolism , Hepatic Stellate Cells/metabolism , Liver Cirrhosis, Experimental/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Acetaminophen , Animals , Carbon Tetrachloride , Cell Proliferation , Cells, Cultured , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/pathology , Collagen/metabolism , DNA Repair , DNA Replication , Hepatic Stellate Cells/pathology , Liver Cirrhosis, Experimental/genetics , Liver Cirrhosis, Experimental/pathology , Male , Methyl-CpG-Binding Protein 2/deficiency , Methyl-CpG-Binding Protein 2/genetics , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Serine , Signal Transduction
The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis.
Adenosine/analogs & derivatives , Epigenesis, Genetic/drug effects , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Adenosine/administration & dosage , Adenosine/pharmacology , Animals , Biomarkers , Carbon Tetrachloride/adverse effects , Collagen Type I/genetics , Collagen Type I/metabolism , Disease Models, Animal , Disease Progression , Gene Expression Profiling , Gene Expression Regulation/drug effects , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histones/metabolism , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Male , Mice , Myofibroblasts/cytology , Myofibroblasts/drug effects , Myofibroblasts/metabolism
BACKGROUND & AIMS: DNA methylation (5-mC) is an epigenetic mark that is an established regulator of transcriptional repression with an important role in liver fibrosis. Currently, there is very little knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cell (HSC), the key cell type responsible for onset and progression of liver fibrosis. Moreover, recently discovered DNA hydroxymethylation (5-hmC) is involved in transcriptional activation and its patterns are often altered in human diseases. The aim of this study is to investigate the role of DNA methylation/hydroxymethylation in liver fibrosis. METHODS: Levels of 5-mC and 5-hmC were assessed by slot blot in a range of animal liver fibrosis models and human liver diseases. Expression levels of TET and DNMT enzymes were measured by qRT-PCR and Western blotting. Reduced representation bisulfite sequencing (RRBS) method was used to examine 5-mC and 5-hmC patterns in quiescent and in vivo activated rat HSC. RESULTS: We demonstrate global alteration in 5-mC and 5-hmC and their regulatory enzymes that accompany liver fibrosis and HSC transdifferentiation. Using RRBS, we show exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. In addition, we demonstrate that reduction in DNMT3a expression leads to attenuation of pro-fibrogenic phenotype in activated HSC. CONCLUSIONS: Our data suggest that DNA 5-mC/5-hmC is a crucial step in HSC activation and therefore fibrogenesis. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.
Cell Transdifferentiation , DNA Methylation , Hepatic Stellate Cells/cytology , Liver Cirrhosis/etiology , Animals , DNA (Cytosine-5-)-Methyltransferases/physiology , DNA Methyltransferase 3A , Hepatic Stellate Cells/physiology , Humans , Rats , Rats, Sprague-Dawley , DNA Methyltransferase 3B
Fibrosis is a common and important pathology associated with progressive chronic liver diseases and underlies the development of cirrhosis and hepatocellular carcinoma. Research into the molecular regulation of fibrosis has discovered that it is under the control of a number of epigenetic mechanisms including DNA methylation, histone modifications and the activities of non-coding RNAs. A deeper understanding of how epigenetic regulators such as DNA methyltranserases, methyl-DNA binding proteins, histone modifying enzymes and regulatory RNA molecules impact on the fibrogenic process is expected to result in new biomarkers for disease progression as well as novel therapeutic targets. The aim of this mini-review is to briefly introduce the reader to the major epigenetic regulators so far identified as being implicated in fibrosis.
Epigenesis, Genetic/physiology , Liver Cirrhosis/therapy , DNA Methylation/physiology , Disease Progression , Histones/metabolism , Humans , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , RNA, Untranslated/physiology
BACKGROUND & AIMS: Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. METHODS: HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patient's liver and through precision cut liver slices. RESULTS: HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. CONCLUSIONS: Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.
DNA/genetics , Epigenesis, Genetic , Ethanol/adverse effects , Extracellular Matrix Proteins/genetics , Gene Expression Regulation/drug effects , Hepatic Stellate Cells/drug effects , Liver Cirrhosis, Alcoholic/genetics , Animals , Cell Transdifferentiation , Cells, Cultured , Disease Models, Animal , Disease Progression , Extracellular Matrix Proteins/biosynthesis , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Immunoblotting , Immunohistochemistry , Liver Cirrhosis, Alcoholic/metabolism , Liver Cirrhosis, Alcoholic/pathology , Male , Mice , Polymerase Chain Reaction , Rats , Rats, Sprague-Dawley
Epigenetics is a dynamically expanding field of science entailing numerous regulatory mechanisms controlling changes of gene expression in response to environmental factors. Over the recent years there has been a great interest in epigenetic marks as a potential diagnostic and prognostic tool or future target for treatment of various human diseases. There is an increasing body of published research to suggest that epigenetic events regulate progression of chronic liver disease. Experimental manipulation of epigenetic signatures such as DNA methylation, histone acetylation / methylation and the activities of proteins that either annotate or interpret these epigenetic marks can have profound effects on the activation and phenotype of HSC, key cells responsible for onset and progression of liver fibrosis. This review presents recent advances in epigenetic alterations, which could provide mechanistic insight into the pathogenesis of chronic liver disease and provide novel clinical applications.
