Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic "scarring" of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

Dysregulation of the cohesin subunit RAD21 by Hepatitis C virus mediates host-virus interactions.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

Selective immunotargeting of diabetogenic CD4 T cells by genetically redirected T cells.

The key role played by islet-reactive CD8 and CD4 T cells in type 1 diabetes calls for new immunotherapies that target pathogenic T cells in a selective manner. We previously demonstrated that genetically linking the signalling portion of CD3-ζ onto the C-terminus of ß2 -microglobulin and an autoantigenic peptide to its N-terminus converts MHC-I complexes into functional T-cell receptor-specific receptors. CD8 T cells expressing such receptors specifically killed diabetogenic CD8 T cells, blocked T-cell-induced diabetes in immunodeficient NOD.SCID mice and suppressed disease in wild-type NOD mice. Here we describe the immunotargeting of CD4 T cells by chimeric MHC-II receptors. To this end we chose the diabetogenic NOD CD4 T-cell clone BDC2.5, which recognizes the I-A(g7) -bound 1040-31 mimotope. We assembled several constructs encoding I-A(g7) α- and ß-chains, the latter carrying mim or hen egg lysozyme peptide as control, each supplemented with CD3-ζ intracellular portion, either with or without its transmembrane domain. Following mRNA co-transfection of reporter B3Z T cells and mouse CD8 and CD4 T cells, these constructs triggered robust activation upon I-A(g7) cross-linking. A BDC2.5 T-cell hybridoma activated B3Z transfectants expressing the mimotope, but not the control peptide, in both configurations. Potent two-way activation was also evident with transgenic BDC2.5 CD4 T cells, but peptide-specific activation required the CD3-ζ transmembrane domain. Chimeric MHC-II/CD3-ζ complexes therefore allow the selective immunotargeting of islet-reactive CD4 T cells, which take part in the pathogenesis of type 1 diabetes.

High-Resolution Genomic Profiling of Liver Cancer Links Etiology With Mutation and Epigenetic Signatures.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a model of a diverse spectrum of cancers because it is induced by well-known etiologies, mainly hepatitis C virus (HCV) and hepatitis B virus. Here, we aimed to identify HCV-specific mutational signatures and explored the link between the HCV-related regional variation in mutations rates and HCV-induced alterations in genome-wide chromatin organization. METHODS: To identify an HCV-specific mutational signature in HCC, we performed high-resolution targeted sequencing to detect passenger mutations on 64 HCC samples from 3 etiology groups: hepatitis B virus, HCV, or other. To explore the link between the genomic signature and genome-wide chromatin organization we performed chromatin immunoprecipitation sequencing for the transcriptionally permissive H3K4Me3, H3K9Ac, and suppressive H3K9Me3 modifications after HCV infection. RESULTS: Regional variation in mutation rate analysis showed significant etiology-dependent regional mutation rates in 12 genes: LRP2, KRT84, TMEM132B, DOCK2, DMD, INADL, JAK2, DNAH6, MTMR9, ATM, SLX4, and ARSD. We found an enrichment of C->T transversion mutations in the HCV-associated HCC cases. Furthermore, these cases showed regional variation in mutation rates associated with genomic intervals in which HCV infection dictated epigenetic alterations. This signature may be related to the HCV-induced decreased expression of genes encoding key enzymes in the base excision repair pathway. CONCLUSIONS: We identified novel distinct HCV etiology-dependent mutation signatures in HCC associated with HCV-induced alterations in histone modification. This study presents a link between cancer-causing mutagenesis and the increased predisposition to liver cancer in chronic HCV-infected individuals, and unveils novel etiology-specific mechanisms leading to HCC and cancer in general.

Detection of gene mutations and gene-gene fusions in circulating cell-free DNA of glioblastoma patients: an avenue for clinically relevant diagnostic analysis.

Glioblastoma (GBM) is the most common type of glioma and is uniformly fatal. Currently, tumour heterogeneity and mutation acquisition are major impedances for tailoring personalized therapy. We collected blood and tumour tissue samples from 25 GBM patients and 25 blood samples from healthy controls. Cell-free DNA (cfDNA) was extracted from the plasma of GBM patients and from healthy controls. Tumour DNA was extracted from fresh tumour samples. Extracted DNA was sequenced using a whole-genome sequencing procedure. We also collected 180 tumour DNA datasets from GBM patients publicly available at the TCGA/PANCANCER project. These data were analysed for mutations and gene-gene fusions that could be potential druggable targets. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL-1 ), as compared to healthy controls (1.4 ± 0.4 ng·mL-1 ) of the same average age. We identified unique mutations in the cfDNA and tumour DNA of each GBM patient, including some of the most frequently mutated genes in GBM according to the COSMIC database (TP53, 18.75%; EGFR, 37.5%; NF1, 12.5%; LRP1B, 25%; IRS4, 25%). Using our gene-gene fusion database, ChiTaRS 5.0, we identified gene-gene fusions in cfDNA and tumour DNA, such as KDR-PDGFRA and NCDN-PDGFRA, which correspond to previously reported alterations of PDGFRA in GBM (44% of all samples). Interestingly, the PDGFRA protein fusions can be targeted by tyrosine kinase inhibitors such as imatinib, sunitinib, and sorafenib. Moreover, we identified BCR-ABL1 (in 8% of patients), COL1A1-PDGFB (8%), NIN-PDGFRB (8%), and FGFR1-BCR (4%) in cfDNA of patients, which can be targeted by analogues of imatinib. ROS1 fusions (CEP85L-ROS1 and GOPC-ROS1), identified in 8% of patient cfDNA, might be targeted by crizotinib, entrectinib, or larotrectinib. Thus, our study suggests that integrated analysis of cfDNA plasma concentration, gene mutations, and gene-gene fusions can serve as a diagnostic modality for distinguishing GBM patients who may benefit from targeted therapy. These results open new avenues for precision medicine in GBM, using noninvasive liquid biopsy diagnostics to assess personalized patient profiles. Moreover, repeated detection of druggable targets over the course of the disease may provide real-time information on the evolving molecular landscape of the tumour.

Ezh2 harnesses the intranuclear actin cytoskeleton to remodel chromatin in differentiating Th cells.

Following their first interaction with the antigen, quiescent naive T-helper (Th; CD4+) cells enlarge, differentiate, and proliferate; these processes are accompanied by substantial epigenetic alterations. We showed previously that the epigenetic regulators the polycomb-group (PcG) proteins have a dual function as both positive and negative transcriptional regulators; however, the underlying mechanisms remain poorly understood. Here, we demonstrate that during Th cell differentiation the methyltransferase activity of the PcG protein Ezh2 regulates post-transcriptionally inducible assembly of intranuclear actin filaments. These filaments are colocalized with the actin regulators Vav1 and WASp, vertically oriented to the T cell receptor, and intermingle with the chromatin fibers. Ezh2 and Vav1 are observed together at chromatin-actin intersections. Furthermore, the inducible assembly of nuclear actin filaments is required for chromatin spreading and nuclear growth. Altogether these findings delineate a model in which the epigenetic machinery orchestrates the dynamic mechanical force of the intranuclear cytoskeleton to reorganize chromatin during differentiation.

Studying the Hepatitis C Virus-Induced Epigenetic Signature After Cure with Direct-Acting Antivirals.

Hepatitis C virus (HCV) is the leading cause of hepatocellular carcinoma (HCC). While direct-acting antiviral (DAA) therapy efficiently eradicates HCV infection, epidemiological studies show that sustained virological response (SVR) following anti-HCV treatment reduces, but does not eliminate, the risk for HCC. We have recently demonstrated that HCV infection induces genome-wide epigenetic changes that reprogram host gene expression and persist as "epigenetic signature" following virus eradication by DAAs. We suggest that this epigenetic signature underlie the residual risk for HCC post-SVR. Here, we provide a methodology to study the HCV-induced epigenetic signature. We describe a ChIP-seq protocol to evaluate changes in epigenome profile following HCV infection, its cure with DAA, and after treatment with epigenetic modifier inhibitor. We also describe evaluation of changes in the gene expression profile using RNA-seq. The integration between detected alterations in epigenetic marks and gene expression allows for identification of biological processes that are involved in HCV-driven oncogenesis before and after cure.

[The impact of the annual scientific meetings of the Israel Society of Rheumatology as measured by publication rates of the abstracts in peer-reviewed journals].

OBJECTIVES: We aimed to examine the impact and quality of the research presented in the Israel Society of Rheumatology (ISR) annual scientific meetings by measuring publication rates of the abstracts in peer-reviewed journals and investigating the factors that influenced publication. METHODS: We examined the outcome of all 79 abstracts submitted to the ISR for the 1998-2000 annual meetings. A MEDLINE search of all abstracts, by authors, topics and keywords was performed. Senior authors of abstracts not found to be published in this search were interviewed regarding publication and factors influencing submission. We described the effect of variable factors on the rate of publication. RESULTS: As of September 2002, 63 (80%) abstracts were published in peer-reviewed journals or are currently in-press. Most abstracts were published in prominent journals (with a high impact factor). The majority of the abstracts (61%) were published in rheumatologic journals, 65% of the studies originated from tertiary centers and 19% of the studies were multicenter. The most common diseases studied were antiphospholipid syndrome (20%), systemic lupus erythematosus (19%) and inflammatory arthritis (18%). Most of the studies were of disease pathogenesis (35%) and clinical manifestations (33%). The most common study designs were basic science (34%). An overall 57% of the studies reported "positive" results and 9% reported "negative" results. None of the factors studied were associated with publication or non-publication. The main cause cited by authors for not publishing their abstract was lack of time to prepare a full paper or a desire to further expand the study. Within this group of 16 authors of abstracts, 11 authors still plan to submit a paper. CONCLUSION: The ISR annual meetings have an important clinical scientific impact as measured by the high rate of abstracts published as full length articles in leading peer-reviewed journals.

Levetiracetam in children, adolescents and young adults with intractable epilepsy: efficacy, tolerability and effect on electroencephalogram--a pilot study.

Levetiracetam has been authorized for use in Israel as an add-on therapy for intractable epilepsy since May 2006. The aim of the present study was to document its effectiveness for this indication in children, adolescents, and young adults. The medical files of 78 patients aged 0.5-39 years (mean, 14.2 years) treated at our center for intractable epilepsy were reviewed. All received levetiracetam as add-on therapy following a failure to respond to at least 3 anti-epileptic drugs. Fifty-two patients (67%) had partial epilepsy and the remainder had primary generalized epilepsy. The epilepsy was symptomatic in 57%, cryptogenic in 27%, and idiopathic in 15%. Average age at first seizure was 4.1 years. In 45% of patients, the number of seizures was reduced by half with levetiracetam treatment; 11.5% of the cohort achieved complete remission. There was a statistically significant correlation between clinical seizure control and improvement in the electroencephalography findings (p = 0.0012). The drug was well tolerated, with a retention rate of 69% after one year. The most common adverse effects were irritability and impulsiveness, in 26.9% of patients. Severe behavioral side effects (psychosis, confusion) were experienced by 6.4%. In conclusion, levetiracetam is an effective and tolerable add-on agent for use in most epileptic children, adolescents, and young adults who fail to respond to at least 3 antiepileptic drugs and should be the treatment of choice in this setting. Despite the relatively high rate of behavioral side effects in this study, the retention rate at one year was high.