CSL362 potently and specifically depletes pDCs invitro and ablates SLE-immune complex-induced IFN responses.

Systemic lupus erythematosus (SLE) is an autoimmune disease with significant morbidity and mortality. Type I interferon (IFN) drives SLE pathology and plasmacytoid dendritic cells (pDCs) are potent producers of IFN; however, the specific effects of pDC depletion have not been demonstrated. We show CD123 was highly expressed on pDCs and the anti-CD123 antibody CSL362 potently depleted pDCs in vitro. CSL362 pre-treatment abrogated the induction of IFNα and IFN-induced gene transcription following stimulation with SLE patient-derived serum or immune complexes. RNA transcripts induced in pDCs by ex vivo stimulation with TLR ligands were reflected in gene expression profiles of SLE blood, and correlated with disease severity. TLR ligand-induced protein production by SLE patient peripheral mononuclear cells was abrogated by CSL362 pre-treatment including proteins over expressed in SLE patient serum. These findings implicate pDCs as key drivers in the cellular activation and production of soluble factors seen in SLE.

TEAD Inhibitors Sensitize KRASG12C Inhibitors via Dual Cell Cycle Arrest in KRASG12C-Mutant NSCLC.

KRASG12C is one of the most common mutations detected in non-small cell lung cancer (NSCLC) patients, and it is a marker of poor prognosis. The first FDA-approved KRASG12C inhibitors, sotorasib and adagrasib, have been an enormous breakthrough for patients with KRASG12C mutant NSCLC; however, resistance to therapy is emerging. The transcriptional coactivators YAP1/TAZ and the family of transcription factors TEAD1-4 are the downstream effectors of the Hippo pathway and regulate essential cellular processes such as cell proliferation and cell survival. YAP1/TAZ-TEAD activity has further been implicated as a mechanism of resistance to targeted therapies. Here, we investigate the effect of combining TEAD inhibitors with KRASG12C inhibitors in KRASG12C mutant NSCLC tumor models. We show that TEAD inhibitors, while being inactive as single agents in KRASG12C-driven NSCLC cells, enhance KRASG12C inhibitor-mediated anti-tumor efficacy in vitro and in vivo. Mechanistically, the dual inhibition of KRASG12C and TEAD results in the downregulation of MYC and E2F signatures and in the alteration of the G2/M checkpoint, converging in an increase in G1 and a decrease in G2/M cell cycle phases. Our data suggest that the co-inhibition of KRASG12C and TEAD leads to a specific dual cell cycle arrest in KRASG12C NSCLC cells.

Cytotoxic T lymphocyte antigen-4 regulates development of xenogenic graft versus host disease in mice via modulation of host immune responses induced by changes in human T cell engraftment and gene expression.

Graft versus host disease (GvHD) is a major clinical problem with a significant unmet medical need. We examined the role of cytotoxic T lymphocyte antigen-4 (CTLA-4) in a xenogenic GvHD (xeno-GvHD) model induced by injection of human peripheral mononuclear cells (hPBMC) into irradiated non-obese diabetic (NOD) SCID gamma (NSG) mice. Targeting the CTLA-4 pathway by treatment with CTLA-4 immunoglobulin (Ig) prevented xeno-GvHD, while anti-CTLA-4 antibody treatment exacerbated the lethality and morbidity associated with GvHD. Xeno-GvHD is associated with infiltration of hPBMCs into the lungs, spleen, stomach, liver and colon and an increase in human proinflammatory cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-5. Infiltration of donor cells and increases in cytokines were attenuated by treatment with CTLA-4 Ig, but remained either unaffected or enhanced by anti-CTLA-4 antibody. Further, splenic human T cell phenotyping showed that CTLA-4 Ig treatment prevented the engraftment of human CD45+ cells, while anti-CTLA-4 antibody enhanced donor T cell expansion, particularly CD4+ (CD45RO+ ) subsets, including T box transcription factor TBX21 (Tbet)+ CXCR3+ and CD25+ forkhead box protein 3 (FoxP3) cells. Comprehensive analysis of transcriptional profiling of human cells isolated from mouse spleen identified a set of 417 differentially expressed genes (DEGs) by CTLA-4 Ig treatment and 13 DEGs by anti-CTLA-4 antibody treatment. The CTLA-4 Ig regulated DEGs mapped to down-regulated apoptosis, inflammasome, T helper type 17 (Th17) and regulatory T cell (Treg ) pathways and enhanced Toll-like receptor (TLR) receptor signaling, TNF family signaling, complement system and epigenetic and transcriptional regulation, whereas anti-CTLA-4 antibody produced minimal to no impact on these gene pathways. Our results show an important role of co-inhibitory CTLA-4 signaling in xeno-GvHD and suggest the therapeutic utility of other immune checkpoint co-inhibitory pathways in the treatment of immune-mediated diseases driven by hyperactive T cells.

Novel signatures associated with systemic lupus erythematosus clinical response to IFN-α/-ω inhibition.

OBJECTIVES: We aimed to identify transcriptional gene signatures predictive of clinical response, for pharmacodynamic evaluation, and to provide mechanistic insight into JNJ-55920839, a human IgG1κ neutralizing mAb targeting IFN-α/IFN-ω, in participants with systemic lupus erythematosus (SLE). METHODS: Blood samples were obtained from SLE participants at baseline and up to Day 130, who received six 10 mg/kg IV doses of JNJ-55920839/placebo every 2 weeks. Participants with mild-to-moderate SLE who achieved clinical responses using SLE Disease Activity Index 2000 Responder Index 4-point change were considered responders. Transcriptional signatures from longitudinally collected blood were generated by RNA-Seq; signatures were generated by microarray from baseline blood samples exposed in vitro to JNJ-55920839 versus untreated. RESULTS: Two gene signatures (IFN-I Signaling and Immunoglobulin Immune Response) exhibited pharmacodynamic changes among JNJ-55920839 responders. The Immunoglobulin signature, but not the IFN-I signature, was elevated at baseline in JNJ-55920839 responders. A gene cluster associated with neutrophil-mediated immunity was reduced at baseline in JNJ-55920839 responders, substantiated by lower neutrophil counts in responders. An IFN-I signature was suppressed by JNJ-55920839 in vitro treatment versus untreated blood to a greater extent in responders before in vivo dosing. CONCLUSIONS: These signatures may enable enrichment for treatment responders when using IFN-I-suppressing treatments in SLE.

Suppression of Serum Interferon-γ Levels as a Potential Measure of Response to Ustekinumab Treatment in Patients With Systemic Lupus Erythematosus.

OBJECTIVE: In a previously reported phase II randomized, placebo-controlled, interventional trial, we demonstrated that treatment with ustekinumab, an anti-interleukin-12 (IL-12)/IL-23 p40 neutralizing monoclonal antibody, improved global and organ-specific measures of disease activity in patients with active systemic lupus erythematosus (SLE). Utilizing the biomarker data from this phase II clinical study, we sought to determine whether modulation of the expression of IL-12, IL-23, or both cytokines by ustekinumab is associated with clinical efficacy in patients with SLE. METHODS: This phase II randomized, placebo-controlled study enrolled 102 patients with autoantibody-positive SLE whose disease remained active despite standard-of-care therapy. Patients were randomized at a 3:2 ratio to receive ~6 mg/kg ustekinumab intravenously or placebo at week 0, followed by subcutaneous injections of 90 mg ustekinumab or placebo every 8 weeks, with placebo crossover to 90 mg ustekinumab every 8 weeks. The SLE Responder Index 4 (SRI-4) at week 24 was used to determine which patients could be classified as ustekinumab responders and which could be classified as nonresponders. In addition to measurements of p40 and IL-23, serum levels of interferon-γ (IFNγ), IL-17A, IL-17F, and IL-22, as a proxy for the IL-12 and IL-23 pathways, were quantified by immunoassay. RESULTS: Changes in the serum levels of IL-17A, IL-17F, and IL-22 at different time points after treatment were not consistently significantly associated with an SRI-4 clinical response to ustekinumab in patients with SLE. In contrast, an SRI-4 response to ustekinumab was significantly associated (P < 0.01) with durable reductions in the serum IFNγ protein levels at several time points relative to baseline, which was not observed in ustekinumab nonresponders or patients who received placebo. CONCLUSION: While not diminishing a potential role of IL-23, these serum biomarker assessments indicate that IL-12 blockade has an important role in the mechanism of action of ustekinumab treatment in patients with SLE.

First-in-Human study of JNJ-55920839 in healthy volunteers and patients with systemic lupus erythematosus: a randomised placebo-controlled phase 1 trial.

BACKGROUND: Activation of the type I interferon (IFN) pathway is associated with systemic lupus erythematosus (SLE). We assessed the safety and tolerability of JNJ-55920839, a human monoclonal antibody that selectively neutralises most human IFNα subtypes and IFNω, in healthy participants and those with SLE. METHODS: This was a two-part, first-in-human, phase 1, randomised, double-blind, placebo-controlled, multicentre study of single-ascending intravenous doses of 0·3-15 mg/kg or a single subcutaneous dose of 1 mg/kg JNJ-55920839 administered to healthy participants (part A) and multiple intravenous doses of 10 mg/kg JNJ-55920839 administered to participants with SLE (part B). Healthy men and women (women had to be postmenopausal or surgically sterile) aged 18-55 years; bodyweight of 50-90 kg; and body-mass index (BMI) of 18-30 kg/m2 were eligible for inclusion in part A. Men and women with SLE were recruited to part B, fertile female participants were required to have a negative pregnancy test result before and during the study and be using two highly effective methods of birth control. The inclusion criteria for participants with SLE in part B matched part A, except for bodyweight (40-100 kg). In both parts, participants were randomly assigned (3:1) to receive JNJ-55920839 or placebo; a computer-generated randomisation schedule was used in part A, and randomisation was stratified by racial and ethnic subpopulation and elevated levels of serological disease activity in part B. The primary outcome was evaluation of safety and tolerability of the study regimen assessed using clinical and laboratory tests compared with placebo. This study is registered with ClinicalTrials.gov, NCT02609789. FINDINGS: Between Dec 11, 2015, and Sept 20, 2018, 48 healthy participants from a single site and 28 participants with mild-to-moderate SLE from 19 participating centres in seven countries were enrolled in the study. 12 healthy volunteers in part A and eight participants with SLE in part B received placebo. The most common treatment-emergent adverse events in both part A and B were in the system organ class of infections and infestations with a higher percentage of participants administered JNJ-55920839 with infections (ten [28%] of 36 in part A and nine [50%] of 18 in part B) than those exposed to placebo (two [17%] of 12 in part A and one [13%] of eight in part B). Particpants in part B were permitted to continue on defined ongoing standard of care medications. In two participants with SLE, locally disseminated herpes zoster of the skin was reported. No other clinically significant safety or tolerability issues were identified beyond the infections observed in participants treated with JNJ-55920839. INTERPRETATION: JNJ-55920839 was well tolerated and safe. Additional studies are warranted to determine optimal dosing of patients and further explore safety. FUNDING: Janssen.

Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and associated vulnerabilities.

Kras-driven non-small-cell lung cancers (NSCLCs) are a leading cause of death with limited therapeutic options. Many NSCLCs exhibit high levels of Ezh2, the enzymatic subunit of polycomb repressive complex 2 (PRC2). We tested Ezh2 inhibitors as single agents or before chemotherapy in mice with orthotopic Kras-driven NSCLC grafts, which homogeneously express Ezh2. These tumors display sensitivity to EZH2 inhibition by GSK126 but also amplify an inflammatory program involving signaling through NF-κB and genes residing in PRC2-regulated chromatin. During this process, tumor cells overcome GSK126 antiproliferative effects. We identified oncogenes that may mediate progression through an in vivo RNAi screen aimed at targets of PRC2/NF-κB. An in vitro compound screening linked GSK126-driven inflammation and therapeutic vulnerability in human cells to regulation of RNA synthesis and proteostasis. Interestingly, GSK126-treated NSCLCs in vivo also showed an enhanced response to a combination of nimesulide and bortezomib. Thus, Ezh2 inhibition may restrict cell proliferation and promote defined adaptive responses. Targeting these responses potentially improves outcomes in Kras-driven NSCLCs.

Digital Restriction Enzyme Analysis of Methylation (DREAM).

Digital Restriction Enzyme Analysis of Methylation (DREAM) is a method for quantitative mapping of DNA methylation across genomes using next-generation sequencing (NGS) technology. The method is based on sequential cuts of genomic DNA with a pair of restriction enzymes (SmaI and XmaI) at CCCGGG target sites. Unmethylated sites are first digested with SmaI. This enzyme cuts the sites in the middle at CCC^GGG, leaving behind blunt ended fragments. CpG methylation completely blocks SmaI; therefore, only unmethylated sites are cleaved. The remaining methylated sites are digested with XmaI in the next step. This enzyme is not blocked by CpG methylation. It cuts the recognition site sideways at C^CCGGG forming 5'-CCGG overhangs. The sequential cuts thus create distinct methylation-specific signatures at the ends of restriction fragments: 5'-GGG for unmethylated CpG sites and 5'-CCGGG for methylated sites. The DNA fragments resulting from the digestions are ligated to NGS adapters. Sequencing libraries are prepared using hexanucleotide barcodes for sample identification. Individual libraries with distinct barcodes are pooled and sequenced using a paired ends protocol. The sequencing reads are aligned to the genome and mapped to unique CCCGGG target sites. Methylation at individual CpG sites is calculated as the ratio of sequencing reads with the methylated signature to the total number of reads mapping to the site. Sequencing of 25 million reads per sample typically yields 50,000 unique CpG sites covered with hundreds of reads enabling accurate determination of DNA methylation levels. DREAM does not require bisulfite conversion, has a very low background, and has high sensitivity to low levels of methylation. The method is simple, cost-effective, quantitative, highly reproducible, and can be applied to any species.

Estrogen Receptor α Signaling Exacerbates Immune-Mediated Nephropathies through Alteration of Metabolic Activity.

Glomerulonephritis is one of the most serious manifestations of systemic lupus erythematous (SLE). Because SLE is ≥10 times more common in women, a role for estrogens in disease pathogenesis has long been suspected. Estrogen receptor α (ERα) is highly expressed in renal tissue. We asked whether ERα expression contributes to the development of immune-mediated nephropathies like in lupus nephritis. We tested the overall effects of estrogen receptors on the immune response by immunization with OVA and induction of chronic graft-versus-host disease in female ERα-knockout mice. We used nephrotoxic serum nephritis as a model of immune-mediated nephropathy. We investigated the influence of ERα on molecular pathways during nephritis by microarray analysis of glomerular extract gene expression. We performed RNA sequencing of lupus patient whole blood to determine common pathways in murine and human nephritis. Absence of ERα protects female mice from developing nephritis, despite the presence of immune complexes and the production of proinflammatory cytokines in the kidneys and normal humoral responses to immunization. Time-course microarray analysis of glomeruli during nephrotoxic serum nephritis revealed significant upregulation of genes related to PPAR-mediated lipid metabolism and downregulation of genes in the retinol metabolism in wild-type females compared with ERα-knockout females. Similarly, RNA sequencing of lupus patient blood revealed similar expression patterns of these same pathways. During nephritis, the altered activity of metabolic pathways, such as retinol metabolism, occurs downstream of ERα activation and is essential for the progression to end-stage renal failure.

Activated plasmacytoid dendritic cells regulate type 2 innate lymphoid cell-mediated airway hyperreactivity.

BACKGROUND: Allergic asthma is a prevalent inflammatory disease of the airways caused by dysregulated immune balance in the lungs with incompletely understood pathogenesis. The recently identified type 2 innate lymphoid cells (ILC2s) play significant roles in the pathogenesis of asthma. Although ILC2-activating factors have been identified, the mechanisms that suppress ILC2s remain largely unknown. Plasmacytoid dendritic cells (pDCs) are important in antiviral immunity and in maintaining tolerance to inert antigens. OBJECTIVE: We sought to address the role of pDCs in regulating ILC2 function and ILC2-mediated airway hyperreactivity (AHR) and lung inflammation. METHODS: We used several murine models, including BDCA-2-diphtheria toxin receptor (DTR) transgenic and IFN-α receptor 1-deficient mice, as well as purified primary ILC2s, to reach our objective. We extended and validated our findings to human ILC2s. RESULTS: We show that activation of pDCs through Toll-like receptor 7/8 suppresses ILC2-mediated AHR and airway inflammation and that depletion of pDCs reverses this suppression. We further show that pDCs suppress cytokine production and the proliferation rate while increasing the apoptosis rate of ILC2s through IFN-α production. Transcriptomic analysis of both human and murine ILC2s confirms the activation of regulatory pathways in ILC2s by IFN-α. CONCLUSION: Activation of pDCs alleviates AHR and airway inflammation by suppressing ILC2 function and survival. Our findings reveal a novel regulatory pathway in ILC2-mediated pulmonary inflammation with important clinical implications.

Correction: Human mutations in integrator complex subunits link transcriptome integrity to brain development.

[This corrects the article DOI: 10.1371/journal.pgen.1006809.].

Human mutations in integrator complex subunits link transcriptome integrity to brain development.

Integrator is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. Importantly, its role in human development and disease is so far largely unexplored. Here, we provide evidence that biallelic Integrator Complex Subunit 1 (INTS1) and Subunit 8 (INTS8) gene mutations are associated with rare recessive human neurodevelopmental syndromes. Three unrelated individuals of Dutch ancestry showed the same homozygous truncating INTS1 mutation. Three siblings harboured compound heterozygous INTS8 mutations. Shared features by these six individuals are severe neurodevelopmental delay and a distinctive appearance. The INTS8 family in addition presented with neuronal migration defects (periventricular nodular heterotopia). We show that the first INTS8 mutation, a nine base-pair deletion, leads to a protein that disrupts INT complex stability, while the second missense mutation introduces an alternative splice site leading to an unstable messenger. Cells from patients with INTS8 mutations show increased levels of unprocessed UsnRNA, compatible with the INT function in the 3'-end maturation of UsnRNA, and display significant disruptions in gene expression and RNA processing. Finally, the introduction of the INTS8 deletion mutation in P19 cells using genome editing alters gene expression throughout the course of retinoic acid-induced neural differentiation. Altogether, our results confirm the essential role of Integrator to transcriptome integrity and point to the requirement of the Integrator complex in human brain development.

Transcriptional Selectivity of Epigenetic Therapy in Cancer.

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.

Prion-like Aggregation of Mitochondrial Antiviral Signaling Protein in Lupus Patients Is Associated With Increased Levels of Type I Interferon.

OBJECTIVE: Increased levels of type I interferon (IFN) and type I IFN-regulated genes are found in patients with systemic lupus erythematosus (SLE) and may be central to its pathogenesis. Mitochondrial antiviral signaling protein (MAVS) is a key regulator of type I IFN that undergoes a dramatic prion-like aggregation and self propagates the activation signal from viral RNA to amplify downstream IFN production. We undertook this study to determine whether such MAVS aggregates might play a role in the sustained increased production of type I IFN in SLE. METHODS: Peripheral blood mononuclear cells were isolated and mitochondrial extracts were prepared. MAVS aggregation was detected by semidenatured agarose gel electrophoresis and confirmed by immunofluorescence staining. MAVS-associated signaling proteins were analyzed by Western blotting. MAVS aggregation-associated gene expression signature was analyzed by microarray. RESULTS: In blood cells from 22 of 67 SLE patients, essentially all MAVS was in a high molecular weight aggregated form. None of 6 rheumatoid arthritis patients and only 3 of 33 healthy controls had abnormal MAVS. Compared to MAVS aggregate-negative patients, MAVS aggregate-positive SLE patients had significantly higher serum levels of IFNß and significantly increased levels of autoantibodies against Sm and U1 RNP. Gene array data revealed a characteristic gene expression pattern in these patients, with altered expression of genes involved in IFN signaling and membrane trafficking. CONCLUSION: Persistent MAVS aggregates may lead to increased type I IFN production and result in unmitigated signals leading to autoimmunity.

Polycomb Repressive Complex 2 Is a Barrier to KRAS-Driven Inflammation and Epithelial-Mesenchymal Transition in Non-Small-Cell Lung Cancer.

Polycomb repressive complexes (PRC) are frequently implicated in human cancer, acting either as oncogenes or tumor suppressors. Here, we show that PRC2 is a critical regulator of KRAS-driven non-small cell lung cancer progression. Modulation of PRC2 by either Ezh2 overexpression or Eed deletion enhances KRAS-driven adenomagenesis and inflammation, respectively. Eed-loss-driven inflammation leads to massive macrophage recruitment and marked decline in tissue function. Additional Trp53 inactivation activates a cell-autonomous epithelial-to-mesenchymal transition program leading to an invasive mucinous adenocarcinoma. A switch between methylated/acetylated chromatin underlies the tumor phenotypic evolution, prominently involving genes controlled by Hippo/Wnt signaling. Our observations in the mouse models were conserved in human cells. Importantly, PRC2 inactivation results in context-dependent phenotypic alterations, with implications for its therapeutic application.

Identification of MEF2B, EBF1, and IL6R as Direct Gene Targets of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Critical for EBV-Infected B-Lymphocyte Survival.

UNLABELLED: Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome and can provide a host cell survival function, but it is not yet known how EBNA1 sequence-specific binding is responsible for host cell survival. Here, we integrate EBNA1 chromatin immunoprecipitation sequencing (ChIP-Seq) with transcriptome sequencing (RNA-Seq) after EBNA1 depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV-positive cell types, including Burkitt lymphoma (BL) cells, nasopharyngeal carcinoma (NPC) cells, and lymphoblastoid cell lines (LCLs). EBNA1 binds to ~1,000 sites that are mostly invariant among cell types and share a consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, and EBF1. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1 depletion by decreasing the cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells. IMPORTANCE: Epstein-Barr virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is consistently expressed in all EBV-associated cancers. EBNA1 is known to provide a host cell survival function, but the mechanism is not known. EBNA1 is a sequence-specific binding protein important for viral genome maintenance during latency. Here, by integrating ChIP-Seq and RNA-Seq, we demonstrate that EBNA1 binds directly to the promoter regulatory regions and upregulates the transcription of host genes that are important for the survival of EBV-infected cells. Identification of EBNA1 target genes provides potential new targets for therapeutic intervention in EBV-associated disease.

Hypomethylation of TET2 Target Genes Identifies a Curable Subset of Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is curable in a subset of cases. The DNA methylation regulator TET2 is frequently mutated in AML, and we hypothesized that studying TET2-specific differentially methylated CpGs (tet2-DMCs) improves AML classification. METHODS: We used bisulfite pyrosequencing to analyze the methylation status of four tet2-DMCs (SP140, MCCC1, EHMT1, and MTSS1) in a test group of 94 consecutive patients and a validation group of 92 consecutive patients treated with cytarabine-based chemotherapy. Data were analyzed with hierarchical clustering, Cox proportional hazards regression, and Kaplan-Meier analyses. All statistical tests were two-sided. RESULTS: In the test cohort, hierarchical clustering analysis identified low levels of tet2-DMC methylation in 31 of 94 (33%) cases, and these had markedly longer overall survival (median survival 72+ vs 14 months, P = .002). Similar results were seen in the validation cohort. tet2-DMC-low status was shown to be an independent predictor of overall survival (hazard ratio = 0.29, P = .0002). In The Cancer Genome Atlas (TCGA) dataset where DNA methylation was analyzed by a different platform, tet2-DMC-low methylation was also associated with improved outcome (median survival = 55 vs 15 months, P = .0003) and was a better predictor of survival than mutations in TET2, IDH1, or IDH2, individually or combined. CONCLUSIONS: Low levels of tet2-DMC methylation define a subgroup of AML that is highly curable and cannot be identified solely by genetic and cytogenetic analyses.

G9a is essential for epigenetic silencing of K(+) channel genes in acute-to-chronic pain transition.

Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K(+) channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. We found that nerve injury increased dimethylation of Lys9 on histone H3 (H3K9me2) at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect levels of DNA methylation on these genes in DRGs. Nerve injury increased activity of euchromatic histone-lysine N-methyltransferase-2 (G9a), histone deacetylases and enhancer of zeste homolog-2 (EZH2), but only G9a inhibition consistently restored K(+) channel expression. Selective knockout of the gene encoding G9a in DRG neurons completely blocked K(+) channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K(+) channels but also normalized 638 genes down- or upregulated by nerve injury. Thus G9a has a dominant function in transcriptional repression of K(+) channels and in acute-to-chronic pain transition after nerve injury.

Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2.

Posttranslational modifications, such as poly(ADP-ribosyl)ation (PARylation), regulate chromatin-modifying enzymes, ultimately affecting gene expression. This study explores the role of poly(ADP-ribose) polymerase (PARP) on global gene expression in a lymphoblastoid B cell line. We found that inhibition of PARP catalytic activity with olaparib resulted in global gene deregulation, affecting approximately 11% of the genes expressed. Gene ontology analysis revealed that PARP could exert these effects through transcription factors and chromatin-remodeling enzymes, including the polycomb repressive complex 2 (PRC2) member EZH2. EZH2 mediates the trimethylation of histone H3 at lysine 27 (H3K27me3), a modification associated with chromatin compaction and gene silencing. Both pharmacological inhibition of PARP and knockdown of PARP1 induced the expression of EZH2, which resulted in increased global H3K27me3. Chromatin immunoprecipitation confirmed that PARP1 inhibition led to H3K27me3 deposition at EZH2 target genes, which resulted in gene silencing. Moreover, increased EZH2 expression is attributed to the loss of the occupancy of the transcription repressor E2F4 at the EZH2 promoter following PARP inhibition. Together, these data show that PARP plays an important role in global gene regulation and identifies for the first time a direct role of PARP1 in regulating the expression and function of EZH2.

TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites.