Epigenetic analysis of the IFNλ3 gene identifies a novel marker for response to therapy in HCV-infected subjects.

Chronic hepatitis C virus (HCV) infection is characterized by high interindividual variability in response to pegylated interferon and ribavirin. A genetic polymorphism on chromosome 19 (rs12979860) upstream of interferon-λ3 (IFNλ3) is associated with a twofold change in sustained virologic response rate after 48 weeks of treatment with pegylated interferon/ribavirin in HCV genotype 1 (GT1) treatment-naïve patients. We conducted epigenetic analysis on the IFNλ3 promoter to investigate whether DNA methylation is associated with response to HCV therapy. DNA samples from HCV GT1-infected subjects receiving an interferon-free paritaprevir-containing combination regimen (N=540) and from HCV-uninfected, healthy controls (N=124) were analysed for IFNλ3 methylation levels. Methylation was strongly associated with rs12979860 allele status whether adjusting for HCV status (r=65.0%, 95% CI: [60.2%, 69.5%]), or not (r=64.4%), both with P<2.2×10-16 . In HCV GT1-infected subjects, C/C genotypes had significantly lower methylation levels relative to C/T or T/T genotypes (P<1×10-14 ), with each T allele resulting in a nine-unit increase in mean methylation level. Methylation levels did not correlate with response in subjects treated for 12 or 24 weeks. However, non-C/C subjects with higher methylation levels were more likely to relapse when treatment duration was 8 weeks. This analysis suggests that methylation status of the IFNλ3 promoter region may be a useful parameter that identifies patients more likely to relapse following HCV therapy; however, continuing therapy for a sufficient duration can overcome this difference. These findings may provide mechanistic insight into the role of IFNλ3 genetic variants in HCV treatment response.

Serum miR-122 may serve as a biomarker for response to direct acting antivirals: effect of paritaprevir/R with dasabuvir or ombitasvir on miR-122 in HCV-infected subjects.

Circulating microRNAs (miRNA) have been intensely investigated as biomarkers in disease and therapy. Several studies have identified miR-122 as an important regulator of HCV replication. The effect of new therapies that directly target the HCV replication life cycle on circulating microRNA levels has not been elucidated. We performed expression profiling of circulating miRNA in serum in subjects treated with HCV direct-acting antiviral agents (DAAs). Serum miRNA levels were evaluated from two studies in HCV GT1-infected treatment-naïve subjects and prior nonresponders to pegylated interferon (pegIFN) and ribavirin (RBV) who received paritaprevir/ritonavir + dasabuvir + RBV for 12 weeks, and in treatment-naïve genotype (GT)1-3-infected subjects who received paritaprevir/ritonavir + ombitasvir ± RBV for 12 weeks. Over 100 different miRNA species were detected in serum. Of these, levels of miR-122 showed the most consistent change in response to treatment across all HCV genotypes. In all subjects, miR-122 showed an average four-fold reduction between baseline and week 2, and remained below baseline through post-treatment week 12 in subjects who achieved sustained virological response. In contrast, in subjects who did not achieve SVR, miR-122 levels began to return to baseline levels after the second week of treatment. The change in miR-122 levels was similar across genotypes, and was comparable with or without RBV. This is the first report comparing expression levels of circulating miRNA in HCV GT1-3 subjects treated with IFN-free combinations of DAAs. The results suggest that serum levels of miR-122 are reduced following treatment in subjects who achieve SVR, and correlate with HCV RNA levels across genotypes.

Clustering of hepatotoxins based on mechanism of toxicity using gene expression profiles.

Microarray technology, which allows one to quantitate the expression of thousands of genes simultaneously, has begun to have a major impact on many different areas of drug discovery and development. The question remains of whether microarray analysis and gene expression signature profiles can be applied to the field of toxicology. To date, there are very few published studies showing the use of microarrays in toxicology and important questions remain regarding the predictability and accuracy of applying gene expression profiles to toxicology. To begin to address these questions, we have treated rats with 15 different known hepatotoxins, including allyl alcohol, amiodarone, Aroclor 1254, arsenic, carbamazepine, carbon tetrachloride, diethylnitrosamine, dimethylformamide, diquat, etoposide, indomethacin, methapyrilene, methotrexate, monocrotaline, and 3-methylcholanthrene. These agents cause a variety of hepatocellular injuries including necrosis, DNA damage, cirrhosis, hypertrophy, and hepatic carcinoma. Gene expression analysis was done on RNA from the livers of treated rats and was compared against vehicle-treated controls. The gene expression results were clustered and compared to the histopathology findings and clinical chemistry values. Our results show strong correlation between the histopathology, clinical chemistry, and gene expression profiles induced by the agents. In addition, genes were identified whose regulation correlated strongly with effects on clinical chemistry parameters. Overall, the results suggest that microarray assays may prove to be a highly sensitive technique for safety screening of drug candidates and for the classification of environmental toxins.

Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity.

A rate-limiting step that occurs in the drug discovery process is toxicological evaluation of new compounds. New techniques that use small amounts of the experimental compound and provide a high degree of predictivity would greatly improve this process. The field of microarray technology, which allows one to monitor thousands of gene expression changes simultaneously, is rapidly advancing and is already being applied to numerous areas in toxicology. However, it remains to be determined if compounds with similar toxic mechanisms produce similar changes in transcriptional expression. In addition, it must be determined if gene expression changes caused by an agent in vitro would reflect those produced in vivo. In order to address these questions, we treated rat hepatocytes with 15 known hepatoxins (carbon tetrachloride, allyl alcohol, aroclor 1254, methotrexate, diquat, carbamazepine, methapyrilene, arsenic, diethylnitrosamine, monocrotaline, dimethyl-formamide, amiodarone, indomethacin, etoposide, and 3-methylcholanthrene) and used microarray technology to characterize the compounds based on gene expression changes. Our results showed that gene expressional profiles for compounds with similar toxic mechanisms indeed formed clusters, suggesting a similar effect on transcription. There was not complete identity, however, indicating that each compound produced a unique signature. These results show that large-scale analysis of gene expression using microarray technology has promise as a diagnostic tool for toxicology.

A 3'-transcribed region of the HLA-A2 gene mediates posttranscriptional stimulation by IFN-gamma.

The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-gamma. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-gamma stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5' IFN-stimulated response element consensus sequence, is not sufficient for IFN-gamma response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5'- and 3'-flanking sequences, resulted in stimulation of the gene by IFN-gamma. Nuclear run-on assays revealed that, unlike other class I genes, IFN-gamma stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3' end was unaffected by IFN-gamma treatment. Sequences that mediate the majority of IFN-gamma induction of HLA-A2 mRNA reside in a 127-bp 3'-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-gamma treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-gamma.

Identification of CCAAT displacement protein (CDP/cut) as a locus-specific repressor of major histocompatibility complex gene expression in human tumor cells.

Human major histocompatibility (MHC) class I antigen expression is important in controlling the metastatic growth of malignant tumors. Locus-specific down-regulation of MHC class I gene expression is frequently observed in human tumors, leading to decreased susceptibility to cytotoxic T-cell-mediated lysis. The mechanism of this down-regulation is incompletely understood. Here, we describe the identification of human CCAAT displacement protein (CDP/cut) as a locus-specific repressor of HLA-B and C gene expression. Transient and stable transfections in HeLa and K562 cells demonstrated the presence of a repressor element 650 base pairs upstream of the first exon of HLA-B7. A specific binding complex with the HLA-B7 and Cw2 repressor elements was demonstrated by EMSA. Formation of the EMSA complex was inhibited specifically with polyclonal antiserum to human CDP/cut, demonstrating that CDP/cut binds the HLA-B7 repressor element. The corresponding region of the HLA-A2 promoter neither repressed HLA-A2 gene expression nor bound CDP/cut. Overexpression of CDP/cut in cell lines deficient in CDP/cut resulted in a nearly 4-fold repression of reporter constructs containing the HLA-B7 repressor element but not the corresponding region of the HLA-A2 promoter. Repression of HLA-B and C gene expression by CDP/cut does not involve displacement of NF-Y, nor is CDP/cut associated with the histone deacetylase HDAC1 when bound to the HLA-B7 repressor element. To our knowledge, these results identify CDP/cut as the first example of a locus-specific repressor of MHC class I gene transcription in human tumor cells.

The impact of genomics-based technologies on drug safety evaluation.

Determining the potential toxicity of compounds early in the drug discovery process can be extremely beneficial in terms of both time and money conservation. Because of the speed of modern chemical synthesis and screening, to accurately evaluate the large number of compounds being produced, toxicology assays must have both high-fidelity and high-throughput capabilities. In addition, assays must be performed using limited amounts of compound. In the past decade, several new and innovative techniques have been developed that not only allow for high-throughput screening but can also provide detailed information concerning the molecular mechanisms behind toxic effects. Techniques such as hybridization microarrays, real-time polymerase chain reaction, and large-scale sequencing are some of the methods that have been or are starting to be used routinely in pharmaceutical companies. This review examines the contributions of these and related techniques toward toxicity evaluation of potential drug candidates and their future role in the discovery of new therapeutics.

Interferon consensus sequence binding protein and interferon regulatory factor-4/Pip form a complex that represses the expression of the interferon-stimulated gene-15 in macrophages.

Interferon consensus sequence binding protein (ICSBP), a transcription factor of the interferon (IFN) regulatory factor (IRF) family, binds to the IFN-stimulated response element (ISRE) in the regulatory region of IFNs and IFN-stimulated genes (ISG). To identify target genes, which are deregulated by an ICSBP null-mutation in mice (ICSBP-/-), we have analyzed transcription of an ISRE-bearing gene, ISG15. We have found that although ISG15 expression is unchanged in B cells, it is upregulated in macrophages from ICSBP-/- mice. Three factors, ICSBP, IRF-2, and IRF-4/Pip interact with the ISRE in B cells, however only ICSBP and IRF-4/Pip were found to bind this sequence in macrophages of wild-type mice. Although IRF-4 was considered to be a lymphoid-specific factor, we provide evidence for its role in macrophage gene regulation. Our results suggest that the formation of cell-type-specific heteromeric complexes between individual IRFs plays a crucial role in regulating IFN responses.

Altered development and cytokine responses of myeloid progenitors in the absence of transcription factor, interferon consensus sequence binding protein.

Mice deficient for the transcription factor, interferon consensus sequence binding protein (ICSBP), are immunodeficient and develop disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of ICSBP(-/-) mice, we investigated the growth, differentiation, and leukemogenic potential of ICSBP(-/-) myeloid progenitor cells in vitro, as well as by cell-transfers in vivo. We report that adult bone marrow, as well as fetal liver of ICSBP-deficient mice harbor increased numbers of progenitor cells, which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP(-/-) colonies formed in the presence of M-CSF are mostly of granulocytic morphology. This disproportional differentiation toward cells of the granulocytic lineage in vitro parallels the expansion of granulocytes in ICSBP(-/-) mice and correlates with a 4-fold reduction of M-CSF receptor expressing cells in bone marrow. Cell transfer studies showed an intrinsic leukemogenic potential and long-term reconstitution capability of ICSBP(-/-) progenitors. Further experiments demonstrated strongly reduced adhesion of colony-forming cells from ICSBP(-/-) bone marrow to fibronectin. In summary, ICSBP(-/-) myeloid progenitor cells share several abnormal features with CML progenitors, suggesting that the distal parts of signaling pathways of these two disorders are overlapping.

Lack of interferon consensus sequence binding protein (ICSBP) transcripts in human myeloid leukemias.

Interferon consensus sequence binding protein (ICSBP) was first identified as a transcription factor of the interferon (IFN) regulatory factor family (IRF) which regulates expression of IFN-dependent genes by binding to DNA at specific sites, IFN-stimulated responsive elements. Analysis of ICSBP-deficient mice showed hematologic alterations similar to chronic myelogenous leukemia (CML) in humans and suggested a novel role for ICSBP in regulating proliferation and differentiation of hematopoietic progenitor cells. Here we show that ICSBP-mRNA expression is impaired in human myeloid leukemias: 27 of 34 CML patients (79%) and 21 of 32 patients with acute myeloid leukemia (AML) (66%) showed very low or absent transcript numbers of ICSBP. In contrast, only 2 of 33 normal volunteers (6%) showed low transcription of ICSBP (P < . 0001 both for CML and AML values). The lack of expression was not associated with lack of lymphatic cells, which normally have been shown to express ICSBP at the highest level. More detailed analysis showed an absence of ICSBP-mRNA also in sorted B cells derived from CML patients. To analyze whether ICSBP may be induced in leukemic cells, ex vivo experiments using a known inducer of ICSBP, IFN-gamma, were performed. Ex vivo treatment of primary CML cells using IFN-gamma resulted in induction of ICSBP transcripts. Furthermore, samples of CML patients during IFN-alpha treatment were analyzed. In 11 of 12 CML patients ICSBP-mRNA was inducible upon in vivo treatment with IFN-alpha, but decreased with progression of CML. Stable transfection of K-562 cell line with ICSBP led to no difference in bcr-abl expression in vitro, although two patients showed an inverse correlation between bcr-abl and ICSBP in vivo. These data suggest that lack of ICSBP may have an important role also in human myeloid leukemogenesis.

Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene.

Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Mice with a null mutation of ICSBP exhibit two prominent phenotypes related to previously described activities of the IRF family. The first is enhanced susceptibility to virus infections associated with impaired production of IFN(gamma). The second is deregulated hematopoiesis in both ICSBP-/- and ICSBP+/- mice that manifests as a syndrome similar to human chronic myelogenous leukemia. The chronic period of the disease progresses to a fatal blast crisis characterized by a clonal expansion of undifferentiated cells. Normal mice injected with cells from mice in blast crisis developed acute leukemia within 6 weeks of transfer. These results suggest a novel role for ICSBP in regulating the proliferation and differentiation of hematopoietic progenitor cells.

The human leukocyte antigen A2 interferon-stimulated response element consensus sequence binds a nuclear factor required for constitutive expression.

Both constitutive and interferon-inducible enhancer-like elements have been identified previously in the promoter of human leukocyte antigen (HLA) class I genes. One of these sites is termed the interferon-stimulated response element (ISRE). We have tested the function of an ISRE consensus sequence in the human HLA class I gene HLA-A2 and confirmed previous studies that showed that the HLA-A2 ISRE consensus sequence does not mediate a response to interferons. However, deletion of the ISRE consensus sequence caused a several-fold reduction in the constitutive expression of the HLA-A2 gene in K562 and Jurkat cells. Mobility shift assays performed with the HLA-A2 ISRE revealed the presence of a constitutive binding protein (ISRE/CBP). This protein binds specifically to the HLA-A2 ISRE sequence, and binding is not efficiently competed by the ISRE sequences of the HLA-B7 or ISG54 genes. Substitution of the HLA-B7 or ISG54 ISRE sequences for the HLA-A2 ISRE sequence caused a severalfold reduction in the constitutive expression of the HLA-A2 gene. Mass determinations showed the ISRE/CBP to be 105 kDa, different than any previously characterized ISRE binding proteins. We propose that ISRE/CBP is a novel positive transcriptional regulatory factor for the HLA-A2 gene that may contribute to the differential expression of HLA-A versus HLA-B genes.

Stimulation of MHC class I transcription by interferon-gamma involves a non-A, non-C kinase in addition to protein kinase C.

The signal pathways by which interferon-gamma (IFN-gamma) is able to up-regulate major histocompatibility complex (MHC) class I transcription were studied in two human hematopoietic tumor cell lines, K562 and Ramos. These studies suggest that the IFN-gamma signal is transduced via an H7- and staurosporine-sensitive kinase that is distinct from protein kinase C (PKC) and protein kinase A (PKA) in both cell types. Ramos cells appear to utilize an additional pathway involving double-stranded RNA-dependent protein kinase. PKC and possibly PKA appear to be involved in one or more intersecting pathways by which agonists of these kinases are able to act synergistically with IFN-gamma, but activation of these latter pathways is neither necessary nor sufficient for induction of MHC class I transcription. Modulation of G-protein- and Ca2+-calmodulin-associated pathways and arachidonic acid metabolism had no effect on constitutive or IFN-gamma-stimulated class I transcription. The class I stimulatory factor produced in response to IFN-gamma treatment appears to have a short t1/2. The identity of this factor is unknown, but is likely to be distinct from known mediators of IFN-stimulated transcription. Gene and cell-type specificity in the signal transduction pathways utilized by IFN-gamma implies that such pathways may be useful targets for experimental and therapeutic manipulation.

Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediate early genes.

The immediate early (IE) genes of human cytomegalovirus (HCMV) are expressed in lymphocytes and are known to transactivate both viral and cellular promoters. The mechanism by which IE gene products of HCMV transactivate expression of the HLA A2 gene promoter in Jurkat cells, a T-lymphocyte cell line, was investigated. Transient expression assays were performed using plasmids containing the HLA A2 promoter-regulatory region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE genes. The upregulation of the HLA A2 promoter by HCVM IE gene products was shown not to be secondary to either interferon-gamma or -alpha. Previously described MHC class I regulatory or enhancer elements such as the interferon-stimulated response element (ISRE), NF-kappa B and H2TF1 binding sequences, and the interferon consensus sequence (ICS) were not required for transactivation of the A2 promoter. Rather, the only known regulatory elements in the HLA A2 promoter necessary for both basal expression and transactivation by HCVM IE gene products are the CCAAT box and TATA box motifs. These results support a model in which HCVM IE gene products act through the minimal HLA A2 promoter elements to increase gene expression.