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Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.

The tick-borne encephalitis (TBE) complex of viruses, genus Flavivirus, can cause severe encephalitis, meningitis, and/or hemorrhagic fevers. Effective interferon (IFN) responses are critical to recovery from infection with flaviviruses, and the mosquito-borne flaviviruses can inhibit this response. However, little is known about interactions between IFN signaling and TBE viruses. Langat virus (LGTV), a member of the TBE complex of viruses, was found to be highly sensitive to the antiviral effects of IFN. However, LGTV infection inhibited IFN-induced expression of a reporter gene driven by either IFN-α/β- or IFN-γ-responsive promoters. This indicated that LGTV can inhibit the IFN-mediated JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway of signal transduction. The mechanism of inhibition was due to blocks in the phosphorylation of both Janus kinases, Jak1 and Tyk2, during IFN-α signaling and at least a failure of Jak1 phosphorylation following IFN-γ stimulation. To determine the viral protein(s) responsible, we individually expressed all nonstructural (NS) proteins and examined their ability to inhibit signal transduction. Expression of NS5 alone inhibited STAT1 phosphorylation in response to IFN, thus identifying NS5 as a potential IFN antagonist. Examination of interactions between NS5 and cellular proteins revealed that NS5 associated with IFN-α/β and -γ receptor complexes. Importantly, inhibition of JAK-STAT signaling and NS5-IFN receptor interactions were demonstrated in LGTV-infected human monocyte-derived dendritic cells, important target cells for early virus replication. Because NS5 may interfere with both innate and acquired immune responses to virus infection, this protein may have a significant role in viral pathogenesis.

The continuous emergence of virus that are resistant to current anti-viral drugs, combined with the introduction of new viral pathogens for which no therapeutics are available, creates an urgent need for the development of novel broad spectrum antivirals. Type I interferon (IFN) can, by modulating the cellular expression profile, stimulate a non-specific antiviral state. The antiviral and adjuvant properties of IFN have been extensively demonstrated; however, its clinical application has been so far limited. We have developed a human cell-based assay that monitors IFN-β production for use in a high throughput screen. Using this assay we screened 94,398 small molecules and identified 18 compounds with IFN-inducing properties. Among these, 3 small molecules (C3, E51 and L56) showed activity not only in human but also in murine and canine derived cells. We further characterized C3 and showed that this molecule is capable of stimulating an anti-viral state in human-derived lung epithelial cells. Furthermore, the IFN-induction by C3 is not diminished by the presence of influenza A virus NS1 protein or hepatitis C virus NS3/4A protease, which make this molecule an interesting candidate for the development of a new type of broad-spectrum antiviral. In addition, the IFN-inducing properties of C3 also suggest its potential use as vaccine adjuvant.

Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis. Our previous study showed that gamma interferon (IFN-γ) added prior to or at early stage of infection inhibited infection of human monocytes with E. chaffeensis; however, after 24 h of infection, IFN-γ had no antiehrlichial effect. To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-γ, tyrosine phosphorylation of Stat1, Jak1, and Jak2 in E. chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis. Viable E. chaffeensis organisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-γ within 30 min of infection. Similar results were obtained with human peripheral blood monocytes infected with E. chaffeensis. Heat or proteinase K treatment but not periodate treatment of E. chaffeensis abrogated the inhibitory effect, suggesting that protein factor(s) of E. chaffeensis is responsible for the inhibition of IFN-γ-induced tyrosine phosphorylation. Preincubation of E. chaffeensis with the Fab fragment of dog anti-E. chaffeensis immunoglobulin G also abrogated the inhibitory effect. On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation. These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-γ. Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection with E. chaffeensis. In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3′,5′-cyclic phosphorothioate, E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated. These results suggest that E. chaffeensis blocks IFN-γ-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell.

A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-α. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. In order to enhance the antiproliferative activity of IFN-α, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-α and -γ, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-α among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-α, while it partially inhibited that of IFN-γ. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via TRAIL-R (receptor) 1 and TRAIL-R2. In IFN-α-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL while Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-α predominantly required IRF9. Furthermore, IFN-stimulated response element (ISRE)-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex following IFN-α treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-α and TRAIL, suggesting that TRAIL-R2 mediated both IFN-α and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-α-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-α. Thus, our present study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-α and TRAIL may be one of the potential mediators.

Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated and contributes to malignant progression in various cancers. Janus kinases (JAKs) phosphorylate STAT3 in response to stimulation with cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anti-cancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO) mediated growth inhibition of human melanoma cells and assessed 6BIO as an anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induced apoptosis of human melanoma cells. 6BIO directly inhibited JAK family kinase activity both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both a dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, the anti-apoptotic protein Mcl-1 was down-regulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the AKT and MAPK signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results demonstrate that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induced tumor cell apoptosis. Our findings support further development of 6BIO as a potential anti-cancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.

Suppressor of cytokine signaling–1 (SOCS1) is an intracellular inhibitor of the Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathway that couples interferon-γ (IFN-γ) signaling to the nucleus. Because several inflammatory diseases are associated with uncontrolled IFN-γ signaling, we engineered a recombinant cell-penetrating SOCS1 (CP-SOCS1) to target this pathway. Here, we show that CP-SOCS1, analogous to endogenous SOCS1, interacted with components of the IFN-γ signaling complex and functionally attenuated the phosphorylation of STAT1, which resulted in the subsequent inhibition of the production of proinflammatory chemokines and cytokines. Thus, controlled, intracellular delivery of recombinant CP-SOCS1 boosted the anti-inflammatory potential of the cell by restoring the homeostatic balance between pro- and anti-inflammatory signaling. This approach to controlling signal transduction has potential use for therapeutic targeting of signaling pathways associated with inflammatory diseases.

The type I IFNs (IFN-α and IFN-β), which are crucial in antiviral defense and immune regulation, signal via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway with activation of STAT1 and STAT2. Here, the function of STAT2 was studied in transgenic mice (termed GIFN/STAT2–/–) with CNS production of IFN-α. Surprisingly, GIFN/STAT2–/–, but not GIFN/STAT1-null, transgenic mice, with CNS production of IFN-α, died prematurely with medulloblastoma. An immune response also induced in the brain of the GIFN/STAT2–/– mice was associated with IFN-γ gene expression by CD3+ T cells and the activation of the STAT1, STAT3, STAT4, and STAT5 molecules. Expression of the Sonic hedgehog (Shh) and the downstream transcriptional factor Gli-1 genes, implicated in the pathogenesis of medulloblastoma, was found to be significantly increased and cotranscribed in cerebellar granule neurons of the GIFN/STAT2–/– mice. IFN-γ, but not IFN-α, induced STAT1-dependent expression of the Shh gene in cultured cerebellar granule neurons. Thus, there is an unexpected and extraordinarily adverse biological potency of IFN-α in the CNS when the primary signal transduction molecule STAT2 is absent. Moreover, a hitherto unknown role is indicated for the immune system in the pathogenesis of developmental disorders and tumorigenesis of the CNS via dysregulated Shh signaling mediated by IFN-γ.

The induction of alpha/beta interferon (IFN-α/β) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-α had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-α in the cultured cell system. JEV infection appeared to render cells resistant to IFN-α since the IFN-α-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-α/β receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-α stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-α/β by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway.

Adenoviral evolution has generated mechanisms to resist host cell defense systems, but the biochemical basis for evasion of multiple antiviral pathways in the airway by adenoviruses is incompletely understood. We hypothesized that adenoviruses modulate airway epithelial responses to type I interferons by altering the levels and activation of specific Janus family kinase-signal transducer and activator of transcription (JAK-STAT) signaling components. In this study, specific effects of adenovirus type 5 (AdV) on selected JAK-STAT signal transduction pathways were identified in human tracheobronchial epithelial cells, with focus on type I interferon–dependent signaling and gene expression. We found that wild-type AdV infection inhibited IFN-α–induced expression of antiviral proteins in epithelial cells by blocking phosphorylation of the Stat1 and Stat2 transcription factors that are required for activation of type I interferon–dependent genes. These effects correlated with AdV-induced down-regulation of expression of the receptor-associated tyrosine kinase Jak1 through a decrease in Jak1 mRNA levels. Phosphorylation of Stat3 in response to IL-6 and oncostatin M was also lost in AdV-infected cells, indicating loss of epithelial cell responses to other cytokines that depend on Jak1. In contrast, IL-4– and IL-13–dependent phosphorylation of Stat6 was not affected during AdV infection, indicating that the virus modulates specific signaling pathways, as these Stat6-activating pathways can function independent of Jak1. Taken together, the results indicate that AdV down-regulates host epithelial cell Jak1 to assure inhibition of the antiviral effects of multiple mediators to subvert airway defense responses and establish a productive infection.

In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-γ (IFN-γ). JAK inhibitors AG-490 and WHI-P154 decreased IFN-γ-induced nuclear levels of signal transducer and activator of transcription 1α (STAT1α). JAK inhibitors AG-490 and WHI-P154 decreased also iNOS protein and mRNA expression and NO production in a concentration-dependent manner. Neither of the JAK inhibitors affected the decay of iNOS mRNA when determined by actinomycin D assay. Our results suggest that the inhibition of JAK-STAT1-pathway by AG-490 or WHI-P154 leads to the attenuation of iNOS expression and NO production in IFN-γ-stimulated macrophages.

Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-γ (IFN-γ) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription–dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-γ and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-γ exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-γ more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.

The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.

Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. Injection of the secretory isoform of PAP has potent antinociceptive effects in mouse models of chronic pain. These data suggested that a small molecule activator of PAP may have utility as a novel therapeutic for chronic pain, while inhibitors could be used to acutely inhibit PAP in vitro and in vivo. To identify small molecule modulators of PAP activity, we validated a high throughput, fluorescence-based biochemical assay and then used this assay to screen a compound library. We decreased the frequency of false positive activators by subtracting compound fluorescence from the final assay fluorescence. This approach significantly reduced the number of false positive activators found in the screen. While no activators were confirmed, seven novel inhibitors of PAP were identified. Our results suggest this high throughput assay could be used to identify small molecule modulators of PAP activity.

Cytokines in the bone marrow of multiple myeloma patients activate Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways in tumor cells and promote tumor growth, survival, and drug resistance. INCB16562 was developed as a novel, selective, and orally bioavailable small-molecule inhibitor of JAK1 and JAK2 markedly selective over JAK3. The specific cellular activity of the inhibitor was demonstrated by its potent and dose-dependent inhibition of cytokine-dependent JAK/STAT signaling and cell proliferation in the absence of effects on Bcr-Abl-expressing cells. Treatment of myeloma cells with INCB16562 potently inhibited interleukin-6 (IL-6)-induced phosphorylation of STAT3. Moreover, the proliferation and survival of myeloma cells dependent on IL-6 for growth, as well as the IL-6-induced growth of primary bone marrow-derived plasma cells from a multiple myeloma patient, were inhibited by INCB16562. Induction of caspase activation and apoptosis was observed and attributed, at least in part, to the suppression of Mcl-1 expression. Importantly, INCB16562 abrogated the protective effects of recombinant cytokines or bone marrow stromal cells and sensitized myeloma cells to cell death by exposure to dexamethasone, melphalan, or bortezomib. Oral administration of INCB16562 antagonized the growth of myeloma xenografts in mice and enhanced the antitumor activity of relevant agents in combination studies. Taken together, these data suggest that INCB16562 is a potent JAK1/2 inhibitor and that mitigation of JAK/STAT signaling by targeting JAK1 and JAK2 will be beneficial in the treatment of myeloma patients, particularly in combination with other agents.

Type I (e.g., IFN-α, IFN-β) and type II IFNs (IFN-γ) have antiviral, antiproliferative, and immunomodulatory properties. Both types of IFN signal through the Jak/STAT pathway to elicit antiviral activity, yet IFN-γ is thought to do so only through STAT1 homodimers, whereas type I IFNs activate both STAT1- and STAT2-containing complexes such as IFN-stimulated gene factor 3. In this study, we show that IFN-stimulated gene factor 3 containing unphosphorylated STAT2 (ISGF3II) also plays a role in IFN-γ–mediated antiviral activity in humans. Using phosphorylated STAT1 as a marker for IFN signaling, Western blot analysis of IFN-α2a–treated human A549 cells revealed that phospho-STAT1 (Y701) levels peaked at 1 h, decreased by 6 h, and remained at low levels for up to 48 h. Cells treated with IFN-γ showed a biphasic phospho-STAT1 response with an early peak at 1–2 h and a second peak at 15–24 h. Gene expression microarray following IFN-γ treatment for 24 h indicated an induction of antiviral genes that are induced by IFN-stimulated gene factor 3 and associated with a type I IFN response. Induction of these genes by autocrine type I and type III IFN signaling was ruled out using neutralizing Abs to these IFNs in biological assays and by quantitative RT-PCR. Despite the absence of autocrine IFNs, IFN-γ treatment induced formation of ISGF3II. This novel transcription factor complex binds to IFN-stimulated response element promoter sequences, as shown by chromatin immunoprecipitation analysis of the protein kinase R promoter. STAT2 and IFN regulatory factor 9 knockdown in A549 cells reversed IFN-γ–mediated IFN-stimulated response element induction and antiviral activity, implicating ISGF3II formation as a significant component of the cellular response and biological activity of IFN-γ.

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Studies conducted over the past 10–15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus Kinases (JAKs) and their downstream transcription factors, termed STATs (signal transducers and activators of transcription). Aberrations in these pathways, such as those caused by the recently identified JAK2V617F mutation and translocations of the JAK2 gene, are underlying causes of leukemias and other myeloproliferative disorders. This review discusses the role of JAK/STAT signaling in normal hematopoiesis as well as myeloproliferative and myelodisplastic syndromes. This review also summarizes the status of several small molecule JAK2 inhibitors that are currently at various stages of clinical development. Several of these compounds appear to improve the quality of life of patients with myeloproliferative disorders by palliation of disease-related symptoms. However, to date, these agents do not seem to significantly affect bone marrow fibrosis, alter marrow histopathology, reverse cytopenias, reduce red cell transfusion requirements or significantly reduce allele burden. These results suggest that additional mutational events might be associated with the development of these neoplasms and indicate the need for additional therapeutic approaches as the nature of these additional molecular events is better understood.

Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Studies conducted over the past 10 to 15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors, termed STATs (signal transducers and activators of transcription). Aberrations in these pathways, such as those caused by the recently identified JAK2V617F mutation and translocations of the JAK2 gene, are underlying causes of leukemias and other myeloproliferative disorders. This review discusses the role of JAK/STAT signaling in normal hematopoiesis as well as genetic abnormalities associated with myeloproliferative and myelodisplastic syndromes. This review also summarizes the status of several small molecule JAK2 inhibitors that are currently at various stages of clinical development. Several of these compounds appear to improve the quality of life of patients with myeloproliferative disorders by palliation of disease-related symptoms. However, to date, these agents do not seem to significantly affect bone marrow fibrosis, alter marrow histopathology, reverse cytopenias, reduce red cell transfusion requirements, or significantly reduce allele burden. These results suggest the possibility that additional mutational events might be associated with the development of these neoplasms, and indicate the need for combination therapies as the nature and significance of these additional molecular events is better understood.

Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK/STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11,693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 uM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and nonselective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK/STAT activation.

Type I interferon (IFN) signaling is mediated through several signaling pathways, including the Janus kinase and signal transducer and activator (JAK-STAT) and p38 mitogen-activated protein (MAP) kinase pathways. The VP24 protein of Ebolavirus is an IFN antagonist, blocking type I IFN signaling through the JAK-STAT pathway. Here, we show that, in 293 cells, VP24 also interferes with the p38 MAP kinase pathway by blocking IFN-β–stimulated phosphorylation of p38-α. Similar inhibition was not observed in HeLa cells, suggesting cell type–specific differences in signal transduction.

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.

Author Summary

Influenza viruses are rapidly developing resistance against available anti-influenza drugs and consequently there is an urgent demand for new treatment approaches. We identified compound ASN2 in a high-throughput screen for molecules that are capable of inducing the antiviral cytokine interferon (IFN) in the presence of influenza virus infection. Normally, influenza virus blocks IFN production, an activity that is dependent on the viral NS1 protein and contributes to the ability of the virus to cause disease in an infected host. We show that ASN2 is a potent inhibitor of influenza A virus and can partially protect infected animals from disease and death. ASN2 acts by targeting influenza virus polymerase function which results in inhibition of virus replication, and as a consequence, NS1 expression. Thus the ability of ASN2 to induce IFN is a “side-effect”, albeit a desirable one, of polymerase inhibition. This combination of directly inhibiting the virus while also stimulating the host immune response is a novel property for an antiviral compound.

Type I interferons (IFNs) activate Janus tyrosine kinase-signal transducer and activator of transcription pathway for exerting pleiotropic biological effects, including antiviral, antiproliferative, and immunomodulatory responses. Here, we demonstrate that filamin B functions as a scaffold that links between activated Rac1 and a c-Jun NH2-terminal kinase (JNK) cascade module for mediating type I IFN signaling. Filamin B interacted with Rac1, mitogen-activated protein kinase kinase kinase 1, mitogen-activated protein kinase kinase 4, and JNK. Filamin B markedly enhanced IFNα-dependent Rac1 activation and the sequential activation of the JNK cascade members. Complementation assays using M2 melanoma cells revealed that filamin B, but not filamin A, is required for IFNα-dependent activation of JNK. Furthermore, filamin B promoted IFNα-induced apoptosis, whereas short hairpin RNA-mediated knockdown of filamin B prevented it. These results establish a novel function of filamin B as a molecular scaffold in the JNK signaling pathway for type I IFN-induced apoptosis, thus providing the biological basis for antitumor and antiviral functions of type I IFNs.

Vaccinia virus (VACV) replicates in mouse and human fibroblasts with comparable kinetics and efficiency, yielding similar titers of infectious progeny. Here we demonstrate that gamma interferon (IFN-γ) but not IFN-α or IFN-β pretreatment of mouse fibroblasts prior to VACV infection induces a long-lasting antiviral state blocking VACV replication. In contrast, high doses of IFN-γ failed to establish an antiviral state in human fibroblasts. In mouse fibroblasts, IFN-γ impeded the viral replication cycle at the level of late gene transcription and blocked the multiplication of VACV genomes. The IFN-γ-induced antiviral state invariably prevented the growth of different VACV strains but was not effective against the replication of ectromelia virus. The IFN-γ effect required intact IFN-γ receptor signaling prior to VACV infection through Janus kinase 2 (Jak2) and signal transducer and activator of transcription 1 (STAT1). The permissive state of IFN-γ-treated human cells was unrelated to the VACV-encoded IFN decoy receptors B8 and B18 and associated with a complete disruption of STAT1 homodimer formation and DNA binding. Unlike human fibroblasts, mouse cells responded with long-lasting STAT1 activation which was preserved after VACV infection. The deletion of the IFN regulatory factor 1 (IRF-1) gene from mouse cells rescued efficient VACV replication, demonstrating that IRF-1 target genes have a critical role in VACV control. These data have implications for the understanding of VACV pathogenesis and identify an incongruent IFN-γ response between the human host and the mouse model.