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Glioblastoma (GBM) contains abundant tumor-associated macrophages (TAMs). The majority of TAMs are tumor-promoting macrophages (pTAMs), while tumor-suppressive macrophages (sTAMs) are the minority. Thus, reprogramming pTAMs into sTAMs represents an attractive therapeutic strategy. By screening a collection of small-molecule compounds, we find that inhibiting β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) with MK-8931 potently reprograms pTAMs into sTAMs and promotes macrophage phagocytosis of glioma cells; moreover, low-dose radiation markedly enhances TAM infiltration and synergizes with MK-8931 treatment to suppress malignant growth. BACE1 is preferentially expressed by pTAMs in human GBMs and is required to maintain pTAM polarization through trans-interleukin 6 (IL-6)–soluble IL-6 receptor (sIL-6R)–signal transducer and activator of transcription 3 (STAT3) signaling. Because MK-8931 and other BACE1 inhibitors have been developed for Alzheimer’s disease and have been shown to be safe for humans in clinical trials, these inhibitors could potentially be streamlined for cancer therapy. Collectively, this study offers a promising therapeutic approach to enhance macrophage-based therapy for malignant tumors. Zhai et al. identify clinical brain-penetrant BACE1 inhibitors as regulators of macrophage-dependent phagocytosis in glioblastoma through IL-6–STAT3 signaling and demonstrate preclinical therapeutic efficacy in orthotopic mouse models and PDXs.

Individuals with germline mutations in the gene encoding phosphatase and tensin homolog on chromosome ten (PTEN) are diagnosed with PTEN hamartoma tumor syndrome (PHTS) and are at high risk for developing breast, thyroid and other cancers and/or autoimmunity or neurodevelopmental issues including autism spectrum disorders. Although well recognized as a tumor suppressor, involvement of PTEN mutations in mediating such a diverse range of phenotypes indicates a more central involvement for PTEN in immunity than previously recognized. To address this, sequencing of the T-cell receptor variable-region β-chain was performed on peripheral blood from PHTS patients. Based on patient findings, we performed mechanistic studies in two Pten knock-in murine models, distinct from each other in cell compartment-specific predominance of Pten. We found that PTEN mutations in humans and mice are associated with a skewed T- and B-cell gene repertoire, characterized by increased prevalence of high-frequency clones. Immunological characterization showed that Pten mutants have increased B-cell proliferation and a proclivity towards increased T-cell reactivity upon Toll-like-receptor stimulation. Furthermore, decreases in nuclear but not cytoplasmic Pten levels associated with a reduction in expression of the autoimmune regulator (Aire), a critical mediator of central immune tolerance. Mechanistically, we show that nuclear PTEN most likely regulates Aire expression via its emerging role in splicing regulation. We conclude that germline disruption of PTEN, both in human and mouse, results in compromised central immune tolerance processes that may significantly impact individual stress responses and therefore predisposition to autoimmunity and cancer.

Significance Tamoxifen is beneficial in treating estrogen receptor–positive breast cancer, but resistance to this treatment eventually ensues. A method to identify mechanisms of tamoxifen resistance identified the histone deacetylase ZIP, leading to the finding that increased expression of the tyrosine kinase JAK2 is one important factor. As a result of this discovery, it may be possible to use an inhibitor of JAK2 to block the aberrant activation of STAT3 caused by ZIP deficiency to help overcome or prevent tamoxifen resistance., Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2. By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ER-positive breast cancer.

Highlights • High STAT3 is correlated with poor prognosis in GBM. • Bazedoxifene targets IL-6-mediated sustained STAT3 activation. • STAT3 drives glioma stem cell maintenance, bazedoxifene targets this pathway. • Bazedoxifene crosses the BBB and prolongs survival in GBM bearing mice. • Bazedoxifene-treated tumors have less activated STAT3., An important factor correlated with poor survival in glioblastoma (GBM) is the aberrant and persistent activation of STAT3, a critical transcription factor that regulates multiple genes with key roles in cell survival, proliferation, resistance to chemotherapy, and stem cell maintenance. The Interleukin-6 (IL6)-STAT3 signaling axis has been studied extensively in inflammation and cancer. However, it is not completely understood how high levels of activated STAT3 are sustained in tumors. Previously, we identified a novel mechanism of biphasic activation of STAT3 in response to gp130-linked cytokines, including IL6, in which activation of STAT3 is prolonged by circumventing the negative regulatory mechanisms induced by its initial activationTo target prolonged STAT3 activation, we used the small molecule inhibitor bazedoxifene (BZA), which blocks formation of the IL6 receptor-gp130 complex. Glioma stem-like cells (GSCs) are more tumorigenic and more resistant to therapy. STAT3 is a key driver of the expression of stem cell transcription factors, making it a therapeutically important target in GBM. We show that treating GSCs with BZA decreases their self-renewal capacity and the expression of GSC markers in vitro. Additionally, BZA crosses the blood-brain barrier and confers a survival advantage in an orthotopic syngeneic mouse model of GBM. Although IL6-STAT3 signaling is important for GSC survival, a therapeutic agent that inhibits this pathway without toxicity has yet to be identified. Our findings reveal a mechanism of sustained STAT3 signaling in GBM and reveal its role in GSC maintenance, and we identify BZA as a novel candidate for treating GBM.

High expression of programmed death-ligand 1 (PD-L1) in cancer cells drives immune-independent, cell-intrinsic functions, leading to resistance to DNA-damaging therapies. We find that high expression of the ubiquitin E3 ligase FBXO22 sensitizes nonsmall cell lung cancer (NSCLC) cells to ionizing radiation (IR) and cisplatin, and that activation of FBXO22 by phosphorylation is necessary for this function. Importantly, FBXO22 activates PD-L1 ubiquitination and degradation, which in turn increases the sensitivity of NSCLC cells to DNA damage. Cyclin-dependent kinase 5 (CDK5), aberrantly active in cancer cells, plays a crucial role in increasing the expression of PD-L1 in medulloblastoma [R. D. Dorand et al., Science 353, 399–403 (2016)]. We show in NSCLC cells that inhibiting CDK5 or reducing its expression increases the level of FBXO22, decreases that of PD-L1, and increases the sensitivity of the cells to DNA damage. We conclude that FBXO22 is a substrate of CDK5, and that inhibiting CDK5 reduces PD-L1 indirectly by increasing FBXO22. Pairing inhibitors of CDK5 with immune checkpoint inhibitors may increase the efficacy of immune checkpoint blockade alone or in combination with DNA-damaging therapies.

Programmed death ligand 1 (PD-L1), an immune-checkpoint protein expressed on cancer cells, also functions independently of the immune system. We found that PD-L1 inhibits the killing of cancer cells in response to DNA damage in an immune-independent manner by suppressing their acute response to type I interferon (IFN; IFN-I). In addition, PD-L1 plays a critical role in sustaining high levels of constitutive expression in cancer cells of a subset of IFN-induced genes, the IFN-related DNA damage resistance signature (IRDS) which, paradoxically, protects cancer cells. The cyclic GMP-AMP synthase-stimulator of the IFN genes (cGAS-STING) pathway is constitutively activated in a subset of cancer cells in the presence of high levels of PD-L1, thus leading to a constitutive, low level of IFN-β expression, which in turn increases IRDS expression. The constitutive low level of IFN-β expression is critical for the survival of cancer cells addicted to self-produced IFN-β. Our study reveals immune-independent functions of PD-L1 that inhibit cytotoxic acute responses to IFN-I and promote protective IRDS expression by supporting protective chronic IFN-I responses, both of which enhance the resistance of cancer cells to DNA damage.

Small cell lung cancer (SCLC) is characterised by high relapse rates. Tumour-initiating cells (TICs) are responsible for drug resistance and recurrence of cancer. Rovalpituzumab tesirine (Rova-T), a potent humanised antibody–drug conjugate, selectively targets delta-like protein 3, which is highly expressed in SCLC TICs. The experimental drug CBL0137 (CBL) inhibits the histone chaperone FACT (facilitates chromatin transcription), which is required for the expression of transcription factors that are essential for TIC maintenance. Rova-T and CBL each target SCLC TICs as single agents. However, acquired or intrinsic resistance to single agents is a major problem in cancer. Therefore, we investigated the potential effect of combining Rova-T and CBL in SCLC to eradicate TICs more effectively. Our preclinical studies report a novel and highly translatable therapeutic strategy of dual targeting TICs using Rova-T in combination with CBL to potentially increase survival of SCLC patients.

Type I interferons (IFN-I) protect us from viral infections. Signal transducer and activator of transcription 2 (STAT2) is a key component of interferon-stimulated gene factor 3 (ISGF3), which drives gene expression in response to IFN-I. Using electron microscopy, we found that, in naive cells, U-STAT2, lacking the activating tyrosine phosphorylation, forms a heterodimer with U-STAT1 in an inactive, anti-parallel conformation. A novel phosphorylation of STAT2 on T404 promotes IFN-I signaling by disrupting the U-STAT1-U-STAT2 dimer, facilitating the tyrosine phosphorylation of STATs 1 and 2 and enhancing the DNA-binding ability of ISGF3. IKK-ε, activated by virus infection, phosphorylates T404 directly. Mice with a T-A mutation at the corresponding residue (T403) are highly susceptible to virus infections. We conclude that T404 phosphorylation drives a critical conformational switch that, by boosting the response to IFN-I in infected cells, enables a swift and efficient antiviral defense.

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

The transcription factor ISGF3, comprised of IRF9 and tyrosine‐phosphorylated STATs 1 and 2, transmits the signal from the type I interferon receptor to the genome. We have discovered a novel phosphorylation of STAT2 on T387 that negatively regulates this response. In most untreated cell types, the majority of STAT2 is phosphorylated on T387 constitutively. In response to interferon‐β, the T387A mutant of STAT2 is much more effective than wild‐type STAT2 in mediating the expression of many interferon‐stimulated genes, in protecting cells against virus infection, and in inhibiting cell growth. Interferon‐β‐treated cells expressing wild‐type STAT2 contain much less ISGF3 capable of binding to an interferon‐stimulated response element than do cells expressing T387A STAT2. T387 lies in a cyclin‐dependent kinase (CDK) consensus sequence, and CDK inhibitors decrease T387 phosphorylation. Using CDK inhibitors to reverse the constitutive inhibitory phosphorylation of T387 of U‐STAT2 might enhance the efficacy of type I interferons in many different clinical settings.

Background Highly aggressive, metastatic and therapeutically resistant triple-negative breast cancers (TNBCs) are often enriched for cancer stem cells (CSC). Cytokines within the breast tumor microenvironment (TME) influence the CSC state by regulating tumor cell differentiation programs. Two prevalent breast TME cytokines are oncostatin-M (OSM) and interferon-β (IFN-β). OSM is a member of the IL-6 family of cytokines and can drive the de-differentiation of TNBC cells to a highly aggressive CSC state. Conversely, IFN-β induces the differentiation of TNBC, resulting in the repression of CSC properties. Here, we assess how these breast TME cytokines influence CSC plasticity and clinical outcome. Methods Using transformed human mammary epithelial cell (HMEC) and TNBC cell models, we assessed the CSC markers and properties following exposure to OSM and/or IFN-β. CSC markers included CD24, CD44, and SNAIL; CSC properties included tumor sphere formation, migratory capacity, and tumor initiation. Results There are three major findings from our study. First, exposure of purified, non-CSC to IFN-β prevents OSM-mediated CD44 and SNAIL expression and represses tumor sphere formation and migratory capacity. Second, during OSM-induced de-differentiation, OSM represses endogenous IFN-β mRNA expression and autocrine/paracrine IFN-β signaling. Restoring IFN-β signaling to OSM-driven CSC re-engages IFN-β-mediated differentiation by repressing OSM/STAT3/SMAD3-mediated SNAIL expression, tumor initiation, and growth. Finally, the therapeutic use of IFN-β to treat OSM-driven tumors significantly suppresses tumor growth. Conclusions Our findings suggest that the levels of IFN-β and OSM in TNBC dictate the abundance of cells with a CSC phenotype. Indeed, TNBCs with elevated IFN-β signaling have repressed CSC properties and a better clinical outcome. Conversely, TNBCs with elevated OSM signaling have a worse clinical outcome. Likewise, since OSM suppresses IFN-β expression and signaling, our studies suggest that strategies to limit OSM signaling or activate IFN-β signaling will disengage the de-differentiation programs responsible for the aggressiveness of TNBCs. Electronic supplementary material The online version of this article (10.1186/s13058-019-1136-x) contains supplementary material, which is available to authorized users.

In normal cells exposed to stress, the central transcription factor NF-κB is activated only transiently, to modulate the activation of downstream immune responses. However, in most cancers, NF-κB is abnormally activated constitutively, contributing thus to oncogenesis and tumor progression. Therefore, downregulating NF-κB activity is an important goal of cancer treatment. In order to control NF-κB activity therapeutically, it is helpful to understand the molecular mechanisms that normally govern its activation and how dysregulated NF-κB activity may aid the development of disease. Recent evidence from our laboratories and others indicates that, in addition to various posttranslational modifications of NF-κB that have been observed previously, including phosphorylation, ubiquitination, and acetylation, NF-κB can be methylated reversibly on lysine or arginine residues by histone-modifying enzymes, including lysine and arginine methyl transferases and demethylases. Furthermore, these methylations are required to activate many downstream genes. Interestingly, amplifications and mutations of several such enzymes have been linked to cancer. We propose that some of these mutations may alter the methylation not only of histones but also of NF-κB, making them attractive therapeutic targets. Cancer Res; 75(18); 3692–5. ©2015 AACR.

Up-regulation of IFN-stimulated genes (ISGs) is sustained in hepatitis C virus (HCV)-infected livers. Here, we investigated the mechanism of prolonged ISG expression and its role in IFN responsiveness during HCV infection in relation to unphosphorylated IFN-stimulated gene factor 3 (U-ISGF3), recently identified as a tripartite transcription factor formed by high levels of IFN response factor 9 (IRF9), STAT1, and STAT2 without tyrosine phosphorylation of the STATs. The level of U-ISGF3, but not tyrosine phosphorylated STAT1, is significantly elevated in response to IFN-λ and IFN-β during chronic HCV infection. U-ISGF3 prolongs the expression of a subset of ISGs and restricts HCV chronic replication. However, paradoxically, high levels of U-ISGF3 also confer unresponsiveness to IFN-α therapy. As a mechanism of U-ISGF3-induced resistance to IFN-α, we found that ISG15, a U-ISGF3-induced protein, sustains the abundance of ubiquitin-specific protease 18 (USP18), a negative regulator of IFN signaling. Our data demonstrate that U-ISGF3 induced by IFN-λs and -β drives prolonged expression of a set of ISGs, leading to chronic activation of innate responses and conferring a lack of response to IFN-α in HCV-infected liver.

Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinib-resistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGF-dependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation.

The activation of STAT3 by tyrosine phosphorylation, essential for normal development and for a normal inflammatory response to invading pathogens, is kept in check by negative regulators. Abnormal constitutive activation of STAT3, which contributes to the pathology of cancer and to chronic inflammatory diseases such as rheumatoid arthritis, occurs when negative regulation is not fully effective. SOCS3, the major negative regulator of STAT3, is induced by tyrosine-phosphorylated STAT3 and terminates STAT3 phosphorylation about 2 h after initial exposure of cells to members of the IL-6 family of cytokines by binding cooperatively to the common receptor subunit gp130 and JAKs 1 and 2. We show here that when the epidermal growth factor receptor (EGFR) is present and active, STAT3 is rephosphorylated about 4 h after exposure of cells to IL-6 or oncostatin M and remains active for many hours. Newly synthesized IL-6 drives association of the IL-6 receptor and gp130 with EGFR, leading to EGFR-dependent rephosphorylation of STAT3, which is not inhibited by the continued presence of SOCS3. This second wave of STAT3 activation supports sustained expression of a subset of IL-6-induced proteins, several of which play important roles in inflammation and cancer, in which both IL-6 secretion and EGFR levels are often elevated.

A single high dose of interferon-β (IFNβ) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNβ-induced proteins, and to constitutive resistance to DNA damage., IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage IFNβ induces the formation of a novel transcriptional complex, U-ISGF3, which contains un-phosphorylated STATs. U-ISGF3 regulates the expression of a subset of IFNβ-stimulated genes to promote resistance to virus infection and DNA damage.

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ∼85% of the NF-κB–inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB–inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.

The signal transducers and activators of transcription (STAT)1 and STAT3 genes are specifically activated by phosphorylated STATs 1 and 3, respectively, resulting in large and prolonged increases in the levels of unphosphorylated STATs (U-STATs) in response to interferons (for STAT1) or ligands that activate gp130, such as IL-6 (for STAT3). U-STATs 1 and 3 are transcription factors that drive gene expression by mechanisms distinct from those used by phosphorylated STATs. U-STAT3 drives expression of many proteins not induced by phospho-STAT3, including several that are important in tumorigenesis. U-STAT1 prolongs and increases expression of a subset of proteins induced initially in response to phospho-STAT1, leading to antiviral and immune responses that are long-lived. U-STAT1 levels are also high in some cancers, and the protein products of genes induced by U-STAT1 enhance resistance to DNA damage. Therefore, interferons not only drive short-term expression of proteins that inhibit growth and promote apoptosis and immune surveillance, but also promote long-term expression of proteins that facilitate tumor survival.

Transforming growth factor β2 (TGFβ2) is highly expressed in a variety of different cancer cell lines. Using Z12 cells, a mutant of 293 cells with overexpression of TGFβ2, we found that the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) sequence in the promoter of the TGFβ2 gene is crucial for its increased expression. Further, constitutive phosphorylation of CRE-binding protein (CREB) is increased in these cells. Treating Z12 cells with either the PI3 kinase inhibitor LY294002 or the p38 MAPK inhibitor SB203580 significantly inhibited both the phosphorylation of CREB and expression of TGFβ2. In addition, treating Z12 or cancer cell lines with either of these 2 inhibitors significantly decreased their secretion of TGFβ2. These data suggest that activated PI3 kinase and p38 MAPK play important roles in high expression of TGFβ2 in cancer cells by stimulating the phosphorylation of CREB, which activates the CRE in the promoter of the TGFβ2 gene. We have identified an important link between PI3 kinase, p38 MAPK, and TGFβ2, providing an additional rationale for using inhibitors of these kinases as therapeutic drugs in cancer.

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10 −6 or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFκB. High levels of FBXL11 block the ability of NFκB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFκB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFκB-dependent signaling.

Some tumor cell lines secrete high concentrations of TGFbeta or IL-1. Similarly high concentrations of each of these cytokines cross-activate the other pathway: TGFbeta activates NFkappaB, and IL-1beta activates Smads. The IL-1 signaling components IRAK, MyD88, TRAF6, and TAK1 are all required for cross-activation of NFkappaB by TGFbeta. Knockdown experiments revealed that both TGFbeta receptor subunits are required for IL-1beta to activate Smads, and the IL-1 receptor is required for TGFbeta to activate NFkappaB. Coimmunoprecipitations showed that either TGFbeta or IL-1beta stimulate ligand-dependent association of all three receptor subunits. Furthermore, cross-talk between the TGFbeta and IL-1 signaling pathways leads to dose-dependent cross-control of gene expression. These interactions provide new insight into biological responses to IL-1 and TGFbeta in the proximity of tumors that secrete high concentrations of these factors and probably also at sites of inflammation, where the local concentrations of these cytokines are likely to be high.

Significance Protein lysine methyl transferases and demethylases, previously identified for histone modification, now are known to modify several nonhistone proteins as well. Their deregulation leads to the development and progression of various diseases, including cancer. Thus these enzymes now stand out as attractive therapeutic targets. We present a detailed study of STAT3 posttranslational modification at K49 by the histone methyl transferase, enhancer of zeste homolog 2 (EZH2). Dimethylation of K49 modulates IL-6–responsive transcription, revealing a previously unrecognized important functional modification of STAT3 activity. Because STAT3 is a major oncogene, and EZH2 function is modified in many cancers, this functional connection between the two raises the possibility that modulation of EZH2 activity might be useful in inhibiting the oncogenic activity of STAT3.

The NFkappaB family of transcription factors, central mediators of immune responses, are also involved in oncogenesis. Loss of regulation of the normally latent NFkappaB contributes importantly to the deregulated growth, resistance to apoptosis and propensity to metastasize observed in many cancers. Thus, pathways for activation of NFkappaB are promising targets for new agents that may help to prevent or treat cancer. We find that the abnormal secretion of multiple cytokines that activate NFkappaB by binding to cell-surface receptors is one of the major causes of constitutive NFkappaB activity in cancer. A novel finding is that the latent form of TGFbeta, secreted by some of these cells, can activate NFkappaB. To understand the basis of this abnormal cytokine secretion, we are using forward genetic methods to identify specific causative mutations in cancer cells.

IkappaB kinase (IKK), discovered as the major activator of NF-kappaB, plays additional roles in signaling. By using mouse embryo fibroblasts (MEFs) lacking both the alpha and beta subunits of IKK, we find that these proteins are required for induction of a major subset of IFNgamma-stimulated genes and that this requirement is independent of NF-kappaB activation. Furthermore, there is no defect in IFNgamma-stimulated signal transducer and activator of transcription 1 (Stat1) activation or function in the IKKalpha/beta-null MEFs. Therefore, although activated Stat1 dimers are necessary for the activation of these genes in response to IFNgamma, they are not sufficient. These results reveal an important additional pathway for IFNgamma-stimulated gene expression in which an NF-kappaB-independent function of IKK is required.

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-β- and retinoic acid-induced cell death, codes for a ≈16-kDa protein that induces apoptosis in a number of cell lines. Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19. GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding. Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.

Defects in expression or activation signal transducer and activator of transcription-1 (Stat1) in response to interferon-alpha2 (IFN-alpha2) have been implicated as a mechanism for IFN resistance in melanoma cells. To further determine the significance of this observation, 17 melanoma cell lines sensitive or resistant to the antiproliferative effects of IFN-alpha2 and IFN-beta, as well as 30 melanoma patient samples, were analyzed for Stat1 levels by either Western blot analysis or immunohistochemistry. Although the expression level varied between samples, all the cell lines except one and all melanoma biopsy specimens expressed Stat1. IFN-stimulated levels of Stat1 and Stat2, which constitute the transcriptional activation complexes, such as, gamma activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), for IFN-stimulated gene (ISG) induction were assessed in melanoma cell lines. Both IFN-alpha2 and INF-beta induced equivalent amounts of Stat1 and Stat2 proteins in cell lines, although compared with IFN-alpha2, IFN-beta had greater antiproliferative effects. No significant differences were observed in tyrosine or serine phosphorylation of Stat1 or the formation of GAF or ISGF3 complexes following IFN-alpha2 or IFN-beta treatment of IFN-resistant or IFN-sensitive cell lines. Comparable induction of two ISGs, ISG54 and IFN regulatory factor-1 (IRF-1), was observed in both sensitive WM9 and resistant A375 cells. Therefore, we report that defects in expression or activation of Stat1 or Stat2 were infrequent in melanoma cell lines and tumor samples and did not correlate with IFN resistance. Cellular resistance to IFNs likely results from defective quantitative or qualitative expression of specific ISGs.

The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs). STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors. Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have been associated with cellular resistance to IFNs and mycobacterial infection in humans. Thus, given the relative importance of STAT1, we isolated and characterized a human STAT1 intronic enhancer region displaying IFN-regulated activity. Functional analyses by transient expression identified a repressor region and type I and II IFN-inducible elements within the STAT1 enhancer sequence. A candidate IRF-E/GAS/IRF-E (IGI) sequence containing GAAANN nucleotide repeats was shown by gel shift assay to bind to IFN regulatory factor-1 (IRF-1), but not to IFN-stimulated gene factor-3 (ISGF-3) or STAT1–3. An additional larger IGI-binding complex containing IRF-1 was identified. Mutation of the GAAANN repeats within the IGI DNA element eliminated IRF-1 binding and the IFN-regulated activity of the STAT1 intronic enhancer region. Transfection of the IFN-resistant MM96 cell line to express increased levels of IRF-1 protein also elevated STAT1, STAT2, and p48/IRF-9 expression and enhanced cellular responsiveness to IFN-β. Reciprocating regulation between IRF-1 and STAT1 genes and encoded proteins indicates that an intracellular amplifier circuit exists controlling cellular responsiveness to the IFNs.

The transcription factor NFkappaB is activated by numerous stimuli. Once NFkappaB is fully activated, it participates in the regulation of various target genes in different cells to exert its biological functions. NFkappaB has often been referred to as a central mediator of the immune response, since a large variety of bacteria and viruses can lead to the activation of NFkappaB, which in turn controls the expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. Recent studies have shown that NFkappaB may function more generally as a central regulator of stress responses, since different stressful conditions, including physical stress, oxidative stress, and exposure to certain chemicals, also lead to NFkappaB activation. Furthermore, NFkappaB blocks cell apoptosis in several cell types. Taken together, these findings make it clear that NFkappaB plays an important role in cell proliferation and differentiation. It is the intention of this review to cover the various NFkappaB-dependent signaling pathways, thereby to achieve a better understanding of the mechanisms of NFkappaB activation and the physiological functions of activated NFkappaB.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

Tumor recurrence remains a major therapeutic obstacle to curing breast cancer. Cytokines within the breast tumor microenvironment (TME) influence recurrence following chemotherapy by altering the balance between mesenchymal CSC (MES/CSC) and epithelial non-CSC (EP/non-CSC). Here, we find that transformed breast MES/CSC express elevated levels of Un-phosphorylated Interferon Stimulated Gene Factor 3 (U-ISGF3), which is composed of STAT1 and STAT2 lacking phosphorylation of their tyrosine residues, and IRF9. Elevated expression of the U-ISGF3 proteins in breast cancer has previously been linked with resistance to ionizing radiation, thus we hypothesized that the U-ISGF3 complex is important for maintaining MES/CSC properties. However, knockdown of IRF9, the critical DNA binding component of this complex, or STAT1 did not alter the MES/CSC phenotype. Moreover, ISGF3 transcriptional targets were actually repressed in MES/CSC relative to EP/non-CSC, suggesting that elevated U-ISGF3 may act as a repressor in MES/CSC. Interestingly, we found that an MES/CSC program independent of IFN-B, was responsible for mediating U-ISGF3 expression. We therefore hypothesized that stimulating MES/CSC with IFN-B would phosphorylate the ISGF3 proteins and engage canonical IFN-B mediated inhibition of MES/CSC properties. Acute exposure to IFN-B led to the phosphorylation of ISGF3 in MES/CSC and induced the transcription of IFN-B responsive genes. Continuous exposure to IFN-B maintained Phosphorylated ISGF3 (P-ISGF3) which partially reverted the MES/CSC phenotype, resulting in the repression of mesenchymal markers (Vimentin and Slug), re-expression of Ep/non-CSC markers (CD24) and functionally inhibited colony formation in soft agar and migratory capability. In line with these observations, data from triple-negative breast cancer (TNBC) patients confirmed that increased expression of IFN-B responsive genes correlates with decreased expression of a CSC gene signature, and correlates with improved recurrence-free survival. Taken together, our findings demonstrate that IFN-B mediated signaling promotes the differentiation of MES/CSC by phosphorylating U-ISGF3, resulting in the suppression of MES/CSC-driven tumor recurrence. Future studies will examine the mechanism by which IFN-B mediated P-ISGF3 suppresses MES/CSC properties and whether elevation of U-ISGF3 can predict which patients would be candidates for an IFN-B therapy to phosphorylate STAT1 and STAT2, thus suppressing MES/CSC tumor recurrence. Citation Format: Mary R. Doherty, Damian J. Junk, HyeonJoo Cheon, George R. Stark, Mark W. Jackson. Interferon Beta (IFN-B) re-activates canonical type I IFN signaling to differentiate breast cancer stem cells (CSCs) and suppress mesenchymal/CSC driven tumor recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2884. doi:10.1158/1538-7445.AM2017-2884

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, butc-mycandc-junare still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling.

Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is approximately 10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5-8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.

p53 protects mammals from neoplasia by inducing apoptosis, DNA repair and cell cycle arrest in response to a variety of stresses. p53-dependent arrest of cells in the G1 phase of the cell cycle is an important component of the cellular response to stress. Here we review recent evidence that implicates p53 in controlling entry into mitosis when cells enter G2 with damaged DNA or when they are arrested in S phase due to depletion of the substrates required for DNA synthesis. Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3 sigma. Binding of Cdc2 to Cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. p53 also represses the cdc2 gene, to help ensure that cells do not escape the initial block. Genotoxic stress also activates p53-independent pathways that inhibit Cdc2 activity, activation of the protein kinases Chk1 and Chk2 by the protein kinases Atm and Atr. Chk1 and Chk2 inhibit Cdc2 by inactivating Cdc25, the phosphatase that normally activates Cdc2. Chk1, Chk2, Atm and Atr also contribute to the activation of p53 in response to genotoxic stress and therefore play multiple roles. p53 induces transcription of the reprimo, B99, and mcg10 genes, all of which contribute to the arrest of cells in G2, but the mechanisms of cell cycle arrest by these genes is not known. Repression of the topoisomerase II gene by p53 helps to block entry into mitosis and strengthens the G2 arrest. In summary, multiple overlapping p53-dependent and p53-independent pathways regulate the G2/M transition in response to genotoxic stress.

p53 represses the transcription of cdc2 and cyclin B1, causing loss of Cdc2 activity and G(2) arrest. Here we show that the region -22 to -2 of the cdc2 promoter called the R box is required for repression by p53 but not for basal promoter activity. The R box confers p53-dependent repression on heterologous promoters and binds to p130/E2F4 in response to overexpression of p53. R box-dependent repression requires p21/waf1, and overexpression of p21/waf1 also represses the cdc2 promoter. These observations suggest that p53 represses the cdc2 promoter by inducing p21/waf1, which inhibits cyclin-dependent kinase activity, enhancing the binding of p130 and E2F4, which together bind to and repress the cdc2 promoter.

The LMP2 gene, which encodes a protein required for efficient presentation of viral antigens, requires both unphosphorylated Stat1 and IRF1 for basal expression. LMP2 expression is down-regulated by the adenovirus protein E1A, which binds to Stat1 and CBP/p300, and by the mutant E1A protein RG2, which binds to Stat1 but not to CBP/p300, but not by the mutant protein Delta2-36, which does not bind to either Stat1 or CBP/p300. Stat1 and IRF1 associate in untreated cells and bind as a complex to the overlapping ICS-2/GAS element of the LMP2 promoter. E1A interferes with the formation of this complex by occupying domains of Stat1 that bind to IRF1. These results reveal how adenovirus infection attenuates LMP2 expression, thereby interfering with the presentation of viral antigens.

Stat1 is a fascinating and complex protein with multiple, yet contrasting transcriptional functions. Upon activation, it drives the expression of many genes but also suppresses the transcription of others. These opposing characteristics also apply to its role in facilitating crosstalk between signal transduction pathways, as it participates in both synergistic activation and inhibition of gene expression. Stat1 is a functional transcription factor even in the absence of inducer-mediated activation, participating in the constitutive expression of some genes. This review summarizes the well studied involvement of Stat1 in IFN-dependent and growth factor-dependent signaling and then describes the roles of Stat1 in positive, negative and constitutive regulation of gene expression as well as its participation in crosstalk between signal transduction pathways. Oncogene (2000).

Defects in apoptosis have been noted in signal transducer and activator of transcription (Stat) 1-null cells in vitro. The purpose of this study was to analyse the keratocyte apoptosis response that occurs in vivo in response to corneal epithelial injury in Stat 1null compared with control mice and to determine whether Stat 1null corneal fibroblasts have a defective response to death receptor activation in vitro. Corneal epithelial scrape injuries were performed in Stat 1-null and wild-type mice. Keratocyte apoptosis was monitored with the quantitative TUNEL assay and confirmed using transmission electron microscopy. Corneal fibroblast apoptosis in response to tumor necrosis factor (TNF) alpha, with and without inhibitors of nuclear factor kappa B (NF-kappaB) activation, was monitored using DNA laddering and the methylene blue assay. Significantly less keratocyte apoptosis was noted in Stat 1-null mice compared with wild-type controls. TNF alpha-induced apoptosis only occurred in wild-type mice in the presence of inhibitors of NF-kappaB activation. Corneal fibroblast TNF alpha-induced apoptosis was defective in Stat 1null corneal fibroblasts whether NF-kappaB activation was blocked or not. Stat 1 has an important role in the keratocyte apoptosis that occurs in response to corneal epithelial injury. Previous studies suggest that the defect is due to a lack of constitutive expression of caspases. This study demonstrates that this defect in apoptosis in Stat 1-null mice is present in vivo in Stat 1-null mice and suggests that Stat 1 could be a therapeutic target for transient inhibition of keratocyte apoptosis to modulate corneal wound healing.

Interferons (IFNs) inhibit cell growth in a Stat1‐dependent fashion that involves regulation of c‐ myc expression. IFN‐γ suppresses c‐ myc in wild‐type mouse embryo fibroblasts, but not in Stat1‐null cells, where IFNs induce c‐ myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c‐ myc is likely to contribute to the proliferation of Stat1‐null cells in response to IFNs. IFNs also suppress platelet‐derived growth factor (PDGF)‐induced c‐ myc expression in wild‐type but not in Stat1‐null cells. A gamma‐activated sequence element in the promoter is necessary but not sufficient to suppress c‐ myc expression in wild‐type cells. In PKR‐null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c‐ myc mRNA is induced, not suppressed, in response to IFN‐γ. A role for Raf‐1 in the Stat1‐independent pathway is revealed by studies with geldanamycin, an HSP90‐specific inhibitor, and by expression of a mutant of p50 cdc37 that is unable to recruit HSP90 to the Raf‐1 complex. Both agents abrogated the IFN‐γ‐dependent induction of c‐ myc expression in Stat1‐null cells.

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 andcyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions −287 to −123 of thecyclin B1 promoter and −104 to −74 of thecdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest.

Although much progress has been made in identifying the signaling pathways that mediate the initial responses to interferons (IFNs), much less is known about how IFN-stimulated genes (ISGs) are kept quiescent in untreated cells, how the response is sustained after the initial induction, and how ISG expression is down-regulated, even in the continued presence of IFN. We have used the cell sorter to isolate mutant cells with constitutively high ISG expression. A recessive mutant, P2.1, has higher constitutive ISG levels than the parental U4C cells, which do not respond to any IFN. Unexpectedly, P2.1 cells also are deficient in the expression of ISGs in response to double-stranded RNA (dsRNA). Electrophoretic mobility-shift assays revealed that the defect is upstream of the activation of the transcription factors NFκB and IFN regulatory factor 1. Analysis of the pivotal dsRNA-dependent serine/threonine kinase PKR revealed that the wild-type kinase is present and is activated normally in response to dsRNA in P2.1 cells. Together, these data suggest that the defect in P2.1 cells is either downstream of PKR or in a component of a distinct pathway that is involved both in activating multiple transcription factors in response to dsRNA and in regulating the basal expression of ISGs.

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA. The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immuno-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor alpha-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) non-activated STAT1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

The proliferation and differentiation of neutrophils is regulated by granulocyte-specific colony-stimulating factor (G-CSF ). G-CSF uses a receptor of the cytokine receptor superfamily and, in common with all members of the family, induces the tyrosine phosphorylation and activation of members of the Janus protein tyrosine kinase (Jak) family. In both myeloid cells and a human fibrosarcoma cell line expressing the G-CSF receptor, G-CSF induces the tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. In addition, G-CSF induces the tyrosine phosphorylation of the receptor and members of the signal transducers and activators of transcription (Stat) family, including Stat3, as well as Stat1 and Stat5, depending on the cells involved. Using mutant cell lines lacking various Jaks, we show here that Jak1 is critical for G-CSF–mediated Stat activation, whereas Jak2 or Tyk2 are either not required or play redundant or ancillary roles. In the absence of Jak1, G-CSF induces activation of Jak2 and Tyk2, but fails to induce receptor tyrosine phosphorylation and induces dramatically reduced levels of Stat activation. A kinase-inactive Jak2, when overexpressed in cells lacking endogenous Jak2, can suppress Jak1 activation, receptor phosphorylation, and Stat activation, suggesting competition in the receptor complex either for Jak1 binding or substrates. Because the requirement for Jak1 is very similar to that previously shown for interleukin-6 signaling, the data support the concept that the G-CSF receptor and gp130 are both structurally and functionally similar.

Several different Janus kinases (JAKs) and signal transducers and activation of transcription (STATs) have been implicated in mediating the biological responses induced by PRL, based on their ligand-dependent tyrosine phosphorylation and activation. However, these criteria alone do not prove that a particular JAK or STAT is essential for signal transduction. We have used mutant cell lines defective in JAK1, JAK2, or STAT1 to examine their roles in PRL-dependent signaling. JAK2 is absolutely required for PRL-dependent phosphorylation of the receptor, activation of STATs, and induction of beta-lactoglobulin. Wild type, but not kinase-negative JAK2, restores all responses to PRL in JAK2-defective cells, suggesting that JAK2 function, not merely the protein, is required. In contrast, JAK1, which is phosphorylated in response to PRL, is not required for any of these functions. Although STAT1 homodimers do form in response to PRL, no defect in PRL-dependent signaling is apparent when STAT1 is missing, suggesting that STAT5, which is strongly activated in response to PRL, is primarily responsible for driving the expression of PRL-responsive genes.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.

Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the REL homology domain of p65 on the response to IL-1β in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.

The first STAT-containing transcription factor to be studied, the alpha-interferon-induced ISGF3, is composed of a Stat1:2 heterodimer and a weak DNA-binding protein, p48, that is a member of a growing family of proteins similar to the so-called interferon regulatory factor (IRF-1). The p48 and Stat1:2 heterodimer do not associate stably in the absence of DNA, but we show that amino acids approximately 150 to 250 of Stat1 and a COOH-terminal portion of p48 exhibit physical interaction, implying contact that stabilizes ISGF3. Moreover, amino acid exchanges within the Stat1 contact region diminish or abolish the functional activity of Stat1. This protein interaction domain may be important in other STAT proteins to recruit partners to multiprotein transcription factors.

We report preliminary characterization of a gene designated β-R1, which is selectively expressed in response to interferon β (IFN-β) compared with IFN-α. In human astrocytoma cells, β-R1 was induced to an equivalent extent by 10 IU/mL IFN-β or 2500 IU/mL IFN-α2. To address the mechanism of this differential response, we analyzed induction of the β-R1 gene in fibrosarcoma cells and derivative mutant cells lacking components required for signaling by type I IFNs. β-R1 was readily induced by IFN-β in the parental 2fTGH cell line, but not by recombinant IFN-α2, IFN-α Con1, or a mixture of IFN-α subtypes. IFN-α8 induced β-R1 weakly. β-R1 was not induced by IFN-β in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-α and -β receptor, also failed to express β-R1. U1A cells are partially responsive to IFN-β and IFN-α8 but lacked β-R1 expression, indicating that TYK2 protein is essential for induction of this gene. Taken together, these results suggest that the expression of β-R1 in response to type I IFN requires IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-β compared with IFN-α.

The isolation and complementation of mutant human cell lines has established an essential role for the JAK (Janus kinase) family of protein tyrosine kinases and STAT (signal transduction and transcription) factors in the Interferon response pathways. Activation of STATs by JAKs occurs in receptor complexes at the cell membrane. Activated STATs form homo- or heterodimers and, with or without additional factors, migrate to the nucleus to initiate transcription. Different STAT combinations interact differentially with related DNA response elements. Signalling pathways of this novel type are likely utilized by a wide variety of polypeptide ligands. Data from the IL2, IL6 and IFN systems indicate a major role for the tyrosine phosphorylated receptor/JAK complexes (rather than substrate specificity of the JAKs per se) in STAT selection. The mutant cell lines lacking individual JAKs and STATs are being used together with kinase-negative JAK mutants which differentially affect the IFN- $\gamma $ , and IFN- $\alpha \beta $ and IL-6 pathways in the further analysis of these and additional systems.