STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity.

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.

RIG-I-Mediated STING Upregulation Restricts Herpes Simplex Virus 1 Infection.

UNLABELLED: STING has emerged in recent years as a key player in orchestrating innate immune responses to cytosolic DNA and RNA derived from pathogens. However, the regulation of STING still remains poorly defined. In the present study, we investigated the mechanism of the regulation of STING expression in relation to the RIG-I pathway. Our data show that signaling through RIG-I induces STING expression at both the transcriptional and protein levels in various cell types. STING induction by the RIG-I agonist 5'triphosphorylated RNA (5'pppRNA) was recognized to be a delayed event resulting from an autocrine/paracrine mechanism. Indeed, cotreatment with tumor necrosis factor alpha and type I/II interferon was found to have a synergistic effect on the regulation of STING expression and could be potently decreased by impairing NF-κB and/or STAT1/2 signaling. STING induction significantly contributed to sustainment of the immune signaling cascade following 5'pppRNA treatment. Physiologically, this cross talk between the RNA- and DNA-sensing pathways allowed 5'pppRNA to efficiently block infection by herpes simplex virus 1 (HSV-1) both in vitro and in vivo in a STING-dependent fashion. These observations demonstrate that STING induction by RIG-I signaling through the NF-κB and STAT1/2 cascades is essential for RIG-I agonist-mediated HSV-1 restriction. IMPORTANCE: The innate immune system represents the first line of defense against invading pathogens. The dysregulation of this system can result in failure to combat pathogens, inflammation, and autoimmune diseases. Thus, precise regulation at each level of the innate immune system is crucial. Recently, a number of studies have established STING to be a central molecule in the innate immune response to cytosolic DNA and RNA derived from pathogens. Here, we describe the regulation of STING via RIG-I-mediated innate immune sensing. We found that STING is synergistically induced via proinflammatory and antiviral cytokine cascades. In addition, we show that in vivo protection against herpes simplex virus 1 (HSV-1) by a RIG-I agonist required STING. Our study provides new insights into the cross talk between DNA and RNA pathogen-sensing systems via the control of STING.

Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists.

UNLABELLED: The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5'-triphosphate (5'ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5'ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5'pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5'pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5'pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE: The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5'pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

Triptolide-mediated inhibition of interferon signaling enhances vesicular stomatitis virus-based oncolysis.

Preclinical and clinical trials demonstrated that use of oncolytic viruses (OVs) is a promising new therapeutic approach to treat multiple types of cancer. To further improve their viral oncolysis, experimental strategies are now combining OVs with different cytotoxic compounds. In this study, we investigated the capacity of triptolide - a natural anticancer molecule - to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. Triptolide treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose- and time-dependent manner in vitro and in vivo. Mechanistically, triptolide (TPL) inhibited the innate antiviral response by blocking type I interferon (IFN) signaling, downstream of IRF3 activation. Furthermore, triptolide-enhanced VSV-induced apoptosis in a dose-dependent fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3, and B-cell lymphoma 2 staining. In vivo, using the TSA mammary adenocarcinoma and PC3 mouse xenograft models, combination treatment with VSV and triptolide delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate that triptolide inhibition of IFN production sensitizes prostate cancer cells to VSV replication and virus-mediated apoptosis.

Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5' triphosphate (5'ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5'pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5'pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5'pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5'pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5'pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.

BCL-2 inhibitors sensitize therapy-resistant chronic lymphocytic leukemia cells to VSV oncolysis.

Many primary cancers including chronic lymphocytic leukemia (CLL) are resistant to vesicular stomatitis virus (VSV)-induced oncolysis due to overexpression of the antiapoptotic and antiautophagic members of the B-cell lymphoma-2 (BCL-2) family. In the present study, we investigated the mechanisms of CLL cell death induced as a consequence of VSV infection in the presence of BCL-2 inhibitors, obatoclax, and ABT-737 in primary ex vivo CLL patient samples. Microarray analysis of primary CD19⁺ CD5⁺ CLL cells treated with obatoclax and VSV revealed changes in expression of genes regulating apoptosis, the mechanistic target of rapamycin (mTOR) pathway, and cellular metabolism. A combined therapeutic effect was observed for VSV and BCL-2 inhibitors in cells from untreated patients and from patients unresponsive to standard of care therapy. In addition, combination treatment induced several markers of autophagy--LC3-II accumulation, p62 degradation, and staining of autophagic vacuoles. Inhibition of early stage autophagy using 3-methyladenine (3-MA) led to increased apoptosis in CLL samples. Mechanistically, a combination of BCL-2 inhibitors and VSV disrupted inhibitory interactions of Beclin-1 with BCL-2 and myeloid cell leukemia-1 (MCL-1), thus biasing cells toward autophagy. We propose a mechanism in which changes in cellular metabolism, coupled with pharmacologic disruption of the BCL-2-Beclin-1 interactions, facilitate induction of apoptosis and autophagy to mediate the cytolytic effect of VSV.

Ly49Q positively regulates type I IFN production by plasmacytoid dendritic cells in an immunoreceptor tyrosine-based inhibitory motif-dependent manner.

Plasmacytoid dendritic cells (pDC) are the major producers of type I IFN during the initial immune response to viral infection. Ly49Q, a C-type lectin-like receptor specific for MHC-I, possesses a cytoplasmic ITIM and is highly expressed on murine pDC. Using Ly49Q-deficient mice, we show that, regardless of strain background, this receptor is required for maximum IFN-α production by pDC. Furthermore, Ly49Q expression on pDC, but not myeloid dendritic cells, is necessary for optimal IL-12 secretion, MHC-II expression, activation of CD4(+) T cell proliferation, and nuclear translocation of the master IFN-α regulator IFN regulatory factor 7 in response to TLR9 agonists. In contrast, the absence of Ly49Q did not affect plasmacytoid dendritic cell-triggering receptor expressed on myeloid cells expression or pDC viability. Genetic complementation revealed that IFN-α production by pDC is dependent on an intact tyrosine residue in the Ly49Q cytoplasmic ITIM. However, pharmacological inhibitors and phosphatase-deficient mice indicate that Src homology 2 domain-containing phosphatase 1 (SHP)-1, SHP-2, and SHIP phosphatase activity is dispensable for this function. Finally, we observed that Ly49Q itself is downregulated on pDC in response to CpG exposure in an ITIM-independent manner. In conclusion, Ly49Q enhances TLR9-mediated signaling events, leading to IFN regulatory factor 7 nuclear translocation and expression of IFN-I genes in an ITIM-dependent manner that can proceed without the involvement of SHP-1, SHP-2, and SHIP.

Linear ubiquitination of NEMO negatively regulates the interferon antiviral response through disruption of the MAVS-TRAF3 complex.

The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.

Vesicular stomatitis virus oncolytic treatment interferes with tumor-associated dendritic cell functions and abrogates tumor antigen presentation.

Oncolytic virotherapy is a promising biological approach to cancer treatment that contributes to tumor eradication via immune- and non-immune-mediated mechanisms. One of the remaining challenges for these experimental therapies is the necessity to develop a durable adaptive immune response against the tumor. Vesicular stomatitis virus (VSV) is a prototypical oncolytic virus (OV) that exemplifies the multiple mechanisms of oncolysis, including direct cell lysis, cellular hypoxia resulting from the shutdown of tumor vasculature, and inflammatory cytokine release. Despite these properties, the generation of sustained antitumor immunity is observed only when VSV is engineered to express a tumor antigen directly. In the present study, we sought to increase the number of tumor-associated dendritic cells (DC) in vivo and tumor antigen presentation by combining VSV treatment with recombinant Fms-like tyrosine kinase 3 ligand (rFlt3L), a growth factor promoting the differentiation and proliferation of DC. The combination of VSV oncolysis and rFLt3L improved animal survival in two different tumor models, i.e., VSV-resistant B16 melanoma and VSV-sensitive E.G7 T lymphoma; however, increased survival was independent of the adaptive CD8 T cell response. Tumor-associated DC were actively infected by VSV in vivo, which reduced their viability and prevented their migration to the draining lymph nodes to prime a tumor-specific CD8 T cell response. These results demonstrate that VSV interferes with tumor DC functions and blocks tumor antigen presentation.

Caspase-12 dampens the immune response to malaria independently of the inflammasome by targeting NF-kappaB signaling.

Pathogen sensing by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. Caspase-12 antagonizes the inflammasome and NF-κB and is associated with susceptibility to bacterial sepsis. A single-nucleotide polymorphism (T(125)C) in human Casp12 restricts its expression to Africa, Southeast Asia, and South America. Here, we investigated the role of caspase-12 in the control of parasite replication and pathogenesis in malaria and report that caspase-12 dampened parasite clearance in blood-stage malaria and modulated susceptibility to cerebral malaria. This response was independent of the caspase-1 inflammasome, as casp1(-/-) mice were indistinguishable from wild-type animals in response to malaria, but dependent on enhanced NF-κB activation. Mechanistically, caspase-12 competed with NEMO for association with IκB kinase-α/ß, effectively preventing the formation of the IκB kinase complex and inhibiting downstream transcriptional activation by NF-κB. Systemic inhibition of NF-κB or Ab neutralization of IFN-γ reversed the increased resistance of casp12(-/-) mice to blood-stage malaria infection.

Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC.

Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2K(b). Conversely, CpG-ODN-dependent IFN-alpha production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2K(b) ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo.

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function.

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.