STAT3 is a genetic modifier of TGF-beta induced EMT in KRAS mutant pancreatic cancer.

Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here, we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of the STAT3-responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.

Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Oncogenic dependency plays a dominant role in the immune response to cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Advanced PDAC is considered incurable. Nearly 90% of pancreatic cancers are caused by oncogenic KRAS mutations. The mechanisms of primary or acquired resistance to KRAS inhibition are currently unknown. Here, we propose that oncogenic dependency, rather than KRAS mutation per se, plays a dominant role in the immune response to cancer, including late-stage PDAC. Classifying tumor samples according to KRAS activity scores allows accurate prediction of tumor immune composition and therapy response. Dual RAS/MAPK pathway blockade combining KRAS and MEK inhibitors is more effective than the selective KRAS inhibitor alone in attenuating MAPK activation and unblocking the influx of T cells into the tumor. Lowering KRAS activity in established tumors promotes immune infiltration, but with a limited antitumor effect, whereas combining KRAS/MEK inhibition with immune checkpoint blockade achieves durable regression in preclinical models. The results are directly applicable to stratifying human PDAC based on KRAS dependency values and immune cell composition to improve therapeutic design.

Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice.

Gammaherpesviruses (GHVs) are oncogenic viruses that establish lifelong infections and are significant causes of human morbidity and mortality. While several vaccine strategies to limit GHV infection and disease are in development, there are no FDA-approved vaccines for human GHVs. As a new approach to gammaherpesvirus vaccination, we developed and tested a replication-dead virus (RDV) platform, using murine gammaherpesvirus 68 (MHV68), a well-established mouse model for gammaherpesvirus pathogenesis studies and preclinical therapeutic evaluations. We employed codon-shuffling-based complementation to generate revertant-free RDV lacking expression of the essential replication and transactivator protein (RTA) encoded by ORF50 to arrest viral gene expression early after de novo infection. Inoculation with RDV-50.stop exposes the host to intact virion particles and leads to limited lytic gene expression in infected cells. Prime-boost vaccination of mice with RDV-50.stop elicited virus-specific neutralizing antibody and effector T cell responses in the lung and spleen. Vaccination with RDV-50.stop resulted in a near complete abolishment of virus replication in the lung 7 days post-challenge and virus reactivation from spleen 16 days post-challenge with WT MHV68. Ifnar1-/- mice, which lack the type I interferon receptor, exhibit severe disease upon infection with WT MHV68. RDV-50.stop vaccination of Ifnar1-/- mice prevented wasting and mortality upon challenge with WT MHV68. These results demonstrate that prime-boost vaccination with a GHV that is unable to undergo lytic replication offers protection against acute replication, reactivation, and severe disease upon WT virus challenge.

STAT3 is a genetic modifier of TGF-beta induced EMT in KRAS mutant pancreatic cancer.

Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of STAT3 responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.

B cell-intrinsic STAT3-mediated support of latency and interferon suppression during murine gammaherpesvirus 68 infection revealed through an in vivo competition model.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak latency approximately 7-fold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.

IKKα-Mediated Noncanonical NF-κB Signaling Is Required To Support Murine Gammaherpesvirus 68 Latency In Vivo.

Noncanonical NF-κB signaling is activated in B cells via the tumor necrosis factor (TNF) receptor superfamily members CD40, lymphotoxin ß receptor (LTßR), and B-cell-activating factor receptor (BAFF-R). The noncanonical pathway is required at multiple stages of B cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt noncanonical NF-κB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IκB kinase α (IKKα), named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NF-κB-inducing kinase (NIK). We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTßR-mediated activation of NF-κB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in the colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and wild-type (WT) MHV68 at 16 days postinfection (dpi). Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the noncanonical NF-κB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68. IMPORTANCE The latency programs of the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block noncanonical NF-κB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKα that is nonresponsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the noncanonical NF-κB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.

Interferon-Lambda Intranasal Protection and Differential Sex Pathology in a Murine Model of SARS-CoV-2 Infection.

Outbreaks of emerging viral pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a major medical challenge. There is a pressing need for antivirals that can be rapidly deployed to curb infection and dissemination. We determined the efficacy of interferon lambda-1 (IFN-λ) as a broad-spectrum antiviral agent to inhibit SARS-CoV-2 infection and reduce pathology in a mouse model of disease. IFN-λ significantly limited SARS-CoV-2 production in primary human bronchial epithelial cells in culture. Pretreatment of human lung cells with IFN-λ completely blocked infectious virus production, and treatment with IFN-λ at the time of infection inhibited virus production more than 10-fold. To interrogate the protective effects of IFN-λ in response to SARS-CoV-2 infection, transgenic mice expressing the human angiotensin-converting enzyme 2 (ACE-2) were tested. One dose of IFN-λ administered intranasally was found to reduce animal morbidity and mortality. Our study with SARS-CoV-2 also revealed a sex differential in disease outcome. Male mice had higher mortality, reflecting the more severe symptoms and mortality found in male patients infected with SARS-CoV-2. The results indicate that IFN-λ potentially can treat early stages of SARS-CoV-2 infection and decrease pathology, and this murine model can be used to investigate the sex differential documented in COVID-19. IMPORTANCE The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease. Specific vaccines have been responsible for curbing the transmission of SARS-CoV-2 in developed nations. However, vaccines require time to generate and keep pace with antigenic variants. There is a need for broad-spectrum prophylactic and therapeutic agents to combat new emerging viral pathogens. Our mouse model suggests IFN-λ has clinical utility, and it reflects the well-documented finding that male COVID-19 patients manifest more severe symptoms and mortality. Understanding this sex bias is critical for considering therapeutic approaches to COVID-19.

RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

A role of hypoxia-inducible factor 1 alpha in Murine Gammaherpesvirus 68 (MHV68) lytic replication and reactivation from latency.

The hypoxia-inducible factor 1 alpha (HIF1α) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses (γHV), Kaposi sarcoma herpes virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIF1α during the virus life cycle and its biological relevance in the context of host has been challenging due to the lack of animal models for human γHV. To study the role of HIF1α, we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIF1α protein and HIF1α-responsive gene expression in permissive cells. siRNA silencing or drug-inhibition of HIF1α reduce virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia-responsive elements (HREs) such as the viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1α transcriptional activity during KSHV infection. We found that the promoter of MHV68 ORF74 is responsive to HIF1α and MHV-68 RTA. Moreover, Intranasal infection of HIF1αLoxP/LoxP mice with MHV68 expressing Cre- recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Low oxygen concentrations accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIF1α plays a critical role in promoting virus replication and reactivation from latency by impacting viral gene expression. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis.

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-ß1 (TGF-ß1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. OBJECTIVES: We aimed to define an association between mtDNA and fibroblast responses in IPF. METHODS: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-ß1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. MEASUREMENTS AND MAIN RESULTS: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-ß1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. CONCLUSIONS: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency.

RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.

Sustained activation of toll-like receptor 9 induces an invasive phenotype in lung fibroblasts: possible implications in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive scarring of the lung parenchyma, resulting in a steady decline of lung function and ultimately respiratory failure. The disease course of IPF is extremely variable, with some patients exhibiting stability of symptoms for prolonged periods of time, whereas others exhibit rapid progression and loss of lung function. Viral infections have been implicated in IPF and linked to disease severity; however, whether they directly contribute to progression is unclear. We previously classified patients as rapid and slow progressors on the basis of clinical features and expression of the pathogen recognition receptor, Toll-like receptor 9 (TLR9). Activation of TLR9 in vivo exacerbated IPF in mice and induced differentiation of myofibroblasts in vitro, but the mechanism of TLR9 up-regulation and progression of fibrosis are unknown. Herein, we investigate whether transforming growth factor (TGF)-ß, a pleiotropic cytokine central to IPF pathogenesis, regulates TLR9 in lung myofibroblasts. Results showed induction of TLR9 expression by TGF-ß in lung myofibroblasts and a distinct profibrotic myofibroblast phenotype driven by stimulation with the TLR9 agonist, CpG-DNA. Chronic TLR9 stimulation resulted in stably differentiated α-smooth muscle actin(+)/platelet-derived growth factor receptor α(+)/CD44(+)/matrix metalloproteinase-14(+)/matrix metalloproteinase-2(+) myofibroblasts, which secrete inflammatory cytokines, invade Matrigel toward platelet-derived growth factor, and resist hypoxia-induced apoptosis. These results suggest a mechanism by which TGF-ß and TLR9 responses in myofibroblasts collaborate to drive rapid progression of IPF.