Proteasome α6 Subunit Negatively Regulates the JAK/STAT Pathway and Blood Cell Activation in Drosophila melanogaster

JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. Using mass spectrometry to analyze proteins associated with a negative regulator of the JAK/STAT pathway, and by performing a genome-wide RNAi screen, we identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. Our results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo.

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Downregulation of Graft-Versus-Host-Disease (GVHD) by Treatment with Histone Deacetylase (HDAC) Inhibitors Involves Modulation of the JAK/STAT Signaling Pathway.

Blood ◽

10.1182/blood.v104.11.3050.3050 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3050-3050

Author(s):

Corinna Leng ◽

Margathe Gries ◽

Suzanne Lentzsch ◽

Simone Lusatis ◽

Paolo Mascagni ◽

...

Keyword(s):

T Cell ◽

Histone Deacetylase ◽

Graft Versus Host Disease ◽

Hdac Inhibitors ◽

Inhibitory Effect ◽

Graft Versus Host ◽

Stat Signaling ◽

Stat Pathway

Abstract Graft-versus-host disease (GVHD) mediated by alloreactive donor T cells is the most dreaded complication after allogeneic bone marrow transplantation (BMT). Conditioning therapy in the context of BMT creates a proinflammatory milieu, which is thought to be central to the development of GVHD. Interfering with the conditioning-induced inflammatory response could be an approach to prevent GVHD without compromising the graft-versus-malignancy reaction. Histone deacetylase (HDAC) inhibitors belong to a new family of anti-cancer drugs with potent anti-inflammatory properties and have recently been shown to reduce the development of GVHD. The aim of this study was understand the mechanisms underlying the downregulation of GVHD after treatment with the HDAC inhibitor suberonylanilide hydroxamic acid (SAHA). Using the fully MHC-mismatched strain combination B6 to BALB/c, treatment with SAHA resulted in a significantly reduced GVHD mortality. Thus, at days +10 or +37 post-BMT survival for vehicle-treated or SAHA-treated mice was 33 % versus 86 % and 8 % versus 57 % respectively (Chi2 test, p = 0,027 and p = 0,02, respectively). This was associated with a significant reduction in IFN-g and IL-5 serum levels of SAHA-treated animals. As we could not detect any effect of SAHA treatment on T cell activation or T cell expansion in vitro and in vivo, we hypothesized that the inhibitory effect of SAHA treatment on the development of GVHD might be primarily due to an interference in the early events of the inflammatory cascade occurring after conditioning and initial alloactivation. Therefore, we performed gene expression profiling studies in classical GVHD target organs of animals treated with SAHA or vehicle to further understand the mechanisms underlying this effect. SAHA treated animals revealed a significant upregulation of the mRNA expression of the Protein inhibitor of activated stat 1 (PIAS1) gene in the liver compared to vehicle-treated animals. To further strengthen the hypothesis that SAHA might exert its action by interfering with inflammatory reaction and subsequent signaling through the JAK/STAT pathway, we analyzed the effects of SAHA on STAT-1, 3, and 5 activation and expression of SOCS-1 and SOCS-3 in vitro and in vivo. Thus, BALB/c responder splenocytes were incubated with or without irradiated B6 stimulators in the presence or absence of LPS in order to allow for the separate analysis of LPS and alloactivation-induced JAK/STAT activation. Treatment for 24 hours with SAHA completely inhibited phosphorylation of STAT-1 and STAT-3 in response to LPS and alloactivation using western blot analysis. Furthermore, analysis of liver tissue from GVHD animals showed a sustained expression of SOCS-1 protein in SAHA treated animals whereas SOCS-1 was downregulated in the absence of SAHA. In conclusion our data suggest that the inhibitory effect of SAHA on the development of GVHD is associated with an inhibition of the JAK/STAT signaling pathway. Further studies are warranted to understand the precise mechanisms how SAHA interferes with JAK/STAT signaling and how this leads to inhibition of GVHD. However, it is conceivable that interfering with inflammatory signaling pathways using pharmacological inhibitors of the JAK/STAT pathway might provide a highly attractive treatment strategy for the prevention of GVHD.

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550 An Axl-targeting monoclonal antibody that inhibits Axl activity and potently stimulates the innate immune response

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0550 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A587-A587

Author(s):

Diego Alvarado ◽

Laura Vitale ◽

Mike Murphy ◽

Thomas O’Neill ◽

Edward Natoli ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Cell Activation ◽

Human Cancer ◽

Cell Activity ◽

Negative Regulator

BackgroundAxl is a member of the TAM (Tyro3/Axl/MerTK) family of receptor tyrosine kinases and a negative regulator of innate immunity. Activation of Axl through its ligand Gas6 leads to suppression of myeloid cell activity, while its activation in tumor cells drives tumor growth, metastasis, and is associated with acquired resistance to targeted therapies, radiotherapy and chemotherapy.MethodsPurified monoclonal antibodies and variants thereof were tested in human cancer lines and primary human myeloid cells for effects on Axl signaling and immune activation, respectively.ResultsWe describe a humanized IgG1 Axl-targeting monoclonal antibody (mAb), CDX-0168, that binds to the ligand-binding domain of Axl with sub-nanomolar affinity and potently inhibits Gas6 binding. In tumor cells, CDX-0168 inhibits Gas6-dependent Axl phosphorylation and signaling and elicits tumor cell killing via ADCC in vitro and in vivo. In primary human immune cells, CDX-0168 treatment induces potent release of pro-inflammatory cytokines and chemokines from dendritic cells, monocytes and macrophages through an Fc receptor-dependent mechanism and enhanced T cell activation in mixed lymphocyte reactions. Axl inhibition may further enhance antitumor activity associated with PD-(L)1 blockade. To this end, we generated a tetravalent bispecific Axl x PD-L1 antibody combining CDX-0168 with a potent anti-PD-L1 mAb (9H9) using an IgG-scFv format. The bispecific antibody elicits greater cytokine release and T cell activation in vitro than the combination of the parental antibodies, while maintaining robust Axl and PD-L1 blockade.ConclusionsAdditional studies investigating simultaneous blockade of the Axl and PD-L1 pathways with other agents may further exploit the potential for this novel anti-cancer therapeutic approach.

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Genetic variation at RAB3GAP2 and its role in exercise-related adaptation and recovery

10.21203/rs.3.rs-701652/v1 ◽

2021 ◽

Author(s):

Kristoffer Ström ◽

Nikolay Oskolkov ◽

Tugce Karaderi ◽

Sebastian Kalamajski ◽

Ola Ekström ◽

...

Keyword(s):

Skeletal Muscle ◽

T Cell Activation ◽

Cell Activation ◽

Genome Wide Association Study ◽

Fiber Type ◽

Whole Body ◽

Protein Subunit ◽

A Genome

Abstract Skeletal muscle fiber composition and capillary density influence physical performance and whole-body metabolic properties. ~45% of the variance in fiber type is heritable, which motivated us to perform a genome-wide association study of skeletal muscle histology from 656 Swedish men. Four independent variants were associated (p < 5x10− 8) with proportion of type IIx fibers or capillary-to-fiber ratio (C:F). The strongest signal localized to the rs115660502 variant, where the G-allele corresponded with increased C:F and reduced skeletal muscle expression of the proximal gene, RAB3 GTPase Activating Non-Catalytic Protein Subunit 2 (RAB3GAP2). The G-allele was less frequent in elite short-track sprinters and more frequent in endurance athletes than in matched non-athlete (population) controls; RAB3GAP2 expression was reduced by high-intensity intermittent training. RAB3GAP2 protein was not uniformly expressed in muscle tissue but localized to the endothelium and capillaries. Experimental reduction of RAB3GAP2 in human endothelial cells led to increased tube formation in vitro, to regulation of secreted factors promoting angiogenesis and T-cell activation, to reduced intracellular levels of von Willebrand factor (VWF) and, post-implantation, to increased endothelial cell density in vivo in mice. The amount of RAB3GAP2 in skeletal muscle was positively associated with exercise-induced release of VWF in vivo in humans. By regulating the release of protein factors (VWF, CD70, TNC, TNXB, MCP1, IGFBP3, COL1A1, TFPI2 and tPA), RAB3GAP2 influences fitness adaptation after exercise by improving muscle healing and promotion of capillary formation.

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JAK/STAT pathway inhibition sensitizes CD8 T cells to dexamethasone-induced apoptosis in hyperinflammation

Blood ◽

10.1182/blood.2020006075 ◽

2020 ◽

Vol 136 (6) ◽

pp. 657-668 ◽

Cited By ~ 7

Author(s):

Lauren K. Meyer ◽

Katherine C. Verbist ◽

Sabrin Albeituni ◽

Brooks P. Scull ◽

Rachel C. Bassett ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Immune System Activation ◽

Stat Signaling ◽

Induced Apoptosis ◽

Stat Pathway

Abstract Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.

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The Ups and Downs of STAT Inhibition in Acute Myeloid Leukemia

Biomedicines ◽

10.3390/biomedicines9081051 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1051

Author(s):

Bernhard Moser ◽

Sophie Edtmayer ◽

Agnieszka Witalisz-Siepracka ◽

Dagmar Stoiber

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Janus Kinase ◽

Hematopoietic Malignancy ◽

Stat Signaling ◽

Acute Myeloid ◽

Stat Pathway

Aberrant Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling is implicated in the pathogenesis of acute myeloid leukemia (AML), a highly heterogeneous hematopoietic malignancy. The management of AML is complex and despite impressive efforts into better understanding its underlying molecular mechanisms, survival rates in the elderly have not shown a substantial improvement over the past decades. This is particularly due to the heterogeneity of AML and the need for personalized approaches. Due to the crucial role of the deregulated JAK-STAT signaling in AML, selective targeting of the JAK-STAT pathway, particularly constitutively activated STAT3 and STAT5 and their associated upstream JAKs, is of great interest. This strategy has shown promising results in vitro and in vivo with several compounds having reached clinical trials. Here, we summarize recent FDA approvals and current potential clinically relevant inhibitors for AML patients targeting JAK and STAT proteins. This review underlines the need for detailed cytogenetic analysis and additional assessment of JAK-STAT pathway activation. It highlights the ongoing development of new JAK-STAT inhibitors with better disease specificity, which opens up new avenues for improved disease management.

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ZAP-70 Regulates Autoimmune Arthritis via Alterations in T Cell Activation and Apoptosis

Cells ◽

10.3390/cells8050504 ◽

2019 ◽

Vol 8 (5) ◽

pp. 504 ◽

Cited By ~ 1

Author(s):

Réka Kugyelka ◽

Lilla Prenek ◽

Katalin Olasz ◽

Zoltán Kohl ◽

Bálint Botz ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Severe Disease ◽

Negative Regulator ◽

Human Rheumatoid Arthritis ◽

Autoantibody Production

T cells play an essential role in the pathogenesis of both human rheumatoid arthritis (RA) and its murine models. A key molecule in T cell activation is ZAP-70, therefore we aimed to investigate the effects of partial ZAP-70 deficiency on the pathogenesis of recombinant human G1(rhG1)-induced arthritis (GIA), a well-established mouse model of RA. Arthritis was induced in BALB/c and ZAP-70+/− heterozygous mice. Disease progression was monitored using a scoring system and in vivo imaging, antigen-specific proliferation, cytokine and autoantibody production was measured and T cell apoptotic pathways were analyzed. ZAP-70+/− mice developed a less severe arthritis, as shown by both clinical picture and in vitro parameters (decreased T cell proliferation, cytokine and autoantibody production). The amount of cleaved Caspase-3 increased in arthritic ZAP-70+/− T cells, with no significant changes in cleaved Caspase-8 and -9 levels; although expression of Bim, Bcl-2 and Cytochrome C showed alterations. Tyrosine phosphorylation was less pronounced in arthritic ZAP-70+/− T cells and the amount of Cbl-b—a negative regulator of T cell activation—decreased as well. We hypothesize that the less severe disease seen in the partial absence of ZAP-70 might be caused by the decreased T cell activation accompanied by increased apoptosis.

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SAT0246 TARGETING JAK/STAT PATHWAY IN TAKAYASU’S ARTERITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4987 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1066.1-1066

Author(s):

P. Régnier ◽

A. Le Joncour ◽

A. Maciejewski-Duval ◽

A. C. Desbois ◽

C. Comarmond ◽

...

Keyword(s):

T Cells ◽

Signaling Pathway ◽

Cd8 T Cells ◽

Takayasu’S Arteritis ◽

Type I ◽

Cd25 Expression ◽

Stat Signaling ◽

Stat Pathway

Background:Takayasu’s arteritis (TAK) is a large vessel vasculitis (LVV) in which the aorta and its main branches are greatly inflamed, leading to wall thickening, fibrosis, stenosis and to artery occlusion(1). The disease is more common in women mostly between 20 and 30 years old. TAK has a high morbidity rate: 50% of patients will relapse within 10 years after diagnosis(2, 3). This inflammation is essentially mediated by infiltration with macrophages and pro-inflammatory Th1/Th17 effector subsets(4–8). But the mechanisms behind these phenomena are essentially unknown. TAK is mainly treated with non-specific steroids(1) which are associated with potential side effects when used for a long-time course.Objectives:Our work aims to explore the involvement of JAK/STAT signaling pathway and its downstream biological cascades in pro-inflammatory T cells differentiation and disease activity of TAK. Plus, our work allows to consider targeting the JAK/STAT pathway in TAK using JAK inhibitors (JAKinibs).Methods:We analyzed transcriptome of FACS-sorted CD4+ and CD8+ T cells from healthy donors (HD) and TAK, using differential gene, pathway and network analysis. Then, we assessed in vitro and in vivo effects of JAKinibs in TAK by flow cytometry (FC).Results:Transcriptome analysis showed hundreds of significantly dysregulated genes/pathways for CD4+ and CD8+ samples between HD and TAK. Among these, we noticed in TAK a great enrichment for pathways linked to type I and II interferons (IFN), JAK/STAT and cytokines/chemokines-related signaling. We confirmed by RT-qPCR the upregulation of a type I IFN-specific gene signature in TAK T cells as compared to HD. Using genes coming from the previous pathways, we constructed networks connecting them according to their respective protein interactions. This representation showed for both CD4+ and CD8+ T cells that JAK and STAT genes were densely connected, thus representing core genes/proteins in the TAK physiopathology.We then performed in vitro cell cultures of PBMCs from HD or TAK supplemented with Ruxolitinib (JAK1/2 inhibitor) or PBS. We observed by FC that JAKinibs significantly induced in TAK CD4+ and CD8+ T cells reduction of CD25 expression, decrease of Th1/Th17 pro-inflammatory cells and increase of Tregs.Next, we followed by FC 3 TAK (refractory to conventional treatments) treated with JAKinibs. We also observed in their PBMCs a reduction of CD25 expression by CD4+ T cells, a decrease of Th1 and Th17 cells and an increase of Tregs, accompanied by an increase of the Tregs/Teffs ratio. JAKinibs also decreased C-Reactive Protein level, NIH score and co-administered steroids doses (present before JAKinibs introduction) in these 3 in vivo-treated TAK.Conclusion:JAK/STAT signaling pathway is critical in the pathogenesis of TAK and JAKinibs may be promising in its treatment.References:[1]F. Numano, M. Okawara, H. Inomata, Y. Kobayashi, The Lancet. 356, 1023–1025 (2000).[2]C. Comarmond et al., Circulation. 136, 1114–1122 (2017).[3]A. Mirouse et al., J. Autoimmun. 96, 35–39 (2019).[4]C. M. Weyand, J. J. Goronzy, Nat. Rev. Rheumatol. 9, 731–740 (2013).[5]C. M. Weyand et al., Clin. Immunol. 206, 33–41 (2019).[6]D. Saadoun et al., Arthritis Rheumatol. 67, 1353–1360 (2015).[7]T. Mirault, H. Guillet, E. Messas, Presse Médicale. 46, e189–e196 (2017).[8]D. P. Misra, S. Chaurasia, R. Misra, Autoimmune Dis. 2016, 1–8 (2016).Disclosure of Interests:Paul Régnier: None declared, Alexandre Le Joncour: None declared, Anna Maciejewski-Duval: None declared, Anne-Claire DESBOIS: None declared, Cloé Comarmond: None declared, Michelle Rosenzwajg: None declared, David Klatzmann Consultant of: ILTOO Pharma, Patrice cacoub: None declared, David Saadoun: None declared

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Regulation of c-Jun NH2-terminal Kinase ( Jnk) Gene Expression during T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.191.1.139 ◽

2000 ◽

Vol 191 (1) ◽

pp. 139-146 ◽

Cited By ~ 72

Author(s):

Linda Weiss ◽

Alan J. Whitmarsh ◽

Derek D. Yang ◽

Mercedes Rincón ◽

Roger J. Davis ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

T Cell Activation ◽

Map Kinases ◽

Cell Activation ◽

Cell Receptor ◽

Jnk Pathway ◽

Gene And Protein Expression

The c-Jun NH2-terminal kinases (JNKs) are a group of mitogen-activated protein (MAP) kinases that participate in signal transduction events mediating specific cellular functions. Activation of JNK is regulated by phosphorylation in response to cellular stress and inflammatory cytokines. Here, we demonstrate that JNK is regulated by a second, novel mechanism. Induction of Jnk gene expression is required in specific tissues before activation of this signaling pathway. The in vivo and in vitro ligation of the T cell receptor (TCR) leads to induction of JNK gene and protein expression. TCR signals are sufficient to induce JNK expression, whereas JNK phosphorylation also requires CD28-mediated costimulatory signals. Therefore, both expression and activation contribute to the regulation of the JNK pathway to ensure proper control during the course of an immune response.

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The E3 Ubiquitin Ligase NARF Promotes Colony Formation in vitro and Exhibits Enhanced Expression Levels in Glioblastoma Multiforme in vivo

American Journal of Undergraduate Research ◽

10.33697/ajur.2010.017 ◽

2010 ◽

Vol 9 (2 and 3) ◽

Cited By ~ 1

Author(s):

Tucker Anderson ◽

Christopher Wright ◽

William Brooks

Keyword(s):

Glioblastoma Multiforme ◽

Ubiquitin Ligase ◽

Colony Formation ◽

Ring Finger ◽

Negative Regulator ◽

Low Grade ◽

Dependent Manner ◽

Enhanced Expression

The ubiquitin ligase NARF is a newly identified protein belonging to a small family of structurally similar E3 proteins. NARF is a negative regulator of the canonical Wnt-β-catenin pathway, targeting TCF/LEF family members for proteolytic degradation through poly-ubiquitination. We examined the role that NARF plays in cell division and found that overexpression of NARF in a colony forming assay increases colony formation in a RING finger-dependent manner. Furthermore, we demonstrate that NARF transcripts are expressed at a higher level in the grade IV brain tumor glioblastoma multiforme as compared with low grade astrocytomas. Our data thus indicate that NARF is a positive regulator of cell growth and may be involved in the tumorigenic process.

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A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability

10.1101/187286 ◽

2017 ◽

Author(s):

Katherine H Fisher ◽

Maria Fragiadaki ◽

Dhamayanthi Pugazhendhi ◽

Nina Bausek ◽

Stephen Brown ◽

...

Keyword(s):

Rnai Screen ◽

Janus Kinase ◽

Cytokine Receptor ◽

Cytokine Receptors ◽

Positive Regulator ◽

Genome Wide ◽

A Genome ◽

Stat Pathway

AbstractIn order for cells to sense and thus respond to their environment, they require transmembrane receptors, which bind extracellular ligands and then transduce this signal within the cell. A subset of receptors, with single-pass transmembrane domains are known as cytokine receptors and act via the Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) pathway. These receptors are essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are largely unknown. In order to better understand the processes regulating cytokine receptor levels, activity and dimerisation, we used the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We have performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. We show that MASK is able to regulate JAK/STAT signalling both in vitro and in vivo. We go on to show that MASK is able to bind to Domeless via its Ankyrin repeat domains and alters the stability of the receptor. Finally, we extend our observations to the human homologue, ANKHD1, and demonstrate functional conservation, with ANKHD1 able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, we have identified MASK as a conserved regulator of cytokine receptor levels, which may have implications for human health.

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