JAK-STAT signaling maintains homeostasis in T cells and macrophages.

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.

Oncogenic TYK2 P760L kinase is effectively targeted by combinatorial TYK2, mTOR and CDK4/6 kinase blockade.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALL) or precursor B-ALL. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2 P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2 P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2 P760L-transformed cell models and ex vivo cultured TYK2 P760L-mutated patient- derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.

Temporal stability of fecal cortisol metabolites in mountain-dwelling ungulates.

Fecal cortisol metabolites (FCMs) are widely used to track stress responses in wildlife and captive species. Rules of thumb suggest that samples should be collected as soon as possible after defecation, to avoid decay of FCMs. To date, however, only a few studies investigated the stability of defecated FCMs over time, and most of them were conducted in controlled laboratory conditions. Here, we investigated the stability of FCMs over seven consecutive days, in two mountain-dwelling ungulates, under natural environmental conditions using a semi-experimental approach. Fecal samples from Northern chamois Rupicapra rupicapra (n = 24) and red deer Cervus elaphus (n = 22) were collected in summer of 2020 within the Stelvio National Park, Italy, and placed in an open area above 2000 m a.s.l. For the next 7 days, we collected a portion of each sample, and all sub-samples were analyzed with an 11-oxoetiocholanolone enzyme immunoassay. Exposure, temperature, and precipitation were fitted as covariates in non-linear generalized mixed models to assess FCM variation over time, and competing models were selected using AICc. For chamois, the best model included only time as a predictor, while for red deer, it included time, precipitation, and exposure. For both species, FCM values decreased rapidly from the first days after deposition until the fourth day. For red deer, in northern-exposed samples, FCM values decreased slower than in south-exposed ones; furthermore, FCM values increased with increasing precipitation. Our results offer a solid methodological basis to wildlife researchers and practitioners interested in the investigation of the ecological factors affecting stress variation in wildlife and support the recommendation to collect samples as fresh as possible, to avoid misleading inference. Further studies are necessary to evaluate the stability of FCMs when other enzyme immunoassays are used.

Upregulation of signal transducer and activator of transcription 3 in dogs with chronic inflammatory enteropathies.

BACKGROUND: In inflammatory bowel disease (IBD) in humans, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) is upregulated in mucosal epithelial cells and correlates with clinical severity. HYPOTHESIS/OBJECTIVE: To investigate the expression pattern of pSTAT3 in the mucosa of dogs with chronic inflammatory enteropathy (CIE) and explore correlations between its expression and clinical and histopathological severity scoring. ANIMALS: Twenty-eight canine CIE patients grouped into food-responsive enteropathy (FRE; 9), steroid-responsive enteropathy (SRE; 10), and protein-losing enteropathy (PLE; 9). Ten healthy beagle dogs served as controls (CO). METHODS: Retrospective case control study. Immunohistochemistry was used to detect pSTAT3 in canine duodenal mucosa samples. RESULTS: Compared to CO, SRE (P < .001) and PLE (P < .001) dogs had significantly higher pSTAT3 expression in the villus epithelium. The SRE group had a significantly higher expression in the villus lamina propria (VLP) compared to controls (P = .009). In the crypt epithelium (CE), all CIE dogs had significantly higher pSTAT3 expression (FRE, P = .002; SRE, P = .003; PLE, P < .001) compared to CO. In the lamina propria crypt region (CLP), dogs with FRE (P = .04) and SRE (P = .03) had significantly upregulated pSTAT3 compared to controls. A positive correlation was found between canine chronic enteropathy clinical activity index (CCECAI) scoring and pSTAT3 expression for both epithelial (rho = .541; P < .001) and crypt regions (rho = .32; P = .02). CONCLUSIONS AND CLINICAL IMPORTANCE: pSTAT3 is upregulated in CIE in dogs, correlates with clinical severity, and may be helpful as a clinical marker in dogs with CIE.

TYK2 in Tumor Immunosurveillance.

We review the history of the tyrosine kinase 2 (TYK2) as the founding member of the Janus kinase (JAK) family and outline its structure-function relation. Gene-targeted mice and hereditary defects of TYK2 in men have established the biological and pathological functions of TYK2 in innate and adaptive immune responses to infection and cancer and in (auto-)inflammation. We describe the architecture of the main cytokine receptor families associated with TYK2, which activate signal transducers and activators of transcription (STATs). We summarize the cytokine receptor activities with well characterized dependency on TYK2, the types of cells that respond to cytokines and TYK2 signaling-induced cytokine production. TYK2 may drive beneficial or detrimental activities, which we explain based on the concepts of tumor immunoediting and the cancer-immunity cycle in the tumor microenvironment. Finally, we summarize current knowledge of TYK2 functions in mouse models of tumor surveillance. The biology and biochemistry of JAKs, TYK2-dependent cytokines and cytokine signaling in tumor surveillance are well covered in recent reviews and the oncogenic properties of TYK2 are reviewed in the recent Special Issue 'Targeting STAT3 and STAT5 in Cancer' of Cancers.

TYK2: An Upstream Kinase of STATs in Cancer.

In this review we concentrate on the recent findings describing the oncogenic potential of the protein tyrosine kinase 2 (TYK2). The overview on the current understanding of TYK2 functions in cytokine responses and carcinogenesis focusses on the activation of the signal transducers and activators of transcription (STAT) 3 and 5. Insight gained from loss-of-function (LOF) gene-modified mice and human patients homozygous for Tyk2/TYK2-mutated alleles established the central role in immunological and inflammatory responses. For the description of physiological TYK2 structure/function relationships in cytokine signaling and of overarching molecular and pathologic properties in carcinogenesis, we mainly refer to the most recent reviews. Dysregulated TYK2 activation, aberrant TYK2 protein levels, and gain-of-function (GOF) TYK2 mutations are found in various cancers. We discuss the molecular consequences thereof and briefly describe the molecular means to counteract TYK2 activity under (patho-)physiological conditions by cellular effectors and by pharmacological intervention. For the role of TYK2 in tumor immune-surveillance we refer to the recent Special Issue of Cancers "JAK-STAT Signaling Pathway in Cancer".

Myeloid Cells Restrict MCMV and Drive Stress-Induced Extramedullary Hematopoiesis through STAT1.

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection-associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen.

The good and the bad faces of STAT1 in solid tumours.

Signal transducer and activator of transcription (STAT) 1 is part of the Janus kinase (JAK)/STAT signalling cascade and is best known for its essential role in mediating responses to all types of interferons (IFN). STAT1 regulates a variety of cellular processes, such as antimicrobial activities, cell proliferation and cell death. It exerts important immune modulatory activities both in the innate and the adaptive arm of the immune system. Based on studies in mice and data from human patients, STAT1 is generally considered a tumour suppressor but there is growing evidence that it can also act as a tumour promoter. This review aims at contrasting the two faces of STAT1 in tumourigenesis and providing an overview on the current knowledge of the underlying mechanisms or pathways.

High-throughput mRNA and miRNA profiling of epithelial-mesenchymal transition in MDCK cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an important process in embryonic development, especially during gastrulation and organ formation. Furthermore EMT is widely observed in pathological conditions, e.g., fibrosis, tumor progression and metastasis. Madin-Darby Canine Kidney (MDCK) cells are widely used for studies of EMT and epithelial plasticity. MDCK cells show an epithelial phenotype, while oncogenic Ras-transformed MDCK (MDCK-Ras) cells undergo EMT and show a mesenchymal phenotype. METHODS: RNA-Seq and miRNA-Seq analyses were performed on MDCK and MDCK-Ras cells. Data were validated by qRT-PCR. Gene signature analyses were carried out to identify pathways and gene ontology terms. For selected miRNAs target prediction was performed. RESULTS: With RNA-Seq, mRNAs of approximately half of the genes known for dog were detected. These were screened for differential regulation during Ras-induced EMT. We went further and performed gene signature analyses and found Gene Ontology (GO) terms and pathways important for epithelial polarity and implicated in EMT. Among the identified pathways, TGFß1 emerged as a central signaling factor in many EMT related pathways and biological processes. With miRNA-Seq, approximately half of the known canine miRNAs were found expressed in MDCK and MDCK-Ras cells. Furthermore, among differentially expressed miRNAs, miRNAs that are known to be important regulators of EMT were detected and new candidates were predicted. New dog miRNAs were discovered after aligning our reads to that of other species in miRBase. Importantly, we could identify 25 completely novel miRNAs with a stable hairpin structure. Two of these novel miRNAs were differentially expressed. We validated the two novel miRNAs with the highest read counts by RT-qPCR. Target prediction of a particular novel miRNA highly expressed in mesenchymal MDCK-Ras cells revealed that it targets components of epithelial cell junctional complexes. Combining target prediction for the most upregulated miRNAs and validation of the targets in MDCK-Ras cells with pathway analysis allowed us to identify two novel pathways, e.g., JAK/STAT signaling and pancreatic cancer pathways. These pathways could not be detected solely by gene set enrichment analyses of RNA-Seq data. CONCLUSION: With deep sequencing data of mRNAs and miRNAs of MDCK cells and of Ras-induced EMT in MDCK cells, differentially regulated mRNAs and miRNAs are identified. Many of the identified genes are within pathways known to be involved in EMT. Novel differentially upregulated genes in MDCK cells are interferon stimulated genes and genes involved in Slit and Netrin signaling. New pathways not yet linked to these processes were identified. A central pathway in Ras induced EMT is TGFß signaling, which leads to differential regulation of many target genes, including miRNAs. With miRNA-Seq we identified miRNAs involved in either epithelial cell biology or EMT. Finally, we describe completely novel miRNAs and their target genes.

Targeting a cell state common to triple-negative breast cancers.

Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

Interleukin-like epithelial-to-mesenchymal transition inducer activity is controlled by proteolytic processing and plasminogen-urokinase plasminogen activator receptor system-regulated secretion during breast cancer progression.

INTRODUCTION: Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive. METHODS: The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients. RESULTS: We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer. CONCLUSIONS: Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.