ag-490 and tofacitinib

Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-β signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-β signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-β and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-β triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-β receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-β-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-β-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.

Topics: Brain Neoplasms; Catechin; Cell Line, Tumor; Concanavalin A; Epithelial-Mesenchymal Transition; Fibronectins; Glioblastoma; Humans; Matrix Metalloproteinase 14; Piperidines; Pyrazoles; Pyrimidines; Quinolines; Receptors, Transforming Growth Factor beta; Signal Transduction; Snail Family Transcription Factors; STAT3 Transcription Factor; Transforming Growth Factor beta1; Tyrphostins

We studied the involvement of individual JAK kinases in the realization of the growth potential of mesenchymal precursors under the effect of fibroblast growth factor. The important role of JAK2 and JAK3 in determining the initial level of mitotic activity of progenitor cells and participation of JAK1 in this process under conditions of cytokine stimulation of progenitor cells were demonstrated. Specific inhibitors of these kinases reduced the yield of fibroblast CFU and the rate of their division. Moreover, blockade of JAK1, JAK2, and JAK3 under the effect of fibroblast growth factor was accompanied by an increase in the intensity of progenitor cell differentiation.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Colony-Forming Units Assay; Fibroblast Growth Factors; Fibroblasts; Gene Expression Regulation; Janus Kinase 1; Janus Kinase 2; Janus Kinase 3; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred CBA; Piperidines; Primary Cell Culture; Protein Kinase Inhibitors; Pyrimidines; Pyrroles; Signal Transduction; Tyrphostins

Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the Janus-activated kinase (JAK) tyrosine kinase family, TYK2, and its downstream effector STAT1, in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently showed TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway in T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of interleukin (IL)-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the antiapoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway.. In recent years, "pathway dependence" has been revealed in specific types of human cancer, which can be important because they pinpoint proteins that are particularly vulnerable to antitumor-targeted inhibition (so-called Achilles’ heel proteins). Here, we use RNAi technology to identify a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression

Topics: Animals; Antineoplastic Agents; Bone Marrow Cells; Cell Line; Cell Survival; Cells, Cultured; Humans; Interleukin-10; Janus Kinase 3; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Piperidines; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Pyrroles; RNA Interference; Signal Transduction; STAT1 Transcription Factor; TYK2 Kinase; Tyrphostins

Human bone marrow-derived mesenchymal stromal cells (MSCs) express Toll-like receptors (TLRs) and produce cytokines and chemokines, all of which contribute to these cells' immunomodulatory and proangiogenic properties. Among the secreted cytokines, colony-stimulating factors (CSFs) regulate angiogenesis through activation of endothelial cell proliferation and migration. Since MSC are recruited within hypoxic tumors where they signal paracrine-regulated angiogenesis, the aim of this study was to evaluate which CSF members are expressed and are inducible in activated MSC. Furthermore, we investigated the JAK/STAT signal transducing pathway that may impact on CSF transcription. MSC were activated with Concanavalin-A (ConA), a TLR-2/6 agonist as well as a membrane type-1 matrix metalloproteinase (MT1-MMP) inducer, and we found increased transcription of granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP. Gene silencing of either STAT3 or MT1-MMP prevented ConA-induced phosphorylation of STAT3, and reversed ConA effects on CSF-2 and CSF-3. Treatment with the Janus Kinase (JAK)2 inhibitor AG490 antagonized the ConA induction of MT1-MMP and CSF-2, while the pan-JAK inhibitor Tofacitinib reversed ConA-induced CSF-2 and -3 gene expression. Silencing of JAK2 prevented the ConA-mediated increase of CSF-2, while silencing of JAK1, JAK3 and TYK2 prevented the increase in CSF-3. Given that combined TLR-activation and locally-produced CSF-2 and CSF-3 could regulate immunomodulation and neovascularization, pharmacological targeting of TLR-2/6-induced MT1-MMP/JAK/STAT3 signalling pathway may prevent MSC contribution to tumor development.

Topics: Cell Movement; Cell Proliferation; Cells, Cultured; Concanavalin A; Enzyme Inhibitors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Janus Kinases; Macrophage Colony-Stimulating Factor; Matrix Metalloproteinase 1; Mesenchymal Stem Cells; Neovascularization, Physiologic; Phosphorylation; Piperidines; Protein Kinase Inhibitors; Pyrimidines; Pyrroles; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Toll-Like Receptor 2; Toll-Like Receptor 6; Transcription, Genetic; TYK2 Kinase; Tyrphostins

New immunosuppressive compounds with less systemic toxicity that could replace calcineurin inhibitors are urgently needed. For identification of specific inhibitors of JAK3, a potential new drug target, from large chemical libraries we developed a cell-based screening system. TEL-JAK fusion proteins composed of an oligomerization domain of TEL and kinase and/or pseudokinase domains of JAKs provided constitutive activation of JAKs without receiving a signal from the cytokine receptors. These fusion proteins also induced STAT5b phosphorylation in the absence of cytokine receptors. Both the kinase and pseudokinase domains of JAKs were required for full activation of the JAKs, and four copies of STAT5 response elements provided the greatest luciferase activity. The sensitivity and specificity of the system was evaluated using specific JAK3, JAK2, or MEK inhibitors. Thus, we generated a receptor-independent, cell-based selective screening system for specific JAK3 inhibitors, which is easily convertible to a high-throughput screening platform.

Topics: Binding Sites; Butadienes; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; ETS Translocation Variant 6 Protein; Genes, Reporter; High-Throughput Screening Assays; Humans; Immunosuppressive Agents; Janus Kinase 2; Janus Kinase 3; MAP Kinase Kinase Kinases; Nitriles; Phosphorylation; Piperidines; Protein Kinase Inhibitors; Protein Structure, Tertiary; Proto-Oncogene Proteins c-ets; Pyrimidines; Pyrroles; Recombinant Fusion Proteins; Repressor Proteins; Reproducibility of Results; Response Elements; STAT5 Transcription Factor; Transfection; Tyrphostins

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

Topics: Animals; Enzyme Inhibitors; Gene Expression Regulation; Graft Rejection; Graft Survival; Heart Transplantation; Humans; Immunosuppressive Agents; Interleukin-2; Janus Kinase 3; Kidney Transplantation; Lymphocyte Activation; Lymphocyte Count; Lymphocyte Culture Test, Mixed; Lymphocyte Subsets; Macaca fascicularis; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Myocardium; Piperidines; Protein-Tyrosine Kinases; Pyrimidines; Pyrroles; Transplantation, Heterotopic; Transplantation, Homologous; Tumor Cells, Cultured