Dissecting the role of Janus Kinases in Common Cytokine Receptor y-chain signaling in T-cells using chemical genetics

使用化学遗传学剖析 Janus 激酶在 T 细胞常见细胞因子受体 y 链信号传导中的作用

• 批准号: 9105153
• 负责人: Geoffrey Alexander Smith
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9105153
• 关键词: Acute; Address; American; Autoimmune Diseases; Binding; Blast Cell; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Cycle; Cell Proliferation; Cells; Chemicals; Complex; Cysteine; Cytokine Receptors; Cytokine Signaling; Data; Development; Disease; Dose; Effectiveness; Event; Exhibits; Family member; Future; Genes; Genetic Techniques; Genetic Transcription; Health; Hour; IL2RA gene; Immune; Immune system; Interleukin 2 Receptor Gamma; Interleukin-2; Janus kinase; Janus kinase 1; Kinetics; Lymphocyte Function; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Modeling; Molecular Immunology; Monitor; Mus; Pattern; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Play; Pre-Clinical Model; Proteins; Research; Rheumatoid Arthritis; Role; S Phase; Signal Transduction; Signaling Molecule; Stat5 protein; T-Lymphocyte; Testing; Therapeutic; Therapeutic Effect; Transcript; Up-Regulation; Work; Zymosan; cell type; chemical genetics; cytokine; differential expression; follow-up; genetic approach; in vivo; inhibitor/antagonist; knock-down; melanoma; overexpression; receptor; response; scaffold; small molecule

DESCRIPTION (provided by applicant): γ-chain cytokines, such as IL-2, signal through a receptor complex that contains both the Jak1 and Jak3 kinases. Whether these two kinases play distinct or redundant roles remains unclear, with recent work suggesting that while Jak1 kinase activity is essential, Jak3 acts primarily as a scaffold to bring together other parts of th signaling complex. To address the catalytic role of Jak3, a highly selective and potent Jak3 inhibitor was developed by targeting a cysteine found in Jak3, but not in family members Jak1, Jak2 or Tyk2. In murine CD4+ T-cell blasts, the Jak3 inhibitor potently blocked IL-2 driven proliferation and up-regulation of CD25. Unlike the Jak1/Jak2 inhibitor ruxolitinib or the pan-Jak inhibitor tofacitinib, this Jak3 inhibitor was not nearly as potent at blocking STAT5 phosphorylation after acute IL-2 stimulation. Most research on IL-2 signaling has focused on phosphorylation changes that occur in the first hour. However, preliminary data suggests that there are two "waves" of STAT5 phosphorylation following IL-2 stimulation: one that peaks 15 minutes after stimulation and reduces nearly to baseline by an hour and a second, weaker wave that peaks around 6 hours. Interestingly, although Jak1 blockade blocks phosphorylation in both of these waves, the Jak3 inhibitor potently blocks only this second wave of phosphorylation, suggesting a non-redundant role for Jak3 in sustaining signaling. The proposed work aims to explore the consequences and generality of this signaling pattern. Aim 1 will use mRNA-seq to determine whether a Jak3 inhibitor blocking just this second wave of signaling blocks all IL-2 driven transcriptional changes or a specific subset of those changes. Preliminary studies suggest that CD8+ T-cell blasts do not display these two waves of STAT5 phosphorylation but rather a single sustained peak of STAT5 phosphorylation. Aim 2 will exploit this difference to elucidate the mechanism and consequences of these distinct signaling patterns. The Jak3 inhibitor and the Jak1/2 inhibitor ruxolitinib will be used to probe the temporal roles of each kinase, and the expression of negative signaling regulators, such as SOCS proteins, will be compared to identify potential mechanisms. Aim 3 will assess the in vivo consequences of this signaling pattern in the SKG model of rheumatoid arthritis. This model is known to respond to tofacitinib, which blocks nearly all cytokine signaling, but it is unknown whether selectively blockingγ-chain cytokines is sufficient. To address this question, a high dose of the Jak3 inhibitor, sufficient to block all STAT5 phosphorylation, will be given and disease score monitored. In parallel, a low dose of the inhibitor will be used to assess whether blocking only sustained signaling is sufficient for a therapeutic effect. Completion of this project will both further our basic understanding of cytokine signaling and have immediate translational implications for the treatment of rheumatoid arthritis.

描述（由适用提供）：γ链细胞因子（例如IL-2）通过接收器复合物信号，含有JAK1和JAK3激酶。这两个激酶是否起着独特的或冗余的作用，最近的工作表明，尽管JAK1激酶活性是必不可少的，但JAK3作为脚手架起主要作用，作为将TH信号复合物的其他部分汇总在一起的脚手架。为了解决JAK3的催化作用，通过靶向JAK3中发现的半胱氨酸，而不是在家庭成员JAK1，JAK2或TYK2中开发高度选择性和潜在的JAK3抑制剂。在鼠CD4+ T细胞爆炸中，JAK3抑制剂可能阻断IL-2驱动的增殖和CD25的上调。与JAK1/JAK2抑制剂r氧替尼或PAN-JAK抑制剂tofacitinib不同，该JAK3抑制剂在急性IL-2刺激后阻止STAT5磷酸化的潜力并不是潜力。关于IL-2信号传导的大多数研究都集中在第一个小时内发生的磷酸化变化。然而，初步数据表明，IL-2刺激后有两个“ STAT5磷酸化”的“波浪”：一个在刺激后15分钟达到峰值，几乎将基线降低到一个小时，第二个较弱的波浪峰值约为6小时。有趣的是，尽管JAK1阻断了这两个波中的磷酸化，但JAK3抑制剂可能仅阻止第二波光基化，这表明JAK3在维持信号传导中起着非冗余作用。拟议的工作旨在探讨这种信号传导模式的后果和普遍性。 AIM 1将使用mRNA-SEQ来确定JAK3抑制剂是否仅阻止第二波信号传导阻塞所有IL-2驱动的转录变化或这些变化的特定子集。初步研究表明，CD8+ T细胞爆炸没有显示这两个STAT5磷酸化的波，而是STAT5磷酸化的单个持续峰。 AIM 2将探索这种差异，以阐明这些不同的信号传导模式的机制和后果。将使用JAK3抑制剂和JAK1/2抑制剂r氧替尼来探测每种激酶的临时作用，并且将比较负信号调节剂（例如SOCS蛋白）的表达，以识别潜在的机制。 AIM 3将评估类风湿关节炎SKG模型中这种信号传导模式的体内后果。已知该模型对tofacitinib的反应，该模型几乎阻止了所有细胞因子信号传导，但是未知是否有选择地阻断γ链细胞因子就足够了。为了解决这个问题，将给出一定剂量的JAK3抑制剂，足以阻止所有STAT5磷酸化并监测疾病评分。同时，将使用低剂量的抑制剂来评估仅阻断持续信号是否足以产生治疗作用。该项目的完成将进一步我们对细胞因子信号传导的基本理解，并且对类风湿关节炎的治疗具有直接的翻译意义。