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

描述（由申请人提供）：γ链细胞因子，如IL-2，通过包含Jak1和Jak3激酶的受体复合物发出信号。目前尚不清楚这两种激酶是否发挥着不同的或冗余的作用，最近的研究表明，虽然Jak1激酶的活性是必不可少的，但Jak3主要是作为一个支架，将信号复合物的其他部分聚集在一起。为了解决Jak3的催化作用，一种高选择性和有效的Jak3抑制剂通过靶向Jak3中发现的半胱氨酸，而不是在Jak1， Jak2或Tyk2家族成员中发现。在小鼠CD4+ t细胞中，Jak3抑制剂能有效阻断IL-2驱动的增殖和CD25的上调。与Jak1/Jak2抑制剂ruxolitinib或pan-Jak抑制剂tofacitinib不同，这种Jak3抑制剂在急性IL-2刺激后阻断STAT5磷酸化的作用几乎没有那么有效。大多数关于IL-2信号传导的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，在IL-2刺激后，STAT5磷酸化有两个“波”：一个在刺激后15分钟达到峰值，并在接近基线时减少一个小时，第二个较弱的波在6小时左右达到峰值。有趣的是，尽管Jak1阻断剂阻断了这两波磷酸化，但Jak3抑制剂仅有效阻断了这第二波磷酸化，这表明Jak3在维持信号传导方面具有非冗余作用。提出的工作旨在探索这种信号模式的后果和普遍性。Aim 1将使用mRNA-seq来确定Jak3抑制剂阻断这第二波信号是否阻断了所有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细胞原始细胞不表现出这两波STAT5磷酸化，而是一个持续的STAT5磷酸化峰值。目的2将利用这种差异来阐明这些不同信号模式的机制和后果。Jak3抑制剂和Jak1/2抑制剂ruxolitinib将用于探测每种激酶的时间作用，并将比较负信号调节因子（如SOCS蛋白）的表达以确定潜在机制。目的3将评估这种信号模式在类风湿关节炎SKG模型中的体内后果。已知该模型对tofacitinib有反应，tofacitinib阻断几乎所有细胞因子信号，但尚不清楚选择性阻断γ链细胞因子是否足够。为了解决这个问题，将给予高剂量的Jak3抑制剂，足以阻断所有STAT5磷酸化，并监测疾病评分。同时，低剂量抑制剂将用于评估仅阻断持续信号传导是否足以产生治疗效果。该项目的完成将进一步加深我们对细胞因子信号传导的基本理解，并对类风湿关节炎的治疗具有直接的转化意义。

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.