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

描述（由申请方提供）：γ链细胞因子，如IL-2，通过含有Jak 1和Jak 3激酶的受体复合物发出信号。这两种激酶是否发挥不同或冗余的作用仍不清楚，最近的工作表明，虽然Jak 1激酶活性是必不可少的，但Jak 3主要作为支架将信号复合物的其他部分聚集在一起。为了解决Jak 3的催化作用，开发了一种高度选择性和有效的Jak 3抑制剂，靶向Jak 3中发现的半胱氨酸，而不是家族成员Jak 1，Jak 2或Tyk 2。在鼠CD 4 + T细胞母细胞中，Jak 3抑制剂有效阻断IL-2驱动的增殖和CD 25的上调。与Jak 1/Jak 2抑制剂ruxolitinib或pan-Jak抑制剂tofacitinib不同，这种Jak 3抑制剂在急性IL-2刺激后阻断STAT 5磷酸化方面几乎没有那么有效。大多数关于IL-2信号传导的研究都集中在第一个小时发生的磷酸化变化上。然而，初步数据表明，IL-2刺激后有两个STAT 5磷酸化的“波”：一个在刺激后15分钟达到峰值，并在一小时后降至接近基线，第二个较弱的波在6小时左右达到峰值。有趣的是，尽管Jak 1阻断剂阻断了这两个波中的磷酸化，但Jak 3抑制剂仅有效地阻断了第二波磷酸化，这表明Jak 3在维持信号传导中的非冗余作用。拟议的工作旨在探讨这种信号模式的后果和一般性。Aim 1将使用mRNA-seq来确定Jak 3抑制剂是否阻断了第二波信号传导，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，CD 8 + T细胞母细胞不显示这两个波的STAT 5磷酸化，而是一个单一的持续峰值的STAT 5磷酸化。目标2将利用这种差异来阐明这些不同的信号模式的机制和后果。Jak 3抑制剂和Jak 1/2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剂（如SOCS蛋白）的表达，以确定潜在的机制。目的3将评估在类风湿性关节炎的SKG模型中这种信号模式的体内后果。已知该模型对托法替尼有反应，托法替尼阻断几乎所有细胞因子信号传导，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，将给予足以阻断所有STAT 5磷酸化的高剂量Jak 3抑制剂，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的信号传导是否足以达到治疗效果。这个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，并对类风湿性关节炎的治疗有直接的翻译意义。

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.