?-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 blocking?-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.
Approximately 23 million Americans suffer from autoimmune diseases, including 1.5 million with rheumatoid arthritis. Using a new small molecule drug we have developed, this project will first help us better understand the role of the protein Jak3 in immune cells and ultimately will test the effectiveness of selectively inhibiting this target in preclinical models of rheumatoid arthritis.
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|Smith, Geoffrey A; Uchida, Kenji; Weiss, Arthur et al. (2016) Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling. Nat Chem Biol 12:373-9|