Inhibition of JAK2/STAT Signaling by Degrasyn through a Novel Mechanism.

Janus Kinase 2 (JAK2) is a cytokine receptor associated tyrosine kinase. Cytokine stimulation results in JAK2 activation and tyrosine phosphorylation of the cytokine receptor. Cytosolic SH2 domain containing proteins, such as the signal transducer and activator of transcription 3 (STAT3) are recruited to phospho-tyrosine residues on the activated cytokine receptor, and phosphorylated by JAK2 to form stable dimers, followed by their translocation to the nucleus where they function as transcription factors. Deregulation of the JAK-STAT pathway is seen in several epithelial tumors and many hematological malignancies. Recent studies demonstrate that an activating mutation in the pseudokinase domain of JAK2 (V617F) underlies hematological disorders like polycythemia vera. Therefore, inhibition of JAK2 may have therapeutic significance in many cancers. The tryphostin AG490 is the most widely studied inhibitor of JAK2, which inhibits tumor cell growth and increases sensitivity to apoptotic stimuli in vitro. However, in vivo studies with AG490 have been less promising due to its poor pharmacology and requirement for high concentrations to achieve significant anti-tumor activity. To identify a more effective inhibitor of the JAK2-STAT pathway, we screened over 300 analogues of AG490 for their ability to inhibit IL-6 dependent activation of STAT3. A lead compound, Degrasyn (WP1130), was identified from this screen that inhibited IL-6 mediated STAT3 activation at low microM concentrations. Preliminary studies using in vitro kinase assays revealed that Degrasyn is a weak JAK2 kinase inhibitor despite being a strong suppressor of STAT3 activation, suggesting a different mechanism of inhibition of the JAK2-STAT pathway. We show that Degrasyn inhibits STAT3 phosphorylation by inducing the down-regulation of JAK2 protein without affecting STAT3. The loss of JAK2 protein via Degrasyn is a rapid and irreversible process. A decrease in JAK2 protein levels is observed as early as 30 minutes after treatment with near complete loss of JAK2 after 2 hours of Degrasyn incubation. While other tyrosine kinases are not affected, both wild type and mutant (V617F) forms of JAK2 are equally down-regulated by Degrasyn. Loss of JAK2 protein by Degrasyn is not blocked by inhibition of calpain or serine/threonine proteases or by inhibition of the proteosomal or lysosomal pathway. Real-Time PCR analysis of JAK2 transcript levels after Degrasyn treatment showed no significant change, suggesting a direct effect of Degrasyn on the JAK2 protein itself. Recent studies suggest that Degrasyn alters the cytoplasmic compartmentalization of JAK2, sequestering the kinase in an insoluble fraction. In vivo studies show that Degrasyn has significant anti-tumor effects against models of leukemia and lymphoma. These results suggest that Degrasyn induces JAK2 degradation by a unique mechanism and may be useful in treating tumors and diseases where the JAK2 kinase plays a pivotal role.