Inhibition of Cytokine Signaling through Activation of Jak2 Ubiquitination by WP1130.

Abstract 2907

Poster Board II-883

Constitutive activation of Jak2 through mutations or cytokine stimulation leads to abnormal expansion of the myeloid lineage and plays a key role in multiple myeloproliferative disorders. Kinase-specific inhibitors that prevent substrate phosphorylation can provide effective therapy for these disorders. However, this approach is sometimes inadequate as additional mutations and target-specific modifications can alter the kinase in a sufficient fashion to reduce kinase inhibitor affinity. Therefore additional approaches and molecules may be needed to reduce kinase activity through alternate mechanisms. In this report we describe a novel small molecule-mediated approach to block the Jak2/Stat signaling pathway. Using a previously identified small molecule with Jak2 inhibitory activity, we performed structure-activity relationship studies of AG490 derivatives in a cell-based analysis for inhibition of cytokine-stimulated Stat activation. This effort lead to the synthesis of WP1130 which suppressed Jak2 driven Stat activation with nM to μM potency. However, WP1130 did not act as a direct Jak2 kinase inhibitor. Rather, WP1130 treatment induced a rapid, time and dose-dependent trafficking of Jak2 from the detergent soluble to insoluble cellular fraction. We observed a robust induction of protein ubiquitination in WP1130 treated cells which was not directly or indirectly due to proteosome inhibition. Further, WP1130-mediated ubiquitination was unique with respect to other compounds as it did not reduce association of Jak2 with chaperones, did not induce ER stress or increase reactive oxygen species to activate protein ubiquitination. Jak2 was poly-ubiquitinated, primarily with lysine63-linked polymers, after WP1130 treatment and ubiquitination correlated with Jak2 mobilization without its proteolysis. Sub-cellular fractionation and analysis using confocal microscopy suggested rapid trafficking of Jak2 into cellular structures called aggresomes, in complex with Hsp90, Hsp70 and HDAC6. Lysates from WP1130 treated cells had diminished capacity to hydrolyze ubiquitinated substrates, suggesting the involved of reduced deubiquitinase activity as a mediator of Jak2 ubiquitination in WP1130-treated cells. Together, these results suggest that WP1130 enhances a resident ubiquitination cascade that results in Jak2 ubiquitination and its displacement from its peri-membranous, signaling competent sites. The results also suggest that Jak2 is able to undergo K63-linked poly-ubiquitination and may be exploitable as an alternate approach to interrupt Jak/Stat signaling.