Abstract #4773: Activation of a novel Jak-Stat inhibitory pathway by a small molecule

Effective cytokine signaling requires the participation of multiple kinases, adaptor molecules and receptors and many of these participants are modified by mutations and other changes that are detected in diseased cells. One of the most important cytokine signaling events involves activation of receptor-associated kinases, such as Jak2, which has been recently shown to be involved in multiple myeloproliferative disorders and cancers of epithelial origin. One highly investigated and exploited approach to control aberrantly activated or mutated Jak2 is to utilize small molecule kinase inhibitors that block the ATP binding pocket, preventing substrate phosphorylation. However, this approach has uncovered some obstacles that prevent continual effective use of these inhibitors as additional mutations and target-specific modifications alter the structure of the ATP binding pocket and reduce inhibitor binding or affinity. Therefore additional approaches and molecules should be designed that reduce kinase activity through alternate mechanisms. In this report we describe a novel small molecule-mediated approach to block Jak2 activation. Using a previously identified small molecule with Jak2 inhibitory activity, we performed extensive 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 and other molecules with more potent inhibition (>100-fold) of cytokine-mediated Stat activation than AG490. However, WP1130 had no direct kinase inhibitory activity but Stat inhibition correlated with rapidly increased poly-ubiquitin levels through an unknown mechanism. WP1130 did not inhibit 20S proteosome activity or increase reactive oxygen species. Glutathione levels and ER stress markers were also unchanged in WP1130 treated cells. WP1130 increased upstream events (UBE1) in the ubiquitin activation cascade, co-incident with aggresomal trafficking of Jak2 in complex with Hsp90, Hsp70 and Hsc70. Assessment of Jak2 ubiquitination in WP1130 treated cells demonstrated the presence of K63-linked Ub oligomers on Jak2, which correlated with Jak2 mobilization without proteolysis, in agreement with previous reports of the role played by K63-linked poly-ubiquitination in aggresomal sequestration. Aggresomal compartmentalization of Jak2 was confirmed by biochemical and confocal imaging experiments. Together these results suggest that WP1130 activates a resident ubiquitination cascade that results in Jak2 poly-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 for therapeutic interruption of Jak-Stat signaling.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4773.