Ruxolitinib mediated paradox JAK2 hyperphosphorylation is due to the protection of activation loop phosphotyrosines from phosphatases

Ruxolinitib was approved by the FDA for the treatment of JAK2-V617F positive myelofibrosis. Unexpectedly, treatment of JAK2-V617F expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at Tyr1007/1008 sites. The mechanism of ruxolitinib-induced JAK2 hyperphosphorylation is not well understood. A ruxolitinib-resistant JAK2 variant (V617F+L983F) and a JAK2-V617F kinase dead mutant (JAK2-V617F+K882R) did not show paradox hyperphosphorylation after ruxolitinib treatment indicating that ruxolitinib-mediated JAK2 hyperphosphorylation is mediated by a JAK2 intrinsic mechanism. Native immunocomplexes of JAK2-V617F with an antibody against pTyr1007/1008 could be blocked by the presence of ruxolitinib, although JAK2-V617F was hyperphosphorylated at these sites, suggesting that in the presence of ruxolitinib the JAK2 activation loop is buried within the kinase domain. This stabilization of the activation loop conformation results in the protection of pTyr1007/1008 sites from phosphatases. When JAK2 is in an inhibited conformationally restricted state, the phosphate group of Tyr1007 and Tyr1008 forms an intermolecular interaction with Arg975 and Lys999, respectively. Mutation of Arg975 and Lys999 to Ala reduced the phosphorylation at both Tyr1007/1008 residues, and importantly, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation in these variants. Hyperphosphorylated JAK2 is hyperactive after ruxolitinib dissociation and activates STAT5 target genes PIM2, ID1, and MPL. Our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolitinib.