Ruxolitinib Mediated Paradox JAK2 Hyperphosphorylation Is Due To The Protection Of Activation Loop Phosphotyrosines From Phosphatases

The JAK2 V617F mutation has been reported in the majority of bcr-abl negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV:90%), essential thrombocythemia (ET:50%) and idiopathic myelofibrosis (IMF:50%). Recently, ruxolitinib (Jakavi, INCB018424) a JAK2/JAK1 specific inhibitor has been approved by the FDA for the treatment of myelofibrosis. Unexpectedly, treatment of V617FJAK2 expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at its two critical sites (tyr1007/tyr1008). The mechanism of ruxolitinib induced JAK2 hyperphosphorylation is not understood. It has been demonstrated that also the pseudokinase domain of JAK2 display kinase activity and plays a major role in regulating the kinase activity of JAK2. Deletion of the pseudokinase domain leads to hyperphosphorylation of JAK2. We thus hypothesized that ATP-competitive inhibitors might target the kinase activity of the pseudokinase domain. However, treatment of kinase dead pseudokinase domain JAK2V617F mutants with ruxolitinib also causes JAK2 hyperphosphorylation indicating that ruxolitinib mediated JAK2 hyperphosphorylation is not due to inhibition of the pseudokinase domain. We then reasoned that treatment with ruxolitinib might protect JAK2 from tyrosine phosphatases. Co-immunoprecipitation results however did not suggested that ruxolitinib treatment leads to the dissociation of phosphatases such as SHP-2. A Ruxolitinib resistant JAK2V617F mutant (V617F+L902QJAK2) and a JAK2V617F kinase dead mutant (V617F+K882RJAK2) both did not show paradox hyperphoshorylation after ruxolitinib treatment. This indicated that ruxolitinib mediated JAK2 hyperphosphorylation is mediated by a JAK2 intrinsic mechanism. Finally, native-immunocomplexes (non-denaturing IP) of JAK2V617F generated with a monoclonal antibody against ptyr1007/1008 could be blocked by the presence of ruxolitinib, although JAK2V617F was hyperphosphorylated at these sites. We therefore believe now that in the presence of ruxolitinib the JAK2 activation loop folds inside the kinase domain. This leads to the protection of ptyr1007/1008 sites from phosphatases. Most tyrosine kinases are structurally very plastic enzymes with large conformational differences observed between the inactive and active states of the kinase. These conformational changes are largely governed by disorder to order changes in N-lobe, activation loop and hydrophobic motif. When JAK2 is in an inhibited conformational restricted state, the phosphate group of tyr1007 and tyr1008 have an intermolecular interaction with Arg975 and Lys999. These interactions play a major role in the stabilization of the activation loop conformation inside the kinase domain. Mutation of Arg975 and Lys999 to Ala (V617F+R975A), (V617F+L999A) reduced the phosphorylation at both tyr1007/1008 residues. Importantly, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation in this mutants. Taken together our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolutinib.