Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2V617F mutant CD34+ progenitors after drug washout. Type I inhibitor–induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients.
License type:
http://creativecommons.org/licenses/by-nc/4.0/
Funding Info:
Funding for this project was provided through a National Health and Medical Research Council of Australia (NHMRC) Program grant to A.F.L., M.W.P., and T.P.H. (APP 1071897). We acknowledge funding from the Victorian Government Operational Infrastructure Support Scheme to St. Vincent’s Institute. This work was also supported by CSL Limited, Australia. T.P.H. is an NHMRC Practitioner Fellow and M.W.P. is an NHMRC Research Fellow. D. Thomas was funded by NIH/NCI Pathway-to-Independence (K99) grant number 5K99CA207731-02. Author contributions: D. Tvorogov designed and executed experiments, analyzed data, and wrote the manuscript. D. Thomas designed and performed experiments, analyzed data, and wrote the manuscript. N.P.D.L. and J.J.B. designed and performed experiments and supplied critical reagents. M.D., E.F.B., W.L.K., and F.S. performed experiments, analyzed data, and reviewed the manuscript. M.L. performed database searches and data analysis. T.R.H. performed DNA manipulations and reviewed the manuscript. D.M.R. provided clinical material and reviewed the manuscript. M.W.P. discussed the structural biology aspects and reviewed the manuscript. T.P.H., V.T., and R.M. provided advice and reviewed the manuscript. A.F.L. and D. Tvorogov conceived the study and wrote the manuscript. Competing interests: D.M.R. receives honorarium and research funding from Novartis. T.P.H. receives honorarium and research funding from Novartis, BMS, and Ariad.