Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis

Abstract

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JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn.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 JAK2^V617F and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2^V617F samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2^V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr^1007/1008 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 JAK2^V617F 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 JAK2^V617F patients.