Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations

Background: Janus kinases (JAKs; JAK1 to JAK3 and tyrosine kinase 2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia, and the mutations strongly concentrate in the regulatory pseudokinase domain Janus kinase homology (JH) 2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation and pathway selectivity. Objective: We sought to characterize mechanisms and differences for pathogenic and cytokine-induced JAK2 activation to enable design of novel selective JAK inhibitors. Methods: We performed a systematic analysis of JAK2 activation requirements using structure-guided mutagenesis, cell-signaling assays, microscopy, and biochemical analysis. Results: Distinct structural requirements were identified for activation of different pathogenic mutations. Specifically, the predominant JAK2 mutation, V617F, is the most sensitive to structural perturbations in multiple JH2 elements (C helix [aC], Src homology 2-JH2 linker, and ATP binding site). In contrast, activation of K539L is resistant to most perturbations. Normal cytokine signaling shows distinct differences in activation requirements: JH2 ATP binding site mutations have only a minor effect on signaling, whereasJH2aCmutations reduce homomeric (JAK2-JAK2) erythropoietin signaling and almost completely abrogate heteromeric (JAK2-JAK1) IFN-gamma signaling, potentially by disrupting a dimerization interface on JH2. Conclusions: These results suggest that therapeutic approaches targeting the JH2 ATP binding site and aC could be effective in inhibiting most pathogenic mutations. JH2 ATP site targeting has the potential for reduced side effects by retaining erythropoietin and IFN-gamma functions. Simultaneously, however, we identified the JH2 aC interface as a potential target for pathway-selective JAK inhibitors in patients with diseases with unmutated JAK2, thus providing new insights into the development of novel pharmacologic interventions.Peer reviewe