Differential effect of inhibitory strategies of the V617 mutant of JAK2 on cytokine receptor signaling

Abstract

Background: Janus kinase (JAK) 2 plays pivotal roles in signaling by several cytokine receptors. The mutant JAK2 V617F is the most common molecular event associated with myeloproliferative neoplasms. Selective targeting of the mutant would be ideal for treating these pathologies by sparing essential JAK2 functions. Objective: We characterize inhibitory strategies for JAK2 V617F and assess their effect on physiologic signaling by distinct cytokine receptors. Methods: Through structure-guided mutagenesis, we assessed the role of key residues around F617 and used a combination of cellular and biochemical assays to measure the activity of JAKs in reconstituted cells. We also assessed the effect of several specific JAK2 V617F inhibitory mutations on receptor dimerization using the NanoBiT protein complementation approach. Results: We identified a novel Janus kinase homology 2 (JH2) aC mutation, A598F, which is suggested to inhibit the aromatic stacking between F617 with F594 and F595. Like other JAK2 V617F inhibitory mutations, A598F decreased oncogenic activation and spared cytokine activation while preventing JAK2 V617F-promoted erythropoietin receptor dimerization. Surprisingly, A598F and other V617F-inhibiting mutations (F595A, E596R, and F537A) significantly impaired IFN-g signaling. This was specific for IFN-g because the inhibitory mutations preserved responses to ligands of a series of receptor complexes. Similarly, homologous mutations in JAK1 prevented signaling by IFN-g. Conclusions: The JH2 aC region, which is required for JAK2 V617F hyperactivation, is crucial for relaying cytokine-induced signaling of the IFN-g receptor. We discuss how strategies aiming to inhibit JAK2 V617F could be used for identifying inhibitors of IFN-g signaling.

Characterization of JAK1 Pseudokinase Domain in Cytokine Signaling

“…The αC resides in the JH1-JH2 interface but also lines the ATP-binding pocket. The region is important in cytokine-induced activation of kinases, and modulating it with mutations can inhibit constitutive activation of JAKs [17][18][19]. However, the mechanisms of function for many mutations is yet unknown.…”

Janus Kinases in Leukemia

“…Furthermore, many JAK2 mutations in JH2 result in hyperactivation, leading to various malignancies (discussed in the later sections) indicating a negative regulatory role of the JH2 domain. Mutagenesis studies at different regions of the JH2 domain have been shown to suppress the gain-of-function (GOF) disease mutation-induced hyperactivation of JAK, confirming the regulatory role of the JH2 domain [27][28][29][30][31]. A plausible structural basis of the negative regulatory function is that the domain interacts directly with JH1 physically restricting the JH1 trans-phosphorylation thus maintaining the domain in an inactive state.…”

“…Recent studies using a single molecule co-tracking approach demonstrated a critical role for the JAK2 JH2 domain in thrombopoietin receptor dimerization and stabilization of the ligand-induced dimer [18]. The regulatory role of JH2 makes it an intriguing target for inhibitor discovery [8], and importantly, JAK hyperactivation can be suppressed by mutations inhibiting ATP binding to JH2 [27,30] or targeting the αC helix of JH2 [28,29,31].…”

JAK Inhibition for the Treatment of Myelofibrosis: Limitations and Future Perspectives

“…Recent structure-guided mutagenesis studies have provided key insights that may inform the development of highly selective inhibitors of JAK2 V617F. 103 …”