ALL-Associated JAK1 Mutants Activate the JAK/STAT Pathway Via IL-9Rα Homodimers

Point mutations in the JAK2 tyrosine kinase play a major role in the development of myeloproliferative syndromes by activating preformed homodimers of cytokine receptors such as EpoR, TpoR and GM-CSFR. More recently, a series of activating mutations in JAK1 have also been shown to be associated with B and T cell acute lymphoblastic leukemias, but little is known about the mechanisms involved in the constitutive activation of these mutants and about their mode of interaction with cytokine receptors. Here, we studied the ability of several ALL-associated JAK1 mutants (V658F, A634D, R724H, R879C) to activate the JAK/STAT pathway upon ectopic expression in HEK293 cells, alone or together with the other components of the IL-9 receptor complex (IL-9Rα, γc and JAK3). Expression of the JAK1 mutants alone failed to trigger STAT activation, but coexpression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STATs activation, even without γc and JAK3 which are required for IL-9 responsiveness. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with IL-9R and phosphorylate this tyrosine residue to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with deletion or mutation of the JAK-interacting BOX1 region not only failed to promote JAK1 activation, but acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, this process was partially inhibited by the expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Coexpression of wild-type JAK3 partially reverted the inhibitory effect of γc, indicating that JAK3 can cooperate with JAK1 mutants within the IL-9 receptor complex, even in the absence of the cytokine. Similar results were also observed with IL-2Rβ, which binds JAK1, but not for JAK2-associated homodimeric receptors such as TpoR or EpoR. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers can efficiently mediate constitutive activation of ALL-associated JAK1 mutants, even in cells lacking the expression of γc, which is required for the activation of wild-type JAK1 to their respective physiological ligands, IL-9 and IL-2.