Interdiction of Src Family Kinase Activity as a Therapeutic Option for Aberrant JAK-STAT Signaling: Potential Targeted Therapy of JAK2 V617F in Myeloproliferative Disorders with Currently Available Kinase Inhibitors.

Abstract

Cytokine receptors preferentially associate with particular JAK-STAT combinations to transduce specific signals. For example, erythropoietin receptor (Epo-R) preferentially interacts with JAK2 to initiate signaling pathways via STAT5. The JAK2 V617F mutation (mJAK2) found in some myeloproliferative disorders (MPD) still requires binding to type I cytokine receptors to initiate signaling. Consequently, aberrant JAK-STAT signaling in MPD may require physiologic interactions with other pathways. Src family kinases (SFK) interact with various cytokine receptors resulting in close association between SFK and JAK-STAT pathways. We hypothesized that SFK activity plays a role in the activation of STAT5 and, given the importance of STAT5 in the pathogenesis of MPD, targeted inhibition of SFK could provide a novel therapeutic approach. First, we examined the effect the SFK inhibitors PP2 and SU6656 on the proliferation of the HEL cell line harboring mJAK2 and the Epo-dependent AML line UT7/Epo; SFK inhibition significantly diminished proliferation in both cell lines. These results imply that despite of the constitutive activity of mJAK2 or in the presence of Epo stimulated JAK2-STAT5 induction, SFK activation is required for proliferation. Since mJAK2 requires a functionally intact type I cytokine receptor, we examined Epo-R signaling in greater detail via phospho-specific immunoblotting. SFK inhibition resulted in diminished levels of phospho-SFK, coinciding with a similar degree of diminished phospho-STAT5. Simultaneously decreased induction of AKT and ERK pathways after SFK inhibition suggested SFK activity is also regulating a more global signaling network through the Epo-R. Inhibition of JAK2 activity potently suppressed phospho-STAT5, as well as ERK and AKT, without affecting SFK phosphorylation. Thus, SFK lies upstream of JAK2, or SFK and JAK2 may be regulating the second messenger pathways in parallel. In electrophoretic mobility shift assays to examine the effect of SFK inhibition on STAT5 DNA binding, SFK inhibition resulted in decreased STAT5 DNA binding despite constitutive activity of mJAK2. Analogous results were obtained after Epo stimulation in UT7/Epo cells. As expected, inhibition of JAK2 resulted in almost complete loss of STAT5 DNA binding. To confirm these results in primary cells, we examined the effects of SFK inhibition on primary monocytes from a patient with mJAK2. Stimulation with GM-CSF resulted in increased STAT5A DNA binding, but not STAT5B. In the presence of the SFK inhibitor PP2, GM-CSF induction of STAT5A DNA binding activity was completely inhibited. It is interesting to note that a key difference between STAT5A and STAT5B is the potential for ERK regulation of STAT5A DNA binding activity. Thus, showing here that SFK regulates ERK activity, and knowing that ERK activity can positively regulate STAT5A DNA binding, we propose a model in which SFK activity may modulate the JAK2-STAT5 signaling axis via the ERK pathway. In summary, our results demonstrate that while JAK2 is essential for this process, SFK activity appears to be necessary for full activation by positively modulating the JAK2-STAT5 axis. SFK inhibitors recently approved and in clinical trials may demonstrate efficacy in hematologic diseases characterized by aberrant JAK-STAT signaling, such as MPD.