Dysregulated Signaling Networks in Chronic Myeloproliferative Disorders: Co-Operation of JAK2 and Src Family Kinases (SFK)

Abstract

The JAK2-STAT5 pathway plays an key role in chronic MPD (cMPD) with the JAK2 V617F mutation. However, JAK2 also initiates several second messenger pathways, including the Ras-ERK and PI3K-AKT pathways. Since these essential second messenger pathways are also regulated by SFK activity, and crosstalk between JAK and SFK has been demonstrated, we investigated the potential SFK-dependent regulation of STAT5, Ras-ERK and PI3K-AKT in primary cells from patients with cMPD and myeloid cell lines with the JAK2 V617F mutation (HEL) or Epo-dependent (UT-7/Epo). In primary cells from JAK2 V617F patients, we show that SFK activity is required for optimal JAK2 induced phospho (p)-STAT5, nuclear targeting of p-STAT5, and STAT5-DNA binding. Both endogenous and Epo-induced colony formation in methylcellulose by peripheral blood cells derived from polycythemia vera (PV) patients was markedly suppressed with the SFK inihibitor dasatinib; endogenous colony formation was abrogated while both the number and size of Epo-induced colonies was markedly reduced (> 70% inhibition at 1 uM), suggesting that despite of the presence of activating JAK2 V617F in primary cells, SFK activity is required for optimal proliferation. Interestingly, a similar observation was also made in a PV patient lacking the JAK2 V617F mutation. When HEL or Epo-stimulated UT-7/Epo cells were treated with a JAK inhibitor (P6), there was significant inhibition of p-STAT5 induction and second messenger pathways, demonstrating the importance of this proximal kinase. Inhibition of JAK2 did not affect SFK activation however, suggesting that SFK may lie upstream of JAK2. Interestingly, when SFK activity was suppressed (PP2 or dasatinib), a marked inhibition of p-STAT5 (and STAT5 DNA-binding), p-AKT, p-ERK, p-90RSK, p-S6 ribosomal protein(rp), and global protein tyrosine phosphorylation was observed in cell lines and primary cells. These findings suggest that SFK activity is involved in the most proximal aspects of Epo signaling and facilitates JAK2 activation of key second messenger pathways essential for proliferation, differentiation, and survival. Since JAK2 and SFK coordinately regulate multiple second messenger pathways, we next examined the relative importance of each by specifically targeting either the Ras-ERK pathway (MEK inhibition) or the PI3K-AKT pathway (PI3K and mTOR inhibition) downstream from JAK2 and SFK in both primary cells and cell lines. Using intracellular phospho-flow cytometry and immunoblotting, we show that the SFK-dependent Ras-ERK-RSK axis can directly induce p-S6rp, a key component in translational control and cell cycle progression. To determine whether this finding can be reproduced in vivo, we examined p-S6rp expression in bone marrow biopsies from PV patients (n=16); a marked hyperphosphorylation S6rp was consistently seen in patients' megakaryocytes as compared to control specimens obtained from reactive processes, lymphoma staging, CMML, or even a case of hypoxia-induced polycythemia (all showing low/absent p-S6rp). Simultaneous staining for p-mTOR revealed that the level of p-mTOR was not increased. In sum, our findings suggest that SFK activation is necessary for optimal signal transduction along of the JAK2-STAT5 axis. In addition, the Ras-ERK-RSK pathway, requiring both JAK and SFK, may play a key role in S6rp hyperphosphorylation leading to increased cell cycling. Both findings point towards potential therapeutic targets, along with JAK2, for treating cMPD.