Abstract

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) caused by a valine to phenylalanine mutation in the inhibitory JH2 “pseudokinase” domain of the signaling protein Janus Kinase 2 (Jak2). While Jak2 has several known regulators (e.g. SOCS1, SOCS3, PP2A, and SHP-1), the role played by these signaling molecules in the development of PV is still largely unclear. One of these regulators, the tumor suppressor phosphatase PP2A, has been found functionally inactivated in different hematologic myeloid and lymphoid malignancies characterized by the expression of constitutively activated oncogenic tyrosine kinases (e.g. BCR/ABL). To investigate the role of PP2A in the pathogenesis of Jak2 V617F+ MPDs and the potential therapeutic relevance of PP2A activating drugs (e.g. FTY720), we determined the effects of wild type and V617F Jak2 expression on PP2A activity and assessed the molecular and biological effects of FTY720 in hematopoietic precursor cell lines and/or primary lineage-negative bone marrow cells engineered to expressed either wild type or V617F Jak2 protein. Herein we report that PP2A activity is significantly reduced by about 82% and 78% (P< 0.01) in mJak2 V617F-transduced growth factor-dependent and erythropoietin receptor-expressing 32Dcl3 (32D-EPO) and Ba/F3 cells, respectively, compared to MigR1-transduced controls. Furthermore, addition of the PP2A activator FTY720 (2.5μM) for 10 hours restored PP2A activity to 66% and 75% respectively compared to that of MigR1 controls. Mechanistically, we demonstrated that inactivation of PP2A was due to constitutive Jak2 activity. In fact, treatment of V617F Jak2-expressing cells with Jak inhibitor I (1μM; 10 hours) restored PP2A activity to 80% of controls in 32Dcl3 cells, while 600nM was sufficient to restore activity to 108% of controls in Ba/f3 cells. Likewise, transduction of murine lineage-negative bone marrow cells with wild-type Jak2 produced a 62% reduction in PP2A activity (P<0.01), while expression of the Jak2 V617F mutant produced a 97% reduction (P< 0.01). Moreover, as we previously reported for BCR/ABL, pharmacologic restoration of PP2A activity by treatment of Ba/F3 or 32D-EPO cells with the PP2A activator FTY720 (2.5μM for 10 hours) led to reduced expression and dephosphorylation of wild type and V617F Jak2. This, in turn, resulted in a 60% reduction in the colony forming ability of IL-3 cultured cells overexpressing wild type Jak2, while a 94% suppression of colony formation was evident in Jak2 V617-expressing cells maintained in the absence of cytokines. Moreover, addition of IL-3 to the FTY720-containing semisolid medium restored viability to levels similar to those of FTY720-treated wild type Jak2-expressing cells (70% suppression of colony formation upon treatment), indicating that restoration of PP2A activity counteracts cytokine-independent pathways triggered by the V617F Jak2 mutated tyrosine kinase. Altogether, these results suggest that inactivation of PP2A is essential for Jak2 (V617F mutant included) -driven cell/proliferation and survival and that pharmacologic activation of PP2A might represent a potential avenue for treatment of PV and, perhaps, of other MPDs characterized by the expression of a mutated Jak2 kinase.

D.P. is a Scholar of the Leukemia and Lymphoma Society.

Author notes

Disclosure: No relevant conflicts of interest to declare.