FTY720 Restores PP2A Tumor Suppressor Activity in Polycythemia Vera CD34⁺ Progenitors Through Inhibition of Jak2 V617F- and PI-3Kγ-Dependent SET Serine Phosphorylation and Enhancement of NOS-Dependent PP2A Tyrosine Nitration

Abstract 2494

In CD34⁺ bone marrow (BM) progenitors from Polycythemia Vera (PV) patients and Jak2^V617F-expressing cell lines, loss of protein phosphatase 2A (PP2A) tumor suppressor activity is essential for their survival and clonogenic potential, and it depends on signals generated by the mutated Jak2 oncogenic kinase that, in turn, is also negatively regulated by PP2A. Additionally, we showed (Oaks J.J. et al., ASH 2010) that suppression of PP2A requires expression of the Jak2-regulated PP2A inhibitor SET, and it can be reverted by clinically-relevant Jak2 inhibitors or by the sphingosine analog FTY720, which reactivates PP2A and inhibits Jak2^V617F expression/activity upon binding/sequestration of SET.

Here we report that, unlike the effect of BCR-ABL1 in CML, SET levels do not increase in normal CD34⁺ BM progenitors engineered to express Jak2^V617F; however, SET knock-down restores PP2A activity in both Jak2^V617F cell lines and CD34⁺ PV progenitors, suggesting a post-translational control of SET activity. By using Jak2 and PI-3K kinase inhibitors and SET mutants, we found that PP2A inactivation in PV depends on the Jak2^V617F- and PI-3Kγ-induced SET phosphorylation on serine 24. Indeed, a ∼93% restoration of PP2A activity and suppression of SET phosphorylation is achieved upon exposure of Jak2^V617F+ cells to the specific PI-3Kγ (AS-604850, 1μM) inhibitor. Likewise, ∼80% loss of SET inhibitory activity is observed upon transduction of S24A but not S9A SET mutant that, albeit essential for PP2A inhibition in other cell types, behaved as wild type SET.

Because both Jak2 and PI-3Kγ may regulate nitric oxide synthase (NOS2) that, in turn, inhibits PP2A activity upon induction of peroxynitrate (PN)-mediated nitration of PP2A tyrosine residues with negative regulatory function, we assessed the effect of NOS/PN modulation on the Jak2^V617F-dependent inactivation of PP2A. Treatment of Jak2^V617F- expressing BaF3 cells with NO donors (1 mM DPTA NONOate and 10μM SIN-1) or PN (500 μM) resulted in 2.7- and 28-fold induction of PP2A activity, respectively. Accordingly, 80% inhibition of PP2A activity was observed in parental BaF3 cells upon inhibition of NOS2 by 3-bromo-7-nitroindazole (5μM). Thus, Jak2^V617F inhibits PP2A by inducing SET phosphorylation and inhibiting PP2A tyrosine nitration. Notably, co-existence of both mechanisms of Jak2^V617F-dependent PP2A inactivation is also supported by the evidence that NOS2 levels increase upon shRNA-mediated downregulation of SET expression, and by the marked decrease in SET phosphorylation and increase in PP2A tyrosine nitration detected in Jak2^V617F cells treated with FTY720 (2.5 mM). Interestingly, similar effects were observed upon exposure of Jak2^V617F cells to non-phosphorylatable and non-immunosuppressive FTY720 derivatives (e.g. OSU-2S, QC-FTY720 and SR-FTY720) but not in cells treated with the immunosuppressive phosphorylated FTY720 (FTY720-P).

In agreement with our findings indicating that the anti-leukemic activity of FTY720 in Jak2^V617F+ primary progenitors and cell lines does not require the sphingosine kinase 2-dependent conversion of FTY720 into FTY720-P and, consequently, its interaction with the S1PR1 receptor (Oaks JJ. et al., ASH 2010), we have now evidence showing that a synthetic FTY720-P, unlike FTY720, impairs (81% inhibition) PP2A activity in normal hematopoietic progenitors, and that the major part of the intracellular FTY720 remains non-phosphorylated. By using S1PR1-selective agonists and S1PR1 receptor knock-down, we also found that inhibition of PP2A activity by synthetic FTY720-P is mediated by the S1PR1-dependent triggering and activation of Jak2 and PI-3Kγ. Accordingly, co-treatment of normal hematopoietic progenitor cells with FTY720-P and either the Jak2 inhibitor AG490 or the PI-3Kγ inhibitor AS-604850 antagonized the inhibitory effects of FTY720-P and restored PP2A activity to levels similar to those detected in untreated cells.

Altogether our data indicate that the anti-leukemic effects of FTY720 in CD34⁺ PV progenitors depends on its ability to antagonize the Jak2^V617F- and S1PR1-induced and PI-3Kγ-mediated inactivation of PP2A through inhibition of the PI-3Kγ-dependent SET serine 24 phosphorylation, and via binding/sequestration of phosphorylated SET that, in turn, leads to enhancement of NOS/PN-mediated PP2A activation by tyrosine nitration.