Effects of the JAK2 Inhibitor, AZ960, on Pim/BAD/BCL-xL Survival Signaling in the Human JAK2 V617F Cell Line SET-2

Joseph M Gozgit,Geraldine Bebernitz,Pankaj Patil,Minwei Ye,Julie Parmentier,Jiaquan Wu,Nancy Su,Tao Wang,Stephanos Ioannidis,Audrey Davies,Dennis Huszar,Michael Zinda

doi:10.1074/jbc.m803813200

Joseph M Gozgit, Geraldine Bebernitz + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m803813200

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Abstract

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a K(i) of 0.00045 microm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI(50)=0.025 microm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI(50)=0.033 microm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser(112). Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.

Highlights

Acquired mutation in the Janus-associated kinase 2 (JAK2) gene encoding a valine to phenylalanine substitution, V617F, which results in constitutive kinase activity and has been shown to promote deregulated hematopoiesis [2,3,4]
JAK2 V617F is frequently detected in myeloproliferative disorders (MPDs), a group of clonal hematopoietic stem cell disorders that include polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF), all of which have the potential to transform to acute myeloid leukemia [2]
Several groups have shown that hematopoietic stem cell expression of JAK2 V617F in the mouse adoptive transfer model results in a polycythemic phenotype followed by myelofibrosis, demonstrating a critical role for aberrant JAK2 signaling in the pathogenesis of the disease [2]

Summary

Introduction

Acquired mutation in the JAK2 gene encoding a valine to phenylalanine substitution, V617F, which results in constitutive kinase activity and has been shown to promote deregulated hematopoiesis [2,3,4]. Similar to the effects on STAT3/5 phosphorylation, AZ960 potently inhibited SET-2 cell proliferation with an average (n ϭ 3) GI50 of 0.033 Ϯ 0.020 ␮M (Fig. 2B), consistent with its activity in the TEL-JAK2 Ba/F3 better understand the phenotype of JAK2 inhibition, we evaluated the DNA content in SET-2 cells in response to AZ960. BCL-xL Is Regulated by JAK2 and Is Important for Cell Survival—The anti-apoptotic protein BCL-xL is a well known STAT3/5 transcriptional target [8, 12], and may play a key role in the pathogenesis of MPDs by driving cell survival [14, 15].

Results

Conclusion