title: Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2.
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.
authors.affiliation: Cancer Biosciences, AstraZeneca R&D Boston, Waltham, Massachusetts 02451, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Gozgit
authors.forename: Geraldine
authors.initials: G
authors.lastname: Bebernitz
authors.forename: Pankaj
authors.initials: P
authors.lastname: Patil
authors.forename: Minwei
authors.initials: M
authors.lastname: Ye
authors.forename: Julie
authors.initials: J
authors.lastname: Parmentier
authors.forename: Jiaquan
authors.initials: J
authors.lastname: Wu
authors.forename: Nancy
authors.initials: N
authors.lastname: Su
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Stephanos
authors.initials: S
authors.lastname: Ioannidis
authors.forename: Audrey
authors.initials: A
authors.lastname: Davies
authors.forename: Dennis
authors.initials: D
authors.lastname: Huszar
authors.forename: Michael
authors.initials: M
authors.lastname: Zinda
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D017209:Apoptosis; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors*; D010641:Phenotype; D051573:Proto-Oncogene Proteins c-pim-1 / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D051025:bcl-Associated Death Protein / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: C534391:5-fluoro-2-(1-(4-fluorophenyl)ethylamino)-6-(5-methyl-1H-pyrazol-3-ylamino)nicotinonitrile; D000631:Aminopyridines; C494819:BAD protein, human; C494811:BCL2L1 protein, human; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D053614:Janus Kinase 2; C496634:PIM1 protein, human; D051573:Proto-Oncogene Proteins c-pim-1