Atiprimod Inhibits the JAK-STAT Pathway and Induces Apoptosis in Human Cells Carrying the JAK2^V617F or JAK3 Mutation.

Janus Kinases (JAK), including JAK1, JAK2, TYKa, and JAK3, are a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events by signal transducer and activator of transcription (STAT) proteins, via phosphorylation. The recent reports of an activating somatic mutation in codon 617 of the gene encoding JAK2 (JAK2^V617F) in patients with myeloproliferative disorders (MPDs) and 3 mutations in JAK3 (A572V, V722I, P132T) in acute megakaryoblastic leukemia patients, has opened new avenues for the development of targeted therapies for these malignancies. We report here the activity of Atiprimod, a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2 (JAK2^V617F) mutant-expressing murine FDCP-EpoR cells, set-2 cells, CMK cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2 (JAK2^WT) or mutant JAK2 (JAK2^V617F), and human megakaryoblastic leukemia cells with mutated JAK2^V617F (set-2 cells) or mutated JAK3 (CMK cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2 (JAK2^V617F) cells (Ic50 0.42 μM) and set-2 cells (Ic50 0.53 μM) than the CMK cells with mutant JAK3 (Ic50 0.79 μM) and FDCP wild-type JAK2 (JAK2^WT) cells (Ic50 0.69 μM). Atiprimod, inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose-and time-dependent manner. It exerted its inhibitory activity against cells with mutant JAK2 (JAK2^V617F) by affecting the cell cycle progression and induced apoptosis, as evidenced by increase in mitochondrial membrane potential and caspase3 activity. Atiprimod also cleaved PARP protein, increased turnover of the anti-apoptotic X-linked mammalian inhibitor of apoptosis (XIAP) protein, and inhibited the apoptotic protein BCL2 in a time- and dose-dependent manner. In addition, Atiprimod inhibited the proliferation of peripheral blood hematopoietic progenitors from three patients with PV carrying the JAK2 (JAK2^V617F) mutation as compared to hematopoietic progenitors from normal individuals, by 2-days MTS assay (p= 0.0013). Our data suggest that Atiprimod is active both in vitro and ex vivo against cells with activated tyrosine kinase of the JAK family.