Abstract

A role for Jak2 in the etiology of the myeloproliferative diseases (MPDs) was discovered with the identification of a single activating point mutation, V617F, in the pseudokinase domain of JAK2. We have developed a Jak2 inhibitor, AZ60, which inhibits in vitro JAK2 enzyme activity with a Ki of 0.45 nM. AZ60 demonstrates inhibition of STAT5 phosphorylation and proliferation in a Tel-Jak2 engineered cell line with IC50 values of 18 and 23 nM, respectively. To understand the selectivity versus other Jak kinase family members we engineered three additional cell lines containing Tel fusions with the kinase domains of Jak1, Jak3 and Tyk2. Under these settings, AZ60 demonstrates a 15 to 30-fold selectivity for Tel-Jak2 driven STAT5 phosphorylation when compared to other Jak kinase family members. AZ60 was also tested for its ability to inhibit STAT5 phosphorylation and cellular proliferation in two human hematological cell lines, Set-2 and Hel. Set-2 expresses both wt and V617F Jak2, while Hel is homozygous for the Jak2 V617F mutation. AZ60 decreased phospho-STAT5 levels in a dose-dependent manner in both Set-2 and Hel cells with IC50 values of 15 and 25 nM, respectively. Complete inhibition of proliferation and a marked induction of apoptosis were observed in both cell lines following treatment with AZ60. Induction of apoptosis by AZ60 was characterized by a time- and dose-dependent increase in caspase 3/7 activities and PARP-cleavage. These data demonstrate AZ60 is a potent and selective inhibitor of Jak2 and may help decipher the mechanisms underlying Jak2-driven myeloproliferative disease.

Author notes

Disclosure: Employment: All authors are employees of AstraZeneca. Ownership Interests:; Several authors have ownership interest in AstraZeneca.