JAK2 signaling as a therapeutic target. (A) Chemical structure of BVB808. (B) Kinase assays were performed with recombinant kinase (JH1) domains of the respective JAKs to determine the relative JAK-family selectivity of BVB808. (C) BVB808 activity against JAK2-dependent and JAK2-independent cell lines. GI50 values represent means of at least two independent experiments (n = 2–4). (D) JAK2 V617F mutant MB-02 cells were treated with increasing concentrations of BVB808 for 30 min. Inhibition of constitutive pSTAT5 was analyzed by Western blotting using a Tyr694 phospho-specific antibody. Total STAT5 is included as a loading control. (E) JAK2 V617F mutant SET-2 and JAK3 A572V mutant CMK cells were treated with increasing concentrations of BVB808 for 1 h, and then extracted for immunoblotting. (F and G) MB-02 and SET-2 cells were treated with 1 µM BVB808 for up to 24 h. Cell extracts were prepared at different time points as indicated and probed for pSTAT5. Activation of cell death was assessed by detection of cleaved PARP (arrowhead). β-Tubulin was used as a loading control. (H) Efficacy of BVB808 was evaluated in a mouse bone marrow transplant model of Jak2 V617F–driven MPN after 3 wk of dosing. Bar, 100 µm. (I) In a separate experiment, CT imaging of mice before treatment and after 3 and 50 d of vehicle or BVB808. After imaging on day 50, the mice were sacrificed and the spleens were dissected and weighed. Spleen weight is shown on the right. Bar, 1 cm. (J) Immunohistochemistry for pStat5 in spleen and bone marrow sections from samples collected 2 h after the last dose of vehicle or BVB808. Bar, 50 µm.
