Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805

NVP-BSK805 potently suppresses STAT5 phosphorylation in JAK2^V617F mutant cell lines and displays a bias for JAK2 over JAK1 and JAK3 inhibition. A, JAK1 or JAK2 were depleted in JAK2^V617F mutant MB-02 and SET-2 cells by RNAi followed by Western blotting to detect levels of STAT5 phosphorylation. Controls were treated with nontargeting siRNA oligos. The degree of JAK knockdown was verified by immunoprecipitation and Western blotting. B, JAK family members were depleted in JAK3^A572V mutant CMK cells by RNAi. Levels of STAT5 phosphorylation and the degree of JAK knockdown were assessed as described above. C, MB-02 cells were treated with increasing concentrations of NVP-BSK805 for 30 min and levels of phosphorylated STAT5 were determined as described above. D, SET-2 cells and CMK cells were treated with increasing concentrations of NVP-BSK805 for 1 h. Levels of phosphorylated STAT5 and total STAT5 were determined by Western blotting.

NVP-BSK805 induces apoptosis in JAK2^V617F-mutant SET-2 cells in a dose- and time-dependent manner. A, JAK2^V617F mutant SET-2 cells were treated with DMSO, 150 nmol/L of NVP-BSK805, or 1 μmol/L of NVP-BSK805 for 24 and 48 h. PARP cleavage as well as Bcl-xL levels were assessed by Western blotting. β-Tubulin was probed as a loading control. B, DNA content in SET-2 cells was measured by fluorescence-activated cell sorting using propidium iodide staining following treatment of cells with DMSO, 150 nmol/L of NVP-BSK805, or 1 μmol/L of NVP-BSK805 for 24, 48, and 72 h. The X and Y-axes represent FL2-A fluorescence intensity for propidium iodide staining and cell count, respectively. Results depict a representative experiment. C, percentage of cells with <2N DNA content at each dose and time point (columns, mean of four independent experiments; bars, SD). *, significantly different from DMSO control at respective time points using t test or, if not applicable, ^†Mann-Whitney rank sum test (P < 0.05). Significant difference between time points (#) or doses (‡) as assessed by t test (P < 0.05).

NVP-BSK805 suppresses STAT5 phosphorylation, splenomegaly, and leukemic cell spreading in a Ba/F3 JAK2^V617F cell–driven mouse model. A, SCID beige mice (bearing Ba/F3 JAK2^V617F cells) were given 150 mg/kg NVP-BSK805 orally or vehicle and sacrificed after 2, 6, and 12 h. Spleens were extracted for detection of phosphorylated STAT5 and total STAT5 levels by quantitative Western blotting. B, in SCID beige mice bearing Ba/F3 JAK2^V617F cells, STAT5 phosphorylation was readily detectable throughout the spleen sections by immunohistochemistry. Inset, the phosphorylated STAT5 staining is predominantly nuclear. Phosphorylated STAT5 is strongly reduced in mice that were given 150 mg/kg NVP-BSK805 orally after 12 h. C, Ba/F3 JAK2^V617F cell leukemic burden was detected on day 4 post-cell injection by measuring bioluminescence in vivo (mean, 3.8 × 10⁷ ± 5.0 × 10⁶ photons/s ± SEM), and mice were randomized into treatment groups of 9 to 10 animals. Treatment was initiated with vehicle or with 50 and 150 mg/kg of NVP-BSK805 on day 5. Whole body bioluminescence reading on day 9 reveals suppression of leukemic cell spreading: 36% and 22% T/C were achieved with 50 and 150 mg/kg of NVP-BSK805, respectively (*, P < 0.05 versus vehicle treated animals; one-way ANOVA followed by post hoc test Dunnett's using log₁₀ transformed values). D, assessment of spleen weight at the time of necropsy in the frame of the efficacy experiment outlined above showed dose-dependent suppression of splenomegaly by NVP-BSK805 (*, P < 0.05 versus vehicle-treated animals; one-way ANOVA followed by post hoc test Dunnett or Tukey for multiple comparison using log₁₀ transformed values). Note that the spleen weight in naïve SCID beige mice is in the range of 50 mg.