American Society of Hematology

Figure 5

$Figure 5. Sensitivity of JAK2 R867Q and JAK2 S755R/R938Q mutants to JAK2 and HSP90 inhibitors. (A) Ba/F3-MPL cells expressing either JAK2 R867Q, JAK2 S755R/R938Q, or JAK2 V617F could be maintained in WEHI-supplemented medium but also exhibited cytokine-independent growth (Autonomous). Autonomous or WEHI-maintained cells were serum-starved for 6 hours and stimulated, or not (no TPO), with 5 ng/mL TPO for 15 minutes (+TPO). The constitutive phosphorylation level of each of the JAK2 constructs was analyzed by western blotting. The level of P-STAT5 was also detected, and expression of HSC70 served as a loading control. Blots are representative of a typical experiment. (B) Growth of autonomous Ba/F3-MPL cells expressing JAK2 V617F (×), R867Q (▽), and S755R/R938Q mutants (□), as well as WEHI-dependent Ba/F3-MPL expressing JAK2 WT (gray circle), was determined in response to treatment with various concentrations of INCB018424, TG101348, CYT-387, AZ960, (C) the PI3K inhibitor LY-294002, or (D) AUY922. A WST-1 proliferation assay was performed after 72 hours of exposure to the inhibitors, in the presence of 5 ng/mL TPO. Data (means ± SEM) were calculated as percentages of vehicle-treated cells and were conducted in duplicate in 4 independent experiments. Significant differences for R867Q and S755R/R938Q mutants compared with JAK2 WT were found for all inhibitors except for LY-294002 (P < .05 using the 2-tailed Student t test). (E) The 50% inhibitory concentration (IC50) values of cytokine-independent Ba/F3-MPL cells exposed to inhibitors for 72 hours were calculated using GraphPad PRISM software. (F) Coimmunoprecipitation (IP) was performed in Ba/F3-MPL cells using an anti-JAK2 antibody and blotted for the presence of HSP90 and MPL. Cell homogenates (H) show the amount of proteins before the IP. A quantification of both HSP90 and JAK2 in the IP fractions using ImageJ software (National Institutes of Health) was performed, and the histogram represents the HSP90/JAK2 ratios in AU. Blots shown were reproduced in 2 independent experiments.$

Sensitivity of JAK2 R867Q and JAK2 S755R/R938Q mutants to JAK2 and HSP90 inhibitors. (A) Ba/F3-MPL cells expressing either JAK2 R867Q, JAK2 S755R/R938Q, or JAK2 V617F could be maintained in WEHI-supplemented medium but also exhibited cytokine-independent growth (Autonomous). Autonomous or WEHI-maintained cells were serum-starved for 6 hours and stimulated, or not (no TPO), with 5 ng/mL TPO for 15 minutes (+TPO). The constitutive phosphorylation level of each of the JAK2 constructs was analyzed by western blotting. The level of P-STAT5 was also detected, and expression of HSC70 served as a loading control. Blots are representative of a typical experiment. (B) Growth of autonomous Ba/F3-MPL cells expressing JAK2 V617F (×), R867Q (▽), and S755R/R938Q mutants (□), as well as WEHI-dependent Ba/F3-MPL expressing JAK2 WT (gray circle), was determined in response to treatment with various concentrations of INCB018424, TG101348, CYT-387, AZ960, (C) the PI3K inhibitor LY-294002, or (D) AUY922. A WST-1 proliferation assay was performed after 72 hours of exposure to the inhibitors, in the presence of 5 ng/mL TPO. Data (means ± SEM) were calculated as percentages of vehicle-treated cells and were conducted in duplicate in 4 independent experiments. Significant differences for R867Q and S755R/R938Q mutants compared with JAK2 WT were found for all inhibitors except for LY-294002 (P < .05 using the 2-tailed Student t test). (E) The 50% inhibitory concentration (IC50) values of cytokine-independent Ba/F3-MPL cells exposed to inhibitors for 72 hours were calculated using GraphPad PRISM software. (F) Coimmunoprecipitation (IP) was performed in Ba/F3-MPL cells using an anti-JAK2 antibody and blotted for the presence of HSP90 and MPL. Cell homogenates (H) show the amount of proteins before the IP. A quantification of both HSP90 and JAK2 in the IP fractions using ImageJ software (National Institutes of Health) was performed, and the histogram represents the HSP90/JAK2 ratios in AU. Blots shown were reproduced in 2 independent experiments.

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