STAT5 transcriptional activity, the status of activation of downstream signaling by human JAK2 V617F and R1063H in the presence of dimeric myeloid cytokine receptors, the binding affinities of JAK2 mutants to G-CSFR and in vitro drug sensitivity assay. Constitutive and cytokine-dependent STAT5 transcriptional activity, as assessed by dual-luciferase assay, in γ2A cells transfected with JAK2 WT, JAK2 V617F, JAK2 R1063H, and JAK2 V617F/R1063H double mutant in the presence of EPOR (A), TPOR (B), and G-CSFR (C). Homozygous and heterozygous states of JAK2 mutants are mimicked. Shown are the averages of 9 replicates from 3 independent experiments ± standard error of the mean (SEM). *P < .05, **P < .01, ***P < .001, ****P < .0001, 1-way analysis of variance (ANOVA), followed by the post hoc Tukey test. (D) Western blot analysis of constitutive JAK2, STAT5, and ERK 1/2 phosphorylation levels (indicative of activated status) induced by human JAK2 mutants coexpressed with empty vector/cytokine receptors in HEK 293T cells. β-actin antibody was used as a loading control. Higher levels of p-JAK2 (p-Tyr1007/1008), p-STAT5 (p-Tyr694), and p-ERK 1/2 (p-Thr 202/p-Tyr 204) are observed in cells expressing the JAK2 V617F/R1063H double mutant in comparison with JAK2 V617F. Image shown is representative of 3 independent experiments. (E) The effect of E596R mutation on constitutive STAT5 activation induced by JAK2 V617F and JAK2 V617F/R1063H, as evaluated by dual-luciferase assay, in γ2A cells in the presence of TPOR. Both mutated proteins exhibit a similar decline in constitutive activity. The graph displays the averages of 9 replicates from 3 independent experiments ± SEM. **P < .01, ****P < .0001, 1-way ANOVA, followed by the post hoc Tukey test. (F) JAK2 mutants bind to the cytokine receptor G-CSFR with different affinities. Flag-tagged JAK2 mutants were transiently expressed in HEK 293 cells in which hemagglutinin-tagged G-CSFR was stably expressed. Interaction was examined by coimmunoprecipitation with anti-Flag affinity gel. Immunoblot band intensity was quantified by ImageJ software and normalized to a loading control, and WT JAK2 intensity was set to 100%. The data represent the mean of 3 independent experiments; T bars designate SEM. See also supplemental Figure 4. P values < .05 were considered statistically significant. *P < .05, Student paired t test with equal variance. (G) In vitro drug sensitivity assay. Stably transfected Ba/F3/EPOR cells expressing JAK2 WT, JAK2 V617F, JAK2 R1063H, and JAK2 V617F/R1063H were cultivated for 72 hours with decreasing concentrations of the JAK2 inhibitor ruxolitinib (1, 0.5, 0.25, 0.1, 0.05, 0.01, 0.001, and 0 µM). IC₅₀ was defined as the drug concentration needed to inhibit 50% of cell growth (using GraphPad Prism 6.01 software). The data represent the mean of 7 independent experiments performed in triplicates (see also supplemental Material and methods for details). T bars designate the standard deviation. When the ruxolitinib sensitivity of mutant cells is compared with WT cells, the sensitivity of V617F⁺ cells is not statistically significant, whereas R1063H⁺ and V617F/R1063H double-mutant cells are significantly more sensitive to ruxolitinib compared with WT cells. Experiments with AZ-960 revealed comparable results (data not shown). P < .05 was considered statistically significant. *P < .05, **P < .01, 1-way ANOVA, followed by the post hoc Tukey test. IB, immunoblot; IP, immunoprecipitation; RH, R1063H; rlu, relative light unit; VF, V617F; VF/RH, V617F/R1063H.