Effectiveness of CAMK2 inhibition in MF model cells. (A) Growth curve of BaF/3_MPLmu_S cells treated with DMSO, CAMK2 inhibitors (KN93 and TFP), ruxolitinib, and ruxolitinib in combination with CAMK2 inhibitors. Results are means ± SD. N = 3, independent experiments. ANOVA test: ****P < .0001. (B) BaF/3 cells expressing MPL W515L become resistant against JAK2 inhibitor 1 month after the exposure. Growth curve of BaF/3_MPLmu_R cells treated with DMSO, CAMK2 inhibitors (KN93 and TFP), and ruxolitinib and ruxolitinib in combination with CAMK2 inhibitors is shown. Results are means ± SD. N = 3, independent experiments. ANOVA test: *P < .05, ***P < .001. (C-D) Apoptosis of BaF/3_MPLmu_S cells and BaF/3_MPLmu_R cells was measured by Annexin V staining and FACS analysis when treated with INCB, KN93, TFP, or both. Results are means ± SD. N = 3, independent experiments. (E) BaF/3_MPLmu_S cells and BaF/3_MPLmu_R cells were examined by immunoblot for phosphorylation of STAT5 when treated with ruxolitinib. β-Actin was used as a loading control. (F) BaF/3_MPLmu_S cells and BaF/3_MPLmu_R cells were examined by immunoblot for phosphor-STAT5 when treated with ruxolitinib, CAMK2 inhibitor, or both. β-Actin was used as a loading control. (G) Colony-forming cell assay was performed with CD34⁺ cells from MF patients harboring CALR type 1 mutation and JAK2 V617F in the presence of KN93, ruxolitinib, or both. Results are means ± SD. N = 2, experimental replicates.