(A) Basal phosphorylation of signaling proteins was analyzed by phospho-flow in preleukemic (n = 4) and leukemic E2A-PBX1 (n = 18) BM cells. Results are shown as the MFI of CD19⁺GFP⁺ cells compared with that of CD19⁺GFP^– cells. Horizontal bars denote the mean. Statistical analysis was performed using a 2-sided Student’s t test. (B) BM leukemia cells were stimulated with cytokines, and the phosphorylation of signaling proteins was analyzed. Heatmap shows the MFI fold induction of a representative of 2 experiments. (C) BM cells were stimulated with IL-7 at increasing concentrations, and p-STAT5 was analyzed. Heatmap shows the MFI fold induction of a representative of 3 experiments. (D) BM cells were pretreated with ruxolitinib (1 μM) or vehicle (DMSO) and then stimulated with IL-7 (10 ng/μl). p-STAT5 MFI was determined and compared with unstimulated/untreated cells. Results from leukemic E2A-PBX1 mice (n = 6) are shown. (E) Lin^–CD19⁺CD43⁺ cells from the BM of WT (n = 4) and leukemic E2A-PBX1 mice (n = 5) were FACS sorted and cultured at increasing concentrations of ruxolitinib. Colonies were enumerated, and results are expressed as the mean ± SEM. Statistical analysis was performed using the F test. (F) E2A-PBX1 leukemia cells with a JAK1 L652E mutation were transplanted into sublethally irradiated secondary recipient mice (n = 5 in each cohort). Mice were treated with either vehicle or ruxolitinib (200 μg, i.p.) daily for 20 days. Statistical analysis was performed using the log-rank test. (G) Dose-response curves are shown for leukemia cells (n = 4) cultured in the presence of vehicle or ruxolitinib at increasing concentrations of dexamethasone. Colonies were enumerated, and results are expressed as the mean ± SEM. Statistical analysis was performed using the F test.