(A) Lnk^–/–p53^–/– leukemic blasts show elevated basal activation of pAKT, pS6, and pERK. Representative phosphoflow shows the activation status of indicated proteins in BM B220^loICμ^med cells in comparison with leukemic cells after 1 hour of starvation. The dot plots show relative MFI of phosphoflow signals. n = 5–10. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t test. n = 5–10 mice per group. (B) Growth response curves for B-ALL cells treated with various concentrations of JAK and PI3K inhibitors. Lnk^–/–p53^–/– leukemic blasts were cultured in liquid culture supplemented with SCF and IL-7 in the presence of different concentrations of inhibitors, as indicated. Live cell numbers after 3 days of culture were determined by MTT absorbance. Representative results from 3 independent B-ALLs. (C) WB analyses for effector proteins of the STAT5, ERK, and PI3K/AKT pathways. Lnk^–/–p53^–/– leukemic blasts were stimulated with SCF and IL-7 for 20 minutes in the absence or presence of pretreatment with different concentrations of inhibitors. Cells were lysed, and the protein lysates were subjected to WB analysis with indicated antibodies. (D) Cohort of mice transplanted with Lnk^–/–p53^–/– B-ALLs were treated either with vehicle alone, ruxolitinib (JAKi), BEZ (PI3Ki), or combined 12 days after transplant. Kaplan-Meier survival curves are shown. Log-rank t test, *P < 0.001; **P < 0.0001; drug treatment groups compared with vehicle group. ^#P < 0.01; ^##P < 0.005, single drug treatment groups compared with combined drug treatment group. n = 7–9 mice per group. (E and F) Cohort of mice transplanted with Lnk^–/–p53^–/– B-ALLs were treated either with vehicle alone or ruxolitinib, BEZ, or combined daily for 10 days. The spleen weight along with the spleen images (E), and the donor leukemia percentage in the BM (F, left) and the spleen (F, right) are shown. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Students’ t test. n = 4 mice per group.