Inhibition of IL-7R/JAK signaling diminishes IM resistance of BCR-ABL⁺ ALL cells. (A) Schematic presentation of the generation of IL-7R–deficient ALL cells. (B) Relative IL-7R and Cre-recombinase gene expression in IL-7R KO ALL cells compared with that in IL-7R^fl/fl ALL cells. Note that Cre is undetectable in KO cells owing to the use of self-excising Cre. (C) Microscopic visualization of IL-7R^fl/fl and IL-7R–deficient ALL cell clusters 24 hours after seeding of leukemic cells onto OP9 cells pretreated with the vehicle (IM−) or IM for 4 days (left). Quantification of the ALL cell clusters (middle). Representative bioluminescence images of IL-7R^fl/fl and IL-7R–deficient (IL-7R KO) ALL cells 24 hours after seeding onto the vehicle-pretreated (IM−) or IM-pretreated OP9 cells (right). (D) Microscopic visualization of IL-7R^fl/fl ALL cell clusters after treatment with control IgG or IL-7–neutralizing antibody for 48 hours. OP9 cells had been treated with the vehicle (IM−) or IM for 4 days before the leukemic cells were seeded and antibodies (6 µg/ml) were added (left). Quantification of the ALL cell clusters (middle). Bioluminescence images of IL-7R^fl/fl ALL cells cultured with vehicle- (IM−) or IM-pretreated OP9 cells and treated with control IgG or IL-7 neutralizing antibody for 48 hours (right). (E) Bioluminescence imaging of non-irradiated NOD-SCID mice transplanted with IL-7R^fl/fl or IL-7R–deficient ALL cells (4 million cells per mouse) and treated with dasatinib from day 2 to day 8 posttransplantation. Images from day 5 and day 13 are shown (n = 5 per group). (F) Kaplan-Meier survival analysis of leukemic mice transplanted with IL-7R^fl/fl or IL-7R–deficient ALL cells and treated with vehicle (n = 3) or dasatinib (n = 5). The arrows indicate 7-day treatment from day 2 to day 8 posttransplantation. P values were determined by log-rank test. (G) Bioluminescence imaging of non-irradiated NOD-SCID mice transplanted with ALL cells (3 million cells per mouse) and treated with dasatinib alone or in combination with tofacitinib from posttransplantation day 4 onwards. Images from day 1 and day 29 are shown (n = 6 per group). Note that combinatorial treatment with dasatinib and tofacitinib markedly reduced BCR-ABL⁺ ALL progression. (H) Morphology of peripheral blood smears from leukemic mice at day 30 posttransplantation. Note that there are fewer leukemic blasts in the mice with combinatorial treatment than in the mice with dasatinib treatment alone. Insets, magnified view of blasts. (I) Percentage of leukemic cell blasts in total white blood cells from the peripheral blood smears in (H). (J) Kaplan-Meier survival analysis of leukemic mice treated with vehicle or dasatinib or with the combination of dasatinib and tofacitinib. Arrow indicates the beginning of the treatment at 4 days posttransplantation. P values were determined by log-rank test. (K) Schematic illustration of IM-induced alterations in MSCs and MSC-mediated IM resistance of leukemic cells. In the absence of IM, the growth of BCR-ABL⁺ ALL cells is driven by BCR-ABL signaling and does not require support from MSCs. In the presence of IM, MSCs undergo morphologic and functional changes and produce a multitude of supporting molecules, including IL-7, thus activating IL-7R/JAK and other growth factor signaling pathways in ALL cells, whereas BCR-ABL signaling is blocked by IM. As a result, growth of the BCR-ABL⁺ ALL cells switches from being dependent on BCR-ABL signaling to being dependent on growth factor signaling. Note that JAK1 and JAK3, but not JAK2, are likely involved in the IL-7/IL-7R pathway. Scale bars, 50 µm. Data are shown as the mean ± SEM. *P < .05 by Student t test. ND, not detectable.