Ruxolitinib induces apoptosis and cytostatic effects in IL-7R⁺ T-ALL, irrespective of the IL-7R pathway mutational status. (A) PDX cells were cultured for 96 hours in the presence of rising concentrations of ruxolitinib. Cell viability was assessed on day 4 by FC (annexin V/propidium iodide [PI] staining). (Left) Viability curves. Results are expressed as the percentage of cell survival normalized to a dimethyl sulfoxide (DMSO)-treated condition (control). (Right) histograms depict cell survival for each category in presence of ruxolitinib (1 μM). Means and SEM are represented. Mann-Whitney test. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) PDX cells from 4 T-ALL^IL7R+/WT were cultured in complete medium deprived of IL-7 (green) or supplemented with increasing concentrations of IL-7 (orange, 1 ng/mL; red, 5 ng/mL; and purple, 10 ng/mL). For each condition, cells were exposed to increasing concentrations of ruxolitinib, and cell viability was assessed as in panel A after 72 hours of culture. (Top) Bars depict raw viability (mean with standard deviation) for control-treated conditions (DMSO). (Bottom) Cell viability was normalized for each condition to the corresponding control-treated condition. Results are shown as mean with SD for each ruxolitinib concentration. (C) PDX cells derived from 1 T-ALL^IL-7R–/low, 1 T-ALL^Mut, and 2 T-ALL^IL7R+/WT were stained with CellTrace Violet cell proliferation kit and cultured in medium supplemented with IL-7 (black histograms), with IL-7 and ruxolitinib 1 μM (yellow-filled histograms), or without IL-7 (pink-filled histograms). On day 7, cells were analyzed using FC to assess cell generations from living cells. (D) Bar plot depicts the relative proportions of cell generations among living cells for 2 T-ALL^IL7R−/low, 2 T-ALL^Mut, and 2 T-ALL^IL7R+/WT following the same experimental procedure as in panel C. Note that the cell viability is affected by the absence of IL-7 or the presence of ruxolitinib in T-ALL^Mut and T-ALL^IL7R+/WT in contrast to T-ALL^IL7R−/low.