Antileukemic activity of pimozide in human T-ALL. (A) Levels of STAT5 activity (pSTAT5) in ETP-ALL and mature T-ALL cells from human xenografts (top) treated with vehicle and pimozide, after in vitro stimulation with IL-7 (light area). Levels in unstimulated xenograft cells treated with vehicle were used as control (solid contour line). MFI from 3 technical replicates, Student t test; ∗∗∗P < .001 as compared with stimulated cells. Mutations of growth factor-induced signaling pathways and stimulation-inhibition (S/I) ratio of pimozide for each patient-derived ETP-ALL and mature T-ALL xenografts (bottom). The amino acid position for each mutation is shown, fs means frameshift, del means deletion. For the complete list of mutations in each patient-derived xenograft (PDX) refer to the supplemental Table 1. (B) Experimental schematic for testing the efficacy of pimozide in different ETP-ALL PDX models. Sublethally irradiated NSG recipients were randomized and subsequently treated when the average proportion of human leukemic cells reached 1% in the peripheral blood. Proportion of patient-derived leukemic cells (%hCD45⁺) in the peripheral blood of recipients at 24 hours after the last dose of pimozide was administered. 2-way ANOVA with Tukey correction test; ∗P < .05 as compared with vehicle. (C) Flow sorting strategy for transplantation of purified leukemic KIT⁻CD38⁻CD34⁻ (TN), KIT⁺CD38⁻CD34⁻ (KDN), KIT⁺CD38⁺CD34⁻ (K38⁺), and KIT⁺CD38⁺CD34⁺ (K38⁺34⁺) populations of ETP6 xenograft cells. Indicated leukemic populations were purified and injected (3 × 10⁴ cells) into sublethally irradiated immunocompromised NSG recipients. Kaplan-Meier curves of mice injected with purified TN, KDN, K38⁺, and K38⁺34⁺ populations (N = 5) from ETP6 human xenografts. Log-rank (Mantel-Cox) test; ∗∗P < .01 compared with K38⁺. (D) Levels of pSTAT5 in TN, KDN, K38⁺, and K38⁺34⁺ populations from ETP6 cells. MFI ± SD, 2-way ANOVA with Tukey correction test; ∗P < .05. (E) Experimental schematic for testing the efficacy of pimozide, VXL, and combination therapy in the ETP6 xenograft model of human ETP-ALL. Sublethally irradiated NSG recipients were randomized after engraftment was confirmed in the peripheral blood, and subsequently treated when the average proportion of human leukemic cells in the peripheral blood reached 1%. (F) Levels of pSTAT5 in ETP6 patient-derived cells, harvested from the bone marrow of recipients 24 hours after the last administration of pimozide, assessed by flow cytometry. MFI ± SEM, Student t test; ∗∗P < .01. (G-H) Absolute number of ETP6 (G) and K38⁺34⁺ (H) leukemic cells in the bone marrow and spleen of recipients analyzed 24 hours after the last drug injection. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗∗P < .01 and ∗∗∗P < .001 as compared with vehicle; ^##P < .01 and ^###P < .001 compared with VXL. (I) Kaplan-Meier curves of sublethally irradiated recipients injected with ETP6, administered with either vehicle or pimozide, as a single agent or in combination with VXL chemotherapy. Log-rank (Mantel-Cox) test; ∗∗P < .01 and ∗∗∗P < .001 compared with vehicle; ^###P < .001 compared with VXL. The period of administration is indicated in light gray, with the number of recipients for each cohort indicated.