Effective control of AML through targeting RUNX-GST-ROS axis in vivo. (A) Schematic diagram showing the xenotransplantation AML model in NOG mice. Mice were transplanted with 2 × 106 cells/body of MV4-11 cells stably transduced with indicated lentivirus via tail veins (day 1). At day 7, po doxycycline administration through drinking water was started. In experiments using ethacrynic acid, mice were treated either by daily po ethacrynic acid or control until they showed any physical sign of AML development. (B) Overall survival of NOG mice transplanted with MV4-11 cells properly transduced with control (sh_Luc.) or with RUNX1 shRNAs (sh_Rx1_moderate 1 and sh_Rx1_profound 1) (n = 8). P value by log-rank (Mantel-Cox) test. (C) Representative microscopic images of bone marrow prepared from AML xenograft mice as in panel B (21 d posttransplantation). Results obtained from hematoxylin and eosin (HE) staining and immunohistochemical staining with anti-human CD45 antibody were shown (original magnification ×4 and ×20 (insets); scale bars, 100 μm). (D) Intracellular ROS content was measured in MV4-11 cells treated with ethacrynic acid at 25 to 50 μM or DMSO for 72 h (n = 3). (E) The number of cells with S + G2/M-phase DNA content was determined in MV4-11 cells as in panel D. Seventy-two hours after treatment, cells were harvested and subjected to flow cytometric analysis (n = 3). (F) Overall survival of NOG mice transplanted with MV4-11 cells properly transduced with sh_Rx1_moderate 1. Mice were treated either by daily po ethacrynic acid (10 to 25 mg/kg body weight) or by control solvents (n = 5). P value by log-rank (Mantel-Cox) test. *P < .05; **P < .01.
