Figure 4.

OPB-31121 selectively reduced human primary leukemia cells in mice. MAE cells (1 × 10⁶), primary FLT3/ITD-positive human AML cells, were intravenously transplanted into NOG mice. On day 29, the rate of human leukemia cells in femoral bone marrow of the pretreatment mice was measured by flow cytometry using anti-human CD45 antibody, and daily oral administration of 5% gum arabic or OPB-31121 (200 mg/kg) for 10 days was started. On day 32, daily intraperitoneal injection of cytarabine (400 mg/kg) for 7 days was started. On day 38, bone marrow cells were collected from one femur and the other femur was fixed and subjected to immunohistochemistry with anti-human CD45 antibody. The rate of human leukemia cells in bone marrow was analyzed as above. (a) The average human leukemia cell rate of three mice was plotted on the bar chart with standard deviation. (b) Bone marrow cells collected from a femur were counted. The leukemia cell number was calculated by multiplying the total cell number by the human CD45-positive cell rate. The average value of three mice was plotted on the bar chart with standard deviation. OPB-31121 did not reduce the total cell number. (c) Immunohistochemistry of bone marrow with anti-human CD45 antibody. Cells stained in brown and blue were human CD45-positive cells (leukemia cells) and negative cells (mouse normal hematopoietic cells), respectively. Normal hematopoietic cells recovered by OPB-31121 treatment. (d) Survival benefit of OPB-31121 treatment in mice transplanted with primary CML BC cells harboring BCR–ABL T315I. INH cells (5 × 10⁶), primary BCR–ABL T315I-positive CML BC cells, were intravenously transplanted into NOG mice. Daily oral administration of 5% gum arabic or the indicated dose of OPB-31121 was started on day 2 and continued until mice died. Each group consisted of nine mice. Survival curve were plotted according to the Kaplan–Meier method. Statistical difference of survival was analyzed by the log-rank test. (e) OPB-31121 did not affect the growth of normal hematopoietic cells. CD34-positive human cord blood cells (1 × 10⁵) were transplanted, treated and analyzed as described above except that mice were irradiated (2.5 Gy) the day before transplantation and that OPB-31121 and gum arabic were administered daily from days 50–59. The average human cell rate of two mice was plotted on the bar chart with standard deviation. (f) Immunohistochemistry of bone marrow of mice transplanted with cord blood cells were performed as in (c). (g) Summary of OPB-31121 effect on primary leukemia and normal hematopoietic cells.