In vivo administration of AZD1480 reduces primary human AML but not normal stem cells in NSG mice. (A) AML or CB mononuclear cells were transplanted into NSG mice. After 6 to 10 weeks, engraftment was confirmed, and mice were treated with AZD1480 (50 mg/kg) or vehicle daily for 2 weeks and evaluated for human cell engraftment. In 1 experiment, secondary transplant of BM cells to NSG mice was performed, and human cell engraftment was evaluated after 8 weeks. (B) Human CD45⁺ cells in BM of mice engrafted with AML cells (AML566, AML600, AML666, AML866, and AML1274) following 2 weeks of treatment with AZD1480 or DMSO. The pSTAT3 and pSTAT5 expression levels for each sample are indicated in the figure. (C-E) Pooled AML CD34⁺ cells from 2 patients (AML 404 and 755) were transplanted into NSG mice that were subsequently treated with AZD1480 or vehicle for 2 weeks, following which BM cells from primary recipient mice were transplanted in limiting dilutions (1.6 × 10⁶, 0.8 × 10⁶, 0.2 × 10⁶ cells per mouse × 8 mice each) into secondary recipient mice, and human CD45⁺ cells in PB and BM (1.8 × 10⁶ only) were evaluated after 8 weeks. (C) Human AML CD45⁺ cells and expression of differentiation markers on engrafted CD45⁺ cells in BM of primary recipients following AZD1480 or DMSO treatment. (D) Human CD45⁺ cells in PB cells of secondary recipients 8 weeks after limiting dilution transplant of BM cells from primary recipients. (E) Stem cell frequency in AZD1480- and DMSO-treated mice calculated using Poisson statistics. (F) Engraftment of human AML CD45⁺ cells and expression of differentiation markers on engrafted CD45⁺ cells in BM of secondary recipients at 8 weeks after transplant. (G) Human CD45⁺ cells in BM of mice engrafted with CB cells and treated for 2 weeks with AZD1480 or DMSO. (H) Expression of differentiation markers on human CB CD45⁺ cells in BM after AZD1480 or DMSO treatment. (I) Engraftment of human CD45⁺ cells and expression of differentiation markers on engrafted CD45⁺ cells in BM of secondary recipients at 8 weeks after transplantation of BM cells from AZD1480-treated and control mice. Results represent mean ± SEM. Significance values: *P < .05, **P < .01, ***P < .001.