Figure 6.
Treatment with FHD-286 exhibits in vivo efficacy against AML-initiating stem cells. (A) PD mtNPM1 + FLT3-ITD Luc/GFP cells (AML number 1 in the oncoplot) were ex vivo treated with 10 and 30 nM of FHD-286 for 96 hours. After this, equal numbers of cells (2.5e6 cells/mouse) were tail vein infused into preirradiated (2.5 Gy) NSG mice (n = 5 per cohort). Mice were monitored daily for symptoms of acute leukemia. Luciferase signal in the mice was determined by IVIS imaging (Xenogen) at 1, 2, and 4 weeks after infusion of AML cells. The box plots show the total bioluminescent flux (photons/second) at 1, 2, and 4 weeks after infusion of the AML cells in the mice. Significance between cohorts was determined by a 2-tailed, unpaired t test using GraphPad Prism V9. P values <.05 were considered significant. (B) Kaplan-Meier survival curve of NSG mice infused with ex vivo treated PD mtNPM1 + FLT3-ITD Luc/GFP cells. Significance between cohorts was determined by a Mantel-Cox log-rank test. P values <.05 were considered significant. (C) PD mtNPM1 + FLT3-ITD Luc/GFP cells (2.5e6 cells/mouse) were tail vein infused into preirradiated (2.5 Gy) NSG mice (n = 4 per cohort). Mice were monitored for 5 days, and leukemia engraftment was documented by IVIS imaging. Mice were randomized to equivalent bioluminescence and treated with vehicle or 1.5 mg/kg of FHD-286 for 5 weeks. The box plots show the total bioluminescent flux (photons/second) at 5 weeks after infusion of the AML cells. Significance between cohorts was determined by a 2-tailed, unpaired t test using GraphPad Prism V9. P values <.05 were considered significant. (D) After 5 weeks of treatment when vehicle mice required euthanasia, all mice were euthanized, and the spleens and bone marrow were harvested. The panel shows 2 representative spleens from vehicle and 1.5 mg/kg FHD-286–treated mice. (E) Representative bioluminescent images of mice from panel C. (F) Viable human AML cells from the spleens and bone marrow of vehicle and FHD-286–treated mice were reinfused into preirradiated (2.5 Gy) NSG mice (n = 6 per cohort). The box plots show the total bioluminescent flux (photons/second) 3 weeks after reinfusion of the AML cells. Significance between cohorts was determined by a 2-tailed, unpaired t test using GraphPad Prism V9. P values <.05 were considered significant. (G) Representative bioluminescent images of mice from panel F. (H) Kaplan-Meier survival curve of NSG mice infused with equal numbers of previously in vivo treated PD mtNPM1 + FLT3-ITD Luc/GFP cells. Significance between cohorts was determined by a Mantel-Cox log-rank test. P values <.05 were considered significant. ∗∗∗P < .005; ∗∗∗∗P < .001.

Treatment with FHD-286 exhibits in vivo efficacy against AML-initiating stem cells. (A) PD mtNPM1 + FLT3-ITD Luc/GFP cells (AML number 1 in the oncoplot) were ex vivo treated with 10 and 30 nM of FHD-286 for 96 hours. After this, equal numbers of cells (2.5e6 cells/mouse) were tail vein infused into preirradiated (2.5 Gy) NSG mice (n = 5 per cohort). Mice were monitored daily for symptoms of acute leukemia. Luciferase signal in the mice was determined by IVIS imaging (Xenogen) at 1, 2, and 4 weeks after infusion of AML cells. The box plots show the total bioluminescent flux (photons/second) at 1, 2, and 4 weeks after infusion of the AML cells in the mice. Significance between cohorts was determined by a 2-tailed, unpaired t test using GraphPad Prism V9. P values <.05 were considered significant. (B) Kaplan-Meier survival curve of NSG mice infused with ex vivo treated PD mtNPM1 + FLT3-ITD Luc/GFP cells. Significance between cohorts was determined by a Mantel-Cox log-rank test. P values <.05 were considered significant. (C) PD mtNPM1 + FLT3-ITD Luc/GFP cells (2.5e6 cells/mouse) were tail vein infused into preirradiated (2.5 Gy) NSG mice (n = 4 per cohort). Mice were monitored for 5 days, and leukemia engraftment was documented by IVIS imaging. Mice were randomized to equivalent bioluminescence and treated with vehicle or 1.5 mg/kg of FHD-286 for 5 weeks. The box plots show the total bioluminescent flux (photons/second) at 5 weeks after infusion of the AML cells. Significance between cohorts was determined by a 2-tailed, unpaired t test using GraphPad Prism V9. P values <.05 were considered significant. (D) After 5 weeks of treatment when vehicle mice required euthanasia, all mice were euthanized, and the spleens and bone marrow were harvested. The panel shows 2 representative spleens from vehicle and 1.5 mg/kg FHD-286–treated mice. (E) Representative bioluminescent images of mice from panel C. (F) Viable human AML cells from the spleens and bone marrow of vehicle and FHD-286–treated mice were reinfused into preirradiated (2.5 Gy) NSG mice (n = 6 per cohort). The box plots show the total bioluminescent flux (photons/second) 3 weeks after reinfusion of the AML cells. Significance between cohorts was determined by a 2-tailed, unpaired t test using GraphPad Prism V9. P values <.05 were considered significant. (G) Representative bioluminescent images of mice from panel F. (H) Kaplan-Meier survival curve of NSG mice infused with equal numbers of previously in vivo treated PD mtNPM1 + FLT3-ITD Luc/GFP cells. Significance between cohorts was determined by a Mantel-Cox log-rank test. P values <.05 were considered significant. ∗∗∗P < .005; ∗∗∗∗P < .001.

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