FLT3-KO prevents engraftment of FLT3-ITD LSCs but not of FLT3-WT LSCs. (A) Overview of the in vivo assay of FLT3-KO in AML. (B) Human engraftment of leukemic samples at 12 weeks, in sublethally irradiated NSG female mice injected with FLT3-KO or control gene (OR2W5)-KO cells; the experiment with sample ITD 3 was humanely terminated at 8 weeks; 4 to 10 mice per condition; results refer to the percentages of engraftment in the femurs that were originally injected with the cells (injected femur). (C) KO percentage in the cells pretransplantation (pre-T) and in the engrafted (graft) cells from the FLT3-KO group (top) and control group (bottom), corresponding to the experiment in panel B, determined by Sanger sequencing and indel analysis. (D) FLT3-ITD allele ratio (ITD/WT) determined by polymerase chain reaction (PCR) and capillary electrophoresis in AML samples with FLT3-ITD mutation (ITD1-7), in the bulk sample and in CD34⁺ sorted population. (E) FLT3-ITD allele ratio (ITD/WT) in AML samples ITD1, 2, 6, and 7, in the bulk sample, CD34⁺ sorted population and 12-week engrafted samples. (F) Short-term (2 weeks) transplants of ITD-mutated AMLs (ITD1, 2, and 6) in sublethally irradiated NSG female mice, KO percentage in the cells pre-T and in the engrafted (graft) cells from FLT3 KO (left) and control groups (right), determined by Sanger sequencing and indel analysis; experiment with ITD2 was performed 3 times, whereas experiments with ITD1 and ITD6 were performed once; results refer to the injected femur; in the engrafted samples (graft) each symbol represents 1 mouse; engraftment levels are depicted in supplemental Figure 1H. Positive engraftment was considered if ≥0.1% human cells. Unpaired Student t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; mean ± standard deviation values are reported in the graphs. ns, nonsignificant.