Figure 3.
FLT3 deletion disrupts ITD-positive LSC-specific pathways and favors normal hematopoietic reconstitution. (A) Experimental overview of the competition assay where CB and sample ITD3 were co-transplanted after FLT3-KO and control gene KO; experiment performed twice: in experiment 1 (Expt-1), the % of FLT3-KO in AML cells pretransplant was 71%, and in experiment 2 (Expt-2), the % of FLT3-KO in AML cells pretransplant was 81%. (B) KO percentages of human grafts in each mouse transplanted with the mixture (CB and ITD3 cells) from Expt-1 and Expt-2; 18 to 20 mice per group; sublethally irradiated NSG female mice; 7 to 8 weeks of engraftment. (C) Human engraftment in the competition assay in Expt-1 and Expt-2; results refer to the injected femur. (D) Analysis of human cells isolated from mice BM from panel B, focused on grafts with high percentages of FLT3-KO (>80%, as indicated in the figure). Top: mouse-depleted BM cytospins stained with Giemsa, original magnification ×1000. Bottom left: blast gate (SSC/FSC) and expression of myeloid (CD33) and B-lymphoid (CD19) markers by flow cytometry, representative example. Bottom right: quantification of leukemic blasts and normal cells based on cytomorphology of Giemsa-stained slides from mouse-depleted BM specimens, FLT3-KO average 82%, n = 3 for each condition. (E) Photograph representing spleen size of the mice from the Expt-2 from competition assay, representative examples. (F) Secondary transplantation of 125 000 to 1 million human CD45+ cells per mouse, collected from primary FLT3-KOhigh (i.e., FLT3-KO of ≥90% on transplanted cells) and control grafts: human engraftment at 7 weeks in sublethally irradiated sex-matched NSG-SGM3 (left); CD34/CD33 expression in human grafts (right). (G) Secondary transplantation from panel F: assessment of the FLT3-ITD and FLT3-WT alleles in the human engrafted cells in mice from FLT3-KO and control groups, by PCR and gel electrophoresis. (H) Gene pathways downregulated by FLT3-KO specifically in FLT3-ITD AMLs and not in WT AMLs or normal CB cells; pathway enrichment analysis from the list of the genes differentially expressed by FLT3-KO and control groups of CB (3 samples), ITD-mutated AML (3 samples), and FLT3-WT AML (3 samples). (I) Cell cycle, cell death, and H2AX-P expression by flow cytometry in FLT3-KO and control groups of 2-week grafts from sample AML ITD2; 3-4 sublethally irradiated NSG female mice per condition; experimental design in supplemental Figure 3K. Positive engraftment was considered if ≥0.1% human cells; lineage characterization was performed only on grafts with ≥1% of human cells. Unpaired Student t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; mean ± standard deviation values are reported in the graphs. ATR, ataxia telangiectasia mutated and rad3-related; bp, base pair; ctrl, control; IL-12, interleukin-12; irrad., irradiation; ns, nonsignificant; NES, normalized enrichment score; PID, pathway interaction database; snRNP, small nuclear ribonucleoproteins.

FLT3 deletion disrupts ITD-positive LSC-specific pathways and favors normal hematopoietic reconstitution. (A) Experimental overview of the competition assay where CB and sample ITD3 were co-transplanted after FLT3-KO and control gene KO; experiment performed twice: in experiment 1 (Expt-1), the % of FLT3-KO in AML cells pretransplant was 71%, and in experiment 2 (Expt-2), the % of FLT3-KO in AML cells pretransplant was 81%. (B) KO percentages of human grafts in each mouse transplanted with the mixture (CB and ITD3 cells) from Expt-1 and Expt-2; 18 to 20 mice per group; sublethally irradiated NSG female mice; 7 to 8 weeks of engraftment. (C) Human engraftment in the competition assay in Expt-1 and Expt-2; results refer to the injected femur. (D) Analysis of human cells isolated from mice BM from panel B, focused on grafts with high percentages of FLT3-KO (>80%, as indicated in the figure). Top: mouse-depleted BM cytospins stained with Giemsa, original magnification ×1000. Bottom left: blast gate (SSC/FSC) and expression of myeloid (CD33) and B-lymphoid (CD19) markers by flow cytometry, representative example. Bottom right: quantification of leukemic blasts and normal cells based on cytomorphology of Giemsa-stained slides from mouse-depleted BM specimens, FLT3-KO average 82%, n = 3 for each condition. (E) Photograph representing spleen size of the mice from the Expt-2 from competition assay, representative examples. (F) Secondary transplantation of 125 000 to 1 million human CD45+ cells per mouse, collected from primary FLT3-KOhigh (i.e., FLT3-KO of ≥90% on transplanted cells) and control grafts: human engraftment at 7 weeks in sublethally irradiated sex-matched NSG-SGM3 (left); CD34/CD33 expression in human grafts (right). (G) Secondary transplantation from panel F: assessment of the FLT3-ITD and FLT3-WT alleles in the human engrafted cells in mice from FLT3-KO and control groups, by PCR and gel electrophoresis. (H) Gene pathways downregulated by FLT3-KO specifically in FLT3-ITD AMLs and not in WT AMLs or normal CB cells; pathway enrichment analysis from the list of the genes differentially expressed by FLT3-KO and control groups of CB (3 samples), ITD-mutated AML (3 samples), and FLT3-WT AML (3 samples). (I) Cell cycle, cell death, and H2AX-P expression by flow cytometry in FLT3-KO and control groups of 2-week grafts from sample AML ITD2; 3-4 sublethally irradiated NSG female mice per condition; experimental design in supplemental Figure 3K. Positive engraftment was considered if ≥0.1% human cells; lineage characterization was performed only on grafts with ≥1% of human cells. Unpaired Student t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; mean ± standard deviation values are reported in the graphs. ATR, ataxia telangiectasia mutated and rad3-related; bp, base pair; ctrl, control; IL-12, interleukin-12; irrad., irradiation; ns, nonsignificant; NES, normalized enrichment score; PID, pathway interaction database; snRNP, small nuclear ribonucleoproteins.

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