Figure 1. The dimerization-defective... | American Society of Hematology

Figure 1.

$The dimerization-defective R1984A NOTCH1-ΔE mutant blocks the expansion and transformation of human HSPCs, mediated by the constitutive activation of NOTCH1 signaling. (A) Schematic overview of the experimental approach. Human CD34+ cells derived from CB were doubly transduced with NOTCH1-ΔE/GFP or NOTCH1-ΔE-R1984A/GFP dimer mutant lentiviruses alone (N-only) or in combination with LMO2/mCherry (N+L) or LMO2-TAL1-BMI1/mCherry (N+LTB) constructs on days 0 and 5. Transduced cells were plated on OP9-DL1 feeders for the in vitro long-term coculture assay and before transplantation by IV injection into immunodeficient NSG mice. (B) Flow cytometric analysis of single (GFP+) or doubly transduced (GFP+Cherry+) CB cells that were transduced with N-only, N+L, or N+LTB viruses and tracked at each serial passage onto OP9-DL1 feeders every 5 days. The mean values ± range for duplicate experiments are plotted. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (C) Limiting dilution growth assays. CB cells were transduced with N-only, N+L, or N+LTB viruses on days 0 and cultured until day 3, followed by FACS into individual wells of a 96-well plate on precoated plastic in T-cell expansion media (StemCell Technologies) and culturing for 3 weeks. The entire content of each well was assayed by flow cytometry, and the frequency of well-initiating cells was calculated using the extreme limiting dilution analysis (ELDA) software. In each ELDA plot, the cell dose vs log fraction of negative cultures is represented. The dotted lines represent the confidence interval of 95%. The estimated frequencies are indicated in the box in the right side. The P values were calculated using the ELDA tool. (D) Kaplan-Meier survival curves for the primary recipient NSG mice that were injected with CB cells transduced with N-only, N+L, or N+LTB viruses and expanded for 10 days (N-only) or 15 days (N + T-ALL oncogenes) in vitro. The number of recipient animals for each cell type is indicated in parentheses. The data were pooled from 2 separate cohorts of animals. ∗∗∗P ≤ .001; log-rank Mantel-Cox test. GFP, green fluorescent protein; ns, not significant.$

The dimerization-defective R1984A NOTCH1-ΔE mutant blocks the expansion and transformation of human HSPCs, mediated by the constitutive activation of NOTCH1 signaling. (A) Schematic overview of the experimental approach. Human CD34⁺ cells derived from CB were doubly transduced with NOTCH1-ΔE/GFP or NOTCH1-ΔE-R1984A/GFP dimer mutant lentiviruses alone (N-only) or in combination with LMO2/mCherry (N+L) or LMO2-TAL1-BMI1/mCherry (N+LTB) constructs on days 0 and 5. Transduced cells were plated on OP9-DL1 feeders for the in vitro long-term coculture assay and before transplantation by IV injection into immunodeficient NSG mice. (B) Flow cytometric analysis of single (GFP+) or doubly transduced (GFP+Cherry+) CB cells that were transduced with N-only, N+L, or N+LTB viruses and tracked at each serial passage onto OP9-DL1 feeders every 5 days. The mean values ± range for duplicate experiments are plotted. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (C) Limiting dilution growth assays. CB cells were transduced with N-only, N+L, or N+LTB viruses on days 0 and cultured until day 3, followed by FACS into individual wells of a 96-well plate on precoated plastic in T-cell expansion media (StemCell Technologies) and culturing for 3 weeks. The entire content of each well was assayed by flow cytometry, and the frequency of well-initiating cells was calculated using the extreme limiting dilution analysis (ELDA) software. In each ELDA plot, the cell dose vs log fraction of negative cultures is represented. The dotted lines represent the confidence interval of 95%. The estimated frequencies are indicated in the box in the right side. The P values were calculated using the ELDA tool. (D) Kaplan-Meier survival curves for the primary recipient NSG mice that were injected with CB cells transduced with N-only, N+L, or N+LTB viruses and expanded for 10 days (N-only) or 15 days (N + T-ALL oncogenes) in vitro. The number of recipient animals for each cell type is indicated in parentheses. The data were pooled from 2 separate cohorts of animals. ∗∗∗P ≤ .001; log-rank Mantel-Cox test. GFP, green fluorescent protein; ns, not significant.

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