Inhibition of Wnt signaling eliminates LICs. (A) Survival of recipient mice after transplantation with leukemias transduced by dnTCF lentivirus to block β-catenin signaling. Primary mouse NOTCH1-ΔE leukemias were explanted and transduced with dnTCF/GFP or empty virus control. Two days later, GFP⁺ leukemia cells were FACS-sorted and injected into each of 4 recipient animals (n = 4) at a dose of 1 × 10⁵ cells per mouse. Results depicted are compiled from 2 separate transplant experiments using independent primary leukemias (#V10-1 and #V12-1). **P < .01 (log-rank test). (B) Proliferation of mouse leukemias following transduction with dnTCF lentivirus vs nontransduced cells present in the same culture as measured by 5-bromo-2′-deoxyuridine incorporation. G1/G0, S, G2/M, and subG1 populations are gated as shown. Results depicted are representative of 2 independent leukemias analyzed (#V10-4 and #V12-1;3). (C) CFC assay for clonogenic activity. Primary mouse leukemias were transduced with dnTCF or empty control lentiviruses, FACS-sorted for the linked GFP marker, and plated in methylcellulose-containing media. *P < .05; **P < .01; ***P < .001 (Student t test). (D) Survival of recipient mice after transplantation with leukemias deleted of β-catenin. Primary mouse NOTCH1-ΔE leukemias on a Ctnnb1^loxP/loxP background were explanted, transduced with CreERT2/GFP lentivirus, and then treated with 4-OHT or vehicle (mock) control in vitro for 2 days. GFP⁺ leukemia cells were then FACS-sorted and injected into each of 4 recipient mice (n = 4) at each of the cell doses indicated in parentheses. Two separate experiments are depicted using independent primary leukemias. ****P < .0001 (log-rank test). (E-F) Cell growth/viability assays. Primary mouse NOTCH1-ΔE leukemias generated on a Ctnnb1^loxP/loxP background were transduced with CreERT2/GFP lentivirus, and then treated with 4-OHT or vehicle (mock) in vitro. In (E), relative cell growth was assessed by tracking the % GFP⁺ cells over time in culture by flow cytometry. A decreasing GFP⁺ fraction indicates transduced (GFP⁺) cells are growth disadvantaged compared with nontransduced (GFP⁻) cells in the same culture. In (F), absolute viable cell numbers were tracked over a 3-day culture period by flow cytometry with admixed polystyrene counting beads. Mean ± SD values are plotted.