β-Catenin and FOXO together modulate the transcriptional and LIC activity in human T-ALL. (A) Schematic map of the FOXO and TCF/β-catenin reporters. The FOXO lentiviral reporter (3DNm8) is composed of 3 DAF-16–binding elements (DBEs) upstream of a minimal promoter encoding a truncated NGFR, followed by a separate SV40 promoter expressing a truncated mouse CD8 marker. The fluorescent TCF/β-catenin lentiviral reporter (7TGC) is composed of 7 Tcf/Lef-binding sites upstream of a minimal promoter and green fluorescent protein (GFP), followed by a separate SV40-mCherry cassette. (B) The TCF/β-catenin and FOXO transcriptional activities were evaluated by the pGL-3xDBE and TOPFlash luciferase reporters, respectively. A construct containing 3 mutated DBE and the FOPFlash reporter with mutant TCF/LEF-binding sites were also included as control. PF382 cell lines were also cotransfected with an active FOXO3-TM alone or in combination with a stable isoform of β-catenin (ΔGSK) as indicated. Data were normalized to the Renilla luciferase reporter vector and given as mean and standard deviation. The graphs report the result of 3 independent experiments performed in triplicate. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (Student t test). (C-D) Flow cytometric analysis of NGFR⁺ and GFP⁺ cell abundance in PF382 cell line transduced respectively with FOXO (3DNm8) or TCF/β-catenin (7TGC) reporters. Leukemia cells were doubly transduced with 3DNm8 and short hairpin RNA/GFP (C) or 7TGC and short hairpin RNA/NGFR (D) lentiviral constructs, FACS sorted and cultured in vitro with PI-103, a PI3K inhibitor, or mock control. NGFR⁺ or GFP⁺ alive cells were measured at the indicated time points by flow cytometry for DRAQ7 exclusion. The graphs report the result of 3 independent experiments performed in triplicate. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (2-way analysis of variance with Dunnett test, comparing the sh-scramble control mean with the other values). (E) Schematic diagram of experimental approach. Leukemia cells of patient-derived xenografts were transduced with 7TGC and 3DNm8 lentiviruses, FACS sorted for Cherry and mouse CD8 expression and then transplanted into recipient mice, all of which subsequently developed leukemia. These secondary 7TGC_3DNm8-transduced leukemias were then analyzed by flow cytometry for GFP and NGFR expression. GFP⁺NGFR^+, GFP⁺NGFR⁻, GFP⁻NGFR⁺, and GFP⁻NGFR⁻ subsets were then FACS sorted and transplanted at limiting dilution into new recipients. (F) Survival of recipient NSG mice after transplantation with FACS-sorted β-catenin active (GFP⁺) and/or FOXO active (NGFR⁺) subsets from 7TGC_3DNm8-transduced xenograft-expanded human leukemias. The cell doses injected in each of 4 recipient animals are indicated in parentheses. Two separate experiments are depicted using independent patient-derived xenograft clones as indicated. The calculated LIC frequency in GFP⁺NGFR⁺ is 1 in 14 241 (95% CI, 1 in 3698-54 839), in GFP⁺NGFR⁻ is 1 in 387 857 (95% CI, 1 in 54 949-2 737 697), and in mock-treated cells is >1 in 146 876. DBEmut, mutated DBE; Empty V, empty vector; NS, not significant; tNGFR, truncated NGFR.