Ctnnd1 knockdown compromises growth of Ikaros-kd, BCR-ABL1⁺ B-ALL in vitro. (A) Strategy for generating tet-on competent, Ikaros-kd, BCR-ABL1⁺ B-ALL, allowing inducible target gene knockdown. (B) Kaplan-Meier survival analysis of mice reconstituted with infected bone marrow cells as shown in A. Recipients of cells co-transduced with MSCV-BCR-ABL1-IRES-Luc2 and MSCV-rtTA3-shIkaros (n = 8) had median survival of 24 d, versus 53 d for MSCV-BCR-ABL1-IRES-Luc2 alone (n = 4; P = 0.022, log-rank test). (C) Representative dsRed and GFP flow cytometry of tet-on competent Ikaros-kd BCR-ABL1⁺ B-ALL derived from B cells infected with TRMPV vectors as shown in A. Cells were cultured on OP9 stroma and Dox treated as indicated, with dsRed marking shRNA expressing cells. (D) RT-qPCR expression of Ctnnd1, Emp1, and Ifitm3 relative to Hprt in ex vivo cultured tet-on competent B-ALL cells infected with TRMPV vectors expressing the indicated shRNAs. Cells were Dox treated for 4 d and GFP⁺dsRed⁺ cells were isolated by FACS. Mean ± SEM for three independent experiments. (E) Western blots of Ctnnd1 expression in splenic tumor cells harvested from two independent tet-on competent primary B-ALL transplant recipients infected with TRMPV vectors expressing shRen.713 or two different Ctnnd1 shRNAs as indicated. Before organ harvest, mice were Dox treated for 4 d and GFP⁺dsRed⁺ cells were isolated by FACS. Actin is a loading control. (F) Schematic of the culture strategy for (G–I) below. (G) Representative dsRed and GFP flow cytometry of primary tet-on competent Ikaros-kd BCR-ABL1⁺ B-ALL cells (viable CD19⁺IgM^– cells) after ex vivo infection with TRMPV vectors, 4 d Dox treatment, and plating at a 1:1 ratio of uninfected (CD19⁺GFP^–mCherry^–) and infected (CD19⁺GFP⁺mCherry⁺) B-ALL cells as shown in F. (H) Proportion of dsRed⁺ B-ALL cells within the viable CD19⁺IgM^– gate as described in G. Mean ± SEM for four experiments in two independent primary tet-on–competent leukemias. (I) Analysis of an established cell line derived from continuous passage of a primary tet-on–competent Ikaros-kd BCR-ABL1⁺ B-ALL, infected with indicated TRMPV-shRNAs as shown in F. After 3 d of Dox treatment on OP9 stroma, 50,000 sorted GFP⁺dsRed⁺ B-ALL cells were replated in Dox on OP9 stroma for a further 7 d before analysis. Plots show overall cell counts (left), the proportion of viable DAPI– cells (middle), and RT-qPCR analysis of Myc expression (right). Mean ± SEM, n = 5 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, relative to Ren.713, unpaired Student’s t test.