PI3Kγ KD suppresses the growth of human AML cells. (A) In silico analysis of the expression of PIK3CG in human AML samples from the curated database. (B) The relationship between the PIK3CG expression level and the overall survival in patients with AML from the curated database. (C) Relative mRNA levels of PIK3CG were determined in the immunophenotypic Lin⁻CD34⁺CD38⁻CD90⁺CD45RA⁻ cord blood HSCs, CD11B⁺ differentiated human leukemia cells (Dif-AMLs), and Lin⁻CD34⁺CD38⁻CD90⁻CD45RA⁺ LSCs (n = 3). (D) PI3Kγ protein levels in Scramble, sh-PIK3CG-1, and sh-PIK3CG-2 PDX cells were measured using western blot. (E-F) CD45⁺ human AML cells in the BM at 4 weeks post transplantation (E, n = 6) and the survival (F, n = 6) of the recipients transplanted with PIK3CG-KD (sh-PIK3CG-1 and sh-PIK3CG-2) PDX1 cells or control cells were shown. (G-H) CD45⁺ human AML cells in the BM at 4 weeks after transplantation (G, n = 5) and the survival (H, n = 5) of the recipients transplanted with PIK3CG-KD PDX2 cells or control cells were shown. (I-J) CD45⁺ human AML cells in the BM at 4 weeks post transplantation (I, n = 8) and the survival (J, n = 8) of the recipients transplanted with PIK3CG-KD PDX3 cells or control cells were shown. (K) Quantification of the frequency of Lin⁻CD34⁺CD38⁻CD90⁻CD45RA⁺ LSCs among the total live BM cells from moribund mice (PDX1, n = 5; PDX2, n = 6; and PDX3, n = 6). (L) Flow cytometric analysis of ROS levels in BM LSCs from moribund mice (PDX1, n = 5; PDX2, n = 6; and PDX3, n = 6). (M) Flow cytometric analysis of BM annexin V⁺ apoptotic LSCs from moribund mice (PDX1, n = 5; PDX2, n = 6; and PDX3, n = 6). (N) Flow cytometric analysis of mean fluorescence intensity (MFI) of CD11B in PDX cells from moribund mice (PDX1, n = 5; PDX2, n = 6; and PDX3, n = 6). (O-Q) NADPH (O) and NADP⁺ (P) levels were measured in PIK3CG-KD CD34⁺ PDX cells and control cells and the ratio of NADPH/NADP⁺ (Q) was calculated 4 weeks after transplantation (n = 3). (R) The protein levels of p-AKT (S473), p-AKT (T308), AKT, G6PD, PGD, and nuclear NRF2 in PIK3CG-KD CD34⁺ PDX cells and control cells were measured using western blot 4 weeks after transplantation. (S) Chromatin immunoprecipitation assays were analyzed with 293T cells transfected with PGD-promoter and NRF2 plasmid or empty vector. Input control and the amplification of the NRF2-binding sequence of PGD were determined. (T) PGD luciferase reporter and different doses of NRF2 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (U-V) The frequencies of leukemia cells in the PB (U, n = 5) 4 weeks after transplantation and overall survival (V, n = 5) were compared among the recipients transplanted with WT, Nrf2-overexpressing WT, Pik3cg-KO, and Nrf2-overexpressing KO AML cells. Data are represented as mean ± SEM. One-way ANOVA with Tukey multiple comparison test for panels A,C,E,G,I,K- Q,T-U and log-rank test for panels F,H,J,V were used for the comparison of statistical significance. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.