Targeting preexisting drug-resistant leukemic cells with Notch/PI3K-directed combination therapy. (A) Heatmap depicts the relative activity of PI3K, Notch, PIM, and MYC activation signatures in single cells of ETP-ALL and T-ALL PDX samples. (B) Relative percentage of cell-cycle phase for root cells in ETP-ALL and T-ALL PDXs demonstrating lower cell-cycle activity in root 1 vs root 2. (C) Violin plots show Notch and PI3K activation in root 1 (PI3K high) and root 2 (Notch high) in ETP and T-ALL PDXs. (D) GSI and buparlisib are synergistic in KOPT-K1 T-ALL cells. Cell survival as assayed by CellTiter-Glo after 7-day culture (error bars reflect standard deviation [SD] from 3 replicates; left). Combination index analyses (3 replicates; right). (E) KOPT-K1 cells were pretreated with 1 μM buparlisib or DMSO for 3 days and then immediately sorted or cultured for an additional 24 hours without drug (Washout). Single cells from each of these populations were sorted into individual wells of 96-well plates and cultured with GSI (1 μM) or DMSO for 6 weeks. Bar plots indicate the fraction of single cells that form colonies in GSI (n = 480 wells). Pretreatment with buparlisib eliminates preexisting GSI-tolerant cells from untreated T-ALL populations, which cannot be reversed by 24-hour washout (error bars reflect SD, averaged from 5 plates, using 2-sided Student t test). (F) Flow cytometry demonstrating subpopulation of CD34⁺ cells with p4E-BP1(S65) and pAKT (Thr308) staining in KOPT-K1 cells (overlay histogram gated on CD34⁺ or CD34⁻ cells, respectively; left). CD34⁺ population decreases with buparlisib treatment (barplot below; error bars reflect SD from 3 replicates). *NOTCH1 mutated, #PTEN deleted (A), **P < .01, ****P < .0001 using Kruskal-Wallis test (C), ****P < .0001 using 2-sided Student t test (E), *P < .05 using 2-sided t test (F). CI, combination index; Fa, fraction affected.