Notch inhibition expands preexisting cells with PI3K signaling activity that coexist with Notch-dependent cells and demonstrate opposing differentiation trajectories. (A) t-SNE plot of leukemic cells colored based on GSI treatment with roots and endpoint highlighted as in Figure 2C. (B) Heatmap depicts the relative activity of various signaling pathways as inferred by PROGENy in all cells comprising the 2 root states. (C) Heatmap demonstrating preexisting cells with PI3K activity in untreated patients. (D) Violin plots show decreasing Notch activity with increasing PI3K activity upon GSI treatment in patient 5 (P5). (E) Violin plots depicting expression of Notch1 target genes (top) and PI3K pathway genes (bottom) in untreated and GSI-treated single cells of P5. (F) Scatter plot depicting negative correlation between relative Notch activity and PI3K activity in leukemic cells from all patients. Cells are colored based on GSI treatment. (G) Projection of RNA velocity vectors onto untreated leukemic cells plotted by PI3K and Notch activity (subclusters defined by monocle; supplemental Figure 14G). (H) Leukemic cells belonging to 2 different root states are highlighted and fall into either high PI3K or high Notch activity clusters. (I) Enrichment of endpoint cells at the interface of converging velocity trajectories. (J) Leukemic cells with high PI3K activity persist after GSI treatment with preserved directionality of RNA velocity (subclusters identified by monocle; supplemental Figure 14G). ****P ≤ .0001 using Kruskal-Wallis test.