Extended Data Figure 6. Pharmacological inhibition of divergent pathways facilitates B-leukemogenesis.

a, IL7-dependent pro-B cells were retrovirally transduced with EV-GFP, BCR-ABL1-GFP (BA-GFP), EV-Orange, or NRAS^G12D-Orange (NRAS-Orange). One week later, BA-GFP B-ALL cells were transduced with NRAS-Orange for concurrent activation of Erk and NRAS-Orange B-ALL cells were transduced with BA-GFP for concurrent activation of Stat5. The ability of oncogenic Stat5 (GFP⁺) and Erk (Orange⁺) signaling to contribute to the dominant clone was monitored by flow cytometry over time. Flow cytometry was performed to monitor the proportions of GFP⁺, Orange⁺, and double-positive cells at various time points following transductions. Representative FACS plots from 3 independent biological replicates. b, Cells from (a) were sorted for double-positive (GFP⁺ and Orange⁺) populations, and 10, 000 cells were seeded in methylcellulose for colony formation assays (10 days, n=3 independent biological replicates; mean ± s.d.). P=0.003 (left panel) and P=0.007 (right panel; two-tailed t-test). c-e, Patient-derived BCR-ABL1 B-ALL cells (MXP2) expressing Tet-on NRAS^G12D and patient-derived KRAS^G12V B-ALL cells (LAX7R) expressing Tet-On BCR-ABL1 were induced with Dox. Viable cell counts (c) were measured upon induction with doxycycline (Dox). Annexin V/7AAD and senescence β-galactosidase (d,e) staining were performed. Levels of p16 and p21 were also assessed (n=3 independent experiments). Shown are representative FACS plots and images from 3 independent experiments. P-values were determined by two-tailed t-test. Quantification for senescence β-galactosidase staining: mean of % cells positive for staining ± s.d. f, Murine wild-type BCR-ABL1 B-ALL cells were primed with vehicle control, DPH (1 μM), trametinib (1 nM) or DPH in combination with trametinib for 10 days prior to colony forming assays (n=3 independent biological replicates, mean ± s.d.) g, Murine wild-type NRAS^G12D B-ALL cells cultured in the presence of IL7 were primed with vehicle control, BCI-215 (0.5 μM), ruxolitinib (10 nM), or BCI-215 in combination with ruxolitinib for 10 days prior to colony forming assays (n=3 independent biological replicates, mean ± s.d.). P-values were determined by two-tailed t-test (f,g). For gel source data, see Supplementary Fig. 1.