The gilteritinib/trametinib combination is effective against IgH-CRLF2-r ALL cells regardless of RAS mutation status. (A) Genome-wide CRISPR/Cas9 screen was performed using KOPN49 cells. Dot graph represents β scores obtained from KOPN49 and MUTZ5 screens. Positive regulators of the RAS pathway (depicted in orange), including RRAS2, GRAB2, SHOC2, and PTPN11, were relevant only in MUTZ5 cells. KRAS depletion had significant effects only in KOPN49 cells, which harbor the KRAS G13D mutation. (B) Cells were treated with vehicle, gilteritinib (Gilt) (1 μM), trametinib (Tram) (1 μM), or their combination, and protein samples were obtained 2 hours after treatment. Western blots were performed using 3 cell lines. (C) Growth curves were generated as described in Figure 3C. (D) The IgH-CRLF2-r ALL (BALL091) PDX model was treated with vehicle (DMSO), gilteritinib (30 mg/kg), trametinib (1 mg/kg), or the gilteritinib/trametinib combination as described in Figure 4A. Proportions of hCD19+ ALL cells in PB were examined by FACS over time. Data are represented as means ± SD. Spleen weight (E), ALL cell numbers in spleen (F), and proportions of ALL cells in BM (E) were assessed the day after the last drug injection. Data are represented as individual values with mean ± SD bars. P values were calculated using one-way ANOVA with Tukey’s posttest for multiple comparisons. *P < .05, **P < .01, ***P < .001, ****P < .0001.