Dasatinib inhibits proliferation, CBL phosphorylation, PI3K/AKT signaling, and CBL-PIK3R1 interaction in CBL-mutant cell lines. (A) Proliferation of 32D-Cbl^KO cells expressing luciferase (KO) or V5-tagged CBL WT, Y371H, C384Y, or R420Q in the presence of DMSO or a range of dasatinib concentrations over 3 days. (B-D) Competition between dTomato-labeled 32D-Cbl^KO cells expressing CBL WT and GFP-labeled 32D-Cbl^KO cells expressing (B) CBL Y371H, (C) CBL C384Y, or (D) CBL R420Q in the presence of DMSO (closed symbols) or 1 μM dasatinib (open symbols). (E) WB for total and phosphorylated CBL, LYN, AKT, and S6 proteins in 32D-Cbl^KO cells expressing luciferase (–) or V5-tagged CBL WT, Y371H, C384Y, or R420Q treated with DMSO or 1 μM dasatinib for 2 hours. WB for vinculin (VCL) was used as a loading control. (F) WB for V5, PIK3R1, and β-actin (ACTB) in anti-V5 IP samples and WCL from DMSO- or dasatinib-treated (1 μM for 2 hours) 32D-Cbl^KO cells expressing luciferase (−) or V5-tagged CBL WT or C384Y. Statistical significance for dasatinib dose-response curves was determined by 2-tailed Student t test comparing the 50% inhibitory concentration for each CBL mutant to CBL WT or KO (see also supplemental Table 8). Statistical significance for competitive proliferation assays was determined by 2-tailed Student t test (*P < .05; ***P < .0001).