Gain-of-function CBL mutations are associated with increased cytokine sensitivity and activation of the LYN and PI3K/AKT-signaling pathway. (A-C) Proliferation of Cbl^KO 32D (A-B) or BaF3 (C) cells expressing WT or mutant CBL after 3-day stimulation with IL3 (A,C) or GM-CSF (B). (D-I) Competition between GFP-labeled 32D-Cbl^KO (D,G) or TF1-CBL^KO (E,H) cells expressing CBL-mutant and dTomato-labeled CBL WT (D-E) or CBL knockout (KO) (G-H) cells. Competition between 32D-Cas9 cells expressing Cbl intron 7 guide RNAs (gRNAs) (RFP657) and nontargeting gRNA (blue fluorescent protein [BFP]; panel F) or Cbl exon 1 gRNAs (BFP; panel I). (J) Volcano plot showing relative quantity of phosphotyrosine sites detected by MS and normalized to proteome-level data in 32D-Cbl^KO cells expressing V5-tagged CBL WT or C384Y. (K) WB for total and phosphorylated CBL, LYN, AKT, and S6 proteins in 32D-Cbl^KO cells expressing V5-tagged CBL WT, Y371H, C384Y, or R420Q. WB for vinculin (VCL) was used as a loading control. Statistical significance for cytokine dose-response curves was determined by a 2-tailed Student t test comparing the 50% effective concentration for each CBL mutant with CBL WT (see also supplemental Table 1). Statistical significance for competitive proliferation assays was determined by 2-tailed Student t test comparing each CBL mutant with CBL WT or KO (*P < .05; **P < .001; ***P < .0001).