DOCK8 depletion selectively decreases proximal BCR signaling, cellular proliferation, and viability in DLBCLs with endogenous MYD88^L265P/CD79B^Y196F alterations and synergizes with chemical BTK inhibition. (A) (Top) Immunoblot of DOCK8 in parental DHL4, LY1, and LY7 cell lines with endogenous MYD88^WT (and CD79B^WT), and TMD8 and HBL1 cell lines with endogenous MYD88^L265P and CD79B^Y196F after shRNA–mediated DOCK8 depletion. Phosphorylation of SYK, BTK, and STAT3 was analyzed at baseline and after BCR crosslinking for 10 minutes. Actin was used as a loading control. (Bottom) Densitometry analysis of protein bands. Data are representative of 1 of 3 independent experiments. (B) Proliferation of the indicated DLBCL cell lines, transduced with an empty vector (EV) or short hairpin DOCK8 (shDOCK8), was detected using the alamarBlue assay. P values for shDOCK8 vs EV were determined with a 2-way analysis of variance (ANOVA); ∗∗∗∗P < .0001. Error bars represent the standard deviation (SD) of 3 independent replicates from a representative experiment. (C) Apoptosis of the indicated cell lines at day 4 (96 hours) is shown as percentage of annexin V⁺ cells ± SD from 3 independent replicates of a representative experiment; ∗P < .05; ∗∗∗∗P < .0001. Asterisks represent P values in the Student t test. (D) Proliferation of TMD8 and HBL1 cells transduced with the EV control or shDOCK8 and treated with the indicated doses of IBRU for 6 days, as detected via the alamarBlue assay. P values were determined using 2-way ANOVA; ∗∗∗∗P < .0001. Error bars represent the SD of 3 independent replicates from a representative experiment. (E) Apoptosis of TMD8 and HBL1 cells (in D) on day 4 (96 hours) is shown as the percentage of annexin V⁺ cells ± SD of 3 independent replicates from a representative experiment: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Asterisks represent P values in the Student t test. IBRU, ibrutinib.