BTK gatekeeper mutations T474I and T474L, but not other observed BTK mutations, can activate proximal BCR signaling. (A) Model of human BTK kinase domain binding modes of covalent inhibitor ibrutinib (orange) and noncovalent inhibitor pirtobrutinib (cyan). Upper domain is in light gray and lower domain is in dark gray. (B-E) B7.10 cells (DT40 cells lacking endogenous BTK) were transfected with BTK C481R, BTK M477I, BTK L528W, T474I BTK, T474L BTK, or C481S BTK, or WT BTK as a control. Cells were starved in medium not supplemented with serum 36 hours after transfection, and treated with 0.1 μM pirtobrutinib for 2.5 hours, or 0.5 μM ibrutinib or DMSO for 1 hour. Subsequently, the samples were washed with medium not supplemented with serum 3 times and activated with medium containing pervanadate and mouse antichicken IgM. Representative western blots are shown in panels B-E. Quantification of phospho-BTK, phospho-PLCγ2, phospho-AKT, and phospho-ERK is in supplemental Figure 8. (F) Untransfected (WT B7.10) cells were treated with the indicated doses of the drugs for 1 hour and activated with pervanadate and mouse antichicken IgM–containing medium. Results from duplicate experiments are shown in panel F.