Pirtobrutinib inhibits BCR signaling in a BTK C481S in vitro model and in cells from patients with BTK C481S CLL. (A-C) HEK293 BTK WT and HEK293 BTK C481S cells were treated with either pirtobrutinib or ibrutinib with a dose range of 1.2 to 300 nM for 2 hours. (A) Representative western blot image from 3 independent experiments. BTK Y223 phosphorylation was normalized to total BTK in HEK293 BTK WT (B) and HEK293 BTK C481S (C) cells. IC₅₀ values were calculated using a 4-parameter fit in GraphPad Prism software version 9.3. (D-G) Cells from patients with BTK C481S CLL (n = 3) were pretreated with indicated dosages of pirtobrutinib, ibrutinib, or acalabrutinib for 1 hour followed by anti-IgM stimulation (10 μg/mL) for 30 minutes. (D) Representative western blot images. Quantification of phospho-BTK (E), phospho-PLCγ2 (F), and phospho-ERK (G). The data points are color coded by patient. P values shown are for drug vs DMSO stimulated (ie, IgM stimulated). Differences assessed using linear mixed effect models. ∗P ≤ .05 and ∗∗P ≤ .01. (H,I) Densitometry plots for phospho-BTK normalized to total BTK. Graphs generated using GraphPad Prism software version 9.3. VAF, variant allele frequency.