Pirtobrutinib reduced BCR signaling and induced apoptosis in cells from patients with BTK WT CLL. (A-C) BTK WT CLL cells were treated with either pirtobrutinib or ibrutinib with a dose range of 1.2 to 300 nM. Data shown are representative results from n = 7. (A) Representative western blot. (B) BTK Y223 phosphorylation was normalized to total BTK and IC₅₀ values were calculated using a 4-parameter fit in GraphPad Prism 9.3 software. (C) Densitometry plots for phospho-PLCγ2 (Y1217), normalized to total PLCγ2. Boxes represent median values with the first and third quartiles; whiskers represent the maximum and minimum values. P values shown are for drug vs dimethyl sulfoxide (control). ^∗P ≤ .05, ^∗∗P ≤ .01, and ^∗∗∗P ≤ .001 by mixed effect analysis of variance, Holm-Šidák test. (D-E) CellTrace Violet–labeled BTK WT CLL cells from patients with CLL were incubated with growth stimulants as indicated in “Methods” and treated with 1 μM of either pirtobrutinib, ibrutinib, or acalabrutinib, and cell proliferation was assayed by flow cytometry after a 10-day culture. (D) Histograms showing CTV profiles of 4 patients with BTK WT. Divided cells as a percent of total cells are automatically calculated by the FlowJo software and included in the upper left of each panel. (E) Inhibition of CLL cell proliferation by the indicated drug treatment, normalized to dimethyl sulfoxide, based on data in panel D. (F) Cells from patients with BTK WT CLL were treated with 1 μM of either pirtobrutinib, ibrutinib, or acalabrutinib for 48 hours. Apoptosis was measured with Annexin V propidium iodide staining. Statistics were performed by one-way analysis of variance with Dunnett posttest; ^∗P ≤ .05. Graphs generated using GraphPad Prism software version 9.3. The data points for panels E and F are color coded by patient. Acala, acalabrutinib; Conc., concentration; CTV, CellTrace Violet; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ibr, ibrutinib; IgM, immunoglobulin M; Pirto, pirtobrutinib; Pt, patient.