Pirtobrutinib inhibits BCR signaling and cytokine secretion and proliferation in responding cells from patients with BTK WT and C481S CLL. (A-D) Proteins were evaluated in defrosted lysates, without in vitro treatment, from cells from 2 patients with BTK WT and 1 patient with BTK C481S CLL, and levels of phospho-BTK, phospho-PLCγ2, and phospho-ERK were analyzed by immunoblotting. (A) Western blots for each patient. (B-D) Densitometry plots for phospho-BTK, phospho-PLCγ2, and phospho-ERK normalized to the corresponding total protein. Differences assessed by analysis of variance followed by Tukey test. (E-F) Plasma CCL3 and CCL4 levels measured by Luminex multiplex assay and enzyme-linked immunosorbent assay, respectively. Graphs generated using GraphPad Prism software version 9.3. Data points in panels B-F are color coded by patient. (G) CellTrace Violet–labeled BTK WT and BTK C481S CLL cells were cocultured with HS-5 green fluorescent protein stroma cells, incubated with growth stimulants as indicated in “Methods” and treated in vitro with 1 μM pirtobrutinib. Cell proliferation was assayed by flow cytometry after a 10-day culture. Divided cells as a percent of total cells are automatically calculated by the FlowJo software and included in the upper left of each panel. ^∗P ≤ .05 and ^∗∗P ≤ .01.