CLL cells from patients with disease progression on pirtobrutinib show increased BCR signaling, increased cytokine secretion, reduced inhibition of proliferation, reduced in vitro cell death, and increased cell viability. (A-J) Primary CLL cells obtained at baseline (day 0), responding, and progression time points during pirtobrutinib treatment were used in the analysis. (A) Western blots of lysates from CLL cells of 3 patients whose disease progressed, looking at total and phospho-BTK, total and phospho-ERK, total and phospho-AKT, and GAPDH without additional in vitro drug treatment. (B-C) Densitometry plots for phospho-BTK, phospho-ERK, and phospho-AKT normalized to the corresponding total protein. (D-E) Plasma CCL3 and CCL4 levels measured by Luminex multiplex assay and enzyme-linked immunosorbent assay, respectively. Data points in panels B to E are color coded by patient. (F) CLL cells at responding and progression time points for each of the 3 patients were incubated with indicated doses of pirtobrutinib for 48 hours and effect on viability was analyzed using CellTitre-Glo reagent. Data reported as mean ± standard error of the mean from n = 3. (G-J) Cells were cocultured with HS-5 green fluorescent protein stroma cells. (G-H) Apoptosis was induced by 1 μM pirtobrutinib for 48 hours and analyzed by flow cytometry. (I-J) CTV-labeled cells were stimulated to proliferate as described in “Methods” and incubated with or without 1 μM pirtobrutinib with proliferation analyzed by flow cytometry. Data in panels G-J represent mean from 3 technical replicates. Graphs generated using GraphPad Prism software version 9.3. Analysis of variance and paired t test were used to calculate significance. ^∗P ≤ .05 and ^∗∗P ≤ .01. Prog, progression; resp, responding.