Differential impact of pirtobrutinib and cBTKis on BTK phosphorylation in Ramos RA1 cells and human CLL cells. Autophosphorylation at BTK Y223 and phosphorylation at BTK Y551 were measured by Simple Western in Ramos RA1 cells (A) and human CLL cells (B and C). (A) Ramos RA1 cells were treated with pirtobrutinib, ibrutinib, acalabrutinib, or zanubrutinib for 2 hours before stimulation with anti-IgM for 10 minutes. A representative Simple Western image shows the differential effect of pirtobrutinib vs the cBTKis on BTK Y551 phosphorylation in these cells. (B) PBMCs from 4 treatment-naive donors with CLL were treated with ascending doses of pirtobrutinib. Simple Western images show the potent inhibition of pirtobrutinib on BTK Y223 phosphorylation, with corresponding dose-response curves and calculated IC₅₀ values shown below. Donor 4 was tested in 2 independent experiments with mean ± SD shown. (C) PBMCs from the 4 donors with CLL were treated with pirtobrutinib or ibrutinib at 10 or 100 nM. Simple Western images and a corresponding bar chart show the differential effect of pirtobrutinib and ibrutinib on Y551 phosphorylation in the PBMCs from the 4 donors with CLL (mean and SD are graphed). DMSO is measured in percentage for all blots. pY223, phospho-BTK Y223; pY551, phospho-BTK Y551; tBTK, total BTK.