BCR inhibition by ibrutinib downregulates ROR1 levels and impairs ROR1-targeted therapies. (A) Effect of ibrutinib (1 µM) treatment on MCL cell line proliferation after 3 days. Fold change is compared with starting cell count. Error bars represent mean plus or minus SD of triplicates. (B) Immunoblot analysis of cytoplasmic and nuclear cell lysates of MCL cell lines plus or minus 1 µM ibrutinib for 3 days. *Loss of protein in ibrutinib-treated Mino nuclear cell lysates. Quantifications shown for ROR1 protein levels were normalized to β-tubulin levels and compared with untreated control for each cell line. (C) Flow cytometry analysis of ROR1 levels (in live cells) after 0.5 µM (Jeko-1 and Mino) or 1 µM (Maver-1 and Z-138) ibrutinib for 3 days compared with untreated sample. (D) Percentage of viable cells (compared with sample without mAb) left untreated or pretreated with ibrutinib (1 µM) for 3 days followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (E) Table summary of ROR1 levels as measured by flow cytometry in live cells of primary samples untreated or treated ex vivo with ibrutinib (1 µM for 48 hours). (F) Comparison of ROR1 expression in MCL samples from panel E plus or minus ibrutinib. Statistical significances: *P < .05, **P < .01, ***P < .001, calculated by a 2-tailed paired Student t test.