Biological and molecular consequences of adding CFZ to IBT in primary CLL lymphocytes. Peripheral blood was obtained from CLL patients who were either IBT-naive or undergoing IBT therapy and who had given written informed consent in accordance with the Declaration of Helsinki and under a protocol approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center. PBMCs were separated by Ficoll-Hypaque density centrifugation (Atlanta Biologicals). Cells were cultured at 1 × 10⁷/mL in complete RPMI medium containing 10% human serum and were either untreated or treated with the indicated dose of drugs. (A) Pharmacological screening of IBT-treated CLL cells with 8 therapeutic agents. CLL cells isolated from a patient treated with IBT for 12 weeks were left untreated or were incubated for 24 hours with vehicle (DMSO) or the indicated agents at which point cell death was assessed by annexin V/PI double positivity. For apoptosis assay, 1 × 10⁶ cells were stained with annexin V (BD Biosciences) and PI (Sigma) and analyzed by flow cytometry as previously described.⁷  CFZ (50 nM, proteasome inhibitor; Selleck Chemicals); ABT-199 (5 nM, Bcl-2 antagonist; Xcess Biosciences); ABT-737 (5 nM, Bcl-2 and Bcl-xL antagonist); IBT (5 μM, BTK inhibitor; Selleck Chemicals); IPI-145 (2.5 μM, PI3Kδ/γ inhibitor); bendamustine (30 μM, alkylating agent); GS-1001 (5 μM, PI3Kδ inhibitor); 17-AAG (5 μM, HPS90 inhibitor; Sigma). (B) Ex vivo cytotoxicity of CFZ in CLL cells post-IBT therapy. PMBCs were isolated from 23 CLL patients who had received IBT for the indicated weeks and were then incubated with CFZ (50 nM) for 24 hours; cell death was assessed by annexin V/PI double positivity (percentage of cell death from DMSO treatment was subtracted from all samples). (C) In vitro cytotoxic effect of IBT, CFZ, or their combination. PBMCs from 7 patients diagnosed with CLL were incubated for 16 hours with CFZ (50 nM) or IBT (2 μM) alone or in combination and cell death was then evaluated by annexin V/PI staining (percentage of cell death from DMSO treatment was subtracted from all samples). (D) Impact of IBT, CFZ, or their combination on the expression level of different proteins. PBMCs from 2 patients with CLL were isolated and treated with the indicated concentrations of CFZ only or in combination with IBT for 16 hours and cell death was assessed by annexin V/PI double positivity (bottom numbers). Treated cells at 16 hours were collected, lysed, and the cell lysates were subjected to immunoblot analysis with the indicated antibodies. Immunoblot analysis was performed as previously described⁸  by using the following antibodies: phospho-BTK (Tyr²²³), BTK, phospho-IκBα (Ser^32/36), C/EBP homologous protein (CHOP), cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3 from Cell Signaling Technology; ubiquitin (Santa Cruz Biotechnology Inc); Noxa (EMD Millipore); β-actin (Cytoskeleton Inc); and Bcl-2 (DAKO). Arrows and rounded arrows indicate cleaved and full-length forms, respectively, of the indicated proteins. (E) Impact of IBT therapy on peripheral WBC count and lymphocyte count. WBC count (K/μL) and ALC (K/μL) were plotted for 8 CLL patients prior to (week 0) and 4 weeks (week 4) or 2 (week 2), 4, and 12 (week 12) weeks (CLL-826) after the initiation of IBT treatment. (F) Comparison of CFZ-induced apoptosis in CLL cells pre- and post-IBT therapy. PBMCs from 8 patients were isolated at baseline (week 0) and after 4 weeks (week 4) or after 2, 4, and 12 weeks (CLL-826) of IBT therapy and treated with the indicated concentrations of CFZ (nM). Cell death was measured by annexin V/PI staining at 24 hours or 16 hours (CLL-826) (percentage of cell death from DMSO treatment was subtracted from all samples). No correlation could be made between prognostic markers (Table 1) and CFZ cytotoxic response. (G) Comparison of CFZ-induced changes on the expression level of proteins in CLL cells pre- and post-IBT therapy. The cells from 2 patients obtained at baseline and 4 weeks after IBT therapy were treated as above for 16 hours and then collected for cell death assessment by annexin V/PI double positivity (bottom numbers). Treated cells were lysed and cell lysates were then subjected to immunoblot analysis with the indicated antibodies as described for Figure 1D. Arrows and rounded arrows indicate cleaved and full-length forms, respectively, of the indicated proteins. (H) Evaluation of the cytotoxic effect of CFZ on CLL cells isolated from patients prior to and following the initiation of IBT therapy. For each patient, PBMCs were isolated before IBT therapy (week 0) and at the treatment weeks indicated. The cells were treated with the indicated concentration of CFZ (nM) for 16 hours, and cell death was assessed by annexin V/PI double positivity (bottom numbers). Treated cells at 16 hours were collected, lysed, and the cell lysates were subjected to immunoblot analysis with the indicated antibodies as described in Figure 1D (caspase 8 and cleaved caspase 9; Cell Signaling). Arrows and rounded arrows indicate cleaved and full-length forms, respectively, of the indicated proteins. CFZ, carfilzomib; D, DMSO, dimethyl sulfoxide; IBT, ibrutinib; LE, long exposure; PBMC, peripheral blood mononuclear cell; SE, short exposure; U, untreated; WBC, white blood cell.