Activity of carfilzomib and bortezomib in patient samples. (A) Purified plasma cells were continuously treated with increasing doses of carfilzomib for 24 hours. Cells were then lysed, and the ChT-L activity was determined (10 μg per reaction). (B) CD138⁺ cells were treated with continuous exposure to the indicated concentrations of carfilzomib, followed by a WST-1 cell viability assay. (C) Purified plasma cells were pulse-treated with 100 nM carfilzomib or bortezomib, followed by recovery in drug-free media for 24 hours. WST-1 was used to assess proliferation. Several of the samples are from patients with chromosome 13 deletions (MM-13, MM-15, MM-17, MM-20, and MM-23). Results are expressed as the percentage (%) inhibition of proliferation of carfilzomib-treated cells relative to bortezomib-treated cells, which were set at 0, with a positive result indicating the amount of enhanced antiproliferative activity of carfilzomib over that of bortezomib. (D) Pulse carfilzomib and bortezomib exposure in an NHL patient sample and determination of antiproliferation activity by WST-1 assay is shown. (E) Flow cytometric analysis of carfilzomib-induced versus bortezomib-induced specific apoptosis in patient-derived CD19⁺ CLL B-cells is shown. Patient cells were pulsed (100 nM) for 1 hour with the indicated drug and allowed to recover for 24 hours. Apoptosis was assessed in cells stained with Annexin V/TO-PRO-3/anti-CD19. Specific apoptosis is shown in the CD19⁺ gated population relative to vehicle controls. (F) AML cells from a patient with progressive disease after multiple chemotherapeutic treatments were pulsed for 1 hour with 100 nM carfilzomib or bortezomib or continuously treated with 1 μM Dox. Apoptosis was measured by DNA fragmentation ELISA and expressed as fold induction over DMSO control in CD33⁺cells purified from peripheral blood mononuclear cells (PBMCs). Error bars in panels A,B,C,D, and F are SD.