CpdA is active in patient-derived neoplastic cells. (A) Plasma cells (CD138⁺) were purified from 3 unique patient bone marrow aspirates and incubated with CpdA or bortezomib for 2 days under the indicated conditions. Viability was determined using the WST-1 assay, and is represented as the mean plus or minus SD from triplicate experiments. (B) The mononuclear cell fraction from a fourth patient with myeloma was cultured with DMSO or the indicated concentration of CpdA for 24 hours; stained with CD138-PE, annexin V–FITC, and TO-PRO-3; and analyzed by flow cytometry. CD138⁺ myeloma cells are gated in the top row, while CD138⁻ cells are gated in the bottom row. The proportion of cells present in the respective quadrants is indicated inside each of the panels, including viable cells (FITC⁻/To Pro3⁻), early apoptotic cells (FITC⁺/To Pro3⁻), and late apoptotic or necrotic cells (FITC⁺/To Pro3⁺). (C) Mononuclear cells isolated from the peripheral blood of a patient with CD34⁺ acute lymphoblastic leukemia were cultured with DMSO and CpdA for 24 hours, then stained with CD34-PE and annexin V–FITC and analyzed by flow cytometry. The proportion of cells present in the respective quadrants is indicated inside each panel as described for panel B. (D) Mononuclear cells isolated from 2 unique patients with AML and one patient with MM were cultured with DMSO, 10 μM CpdA, or 5 nM bortezomib for 24 hours. Whole-cell lysates were subject to Western blotting (top panel) using anti-p27 and β-actin antibodies, and analyzed by densitometry (bottom panel). (E) Isolates from a patient with AML were cultured with the indicated concentration of bortezomib, CpdA, or the combination for 24 hours. Cells were then stained with annexin V–FITC and TO-PRO-3 and analyzed using flow cytometry. The proportion of cells present in the respective quadrants is indicated inside each panel as for panel B. (F) Isobologram analysis of the viability results when leukemia patient samples were treated with combinations of bortezomib and CpdA.