Cotreatment with CFZ and HDACIs leads to synergistic induction of cell death in DLBCL (both GC and ABC) cells and primary DLBCL cells, but not in normal hematopoietic cells. (A) SUDHL16 cells were treated with various CFZ concentrations (1.0-4.0nM) in conjunction with fixed vorinostat (0.5 or 0.75mM) concentrations for 36 hours, after which cell death was monitored by flow cytometry and annexin V/propidium iodide staining. (B) SUDHL16 cells were treated with various vorinostat (0.25-1.0μM) concentrations in the presence or absence of fixed concentrations of CFZ (2.5 or 3.0nM) for 36 hours, after which cell death was monitored by flow cytometry and annexin V/propidium iodide staining. (C) SUDHL16 cells were treated with CFZ 2.5nM with or without vorinostat 0.75μM for the indicated intervals, after which cell death was monitored by flow cytometry and annexin V/propidium iodide staining. (D) Fractional effect values were determined by comparing results obtained for untreated controls and treated cells after exposure to agents administered at a fixed ratio, after which median dose effect analysis was used to characterize the nature of the interaction. Combination index (CI) values less than 1.0 denote a synergistic interaction. (E) Cells were treated with minimally toxic concentrations of CFZ (Raji 5nM, SUDHL4 4nM, OCI-LY10 7nM, OCI-LY3 5nM) in the presence or absence of vorinostat (Raji 2.0μM, SUDHL4, OCI-LY10, OCI-LY3 1.5μM) for 48 hours, after which cell death was monitored by 7-AAD and 3,3-dihexyloxacarbocyanine iodide (DiOC₆) staining. (F) Raji and SUDHL16 cells were treated with minimally toxic concentrations of CFZ (SUDHL16 3nM, Raji 5nM), SBHA (SUDHL16 30μM, Raji 50μM), and SNDX-275 (1.0μM) for 36 to 48 hours, after which cell death was monitored by 7-AAD and DiOC₆ staining. (G) Primary human DLBCL cells were isolated as described in “Methods” and resuspended in medium containing 10% fetal calf serum at a cell density of 0.75 × 10⁶/mL cells. They were then treated with CFZ (ABC sample 2nM, GC sample 100nM, unknown type 4nM) with or without vorinostat (ABC sample 0.5μM, GC sample 1.0μM, unknown type 0.75μM) for 16 hours. The percentage of apoptotic cells was monitored by annexin V/propidium iodide staining, and the percentage of dead cells was normalized to controls. Viability of the 3 primary specimens without treatment was 60% to 70%, 80% to 85%, and 70% to 75% for the ABC, the GC, and the unknown types, respectively. (H) CD34⁺ cells were collected from the bone marrow, isolated by an immunomagnetic bead separation technique as described in “Methods,” and exposed to CFZ with or without vorinostat as indicated for 48 hours. Cell death was monitored by annexin V/propidium iodide staining, and the percentage of apoptotic cells was normalized to controls. For all studies, values represent the means for 3 experiments performed in triplicate plus or minus SD.