LAQ824- and LBH589-induced apoptosis and therapeutic efficacy do not require the death receptor pathway. (A) Eμ-myc/CrmA and Eμ-myc/trail^−/− lymphoma cells were incubated for 24 hours with the indicated concentrations of LAQ824 or LBH589. Cell viability was determined by staining with PI and FACS analysis. (B) Whole cell lysates were prepared from Eμ-myc/CrmA lymphomas that had been treated for 2 hours with vehicle, LAQ824 (25 nM), or LBH589 (4 nM). Western blot analysis was performed with antibodies specific to acetylated histone H3. Equivalent protein loading was confirmed by probing for β-actin. (C) Eμ-myc/CrmA and Eμ-myc/trail^−/− lymphomas were treated in vitro for 24 hours with vehicle (■), LAQ824 (25 nM; □), or LBH589 (4 nM; ▩). The extent of apoptosis was measured by flow cytometric analysis of the loss of Δψm and exposure of phosphatidylserine on the cell surface (annexin V staining). Each dose or time point represents the mean value of 3 individual experiments ± SE. (D) Eμ-myc/CrmA and Eμ-myc/trail^−/− lymphoma cells were treated in vitro for 24 hours with vehicle, LAQ824 (25 nM), or LBH589 (4 nM), and cell-cycle analysis was performed by FACS after staining fixed/permeabilized cells with PI. The percentages of cells residing in the G₀/G₁, S, and G₂/M phases of the cell cycle and those cells with less than 2n DNA content (sub-G₁) are indicated. Cells treated with vehicle are represented by the gray histograms in the middle and bottom panels. (E) C57BL/6 mice bearing palpable Eμ-myc/CrmA lymphomas were injected intravenously with LAQ824 (75 mg/kg) or LBH589 (80 mg/kg), and lymph nodes were harvested at the indicated times. Histologic sections were assessed by hematoxylin and eosin staining (top panel), immunohistochemistry using antibodies to acetyl histone H3 (middle panel), and TUNEL staining (bottom panel). Each time point is of an individual mouse. (F) C57BL/6 mice bearing an Eμ-myc/CrmA lymphoma were injected with vehicle (200 μL 10% lactic acid/D5W; n = 10), LAQ824 (75 mg/kg; n = 10), or LBH589 (80 mg/kg; n = 10). Kaplan-Meier survival curves of mice treated with vehicle (black line), LAQ824 (blue line), or LBH589 (red line) are shown. (G) WBC counts of mice bearing an Eμ-myc/CrmA lymphoma (n = 3) were taken 3 days after treatment with vehicle, LAQ824, or LBH589. Each point represents the mean value of 3 individual mice ± SE (*P = .03; #P = .02).