Apilimod induces noncanonical cytotoxicity in B-NHL. (A) Cytotoxicity of SU-DHL-10 B-NHL treated with either dimethyl sulfoxide (DMSO) or 200 nM apilimod for 3 days and stained with the viability dye 7-AAD and the apoptotic indicator Annexin V. Percentages of single- and double-positive cells are displayed. (B) DEVD-UltraGlo Luciferase cleavage activity in SU-DHL-10 B-NHL after 2-day treatment with the indicated concentration of apilimod. (C) Viability of SU-DHL-10 B-NHL measured under increasing concentrations of apilimod ± 25 μM of the caspase inhibitor Z-VAD-FMK for 3 days. (D) Viability of SU-DHL-10 B-NHL treated with apilimod and the indicated concentration of cathepsin inhibitor (cathepsin inhibitor I, E64d, CA-074 Me, or cathepsin inhibitor III) for 3 days. Apilimod was used at 156 nM in single-agent and combination experiments. (E) Viability of SU-DHL-10 B-NHL that were pretreated for 24 hours with 5 μg/mL of the necroptosis inhibitor necrostatin-1 and subsequently cotreated with 5 μg/mL necrostatin-1 and the indicated concentration of apilimod for 3 days. (F) Representative western blot of SU-DHL-10 B-NHL cells that were treated with vehicle, apilimod (200 nM), the autophagy inducer rapamycin (5 μM) or the combination of apilimod and rapamycin in the absence or presence of the autophagy inhibitor bafilomycin A1 (500 nM added for the last 8 hours) for 24 hours. Cell lysates were probed with antibodies against LC3, p62, or vinculin (loading control).