Synergy between niacinamide and 4 pan-class I/II DAC inhibitors in luminetric assays. (A) “Heat map” representing synergy coefficients for 8 DLCBL cell lines treated with niacinamide in combination with 1 of 4 pan-class I/II DAC inhibitors: romidepsin, vorinostat, belinostat, or panobinostat. Cells were treated with niacinamide, 5 and 10 mM, in combination with both the IC₁₀ and IC₂₀ of each pan-DAC inhibitor (romidepsin, 1 nM, 1.5 nM; vorinostat, 400 nM, 500 nM; belinostat, 100 nM, 300 nM; and panobinostat, 2 nM, 4 nM). Cytotoxicity was measured at 24, 48, and 72 hours. Synergistic RRR values <1 are represented by red boxes, additive RRR values = 1 are represented as white boxes, and antagonistic RRR values >1 are represented as blue boxes. GC-derived DLBCL achieved greater synergistic cytotoxicity compared with ABC-derived DLBCL cell lines. (B) Cell viability as compared with controls in 4 DBLCL cell lines treated with niacinamide in combination with 1 of 4 pan-class DAC inhibitors at 24 and 48 hours. Concentrations of drugs were as follows: niacinamide, 10 mM (N); romidepsin, 1.5 nM (R); vorinostat, 500 nM (V); belinostat, 100 nM (B); and panobinostat, 4 nM (P). Each bar represents the mean of 3 experiments expressed as percentages compared with the untreated control; error bars represent standard deviation. (C) Assessment of apoptosis by Yo-Pro-1 and propidium iodide in DLBCL lines. Two GC- and 2 ABC-DLBCL cell lines were incubated with niacinamide (10 mM) alone, pan-class DAC inhibitor alone, either vorinostat or romidepsin, or the combination of niacinamide plus pan-class DAC inhibitor for 48 hours. All cell lines were treated with 10 mM niacinamide; OCI-Ly1, Su-DHL2, and HBL-1 were treated with 1.5 nM romidepsin; OCI-Ly7 was treated with 1.0 nM romidepsin; OCI-Ly1 and Su-DHL2 were treated with 400 nM vorinostat; OCI-Ly7 and HBL-1 with 500 nM vorinostat. Compared with the untreated control, niacinamide or pan-class DAC inhibitor alone resulted in minimal apoptosis. Combination of niacinamide with romidepsin or vorinostat led to increased apoptosis in GC-derived DLBCL cell lines to a greater degree compared with ABC-derived cell lines. (D) PARP and caspase 3 cleavage was demonstrated for cells treated with romidepsin alone and to a greater degree in combination with niacinamide at 24 hours. C, untreated control; N, niacinamide; NR, combination; R, romidepsin.