Inhibition of antioxidant production renders OxPhos-DLBCLs more sensitive to HDACi treatment. (A) Left: OxPhos-DLBCLs and BCR-DLBCLs were treated with increasing concentrations of BSO, a chemical inhibitor of glutathione production. OxPhos-DLBCLs (red lines) exhibited a greater vulnerability to BSO treatment than BCR-DLBCLs (green lines). Right: IC₅₀ values for OxPhos-DLBCLs (red) and BCR-DLBCLs (green) treated with BSO. (B) Cell cycle analysis of DLBCLs treated with a combination of pracinostat (250 nM) and BSO (10 µM). For OxPhos-DLBCLs (right), dual treatment markedly augmented the sub-G₁ fraction in comparison with single treatment with either drug alone. Preincubation with the antioxidant vitamin E (100 µM) rescued cells from the cytotoxicity of dual treatment. Dual treatment did not increase the sub-G₁ fraction in BCR-DLBCLs (left). (C) Schema illustrating proposed mechanism of reduced OxPhos-DLBCL sensitivity to HDACi-induced cytotoxicity via upregulation of ROS. Left panel: intrinsically high glutathione production in OxPhos-DLBCLs⁴¹  neutralizes ROS produced by HDACi treatment, resulting in reduced OxPhos-DLBCL cell death. Right panel: BSO is a specific inhibitor of γ-glutamyl cysteine ligase (GCL), a crucial enzyme in glutathione synthesis. Dual treatment with BSO and HDACi increases OxPhos-DLBCL cell killing. The increased cytotoxicity of dual BSO-HDACi treatment can be blocked by pretreatment with the antioxidant vitamin E (not shown), reducing OxPhos-DLBCL cell death to the level seen when cells are treated with HDACi alone (as shown in left panel). Experiments were performed at least twice. Error bars denote standard deviation.