PRIMA-1^Met impaired GSH metabolism in HMCLs. (A) BSO synergized with PRIMA-1^Met. Cells were incubated for 48 hours with suboptimal doses of PRIMA-1^Met (10 μM) and BSO (0.5 mM), and cell death was assessed using Apo2.7 staining. Each plot represents the mean cell death observed for each of the 27 HMCLs (obtained with 3 independent experiments). (B) Increasing doses of PRIMA-1^Met restored synergy with BSO. XG7 and JIM3 cells were incubated for 48 hours with serial concentrations of PRIMA-1^Met (10-80 μM) in the presence of 0.5 mM BSO. The data represent the mean ± SEM of 3 independent experiments. (C) Synergy between PRIMA-1^Met and BSO was independent of TP53 status. Median cell death induced by PRIMA-1^Met (10 μM) and BSO (0.5 mM) for each cell line was plotted against the TP53 status. (D) NAC overcame PRIMA-1^Met and BSO synergy. Cells were incubated for 48 hours in the presence of PRIMA-1^Met (10 μM) and BSO (0.5 mM), with or without NAC (5 mM). The data represent the mean ± SEM of 4 independent experiments. (E) BSO increased ROS production induced by PRIMA-1^Met. OPM2 (0.5 × 10⁶ cells/mL) was incubated overnight with 30 μM PRIMA-1^Met or with 10 μM PRIMA-1^Met and 0.5 mM BSO. CellROX reagent (5 μM) was added to the cell culture for the last 30 minutes, at 37°C. Cells were washed in phosphate-buffered saline, and fluorescence was analyzed on FACSCalibur. (F) PRIMA-1^Met induced GSH depletion. Cells (0.5 × 10⁶ cells/mL) were treated overnight with PRIMA-1^Met alone (30 μM), BSO alone (0.5 mM), or a combination (10 μM and 0.5 mM). Cells were lysed in acidic buffer (10 × 10⁶ cells/mL), and GSH content was determined using a colorimetric kit by following the instructions of the manufacturer. The data represent the mean ± SEM of 4 experiments. **P < .01. (G) Sensitivity of myeloma cells to PRIMA-1^Met correlated with GSS expression. Expression of GSS or of the 2 subunits of γGCS (GCLM and GCLC) was plotted against LD₅₀ PRIMA-1^Met values. Correlation was assessed by Spearman test. (H) Silencing of GSS increased sensitivity to PRIMA-1^Met. OPM2 cells were incubated for 3 days with siControl or siGSS RNA and treated with 25 μM PRIMA-1^Met for the last 24 hours. Cell death was assessed by Apo2.7 staining. The data represent the mean ± SEM of 4 independent experiments. Expression of GSS (representative experiment) and GSH content (n = 4) was determined at 72 hours. *P < .05.