Direct TrxR/Trx inhibitors bypass CRBN requirement unlike lenalidomide for MM therapy. (A) MM.1R-control and MM.1R-CRBN-KO cells were seeded and incubated with lenalidomide at the indicated concentration for 3 days, and MTT assays were performed. (B) MM.1R-control and MM.1R-CRBN-KO cells were seeded and incubated with auranofin at the indicated concentration for 3 days, and MTT assays were performed. (C-D) OCIMY5-vector, OCIMY5-CRBN, MM.1S, and RPMI-8226 cells were seeded and incubated with auranofin at the indicated concentration for 3 days, and MTT assays were performed. (E-G) MM.1S, RPMI-8226, OCIMY5 (vector and CRBN), and MM.1R (control and KO-CRBN) cells were seeded and incubated with 20 µM concentration PX12 for 3 days, and cell viability was measured by MTT assay. Each experimental condition was performed in triplicate and repeated at least twice. (H-I) MM.1S and MM.1R (control and KO-CRBN) cell lines were treated with lenalidomide (10 μM), auranofin (1 μM), or PX12 (20 μM) for 24 hours. Cell lysates were prepared, separated by electrophoresis, and immunoblotted with the indicated antibodies. Blots are representative of 3 independent experiments. (J) Schematic representation of lenalidomide activity in MM. MM cells overproduce immunoglobulins, which generate high quantities of H₂O₂ through intramolecular and intermolecular disulfide bond formation. In addition, lenalidomide-bound CRBN also causes intracellular elevation of H₂O₂ by inhibiting intracellular TrxR. Cells with high antioxidative capacity are resistant to apoptosis from H₂O₂-mediated oxidative stress. For cells with lower antioxidative capacity, this stress leads to the degradation of IKZF1 and IKZF3, immunoglobulin dimerization, and subsequent ER stress–mediated, Bim-dependent apoptosis. Aura, auranofin; Len, lenalidomide.