Glutathione S-transferase inhibitor NBDHEX induces ROS-dependent immunogenic cell death and enhances PD-1 blockade in DLBCL

Relapsed and refractory diffuse large B-cell lymphoma (R/R DLBCL) remains a significant clinical challenge, with approximately 30–40% of patients failing first-line R-CHOP therapy and responding poorly to subsequent treatments. Although immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have revolutionized cancer immunotherapy, their efficacy in DLBCL is limited. This underscores the urgent need for novel strategies that not only directly eliminate lymphoma cells but also enhance tumor immunogenicity. NBDHEX (6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol), a glutathione S-transferase (GST) inhibitor, has shown promising antitumor effects in solid tumors. However, its therapeutic potential in DLBCL and its impact on the immunosuppressive tumor microenvironment remain unexplored. DLBCL cell lines were treated with NBDHEX in vitro to evaluate reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, apoptosis, and immunogenic cell death (ICD) markers using flow cytometry, western blotting, and ELISA. In vivo efficacy and immune modulation were assessed in an A20 xenograft BALB/c mouse model, with tumor burden, immune cell infiltration, and synergy with PD-1 blockade analyzed through flow cytometry, histological examination, and RNA sequencing. NBDHEX significantly induced ROS accumulation and ER stress, triggering mitochondrial dysfunction, cytochrome c release, and caspase-dependent apoptosis in DLBCL cells. It also elicited ICD, as evidenced by calreticulin surface exposure, HMGB1 release, and ATP secretion. These damage-associated molecular patterns (DAMPs) promoted dendritic cell maturation and antigen presentation. Flow cytometry revealed increased CD8⁺ T cell infiltration in tumors after NBDHEX treatment. RNA sequencing identified enrichment of immune-related pathways, including T cell receptor signaling and antigen processing. In vivo, NBDHEX reduced tumor burden, while its combination with PD-1 blockades further enhanced antitumor efficacy and CD8⁺ T cell activation without systemic toxicity. These results suggest that NBDHEX reprograms the tumor immune microenvironment and augments the efficacy of PD-1 blockade in DLBCL. This study identifies NBDHEX as a novel dual-function agent in DLBCL that induces ROS-mediated apoptosis and converts immunologically “cold” tumors into “hot” ones via ICD-driven immune activation. Its synergy with PD-1 blockade supports further clinical development in relapsed/refractory DLBCL.