Arginine deprivation activates immunogenic cell death and enhances anti-tumor immunity in diffuse large B-cell lymphoma

Abstract

Abstract L-Arginine, a conditionally essential amino acid, plays critical physiological roles via pathways including the urea cycle. Building on our preliminary untargeted metabolomics data revealing urea cycle dysregulation in diffuse large B-cell lymphoma (DLBCL) patients, we demonstrated that arginine deprivation therapy (ADT) induces immunogenic cell death (ICD) in DLBCL cells. Consequently, we systematically evaluated the therapeutic efficacy of ADT against DLBCL and elucidated its underlying immunomodulatory mechanisms. To characterize metabolic alterations in DLBCL, we performed untargeted metabolomic profiling on serum from treatment-naïve DLBCL patients and healthy volunteers. This analysis revealed significant dysregulation of urea cycle metabolism in DLBCL patients. Corroborating the urea cycle dysregulation, we identified frequent argininosuccinate synthetase (ASS1) deficiency in DLBCL tumors. Furthermore, ELISA confirmed pronounced reductions in serum arginine levels in the DLBCL cohort, indicating tumor cells' critical dependence on exogenous arginine for survival and proliferation. In vitro arginine deprivation in DLBCL models significantly suppressed cell proliferation and induced G0/G1 phase arrest. Mechanistically, RNA-seq revealed substantial activation of reactive oxygen species (ROS) generation and DNA damage response pathways. Experimental validation confirmed that ADT triggered robust ROS production, eliciting immunogenic cell death (ICD) manifested by surface-exposed calreticulin (CALR), extracellularly released high mobility group box 1 (HMGB1), and ATP. Furthermore, qPCR analysis demonstrated enhanced expression of interferon-stimulated genes (ISGs). To assess whether ADT-induced ICD effectively activates systemic antitumor immunity, we further performed a prophylactic vaccination model in which ADT-pretreated tumor cells elicited robust anti-tumor immunity significantly delaying subsequent tumor growth and enhancing tumor-free rates. Notably, while ADT potentiated ICD-mediated immune activation, it simultaneously upregulated PD-L1 expression. Mechanistic investigation revealed that ADT attenuated fatty acid metabolism pathways in tumor cells, resulting in decreased acetyl-CoA levels and global protein acetylation. Critically, co-immunoprecipitation assays established that diminished acetylation of PD-L1 promoted its enhanced membrane translocation. Consequently, combining ADT with PD-1 blockade demonstrated synergistic antitumor efficacy, achieving superior tumor control compared to monotherapies. Collectively, this study systematically elucidates the critical dependence of DLBCL cells on exogenous arginine and validates ADT as a promising therapeutic strategy. ADT exerts anti-tumor effects through dual mechanisms: on one hand by inducing ICD to activate anti-tumor immunity, and on the other hand by reprogramming cellular metabolism to regulate PD-L1 post-translational modifications, thereby enhancing the efficacy of PD-1 blockade efficacy. These findings provide a solid theoretical foundation for ADT combined with PD-1 blockade in DLBCL treatment, demonstrating considerable potential for clinical translation.