Background: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, is characterized by high heterogeneity and aggressiveness[1]. The addition of rituximab (R) to form the R-CHOP regimen has been proven to improve patient overall survival by approximately 10-15% through enhanced antibody-dependent cellular cytotoxicity (ADCC)[2]. However, approximately 30% of DLBCL patients develop resistance to immunotherapy, resulting in relapse or refractory disease[2], with dysregulation of the apoptosis pathway being a key contributing factor[3]. This underscores the urgent need to explore novel cell death pathways and therapeutic targets. Pyroptosis, a newly identified form of programmed cell death mediated by Gasdermin family proteins through plasma membrane pore formation[4], can overcome apoptosis resistance and transform “cold” tumors into “hot” tumors by triggering anti-tumor immunity, demonstrating substantial anti-tumor potential[5,6,7,8]. However, therapeutic pyroptosis strategies remain underexplored in DLBCL.

Methods: Public database analysis revealed elevated GSDMD expression in DLBCL versus normal cohorts. Four DLBCL cell lines (DB, U2932, OCI-LY1, RI-1) were treated with DMB/Nigericin across a concentration gradient to determine dose-response relationships. Viability (CellTiter-Glo), death mechanisms (Annexin V/PI flow cytometry, LDH release), and morphological changes were assessed. RNA-seq and GSEA analyzed DMB-induced pathways using MSigDB hallmark gene sets (FDR q-val<0.25 as significance threshold).

Results: GSDMD emerged as the most prominently expressed pyroptosis gene in DLBCL and showed the greatest differential overexpression among 33 cancer types analyzed in public database. GSDMD expression was significantly elevated in DLBCL patients versus healthy controls, suggesting its potential as a specific therapeutic target. We further compared the effects of a novel GSDMD agonist, DMB, with the classic drug Nigericin in DLBCL. We found DMB inhibited DLBCL proliferation more potently than Nigericin. Morphologically, both agents induced characteristic pyroptotic features including cellular swelling and balloon-like protrusions, though DMB induced significantly higher cell death than Nigericin as evidenced by LDH release and elevated Annexin V+. Mechanistically, DMB dually activated JAK/STAT3 and p53 pathways, explaining its enhanced cytotoxicity.

Conclusion: GSDMD expression was higher in DLBCL cells than in healthy control. DMB effectively triggers pyroptosis and apoptosis in DLBCL. Its unique dual-pathway activation (JAK/STAT3 and p53) overcomes apoptosis resistance mechanisms and demonstrates superior efficacy versus classical pyroptosis inducers. These findings establish DMB as a promising therapeutic candidate in DLBCL.

References: [1] Swerdlow S. H., Campo E., Harris N. L., et al. World Health Organisation Classification of Tumors of Hematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008.

[2] Nowakowski GS, et al. Addition of lenalidomide to R-CHOP improves outcomes in newly diagnosed diffuse large B-cell lymphoma in a randomized phase II US intergroup study ECOG-ACRIN E1412. J Clin Oncol 2021;39(12):1329–38.

[3] Smith MR. Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene. 2003;22(47):7359-68

[4] Li M, Jiang P, Yang Y, Xiong L, Wei S, Wang J, et al. The role of pyroptosis and gasdermin family in tumor progression and immune microenvironment. Experimental hematology & oncology. 2023;12(1):103.

[5] Yang D, He Y, Muñoz-Planillo R, Liu Q, Núñez G. Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic Shock. Immunity. 2015;43(5):923-32.

[6] Hou J, Zhao R, Xia W, Chang CW, You Y, Hsu JM, et al. PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis. Nature cell biology. 2020;22(10):1264-75.

[7] Rogers C, Fernandes-Alnemri T, Mayes L, Alnemri D, Cingolani G, Alnemri ES. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nature communications. 2017;8:14128.

[8] Wang Q, Wang Y, Ding J, Wang C, Zhou X, Gao W, et al. A bioorthogonal system reveals antitumour immune function of pyroptosis. Nature. 2020;579(7799):421-6.

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2025
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