Killer lymphocyte-mediated tumor cell killing is a vital and final step of anti-tumor protective immunity. Killer cells, including cytotoxic CD8 T cells and Natural Killer (NK) cells, deliver granzymes (Gzm) into target cells, which normally activates noninflammatory cell death, but becomes inflammatory when target cells express the gasdermin (GSDM) proteins responsible for pyroptosis. GzmA and GzmB cleave and activate GSDMB and GSDME, respectively. However, the role of GSDMB in anti-tumor immunity is unclear - GSDMB pore formation is controversial and GSDMB expression has been linked to both anti- and pro-tumor functions. Here we found that GSDMB splicing variants are functionally distinct. Cleaved N-terminal (NT) fragments of isoforms 3 and 4 caused pyroptosis, but isoforms 1, 2 and 5, which deleted or modified exon 6, did not. Structural analysis based on AlphaFold predictions and the cryoEM structures of GSDMA3 and GSDMD showed that pore formation required the formation of a functional stabilizing belt structural element near the C-terminus of the cleaved N-terminal fragment, which was disrupted in the non-cytotoxic alternatively spliced isoforms. NK attack of GSDMB3-expressing cells caused pyroptosis, but GSDMB4 cells died mostly by apoptosis, and GSDMB1/2 cells died only by apoptosis. GSDMB4 partially resisted NK cell-triggered cleavage, suggesting that only GSDMB3 is fully functional. GSDMB1-3 were the most abundant isoforms in tested cells and were similarly induced by IFNγ and a chemotherapy drug methotrexate. Expression of cytotoxic GSDMB3/4, but not GSDMB1/2, was associated with better outcome in bladder and cervical cancer, suggesting GSDMB-mediated pyroptosis was protective in those tumors. Our study uncovers a novel role of alternative splicing in modulating caner-associated pyroptosis, and suggests that tumors may manipulate GSDMB splicing to avoid pyroptosis. Thus, modulating GSDMB splicing to increase cytotoxic GSDMB variants but suppress non-cytotoxic GSDMB isoforms could improve anti-tumor immunity and have therapeutic utility.

Citation Format: Qing Kong, Shiyu Xia, Xingxin Pan, Kaixiong Ye, Zhouyihan Li, Haoyan Li, Nidhi Sahni, S. Stephen Yi, Xing Liu, Hao Wu, Judy Lieberman, Zhibin Zhang. Alternative splicing of GSDMB modulates killer lymphocyte-triggered pyroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6145.