Alternative splicing of GSDMB modulates killer lymphocyte–triggered pyroptosis.

Granzyme A from killer lymphocytes cleaves gasdermin B (GSDMB) and triggers pyroptosis in targeted human tumor cells, eliciting antitumor immunity. However, GSDMB has a controversial role in pyroptosis and has been linked to both anti- and protumor functions. Here, we found that GSDMB splicing variants are functionally distinct. Cleaved N-terminal (NT) fragments of GSDMB isoforms 3 and 4 caused pyroptosis, but isoforms 1, 2, and 5 did not. The nonfunctional isoforms have a deleted or modified exon 6 and therefore lack a stable belt motif. The belt likely contributes to the insertion of oligomeric GSDMB-NTs into the membrane. Consistently, noncytotoxic GSDMB-NTs blocked pyroptosis caused by cytotoxic GSDMB-NTs in a dominant-negative manner. Upon natural killer (NK) cell attack, GSDMB3-expressing cells died by pyroptosis, whereas GSDMB4-expressing cells died by mixed pyroptosis and apoptosis, and GSDMB1/2-expressing 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 the tested tumor cell lines and were similarly induced by interferon-γ and the chemotherapy drug methotrexate. Expression of cytotoxic GSDMB3/4 isoforms, but not GSDMB1/2 isoforms that are frequently up-regulated in tumors, was associated with better outcomes in bladder and cervical cancers, suggesting that GSDMB3/4-mediated pyroptosis was protective in those tumors. Our study indicates that tumors may block and evade killer cell-triggered pyroptosis by generating noncytotoxic GSDMB isoforms. Therefore, therapeutics that favor the production of cytotoxic GSDMB isoforms by alternative splicing may improve antitumor immunity. Cytotoxic pores require the right splice: Granzyme A (GzmA) is one of the cytotoxic granule proteins killer lymphocytes deploy to kill targeted tumor cells. Whereas GzmA-dependent cytotoxicity is reported to depend on pyroptosis activated by cleavage of gasdermin B (GSDMB), controversy has arisen over whether all splice variants of GSDMB can support formation of cytotoxic pores. Kong and Xia et al. assessed the ability of GSDMB isoforms to induce tumor cell death and found that only the GSDMB3 and GSDMB4 splice variants retaining exon 6 supported pyroptosis. The other GSDMB isoforms interfered with pyroptosis through a dominant negative mechanism. These findings better define the structural requirements for GzmA-dependent pyroptosis involved in antitumor immunity and reveal a mechanism by which some tumor cells can evade this killing pathway through modified splicing of GSDMB mRNA. —IRW [ABSTRACT FROM AUTHOR]