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.
Additional Information© 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. We thank MD Anderson Sequencing and Microarray Facility (SMF) for Sanger sequencing. We thank X. Ma at Boston Children's Hospital, S. Gu at MD Anderson Cancer Center, and J. Ruan at University of Connecticut Health Center for helpful discussion. This study was supported by the University of Texas Rising STARs Award and Elsa U. Pardee Research Grant to Z.Z.; National Institutes of Health grant R01CA240955 to J.L.; Howard Hughes Medical Institute funding, National Institutes of Health grant R01EB030015, and Rothenberg Innovation Initiative (RI2) at Caltech grant 25570017 to M.B.E.; National Institutes of Health grant R01AI139914 to H.W.; Ovarian Cancer Research Alliance Early Career Investigator Grant 649968 and Department of Defense grant W81XWH-18-PRCRP-CDA CA181455 to N.S.; National Institutes of Health grant R35GM133658, Komen Foundation grant CCR19609287, and the 2022–2023 program in Oncological Data and Computational Sciences sponsored by the Joint Center for Computational Oncology between the Oden Institute, MD Anderson, and TACC to S.S.Y.; postdoctoral fellowship from Jane Coffin Childs Memorial Fund for Medical Research to S.X.; and National Institutes of Health grant P30CA016672 to MD Anderson SMF. Author contributions: Q.K. and Z.Z. conceived the study. Z.Z., Q.K., and S.X. designed experiments and analyzed data. Q.K. performed most of the experiments with the assistance of Z.L., H.L., and X.T. S.X. performed structure analysis and modeling. X.P., N.S., and S.S.Y. performed bioinformatics analysis on tumor patient samples. K.Y. performed SNP genome-wide association studies analysis. J.L., X.L., S.X., M.B.E., and H.W. provided valuable editing and comments. Z.Z., Q.K., J.L., and S.X. wrote the manuscript. The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
Accepted Version - nihms-1914945.pdf
Supplemental Material - sciimmunol.adg3196_data_file_s1.zip
Supplemental Material - sciimmunol.adg3196_mdar_reproducibility_checklist.pdf
Supplemental Material - sciimmunol.adg3196_sm.pdf