Caspase-3 activation during apoptosis can trigger caspase-3 cleavage of gasdermin E (GSDME). The gasdermins (GSDM) are a family of proteins, whose cleavage activates inflammatory death, called pyroptosis. N-terminal GSDM fragments form pores in the cell membrane that cause rapid cell death in which the cell swells, activates and releases inflammatory cytokines and other inflammatory mediators, and eventually bursts. GSDME cleavage converts noninflammatory apoptotic death to more rapid inflammatory pyroptotic death. GSDME is not expressed in most cancer cell lines, is epigenetically inactivated in gastric, colorectal and breast cancer, relative to normal tissue, and mutated in some others. GSDME expression suppresses colony formation in gastric and colorectal cancer and invasivity of breast cancer. Worse 5-year survival and an increase in lymph node metastases are associated with reduced GSDME in breast cancer. Moreover, lack of GSDME promotes drug resistance in melanoma and lung cancer cell lines. We hypothesize that GSDME acts like a tumor suppressor, that some tumor cells avoid pyroptosis by downregulating or mutating GSDME and that the switch from apoptosis to pyroptosis profoundly affects tumor cell survival, anti-tumor immunity and response to chemotherapy and radiation. In preliminary data, cancer-associated GSDME mutations are shown to be primarily loss of function mutations. Gsdme knockout in cancer lines promoted tumor growth while ectopic GSDME expression strongly inhibited tumor growth in immune competent mice. The tumor suppressive role of GSDME was lost in immunodeficient NOD.scid.Il2rg-/- (NSG) and Prf1-/- mice deficient in perforin and strongly inhibited by NK or CD8 T cell depletion. GSMDE expression by the tumor increased infiltrating, functional CD8 T cells and NK cells. Based on these data, we hypothesize that GSDME suppresses tumor growth by recruiting and activating anti-tumor killer lymphocytes. We also found that granzymes B and M, death- inducing proteases of killer lymphocytes, directly cleave GSDME during killer cell attack to activate pyroptosis in a caspase-independent manner, which is amplified by caspase activation. We also hypothesize that direct granzyme cleavage of GSDME in cancer cells triggers pyroptosis and enhances their anti-tumor immunity. Killer lymphocyte mediated death has previously been thought to be non-inflammatory. Our goal is to test these hypotheses and develop mechanistic insights to understand whether and how suppression of GSDME activation of pyroptosis in cancer cells impacts oncogenesis, drug sensitivity and protective immunity. Our specific aims are to investigate the effect of GSDME mutation and expression on GSDME activation, lipid binding, oligomerization and por...