Dissecting a New Pyroptotic Mechanism Driven by Gasdermin A Activation - PROJECT SUMMARY/ABSTRACT Pyroptosis is a form of programmed necrotic cell death mediated by pore-forming Gasdermins (GSDMs), essential for host defense. The human GSDM family includes gasdermin A-E, and the inactive DFNB59. Upon cleavage by proteases, the N-terminal (NT) domains of GSDMs oligomerize to form pores on the plasma membrane, inducing pyroptosis. This process releases cytokines and danger signals, activating the immune response. Our recent studies, along with others, show that killer cells secrete granzyme A (GzmA) and GzmB to cleave and activate GSDMB and GSDME, respectively, triggering pyroptosis in tumor cells, converting immune-cold tumors into hot ones, and promoting tumor-specific immune responses. GSDMA, the first identified GSDM, is selectively expressed in the epithelial tissues and is a susceptibility gene in some inflammatory diseases, presumably due to autoactivation. GSDMA expression is also silenced in gastric and esophageal cancers, likely through promoter methylation. Our recent study indicated that human GSDMA is processed by cysteine protease SpeB from Group A Streptococcus (GAS) in keratinocytes, triggering pyroptosis and defense against GAS infection. However, the GSDMA-related inflammatory diseases and cancers are unlikely to be related to GAS infections. Thus, defining how human GSDMA is activated in sterile conditions will clarify its roles in inflammation and cancer and guide new therapeutic strategies. Our preliminary research has discovered that inhibition of p97 (VCP) activates GSDMA cleavage and pyroptosis. ATPase p97 extracts proteins from complexes, membranes, and chromatin for proteasomal degradation, maintaining protein homeostasis. Elevated p97 expression has been observed in various cancers, such as breast, lung, liver, and colorectal cancers. p97 enables cancer cells to cope with increased proteotoxic stress, supporting rapid proliferation and enhancing resistance to therapies. Therefore, tumor cells are generally more sensitive to p97 inhibition than normal cells, and targeting p97 could selectively activate GSDMA-mediated pyroptosis in tumor cells. We hypothesize that p97 functions as a checkpoint to suppress GSDMA-mediated pyroptosis in cancer cells, thereby conferring immunotherapy resistance in tumors. The overarching objective is to exploit this therapeutic vulnerability as a strategy to selectively activate tumor cell pyroptosis and enhance immunotherapy efficacy (e.g., ICB) in otherwise unresponsive cancers. Specifically, our aims are to: 1) elucidate the molecular mechanisms of p97 inhibition-triggered GSDMA activation and cell death, and 2) evaluate the therapeutic efficacy and safety of p97 inhibition-induced GSDMA-dependent pyroptosis. Completion of this work will elucidate novel mechanisms underlying pyroptosis activation and establish a foundation for developing new therapeutic strategies targeting p97 to selectively activate GSDMA-mediated pyroptosis in tumors, potentially converting immune-cold tumors into immune-hot ones and transforming immunotherapy outcomes in currently unresponsive cancers–an urgent unmet need.
