Systematic genetic analysis of GSDMD, GSDME, and MLKL in PANoptosis

Cell death is a key innate immune effector mechanism. While it is beneficial for pathogen clearance, excess lytic cell death is linked to inflammation, pathology, and disease. PANoptosis is an innate immune, lytic, inflammatory cell death pathway initiated by innate immune sensors and driven by caspases and RIPKs, with roles in infection, inflammatory disease, and cancer. During PANoptosis, caspases and RIPKs activate multiple pore-forming "executioner" proteins, including gasdermins and mixed lineage kinase domain-like protein (MLKL), which lead to membrane damage and the release of damage-associated molecular patterns and cytokines. While some have begun to define cell death pathways by their pore-forming executioners, the specific executioner(s) genetically required to drive PANoptosis remain unclear. Therefore, we performed a comprehensive genetic analysis of gasdermin D (GSDMD), gasdermin E (GSDME), and MLKL across triggers that drive PANoptosis. Deletion of GSDMD, GSDME, or MLKL individually did not fully block PANoptosis, suggesting these molecules are compensatory in executing PANoptosis. Furthermore, combined deletion of GSDMD, GSDME, and MLKL generally provided greater protection than any single or double deletion, but 20%-50% of cells still died, suggesting involvement of additional executioners that remain unidentified. Overall, our study suggests that defining cell death pathways purely by executioners is error-prone, as the activation of different pore-forming executioners is variable, context-dependent, and redundant. This further suggests that targeting individual executioners will not be sufficient to block cell death in disease contexts where PANoptosis drives pathology; targeting the upstream molecules, such as innate sensors or PANoptosome complex components, will be needed for therapeutic efficacy in infection, inflammatory disease, and cancer.