PROJECT SUMMARY The incorporation of Immune checkpoint blockade into anticancer armamentarium has revolutionized cancer therapy in recent years, with FDA approved agents in multiple cancer types. Nevertheless, there is an urgent need to identify additional therapeutic strategies to increase durable response rates. Induction of immunogenic cell death is one of such strategies. The BCL-2 family proteins are central regulators of mitochondrial apoptosis and consist of (1) multidomain antiapoptotic BCL-2, BCL-XL, and MCL-1, (2) multidomain proapoptotic BAX and BAK, and (3) proapoptotic BH3-only molecules (BH3s). BAX and BAK are the essential effectors of MOMP whereas BCL-2, BCL-XL, and MCL-1 preserve mitochondrial integrity. BH3s are death sentinels that relay upstream apoptotic signals to initiate apoptosis by either activating BAX/BAK or inactivating BCL-2/BCL- XL/MCL-1. Through an interconnected hierarchical network of interactions, the BCL-2 family proteins integrate developmental and environmental cues to dictate the survival versus death decision of cells. The research on the BCL-2-regulated apoptotic pathway has not only revealed its importance in both normal physiological and disease processes, but has also resulted in the first anti-cancer drug targeting protein-protein interactions. Recent paradigm-shifting discoveries have shown that BAX/BAK activation in the absence of caspases can trigger the release of mitochondrial DNA to the cytosol through a process called “mitochondrial inner membrane permeabilization” (MIMP), which in turn activates the cGAS/STING pathway and type I interferon response. Hence, the BCL-2 family plays a crucial role not only in the decision of cells to live or commit suicide but also in the decision to die in an immunologically silent or inflammatory manner. The discovery of MIMP and its role in activating immunogenic cell death opens up exciting new avenues for cancer cell death research. However, the molecular and biochemical basis of MIMP remains uncharacterized. Furthermore, it is unclear whether induction of MIMP in tumors will affect the tumor-immune crosstalk and immunotherapy response. In this grant application, we have formulated a comprehensive plan to interrogate the biochemical and molecular basis of MIMP and exploit MIMP as a therapeutic strategy to improve and enhance immunotherapy. Our studies will not only provide novel mechanistic insights into the BCL-2-regulated cell death program but also lay the foundation for targeting the BCL-2 family to induce immunogenic cell death and thereby enhance cancer immunotherapy.