A simple arithmetic of life is this: if cells in a tissue divide more frequently than they die, the tissue grows; if cells die more frequently than they divide, the tissue shrinks. This basic principle is enshrined as a “hallmark” of cancer—for a cancer to exist it must evade cell death mechanisms that would shift this equation to attrition. For three decades my laboratory has worked to understand the core pathways of regulated cell death and how they are controlled at the molecular level. This program of research, the continuation of which is proposed in this application, explores the processes of regulated cell death in the forms of apoptosis and necroptosis, and seeks to understand how they are tied to other cellular physiologies, as they must be. Three general goals of this research are outlined as questions, as follows. A. What are the mechanisms of cell survival in apoptosis/necroptosis and how do these integrate with cell life? Here we use the concept of “flatliners,” cells that survive the activation of core apoptotic or necroptotic pathways, to probe the pathways that, when engaged, restrict these core pathways to enable transient resistance to this and subsequent stimuli. We have found that cancer cell survivors of the apoptotic pathway display increased aggressiveness in vivo and we have found signatures of these cells in minimal residual disease. Our flatliners are tested in cancer allograft and xenograft models. We will extend our studies to cells that survive necroptosis. B. What are the consequences of surviving a cellular “near death experience (flatlining)? Cells that survive engagement of a cell death pathway (flatliners) resemble, in all aspects, cancer persister cells that survive a therapeutic treatment without selection for resistance mutations. While the definitions are distinct, we propose that flatliners can take on the properties of persisters. These include genomic stability. We have found that Gasdermin E, a caspase substrate that causes lytic cell death, limits the generation of flatliners, and thus prevents caspase- induced DNA damage and mutation, with consquences for human cancers. C. How do diverse processes of cellular life integrate with the mechanisms of necroptosis? Necroptosis is a form of regulated necrosis that is actively inhibited by the action of a caspase, normally associated with apoptosis (but here with cell survival). We will continue our studies into regulation of necroptosis in relation to cellular physiology and develop tools to probe its activation in the context of cancer and other pathologies. Surviving necroptosis involves repairing membrane damage inflicted by the necroptosis process (a function of ESCRT) and reversing the activation of its executioner. While the understanding of the core pathways of cell death have led to one approved cancer therapeutic, our continued “life and death” efforts set the stage for future success in this critical arena.