Project Summary: p53 is transcription factor that acts in a pleiotropic manner by inducing gene expression programs that orchestrate cytotoxic responses in certain contexts, but only cytostatic responses in others. However, it is not well-understood how cellular context influences the pleiotropic behavior of p53. Understanding how cellular context influences p53-mediated anti-tumor responses will highlight critical control mechanisms that could be targeted to promote cytotoxic programs that would be more efficacious during cancer therapy than less durable cytostatic programs. Our laboratory has developed genetic tools to reversibly inactivate p53 in distinct lung cancer sub-types in the mouse and tumor-derived cell lines. These systems allow us to identify genetically distinct lung cancer sub-types that harbor cellular context variability to influence p53 function. Using our unique toolset, we determined that reactivating p53 in tumor-derived cell lines from Kras- driven lung adenocarcinoma (LUAC) induces senescence, whereas reactivating p53 in small cell lung cancer (SCLC) induces a non-apoptotic form of cell death. We conducted differential gene expression analysis and identified that autophagy signatures become enriched upon p53 reactivation in LUAC cells. In SCLC cells, we found that p53 reactivation enriches a Regulated Necrosis gene expression program. These findings show that distinct lung cancer sub-types harbor context variability that influences the gene expression programs and anti- tumor responses induced by p53 reactivation. My central hypothesis is that autophagy and programmed necrosis are respectively critical for the senescence and non-apoptotic cell death responses induced by p53 reactivation in LUAC and SCLC. In Aim 1, I will investigate the non-apoptotic form of cell death that is induced by p53 reactivation in SCLC. I will verify that programmed necrosis occurs in SCLC cells after p53 reactivation using cell and molecular biological assays, and by using genetic and pharmacological approaches to inhibit regulated necrosis to determine if cell survival is rescued. In Aim 2, I will establish the importance of autophagy in p53-mediated senescence in LUAC. I will achieve this by measuring autophagic flux after p53 reactivation, and by inhibiting autophagy to determine the extent to which it influences cell survival, senescence induction, and cell cycle progression. The goal of this proposal is expose key determinants of p53 pleiotropic behavior and ultimately therapeutically targetable molecules that enforce cytotoxic outcomes during cancer treatment.