PROJECT SUMMARY/ABSTRACT After decades of cell death study, it is now well accepted that cell death can be highly programmed, including some lytic forms. For example, the plasma membrane (PM) pore-forming executor for necroptosis is MLKL, when phosphorylated by RIPK3. For pyroptosis, gasdermin family members are the PM damaging executors, once processed by caspases or granzymes. Intriguingly, the PM pore-forming is not the “point of no return” for necroptosis or pyroptosis. This is because ESCRT- III complex can repair the damaged PM by membrane remodeling. The broken PM fractions can be shed off. Therefore, for both necroptotic and pyroptotic cells, they can tolerate a limited level of PM damage and survive for a long time if ESCRT-III repairing capacity is not overwhelmed. In this grant, we presented our UNPUBLISHED data showing if a necrotic cell can manage to stay alive, the sub-lethal PM damage is sufficient to initiate a sophisticated signal transduction network. We named this signaling transduction pathway as Plasma Membrane Integrity signaling (PMI signaling). PMI signals are efficient in promoting pro-tumor chemokines/cytokines secretions. We hypothesize that the necrotic “survivors” can stimulate oncogenesis via the PMI signaling pathway and the consequent chemokines/cytokines paracrine. Our ultimate goal is to mechanistically understand and further target these “survivors” to improve cancer treatment. To achieve this end, we will firstly hunt for the sensors for PM integrity loss. We proposed to test a promising ion channel candidate that is very likely to be one of the sensors for PM damage. We have also planned an elegant cell culture system to perform both siRNA and sgRNA screen to search for the PM damage sensors genome-wide. Next, we will use both animal models and human specimens to probe the necrotic “survivors.” We will further test the roles of the chemokines/cytokines secreted by these “survivors” in tumorigenesis and metastasis. Third, we will try to eliminate the necrotic “survivors” directly. Our preliminary data lead us to a candidate RING E3 ligase. We will test whether targeting this E3 ligase can help us suppress the ESCRT- III repair action and eradicate necrotic “survivors.” As the necrotic “survivors” were only discovered not long ago (by us and others), the massive impacts of cell death “survivors” in contributing to cancers, transplantation, and other illnesses are still fresh and needed to be fully defined. Innovative research, like this one, will promote the revelation.

PROJECT NARRATIVE Most cell death studies are focused on how cells die, while this study focuses on the cells that suffer from programmed necrosis but can survive. We hypothesize that the necrotic “survivors” can stimulate oncogenesis via chemokines/cytokines secretions. We will reveal the molecular mechanisms of the “necrotic survivors” and further target these “survivors” to improve cancer treatments.