Most patients with colorectal cancer (CRC) do not respond well to therapeutic treatment. New agents for improving CRC therapy are urgently needed. Induction of programmed cell death or apoptosis is a major effect of anticancer therapy. Recent studies indicate that programmed cell death also includes necroptosis, a regulated form of necrotic death controlled by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like protein (MLKL). Accumulating evidence suggests that necroptosis, similar to apoptosis, functions as a barrier against tumor development and plays an important role in anticancer therapy. Downregulation or mutations of RIP1, RIP3, and MLKL have been frequently found in tumors and contribute to therapeutic resistance. However, few attempts have been made to target defective necroptosis in cancer cells due to insufficient understanding of the regulatory mechanism and functional role of necroptosis in anticancer therapy. Our recent studies identified a new necroptosis pathway mediated by PUMA, a p53 target and a BH3-only Bcl-2 family protein that is essential for cell death induced by a variety of anticancer drugs. This pathway can be utilized by common chemotherapeutics such as 5-fluorouracil (5-FU) to kill a subset of CRC cells. However, this pathway often cannot be engaged in CRC cells due to frequent loss of RIP3 expression, which prompted us to search for agents that can restore necroptosis in RIP3-deficient CRC cells. Our preliminary data show that OSW-1, a natural compound with potent anticancer activity, activates p53 and PUMA to induce necroptosis in CRC cells. Surprisingly, OSW-1-induced and PUMA-mediated necroptosis does not require either RIP1 or RIP3, suggesting a novel mechanism of action. Importantly, the in vivo antitumor activity of OSW-1 needs to be characterized using immuno-competent tumor models, suggesting a critical role of necroptosis-triggered antitumor immunity. Based on these findings, we propose to use OSW-1 as a chemical probe to test the hypothesis that PUMA-mediated and RIP3-independent necroptosis is efficacious against CRC via both cell-intrinsic and immunologic effects, and can be exploited to improve CRC therapy. Aim 1: Mechanism by which OSW-1 induces PUMA-mediated necroptosis in RIP3-deficient CRC cells; Aim 2: Role of PUMA-mediated and RIP3-independent necroptosis in tumor suppression by OSW-1; and Aim 3: Induction of PUMA-mediated and RIP3-independent necroptosis for improving CRC therapy. The proposed studies will delineate a novel necroptosis pathway underlying the potent anticancer activity of OSW-1. Completion of these studies will lay a foundation for identifying new anticancer agents that target defective necroptosis in CRC cells to enhance tumor cell killing and antitumor immune response, which may ultimately lead to improved treatment of CRC and other cancers.