Beyond Lysis: Deciphering the Role of Perforin-Mediated Ferroptosis in Anti-Tumor Immunity - Perforin, a crucial T cell protein, mediates tumor cell death by a poorly understood mechanism. This proposal is based on preliminary data suggesting that perforin can induces ferroptosis in tumor cells by increasing intracellular iron. Ferroptosis is a programmed cell death pathway dependent on iron and lipid peroxidation. The long-term goal is to develop translational therapies to enhance perforin-induced ferroptosis. The overall objective is to determine the mechanism and metabolic regulators of perforin-induced ferroptosis. The central hypothesis is that perforin increases intracellular iron, triggering ferroptosis and inhibiting metastasis and immunotherapy resistance. The central hypothesis will be tested by three specific aims: 1) Define the molecular mechanisms of perforin-mediated ferroptosis; 2) Characterize the contribution of perforin- induced ferroptosis to anti-tumor immunity; and 3) Determine the role of tumor metabolism in perforin-induced ferroptosis (R00). Aim 1 will use genetic, pharmacologic, and dietary approaches to mechanistically define perforin-induced ferroptosis. Aim 2 will study the role of perforin-induced ferroptosis in metastatic, primary, and immunotherapy in vivo models. Aim 2 will also bioinformatically explore how perforin expression contributes to patient immunotherapy response. Aim 3 will metabolically manipulate tumor cells to optimize perforin killing and leverage translational chimeric antigen receptor-T cell (CAR-T) technology. Together, this proposed research will elucidate the ferroptosis-inducing function of perforin and its contribution to immunotherapy response and metastasis control in different metabolic contexts. The research proposed in this application is innovative because it explores a previously unknown mechanism of perforin killing molecularly, translationally, and metabolically. This innovative perspective on CTL-mediated tumor killing has the potential to explain the role of in vivo immune-induced ferroptosis in immunotherapy response and lay a foundation to explore novel cancer immunotherapies based on perforin-induced ferroptosis. This work will be accomplished under the mentorship of Dr. Weiping Zou (tumor immunology) and Dr. Arul Chinnaiyan (translational pathology) alongside a team of Michigan-based experts in immunology, bioinformatics, and metabolism. A comprehensive training plan will provide technical training in mass spectrometry, flow cytometry, bioinformatics, and CAR-T approaches. Attention will be given to facilitating training in mentorship, project management, manuscript preparation and grantsmanship. This comprehensive mentorship plan will facilitate a smooth transition to a position as an independent investigator focused on microenvironmental determinants of immune-mediated cell death.
