A chemical genetics approach to defining the role of iron metabolism in ferroptosis regulation

Ferroptosis, a form of regulated cell death characterized by the lethal buildup of lipid peroxides in cell membranes, is distinct from other known cell death mechanisms such as apoptosis, necroptosis, or necrosis. Its involvement in various physiological and pathological processes, including tumor suppression, immune surveillance, and ischemic organ injuries, underscores its significance. Despite its importance, understanding the intricate interplay between iron metabolism and ferroptosis remains a challenge. This gap persists partly due to the lack of efficient tools for monitoring cellular iron levels in live cells. To address this challenge, we engineered a cell line, 293T-5IRE, expressing an advanced reporter construct that allows tracking of changes in the labile iron pool (LIP) within live cells. Unlike conventional fluorescence dyes, this reporter system offers enhanced specificity and sensitivity in detecting cellular iron levels. Leveraging this tool, we aim to uncover cellular mechanisms and chemical agents influencing LIP levels and ferroptosis sensitivity. Our overarching goal is to elucidate the molecular mechanisms governing ferroptosis regulation by iron metabolism genes and their implications in physiological and pathological contexts. This proposal focuses on investigating the molecular mechanism of iron regulatory protein 2 (IRP2) on ferroptosis regulation and determining the role of salinomycin-induced lysosomal changes in modulating ferroptosis sensitivity. To address these two questions, we set two specific aims: 1) Elucidating ferroptosis resistance mechanisms by IRP2 knockout. We will investigate the impact of IRP2 perturbation on iron metabolism and ferroptosis sensitivity. By analyzing changes in the expression of iron metabolism genes and exploring potential alterations in the hypoxia-inducible factor alpha (HIFα) pathway, we aim to delineate how IRP2 knockout affects cellular capacity to handle LIP levels and confer resistance to ferroptosis. 2) Elucidating iron modulation and ferroptosis regulation by salinomycin treatment. We will explore the effects of salinomycin treatment on lysosomal status, iron metabolism genes, and NCOA4-mediated ferritinophagy to understand its mechanism of action in inducing ferroptosis. By examining whether lysosomal iron sequestration by salinomycin promotes ferritinophagy and enhances ferroptosis sensitivity, we aim to broaden our understanding of lysosomal involvement in ferroptosis regulation. This project fosters interdisciplinary collaboration and provides valuable research opportunities for undergraduate and graduate students at St. John’s University. By engaging in high-quality biomedical research, these students will gain invaluable skills and insights, paving the way for future careers in the field. In summary, our proposed research endeavors to unravel the intricate relationship between iron metabolism and ferroptosis, shedding light on novel therapeutic targets and mechanisms underlying cell death regulation in health and disease. Project Number: 1R16GM153395-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Wan Yang | Institution: ST. JOHN'S UNIVERSITY, QUEENS, NY | Award Amount: $150,000 | Activity Code: R16 | Study Section: Special Emphasis Panel[ZGM1 RCB-7 (SU)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11096144