Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. This study shows that cancer cells in a high mesenchymal therapy-resistant state depend on the lipid hydroperoxidase GPX4 for survival. The researchers found that persister cells, which survive chemotherapy or targeted therapy through nonmutational mechanisms, also depend on GPX4. Loss of GPX4 function leads to selective ferroptotic death of persister cells in vitro and prevents tumor relapse in vivo. These findings suggest that targeting GPX4 could be a therapeutic strategy to prevent acquired drug resistance. The study focused on drug-tolerant persister cells from various cancers, including breast, melanoma, lung, and ovarian cancers. RNA sequencing revealed upregulation of stemness markers and mesenchymal markers in persister cells, along with downregulation of epithelial markers. Antioxidant gene expression was also downregulated, indicating a disabled antioxidant program in persister cells. GPX4 inhibitors, such as RSL3 and ML210, were found to be selectively lethal to persister cells, with minimal effect on parental cells or non-transformed cells. The study confirmed that GPX4 is a selective dependency of persister cells, regardless of p53 status. GPX4 inhibition induced canonical ferroptosis in persister cells, which is an iron-dependent form of cell death. Ferroptosis was rescued by lipophilic antioxidants and lipoxygenase inhibitors, indicating that lipid peroxidation is a key factor in persister cell death. The study also showed that persister cells have reduced levels of glutathione and NADPH, which are essential for protecting against lipid peroxidation. In vivo experiments using a melanoma xenograft model confirmed that GPX4 inhibition prevents tumor relapse. The study also demonstrated that pre-treatment with GPX3 could reduce the number of persister cells, suggesting that GPX4 inhibitors could be used in clinical settings to prevent tumor relapse. The findings highlight the importance of targeting GPX4 in cancer therapy to overcome drug resistance and prevent tumor recurrence.Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. This study shows that cancer cells in a high mesenchymal therapy-resistant state depend on the lipid hydroperoxidase GPX4 for survival. The researchers found that persister cells, which survive chemotherapy or targeted therapy through nonmutational mechanisms, also depend on GPX4. Loss of GPX4 function leads to selective ferroptotic death of persister cells in vitro and prevents tumor relapse in vivo. These findings suggest that targeting GPX4 could be a therapeutic strategy to prevent acquired drug resistance. The study focused on drug-tolerant persister cells from various cancers, including breast, melanoma, lung, and ovarian cancers. RNA sequencing revealed upregulation of stemness markers and mesenchymal markers in persister cells, along with downregulation of epithelial markers. Antioxidant gene expression was also downregulated, indicating a disabled antioxidant program in persister cells. GPX4 inhibitors, such as RSL3 and ML210, were found to be selectively lethal to persister cells, with minimal effect on parental cells or non-transformed cells. The study confirmed that GPX4 is a selective dependency of persister cells, regardless of p53 status. GPX4 inhibition induced canonical ferroptosis in persister cells, which is an iron-dependent form of cell death. Ferroptosis was rescued by lipophilic antioxidants and lipoxygenase inhibitors, indicating that lipid peroxidation is a key factor in persister cell death. The study also showed that persister cells have reduced levels of glutathione and NADPH, which are essential for protecting against lipid peroxidation. In vivo experiments using a melanoma xenograft model confirmed that GPX4 inhibition prevents tumor relapse. The study also demonstrated that pre-treatment with GPX3 could reduce the number of persister cells, suggesting that GPX4 inhibitors could be used in clinical settings to prevent tumor relapse. The findings highlight the importance of targeting GPX4 in cancer therapy to overcome drug resistance and prevent tumor recurrence.