Abstract 6792: Understanding and enhancing cancer persister cell ferroptosis sensitivity

Abstract Cancer persister cells are a diverse subset of tumor cells that survive cytotoxic targeted therapy and contribute to tumor relapse. Identifying and exploiting persister cell vulnerabilities is a promising strategy to prevent acquired drug resistance. We previously showed inhibition of the antioxidant GPX4 selectively induces persister cell ferroptotic death.1 However, the mechanism for persister cell sensitivity to ferroptosis is poorly understood. First, to address whether low cell density underlies persister cell ferroptosis sensitivity, 2 we generated high density persister cells using prolonged cytostatic drug treatments and found chronic drug stress induces ferroptosis sensitivity independent of cell density. We also performed scRNAseq analysis of persister cells treated with GPX4 inhibitor RSL3 and observed downregulation of oxidative phosphorylation (OXPHOS) genes and upregulation of antioxidant NRF2 target genes among persister cells which survive RSL3 treatment, indicating that OXPHOS and ROS are critical for persister cell ferroptosis sensitivity. Consistent with this, we found that inhibition of OXPHOS with metformin protects persister cells from ferroptosis. Therefore, persister cells depend on OXPHOS for survival3 yet OXPHOS sensitizes persister cells to ferroptosis. We also explored how disulfiram, which has a wide variety of reported mechanisms of action, selectively kills persister cells.4 We found that disulfiram-induced persister cell killing cannot be rescued by anti-ferroptosis antioxidants such as ferrostatin, but can be rescued with other antioxidants, indicating that persister cells are vulnerable to additional forms of oxidative death beyond ferroptosis. We then sought to identify additional drugs which could sensitize persister cells to ferroptosis and found that inhibition of the oxidoreductase FSP1, which serves as a backup anti-ferroptosis mechanism to GPX4, enhances GPX4 inhibitor-induced persister cell death. We also found that transient, nontoxic pretreatment with HDAC inhibitors sensitizes persister cells to subsequent single agent GPX4 inhibitor treatment in part by increasing persister cell ROS levels. These findings provide additional mechanistic understanding of the disabled persister cell antioxidant state and reveal therapeutic strategies which may be used to selectively induce ferroptosis in persister cells and potentially spare normal tissue. 1.Hangauer, M. J. et al. Nature 551, 247–250 (2017). 2. Wu, J. et al. Nature 572, 402–406 (2019).3. Shen, S. et al. Cell Rep. 33, 108421 (2020).4. Raha, D. et al. Cancer Res. 74, 3579–3590 (2014). Citation Format: Anna E. Stuhlfire, Masayoshi Higuchi, August F. Williams, Ariel H. Nguyen, David A. Gervasio, Claire E. Turkal, Suejean Chon, Matthew J. Hangauer. Understanding and enhancing cancer persister cell ferroptosis sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6792.