Abstract 2820: Intracellular calcium dynamics affect ferroptosis sensitivity

Abstract Background: Melanoma is the deadliest form of skin cancer, and its incidence continues to rise. While advances in immunotherapy, in the form of anti-PD-1, anti-CTLA-4, and anti-LAG-3 immune checkpoint inhibitors, have led to extended cancer-free outcomes for many patients with advanced disease, most patients do not have durable responses. Therefore, improvements in long-term treatment of melanoma will depend on the identification of methods to enhance existing immunotherapy regimens. Recent studies highlight ferroptosis, an iron-dependent form of regulated cell death, as a necessary component of immunotherapy-induced tumor clearance. Cytosolic calcium influx is a key aspect of ferroptosis, preceding cell bursting and linking increased plasma membrane permeability with the activation of membrane repair mechanisms. Design: This study investigated the ability of calcium modulators to alter sensitivity to ferroptosis in cancer cells. SKF96365, an inhibitor of calcium release-activated channel (CRAC) activity and store-operated calcium entry (SOCE) channels, was selected for its effects on cellular calcium homeostasis and its limited exploration as an anti-melanoma agent and ferroptosis regulator. The ability of SKF96365 to sensitize melanoma cells to ferroptosis induction was assessed by evaluating key ferroptotic hallmarks, including cell death, lipid peroxidation and reduced glutathione levels. To further elucidate the underlying mechanism, three additional calcium modulators—GSK7975A, 2-Aminoethoxydiphenyl borate (2-APB), and Ruthenium Red (RR)—were tested. Results: SKF96365 enhanced sensitivity to ferroptotic cell death, but not apoptosis or autophagy. Melanoma and colon adenocarcinoma cells treated with SKF96365 had increased levels of lipid peroxides and decreased reduced glutathione levels. GSK7975A similarly sensitized cells, supporting the role of store-operated calcium entry (SOCE) channels in ferroptosis. In contrast, 2-APB, which shows a dose-dependent bimodal effect on SOCE, increased resistance to ferroptosis despite elevating lipid peroxides and reducing glutathione. RR, a mitochondrial calcium uniporter inhibitor, also sensitized cells to ferroptosis. Conclusion: SKF96365, GSK7975A, and RR enhance ferroptosis sensitivity, whereas 2-APB confers resistance. These results provide a foundation for further exploration of calcium signaling pathways in ferroptotic cell death. Citation Format: Veronica T. Brooks, Ronan Talty, Simon F. Roy, Marcus W. Bosenberg. Intracellular calcium dynamics affect 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 2820.