PROJECT SUMMARY/ABSTRACT BRAFV600E mutations are common in anaplastic thyroid cancer (ATC, 50-75% positive), which is refractory to standard treatment and has high mortality rates. Monotherapy with BRAF inhibitors is ineffective in patients with BRAFV600E-mutant ATC. Although, combination therapy using a BRAF inhibitor, and a mitogen-activated protein kinase (MEK) inhibitor is more effective than BRAF inhibitor monotherapy, acquired resistance is common because BRAF and MEK act on the same downstream target and largely the same pathway. A feature of resistance to BRAF inhibition or combined BRAF and MEK inhibition is increased sensitivity to agents that induce ferroptosis (a distinct mechanism of programmed cell death dependent on iron). Moreover, it has been shown that treatment-resistant (including BRAF inhibitor-resistant) cancer cells (persister cells) are associated with a mesenchymal state that is dependent on GPX4 (a gatekeeper and suppressor of ferroptosis) and show increased sensitivity to ferroptosis induction. Our unpublished preliminary data show that ferroptosis induction is more pronounced in BRAFV600E-mutant than wild type ATC cells and that combination BRAF inhibition and ferroptosis induction has synergistic activity in causing cell death in BRAFV600E-mutant ATC cells. The long-term goal is the development of novel science-based treatment strategies for ATC that improve patient outcomes. The overall objective in this application is to determine the safety and preclinical therapeutic efficacy of combined BRAF inhibitor and ferroptosis induction treatment in BRAFV600E-mutant ATC. The central hypothesis in this proposal is that combination therapy with BRAF inhibitor and ferroptosis induction will have synergistic/additive anti-cancer activity in BRAFV600E-mutant ATC. The rationale for this project is that determination of the safety and preclinical therapeutic efficacy of ferroptosis induction with BRAF inhibition is likely to offer a strong scientific basis for translation of this novel combinatorial treatment strategy to the clinic. The central hypothesis will be tested by pursuing two specific aims: 1) Evaluate the safety and anticancer activity of targeting ferroptosis in combination with BRAF inhibition in BRAFV600E-mutant ATC in vitro and in vivo; and 2) Evaluate the efficacy of combined ferroptosis induction and BRAF inhibition compared to combination BRAF and MEK inhibition. The research proposed in this application is innovative, in the applicant’s opinion, as it represents a significant departure from the current strategy of targeting BRAF only or a single signaling pathway in BRAFV600E-mutant ATC to the concept that combined treatment with a BRAF inhibitor and ferroptosis induction will result in more robust and synergistic/additive anticancer activity. The proposed research is significant because it is expected to provide a strong scientific justification for future studies that could provide a new mechanism-based treat...