Abstract 5658: DHODH inhibition enhances radiotherapy-induced ferroptosis by promoting mitochondrial lipid peroxidation
Authors: Mao Chao , Horbath Amber , Lei Guang , Gan Boyi 
Published: June 2022

Abstract
Background: Ferroptosis is a form of regulated cell death caused by excessive accumulation of lipid peroxides on the cellular membrane. While impaired ferroptosis has been shown to contribute to tumor development, uncontrolled ferroptosis has been associated with neurodegeneration, acute kidney injury, and cardiovascular disease. To defend against ferroptosis, cells are equipped with several antioxidant systems. Glutathione peroxidase 4 (GPX4) is a major antioxidant enzyme that localizes in both the cytosol and the mitochondria and uses glutathione (GSH) as its cofactor to convert lipid hydroperoxides into non-toxic lipid alcohols. In addition, dihydroorotate dehydrogenase (DHODH) catalyzes the reduction of ubiquinone to ubiquinol in the inner mitochondrial membrane and inhibits mitochondrial lipid peroxidation independently of the GSH-GPX4 mechanism. Radiotherapy (RT) is one of the major therapies used to treat cancers. During RT, the radiolysis of water generates reactive oxygen species (ROS), subsequently leading to lipid peroxidation and ferroptosis. In our study, we aim to determine the role of DHODH in RT-induced ferroptosis and to evaluate the therapeutic potential of DHODH inhibitors as robust radiosensitizers in cancer treatment.
Methods: Lipid peroxidation and mitochondrial lipid peroxidation were evaluated by flow cytometry. Ferroptosis marker PTGS2 expression levels were evaluated by real-time PCR. CRISPR-Cas9 system used to generate knock-out cancer cell lines. Patient-derived xenografts (PDX) co-treated with RT and DHODH inhibitors were employed to access the combination effect. Lipid peroxidation marker 4-HNE levels in tumors were evaluated by immunohistochemistry.
Results: RT induced lipid peroxidation and PTGS2 expression in a variety of cancer cell lines. Genetic knockout of DHODH caused cells more sensitive to RT-induced ferroptosis. Similarly, pharmacological inhibition of DHODH sensitized cancer cells to RT-induced ferroptotic cell death both in vitro and in vivo. Mechanistically, RT induces ROS production in mitochondria, resulting in mitochondrial lipid peroxidation and ferroptosis. DHODH catalyzes the reduction of ubiquinone to ubiquinol, which acts as an antioxidant to inhibit lipid peroxidation in mitochondria. In general, DHODH inhibition significantly sensitizes cancer cells to RT-induced ferroptosis by decreasing ubiquinol levels in mitochondria.
Conclusion: Our study suggests a model in which DHODH inhibition enhances RT-induced ferroptosis. DHODH inhibition significantly decreases the ubiquinol/ubiquinone ratio in mitochondria, thereby sensitizing cancer cells to RT-induced ferroptosis caused by mitochondrial lipid peroxidation. Together, our results demonstrate that DHODH inhibitors can be used as radiosensitizers through inducing ferroptosis and propose a novel combination strategy for cancer therapy.
Citation Format: Chao Mao, Amber Horbath, Guang Lei, Boyi Gan. DHODH inhibition enhances radiotherapy-induced ferroptosis by promoting mitochondrial lipid peroxidation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5658.