Transcriptional regulation of redox pathways

Radiotherapy is a standard care for many cancer patients to achieve local control and to reduce metastatic burden. While radiation kills tumour cells by inducing DNA damage, it was recently found that ferroptosis is another way of cell killing by radiation. Ferroptosis is an iron-dependent cell death, which is caused by oxidative stresses from fat molecules. However, key pathways involved in the regulation of ferroptosis still need to be investigated. In our study, we found that the ferroptosis was significantly dependent on MAFF, a protein that regulates antioxidant responses. We specifically showed that MAFF changed production of reactive oxygen species (ROS) and DNA damage repair during ferroptosis. While identifying how MAFF regulates ferroptosis and radiation responses by focusing on its target gene pathways, we will also examine whether MAFF is a significant factor in radiation-induced ferroptosis by comparing conventional and FLASH radiotherapy. FLASH is given at 100-1000 times faster than conventional treatment, which makes it more beneficial by minimizing normal cell killing while having similar radiation effect on tumour. Therefore, in this study, we will investigate how MAFF-mediated redox pathways regulate ferroptosis, specifically when comparing conventional and FLASH radiotherapy. Through identification of MAFF-target genes and their role in FLASH treatment, our study will enable us to find a novel therapeutic target to enhance radiation and FLASH outcome.

Radiotherapy is effective in treating many types of cancers and generates oxidative stress to induce DNA damage. Ferroptosis has recently been described as a non-apoptotic form of cell death dependent on iron and lipid peroxides that can contribute to radiation-induced cell death. However, before targeting ferroptosis to radiosensitise cells, it is critical that we determine which key pathways are involved in the regulation of ferroptosis in response to radiation. Recently, we found that MAFF plays a significant role in ferroptosis by modulating ROS production and subsequent DNA damage, which will provide a new approach to enhance radiation or FLASH-induced cell killing.
MAFF is a bZIP transcription factor that lacks a transactivation domain. Transactivation or repression of MAFF target genes that are involved in cellular antioxidant responses are determined by MAFF heterodimerizing with partners NRF2 and BACH1 or by forming homodimers with itself. Since ferroptosis depends on the production of reactive oxygen species (ROS) from lipid peroxidation and MAFF plays a significant role in redox pathways, we investigated ROS levels in tumour cells after inducing ferroptosis. Our data showed that ferroptosis significantly increased ROS production in control cells, while MAFF knockdown cells did not show any significant change after treatment, indicating that MAFF plays a role in ferroptosis-mediated redox pathways. In addition, lipid profiling using Mass Spectrometry revealed that loss of MAFF lead to significant changes in specific phospholipids essential for cell membranes.
To investigate the role of MAFF in ferroptosis and radiation responses, 1) we will determine how MAFF regulate to induce ferroptosis. Then 2) we will examine how MAFF transcriptionally activate or repress target genes in response to radiation. Lastly, 3) we will investigate whether MAFF-mediated redox changes can be beneficial to FLASH radiotherapy. While determining MAFF target genes responsible for ferroptosis, we will also investigate how MAFF transcriptionally regulates its target genes by focusing on its chromatin complex, dimerization, and post translational modifications. To deliver our findings to translational studies, we will specifically compare the effect of conventional and FLASH radiation on ferroptosis. FLASH is a novel approach to radiotherapy, which uses ultra-high dose rates to enhance the therapeutic window. By focusing on the redox mechanisms of MAFF after radiation, we will examine whether FLASH effect can be enhanced by regulating MAFF and its target genes. Therefore, this study will provide a new approach to increase radiation or FLASH-induced cell killing through the MAFF regulation of redox pathways.