Overall Summary Approximately 50% of cancer patients are treated with radiation therapy (RT), but local recurrence can still occur even with the use of advanced RT techniques. This local recurrence, which commonly develops in 30-50% of cancer cases, is exacerbated by the acquisition of RT resistance. This RT resistance is especially true for patients with locally advanced thoracic cancers, such as lung and esophageal cancers. RT can lead to an iron- dependent cell death modality, called ferroptosis, but whether ferroptosis resistance occurs within tumors giving rise to acquired RT resistance is not known and is the central theme of the proposed Acquired Resistance to Therapy and Iron (ARTI) Center. The overarching goals of the ARTI Center are: 1) to bridge the basic science mechanisms of ferroptosis in acquired resistance with translational research in preclinical models and human patient samples; 2) to identify cohorts of patients who are at greatest risk to develop acquired RT resistance; and 3) to investigate the ability of novel therapeutic agents to re-sensitize lung and esophageal cancer cells to radiation by inducing ferroptosis. The ARTI Center comprises two basic/mechanistic projects (Project 1 and Project 2), one preclinical/translational project (Project 3), and one shared resource core (Molecular Imaging Core [MIC]). Project 1 will focus on elucidating whether ferroptosis evasion is a key driver in acquired RT resistance using radioresistant lung cancer and esophageal cancer cell lines and xenograft models that will be used in Project 2. Project 2 will test the hypothesis that hypoxia, a long-recognized driver of tumor radioresistance, suppresses ferroptosis induction during RT and contributes to RT-induced acquired resistance to ferroptosis. Furthermore, expression of hypoxia-related genes and other targets of acquired RT resistance will be analyzed by single-cell sequencing in Project 3. Project 3 investigates changes in immune cells in the tumor microenvironment of humanized tumor models derived from chemoradiation therapy-responsive or -non- responsive esophageal adenocarcinoma patients. These ferroptosis-mediated immunologic changes in the tumor microenvironment may serve as prognostic biomarkers for identifying tumors that may acquire RT resistance and predicting cancer patient outcomes, which could, in the future, be modulated by the ferroptosis- inducing agents tested in Projects 1 and 2. Projects 1, 2, and 3 will be supported by the MIC that utilizes bioluminescence imaging to monitor tumor growth, positron emission tomography (PET) tracers to monitor cystine transporter activity and to identify hypoxic regions within tumors, as well as novel, redox-tuned PET tracers for identifying activated innate immune cells. The ARTI Center will develop an Administrative Core for effective communication and collaboration between the ARTI Center Project and Core Leaders and Co-Leaders with National Cancer Institute (NCI) of Acquired Resistance to Therapy Network (ARTNet) program staff as well as other ARTNet centers to synergize ARTI Center-related activities.

Project Narrative The Acquired Resistance to Therapy and Iron (ARTI) Center at The University of Texas MD Anderson Cancer Center will establish a foundation for understanding the role of iron in cancer resistance to therapies and will focus on deciphering the mechanisms of acquired resistance to radiation therapy, which can be extrapolated to other types of cancer therapy. The overarching goals of the ARTI Center are: 1) to bridge the basic science mechanism of a novel type of iron-dependent cell death called ferroptosis in acquired resistance to radiation therapy with translational research in preclinical models and human patient samples; 2) to identify cohorts of patients who are at greatest risk to develop acquired radiation therapy resistance; and 3) to investigate the ability of novel therapeutic agents to re-sensitize cancer cells to radiation by inducing ferroptosis. The long-term outcomes from the ARTI Center will foster collaborations with other centers in the Acquired Resistance to Therapy Network (ARTNet), build upon and iteratively feedback into research projects focused on drug resistance and sensitivities, and ultimately lead to cancer treatment strategies tailored to the individual patient.