PROJECT SUMMARY Overall Section Our ROBIN center focuses on elucidating the genomic and microenvironmental determinants, and temporal dynamics underlying efficacy of radiation-based combination therapies. Radiotherapy (RT), alone or in combination with other treatments, is used to treat about two-thirds of all cancer patients. Despite the widespread use of radiation therapy in oncology, our understanding of the mechanisms driving response and resistance remains poor. Our long-term goal is to understand the mechanisms that underlie efficacy and resistance of radiation-based therapies. New efforts to improve treatment for many cancer types now focus on using combination therapies in which radiation is used with systemic agents, highlighting the urgent need to understand the drivers of efficacy. Among the most promising new biologics being studied for use with radiation are antibody-drug conjugates (ADC) and immune checkpoint inhibitors (ICI). We will use an innovative molecular characterization trial testing radiation plus ADC in bladder cancer and radiation plus ICI in head and neck cancer to characterize the mechanistic drivers underlying these next generation RT-based combinations. The central hypothesis of this U54 application is that specific genetic and immunologic mechanisms underlie sensitivity and resistance to radiation-based combination therapies. We will address these questions through 3 specific aims. In Aim 1, we will work to understand the molecular mechanisms that underlie efficacy of treatment with radiation plus ADC. Here, our working hypothesis is that specific genetic and immunologic events underlie response to RT plus sacituzumab govitecan (SG) treatment. We will leverage our molecular characterization trial (Part A) investigating the use of RT and sacituzumab for bladder preservation therapy. We will determine the differential molecular effects with standard-of-care RT + cisplatin versus RT + SG. In Aim 2, we will improve identification of patients who are sensitive or resistant to RT-based therapies based on new insights into transcriptional dynamics and temporal reprogramming during treatment with radiation-based therapies. Here, we will leverage our molecular characterization trial treating head and neck squamous cell carcinoma (HNSCC) or bladder cancer patients with RT + chemotherapy versus RT + SG or ICI. We will build on recent experimental and clinical breakthroughs led by our research groups, which have identified highly refined gene expression programs associated with RT sensitivity and delta radiomics. In Aim 3, we will identify the differential mechanisms underlying the anti-tumor activities of RT + cisplatin versus RT + immune checkpoint blockade. Here, using our head and neck trial (Part B), we will uncover the unique genetic and immunologic factors that govern response to RT when combined with these two classes of agents. We will elucidate the differential molecular effects of the two approaches, immune reprogra...