Project Summary Cancer is a leading cause of death worldwide. Recent advancements in the use of immune checkpoint blockade have transformed oncology, and immunotherapy has extended the potential application of radiotherapy to systemic disease. More than 200 clinical trials of combined checkpoint blockade and radiotherapy are ongoing or completed. The results of these preliminary trials demonstrate efficacy only in a limited subpopulation of patients. Treatment resistance likely manifested by poor T-cell priming and tumor- mediated immunosuppression continue to constitute significant barriers to optimal patient outcomes; therefore, the opportunity for transformative clinical impact is real in this setting. We propose a new and innovative strategy guided by new findings to improve the interaction of radiotherapy and immunotherapy by incorporating the latest techniques in the emerging field of mRNA modification with well-established radiobiological and immunological approaches. We will leverage collaboration with our Co-Investigator Chuan He, who helped to discover and decipher reversible RNA methylation in post-transcriptional gene expression regulation. We will use the novel techniques to identify the binding sites of Y1 and Y2, and incorporate integrated bioinformatics analysis approaches to investigate the impact of m6A readers in functional pathways of immune cells in irradiated tumors. These techniques are new and, to our knowledge, have yet to be applied in the context of radiotherapy and radioimmunotherapy. We hypothesize that targeting regulation of m6A modifications associated with m6A-binding protein YTHDF1 (for improved antigen presentation and T-cell priming) and YTHDF2 (for alleviation of immunosuppression) will potentiate anti-tumor immunity in the context of both RT alone and RT combined with anti-PD-L1 antibodies. Our proposal focuses on 1) establishing YTHDF1 (Aim 1) and YTHDF2 (Aim 2) as viable targets for RT and radioimmunotherapy, and 2) uncovering underlying pathways. Small molecules from the He Lab will be used to validate our hypothesis. The ultimate goal of this therapeutic approach is to modulate gene expression via targeting m6A methylation related to translation (Y1) or degradation (Y2) of mRNA in order to potentiate immune response. We are uniquely positioned to discover new knowledge and elucidate an unprecedented level of mechanistic understanding of the complex molecular and cellular interplay between radiotherapy and checkpoint inhibition in the context of the immune system. These new findings will provide the mechanistic data required for translational pursuit of superior treatment strategies. Increased local and/or distant control to actualize radio-immunotherapy would be a practice-changing and would broadly enhance cancer care and expand the pool of patients who respond to inhibition of the PD-1/PD-L1 axis.