PROJECT SUMMARY/ABSTRACT Radiation therapy is utilized to treat approximately half of all cancer patients. For some patients, radiation therapy can achieve local tumor control and cure. For other patients, radiation therapy palliates symptoms and alleviates suffering. However, radiation therapy can also cause acute toxicity and late effects that diminish quality of life. The goal of our research program is to widen the therapeutic window of radiation therapy by dissecting mechanisms of normal tissue radiation injury and tumor response to radiotherapy. As I am a sarcoma radiation oncologist, my research group not only studies sarcoma response to radiation therapy, but also sarcoma development and metastasis. During the prior funding period, we adapted CRISPR/Cas9 technology to develop novel genetically engineered mouse models of sarcoma that facilitated lineage tracing of sarcoma clones during tumor progression. This approach identified novel regulators of metastasis, which are potential targets for new cancer therapies. We also used new mouse and in vitro models to discover a novel mechanism for the exquisite radiosensitivity of myxoid liposarcoma: DNA-damage induced phosphorylation of a prion-like domain in the FUS-CHOP fusion protein disrupts interactions with chromatin remodeling complexes that are required for tumor maintenance. We initiated new projects with a novel sarcoma model with high tumor mutation burden that revealed tumor-intrinsic and immune-mediated mechanisms of response and resistance to radiotherapy and immunotherapy. Our findings provided the rationale for a randomized clinical trial in sarcoma patients testing radiation therapy with immune checkpoint inhibition. We also used our genetically engineered mice to uncover mechanisms regulating acute toxicity and late effects of radiation, such as radiation carcinogenesis. Our proposed research will build on the success of our recent work. We will use innovative model systems and sophisticated approaches to make discoveries in three broad areas: 1. Response of sarcomas to radiotherapy 2. Response and resistance of sarcomas to radiation and immunotherapy 3. Normal tissue injury from radiation The knowledge gained from the proposed studies will not only deepen our understanding of radiation and sarcoma biology, but will also inform the design of future clinical trials that aim to widen the therapeutic ratio of radiation therapy to improve the outcome for patients with sarcomas and other cancers.