PROJECT SUMMARY/ABSTRACT The goal of this project is to determine the influence of extrachromosomal DNA (ecDNA) on tumor evolution and resistance and to validate a potential therapeutic strategy based on ecDNA inhibition. The existence of ecDNA was first recognized through pioneering cytopathology studies by Arthur Spriggs over five decades ago. We know that ecDNAs are largely a phenomenon of tumor cells, can be detected in nearly half of all solid cancers, and frequently express oncogenes, making them vehicles for amplification of oncogene expression and consequent tumorigenesis. Our studies in glioblastoma extend this knowledge, showing that ecDNAs are highly pervasive and significantly increase intra-tumoral genomic heterogeneity. Despite this knowledge, research into the mechanisms of ecDNA-driven tumorigenesis is largely non-existent. To advance ecDNA research to the next level of understanding, we must now define its origins, replication mechanisms, and strategies to target it for treatment. At this time, however, there are significant barriers to such studies, such as the absence of profiling technologies and molecular tools to accurately detect and characterize these oncogenic elements. The work proposed here will remove these barriers to progress. We hypothesize that ecDNAs drive tumorigenesis beyond their role as oncogene activators, and that mechanisms of ecDNA formation and replication could serve as potential therapeutic targets. To test our hypothesis, we will develop methods to visualize and trace ecDNA in live cells and use these tools to define the mechanisms of ecDNA localization and transfer within a tumor. We will functionally evaluate whether ecDNA mediated cis-regulation of non-ecDNA gene transcription can be modulated to change tumor cell behavior. Finally, we will explore whether pharmacological depletion of ecDNA affects tumor growth and maintenance in cellular and animal model systems. Our Specific Aims are to: 1) Develop CRISPR-based tracing tools for real-time imaging of ecDNA dynamics; 2) Discovery and functional interrogation of ecDNA mediated oncogene activation; and 3) Target ecDNA through inhibition of ribonucleotide reductase (RNR), the enzyme responsible for generating the deoxyribonucleotide triphosphates that are the building blocks of autosomal and extrachromosomal DNA. Impact: This project is based on the conceptual innovation that ecDNAs are cancer-specific and cancer- enabling genomic alterations. Despite the recognition of ecDNA decades ago, the function of these structures and how to effectively target their genesis, is virtually uncharted territory. In addition to developing new, broadly applicable, tools for ecDNA studies, the results of this work will provide novel insights into the characteristics, behavior and replicative mechanism of ecDNA, and will assess whether ecDNA is an unexploited cancer cell vulnerability that can be therapeutically exploited for patient benefit.