Extrachromosomal DNA (ecDNA) is a circular, megabase-sized vehicle containing the most common oncogenes (e.g., MYC, EGFR, KRAS) found in majority of human cancer types but not in normal tissues. Emerging evidence suggests that ecDNA is co-opted driving massive oncogene expression, enabling intra-tumoral heterogeneity, and conferring drug resistance and therapeutic refractoriness. Therefore, NCI has designated ecDNA as one Cancer Grand Challenge. Despite the central relevance of ecDNA to human cancer, there is at present no ecDNA-targeted approach to either experimental or clinical cancer therapy, largely due to the sparsity of mechanistic understanding of ecDNA regulation. But the diffraction limit of light and lack of quantitative method present challenges to mechanistically study ecDNA in in situ and in living cells. To address this critical need, we propose in this study to develop super-resolution imaging and quantitative analysis platform to study ecDNA organization and dynamics in both interphase and mitosis. The idea of this proposal is based on our past observation of MYC ecDNA forming spatially clustered hubs driving intermolecular oncogene expression. We will leverage our experience in state-of-the-art fluorescence microscopy, imaging analysis, and their application to chromatin biology. In this project, we will investigate the molecular mechanism by which the BET family proteins regulate ecDNA interphase organization and propagation. Our long-term goal is to dissect fundamental mechanisms of ecDNA regulation in the cancer genome, train physician scientists in quantitative biomedical inquiry, identify novel druggable targets, and discover the Achilles’ heel for ecDNA-targeted oncotherapy.