PROJECT SUMMARY / ABSTRACT The devastation wrought by cancer derives primarily from the capacity of tumor cells to evolve. Metastasis, immune evasion, treatment resistance, and even tumorigenesis itself are evolutionary processes. Our understanding of tumor evolution is incomplete, evidenced by the ability of some cancers to evolve more quickly in response to treatment than is compatible with classical genetics. A more comprehensive molecular understanding of how tumors evolve is key to improving cancer treatment. Recent work has shown that oncogene amplification on extrachromosomal DNAs (ecDNAs) is a major driver of tumor evolution, treatment resistance, and poor outcomes in patients. These circular DNAs are acentric and have long been thought to asymmetrically segregate at cell division, leading to intratumoral heterogeneity. We have recently proved this to be the case, but our understanding of the precise mechanisms through which ecDNA drives tumor evolution remains limited. In this project, Dr. John Rose aims to advance our understanding of ecDNAs in cancer evolution through unprecedented, well-controlled experimental studies of ecDNA. First, through a novel approach to image every ecDNA in living cells, I will delineate ecDNA dynamics on the level of single cells and single ecDNAs (Aim 1a), before extending these findings to organoid models and analysis of ecDNA+ patient samples (Aim 1b). Second, I will identify the genes that impact ecDNA, either promoting or inhibiting their accrual in tumor cells, using a high-throughput CRISPR screening strategy (Aim 2). Finally, I will characterize the development of ecDNAs’ uniquely accessible chromatin structure, elucidating its etiology (Aim 3). Together, these studies will dramatically improve our understanding of ecDNA in tumor evolution, while identifying putative avenues for therapeutic intervention. This work will be performed in the world-class training environment at Stanford University, under the mentorship of Dr. Howard Chang, an expert in the application of epigenomics to the study of cancer, and Dr. Paul Mischel, an expert in extrachromosomal DNA. An advisory committee composed of leaders in the fields of tumor evolution, computational biology, advanced cell imaging, high-throughput CRISPR screens, and cancer organoid models will provide additional expertise and mentorship. The first half of each aim will be completed predominantly during the K99 phase of the award, providing a platform for completion of the aims in the R00 phase.

Oncogene amplification on extrachromosomal DNA (ecDNA) is common in human cancers and drives rapid tumor evolution and poor outcomes. Despite recent advances characterizing their biology, the mechanisms by which ecDNA promotes tumor evolution are poorly understood. Through powerful combinations of new and established techniques, this project aims to elucidate how ecDNA drives tumor evolution with the goal of identifying novel therapeutic strategies.