Project description:Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
Project description:Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general strategy to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs) in a spatiotemporally controlled manner in cells and in genetically engineered mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures. We also apply this approach to generate mice harboring Cre-inducible Myc- and Mdm2-containing ecDNAs analogous to those occurring in human cancers. We show that the engineered ecDNAs spontaneously accumulate in primary cells derived from these animals, promoting their proliferation, immortalization, and transformation. Finally, we demonstrate the ability of Mdm2-containing ecDNAs to promote tumor formation in an autochthonous mouse model of hepatocellular carcinoma.
