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Extrachromosomal Oncogene Amplification Involves Rearrangement of Regulatory Elements

NEW YORK – Non-coding regulatory sequences can get co-amplified with extrachromosomal oncogenes in cancer, new research suggests, producing circularized structures with altered connections that may be selected to boost the proliferation of these tumor cells.

"Collectively, our results uncover an additional layer of regulatory complexity in canonical driver events, which may provide avenues for the targeted disruption of key oncogenes that have been considered undruggable to date," senior and co-corresponding author Peter Scacheri, a researcher in the department of genetics and genome sciences at Case Western Reserve University School of Medicine, and his co-authors wrote.

As they reported online today in Cell, Scacheri and his colleagues started with a computational strategy to find co-amplification of oncogene-regulatory sequence in glioblastoma (GBM), using targeted sequencing, CRISPR-based screening, and other methods to take a closer look at EGFR-containing amplifications. They discovered that EGFR tends to get amplified in combination with a pair of enhancer elements that are active in nervous system tissues from which the cancer arises.

But those endogenous enhancers were not the only regulatory sequences capable of making their way into the circularized extrachromosomal sequences, the team found: still other fitness-related regulatory elements turned up in EGFR amplicons scrutinized in a subsequent CRISPR-Cas9-based screen.

"With EGFR as our exemplar," the authors explained, "we found that the oncogenes amplified on circular extrachromosomal DNA select for existing and new regulatory interactions that promote cancer growth."

Using clues from prior studies, the researchers reasoned that regulatory features and genome organization changes may contribute to the cancer-promoting impact of oncogene-containing DNA amplifications.

"[A]mplifications always take additional sequences beyond the body of the [oncogene]," the authors explained, "but it remains unclear whether these additional sequences are functional or are just passengers."

After their computational analyses pointed to an overrepresentation of sequences upstream of EGFR in amplicons from 174 EGFR-amplified GBM cases profiled for the Cancer Genome Atlas project, for example, the investigators performed chromatin immunoprecipitation sequencing (ChIP-seq) focused on the H3K27ac histone mark to profile active regulatory elements upstream of the oncogene, uncovering apparent EGFR enhancers.

The team went on to show that such enhancers could impact the expression of EGFR from the extrachromosomal amplicons using CRISPR-based interference screens focused on two potential enhancer loci in a GBM cell line, and demonstrated that the enhancer patterns in the EGFR amplicons from GBM shared features with nervous system tissues profiled for the Roadmap Epigenomics Consortium.

Two endogenous enhancers appeared particularly prone to co-amplification with EGFR in the researchers' ChIP-seq analyses on nine EGFR-amplified cell cultures, while their fluorescence in situ hybridization, ChIP-seq, and other data confirmed that at least some of the GBM cell lines did carry amplified forms of EGFR on circularized extrachromosomal DNA fragments.

With a series of follow-up experiments, including ChIP-seq, circular chromosome conformation capture sequencing (4C-seq), and additional CRISPR screens, the team examined the EGFR oncogene's three-dimensional interactions and effects on tumor cell viability in amplified and unamplified forms of GBM — analyses that highlighted the role for endogenous and rewired enhancers in cancer fitness at the amplified EGFR "onco-locus."

Such events were not limited to GBM, the team reported, noting that similar oncogene co-amplifications appeared to occur in breast cancer, medulloblastoma, and other cancer types based on additional experiments and analyses. 

"We find that neighboring non-coding regions harboring enhancers co-amplify with oncogenes and maintain the physical contacts present on the linear chromosome," the authors reported. "However, circularization of these amplicons can lead to the incorporation of new regulatory elements and changes to the topological connections to the oncogene."