Enhancer hijacking determines intra- and extrachromosomal circularMYCNamplicon architecture in neuroblastoma

Abstract

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA. The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyzed the MYCN amplicon structure and its chromatin landscape. This revealed two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplified a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons was characterized by high structural complexity, lacked key local enhancers, and instead contained distal chromosomal fragments, which harbored CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

Extrachromosomal DNA—relieving heredity constraints, accelerating tumour evolution

“…46 Interestingly, functional endogenous enhancer activity was maintained across all samples tested. This is in contrast to the characterisation by Helmsauer et al 47 of MYCN amplicons in neuroblastoma cell lines. They describe a structurally complex subset that lacks endogenous enhancers and thus hijack ectopic enhancers from distal regions of the same chromosome into the ecDNA structure.…”

Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers

“…Following DNA copy number level correction, chromatin of Circular and BFB amplicons was significantly more accessible compared to Heavily-rearranged and Linear amplicons (1.2x higher median ATAC-seq signal fold-change; Wilcoxon rank sum test; p-value < 1e-16)( Fig. 3B ), consistent with recent findings that increased accessibility plays a role in dysregulation of ecDNA oncogenes 31 , 32 , 34 . Finally, the frequency of amplicon-derived transcript fusions was increased by fivefold in Circular compared to non-circular amplifications ( Fig.…”

Decoil: Reconstructing extrachromosomal DNA structural heterogeneity from long-read sequencing data

“…To show the versatility of the algorithm, Decoil was applied to shallow whole-genome nanopore sequencing of three neuroblastoma cell lines, i.e. CHP212, STA-NB-10DM and TR14, for which ecDNA elements were previously characterized based on various circular DNA enrichment methods and/or validated using fluorescence in situ hybridization (FISH)[6, 19, 20]. Decoil’s reconstructions recapitulated the previously validated ecDNA element in CHP212 with high fidelity (Suppl.…”

“…To assess Decoil's reconstruction performance, we generated an in-silico collection of ecDNA elements, spanning various sequence complexities for systematic evaluation. We introduced a ranking system and defined seven topologies of increasing computational complexity, based on the SV's contained on the ecDNA element: (1) Simple circularization -no structural variants on the ecDNA template, (2) Simple SV's -ecDNA contains either a series of inversions or deletions, (3) Mixed SV's -ecDNA has a combination of inversions and deletions, (4) Multi-region -ecDNA contains di↵erent genomic regions from the same chromosome (DEL, INV and TRA allowed), (5) Multi-chromosomal -ecDNA originates from multiple chromosomes (DEL, INV and TRA allowed), (6) Duplications -ecDNA contains duplications defined as a region larger than 50 bp repeated on the amplicon (DUP's + other simple rearrangements), (7) Foldbacks -ecDNA contains a foldback defined as a two consecutive fragments which overlap in the genomic space, with di↵erent orientations (INVDUP's + all other simple SV's). Every topology can contain a mixture of all other low-rank topologies.…”

“…As a result, ecDNA can be structurally diverse, with different functional outcomes. The structure of ecDNA can impact gene regulation through the rearrangement of regulatory elements as well as topologically associated domain (TAD) boundaries [6]. To explore ecDNA diversity and complexity, high-resolution computational methods to reconstruct ecDNA with high accuracy from genome sequencing data are required.…”