Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma

Koche, Richard P.; Rodríguez-Fos, Elias; Helmsauer, Konstantin; Burkert, Martin; MacArthur, Ian; Mååg, Jesper L.V.; Chamorro, Rocio; Munoz-Perez, Natalia; Puiggròs, Montserrat; García, Heathcliff Dorado; Bei, Yi; Röefzaad, Claudia; Bardinet, Victor; Szymansky, Annabell; Winkler, Annika; Thole, Theresa; Timme, Natalie; Kasack, Katharina; Fuchs, Steffen; Klironomos, Filippos; Thiessen, Nina; Blanc, Eric; Schmelz, Karin; Künkele, Annette; Hundsdörfer, Patrick; Rosswog, Carolina; Theißen, Jessica; Beule, Dieter; Deubzer, Hedwig E.; Sauer, Sascha; Toedling, Joern; Fischer, Matthias; Hertwig, Falk; Schwarz, Roland F.; Eggert, Angelika; Torrents, David; Schulte, Johannes H.; Henssen, Anton

doi:10.1038/s41588-019-0547-z

Cited by 231 publications

(382 citation statements)

References 40 publications

(37 reference statements)

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“…EcDNA re-integration into chromosomes can lead to intrachromosomal amplification as HSRs 8,9 and act as a general driver of genome remodeling 10 . Our knowledge of the functional relevance of non-coding regions co-amplified on ecDNA, however, is currently limited.…”

Section: Introductionmentioning

confidence: 99%

Enhancer hijacking determines intra- and extrachromosomal circularMYCNamplicon architecture in neuroblastoma

Helmsauer

Валиева

Ali

et al. 2019

Preprint

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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.

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Section: Introductionmentioning

confidence: 99%

Enhancer hijacking determines intra- and extrachromosomal circularMYCNamplicon architecture in neuroblastoma

Helmsauer

Валиева

Ali

et al. 2019

Preprint

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“…Moreover, DMs, in the form of extrachromosomal DNA and another type of chromosomal abnormality, were detected in the amplified region and not in FISH analyses. This could indicate that this structure exists on highly compacted chromosome positions and harbors the amplification of several genes by generating a new chromosomal structure DM and dynamically converting it to another type of chromosomal HSR, which was previously described in neuroblastoma study 55 . This could explain the involvement of chromosomal abnormalities in MTX resistance.…”

Section: Discussionmentioning

confidence: 52%

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Kim

Shin

Seo

2020

Preprint

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DHFR gene amplification is present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. However, little is known about DHFR gene amplification due to difficulties in quantifying amplification size and recognizing the repetitive rearrangements involved in the process. In this study, we have proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). Amplification of the DHFR gene was optimized to generate homogenously amplified patterns. The amplification units of 11 genes, from the DHFR gene to the ATP6AP1L gene position on chromosome 5 (~2.2Mbp), and a twenty-fold tandemly amplified region were verified using longrange genome and RNA sequencing data. In doing so, a novel inversion at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These might stimulate chromosomal breakage and cause the dysregulation of mismatch repair pathways. Using Hi-C technology, high adjusted interaction frequencies were detected on the amplified unit and unsuspected position on 5q, which could have a complex network of spatial contacts to harbor gene amplification. Characterizing the tandem gene-amplified unit and genomic variants as well as chromosomal interactions on intra-chromosome 5 can be critical in identifying the mechanisms behind genomic rearrangements. These findings may give new insight into the mechanisms underlying the amplification process and evolution of drug resistance. Results Determination of MTX-resistant HT-29 cellsWhile generating MTX-resistant HT-29 cells (selected clone: C1-2) from singe-cell selection and MTX sensitization as previously described 15 , dramatic morphological changes in the cells themselves were detected in the form of rounded and circular shapes at the 1 st cycle of sensitization as well as rod and irregular shapes at the 2 nd cycle (Supplementary Fig. 1). The original shapes were again observed at the 3 rd cycle of sensitization, which might indicate that the HT-29 cells were becoming resistant to MTX and rapidly grew up under high MTX concentration.As expected, DHFR expression, which was normalized by the B2M housekeeping gene, was steadily increased from the 1 st to 3 rd cycle, as the HT-29 clones became resistant to MTX (Supplementary Fig. 2a). After confirming these morphological changes and increased DHFR expression, the DHFR copy number was measured in several clones.The clones' cycle quantification values (Ct) were used to compute the rate of copy number. These values dropped from 26.04 to 19.99, as the number of cycles increased ( Supplementary Table 1). Interestingly, there was a dramatic increase in the DHFR copy number between the 1 st (0.97 copies) and 2 nd (54.83 copies) cycles ( Supplementary Fig. 2b).Based on these results, we ascertained that a specific time period and set of conditions were required for clones to su...

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“…Revealing the architecture of fCNAs, particularly at a large scale, is critical to understanding their functional implications. For instance, rearrangements present in fCNAs can directly increase oncogene copy number, disrupt gene structure 41 , and lead to dysregulation of chromatin 17–19 . Thus, understanding the organization and content of fCNAs is essential in predicting the behavior of the underlying sequences.…”

Section: Discussionmentioning

confidence: 99%

AmpliconReconstructor: Integrated analysis of NGS and optical mapping resolves the complex structures of focal amplifications in cancer

Luebeck

Çoruh

Dehkordi

et al. 2020

Preprint

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32Oncogene amplification, a major driver of cancer pathogenicity, is often mediated through 33 focal amplification of genomic segments. Recent results implicate extrachromosomal 34 DNA (ecDNA) as the primary mechanism driving focal copy number amplification (fCNA) 35 -enabling gene amplification, rapid tumor evolution, and the rewiring of regulatory 36 circuitry. Resolving an fCNA's structure is a first step in deciphering the mechanisms of 37 its genesis and the subsequent biological consequences. Here, we introduce a powerful 38 new computational method, AmpliconReconstructor (AR), for integrating optical mapping 39 (OM) of long DNA fragments (>150kb) with next-generation sequencing (NGS) to resolve 40 fCNAs at single-nucleotide resolution. AR uses an NGS-derived breakpoint graph 41 alongside OM scaffolds to produce high-fidelity reconstructions. After validating 42 performance by extensive simulations, we used AR to reconstruct fCNAs in seven cancer 43 cell lines to reveal the complex architecture of ecDNA, breakage-fusion-bridge cycles, 44 and other complex rearrangements. By distinguishing between chromosomal and 45 extrachromosomal origins, and by reconstructing the rearrangement signatures 46 associated with a given fCNA's generative mechanism, AR enables a more thorough 47 understanding of the origins of fCNAs, and their functional consequences. 48 49 Main: 50 Oncogene amplification is a major driver of cancer pathogenicity 1-5 . Genomic signatures 51 of oncogene amplification include somatic focal Copy Number Amplifications (fCNAs) of 52 small (typically < 10Mbp) genomic regions 5,6 . Multiple mechanisms cause fCNAs 53including, but not limited to, extrachromosomal DNA (ecDNA) formation 5,7,8 , 54 chromothripsis 9 , tandem duplications 10,11 and breakage-fusion-bridge (BFB) cycles 12-14 . 55 ecDNA, in particular, enables tumors to achieve far higher oncogene genomic copy 56 numbers and maintain far greater levels of intratumor genetic heterogeneity than 57 previously anticipated, due to their non-chromosomal mechanism of inheritance -58 enabling tumors to evolve rapidly 5,15,16 . In addition, the very high DNA template level 59 generated by ecDNA-based amplification, coupled to its highly accessible chromatin 60 architecture, permits massive oncogene transcription [17][18][19] . 61 62 3While ecDNA elements are a common form of fCNA 5 , other mechanisms can also result 63 in amplification with very different functional consequences 6 . Thus, accurate identification 64 and reconstruction of the fCNA structure not only describes the rearranged genomic 65 landscape, but also represents a first step in identifying the generative mechanism. 66Reconstruction of fCNA architecture involves determining the order and orientation of the 67 genomic segments that constitute the amplicon. There are many methods to detect single 68 genomic breakpoints from sequencing data using a variety of different sequencing 69 technologies 20-23 . However, fewer methods are available to handle the more difficult 70 problem of...

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Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma

Abstract: G.H. contributed to the study design and collection and interpretation of the data. R.P.K. performed the analysis of Circle-seq and whole-genome sequencing. E.R.F. performed the data analysis of the whole-genome sequencing data. I.

Cited by 231 publications

References 40 publications

Enhancer hijacking determines intra- and extrachromosomal circularMYCNamplicon architecture in neuroblastoma

Enhancer hijacking determines intra- and extrachromosomal circularMYCNamplicon architecture in neuroblastoma

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

AmpliconReconstructor: Integrated analysis of NGS and optical mapping resolves the complex structures of focal amplifications in cancer

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