Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond

MYCN Amplifikation im Neuroblastom: Sequenz, Chromatinlandschaft und genomische Remodellierung

MYCN amplification drives half of all high-risk neuroblastomas and confers dismal prognosis. It occurs when MYCN and other chromosomal fragments are joined together to form an amplicon sequence that can be found tens to hundreds of times per cell. In most cases, these copies come as extrachromosomal DNA (ecDNA) in addition to chromosomes. The sequence and chromatin landscape of the MYCN amplicon has not been systematically mapped, gene regulation in the context of amplification has not been explored, and the consequences of extrachromosomal amplification are incompletely understood. Here, we analyze Illumina and Nanopore whole- genome sequencing, RNA-seq, ATAC-seq, ChIP-seq, and Hi-C data of primary neuroblastomas and neuroblastoma cell lines. We map MYCN-driving enhancers in neuroblastoma and identify co- amplification of local enhancers as the main principle governing the non-coding contents of MYCN amplification. We find that loss of local enhancers can be compensated for by the incorporation of distal enhancers and the formation of new chromatin domains through structural variation on the amplicon. We show that MYCN amplification in neuroblastoma is associated with clusters of interchromosomal rearrangements at the MYCN locus indicative of complex amplicon structure or re-integration of ecDNA into chromosomes. This can affect gene expression and is prognostically relevant. Finally, we describe heterogeneous ecDNA populations and show that ecDNA can engage in in trans interactions forming transcriptionally active hubs of individual amplicon copies. Our results demonstrate how non-coding elements shape amplification structure and function. Together, they suggest a model of amplification as building blocks for genome remodeling and nuclear reorganization.MYCN Amplifikation charakterisiert etwa jedes zweite Hochrisiko-Neuroblastom und geht mit besonders aggressivem klinischem Verhalten einher. In solchen Tumoren sind MYCN und andere chromosomale Fragmente zu einer Amplikonsequenz zusammengefügt, die dann meist viele dutzendmal kopiert pro Zelle vorliegt. Typischerweise liegen die Amplikonkopien als ringförmige extrachromosomale DNA (ecDNA) zusätzlich zu den Chromosomen vor. Wie Sequenz und Epigenetik des MYCN-Amplikons zur Genregulation im Kontext von Amplifikation beitragen, wurde bisher noch nicht systematisch untersucht. Auch die Auswirkungen extrachromosomaler Amplifikation auf das chromosomale Genom und dessen Organisation im Zellkern sind nur unvollständig bekannt. In der vorliegenden Arbeit analysieren wir Illumina und Nanopore Whole Genome Sequencing, RNA-seq, ATAC-seq, ChIP-seq und Hi-C-Daten von primären Neuroblastomen und Zelllinien. Wir lokalisieren MYCN-regulierende Enhancer und identifizieren die Ko-Amplifikation von lokalen Enhancern als Prinzip, das die Sequenz von MYCN Amplifikation entscheidend mitbestimmt. Wir stellen fest, dass der Verlust lokaler Enhancer durch den Einbau distaler Enhancer und die Bildung neuer Chromatindomänen durch strukturelle Veränderungen auf dem Amplikon kompensiert werden kann. Außerdem zeigen wir, dass MYCN- Amplifikation mit Clustern von interchromosomalen Bruchpunkten am MYCN-Lokus einhergeht, die auf komplexe ecDNA-Strukturen oder die Re-Integration von ecDNA in Chromosomen hinweisen. Dies kann die Genexpression in der Bruchpunktumgebung beeinflussen und wird als klinisch prognostischer Faktor identifiziert. Zuletzt beschreiben wir heterogene ecDNA- Populationen, die Organisation von ecDNA in transkriptionell aktiven ‚ecDNA hubs‘ im Zellkern und zeigen, dass ecDNA in trans interagieren kann. Unsere Ergebnisse zeigen, wie nicht- kodierende DNA-Elemente die Struktur und Funktion von Genamplifikationen mitbestimmen. Davon ausgehend schlagen wir ein Modell von Genamplifikationen als Bausteine zur Umordnung des Genoms und der Zellkernorganisation vor

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring 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.Open Access funding enabled and organized by Projekt DEAL. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. A.G.H. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 398299703 Helmsauer is supported by Boehringer Ingelheim Fonds. This work was also supported by the TransTumVar project - PN01360

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring 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.We thank the patients and their parents for granting access to the tumor specimen and clinical information that were analyzed in this study. We are grateful to Yingqian Zhan, Natalia Munoz Perez, Jennifer von Stebut, and Victor Bardinet for critical discussions. We thank Elisabeth Baumann and Anna Szymborska-Mell for help with imaging. We are grateful to Peter Van Loo for providing data during peer review and to B. Hero, H. Düren, and N. Hemstedt of the neuroblastoma biobank and neuroblastoma trial registry of the German Society of Pediatric Oncology and Hematology (GPOH) for providing samples and clinical data. Computation has been performed on the HPC for Research cluster of the Berlin Institute for Health. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. R.P.K. is supported by the Berlin Institute of Health visiting professorship program. A.G.H. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–398299703 and the Wilhelm Sander Stiftung. A.G.H. and P.E. are participants in the BIH-Charité Clinical Scientist Program funded by the Charité—Universitätsmedizin Berlin and the Berlin Institute of Health. A.G.H. and K. Helmsauer are supported by Berliner Krebsgesellschaft e.V. K. Helmsauer is supported by Boehringer Ingelheim Fonds. This work was also supported by the TransTumVar project—PN013600.Peer Reviewed"Article signat per 29 autors/es:Konstantin Helmsauer, Maria E. Valieva, Salaheddine Ali, Rocío Chamorro González, Robert Schöpflin, Claudia Röefzaad, Yi Bei, Heathcliff Dorado Garcia, Elias Rodriguez-Fos, Montserrat Puiggròs, Katharina Kasack, Kerstin Haase, Csilla Keskeny, Celine Y. Chen, Luis P. Kuschel, Philipp Euskirchen, Verena Heinrich, Michael I. Robson, Carolina Rosswog, Joern Toedling, Annabell Szymansky, Falk Hertwig, Matthias Fischer, David Torrents, Angelika Eggert, Johannes H. Schulte, Stefan Mundlos, Anton G. Henssen & Richard P. Koche"Postprint (published version

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring 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. MYCN amplification is common in neuroblastomas. Here the authors analyse the MYCN amplicon structure and its epigenetic regulation by integrating short- and longread genomic and epigenomic data and find two classes of MYCN amplicons in neuroblastomas, one driven by local enhancers and the other by hijacking of distal regulatory elements

Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma

Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer. Combined genomic and transcriptomic approaches identify the landscape of extrachromosomal circular DNA in neuroblastoma and reveal that extrachromosomal circular DNA is a major source of somatic rearrangements