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

Re-analysis of public genetic data reveals a rare X-chromosomal variant associated with type 2 diabetes.

The reanalysis of existing GWAS data represents a powerful and cost-effective opportunity to gain insights into the genetics of complex diseases. By reanalyzing publicly available type 2 diabetes (T2D) genome-wide association studies (GWAS) data for 70,127 subjects, we identify seven novel associated regions, five driven by common variants (LYPLAL1, NEUROG3, CAMKK2, ABO, and GIP genes), one by a low-frequency (EHMT2), and one driven by a rare variant in chromosome Xq23, rs146662057, associated with a twofold increased risk for T2D in males. rs146662057 is located within an active enhancer associated with the expression of Angiotensin II Receptor type 2 gene (AGTR2), a modulator of insulin sensitivity, and exhibits allelic specific activity in muscle cells. Beyond providing insights into the genetics and pathophysiology of T2D, these results also underscore the value of reanalyzing publicly available data using novel genetic resources and analytical approaches

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells

Background: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells

Study of complex chromosomal rearrangements in cancer. The role of extrachromosomal circular DNA as a genome remodeler in neuroblastoma

[eng] This thesis illustrates the work I have developed as a Ph.D. student in the computational genomics group lead by Dr. David Torrents at the Barcelona Supercomputing Center. The group’s expertise in the analysis of biological data and the detection of variants to gain more knowledge about the genetic and molecular implications of human diseases, such as cancer, has allowed me to learn and conduct my research. Focusing on the analysis of structural variation in cancer, I have been able to apply different methodologies for sequencing data, retrieving, filtering, and determining the mutational profile foreach ofthe studied samples.Moreover,I have characterized new patterns of genomic rearrangements related to transposase-derived genes and extrachromosomal circular DNA elements in cancer. Therefore, this thesis is centered in the study of the genomic variation and mechanisms associated with oncogenic processes together with the analysis of elements of the human genome that are not generally included in comprehensive cancer studies, such as circular DNA elements. In summary, starting with the introduction, I give an overview of the methodological aspects of the study of cancer through the impact of sequencing technologies, the biological and molecular causes and consequences of this disease, focusing on structural variation, and the description of the circular DNA genomic component and its known implications in cancer. Finally, I introduce neuroblastoma, an example of how structural variants and circular DNA drive tumorigenesis. Next, I present the results of this thesis in three blocks, all of which have in common the study of structural variation in cancer. Two of the blocks correspond to the PGBD5 and neuroblastoma publications, and one corresponds to the continuation of the PGBD5 analysis in ICGC-Pan-Cancer data. As an overview of the trajectory of this thesis, I started with my involvement in a project focused on analyzing the role of PGBD5 —a transposase-derived gene— as an oncogenic mutator with an associated mechanism for site-specific DNA rearrangements. In this study, we describe how the expression of this gene promotes cell transformation and the generation of recurrent rearrangements, presenting a conserved motif in cell lines and childhood tumors. As a logical continuation of this publication and thanks to the access of our group to ICGC-PCAWG data, we expanded the study of these characteristic PGBD5-motif-related rearrangements to different patients and tumor types. The following part of this thesis is focused on the analysis, description, and classification of the genomic somatic rearrangements in neuroblastoma. With the aim of better grouping the patients with different clinical outcomes, we searched for differential patterns of structural variants across the samples. From this analysis, we were able to describe a new phenomenon that connects circular DNA with different integration sites around the genome through complex rearrangement clusters providing evidence on how circular DNA can act as a driver of genomic remodeling in neuroblastoma. To finalize, I present the general discussion of the results and questions addressed in this work to, then, end up disclosing the final conclusions of this thesis

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

Neuroblastoma Risk Assessment and Treatment Stratification with Hybrid Capture-Based Panel Sequencing

For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our neuroblastoma hybrid capture sequencing panel (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000x) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection

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

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells