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Transcription Factors Involved in Tumorigenesis Are Over-Represented in Mutated Active DNA Binding Sites in Neuroblastoma

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Transcription Factors Involved in Tumorigenesis Are Over-Represented in Mutated Active DNA Binding Sites in Neuroblastoma Author Manuscript Published OnlineFirst on November 29, 2019; DOI: 10.1158/0008-5472.CAN-19-2883 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Transcription factors involved in tumorigenesis are over-represented in mutated active DNA 2 binding sites in neuroblastoma 3 4 Mario Capasso1,2,3*^, Vito Alessandro Lasorsa1,2*, Flora Cimmino1,2, Marianna Avitabile1,2, Sueva 5 Cantalupo1,2, Annalaura Montella1,2, Biagio De Angelis4, Martina Morini5, Carmen de Torres6,§, 6 Aurora Castellano4, Franco Locatelli4,7, Achille Iolascon1,2^. 7 8 Authors' information 9 10 * These authors equally contributed to this work. 11 12 ^ Corresponding authors: 13 Mario Capasso 14 Università degli Studi di Napoli “Federico II”, via Gaetano Salvatore 486, 80145 Napoli; 15 e-mail: [email protected] 16 Phone: +390813737889 17 18 Achille Iolascon 19 Università degli Studi di Napoli “Federico II”, via Gaetano Salvatore 486, 80145 Napoli; 20 e-mail: [email protected] 21 Phone: +390813737897 22 23 1 Department of Molecular Medicine and Medical Biotechnology, Università degli Studi di Napoli 24 Federico II. Via Sergio Pansini, 5, 80131 Napoli, Italy. 25 26 2 CEINGE Biotecnologie Avanzate. Via Gaetano Salvatore 486, 80145 Napoli, Italy. 27 28 3 IRCCS SDN. Via Emanuele Gianturco, 113, 80143 Napoli, Italy. 29 30 4 Department of Pediatric Haematology and Oncology, IRCCS Ospedale Pediatrico Bambino Gesù. 31 Piazza di Sant'Onofrio, 4, 00165 Roma, Italy. 32 33 5 Laboratory of Molecular Biology, IRCCS Istituto Giannina Gaslini. Via Gerolamo Gaslini, 5, 34 16147 Genova, Italy. 35 36 6 Hospital Sant Joan de Déu, Developmental Tumor Biology Laboratory and Department of 37 Oncology. Passeig de Sant Joan de Déu, 2, 08950 Esplugues de Llobregat, Barcelona, Spain. 38 39 7 Department of Paediatrics, Sapienza University of Rome. Piazzale Aldo Moro, 5, 00185 Roma, 40 Italy. 41 42 § Deceased. 43 44 Running title 45 Tumorigenic noncoding mutations in neuroblastoma. 46 Conflict of interests 47 The authors declare that they have no competing interests. 1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 29, 2019; DOI: 10.1158/0008-5472.CAN-19-2883 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 48 Abstract 49 50 The contribution of coding mutations to oncogenesis has been largely clarified whereas little is 51 known about somatic mutations in noncoding DNA and their role in driving tumors remains 52 controversial. Here, we used an alternative approach to interpret the functional significance of 53 noncoding somatic mutations in promoting tumorigenesis. Noncoding somatic mutations of 151 54 neuroblastomas (NB) were integrated with ENCODE data to locate somatic mutations in regulatory 55 elements specifically active in NB cells, non-specifically active in NB cells, and non-active. Within 56 these types of elements, transcription factors (TFs) were identified whose binding sites (BS) were 57 enriched or depleted in mutations. For these TFs, a gene expression signature was built to assess 58 their implication in NB. DNA and RNA sequencing data were integrated to assess the effects of 59 those mutations on mRNA levels. The pathogenicity of mutations was significantly higher in TFBS 60 of regulatory elements specifically active in NB cells, as compared to the others. Within these 61 elements, there were 18 over-represented TFs involved mainly in cell cycle phase transitions, and 62 15 under-represented TFs primarily regulating cell differentiation. A gene expression signature 63 based on over-represented TFs correlated with poor survival and unfavourable prognostic markers. 64 Moreover, recurrent mutations in TFBS of over-represented TFs such as EZH2 affected MCF2L 65 and ADPRHL1 expression, among the others. We propose a novel approach to study the 66 involvement of regulatory variants in NB that could be extended to other cancers and provide 67 further evidence that alterations of gene expression may have relevant effects in NB development. 68 69 70 Significance 71 Findings propose a novel approach to study regulatory variants in neuroblastoma and suggest that 72 noncoding somatic mutations have relevant implications in neuroblastoma development. 73 2 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 29, 2019; DOI: 10.1158/0008-5472.CAN-19-2883 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 74 Background 75 76 As most high throughput sequencing studies of cancer focused mainly on the protein-coding part of 77 the genome, the role of somatic mutations in regulatory regions (i.e. TFBSs) remains 78 underestimated. The most recent literature clearly demonstrates that altered transcriptional 79 regulatory circuits play relevant roles in cancer development (1). For instance, a re-analysis of 80 sequencing data from 493 tumors found somatic mutations in TFBSs under positive selection, 81 consistent with the fact that these loci regulate important cancer cell functions (2). Another recent 82 study has demonstrated that noncoding mutations can affect the gene expression of target genes in a 83 large number of tumors (3). Promoter mutations can either create ETS-binding sites activating 84 transcription of oncogenes such as TERT (4) or disrupt the same binding sites disabling 85 transcription of tumour-suppressors such as SDHD (5). 86 87 Despite these recent advances, it remains a challenge to establish the pathogenic repercussions of 88 noncoding variants in cancer development. First, noncoding variants can alter a number of functions 89 including transcriptional and post-transcriptional regulation. Second, noncoding regions accumulate 90 mutations at higher rates than coding regions because of a weaker selective pressure (5). As a result, 91 distinguishing driver noncoding mutations from passengers becomes a difficult statistical and 92 computational task. Third, it is challenging to computationally predict whether a noncoding variant 93 affects gene expression or mRNA stability because the logic involved in regulatory element 94 function has not yet been fully elucidated. In this respect, data from ENCODE and Roadmap 95 projects, which map regulatory elements in several tissues and cell lines can facilitate functional 96 interpretation of noncoding somatic mutations. 97 98 NB is the most common pediatric, extra-cranial solid tumor of neural crest origin accounting for the 99 8–10% of all pediatric cancers. Despite decades of international efforts to improve the outcome, 3 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 29, 2019; DOI: 10.1158/0008-5472.CAN-19-2883 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 100 long-term survivors of high-risk NB are about 30% (6). Genomic and transcriptomic biomarkers, 101 including MYCN amplification, chromosomal aberrations and gene expression signatures are used 102 to stratify patients into different risk groups (6-8). Next generation sequencing studies of NB have 103 documented low somatic mutation rates and few recurrently mutated genes. Indeed, only mutations 104 in ALK, ATRX and TERT have been identified as the most frequent genetic abnormalities (9-12). 105 Recent studies have shown that disease-associated genomic variation is commonly located in 106 regulatory elements in the human population (13). Our genome-wide association studies (GWASs) 107 using DNA from non-disease cells (blood) have revealed that many genetic variants that are 108 associated with NB susceptibility lie in noncoding regions of the genome (14-16). Functional 109 investigations have shown that the cancer genes LMO1 (17), BARD1 (16), LIN28B (18), whose 110 expression is affected by risk variants, play a role in NB tumorigenesis. Importantly, despite 111 considerable efforts, the molecular events that drive NB initiation and progression are still largely 112 unknown. Therefore, a better knowledge of the alterations that shape NB genomes and 113 transcriptomes becomes necessary to understand its aetiology. Detailed genomic information 114 leading to new drug targets is also the starting point to develop more effective and less toxic 115 treatments (19). 116 117 In this work, we propose an alternative method to verify the potential pathogenic consequence of 118 noncoding variants in NB using whole genome sequencing (WGS) data from 151 tumors. First, we 119 tested if the predicted functional impact of somatic variants in TFBS is a feature of a specific cancer 120 tissue. Second, we tested if there is an enrichment of somatic mutations in DNA binding sites for 121 TF involved in tumorigenesis. Third, we verified if recurrent somatic mutations in TFBS are 122 associated with changes in mRNA levels. 123 Overall, our analysis strategy allowed us to find mutated binding sites for TFs that may have key 124 functions in NB initiation. Moreover, for some of these mutated sites, we identified candidate target 125 genes whose deregulation may contribute to NB development. 4 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published
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