Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding

• Published in: American journal of human genetics Vol. 110; no. 3; pp. 427 - 441
• Main Authors: Lee, Olivia W., Rodrigues, Calvin, Lin, Shu-Hong, Luo, Wen, Jones, Kristine, Brown, Derek W., Zhou, Weiyin, Karlins, Eric, Khan, Sairah M., Baulande, Sylvain, Raynal, Virginie, Surdez, Didier, Reynaud, Stephanie, Rubio, Rebeca Alba, Zaidi, Sakina, Grossetête, Sandrine, Ballet, Stelly, Lapouble, Eve, Laurence, Valérie, Pierron, Gaelle, Gaspar, Nathalie, Corradini, Nadège, Marec-Bérard, Perrine, Rothman, Nathaniel, Dagnall, Casey L., Burdett, Laurie, Manning, Michelle, Wyatt, Kathleen, Yeager, Meredith, Chari, Raj, Leisenring, Wendy M., Kulozik, Andreas E., Kriebel, Jennifer, Meitinger, Thomas, Strauch, Konstantin, Kirchner, Thomas, Dirksen, Uta, Mirabello, Lisa, Tucker, Margaret A., Tirode, Franck, Armstrong, Gregory T., Bhatia, Smita, Robison, Leslie L., Yasui, Yutaka, Romero-Pérez, Laura, Hartmann, Wolfgang, Metzler, Markus, Diver, W. Ryan, Lori, Adriana, Freedman, Neal D., Hoover, Robert N., Morton, Lindsay M., Chanock, Stephen J., Grünewald, Thomas G.P., Delattre, Olivier, Machiela, Mitchell J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.03.2023; Elsevier
• Subjects: 6p25.1; Alleles; Bone Neoplasms - genetics; Bone Neoplasms - pathology; Cell Line, Tumor; Ewing sarcoma; EWSR1-FLI1; Gene Expression Regulation, Neoplastic; germline somatic interaction; GGAA microsatellite; GWAS; Humans; long-read sequencing; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - metabolism; Proto-Oncogene Protein c-fli-1 - genetics; Proto-Oncogene Protein c-fli-1 - metabolism; RNA-Binding Protein EWS - genetics; RNA-Binding Protein EWS - metabolism; RREB1; Sarcoma, Ewing - genetics; Sarcoma, Ewing - metabolism; Sarcoma, Ewing - pathology; Ewing sarcoma; GWAS; germline somatic interaction; 6p25.1; GGAA microsatellite; long-read sequencing; RREB1; EWSR1-FLI1
• ISSN: 0002-9297; 1537-6605
• DOI: 10.1016/j.ajhg.2023.01.017

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation. Lee et al. characterized germline variation in the 6p25.1 EwS susceptibility region. Longer GGAA microsatellite alleles at 6p25.1 were associated with EwS risk and the microsatellite showed chromatin features of an EWSR1-FLI1 enhancer. EWSR1-FLI1 binding altered expression of RREB1, promoting proliferation.