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

ABSTRACT: 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.

SUBMITTER: Lee OW

PROVIDER: S-EPMC10027473 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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

Lee Olivia W OW Rodrigues Calvin C Lin Shu-Hong SH Luo Wen W Jones Kristine K Brown Derek W DW Zhou Weiyin W Karlins Eric E Khan Sairah M SM Baulande Sylvain S Raynal Virginie V Surdez Didier D Reynaud Stephanie S Rubio Rebeca Alba RA Zaidi Sakina S Grossetête Sandrine S Ballet Stelly S Lapouble Eve E Laurence Valérie V Pierron Gaelle G Gaspar Nathalie N Corradini Nadège N Marec-Bérard Perrine P Rothman Nathaniel N Dagnall Casey L CL Burdett Laurie L Manning Michelle M Wyatt Kathleen K Yeager Meredith M Chari Raj R Leisenring Wendy M WM Kulozik Andreas E AE Kriebel Jennifer J Meitinger Thomas T Strauch Konstantin K Kirchner Thomas T Dirksen Uta U Mirabello Lisa L Tucker Margaret A MA Tirode Franck F Armstrong Gregory T GT Bhatia Smita S Robison Leslie L LL Yasui Yutaka Y Romero-Pérez Laura L Hartmann Wolfgang W Metzler Markus M Diver W Ryan WR Lori Adriana A Freedman Neal D ND Hoover Robert N RN Morton Lindsay M LM Chanock Stephen J SJ Grünewald Thomas G P TGP Delattre Olivier O Machiela Mitchell J MJ

American journal of human genetics 20230213 3

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 f ...[more]

PMID: 36787739

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Project description:BackgroundThe EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown.MethodsWe used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single cell expression profiling, CyTOF and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting and immunohistochemical staining.ResultsWe defined a workflow to study EWSR1/FLI1 driven transcriptional states and phenotypes. We tracked EWSR1/FLI1 dependent proliferative activity over time to discover sources of intra-tumoral diversity. Single-cell RNA profiling was used to compare expression profiles in exponentially growing populations (si-Control) or in two dormant populations (D1, D2) in which EWSR1/FLI1 had been suppressed. Three distinct transcriptional states were uncovered contributing to ES intra-heterogeneity. Our predictive model identified ~1% cells in a dormant-like state and ~ 2-4% cells with stem-like and neural stem-like features in an exponentially proliferating ES cell line and in ES xenografts. Following EWSR1/FLI1 knockdown, cells re-entering the proliferative cycle exhibited greater stem-like properties, whereas for those cells remaining quiescent, FAM134B-dependent dormancy may provide a survival mechanism.ConclusionsWe show that time-dependent changes induced by suppression of oncogenic EWSR1/FLI1 expression induces dormancy, with different subpopulation dynamics. Cells re-entering the proliferative cycle show enhanced stem-like characteristics, whereas those remaining dormant for prolonged periods appear to survive through autophagy. Cells with these characteristics identified in exponentially growing cell populations and in tumor xenografts may confer drug resistance and could potentially contribute to metastasis.

Dataset Information

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

Publications

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

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