Oncofusion-drivende novoenhancer assembly promotes malignancy in Ewing sarcomaviaaberrant expression of the stereociliary protein LOXHD1

Abstract

Ewing Sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1–ETS (EWSR1–FLI1/EWSR1–ERG) transcription factors drive EwS by orchestrating an oncogenic transcription program through de novo enhancers. Pharmacological targeting of these oncofusions has been challenged by unstructured prion-like domains and common DNA binding domains in the EWSR1 and ETS protein, respectively. Alternatively, identification and characterization of mediators and downstream targets of EWSR1–FLI1 dependent or independent function could offer novel therapeutic options. By integrative analysis of thousands of transcriptome datasets representing pan-cancer cell lines, primary cancer, metastasis, and normal tissues, we have identified a 32 gene signature (ESS32 – Ewing Sarcoma Specific 32) that could stratify EwS from pan-cancer. Of the ESS32, LOXHD1 – that encodes a stereociliary protein, was the most exquisitely expressed gene in EwS. CRISPR–Cas9 mediated deletion or silencing of EWSR1–FLI1 bound upstream de novo enhancer elements in EwS cells led to the loss of LOXHD1 expression and altered the EWSR1–FLI1, MYC, and HIF1; pathway genes, resulting in decreased proliferation and invasion in vitro and in vivo. These observations implicate LOXHD1 as a novel biomarker and a major determinant of EwS metastasis and open up new avenues for developing LOXHD1–targeted drugs or cellular therapies for this deadly disease.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation

“…As a proof-of-concept, this cell line-specific approach identified and refined an EWSR1-ETS-dependent transcriptional core-signature (Suppl. Data 12), which contained a large number of previously identified EWSR1-ETS-regulated and functionally relevant genes 53,55,60,62,67 .…”

“…Data 12). The EWSR1-ETS-induced core-genes comprised CCK, E2F2, IL1RAP, LOXHD1, PPP1R1A, and STEAP1, all of which have been shown to contribute to the malignant phenotype of EwS 36,53,55,[59][60][61] . Conversely, the EWSR1-ETS-suppressed coregenes comprised LOX and FOXO1 that were shown to antagonize EwS malignancy 51,62 .…”

Ewing sarcoma from molecular biology to the clinic

“…As this ability to bind to GGAA microsatellites is not observed for FLI1 alone, this confers on EWS-FLI1 specific functions enabling the activation of de novo enhancers through the recruitment of BAF (BRG1-or BRM-Associated Factors), a chromatin remodeling complex (Boulay et al, 2017). This modulates the expression of numerous target genes, such as LOXHD1 (Deng et al, 2022), or activates the expression of genes not expressed in healthy tissues or other tumor tissues outside ES (Vibert et al, 2022).…”