Abstract B076: ROME, a novel membrane protein in vertebrates, enhances metastatic phenotype of Ewing sarcoma cells

Abstract In order to discover genes that can accelerate metastasis of Ewing sarcoma (ES), we performed a genome-wide CRISPR/Cas9 transcriptional activation screen in zebrafish using human ES cells as xenografts. We identified the human INAFM2 gene as a strong driver of metastatic activity of ES cells. INAFM2 is the human homolog of the Drosophila InaF gene and there were no publications about the protein product of this gene in any vertebrate animals. We named the new protein ROME (Regulator of metastasis) and characterized its expression and its role in ES metastasis using multiple in vitro and in vivo models. We established that the human ROME protein is indeed expressed at the plasma membrane. We demonstrated that the ROME protein is both phosphorylated and glycosylated. The functional significance of these posttranslational modifications remains to be determined. Lack of ROME expression in zebrafish embryos resulted in severe developmental defects and early mortality. We performed single cell RNA sequencing of zebrafish embryos that were injected with ROME targeting morpholino. RNA sequencing of ES cell lines that were either overexpressing ROME or lack ROME expression (by CRISPR). We determined that ROME expression regulates Ca++, Wnt and MAPK signaling pathways. Overexpression of ROME in four ES cell lines caused increased cell migration and chemotaxis in vitro, and increased intravasation in zebrafish xenografts. ROME knockout in two ES cell lines decreased chemotaxis. Rescue of ROME expression restored the cells’ ability to migrate in CRISPR knockout clones. In vivo studies by tail vein injection of ES cells in immunodeficient mice demonstrated significantly increased gross metastases with ROME overexpression and decreased gross metastases with ROME knockout compared to the respective control groups. Additional ES xenograft studies where the primary tumor was removed by amputating the affected leg, showed that overexpression of ROME results in enhanced lung metastasis. Analysis of patient tumor RNA sequencing data from many different types of human cancers revealed correlations between ROME expression and both an increased metastatic phenotype and poor patient survival. This provides strong evidence for a substantial role of ROME in ES and cancer metastasis in general. In summary, we present the first ever characterization of the ROME protein in zebrafish embryos and ES cancer cell lines. Our data suggest that ROME is a membrane protein that can regulate cell motility and invasion through different signaling pathways, which results in increased metastatic potential. Citation Format: Anna Molotkova, Emre Deniz, Matthew Swift, Eric Glasgow, Jeffrey Toretsky, Aykut Uren. ROME, a novel membrane protein in vertebrates, enhances metastatic phenotype of Ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B076.