Abstract 6601: Menin drives oncogenesis in Ewing sarcoma cells by activating transcription of key metastatic factors

Abstract

Abstract Ewing sarcoma (EwS) is a bone and soft tissue tumor that is driven by the fusion of FET and ETS genes, most commonly resulting in creation of the EWS::FLI1 chimeric oncoprotein. The scaffolding protein Menin regulates transcription through interactions with transcription factors and chromatin modifying enzymes and has both oncogenic and tumor suppressive roles, depending on the cellular context. The half-life of Menin is prolonged in EwS cells leading to high levels of the protein that is dependent on EWS::FLI1 expression. To investigate tumorigenic functions of Menin in EwS, we used CRISPR/Cas9 to generate clonal A673 and TC32 Menin knockout cells. Consistent with our earlier studies using shRNA-mediated knockdown, loss of Menin had little effect on proliferation in 2-D culture but resulted in reduced anchorage-independent growth in soft agar assays. When MEN1-KO cells were injected into NSG mice, either subcutaneously or by tail vein, tumor formation was delayed. Injection of MEN1-KO cells into the renal subcapsule of NSG mice resulted in locally invasive tumors but their capacity to metastasize to the liver was reduced compared to control cells. To elucidate how loss of Menin led to diminished tumorigenic and metastatic potential, we performed RNA-seq of control and MEN1-KO cells. This analysis revealed significant and reproducible down-regulation of MYC target genes and up-regulation of the epithelial to mesenchymal transition pathway and extracellular matrix protein-encoding genes in MEN1-KO cells. To determine if genes altered in MEN1-KO cells were dependent on the role of Menin in Menin/MLL methyltransferase complexes, we analyzed transcriptomes of EwS cells that had been exposed to the Menin/MLL interaction inhibitor, VTP-50469. Only a subset of genes altered by the MEN1-KO were also altered by the Menin/MLL inhibitor, suggesting that Menin regulates gene expression in EwS cells by both MLL-dependent and MLL-independent mechanisms. To test this, we performed CUT&RUN to define Menin binding sites and discovered that Menin binds not only at H3K4me3-marked gene promoters but also at intragenic enhancers marked by H3K27Ac. Importantly, comparison of RNA-seq data revealed substantial overlap between Menin-regulated and established EWS::FLI1 target genes and we identified that a subset of EWS::FLI1-bound intragenic enhancers are also bound by Menin. Significantly, these co-bound, co-regulated loci encode for pro-metastatic genes, including IL1RAP, STEAP1, and CAV1. Finally, we found that Menin and EWS::FLI1 co-immunoprecipitate in EwS nuclear extracts suggesting that they may exist in complex with one another at sites of shared gene regulation. Taken together, these data indicate that Menin promotes EwS metastasis and that this is achieved by its function as a scaffolding protein that augments the transcriptional activity of EWS::FLI1 at intragenic enhancers of pro-metastasis genes. Citation Format: Katherine A. Braun, Aya Miyaki, Nicolas M. Garcia, Darleen Tu, Feinan Wu, Jay F. Sarthy, Elizabeth R. Lawlor. Menin drives oncogenesis in Ewing sarcoma cells by activating transcription of key metastatic factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6601.