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ChIP-seq of A673 Ewing sarcoma cells using a specific anti-MYBL2 antibody

ABSTRACT: Deciphering principles of inter-individual tumor heterogeneity is essential for refinement of personalized anti-cancer therapy. Unlike cancers of adulthood, pediatric malignancies including Ewing sarcoma feature a striking paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here we demonstrate in the Ewing sarcoma model how cooperation of a dominant oncogene and regulatory variants determine tumor growth, patient survival and drug response. We show that binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls transcription of MYBL2, whose high expression promotes poor patient outcome via activation of pro-proliferative signatures. Genetic interference with this regulatory element almost abolished MYBL2 transcription, and MYBL2 knockdown decreased proliferation and tumorigenicity of Ewing sarcoma cells. Combined RNA- and ChIP-seq experiments identified CCNF, BIRC5 and AURKB as direct MYBL2 targets and critical mediators of its phenotype. In drug-response experiments high MYBL2 levels sensitized Ewing sarcoma cells for inhibition of its activating cyclin dependent kinase, CDK2, in vitro and in vivo, suggesting MYBL2 as a predictive biomarker for targeted anti-CDK2-therapy.Collectively, our findings establish cooperation of somatic mutations and regulatory variants as a major determinant of tumor progression and indicate the importance of integrating the regulatory genome in the process of developing new diagnostic and/or therapeutic strategies to fully harness the potential of precision medicine.

ORGANISM(S): Homo sapiens

PROVIDER: GSE119971 | GEO | 2019/08/16

REPOSITORIES: GEO

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Project description:Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta- catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells upregulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression.

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ChIP-seq of A673 Ewing sarcoma cells using a specific anti-MYBL2 antibody

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