(P 435) ONCOGENIC FUSION YWHAE::NUTM2 DISRUPT EP300/BCOR INTERACTION

Objective: Several groups of primitive round cell sarcomas harbor YWHAE::NUTM2 oncogenic fusion, resulting in BCOR overexpression. However, YWHAE::NUTM2 mechanisms of oncogenesis and roles in BCOR overexpression have not yet been well described. With the aim to provide a comprehensive description of YWHAE::NUTM2 oncogenic mechanism, we investigated by RNA-sequencing a retrospective series of tumors and performed mass spectrometry across transfected cell lines.

Methods: This study was performed as a Centre Léon Bérard (Lyon, France) monocentric retrospective translational research program on tumor samples from an investigational cohort of 11 YWHAE::NUTM2 sarcomas: 8 endometrial stromal sarcomas and three undifferentiated round-cell sarcoma (two neonatal soft tissue/visceral sarcoma and one adult bone sarcoma). Whole exome RNA sequencing was performed with FFPE material in all cases using TruSeq RNA Access Library Prep Kit (Illumina®, San Diego, USA). After the YWHAE::NUTM2 fusion was cloned and sequenced, we established the expressing FLAG-tagged plasmid pIRES-YWHAE::NUTM2 and transfected it into the human cervical cancer cell line HeLa, using Neon electroporation. Then, we performed protein extraction and FLAG immunoprecipitation followed by mass spectrometry in order to identify and characterize YWHAE::NUTM2 oncoprotein interactions.

Results: RNAseq analysis revealed YWHAE fusions in all 11 patients including 8 involving NUTM2E, two invoving NUTM2B and one involving NUTM2A. Furthermore, it showed significant higher BCOR and BCORL1 expression ranked in the top 5% expressing tumors in a panel of 2642 solid tumors. Unsupervised clustering on the expression profiles issued from this series identified a biologically homogeneous group of tumors, together with BCOR-rearranged sarcomas and characterized by an upregulation of genes involved in the cell cycle/proliferation pathways, morphogenesis/development, translation and spliceosome and a low immune infiltrate. With FLAG immunoprecipitation followed by mass spectrometry, YWHAE::NUTM2 oncoprotein was found to interact with EP300, CREBBP, A-RAF and C-RAF but not BCOR surprisingly, suggesting an indirect dysregulation. With EP300 immunoprecipitation followed by Western Blot, we confirmed the interaction. Furthermore, we also detect that YWHAE::NUTM2 disrupt EP300 interactions with BCOR (Figure 1).

Conclusion: Altogether, our RNA-sequencing findings support that YWHAE::NUTM2 and BCOR rearranged sarcomas might represent a common entity. While YWHAE::NUTM2 does not seem to interact directly with BCOR, it might regulates BCOR expression by disrupting its interaction with EP300. Consequently, it might lead to Non-Canonical PRC1.1 (Polycomb repressive complex) dysregulation and then promote tumorigenesis.