Abstract LB188: RNA-seq and chIP-seq profiling identifies genes and pathways dysregulated by hey1-ncoa2 fusion and shed a light on mesenchymal chondrosarcoma tumorigenesis

RNA-seq and chIP-seq profiling identifies genes and pathways dysregulated by hey1-ncoa2 fusion and shed a light on mesenchymal chondrosarcoma tumorigenesis Wenqing Qi1, Wojciech Rosikiewicz2, Zhaohong Yin1, Beisi Xu2, Shibiao Wan2, Yiping Fan2, Gang Wu1,2 and Lu Wang1 1. Department of Pathology, St. Jude Children9s Research Hospital, Memphis, TN 2. Center for Applied Bioinformatics, St. Jude Children9s Research Hospital, Memphis, TN Correspondence and requests for materials should be addressed to Lu Wang (email: lu.wang2@stjude.org) Mesenchymal chondrosarcoma is a rare and often aggressive cancer that accounts for 2-10% of all chondrosarcomas. Genetically, this tumor is characterized by the recurrent HEY1-NCOA2 fusion. However, the function of HEY1-NCOA2 fusion in mesenchymal chondrosarcoma remains to be elucidated. In this study, we transduced iPSC-derived mesenchymal stem cells (MSCs) with inducible expression HEY1, NCOA2 and HEY1-NCOA2 (HN) construct, respectively. The iPSC-MSCs expressing HEY1-NCOA2 (HN_MSCs) showed significantly accelerated growth rates and higher percentage of cells in S phase compared with cells expressing wild-type HEY1 or NCOA2 (HEY1_MSCs or NCOA2_MSCs). Intriguingly, in 3-D spheroidal growth model, we observed both accelerated cell proliferation and distinct morphological features of the HN_MSCs in contrast to the control cells. To identify direct target genes and the pathways modulated by HEY1-NCOA2, stably transduced iPSC-MSCs with and without the expression of the exogenous HN, HEY1 and NCOA2, were subjected to Chromatin Immunoprecipitation Sequencing and RNA-seq analyses. ChIP-seq data showed a largely overlap in DNA-binding peaks observed in HN_MSCs and HEY1_MSCs (~90% of HN binding peaks). In contrast, very few (0.1%) overlaps observed between HN_MSCs and NCOA2_MSCs. In addition, ~25% of HN binding peaks contain the canonical HEY1 binding E-box motif (CACGTG). Through RNA-seq analysis, HN_MSC and HEY1-MSC can be robustly separated by gene expression profiling, and GSEA pathway enrichment analysis revealed that genes encoding cell cycle related targets of E2F transcription factors and genes involved in the G2/M checkpoint as in progression through the cell division cycle are significantly enriched in genes highly expressed in HN_MSC. Differential expression analysis identified 316 genes that were significantly up-regulated by HN but down-regulated by HEY1, of which CCND1 is one of the most differentially expressed genes. When correlating with ChIP-seq analysis, 59 of the 316 genes showed DNA-binding peaks with the canonical HEY1-binding motif, including HES1 and SOX4 which were reported to be involved in chondrogenesis. We further verified the significant upregulation of CCND1, HES1 and SOX4 in HN_MSCs by Q-RT-PCR. Taken together, our study demonstrated that HN is oncogenic by significantly accelerating cell proliferation. RNA-seq and ChIP-seq profiling revealed that HEY1-NCOA2 directly binds to HEY1 target genes through HEY1-binding motif to dysregulate their expression and consequently rewire the downstream pathways. The combined RNA-seq and ChIP-seq analysis suggested that HES1, SOX4 and CCND1 are critical targets of the transcriptional dysregulation mediated by HN and may play key roles in mesenchymal chondrosarcoma tumorigenesis. Citation Format: Wenqing Qi. RNA-seq and chIP-seq profiling identifies genes and pathways dysregulated by hey1-ncoa2 fusion and shed a light on mesenchymal chondrosarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB188.