EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma.

Xianping Shi,Yueyuan Zheng,Wei Yang,Lingwen Ding,H Phillip Koeffler,Liling Jiang,Sigal Gery,De-Chen Lin,Bo Zhou,Moli Huang,Liyan Li

doi:10.1093/nar/gkaa901

Abstract

Core regulatory circuitry (CRC)-dependent transcriptional network is critical for developmental tumors in children and adolescents carrying few gene mutations. However, whether and how CRC contributes to transcription regulation in Ewing sarcoma is unknown. Here, we identify and functionally validate a CRC ‘trio’ constituted by three transcription factors (TFs): KLF15, TCF4 and NKX2-2, in Ewing sarcoma cells. Epigenomic analyses demonstrate that EWS-FLI1, the primary fusion driver for this cancer, directly establishes super-enhancers of each of these three TFs to activate their transcription. In turn, KLF15, TCF4 and NKX2-2 co-bind to their own and each other's super-enhancers and promoters, forming an inter-connected auto-regulatory loop. Functionally, CRC factors contribute significantly to cell proliferation of Ewing sarcoma both in vitro and in vivo. Mechanistically, CRC factors exhibit prominent capacity of co-regulating the epigenome in cooperation with EWS-FLI1, occupying 77.2% of promoters and 55.6% of enhancers genome-wide. Downstream, CRC TFs coordinately regulate gene expression networks in Ewing sarcoma, controlling important signaling pathways for cancer, such as lipid metabolism pathway, PI3K/AKT and MAPK signaling pathways. Together, molecular characterization of the oncogenic CRC model advances our understanding of the biology of Ewing sarcoma. Moreover, CRC-downstream genes and signaling pathways may contain potential therapeutic targets for this malignancy.

Highlights

As a developmental cancer carrying few genetic alterations, Ewing sarcoma is the second most common malignancy of bone and soft tissue predominantly occurring in adolescents and young adults [1]
We initially focused on the A673 cell line, since it is a wellcharacterized Ewing sarcoma line with available H3K27ac and EWS-FLI1 Chromatin immunoprecipitation (ChIP)
We investigated in-depth the term “Signal transduction” since it was ranked highest in all three RNA sequencing (RNA-Seq) upon knockdown of CRC transcription factors (TFs) (1st in both siKLF15 and siNKX2-2 and 2nd in siTCF4, Fig. 4G)

Summary

Introduction

As a developmental cancer carrying few genetic alterations, Ewing sarcoma is the second most common malignancy of bone and soft tissue predominantly occurring in adolescents and young adults [1]. EWS-FLI1, the primary fusion driver, rewires fundamentally the transcriptome of Ewing sarcoma cells [2,3,4,5]. In many cases, EWS-FLI1 requires the cooperation of other transcriptional cofactors, such as WDR5 and CBP/p300, to regulate chromatin modification and gene expression [6,7,8]. EWS-FLI1-targeting transcription factors (TFs), such as MEIS1, NKX2-2, SOX2, and OTX2 [7, 9,10,11], are required for the fusion driver to fulfil its oncogenic function in Ewing sarcoma cells. Despite having immense capacity in chromatin regulation, EWS-FLI1 still relies on additional cooperators and mediators to orchestrate gene expression programs characterization of such partners and mediators is critical for further understanding the biology of Ewing sarcoma. Despite having immense capacity in chromatin regulation, EWS-FLI1 still relies on additional cooperators and mediators to orchestrate gene expression programs characterization of such partners and mediators is critical for further understanding the biology of Ewing sarcoma. in Ewing sarcoma.

Methods

Results

Conclusion