Therapeutic Targeting Ewing Sarcoma through Inhibition of the FACT Complex (ChIP-Seq)

EWS/ETS fusion transcription factors, most commonly EWSR1-FLI1, drive initiation and progression of Ewing sarcoma (EwS), a highly aggressive childhood cancer of bone and soft tissues. Even though direct targeting EWSR1-FLI1 is a formidable challenge, epigenetic or transcriptional modulators have been recently proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we performed transcriptome and functional genomics dataset analyses, and combined with small molecule screening of EwS lines to identify novel epigenetic/transcriptional-targeted therapeutic strategies. SSRP1 and SUPT16H, two subunits of the Facilitates Chromatin Transcription (FACT) complex, are both found to be EWSR1-FLI1-induced essential oncogenes in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models in vitro and in vivo. Mechanistically, the FACT complex and EWS-FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1-FLI1-FACT circuit, resulting in transcriptional disruption of EWS-FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1-FLI1 and demonstrates FACT inhibition as a novel therapeutic strategy against EwS via indirect targeting the oncogenic fusion TF, providing preclinical support for adding EwS to CBL0137’s future clinical trials. Briefly, 1~2 × 107 SKNMC cells were digested with Micrococcal Nuclease (New England Biolabs, M0247S) and sonicated for Chromatin IP experiment. Then 10 ug chromatin was immunoprecipitated with 10 uL of SSRP1 antibody (Cell Signaling Technology, 13421S), FLI1 antibody (Abcam, ab15289) or H3K27Ac antibody (Active Motif, AM39133,) and 50 uL Pierce™ ChIP-grade Protein A/G Magnetic Beads (Thermo Scientific, 26162). Immunoprecipitated DNA and INPUT DNA were then purified and sequenced by Romics, Shanghai, China. ChIP-seq data were first mapped against the human genome build hg19 using BowTie2 using the default settings. Then Model-based analysis of ChIP-seq (MACS2: v2.2.7.1) was used to identify peak regions of ChIP-seq enrichment comparing to input control with a q threshold of 0.05. Peak annotation and motifs finding was performed by HOMER Analysis. DeepTools was conducted to generate BigWig files and visualization. We explore Integrative Genome Viewer and WashU Epigenome Browse (http://epigenomegateway.wustl.edu/browser/) to visualize BigWig files on the hg19 genome track. Overlapped peaks were identified at least 20% intersected signal between two or three signal tracks.