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

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. mRNA profiling:RNA was prepared from vehicle or CBL0137 (1μM) treated human Ewing sarcoma cells SKNMC for 24 h. Trim galore was used to automatically detect and trim adapters. Reads were mapped to hg19 reference genome using Hisat2. Read count was generated using HTSeq (version 0.11.1). Differentially expressed genes (DEGs) were calculated using DESeq2 (version 1.26.0). Absolute value of fold change (FC) > 1.5 and false discovery rate (FDR) < 0.05 was used as cut-off value to select significant target genes. FPKM (Fragments per Kilobase Million) was calculated from the number of reads that mapped to each particular gene sequence and the gene length and the sequencing depth were taken into account.