The main oncogenic event in approximately 85% of Ewing Sarcoma (ES) tumors is a chromosomal translocation that produces a fusion gene containing the 5’ end of the EWSR1 gene and the 3’ end of the FLI1 gene. In an estimated 30% of tumors, translocations that retain exon 8 of the EWSR1 gene generate an out-of-frame transcript unless the exon is removed by alternative splicing. Previous work has demonstrated ES cells with a genomic breakpoint that retains exon 8 of EWSR1 require the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) to express an in-frame EWS-FLI1 mature mRNA. Thus, the ability to find a small molecule that inhibits alternative splicing by HNRNPH1 would provide an opportunity to uncover new therapies for ES. In this work, we demonstrated that the G-rich regions within exon 8 of EWSR1 pre-mRNA fold into G-quadruplexes in vitro. Additionally, we employed a Small Molecule Microarray (SMM) approach to identify compounds that selectively bind to the EWSR1 G-quadruplex over other DNA and RNA G-quadruplexes. We developed a short and flexible synthetic sequence to access a lead compound as well as a number of analogues to provide a structure-activity relationship for this series. Compounds identified from this approach have shown good affinity for the RNA. Biochemical and cell-based experiments have demonstrated that the lead compound identified from our screen prevents HNRNPH1 binding. Therefore, the SMM approach has allowed for the rapid identification of a selective RNA-binding small molecule that can be developed into a novel therapy for ES.

Targeting the EWS-FLI1 pre-mRNA in Ewing sarcoma through small molecule microarray screening

254th National Meeting (2017)

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