Abstract B45: Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing

Abstract

Abstract MDM2 undergoes complex alternative splicing and one of its spliced isoforms, MDM2-ALT1, comprised of coding exons 3 and 12, is highly expressed in pediatric rhabdomyosarcoma, and its expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress, and this alternative splicing is conserved in the mouse Mdm2 gene. Furthermore, previous research from our lab has reported that the splicing factors SRSF1, FUBP1, and PTBP1 are involved in this regulation; nevertheless, the way that MDM2 splicing is controlled is not entirely understood. In this work, we explore the role of the splicing factor SRSF2/SC35 in the alternative splicing of MDM2, hypothesizing that this factor acts as a positive regulator, in both human and mouse, facilitating the production of the full-length MDM2 mature transcript. This splicing factor has been reported to be mutated in several cancers, including myelodysplastic syndromes and rhabdomyosarcoma. Moreover, using a minigene system that mimics the endogenous Mdm2 splicing in response to UV and cisplatinum-induced DNA damage and the CRISPR-Cas9 technology, we show that mutations in SRSF2 predicted binding sites alter Mdm2 alternative splicing. The CRISPR-engineered mutant cells that express this splice isoform show increased proliferation in an Arf/p19 null context. On the other hand, we show that by using antisense oligonucleotides working as splice-switching molecules that target the SRSF2 binding sites in the MDM2 pre-mRNA, we are able to shift the expression of the MDM2 splice isoforms to drive p53 target gene expression in p53 wild-type cells. Furthermore, in an effort to understand better the effect of the MDM2-ALT1 isoform in carcinogenesis, we performed the CRISPR-Cas9 technique to generate a mouse model expressing the cancer-associated spliced isoform. We expect the mouse to be a cancer-sensitized model that will give rise to RMS tumors and provide a preclinical model for testing splice-switching oligonucleotide therapies. Citation Format: Matias I. Montes, Daniel F. Comiskey, Safiya Khurshid, Dawn S. Chandler. Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B45.