Abstract A14: Use of VEGF-based splicing-sensitive fluorescent reporters to screen for anti-angiogenic molecules

Abstract

Two vascular endothelial growth factor (VEGF) protein families are generated via alternative splicing of the VEGF gene terminal exon. Use of the canonical proximal 3' splice site results in pro-angiogenic VEGF isoforms (VEGFxxx). Anti-angiogenic (VEGFxxxb) isoforms are produced through usage of a distal 3' splice site. In a quest to discover novel anti-angiogenic compounds we performed a screen using molecular tools designed to mimic VEGF splicing. Splicing-sensitive fluorescent reporters (SSFRs) can be used to study specific splicing events. A bichromatic SSFR has been engineered to mimic VEGF terminal exon splicing. Endogenous VEGF intron 7 and parts of exon 8 have been inserted into the reporter. These sequences are followed by the coding sequences for two fluorescent proteins. Depending on which alternative 3' splice site is used, a different fluorescent protein is expressed. This splicing reporter allows VEGF alternative splicing to be followed in vitro and in vivo. Reporter validation was achieved by transfection of the reporter in several cell lines with differential endogenous VEGF splicing as well as by treating stable cell lines expressing the reporter with growth factors or small molecule inhibitors known to alter VEGF alternative splicing. Flow cytometric analysis was used to evaluate the alternative splicing pattern. Following successful validation, several small pilot screens looking for novel regulators of the pro/anti-angiogenic splicing switch were undertaken with growth factors and various chemicals. Following validation and pilot experiments, a large screen of 1280 pharmacologically active compounds (LOPAC library) was performed. Changes to reporter splicing were measured using a fluorescent plate reader. Hits from the screen were investigated further using flow cytometry to confirm alteration in splicing pattern. Several compounds of interest were identified and their effect on endogenous pro/anti-angiogenic VEGF being confirmed by RTPCR. Anti-angiogenic activity is being assessed by several in vitro and in vivo angiogenic assays. Citation Format: Eleanor Star, Steven Harper, David Bates, Sebastian Oltean. Use of VEGF-based splicing-sensitive fluorescent reporters to screen for anti-angiogenic molecules. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A14.