Novel SRPK1 inhibitors specifically alter alternative splicing and switch VEGF-A expression from pro-angiogenic to anti-angiogenic isoforms.

Background Pathological angiogenesis occurs when the balance between pro-angiogenic VEGF-A165a and anti-angiogenic VEGF-A165b is switched by differential splicing of VEGF mRNA to the proangiogenic form. Therapeutic VEGF-A inhibitors are the standard of care for wet age-related macular degeneration (wAMD) but target both pro- and anti-angiogenic isoforms. Specifically altering the balance of VEGF-A from pro-angiogenic VEGF-A165a to anti-angiogenic VEGF-A165b in retinal pigment epithelial (RPE) cells could prove effective in the treatment of wet AMD (1). VEGF-A alternative splicing is controlled through RNA binding protein SRSF1 which when phosphorylated by the serine kinase SRPK1 (2) induces pro-angiogenic VEGF-A165a expression (3). However, the specificity of compounds inhibiting a splicing factor kinase for all its potential downstream targets is unknown. The aim of this investigation was to use novel SRPK1 inhibitors that specifically alter splicing in human primary RPE cells to determine the proportion of mRNAs known to act as downstream targets for SRPK1, or known to have alternative splice variants and expressed in RPE cells, that are affected by SRPK1 inhibition.

Methods Human primary RPE cells were treated with 3 novel SPHINX compounds, synthesized based on the structure of SRPK1 and SPHINX (1), followed by extraction of RNA and protein. Quantity of VEGF-A165a and VEGF-A165b protein was detected in RPE lysates and conditioned media using ELISA and WB. RT-PCR was used to examine isoform expression of MKNK2, HNRNPA2B1, TEAD1, BIM, MSTR1, FLT1, BCL2L1, ARR1, CAMK2D, and FN1. House keeping gene B2M was used to show RNA levels between samples were comparable.

Results ELISA on RPElysates showed that concentrations of VEGF-A165b were increased from 8.5-13.6 pg/mg for DMSO up to 18.6 pg/ml, 23.1 pg/mg and 24.7 pg/mg following treatment with SPHINX inhibitors. Conditioned media collected from RPEs showed a dose response reduction of VEGF-A165a compared to DMSO control, from 361.6 pg/ml down to 197 pg/ml (n=4. P<0.05 vs DMSO treatment) with SPHINX inhibitor treatment. RT-PCR showed that treatment with 5 µM SPHINX inhibitor altered alternative splicing of MKNK2, a known splicing target of SRSF1 (4), resulting in a 1.96± 0.24 (n=4. P<0.005 vs DMSO treatment) fold change to isoform 2 in RPE cells. No change in splicing was observed for other known targets of SRPK1 or mRNAs expressed in the eye including BCL2L1, ARR1, CAMK2D, RAC1, FN1 or HNRNPB1/A2. Other SRSF1 targets such as TEAD1, BIM and MST1R were not expressed in RPE cells.

Discussion These results indicate that SRPK1 inhibitors cantarget alternative splicing of specific downstream targets (e.g. VEGF and MKNK2) within the retina without affecting general alternative splicing, or alternative splicing of mRNAs previously shown to be regulated by SRSF1 phosphorylation in other cell types.

(1) Gammons MV et al. (2013). Invest Ophthalmol Vis Sci. 54: 6052–6062.

(2) Sanford JR et al. (2005) ProcNatlAcadSci 102(42): 15042–15047.

(3) Nowak DGet al. (2010) J BiolChem 285 (8): 5532-40.

(4) Anczukow O et al. (2012). Nat StructMolBiol 19(2): 220-228.