Cancer-relevant alternative RNA splicing events driven by the PRMT5-E2F1 axis in high risk neuroblastoma
Neuroblastoma (NB) is a devastating childhood cancer that remains clinically unmet. There is an urgency to develop new approaches to treat the disease. Protein arginine methyltransferase (PRMT5) is over-expressed in a wide variety of cancers and has been implicated with a key oncogenic role, achieved in part through its control of the master transcription regulator E2F1. Here, we have investigated the relevance of the interplay between PRMT5 and E2F1 in neuroblastoma and found that elevated expression occurs in poor prognosis high-risk disease. Cell lines derived from NB that recapitulate high PRMT5 and E2F1 expression exhibit increased levels of alternative RNA splicing compared to their low expression cell counterparts.  Cancer-relevant exon-skipping events in apoptotic genes were apparent in high expressing NB cells which resulted in protein isoforms with decreased apoptotic activity. Pharmacological inhibition of PRMT5 or inactivation of E2F1 activity reduced exon skipping and reinstated normal apoptotic activity. Our findings show that a cancer relevant alternative splicing programme desensitises high risk NB to apoptosis. The critical role ascribed to PRMT5 and E2F1 in attaining the alterative RNA splicing programme equally identifies PRMT5 as a potential therapeutic target for neuroblastoma disease. Overall design: To investigate the impact of NMYC, E2F1, and PRMT5 on gene expression in neuroblastoma, we performed RNA-seq and downstream gene expression analysis in CHP134 cells (high expressors of NMYC, E2F1 and PRMT5) and GIMEN cells (low expressors of NMYC, E2F1 and PRMT5) treated with either DMSO or a PRMT5 inhibitor (T1-44).  Regulation of alternative splicing was also examined using the rMATS data package.  Subsequent to this, we generated a CHP134 cell line that had the E2F1 gene knocked out by CRISPR (Cr), and performed another RNA-seq and gene expression analysis on wild-type cells, or E2F1 Cr cells treated with DMSO or T1-44