Transcriptome analysis following inhibition of PRMT5 using GSK3203591 in MYCN amplified neuroblastoma
Neuroblastoma (NB) represents one of the most common paediatric solid tumours. Approximately half of NB patients have a poor prognosis, emphasising the need for improved therapies. Previously, our work demonstrated that the MYCN oncoprotein, which drives tumorigenesis in ~40% of high-risk NBs, physically interacts with protein arginine methyltransferase 5 (PRMT5) whereby genetic interference of PRMT5 led to apoptosis of MYCN amplified (MNA) neuroblastoma cells. In this study we have evaluated selective PRMT5 inhibitors GSK3203591 and GSK3326593 as targeted therapeutics for NB. We show that cell-lines with MNA show increased sensitivity to GSK3203591, undergoing MYCN-dependent growth inhibition and apoptosis. In vivo efficacy of the PRMT5 inhibitor was verified by showing increased survival in the Th-MYCN mouse model for MNA NB. Using RNA-sequencing in three MNA cell-lines, we observed altered MYCN-regulated transcriptional programs following PRMT5 inhibition. Together with differential gene expression, extensive alternative splicing was observed, converging on key regulatory pathways such as RNA processing, DNA damage response and cellular metabolism. Genes with altered expression included HNRNPA1, SRPK1, METTL3, MLX, TIMELESS, DONSON, CAD and PKM. PKM2 to PKM1 splice-switching prompted us to define metabolic changes using stable isotope labelling. Although glucose utilization was unaffected by GSK3203591, glutamine metabolism was consistently altered. Notably, GSK3203591 disrupted the MLX/Mondo nutrient sensors via intron retention of MLX. Accordingly, decreased expression of a key glutamine transporter (SLC1A5) and genes associated with glutamine metabolism (GOT1, CAD) was observed. Interestingly, GLS protein was decreased following PRMT5 inhibition despite stable transcript levels. As METTL3 intron retention increased and m6A reader YTHDF3 had an alternative 5'splice site after PRMT5 inhibition, leading to reduced METTL3 and YTHDF3 protein, we examined if GLS mRNA was m6A - methylated. Methylated RNA immunoprecipitation assays verified m6A methylation and its reduction following PRMT5 inhibition. As well as its documented effects on survival pathways such as the DDR, we conclude that PRMT5 is further required in MNA neuroblastoma to augment glutamine addiction via splicing and epitranscriptic mechanisms. Together our study rationalises PRMT5 inhibition as a targeted therapy for neuroblastoma, and supports the spliceosome as a key vulnerability of MNA NB and possibly other MYCN-dependent cancers.