SNRPD3 preserves balanced MYCN-driven alternate splicing and represents a therapeutic vulnerability in neuroblastoma

Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms behind this remain unknown. We found that MYCN up-regulated the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed, while depletion of SNPRD3 in the presence of overexpressed MYCN was sufficient to induce additional increases in differential splicing, particularly genes involved in the cell cycle. SNRPD3 knockdown in the presence of MYCN was accompanied with marked loss of cell viability, suggesting that SNRPD3 maintains splicing balance for MYCN, and prevents detrimental differential splicing of cell cycle genes in neuroblastoma. Further analysis revealed BIRC5 and CDK10 to be among the topmost differentially spliced cell cycle genes, both of which play a role in the G2/M phase of the cell cycle. Flow cytometry analysis revealed that depletion of SNRPD3 resulted in G2/M cell cycle arrest. Furthermore, SNRPD3 was an oncogenic co-factor for MYCN in vitro and in vivo. As such, SNRPD3 represents a therapeutic vulnerability for neuroblastoma cells in the presence of high MYCN expression. Indeed, the PRMT5 inhibitor, JNJ-64619178, that prevents SNRPD3 methylation was cytotoxic in neuroblastoma cell lines with high SNRPD3 and MYCN expression. Overall, our findings demonstrate a selective relationship exhibited between MYCN and SNRPD3 and suggest that SNRPD3 plays a vital role in MYCN-driven tumorigenesis and could serve as a novel therapeutic target. Leafcutter comparison of differential splicing in neuroblastoma cell line SHEP21N for 6 conditions (3 replicates per condition): i) SHEP21N cells + DOX + siControl (+SNRPD3-MYCN), ii) SHEP21N cells + DOX + siSNRPD3#1 (-SNRPD3-MYCN)_1, iii) SHEP21N cells + DOX + siSNRPD3#2 (-SNRPD3-MYCN)_2, iv) SHEP21N cells + DMSO + siControl (+SNRPD3+MYCN), v) SHEP21N cells + DMSO + siSNRPD3#1 (-SNRPD3+MYCN)_1, vi) SHEP21N cells + DMSO + siSNRPD3#2 (-SNRPD3+MYCN)_2