There is a strong clinical need for novel therapeutic options for pancreatic ductal adenocarcinoma (PDAC), where the 5-year overall survival remains poor at 15% with limited treatment options beyond chemotherapy (1, 2). Recent studies suggest alternative splicing drives PDAC aggressiveness and render it vulnerable to splicing inhibition, indicating that this may be a novel therapeutic strategy for this disease (3). Increased expression of PRMT5 is associated with a worse prognosis and interestingly, the more alternative splicing PRMT5 inhibition induces, the greater reduction in PDAC proliferation (4, 5). Whilst commercially available PRMT5 inhibitors have already shown safety in early phase trials, it remains unclear which of PRMT5’s plethora of biological effects is more important in PDAC (6-8). Whole genome CRISPR screen with PRMT5 inhibitor (GSK3266595) treated MiaPaca2 cell line identified four genes (SNRPD1, SNRPA, DHX15, AKAP17A) within the top 20 that enhanced GSK3266595’s growth inhibitory effect was related to spliceosome formation or initiation. Furthermore, knockout of USP39, an initiator of spliceosome assembly, was also identified as required for GSK3266595’s growth inhibitory effect. Alternative splicing analysis utilising rMATS on whole transcriptomes after 6 days of GSK3266595 treatment resulted in alternative splicing of RBM39, a regulator of pre-mRNA alternative splicing. The switch for the shorter isoform without premature stop codon may be a compensatory mechanism where combination inhibition of RBM39 (E7820 inhibitor) and PRMT5 leads to at least an additive inhibitory effect based on proliferation assay. Similarly, inhibition of AKAP17A, an accessory protein associated with protein kinase A’s role in spliceosome assembly, showed at least an increase in growth inhibition with PRMT5 inhibitor. Together, the evidence supports that alternative splicing may underpin part of GSK3266595’s biological effect. Whether differences in splicing pathway dependence can be utilised as a biomarker for GSK3266595 treatment selection needs to be further explored as they have in glioblastoma multiforme (9).