IWS1 phosphorylation promotes cell proliferation and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the cell cycle-regulated U2AF2 RNA splicing.

Our previous studies have shown that IWS1 (Interacts with Spt6) is a phosphorylation target of AKT and regulates the alternative RNA splicing of FGFR-2, linking IWS1 with lung cancer tumorigenesis. To further address the role of IWS1 in alternative RNA splicing and lung cancer, we performed an RNA-seq study using lung adenocarcinoma cells in which IWS1 was knocked down or replaced by its phosphorylation site mutant. The results identified a novel splice variant of the splicing factor U2 Associated-Factor 2 (U2AF2), lacking exon 2 upon loss of phosphorylated IWS1. This exon encodes part of the U2AF65 Serine-Rich (SR) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). Here, we show that the loss of phosphorylated IWS1 hinders histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes on the U2AF2 gene in a cell-cycle specific manner. The latter results in the downregulation of cell cycle division associated 5 (CDCA5), a phosphorylation target and regulator of ERK, leading to impaired cell proliferation, G2/M phase arrest and tumor growth in mouse xenografts models, an effect more pronounced in cells harboring EGFR mutations. Analysis of human lung adenocarcinoma samples revealed robust correlations between IWS1 phosphorylation, U2AF2 RNA splicing, and Sororin/p-ERK levels, especially in EGFR, as opposed to KRAS mutant patients. More importantly, IWS1 phosphorylation and U2AF2 RNA splicing pattern are positively correlated with tumor stage and grade and defines poor survival in lung adenocarcinoma patients, harboring EGFR, and not KRAS, mutations. This work highlights the instrumental role of the AKT/p-IWS1 axis to alternative RNA splicing in governing cell cycle progression and tumorigenesis, and proposes this axis as a novel drug target in lung adenocarcinoma, by concomitantly affecting the epigenetic regulation of RNA processing and oncogenic signals.