Abstract 3649: Alternative RNA splicing of U2AF2, induced by AKT3-phosphorylated IWS1, promotes tumor growth, by activating a CDCA5-pERK positive feedback loop

Abstract

Abstract A phosphoproteomics study of isogenic cell lines expressing the 3 different Akt isoforms identified 606 proteins that are phosphorylated by at least one isoform. About 30 of these proteins were involved in various steps of RNA processing. One of them, IWS1, is a transcription elongation factor, which was originally identified in the yeast Saccharomyces cerevisiae, as a protein that interacts with the histone H3/H4 chaperone Spt6 or as a suppressor of TATA binding protein (TBP) mutations that impair post-recruitment transcriptional activation. The human IWS1 is an 819aa protein, which contains a C-terminal domain that is similar to domain I of the transcription elongation factor TFIIS, and to related domains in Elongin A and the Mediator Complex subunit 26 (Med26). IWS1 was shown to be phosphorylated, primarily by Akt3, at two neighboring sites (Ser720/Thr721). To address the role of phosphorylated IWS1 in RNA processing, we performed an RNA-seq study, using human lung adenocarcinoma cell lines in which IWS1 was knocked down or replaced by its phosphorylation site mutant. This identified the splicing factor U2AF2 as a target of IWS1 phosphorylation. Specifically, phosphorylated IWS1 regulated the alternative splicing of U2AF2 and its loss resulted in U2AF2 transcripts lacking exon 2. This exon encodes part of the U2AF2 Serine-Rich (SR) Domain, required for the binding of U2AF2 with Prp19. Exploring the mechanism of this alternative splicing event revealed that the loss of phosphorylated IWS1 interferes with the recruitment of the histone H3K36 trimethyltransferase SETD2 to an Spt6/IWS1/Aly complex, which assembles on the Ser-2-phosphorylated CTD of RNA-Pol II. The absence of SETD2 recruitment to this complex impairs histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes inthe U2AF2 gene, resulting in the exclusion of exon 2 from the mature U2AF2 mRNA transcript. The loss of the U2AF2/Prp19 interaction results in the downregulation of CDCA5, a component of the cohesin complex, giving rise to genomic instability. Phosphorylation of CDCA5 by p-ERK at Ser79 and Ser209 has a major impact in the regulation of cell proliferation and cancer stem cell renewal, although does not affect its role in the cohesin complex. The effect of phosphorylated CDCA5 on cell proliferation appears to depend on the transcriptional regulation of a set of genes involved in the control of the G2/M phase of the cell cycle, including Cyclin B1 and CDK1. Overall, these data describe a novel pathway, which starts with the phosphorylation of IWS1 by AKT3 and results in the epigenetic modulation of RNA splicing and cell cycle regulation. The importance of this pathway to human cancer was confirmed by meta-analysis of pre-existing patient data, tumor xenograft models and prospective studies on human lung adenocarcinomas. Citation Format: Georgios I. Laliotis, Evangelia Chavdoula, Maria D. Paraskevopoulou, Vollter Anastas, Ioannis Vlachos, Vasiliki Tarasslia, Artemis Hatzigeorgiou, Dario Palmieri, Abdul Kaba, Marina Capece, Arturo Orlacchio, Lalit Sehgal, Vincenzo Coppola, Philip N. Tsichlis. Alternative RNA splicing of U2AF2, induced by AKT3-phosphorylated IWS1, promotes tumor growth, by activating a CDCA5-pERK positive feedback loop [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3649.