Alternate Splicing Factor Fox2 Affects Growth and Survival of Multiple Myeloma Cells

Abstract 1810

Alternative splicing of pre-mRNA plays a critical role in number of cellular processes including cell growth and survival. We have previously demonstrated significant frequency of alternate splicing in multiple myeloma (MM) that points to the need for evaluation of not only the expression level of genes but also post translational modifications. Our data utilizing exon array profile from 170 uniformly treated newly diagnosed patients with MM confirms clinical relevance of splicing as demonstrated by impact of level and extent of alternate splicing on both progression free and overall survival. We have now further studied the molecular mechanisms that may be involved in differential splicing in myeloma. We have evaluated the expression data from 170 uniformly treated newly diagnosed patients with MM and identified Fox-2, an RNA binding protein that is thought to be a key regulator of alternative exon splicing as one of the most important gene predicting clinical outcome in MM. We have now investigated the molecular effects of Fox2 and its role on myeloma cell growth and survival. Using Fox-2-directed shRNA lentivirus, we knocked-out (KO) the expression of Fox2 in MM1S and RPMI8226 cell lines. Real-time PCR and western blot analysis confirmed KO of over 90% of Fox2 expression in both cell lines. We observed significant inhibition of proliferation of both MM1S and RPMI8226 cell lines in Fox-2- KO compared to control cell lines. Using annexin V and PI staining and flow cytometric analysis we also observed induction of apoptosis in Fox-2-KO-MM1S and RPMI8226 cells. The apoptosis was Caspase 3 and caspase 8-mediated in the Fox-2-KO. A similar effect of Fox-2-KO was observed on cell survival and proliferation in Hela and 293 cells. Fox-2 also plays a crucial role in the maintenance of cytoskeleton of MM cells. We observed that actin was significantly downregulated in both MM1S and RPMI8226 knockdown cell lines compare to controls, whereas GAPDH expressions are equal in all conditions. It's been recognized that actin derangement is associated with cell apoptosis. The RNA binding specificity of Fox-2 in MM cells by CLIP-seq and changes in alternate splicing in Fox-2-KO by exon array is underway to understand the molecular events mediated by this transcriptome modifier affecting MM pathobiology. These results identify Fox-2 as a biologically important intermediate with essential function in myeloma with potential biological and clinical implications.