Bone Marrow Microenvironment Affects The Pathogenesis Of Multiple Myeloma Through Downregulation Of Alternative Splicing Factor Fox2 In Myeloma Cells

Alternate splicing is an important post translational change that alters specificity of gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. We have analyzed alternate splicing in myeloma using high throughput GeneChip Human Exon 1.0 ST Arrays in 170 uniformly treated patients and identified pattern of splicing as well as their impact on both overall and event free survival in myeloma. We have now further analyzed this data and identified Fox2, a RNA alternative splicing regulator, as one of the most important genes predicting clinical outcome in these patients. We observe that the expression level of Fox2 correlates with the frequency of RNA splicing and disease prognosis in MM patients. We have now further investigated the molecular role of Fox2 in myeloma. Fox2 expression was detected in all 10 MM cell lines tested at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. We next evaluated impact of IL-6 on Fox2 expression in MM1S and RPMI8226 MM cell lines and observed dose-dependent reduction in Fox2 expression. Importantly, MM cell - bone marrow stromal cells (BMSC) interaction also led to significant inhibition of Fox2 expression in MM1S and RPMI8226 cells. Similar response was also observed using BMSC supernatants. On the other hand, IGF-1 stimulation showed slight upregulation of Fox2 in MM cell lines. We have also evaluated impact of IL-6 on Fox2 and splicing using genomewide RNA-seq and confirmed the results. Fox2 was downregulated 33% in MM1S and 37% in RPMI8226 at gene expression level. To study its role in MM, we knocked down the expression of Fox2 in MM1S and RPMI8226 cell lines by using Fox2-directed siRNA. Compared to control cell lines, Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival, as measured by cell titer glo luminescent cell viability assay and annexin V and PI staining respectively. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Currently, RNA seq data following Fox2 knock down in MM cell lines is being evaluated to define the molecular mechanisms of bone marrow microenvironment-mediated Fox2-regualted alternative splicing events in MM. In summary, our results identify Fox2 as a biologically important splicing factor with essential function and potential clinical implications in multiple myeloma.