siRNA Knockdown of MMSET Disrupts Cell Cycle and Induces Apoptosis in t(4;14) Myeloma.

Multiple myeloma (MM) is characterised by a clonal expansion of plasma cells in the bone marrow. The recurrent immunoglobulin translocation t(4;14)(p16;q32) occurs in 15% of MM patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) translocation up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET (MMSET) genes. While the role of FGFR3 in myelomagenesis has been shown in various in vitro and in vivo studies, the up-regulation of MMSET at the protein level and its involvement in the pathogenesis of t(4;14) myeloma is still elusive. Moreover, about 30% of MM tumours with the t(4;14) translocation are reported to lack expression of FGFR3 due to the loss of der(14). Interestingly, the poor prognosis associated with the t(4;14) translocation in such patients lacking FGFR3 expression remains unchanged. These findings suggest that MMSET, if up-regulated at the protein level may be involved in the pathogenesis of t(4;14) myeloma and be a target for therapeutic manipulation. To assess the involvement of MMSET in the pathogenesis of t(4;14) MM we initially evaluated the expression of MMSET variants in a panel of human MM cell lines with and without the t(4;14) translocation by western blotting. Several isoforms corresponding to the expected molecular weight of MMSET II, MB4-2II, MB4-3II and REIIBP isoforms were expressed in MM cells, the REIIBP being exclusively up-regulated in t(4;14)-positive cell lines. In an attempt to determine the biological function of each MMSET isoform, western blotting was performed on nuclear and cytosol fractions isolated from t(4;14)-positive cell lines. It was found that MMSET II, MB4-2II and MB4-3II localise to the nucleus and not in the cytoplasm, but in contrast, REIIBP is found in both cellular fractions. To further determine the involvement of MMSET in the t(4;14) pathogenesis, MMSET expression was knocked down using siRNAs in t(4;14)-positive and t(4;14)-negative MM cell lines and its effect on cell proliferation, cell cycle and apoptosis was assessed by MTT, propidium iodide and annexin-V staining, respectively. It was found that the knockdown of MMSET expression significantly impairs cell proliferation (p<0.05, two-way ANOVA) and led to a reduction of t(4;14)-positive cells in the S and G₂/M phases of the cell cycle compared to t(4;14)-negative cells. In addition, the knockdown of MMSET expression induced apoptosis in t(4;14)-positive cells when compared with t(4;14)-negative cells. Lastly, in order to determine the targets of MMSET in cells carrying the t(4;14) translocation, expression array analysis on cells in which MMSET was knocked down by siRNAs was performed. It was found that several key genes involved in cell cycle control are deregulated by the knockdown of MMSET expression. Our data suggest that MMSET up-regulation may be playing a critical role in the oncogenic behaviour of MM cells carrying the t(4;14) translocation and validates MMSET as a therapeutic target in t(4;14) MM.