MMSET Stimulates Myeloma Cell Growth Through MicroRNA-Mediated Modulation of c-MYC

Abstract 469

In multiple myeloma recurrent chromosomal translocations lead to the overexpression of oncogenes such as MAF, MAFB and MMSET. An underlying paradox of MM is the continued proliferation and self-renewal of a highly differentiated cell. Plasma cell development requires the BLIMP1 transcription factor, which stimulates terminal B cell differentiation and represses expression of c-MYC, a master regulator of cell growth. By contrast, malignant plasma cells express c-MYC and proliferate while maintaining the specialized machinery required for immunoglobulin production. MMSET gene is overexpressed in about 15% of cases of MM due to chromosomal translocation (4;14). In most cases upregulation of MMSET is accompanied by the overexpresson of FGFR3. Overexpression of FGFR3 would be expected to increase MAP kinase signaling and c-MYC expression. However, in 30% of cases of t(4;14) myeloma, only MMSET is overexpressed. Hence we hypothesized that MMSET might also stimulate c-MYC expression. We showed that MMSET is a histone methyltransferase that has promiscuous activity in vitro. In vivo, MMSET overexpression was correlated with elevated dimethylation of H3K36 and depressed trimethylation of H3K27 levels, altered chromatin structure, gene expression profiles, and cell growth. However, the exact genetic targets underlying the MMSET activity are not well understood. We found that MMSET overexpression was associated with increased c-MYC protein but not mRNA expression. Depletion of MMSET from myeloma cells was associated with decreased levels of c-MYC protein but not mRNA and no change in the half-life of the MYC proteins. This suggested that MMSET controlled MYC at the post-transcriptional level by preventing its translation, an effect reminiscent of the action of microRNAs. miRNA profiling of t(4;14) myeloma cells before and after shRNA-mediated depletion of MMSET showed that MMSET repressed the expression of many miRNAs including miR-126*, which was predicted to target c-MYC. We therefore hypothesized that by repressing miR126* expression MMSET could stimulate c-MYC protein expression. Accordingly, miR126* but not a point mutant of mir126* inhibited translation of a construct fusing the 3' untranslated region of c-MYC to luciferase. Furthermore a point mutation within the 3'UTR of c-MYC abrogated the effect of miR126* overexpression on the reporter. Overexpression of miR126* in t(4;14) myeloma cells suppressed c-MYC expression and cell growth. Furthermore, growth suppression and inhibition of c-MYC expression in myeloma cells after MMSET knock-down was partially blocked by an antagomir directed against mir126*. Importantly, growth suppression mediated by MMSET knockdown was reversed by expression of exogenous miRNA-resistant form of c-MYC. MMSET was recruited to the miR126* promoter along with KAP1 a partner protein identified by affinity chromatography and mass spectroscopic analysis. KAP1 mediated repression has been associated with increased H3K27 and H3K9 methylation at promoter sites and accordingly on the miR126/126* promoter, MMSET expression was associated with MMSET and KAP1 recruitment to the promoter, increased H3K9 and H3K27 methylation and decreased histone acetylation. KAP1 depletion in myeloma cells was associated with increased expression of mir126*, decreased c-MYC levels and decreased myeloma growth. Lastly to model the effects of an MMSET inhibitor in combination with HDAC inhibitors cells were subjected to trichostatin A, MMSET depletion or both. HDAC inhibitor treatment on its own elevated mir126* levels and decreased c-MYC expression but the combination of a sub-optimal dose of the HDAC inhibitor combined with MMSET depletion led to complete silencing of c-MYC expression and increased cell death. Collectively, these data suggest a new mechanism of oncogenesis by MMSET in MM, and emphasize the key role of c-MYC overexpression in this disease.