B582 Unusual Monoclonal Gammopathies Detected in the Clinical Laboratory of a General Hospital

have been described to play roles in solid tumors and hematologic malignancies; however the role of miRNAs in MM has not been yet fully described. Methods: We performed miRNA-expression-profiling of bone marrow-derived CD138 MM cells isolated from patients included in RAD001 clinical trial, compared to their normal cellular counterparts and validated data by qRT-PCR. MM cell lines (MM.1S/ RPMI8226/U266) were also studied. In vitro and in vivo functional studies were performed on miRNA-15aand -16-1-precursorstransfected-MM cells. Effect of miRNA-15a and -16-1 on signaling cascades have been evaluated by western-blot and immunofluorescence. NF-kB activity has been investigated using a DNA-binding-enzymelinked-immunosorbent-assay-based-assay. Angiogenesis has been studied in vitro and in vivo using the chorioallantoic-membrane model. Results: We identified a MM-specific miRNA signature characterized by decreased expression of miRNA-15a, -16-1 and increased expression of miRNA-222/-221/-382/-181a/-181b (P < .01). MM cell lines showed similar miRNA expression pattern to primary MM tumor cells. qRT-PCR was performed on matched samples and showed expression patterns similar to those observed in miRNA analysis. Using algorithms commonly used to predict human miRNA gene targets, predicted targets of decreased miRNAs in MM patients included pro-angiogenic cytokines/oncogenes/cell cycle regulators/ NFkB activators. Conversely, predicted target genes for increased miRNAs in MM included cell cycle inhibitors/suppressors of cytokine signaling/pro-apoptotic factors. Functional studies revealed that miRNA-15a and -16-1 regulate proliferation and growth of MM cells. Indeed, transfected cells showed decreased DNA synthesis; decreased cyclinD1/cyclinD3/cdk6/pRb protein expression; phase-G1-cell-cycle arrest; as compared to either scramble probe-transfected or nontransfected-MM cells. Moreover, transfected cells showed inhibition of NFkB pathway as shown by reduced p65-/p50-/p52-NFkB activities; downregulation of p-p65/p50/p52 nuclear protein level; upregulation of phospho-IkB in the cytoplasm; and decreased translocation of p-p65 from the cytoplasm to the nucleus. We also confirmed antiangiogenic properties of miRNA-15a and -16-1 both in vitro and in vivo. Conclusion: These data indicate that miRNAs play a pivotal role in the biology of MM; and provide the basis for the development of new miRNA-based targeted-therapies in this disease.”