Genomic instability is a hallmark of several types of solid and hematologic malignancies, including multiple myeloma (MM). Although structural and numerical chromosomal abnormalities are common features of MM cells, the underlying molecular basis of MM genomic instability is still largely unknown. To this aim, we have investigated the activity of non-homologous end joining (NHEJ), which represents the most important mechanism of double-strand breaks (DSBs) repair, in MM cells. First, we developed and validated a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene which measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency, in either intact cells (in vivo assay) or in cell free extracts (in vitro assay). The first one is a chemiluminescent assay which allows for direct measurement of LUC and SEAP in the supernatant of the cells 24h after electroporation with the plasmid, while the cell free extract method is a customized TaqMan® approach based on a quantitative evaluation of the plasmid rejoining. Both assays revealed a significant increase in NHEJ in all 6 MM cell lines tested compared to normal peripheral blood mononuclear cells (PB-ND) and bone marrow stromal cells (BMSC). We further confirmed the hyper-activation of the NEHJ pathway by analyzing the binding activity of ku86, a key NHEJ-related protein involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. Six out of 9 MM cell lines showed a significant increase in ku86-binding activity respect to normal cells. We also found a higher phosphorylation at Ser 2056 of DNA-PK, a ku86-partner whitch plays a key role in NHEJ. Next, we evaluated the NHEJ activity in 35 patient samples using the cell free assay. Interestingly, level of NHEJ activity divided patients into two different groups: one with an NHEJ activity similar to normal cells and the other to the MM cell lines. Preliminary correlation analysis between NHEJ activity and the clinical features of the patients indicated that the MGUS and Smoldering MM subgroup fall into the normal cluster while relapsed/refractory disease to the cell lines one. Finally, using the French (IFM) and the Arkansas (GSE2658) dataset, we demonstrate a significant association between NHEJ pathway-related gene expression and overall survival by the Globaltest analysis. We were also able to find a common NHEJ signature of 6 genes whose expression significantly correlates with patient survival in both the datasets. In conclusion, our data indicate an aberrant activation of NHEJ in MM, highlighting its role in the progression of the disease and suggesting this pathway as an important new prognostic marker in myeloma.

Non Homologous End Joining, a Marker Of Genomic Instability Is Elevated In Multiple Myeloma: A New Prognostic Factor

Rossi M;
2013-01-01

Abstract

Genomic instability is a hallmark of several types of solid and hematologic malignancies, including multiple myeloma (MM). Although structural and numerical chromosomal abnormalities are common features of MM cells, the underlying molecular basis of MM genomic instability is still largely unknown. To this aim, we have investigated the activity of non-homologous end joining (NHEJ), which represents the most important mechanism of double-strand breaks (DSBs) repair, in MM cells. First, we developed and validated a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene which measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency, in either intact cells (in vivo assay) or in cell free extracts (in vitro assay). The first one is a chemiluminescent assay which allows for direct measurement of LUC and SEAP in the supernatant of the cells 24h after electroporation with the plasmid, while the cell free extract method is a customized TaqMan® approach based on a quantitative evaluation of the plasmid rejoining. Both assays revealed a significant increase in NHEJ in all 6 MM cell lines tested compared to normal peripheral blood mononuclear cells (PB-ND) and bone marrow stromal cells (BMSC). We further confirmed the hyper-activation of the NEHJ pathway by analyzing the binding activity of ku86, a key NHEJ-related protein involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. Six out of 9 MM cell lines showed a significant increase in ku86-binding activity respect to normal cells. We also found a higher phosphorylation at Ser 2056 of DNA-PK, a ku86-partner whitch plays a key role in NHEJ. Next, we evaluated the NHEJ activity in 35 patient samples using the cell free assay. Interestingly, level of NHEJ activity divided patients into two different groups: one with an NHEJ activity similar to normal cells and the other to the MM cell lines. Preliminary correlation analysis between NHEJ activity and the clinical features of the patients indicated that the MGUS and Smoldering MM subgroup fall into the normal cluster while relapsed/refractory disease to the cell lines one. Finally, using the French (IFM) and the Arkansas (GSE2658) dataset, we demonstrate a significant association between NHEJ pathway-related gene expression and overall survival by the Globaltest analysis. We were also able to find a common NHEJ signature of 6 genes whose expression significantly correlates with patient survival in both the datasets. In conclusion, our data indicate an aberrant activation of NHEJ in MM, highlighting its role in the progression of the disease and suggesting this pathway as an important new prognostic marker in myeloma.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12317/20597
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