Aberrant Non-Homologous End Joining in Multiple Myeloma: A Role in Genomic Instability and As Potential Prognostic Marker.

Abstract 2932

Multiple Myeloma (MM) is a hematologic malignancy characterized by a complex combination of structural and numerical chromosomal abnormalities. However, the underlying molecular basis of the genomic instability remains largely unknown. The ability to repair DNA damages, especially double-strand breaks (DSBs), is essential to suppress genetic instability. Non-homologous end joining (NHEJ) is one of the most important mechanisms responsible for repair of these breaks. Since both impaired and aberrant NHEJ seem to be linked to genomic instability in solid as well as other hematologic tumors, we have investigated its altered function in MM. To confirm involvement of an aberrant NHEJ pathway in MM genomic instability, we measured the end joining (EJ) capacity of 6 different MM cell lines using a plasmid based assay containing both the test gene (Luciferase - LUC) measuring end joining as well as a reporter gene (Alkaline Phosphatase - SEAP) to control for transfection efficiency. MM and normal control cells were transfected with this plasmid and the LUC and SEAP activity was detected directly in the supernatant of the cells at 24 h. Increased EJ activity was observed in all the MM cell lines tested compared to peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs) from healthy donor. To confirm the role of the NHEJ pathway in this increased DNA EJ activity, nuclear extracts from 9 different MM cell lines were used to determine the DNA-binding-activity of Ku86, a key protein of this repair mechanism involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. As in EJ activity, all the MM cell lines showed an increased Ku86 binding respect to normal cells confirming the aberrant activation of the NHEJ pathway in MM cells. Interestingly, we did not observe significant differences in Ku86 level in nuclear extracts between PBMCs and MM cell lines suggesting that the difference in the Ku86 DNA-binding-activity was likely a functional and not due to disparity in the protein levels. We further investigated the link between this aberrant NHEJ activity and MM genomic instability using an immune-fluorescent based assay for DSBs. We observed an increased constitutive DNA damage in the absence of treatment with DSB-inducing agents in 5 of 6 MM cell lines compared with normal PBMCs. Most importantly, we noticed a direct correlation between the basal level of DSBs and the Ku86-binding-affinity. Furthermore, all the MM cell lines showed little or no ability to repair ionizing radiation (IR)-induced DNA damage compared to normal cells as well as no change in the Ku86-binding-affinity after stimulation suggesting that the aberrant NHEJ pathway in MM might represent a response to the constitutive endogenous DNA damage in these cells. We have also observed that 2 key NHEJ genes (Ku86 and Artemis) are overexpressed in MM compared to MGUS and normal plasma cells and their overexpression correlates with a shortened overall survival in MM suggesting that an hyper-activation of this pathway could have a potential role in MM progression and prognosis. Ongoing experiments are assessing the NHEJ activity in primary MM cells to correlate with clinical outcome. In conclusion, our data suggests that an aberrant NHEJ in the context of a constitutive endogenous DNA damage might contribute to the high frequency of chromosome abnormalities in MM cells, thus potentially playing a central role in the tumor progression and as an important prognostic marker in this disease.