Abstract
Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells, carrying abnormal karyotypes, chromosomal translocations, and innumerous DNA copy-number variations. We and others have previously shown that MM cells have constitutive DNA damage and DNA damage response (DDR), while normal plasma cells (NPCs) are negative for these DDR markers. Moreover, we recently observed that markers of replicative stress, such as p-ATR and p-CHK1 together with RPA foci, are also present in MM cells. The MYC (or c-MYC) oncogene is pervasively altered in MM. Since MYC is associated with DNA replication stress, oxidative stress, and DDR, we explored whether MYC is implicated in these pathways in MM. Indeed, by analyzing various DNA damage gene expression signatures, we found a positive correlation between MYC levels and ongoing DNA damage. We next examined whether MYC modulation could alter replicative stress markers, and induce DNA double-strand breaks. In a gain-of-function model, c-MYC was expressed in U266 MM cell line, which has low c-MYC levels and importantly shows low levels of ongoing DNA damage. In parallel, the H929 and MM.1S MM cell lines were used to knock-down c-MYC expression. Re-expression of a functional MYC-EGFP in U266 cells induced replicative stress markers, such as RAD51, RPA, and phospho-CHK1 foci, as well as increased RAD51, RPA and phospho-CHK1 protein levels. To determine whether this phenotype was linked to concomitant oxidative stress, we incubated MM cells with an antioxidant reagent N-Acetylcysteine (NAC). We observed a modest reduction in replicative markers after NAC treatment, which was more evident by MYC overexpression. Taken together, these results suggest that the replicative stress induced by MYC is, at least in part, associated with oxidative stress. Additionally, MYC-EGFP positive U266 cells also show DNA damage, evidenced by appearance of phospho-H2A.X foci (which detect DNA double strand breaks), that in turn triggers an intense DNA damage response, assessed by phospho-ATM/phospho CHK2 positivity. In contrast, all these DDR markers were downregulated by MYC silencing, prior to cell death, in MM.1S and H929 MM cell lines. Finally, we examined whether targeting the replicative stress response may represent a novel therapeutic strategy in MM cells with high expression of MYC. Specifically, we treated U266 cells transduced with MYC or control LACZ cells, as well as MM.1S and H929 transfected with a specific MYC-shRNA or their scrambled shRNA controls, with a small molecule ATR inhibitor VE-821 which prevents proper DNA repair after DNA damage. Cells overexpressing MYC were significantly more sensitive to VE-821 treatment compared to controls; conversely MYC-silenced cells were more resistant to VE-821. These results suggest the potential utility of VE-821 as a novel therapeutic agent in cells with high expression of MYC. In conclusion, our data show that MYC may exert its oncogenic activity partly through its ability to trigger DNA replication stress, leading to DNA damage and genomic instability in MM cells. Given the pervasive deregulation of MYC present in MM cells, its role in DNA replication and DNA damage may correlate with the extensive genomic rearrangements observed in MM cells. Therefore, treatment strategies targeting this Achilles' heel may improve patient outcome in MM.
Disclosures:
Hideshima: Acetylon Pharmaceuticals: Consultancy. Anderson:Acetylon, Oncopep: Scientific Founder, Scientific Founder Other; Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees.
Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression in Escherichia coli
