Nuclease Activity Is Associated with Genomic Instability As Well As Survival in Myeloma; Underlying Mechanisms and Significance

Genomic instability leads to acquisition of mutational changes which underlie development and progression of cancer, including development of drug resistance and poor clinical outcome. Understanding mechanisms of genomic instability is therefore necessary to develop promising strategies for prevention and treatment of disease. Homologous recombination (HR), the most precise DNA repair mechanism, has been previously described to be dysregulated in multiple myeloma (MM) mediating genomic instability. Since nuclease activity, by producing free ends of DNA, can induce DNA recombination leading to genomic rearrangements, we investigated prognostic significance of nuclease activity and nuclease gene expression in MM. We first developed a nuclease gene signature correlating with both the genomic instability and survival in myeloma patients. We used two different myeloma patient datasets (gse26863, n=246 and IFM 170 patient dataset) which had both the gene expression and SNP/CGH array-based copy number information for each patient. Genomic instability in each patient was determined by counting the total number of amplification and/or deletion events; an event was defined as a change in ≥3 and/or 5 consecutive SNPs/probes. We identified 34 nucleases whose elevated expression correlated with increased genomic instability in gse26863 dataset. Of these, the elevated expression of 21 nucleases also correlated with increased genomic instability in 170 dataset. Elevated expression of seven of these genes also correlated with poor overall survival (p=0.00005) as well as event free survival (P=0.0003) in myeloma patients (n=170). We further tested one of these nucleases (APEX2) in both the loss and gain of function studies and found that its suppression significantly reduces DNA breaks and dysregulated HR, an important activity underlying ongoing genomic rearrangements and instability in myeloma. Upregulation of APEX2 was associated with excessive DNA breaks, dysregulation of HR, acquisition of new genomic changes over time in myeloma cells.

We also investigated the prognostic significance of nucleolytic activity in cell lines and patient samples using a plasmid degradation assay in which supercoiled DNA is converted to open circular and linear forms by the MM cell lystae prepared from purified CD138+ patient MM cells or MM cell lines. The ratio of supercoiled to total DNA per lane was graphed across successive time points (0, 3, 6, 12, 24 minutes) and analyzed via nonlinear regression using PRISM (statistical software) to calculate a half-life and k-constant. The longer half life suggests lower nuclease activity in MM cells. This assay was able to differentiate MGUS and smoldering myeloma (SMM) patients with long half-life of plasmid (9499 minutes) versus newly diagnosed MM (9 minutes) in which there was variability with some patients with plasmid degradation pattern closer to SMM versus some with significantly higher activity. A large number of (N = 410) clinically annotated sample patients are currently being evaluted for both functional and clinical correlation of nuclease activity. In summary, we show correlation between nucleases activity and genomic instability with impact on survival in MM. The genes in this signature not only provide novel markers to predict clinical outcome but also potential targets for prevention/reduction of genomic evolution. Investigation of the role of each of the seven genes, separately and in combination, in the overall nucleolytic activity, genomic instability, and pathways involved in the regulation of cell cycle, DNA repair/maintenance checkpoints, apoptosis and survival is currently ongoing.