Elevated APEX1 Endonuclease Is Associated with Increased DNA Breaks and Instability in Myeloma

Abstract

Abstract 1805Exposure to DNA-damaging agents of both endogenous and exogenous origin, including ultraviolet (UV) radiation, give rise to apurinic/apyrimidinic (AP) or abasic sites in genome, probably the most common mutagenic DNA lesions. APEX1, the key protein involved in the repair of AP sites through base excision repair, plays an important role in the maintenance of genomic integrity. We have observed that APEX1 is overexpressed in multiple myeloma (MM) cell lines and primary cells. To evaluate its functional role MM cells were transduced with control (CS) or APEX1-specific lentivirus based shRNAs, targeting two different regions of the gene. Both shRNAs mediated >70% suppression of APEX1 expression. Control and APEX1-Knock out (KO) cells were treated with UV (20 J/m2), cultured for 24 hrs and evaluated for both the cell viability and phosphorylated-H2AX (p-H2AX), a marker for DNA breaks. Although cell viability did not change, the amount of p-H2AX was reduced in APEX1-KO cells, indicating that elevated APEX1 is associated with induction of DNA breaks in MM cells. To directly evaluate DNA breaks, transduced myeloma cells were exposed to UV (20 J/m2), cultured for a short period, and evaluated by comet assay, a sensitive gel-based technique for detection and assessment of DNA breaks in individual cells. The fraction of cells with DNA breaks and the intensity and size distribution of comets indicating the number of DNA breaks, were evaluated. In cells transduced with control shRNA, very large, large, medium and small comets were observed in 24%, 18%, 23% and 23% of cells, respectively. However in APEX1-KO MM cells, very large and large comets were not observed at all, comets of medium size were observed in 13% and small comets observed in 40% of the cells. A large fraction (47%) of APEX1-KO cells did not have any comets, indicating minimal or no DNA breaks even after UV exposure, under the experimental conditions used. These data show that the suppression of APEX1 significantly reduces the acquisition DNA breaks in MM cells following exposure to UV. We also evaluated the impact of reduced APEX1 levels on DNA integrity in MM cells. Control (non targeting shRNA transduced) and APEX1-KO cells were transfected with a plasmid, encoding secretory Gaussia luciferase (GLuc). Cells were plated at equal density in triplicate dishes, and beginning at 8 hrs, GLuc activity was measured in the supernatants at 12-hr intervals. In preliminary experiments we have found that plasmid DNA is more stable in APE-KO, compared to control cells, indicating that suppression of APEX1 in myeloma cells may stabilize DNA. We have also observed that chemical inhibition of endonuclease suppresses acquistionof new genomic change sin MM. We are currently evaluating the impact of APEX1 KO on acquisition of genomewide changes in myeloma cells over time. In summary, our data shows that elevated APEX1 endonuclease makes myeloma cells vulnerable to acquire DNA breaks following exposure to intrinsic or extrinsic DNA damaging agents. APEX-1 thus may be an important target to further understandgenomic instability in MM. Disclosures:No relevant conflicts of interest to declare.