Role Of Base Excision Repair Associated AP Nuclease Activity In The Induction Of Homologous Recombination Repair Pathway and Survival Of MM Cells Following DNA Damage

We have previously shown that endonuclease activity is deregulated in myeloma and suppression of base excision repair (BER) associated apurinic/apyrimidinic endonuclease (APE) activity, mediated chemically or transgenically, reduces homologous recombination (HR) and genomic instability in multiple myeloma (MM). The purpose of this study was to investigate the role of BER-specific AP nucleases APE1 and APE2, separately or together, in the activation of HR pathway following exposure of MM cells to different DNA damaging agents and unravel possible mechanism/s and translational significance of this cross talk between two repair pathways in MM. We transduced MM cells with lentivirus-based shRNAs, either control (CS) or those targeting APE1, APE2, or both (APE1/2; double knockdown) and selected the transduced cells in puromycin. Knockdowns were confirmed by Western blotting and Q-PCR. Using evaluation by Q-PCR we observed that whereas APE2 was suppressed by 80% in APE2- as well as double-knockdown cells, it was upregulated by 70% in APE1 knock down cells. These data indicate that certain level of AP nuclease activity is probably required by MM cell to function and is consistent with a 25-30% reduced proliferation rate of double-knockdown cells under spontaneous condition. To study the impact of these modulations on ability of cells to activate HR-mediated repair pathway in response to DNA damage, the cells were exposed to either UV (20 J/m²) and incubated for 2 and 48 hrs or melphalan (2.5 µM) treatment for 24 hrs, and then incubation for further 1 and 24 hrs and evaluated for RAD51 and γ-H2AX foci. Following UV treatment, RAD51 foci were detected in 91%, 48%, 49%, and 28% of cells transduced with control, APE1, APE2, or both shRNAs, respectively. Similary melphalan treatment induced RAD51 foci in 76% of control shRNA transduced cells whereas only in 46%, 47%, and 27% of APE1, APE2, and APE1/2-knockdown cells. These data show that AP nuclease activity is involved in DNA damaging agent-induced activation of HR repair pathway. Impact of the suppression of AP nucleases was also assessed on cell proliferation at 48 hrs after treatment with melphalan. Viability of cells lacking APE1, APE2, and APE1/2 relative to control shRNA-transduced cells was reduced by 28%, 26%, and 43% (P<0.00005), respectively, within 48 hrs of treatment. In summary, we show that: 1) AP nuclease activity plays a critical role in the activation of HR-mediated DNA repair and survival of MM cells following DNA damage; 2) Although suppression of APE1 or APE2 alone does not significantly affect spontaneous proliferation rates, simultaneous suppression of both reduces proliferation by ∼25-30%; 3) Suppression of APE1 leads to induction of APE2, indicating that certain level of AP nuclease activity (from either APE1 or APE2) is required by MM cell to function and is consistent with the reduced proliferation rate of double-knockdown cells; 4) Simultaneous suppression of both AP nucleases impairs the activation of HR repair following DNA damage. These data combined with our previous observations conclude that AP nucleases (APE1 and APE2) play critical role in HR-mediated repair and survival of MM cells following DNA damage and are important targets to reduce genomic instability as well as to sensitize MM cells to radio/chemotherapy.