Published OnlineFirst April 25, 2014; DOI: 10.1158/1541-7786.MCR-13-0502 Molecular Cancer DNA Damage and Repair Research Base Excision Repair Defects Invoke Hypersensitivity to PARP Inhibition Julie K. Horton, Donna F. Stefanick, Rajendra Prasad, Natalie R. Gassman, Padmini S. Kedar, and Samuel H. Wilson Abstract PARP-1 is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activated synthesizing PAR polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase b (pol b) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, À À À À impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol b / and Xrcc1 / cells resulted in hypersensitivity to the PARP inhibitor 4-AN and reexpression of pol b or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and À À À À veliparib also exhibited hypersensitivity in both pol b / and Xrcc1 / BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency. Implications: BER deficiency represents a new therapeutic opportunity to enhance PARPi efficacy. Visual Overview: http://mcr.aacrjournals.org/content/12/8/1128/F1.large.jpg. Mol Cancer Res; 12(8); 1128–39. Ó2014 AACR. Introduction as a scaffold able to modulate and coordinate the various steps of BER. In this repair pathway, base lesions removed by The base excision repair (BER) pathway is a primary fi mechanism for repair of endogenous and exogenous base a lesion-speci c monofunctional glycosylase produce abasic lesions in DNA. PARP-1, the first discovered member of the sites that are cleaved by AP endonuclease 1. This results in a repair intermediate with a gap containing a 30-OH and 50- PARP family and an abundant nuclear protein, is particu- fl larly important for DNA damage recognition involving deoxyribose phosphate (dRP) ap at the margins. DNA pol b removes the 50-dRP flap through its lyase activity, and binding to strand breaks in DNA (1), including intermedi- fi ates of BER (2). Once bound to damaged DNA, PARP-1 conducts gap- lling DNA synthesis leaving a nicked DNA that is a substrate for DNA ligase. Both XRCC1-deficient becomes catalytically activated synthesizing PAR polymers – fi fi attached to itself and other repair factors (PARylation). As a and pol b de cient mouse broblasts are hypersensitive to the DNA base methylating agent methyl methanesulfonate result of PARP-1 auto-PARylation, BER proteins such as fi DNA polymerase b (pol b) and the XRCC1-DNA ligase (MMS), and this is linked to a repair de ciency as measured IIIa (lig-IIIa) complex are recruited more efficiently to sites by accumulation of strand breaks (3). of DNA damage. In the presence of an inhibitor of PARP catalytic activity XRCC1 is a multidomain protein with no known catalytic (PARPi), PARP-1 binds at sites of DNA damage, but auto- activity. Yet, it interacts with a number of repair proteins, PARylation is prevented. In the absence of PAR polymers, e.g., PARP-1, pol b, and lig-IIIa, and is thought to function BER enzyme recruitment is hindered, but binding of PARP- 1 to DNA is stabilized, impeding DNA repair (4). PARPi- treated wild-type cells are highly sensitive to MMS, whereas Parp-1À/À Authors' Affiliation: Laboratory of Structural Biology, NIEHS, NIH, cells demonstrate only moderate MMS hyper- Research Triangle Park, North Carolina sensitivity (5). Thus, the cellular cytotoxicity phenotype Note: Supplementary data for this article are available at Molecular Cancer associated with inhibition of the PARP-1 enzyme is not Research Online (http://mcr.aacrjournals.org/). equivalent to PARP-1 deletion (4, 5). Previous results Corresponding Author: Samuel H. Wilson, NIH, 111 T.W. Alexander Drive, suggested that the DNA-bound and inhibited PARP-1 Research Triangle Park, NC 27709. Phone: 919-541-4701; Fax: 919-541- protein is cytotoxic as a function of formation of replica- 4724; E-mail: [email protected] tion-dependent double-strand breaks (DSB; ref. 6). This doi: 10.1158/1541-7786.MCR-13-0502 type of DSB is preferentially repaired by the homologous Ó2014 American Association for Cancer Research. recombination (HR) pathway. 1128 Mol Cancer Res; 12(8) August 2014 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst April 25, 2014; DOI: 10.1158/1541-7786.MCR-13-0502 BER Deficiency and Hypersensitivity to PARPi BRCA1 and BRCA2 are components of the HR path- ascertained by Western blotting. Independently derived way, and BRCA1/2-deficient cells were shown to be SV40-transformed wild-type and pol b null cells (36.3 and hypersensitive to treatment with a PARPi (7, 8). This key 38D4) have also been described (16). Stable transfection of combination became known as a form of "synthetic 38D4 pol b null cells with wild-type pol b and its 8-kDa dRP lethality" where there is synergy between two otherwise lyase domain and selection in G418 (600 mg/mL) or puro- nonlethal events, here a PARPi resulting in DSB formation mycin (6 mg/mL), respectively, have been outlined (16). fi and a genetic de ciency resulting in loss of the pathway Cells were routinely grown at 34 C in a 10% CO2 incubator required for DSB repair. A recent model predicts that in high glucose DMEM supplemented with 10% FBS inactivation of both PARP-1 and BRCA activities at the (HyClone), glutaMAX 1, and 80 mg/mL hygromycin same time would result in repair through an error-prone (Invitrogen). þ þ À À nonhomologous end-joining mechanism (9). Because car- Xrcc1 / and Xrcc1 / p53-deficient mouse embryonic riers of germ-line heterozygous BRCA mutations are pre- fibroblasts were obtained from Dr. Robert Tebbs (17). disposed to cancer after loss of the wild-type allele, PARPi These cells were maintained in low-glucose DMEM (Invi- were rapidly considered for targeted monotherapy that trogen) supplemented with 10% FBS at 37C. A clone should not affect other repair-competent cells (10). containing the full-length open reading frame of mouse Screening for determinants of PARPi sensitivity has become XRCC1 (Invitrogen) was subcloned into the pDONR221 critical (11, 12). A high throughput siRNA screen targeting vector and then the pEF-DEST51 vector utilizing Gateway À À 98% of known DNA repair proteins implicated several HR technology (Invitrogen). Xrcc1 / cells were stably trans- proteins, but additionally identified XRCC1, involved in BER fected with the full-length wild-type XRCC1 construct (11). The most striking PARPi-induced enhancement of (WT17 cells). Other stable cell lines with mutant XRCC1 cytotoxicity in mouse cell lines is observed with alkylating proteins have been engineered. V88R XRCC1-transfected agents resulting in damage repaired by a specificBERsubpath- cells (XV2 cells) are known to have interrupted binding to way involving pol b and XRCC1 (13). With MMS and PARPi pol b (14, 18, 19). Reduced C12A XRCC1 protein trans- combination treatment in mouse fibroblasts, the effects of fectants (XRE8 cells), unable to form the C12-C20 disulfide PARPi and BER deficiency are clearly not epistatic, but instead bond and also deficient in interaction with pol b (20), have are additive (5, 14). Thus, PARPi does not target the BER been characterized previously (14). Another XRCC1 deficiency mediators studied, pol b and XRCC1, but instead mutant, L360R, was introduced by site-directed mutagen- works through a separate mechanism. esis of the pDONR221 vector and subcloned into pEF- Sensitization to MMS is consistent with increased PARP DEST51. The resulting vector, pXL, was sequence verified. binding sites in DNA in the absence of efficient BER. Transfection was conducted as described, clones were select- However, the question of PARPi hypersensitivity as a ed with blasticidin (10 mg/mL; Invitrogen), and XRCC1 function of BER deficiency has not been widely studied. expression was confirmed by Western blotting. Mycoplasma Because the BER deficiencies mediated by pol b and XRCC1 testing was performed routinely on all cell lines using a knockouts are additive with combination treatment, we MycoAlert Mycoplasma detection kit (Lonza). proposed that cells deficient in these BER factors would be hypersensitive to treatment with a PARPi. We suspected that Determination of total cellular PAR endogenous DNA damage would effectively substitute for Cellular PAR levels were quantified using the PARP in the alkylating agent used in combination treatment. Here, vivo Pharmacodynamic Assay 2nd Generation (PDA II) we determined whether BER deficiency can lead to hyper- Kit (4520-096-K; Trevigen) as described previously (21). sensitivity to 4-amino-1,8-naphthalimde (4-AN), a com- Cells were seeded in 60-mm dishes at 106 cells per dish mercially available PARPi. Results were compared with and analyzed the following day. Dishes were placed on ice those obtained with clinically used PARPi olaparib and and lysed according to the kit protocol. Cell counts were veliparib. We find that BER deficiency as a result of either verified to ensure no growth differential between cell lines. pol b deletion or XRCC1 deletion is associated with PARPi After cell lysis and DNA digestion, total protein amounts À À hypersensitivity. These results suggest that BER deficiency were determined for each sample, and 4 mgofpol b / and À À þ þ þ þ could represent a therapeutic opportunity for single-agent Xrcc1 / cell extracts and 10 mgofpol b / and Xrcc1 / PARPi therapy.