Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

Abstract

Poly(ADP-ribose) polymerase (PARP) inhibitors are strikingly toxic to cells with defects in homologous recombination (HR). The mechanistic basis for these findings is incompletely understood. Here, we show that PARP inhibitor treatment induces phosphorylation of DNA-dependent protein kinase substrates and stimulates error-prone nonhomologous end joining (NHEJ) selectively in HR-deficient cells. Notably, inhibiting DNA-dependent protein kinase activity reverses the genomic instability previously reported in these cells after PARP inhibition. Moreover, disabling NHEJ by using genetic or pharmacologic approaches rescues the lethality of PARP inhibition or down-regulation in cell lines lacking BRCA2, BRCA1, or ATM. Collectively, our results not only implicate PARP1 catalytic activity in the regulation of NHEJ in HR-deficient cells, but also indicate that deregulated NHEJ plays a major role in generating the genomic instability and cytotoxicity in HR-deficient cells treated with PARP inhibitors.

Fig. 1.

PARP inhibitor synthetic lethality is independent of XRCC1 and BER. (A) Current model explaining synthetic lethality of PARP inhibition and HR deficiency. PARP inhibition is thought to induce accumulation of SSBs, which are converted to DSBs by collisions with replication machinery. The inability of HR-deficient cells to adequately repair DSBs results in genomic instability and eventual cell death. (B) Western blotting analysis of cell lysates from PEO1 and PEO4 cells. Blots were probed for BRCA2, PARP1, and Hsp90 (loading control). (C) Western blots demonstrating siRNA-mediated knockdown with luciferase (control), PARP1, or XRCC1 siRNA in PEO1 or PEO4 cells. (D) Clonogenic viability of cells from C after siRNA knockdown. After knockdown, cells were plated onto triplicate plates and allowed to form colonies. All results are reported as means of triplicate plates ± SEM and are representative of three independent experiments. (E) An alternate model of PARP inhibitor synthetic lethality centering on error-prone NHEJ. In this model, PARP1 catalytic activity regulates NHEJ activity, preventing NHEJ components from binding to sites of DNA damage or DNA ends. In the absence of HR and PARP activity, deregulated NHEJ aberrantly processes DNA and introduces chromosomal instability, leading to cell death.

Fig. 2.

DNA-PK is activated after PARP inhibitor exposure in PEO1 cells. (A) Western blots for poly(ADP-ribose) polymer (pADPr) and phosphorylation of DNA-PK substrates (DNA-PKcs autophosphorylation at Thr²⁶⁰⁹ and histone H2AX at Ser¹³⁹) in PEO1 cells after 72 h of exposure to increasing concentrations of ABT-888 (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM). Hsp90, total DNA-PKcs, and histone H1 are used as loading controls. (B) Phosphorylation of DNA-PK substrates after treatment for 72 h with diluent (0.2% DMSO, lanes 1 and 4), 500 nM DNA-PK inhibitor AZ12594248 (DNA-PKi, lanes 2 and 5), or 5 μM ATM inhibitor KU55933 (ATMi, lanes 3 and 6) alone (lanes 1–3) or in combination with 20 μM ABT-888 (lanes 4–6). (C) Quantitation of cells positive for phospho-H2AX foci in PEO1 and PEO4 cells, after treatment with DMSO, 500 nM DNA-PK inhibitor, 20 μM ABT-888 (ABT), ABT-888 and DNA-PK inhibitor, 50 μM etoposide (Etop), or 5 Gy of ionizing radiation (IR). Cells were exposed to ABT-888 and/or DNA-PK inhibitor for 72 h, etoposide for 1 h, or allowed to recover for 1 h after IR. Results are reported as mean ± SEM of three independent experiments. (D) Confocal images of PEO1 cells treated as in C. Phospho-Ser¹³⁹-H2AX is shown in green, phospho-Thr²⁶⁰⁹-DNA-PKcs is shown in red, and Hoechst 33258 is shown in blue.

Fig. 3.

Error-prone NHEJ activity is enhanced by PARP inhibitors in PEO1 cells. (A) Schematic of the in vivo NHEJ assay. Pem1-Ad2-EGFP is an EGFP-containing vector with a 2.4-kb intron (Pem1) and one exon (Ad2) inserted into the EGFP cassette. Pem1-Ad2-EGFP was cut with either HindIII or I-SceI to produce linearized substrate with compatible overhangs or incompatible inverted overhangs, respectively. Successfully recircularized plasmid will produce intact EGFP, which can be assayed via flow cytometry. Any residual uncut plasmid, caused by the insertion of the Ad2 exon within the EGFP ORF, will be EGFP-negative. A pCherry plasmid was cotransfected with substrate to correct for transfection efficiency. (B and C) Quantitation of NHEJ activity in PEO1 and PEO4 cells transfected with HindIII substrate (B) or I-SceI substrate (C) and exposed to ABT-888 for 72 h. Each data point represents the mean ± SEM from three independent experiments. Representative flow cytometry profiles are shown in Fig. S4.

Fig. 4.

PARP inhibitor-induced chromosomal derangement and genomic instability depend on DNA-PK activity. (A) Representative images of metaphase spreads from cells treated with diluent (0.2% DMSO), 500 nM DNA-PK inhibitor (DNA-PKi), 2.5 μM ABT-888, or both ABT-888 and DNA-PK inhibitor for 72 h. Chromosomal breaks are marked with arrowheads, and radial structures are marked with asterisks. (B) Quantitation of data from A showing average radial chromosomes per cell (n = 100 for each data point pooled from two separate experiments, error bars represent SEM). ^†Zero values. (C) Calculated mutagenesis frequency in BRCA2-mutant CAPAN1 cells after control treatment or exposure to ABT-888 with or without 250 nM DNA-PK inhibitor. Each bar represents the mean ± SEM of five to eight plates. This result is representative of three independent experiments.

Fig. 5.

NHEJ is a major contributor to PARP inhibitor effects in BRCA2-deficient cells. (A) Western blots showing knockdown of Ku80 in PEO1 and PEO4 cells. (B) Clonogenic survival of PEO1 and PEO4 cells from A, which were treated with the indicated ABT-888 concentration for 72 h, washed, and allowed to form colonies. (C) Western blots after treatment with siRNA targeting luciferase (control), Ku80, PARP1, or both Ku80 and PARP1. (D) Clonogenic viability of PEO1 and PEO4 cells from C. After knockdown, cells were plated onto triplicate plates and allowed to form colonies. (E) Clonogenic survival of PEO1 cells after Artemis knockdown. After treatment with the indicated siRNA, plates were treated with indicated concentration of ABT-888 for 72 h, washed, and allowed to form colonies. (Inset) Western blots showing knockdown with luciferase (control) or Artemis siRNAs in PEO1 cells. (F) Clonogenic survival of PEO1 and PEO4 cells treated for 72 h with ABT-888 in combination with diluent or 500 nM DNA-PK inhibitor (DNA-PKi). All results are reported as means of triplicate plates ± SEM, and are representative of three independent experiments.

Fig. 6.

NHEJ contributes to PARP inhibitor-induced effects in other HR-deficient contexts. (A) BRCA1-deficient HCC1937 and BRCA1-reconstituted HCC1937/BRCA1 cells were continuously exposed to ABT-888 in the presence or absence of 125 nM DNA-PK inhibitor (DNA-PKi) and assayed for clonogenic survival. (Inset) Western blots of cell lysates from HCC1937 and HCC1937/BRCA1. (B) Western blots of M059J and reconstituted M059J?PKcs lines showing the restoration of DNA-PKcs expression and the shRNA-mediated knockdown of BRCA1. (C) Clonogenic survival of shRNA–transfected M059J/M059J?PKcs lines treated with ABT-888 for 72 h. (D) Clonogenic survival of ATM-deficient GM16666 or ATM-reconstituted GM16667 fibroblasts. Cells were exposed to ABT-888 for 48 h in the presence or absence of 250 nM DNA-PK inhibitor (DNA-PKi), washed, and allowed to form colonies. (Inset) Western blots of lysates from GM16666 and GM16667 fibroblasts. Data are displayed as mean ± SEM of triplicate plates. Results are representative of three independent experiments.