ATR Inhibitor Synergizes PARP Inhibitor Cytotoxicity in Homologous Recombination Repair Deficiency TK6 Cell Lines

Abstract

The inhibition of poly(ADP-ribose) polymerases (PARPs) and ataxia telangiectasia and Rad3-related (ATR) would be an alternative approach for cancer treatments. The aim of this study is to investigate the synergy of the different combinations of PARP inhibitors (olaparib, talazoparib, or veliparib) and ATR inhibitor AZD6738. A drug combinational synergy screen that combines olaparib, talazoparib, or veliparib with AZD6738 was performed to identify the synergistic interaction, and the combination index was calculated to verify synergy. TK6 isogenic cell lines with defects in different DNA repair genes were used as a model. Cell cycle analysis, micronucleus induction, and focus formation assays of serine-139 phosphorylation of the histone variant H2AX demonstrated that AZD6738 diminished G2/M checkpoint activation induced by PARP inhibitors and allowed DNA damage-containing cells to continue dividing, leading to greater increases in micronuclei as well as double-strand DNA breaks in mitotic cells. We also found that AZD6738 was likely to potentiate cytotoxicity of PARP inhibitors in homologous recombination repair deficiency cell lines. AZD6738 sensitized more genotypes of DNA repair-deficient cell lines to talazoparib than to olaparib and veliparib, respectively. The combinational approach of PARP and ATR inhibition to enhance response to PARP inhibitors could expand the utility of PARP inhibitors to cancer patients without BRCA1/2 mutations.

Figure 1

ATR inhibition facilitates the cell cycle progression of cells containing PARP inhibitor-induced DNA damage. The proportion of cells in G1, S, and G2/M of the cell cycle of indicated genotypes exposed to PARP inhibitor ((a) olaparib, (b) talazoparib, and (c) veliparib) and/or AZD6738. Averages and standard deviations of three independent experiments are displayed. The statistical difference of proportions of cells in G2/M phases between PARP inhibitor with and without AZD6738 treatments within the same genotype was calculated using Student's t-test. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 2

The combination of PARP and ATR inhibitors increases a number of micronuclei. Average numbers and standard deviations of micronuclei of indicated genotypes exposed to PARP inhibitor ((a) olaparib, (b) talazoparib, and (c) veliparib) and/or AZD6738 are displayed. At least three independent experiments were performed. The statistical difference of numbers of micronuclei between treatments within the same genotype was calculated using Student's t-test. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 3

The combination of PARP and ATR inhibitors increases an amount of DNA damage in mitotic cells. A number of gH2AX foci per mitotic nucleus of indicated genotypes treated with PARP inhibitor ((a) olaparib, (b) talazoparib, and (c) veliparib) and/or AZD6738 are shown. Black lines indicate medians. The statistical difference of a number of gH2AX foci per mitotic nucleus between treatments within the same genotype was calculated using the Mann-Whitney test. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 4

The combination of olaparib and AZD6738 decreases the cell survival. Percent survivals are displayed for each indicated genotype treated with olaparib and/or AZD6738. The experiments were performed in triplicate with two independent experiments. Averages and standard deviations are shown.

Figure 5

The combination of talazoparib and AZD6738 decreases the cell survival. Percent survivals are represented for each indicated genotype treated with talazoparib alone, AZD6738 alone, and the combination of talazoparib and AZD6738. The experiments were performed in triplicate with two independent experiments. Averages and standard deviations are displayed.

Figure 6

The combination of veliparib and AZD6738 decreases the cell survival. Percent survivals are exhibited for each indicated genotype exposed to veliparib and/or AZD6738. The experiments were performed in triplicate with two independent experiments. Averages and standard deviations are shown.

Figure 7

AZD6738 synergizes PARP inhibitor cytotoxicity in specific cell lines. CI values were calculated at IC₅₀ of PARP and ATR inhibitors of indicated genotypes using CompuSyn version 1.0. CI < 3 indicates strong synergism; 0.3 < CI < 0.7 indicates synergism; 0.7 < CI < 0.9 indicates slight synergism; 0.9 < CI < 1.1 indicates additive effects; CI > 1.1 indicates antagonism. Averages and standard errors of two independent experiments are displayed.