Synergistic Activity of DNA Damage Response Inhibitors in Combination with Radium-223 in Prostate Cancer

Abstract

Radium-223 (²²³Ra) and Lutetium-177-labelled-PSMA-617 (¹⁷⁷Lu-PSMA) are currently the only radiopharmaceutical treatments to prolong survival for patients with metastatic-castration-resistant prostate cancer (mCRPC); however, mCRPC remains an aggressive disease. Recent clinical evidence suggests patients with mutations in DNA repair genes associated with homologous recombination have a greater clinical benefit from ²²³Ra. In this study, we aimed to determine the utility of combining DNA damage response (DDR) inhibitors to increase the therapeutic efficacy of X-rays, or ²²³Ra. Radiobiological responses were characterised by in vitro assessment of clonogenic survival, repair of double strand breaks, cell cycle distribution, and apoptosis via PARP-1 cleavage. Here, we show that DDR inhibitors increase the therapeutic efficacy of both radiation qualities examined, which is associated with greater levels of residual DNA damage. Co-treatment of ATM or PARP inhibition with ²²³Ra increased cell cycle arrest in the G2/M phase. In comparison, combined ATR inhibition and radiation qualities caused G2/M checkpoint abrogation. Additionally, greater levels of apoptosis were observed after the combination of DDR inhibitors with ²²³Ra. This study identified the ATR inhibitor as the most synergistic inhibitor for both radiation qualities, supporting further pre-clinical evaluation of DDR inhibitors in combination with ²²³Ra for the treatment of prostate cancer.

Keywords: ATM inhibition; ATR inhibition; DNA damage; PARP inhibition; X-rays; cell cycle checkpoints; prostate cancer; radium-223.

Figure 1

Survival curves for PC-3, DU145, and LNCaP human prostate cancer cell lines after exposure to different doses of X-rays (0–8 Gy) (A) or ²²³Ra (0–0.5 Gy) (B) for 24 h. After 7–10 days of culture, colonies were counted and survival fractions calculated. The data were fit to a linear quadratic model. Points represent the mean from three independent experiments and the respective standard error.

Figure 2

Clonogenic survival response for PC-3 (A,D,G), DU145 (B,E,H), and LNCaP (C,F,I) human prostate cancer cell lines to isotoxic doses of X-rays (2 Gy) or ²²³Ra (0.25 Gy) alone or in combination with AZD0156 (100 nM) (A–C), AZD6738 (100 nM) (D–F), or AZD2281 (500 nM) (G–I). After 7–10 days of culture, colonies were counted and survival fractions calculated. Each value represents the mean from three independent experiments and the respective standard error. Differences between two groups were compared by using an unpaired Student’s t-test (**** p < 0. 0001, *** p < 0. 001, ** p < 0.01, * p < 0.05, non-significant (ns)).

Figure 3

Mean 53BP1 foci per cell for PC-3 (A,D,G), DU145 (B,E,H), and LNCaP (C,F,I) human prostate cancer cell lines, corrected for background levels, was plotted at 1 h and 24 h following X-rays (2 Gy) or ²²³Ra (0.25 Gy) alone or in combination with AZD0156 (100 nM) (A–C), AZD6738 (100 nM) (D–F), or AZD2281 (500 nM) (G–I). Each value represents the mean from three independent experiments and the respective standard error. Differences between two groups were compared by using an unpaired Student’s t-test (**** p < 0. 0001, *** p < 0. 001, ** p < 0.01, * p < 0.05, non-significant (ns)).

Figure 4

Cell cycle analysis of PC-3 (A,D,G), DU145 (B,E,H), and LNCaP (C,F,I) human prostate cancer cell lines 24 h after treatment with X-rays (2 Gy) or ²²³Ra (0.25 Gy) alone or in combination with AZD0156 (100 nM) (A–C), AZD6738 (100 nM) (D–F), or AZD2281 (500 nM) (G–I). Bars represent the mean from three independent experiments and the respective standard error. Significant differences for control vs. all groups (white stars), X-rays vs. all groups (red stars), ²²³Ra vs. all groups (purple stars), and X-rays + DDR inhibitors vs. ²²³Ra vs. DDR inhibitors (green stars) are represented by (**** p < 0. 0001, *** p < 0. 001, ** p < 0.01, * p < 0.05).

Figure 5

Western blot for protein expression of full and cleaved PARP-1 in the PC-3 human prostate cancer cell line at 48 h after treatment with X-rays (2 Gy), ²²³Ra (0.25 Gy), AZD0156 (100 nM) (A), AZD6738 (100 nM) (B), or AZD2281 (500 nM) (C) alone or in combination. β-actin was used as a loading control. (D–F) The intensity ratio between Cleaved-PARP and β-actin.