PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells

Abstract

Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR), a DNA double-strand break (DSB) repair pathway, are hypersensitive to PARP inhibitors (PARPi). Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE) assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR), and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold) and chromatid aberrations (2-6-fold). Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

Fig 1. Olaparib increases the SCE frequency in repair-proficient human cells.

A. Cells were exposed with BrdU and 1μM olaparib during two cell cycle periods. Representatives of two metaphase spreads harvested from untreated (left) and olaparib treated (right) primary T cells 2. Arrows, e.g. site of SCE. B. Spontaneous and olaparib-induced SCEs by cell type. The y-axis is the number of SCE per chromosome for each metaphase counted. Approximately 50 metaphases were counted for each cell type. In order to compare each cell type, the number of SCEs per metaphase was divided by chromosome number. Fold increases of SCE per chromosome by olaparib for each cell type are shown. Error bars identify the mean with SD. Asterisks designate statistical significance at P < 0.0001 using unpaired t-test. C. Multiple SCEs per chromosome are observed following olaparib. Histograms show the percentage of chromosomes categorized by number of SCEs per chromosome by cell type and exposure. D. Temporal induction of SCEs by olaparib. (a) Spontaneous SCE, vehicle treated EBV-BL cells (90 hr—BrdU). (b) Acute olaparib-induced SCE (1μM olaparib and BrdU). (c) Olaparib-exposure (1μM) followed by removal and 2 cell cycle BrdU exposure. Fold increases of SCE per chromosome versus (a) of each exposure are shown. Error bars identify the mean with SD. Asterisks designate statistical significance at P < 0.0001 using unpaired t-test.

Fig 2. Olaparib increases chromatid-type aberrations in repair-proficient human cells.

A. Representative chromatid-type aberrations in human cells include chromatid gap (a), chromatid break (b), radial chromosome (c and d) and telomere association (e). B. Olaparib-induced chromatid-type aberrations. Cells were exposed to vehicle or 1μM olaparib for 24 hrs. For each cell type, 100 metaphases were counted and the number of chromatid-type aberrations per metaphase was divided by chromosome number. Bars indicate the mean with SEM. The P-values were calculated using unpaired t-test.

Fig 3. SCE induction increases with dose and associates with inhibition of PARP activity.

Induction of SCEs occurs with increasing dose of olaparib and veliparib but not BSI-201in MCF-10A (A) and EBV-BL (B) cells. Approximately 50 metaphases were counted per cell type. Fold increases of SCE per chromosome are shown compared to vehicle treated cells. Error bars depict mean with SD. One and two asterisks designate statistical significance compared with vehicle treated cells at P < 0.0001 and P < 0.001, respectively. The P-values were calculated using unpaired t-test. Dose response curves for cell survival are depicted on the left y-axis (solid circles) with corresponding SCE frequency on the right y-axis for MCF-10A (C) and EBV-BL (D) cells with olaparib, veliparib and BSI-201. Means with SD represent 3 survival assays per cell type. The IC₅₀ for olaparib, veliparib and BSI-201 were 4.7, 69.1 and 56.0 μM for MCF-10A, and 3.7, 42.5 and 48.9 μM for EBV-transformed B cells, respectively. E. Inhibition of cellular PARP activity in EBV-BL. Cells were incubated with or without increasing concentrations of olaparib, veliparib and BSI-201 24 hr before PARP activity was measured and values were normalized using protein concentration. Bars depict mean with SD of PARP activity for each concentration (μM) (n = 3). One asterisk designate statistical significance compared with untreated cells for paired t-test at P < 0.0001.

Fig 4. BRCA1- and BRCA2-deficient mES cells are hypersensitive to olaparib and veliparib but not BSI-201.

The clonogenic survival assays of wild-type, Brca1^{-/- 236.44} and Brca2^lex1/lex2 mES cells following treatment with olaparib, veliparib and BSI-201. BRCA1- and BRCA2-deficient cells were extremely sensitive to olaparib and veliparib as compared to wild-type cells. The concentration and arrows indicate the IC₅₀ for each cell line. Error bars depict mean with SD (n = 3).

Fig 5. Potentiation of genomic instability with combined exposure of olaparib and cisplatin.

A. Dose-response curve of survival with cisplatin alone and cisplatin with 1μM olaparib of MCF-10A and primary T cell 1. In MCF-10A, cells were grown with indicated concentration of cisplatin for the first 30 hr with or without continuous exposure at 1 μM olaparib. Clonogenic survival was assessed by colony counting at 9 days. In primary T cell 1, cells were grown with indicated concentration of cisplatin with or without 1 μM olaparib for the first 24 hr. Viable cells were scored and enumerated by trypan blue exclusion at 7 days. Means with SD are shown (MCF-10A: n = 3, primary T cell 1: n = 2). B. Induction of SCEs by cisplatin and/or olaparib in MCF-10A and primary T cell 1. In MCF-10A, cells were exposed at 0.5 μM cisplatin with or without 1 μM olaparib for 30 hr. In primary T cell 1, cells were exposed at 0.5 μM cisplatin for the first 24hr with or without 1 μM olaparib for 70 hr. Fold increases of SCE per chromosome compared with no treatment or 0.5 μM cisplatin are shown for each cell type. Approximately 50 metaphases were counted for each cell. Error bars depict the mean with SD. Asterisks designate statistical significance for unpaired t-test at P < 0.0001. C. Chromatid-type aberrations following cisplatin with or without olaparib in primary T cell 1. Cells were exposed at 0.5 μM cisplatin with or without 1 μM olaparib for 24 hr. One hundred metaphases were counted for each exposure. Fold increases of chromatid-type aberrations per chromosome compared with no treatment or 0.5 μM cisplatin are shown for each cell type. The y-axis is the number of total chromatid-type aberrations per chromosome for each metaphase counted. Error bars indicate mean with SEM. The P-values were calculated using unpaired t-test.