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

<div><p>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.</p></div

<i>Arf</i>-null pachytene spermatocytes exhibit synaptic defects.

<p>(A–C) Staining of surface spread spermatocytes for SYCP3 marks the lateral elements of bivalents. (A) In wildtype pachytene spermatocytes, fully synapsed autosomal bivalents are observed, as judged by continuous SYCP3 staining along the axes. (B–C) Representative synaptic defects in <i>Arf</i>-null pachytene spermatocytes include (B) unsynapsed ends (arrow) and interstitial asynaptic “bubbles” (arrow heads), and (C) asynapsis of the X and Y chromosomes (arrow). Synapsed X and Y chromosomes are shown in (A, B). Synaptonemal complexes with segmental disruption of SYCP3 staining were more frequently observed (p = 0.051) in <i>Arf</i>-null cells (C, arrowheads) versus their wild-type counterparts (191 versus 53 such segments in 300 pachytene chromosomal spreads from each genotype). (D) Quantification of synaptic defects observed in wild-type and <i>Arf</i>-null pachytene spermatocytes. “Wide open” ends are illustrated in (B); less extensive asynapsis at telomeres was categorized as “slight” open ends. 100 pachytene spermatocytes from each of three mice of different genotypes yielded highly significant differences in overall defects (p = 0.0017 by Student's t-test). Error bars indicate standard deviation from the mean.</p

Decreased sperm production in <i>Arf</i>-null mice is compensated by loss of <i>Ink4a</i> or <i>p53</i>.

<p>(A, C) Testes were dissected from adult (2–6 month old) mice of the indicated genotypes and weighed as pairs. (B, D) Caudal epididymides were collected from corresponding mice, and recovered sperm were enumerated using a hemocytometer. Relative testes weights (A) and sperm counts (B) are reduced in <i>Arf</i>-null males but increased in <i>Ink4a</i>-null mice. <i>Ink4a-Arf</i> double-null and <i>p53</i>^−/−;<i> Arf</i>^−/− double null mice exhibit increased testes weights (A) and sperm counts (B). While testes weights (C) are not significantly reduced in <i>Arf</i>^Cre/Flox males, reduced sperm counts (D) mimic the <i>Arf</i> loss-of-function phenotype. [N = 20–32 mice (A, B) and 5 mice (C, D)]. Bars represent standard deviations from the mean. P values were determined using a Student's t-test (*p<0.001, **p<0.0001) and designate significant differences from the wild type genotype.</p

Levels of p53 detected in wild-type and <i>Arf</i>-null testis.

<p>Immunoblotting analysis was performed using whole testis lysates from four representative 3–4 month-old mice of each genotype. Actin was used as a loading control.</p

<i>Arf</i> deficiency leads to increased apoptosis of primary spermatocytes.

<p>(A) During spermatogenesis, germ cells move intraluminally as they differentiate, initially yielding primary meiotic spermatocytes (immunostained for Dmc1, green) that have detached from the basement membrane (represented by a dashed line). Dmc1-positive cells are visualized in a longitudinal section of a tubule from a two month-old <i>Arf</i>-null mouse. Spermatocytes within the tubular lumen are TUNEL-positive (red). DAPI (blue) marks the nuclei of all developing germ cells within the tubule. Neither DAPI-positive spermatogonia residing on the basement membrane nor more mature intraluminal cells express Dmc1 or are TUNEL-positive. (B–D) Surface spread spermatocytes from three month old wild-type (B), <i>Arf^−/−</i> (C), and <i>p53</i>^−/−;<i> Arf</i>^−/− (D) mice were immunostained for SYCP3 (green) and assayed for TUNEL (red). Although pachytene and diplotene spermatocytes from <i>Arf^−/−</i> mice were TUNEL-positive, few such cells were identified in spreads from age-matched wildtype or <i>p53^−/−</i>;<i> Arf^−/−</i> mice. (E) TUNEL assays were performed on testis sections from 2–3 month old mice of the indicated genotypes. The total numbers of TUNEL-positive cells were enumerated in 50 tubules within three different sections taken from 3 different mice of each genotype. (F) One hundred surface spread spermatocytes (n = 3 mice) were categorized into the four phases of prophase I based on SYCP3 and γ-H2AX immunostaining patterns, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002157#s4" target="_blank">Materials and Methods</a>. Errors bars represent standard deviations from the mean. ** p<0.0001 and *p<0.003 vs wild-type levels by Student's t-test.</p

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

<p><b>A.</b> Representative chromatid-type aberrations in human cells include chromatid gap (a), chromatid break (b), radial chromosome (c and d) and telomere association (e). <b>B.</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 <i>P</i>-values were calculated using unpaired t-test.</p

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

<p>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 <i>P</i> < 0.0001 and <i>P</i> < 0.001, respectively. The <i>P</i>-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. <b>E.</b> 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 <i>t</i>-test at <i>P</i> < 0.0001.</p

<i>Arf</i> deficiency provokes elevated and persistent γ-H2AX foci.

<p>(A) Testis sections from 3 month-old mice of the indicated genotypes were immunostained for γ-H2AX, as visualized here at low magnification to demonstrate overall relative intensities of staining. The brightest γ-H2AX-positive cells are primary spermatocytes. (B) Immunoblotting analysis of whole testis lysates from 3 month-old mice showing an accumulation of γ-H2AX in <i>Arf</i>-deficient testis. (C) Surface spread spermatocytes from three month old mice were immunostained for SYCP3 (red), γ-H2AX (green), and with DAPI to visualize nuclei (blue). Representative spreads from each stage of prophase I are shown for wild type (top) and <i>Arf</i>-null (bottom) strains, demonstrating persistence of autosome-associated γ-H2AX in pachytene spermatocytes from <i>Arf</i>-deficient mice. Staining of sex bodies persists throughout prophase in both genotypes. Images were captured with the same exposure times using a Zeiss Axioscope fluorescence microscope (A) or Intelligent Imaging Innovations Marianas spinning disc confocal microscope (C). Scale bars: (A) 100 µm.</p

Diminished Dmc1/Rad51 focus formation in <i>Arf</i>-deficient spermatocytes.

<p>(A, B) Surface spread spermatocytes from three month old WT (left panels) and <i>Arf</i>-null (right panels) mice were immunostained for SCP3 (A, B; green) and Dmc1 (A; red) or Rad51 (B; red). Images were captured with the same exposure time using a Marianas spinning disc confocal microscope. (C, D) Histograms showing the distribution of the number of Dmc1 (C) and Rad51 (D) foci found in wild-type (blue bars) and <i>Arf</i>-null (red bars) spermatocytes. Foci were enumerated from one hundred zygotene spermatocytes immunostained for SYCP3 and either Dmc1 (C) or Rad51 (D) using Slidebook 5.0 SDC software. Actual values of foci per cell are plotted within bin ranges to display the distribution of frequencies. (E, F) Histograms showing the distribution of average intensities of Dmc1 (C) and Rad51 (D) foci found in wild-type (blue bars) and <i>Arf</i>-null (red bars) spermatocytes analyzed in panels C and D.</p

Increased frequency of BrdU-incorporating spermatogonia in <i>Ink4a</i>-null mice.

<p>Quantification of BrdU-positive spermatogonia in five month-old wild-type, <i>Arf</i>-null, <i>Ink4</i>a-null, and doubly <i>Ink4a</i> and <i>Arf</i>-null mice was determined two hours after intraperitoneal BrdU administration. BrdU-labeled cells were scored in 100 tubules in testis sections from seven different mice. Error bars indicate standard deviations from the mean. ** p<0.0001 vs wild-type by Student's t-test.</p