Correction: Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

[This corrects the article DOI: 10.1371/journal.pone.0155711.]

Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations

<div><p>Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis) show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR). However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi), become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi)). Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG)–a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF) 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.</p></div

The effect of vorinostat on the sensitivity of cancer cells to ABT-888 is mediated by increased phosphorylation of eIF2α.

<p>a. Vorinostat increased eIF2α phosphorylation in ABT-888 treated MDA-MB-231 cells but not in ABT-888 treated fibroblasts and normal breast cells: ABT-888 (10 μM), vorinostat (0.5 μM) and their combination were added to the cells 48 hours post-plating. The cells were harvested 48 hours later and processed for western blot analysis of changes in the ratio p-eIF2α/eIF2α. Numbers at the bottom of the autoradiograms indicate changes relative to controls of p-eIF2α/ eIF2α and of loaded proteins (Ponceau). The experiment was reproduced once with similar results. b. Salubrinal increases CD of MDA-MB-231 cells in response to ABT-888: Cells were treated with salubrinal, ABT-888 or both. Values are means CD (%) of triplicates ± SEM. The experiment was reproduced once with similar results. Differences between CD (%) of combined treatment and each of the experimental treatments or controls were significant. **<i>p</i><0.01, * <i>p</i><0.05. CI were < 0.9. c. A phosphomimetic eIF2α variant increases clonogenic death in response to ABT-888: The PARPi (10 μM) was added to the cells 18 hours following transfection with 1.5μg/2ml eIF2α S51A or eIF2α S51D. Colonies were processed for analysis 10 days post-treatment initiation. Values are means CD (%) of triplicate samples ± SEM. The experiment was reproduced twice with similar results. Differences between each of the controls and their ABT-888 treated counterparts as well as between the ABT-888 treated S51A and S51D variants were significant. SA—non-phosphorylatable S51A eIF2α, SD—phosphomimetic S51D eIF2α. d. Salubrinal increases eIF2α phosphorylation and 53BP1 expression in ABT-888 treated cells without affecting the level of BRCA1 or RAD51: ABT-888 (10 μM) and salubrinal (4.5 μM) were added to the cells 48 hours post-plating. The cells were harvested 48 hours later and processed for western blot analysis of changes in BRCA1, RAD51, 53BP1 and the ratio p-eIF2α/eIF2α. Numbers at the bottom of the autoradiograms are changes relative to controls of the level of BRCA1, RAD51, 53P1, the ratio p-eIF2α/eIF2α and the amounts of loaded proteins (Ponceau). The experiment was reproduced once with similar results. e. The level of phosphorylated eIF2α is higher and the level of total eIF2α is lower in normal fibroblasts and breast cells than in cancer cells: Cells were harvested for western blot analysis four days post-plating. The experiment was reproduced once with similar results. Numbers at the bottom of the autoradiograms indicate changes relative to control of peIF2α/eIF2α and the amount of loaded proteins (Ponceau). The experiment was reproduced once with similar results. Unabridged images of the MDA autoradiogram in panel (a) of RAD51 and 53BP1 in panel (d) and of the autoradiograms in panel (e) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155711#pone.0155711.s003" target="_blank">S3 Fig</a>.</p

Combined treatment of Vorinostat and 6-TG significantly increases the inhibition of MDA-MB-231 tumor xenograft growth relative to each agent alone.

<p>a. Effect of the drug combination on tumor development: Differences between the various treatment groups were not detected at first measurements but developed over time. Administration of drugs was initiated one day prior to the injection of the cells on day zero. The combined treatment of vorinostat (150 mg/kg) and 6-TG (0.75 mg/kg) had a significant (<i>p</i><0.03) inhibitory effect on the development of tumor growth relative to each agent alone. Values are tumor volumes in mm³ ± SEM. b. Effect of treatments on mice weight: Mice were weighed throughout the experiments. The various drugs did not lead to significant change in weight. c. Samples of mice bearing tumors on last day of the experiment.</p

The effect of vorinostat, ABT-888 and their combination on cellular level of BRCA1, RAD51 and TXNIP.

<p>Combined treatment of vorinostat and ABT-888 reduced the level of BRCA1 by ~ 30% (a), did not affect the level of RAD51 (b), increased the level of 53BP1 relative to control ABT-888 or vorinostat treated cells (c) and decreased the level of TXNIP (d). ABT-888 (10 μM), vorinostat (0.5 μM) and their combination were added to the cells 48 hours post-plating. The cells were harvested 48 hours later and processed for western blot analysis of changes in the levels of BRCA1, RAD51 and TXNIP. Numbers at the bottom of the autoradiograms indicate changes relative to controls in the level of BRCA1, RAD51, TXNIP and of loaded proteins (Ponceau). The experiment was reproduced once with similar results. Unabridged images of the autoradiograms presented in panels (a) and (b) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155711#pone.0155711.s002" target="_blank">S2 Fig</a>.</p

Combined treatment of vorinostat and 6-TG enhances clonogenic cancer cell death and increases phosphorylation of eIF2α.

<p>a. Combined treatment of vorinostat and 6-TG led to enhanced clonogenic cell death in cancer cells. Cells plated for clonogenic survival assays were treated with vorinostat, 6-TG or both. Values are means CD (%) of triplicates ± SEM. Determination of percent cell death (CD) for normal breast cells was performed by trypan blue exclusion assay. Differences between CD (%) of combined treatment and each of the treatments or controls were significant **<i>p</i><<i>0</i>.<i>01</i>, *<i>p</i><0.05. The experiments were reproduced once with similar results. CI values were < 0.9. b. Combined treatment of vorinostat and 6-TG increased phosphorylation of eIF2α: 6-TG (10 nM) and vorinostat (0.5 μM) were added to MDA-MB-231 cells 48 hours post-plating. The cells were harvested 48 hours later and processed for western blot analysis of changes in the ratio p-eIF2α/eIF2α. Numbers at the bottom of the autoradiograms indicate changes relative to controls of p-eIF2α/ eIF2α and of the loaded proteins (Ponceau). The experiment was reproduced once with similar results. An unabridged image of the autoradiogram in presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155711#pone.0155711.s008" target="_blank">S8 Fig</a>.</p

Vorinostat enhances the response of human cancer cell lines to ABT-888.

<p><b>a</b>. Clonogenic death in response to combined treatment of vorinostat and ABT-888: Cancer cell lines and fibroblasts were plated for clonogenic survival assays and treated with vorinostat, ABT-888 or both. Values are means clonogenic death (CD) (%) of triplicates ± SEM. Determination of percent cell death (CD) for normal breast cells was performed by trypan blue exclusion assay. The experiment was reproduced once with similar results. Differences between CD of combined treatment and each of the sole treatments or the controls were significant. *<i>p</i><0.05, **p<0.01. Combination indices (CI) were < 0.9. <b>b</b>. <b>ABT-888 is active within the cells</b>: MDA-MB-231 cells were incubated overnight with 10 μM ABT-888 than 15 min with 1 mM H2O2 before harvesting and processing for detection of poly (ADP-ribose) (PADPR) by western blot.</p