Abstract
Background/Aim: To assess poly (ADP-ribose) polymerase (PARP) inhibitor MK-4827 together with radiation for the treatment of neuroblastoma. Materials and Methods: Clonogenic survival assays were used to assess MK-4827, radiation and combination thereof in four neuroblastoma cell lines. In vivo efficacy was tested in a murine xenograft model of metastatic neuroblastoma. In vivo targeted inhibition and biological effects included measurement of cleaved caspase-3, γ-H2AX, and Ki 67 by immunohistochemistry (IHC) and poly-ADP-ribose by Enzyme-Linked Immunosorbent Assay. Results: Treatment of neuroblastoma cell lines reduced clonogenicity and resulted in additive effects with radiation. In vivo treatment with MK-4827 and radiation prolonged survival (p<0.01) compared to single modalities. In vivo superiority of MK-4827 plus radiation was further documented by significant elevations of cleaved caspase-3 and γ-H2AX in tumors from the combination group compared to single modality cohorts. Conclusion: Combination of MK-4827 and radiation might provide effective therapy for children with high-risk neuroblastoma.
Neuroblastoma is the most common extracranial tumor of childhood, with approximately 650 new cases diagnosed each year in the United States. Approximately 50% of patients present with high-risk disease, and show de novo resistance or relapse after initially responding to treatment. The prognosis for this subset of patients is poor, with survival rates of approximately 20% (1). Hence, there is a compelling need for novel treatment approaches to improve clinical outcomes for patients with metastatic neuroblastoma.
Currently, radiotherapy is used after surgical resection of the primary tumor to prevent local relapse, and for sites of residual metastatic disease after chemotherapy. Radiation causes single-(SSB) and double-strand breaks (DSB) in DNA that are usually repaired by the base excision repair pathway (BER), non-homologous end-joining (NHEJ), or homologous recombination pathway (HR) (2). In response to DNA damage, poly (ADP-ribose) polymerase (PARP)-1, a nuclear enzyme and most abundant member of the PARP family, and PARP-2 rapidly associate with DNA breaks, increasing enzymatic activity by 10- to 500-fold (3). Both histones and PARP are ribosylated in the presence of NAD+, leading to the formation of negatively charged poly-ADP-ribose (PAR) polymer. PARP facilitates repair of radiation-induced DSBs and therefore PARP inhibitors act as radiosensitizing agents (4, 5). PARP expression is higher in tumor cells than in normal tissues, allowing PARP inhibitors to selectively promote cytotoxic effects in tumor cells, thus increasing the therapeutic index (6). We evaluated the PARP-1 and -2 inhibitor MK-4827 in combination with radiation in a model of metastatic neuroblastoma. We hypothesized that combining MK-4827 with radiation would provide an effective treatment approach for high-risk neuroblastoma. We performed in vitro and in vivo studies to test this hypothesis.
Materials and Methods
Clonogenic survival assays. Cell lines SH-SY-5Y, Kelly, NB1691luc and Tet 21 were genotyped prior to their use. SH-SY-5Y, Kelly and Tet 21 were a generous gift from Dr. Weiss, San Francisco, CA. NB1691luc were a generous gift from Dr. Davidoff, Memphis, TN. Clonogenic survival assays were carried out as described previously and clinical grade MK-4827 (Merck, Whitehouse Station, NJ, USA) was used for all experiments (7). Doses of irradiation between 0 and 8 Gy were given three hours after MK-4827 administration. MK-4827 was administered at 13 to 38 nM, depending on the IC50 of each specific cell line; controls were given an equal amount of DMSO. Surviving fractions were normalized to the plating efficiency of each cell line as previously described (8). The data presented are the mean±standard deviation (SD) and cells were plated in quadruplicates.
Western blot analysis. Western blot analysis was carried out as described previously (7). Anti-PAR polyclonal antibody (BD Pharmingen, San Diego, CA, USA) was used as a primary antibody and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody as a secondary antibody. The blot was re-probed for β-actin (1:1000) as a loading control.
Immunofluorescence assay for DNA repair marker γ-H2AX. NB1691luc cells were treated at 3, 6, and 24 hours prior to fixation with 2 Gy of radiation and/or 600 nM clinical grade MK-4827. For the radiation plus MK-4827 combination arm, cells were first treated with MK-4827, followed three hours later by radiation, and then harvested, fixed and permeabilized at the indicated time points after irradiation. Cells were incubated with anti-phospho-histone H2AX antibody (Millipore, Billerica, MA, USA) overnight at 4°C followed by incubation with secondary antibody Alexa-Fluor 488-labeled goat anti-mouse IgG antibody (Invitrogen, Grand Island, NY, USA). Nuclear staining was performed with 1.5 μM propidium iodide solution (Invitrogen). Analyses were performed by an INcell 1000 analyzer (GE Healthcare, Madison, WI, USA) examining five random regions per well at ×10 magnification. Assays were carried out at each time point in triplicate. Student's t-test was used to assess statistically significant differences between treatment groups.
Metastatic neuroblastoma in vivo model and treatment. All procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (UCSF). We used an established model of NB1691luc metastatic neuroblastoma in which disease in lymph nodes and liver develop in 100% of mice (7, 9). Forty 5-6 week old female athymic nude Mice (Simonsen Laboratories, Gilroy, CA) were randomized into one of four groups: Ora-Plus (Paddock Laboratories, Minneapolis, MN, USA) carrier control; MK-4827 alone (50 mg/kg by oral gavage for a total of 10 days, Monday-Friday); radiation alone (0.5 Gy 5 daily fractions administered Monday-Friday for a total dose of 2.5 Gy); or MK-4827 plus radiation on the same schedule as the single modality treatment arms. In the combination group, 0.5 Gy of radiation followed one hour after MK-4827 administration for a total of 2.5 Gy. Treatment started seven days after tail vein injection of 6×106 NB1691luc cells. Each treatment group contained 10 mice. Tumor volume was monitored as outlined previously (7). The Kaplan–Meier estimator was used to generate survival curves, with differences between survival curves calculated using a log-rank test.
PARP pharmacodynamic assay. The HT PARP in vivo pharmacodynamic assay II (Trevigen, Gaithersburg, MD, USA) was performed to quantify PAR in the tumor xenograft samples following the manufacturer's guidelines. Mice with metastatic neuroblastoma were treated with 50 mg/kg of MK-4827 and/or 0.5 Gy of radiation one hour after drug treatment, or with control agent Ora-Plus. Tumors were harvested 24 hours after radiation treatment, and flash frozen in liquid nitrogen to stabilize PAR levels. The assay was carried out in triplicates.
Immunohistochemistry of tumor samples. Tumor samples (multiple liver metastases per mouse) were analyzed from mice treated with 50 mg/kg of MK-4827, 0.5 Gy radiation, 50 mg/kg MK-4827 followed by 0.5 Gy one hour after MK-4827 was given, or Ora-Plus as control. Liver metastases were harvested 24 hours after final treatment, fixed in 4% formalin overnight and processed through ethanolic dehydration series for paraffin embedding. Five micrometer paraffin sections were stained using the following antibodies: anti-γ-H2AX (Cell Signaling, Boston, MA, USA); anti-cleaved caspase-3 (Cell Signaling), and anti-Ki-67 (Dako, Carpinteria, CA, USA); detected with 3,3’-diaminobenzidine tetrahydrochloride (DAB) substrate (Vector Labs, Burlingame, CA, USA), and counterstained with hematoxylin. Image-based quantification was performed and normalized to that of the control group. Data represent measurements from all metastases present in the section (four in the control group, two in the radiation group, three in the MK-4827 group, and five in the combination treatment group). Up to ten images of areas with the highest numbers of positive cells were taken per tumor (×20; Leica, DMLS microscope, Buffalo Grove, IL, USA) and both positively and negatively stained cells were quantified. Percentages of positive cells were averaged per tumor, and a grand mean of all tumors was normalized to the control (set at 100%). To assess the significance of differences between treatment groups, we first used the Kruskal-Wallis test followed by a Wilcoxon rank sum test for pairwise comparisons.
Results
MK-4827 enhances radiation-induced cytotoxicity in neuroblastoma cells. We first assessed the antiproliferative effects of MK-4827 alone and determined that MK-4827 caused a dose-dependent decrease in clonogenicity of SH-SY-5Y, Kelly, NB1691luc and Tet 21 (without and without doxycycline) cells. The half maximal inhibitory concentration (IC50) ranged from 13 to 38 nM (data not shown) and the IC50 for each cell line was used for the combination experiments with radiation (Figure 1). Figure 1 demonstrates at least additive cytotoxicity produced by combinations of MK-4827 and radiation, independent of V-myc myelocytomatosis viral related gene, neuroblastoma derived (MYCN) amplification status.
It is conceivable that higher PARP expression renders cells more sensitive to PARP inhibitors and that PARP expression levels differ between MYCN amplified and MYCN non-amplified tumors. We therefore interrogated a published gene expression data set and analyzed expression of PARP-1 and PARP-2 (Gene Expression Omnibus 3960; GSE3960). We found a significant difference in the expression of PARP-1 between MYCN amplified and non-amplified tumors (two-tailed Student's t-test p<0.002) but no significant difference was seen for PARP-2 (Figure 2).
MK-4827 reduces PAR expression and PARP activity in neuroblastoma cells. When PARP is activated and associates with SSB, it synthesizes PAR polymers, which in turn act as molecular signals to recruit DNA-repair enzymes to mend damaged DNA. As shown in Figure 3, treatment of NB1691luc and Kelly cells with increasing doses of radiation led to increased poly-ADP ribosylation levels, as expected. Treatment with MK-4827 or a combination of MK-4827 and radiation reduced PAR protein expression in Kelly as well as NB1691luc cells, an effect that was present three hours after treatment and lasted up to 24 hours after treatment. PAR polymers appear as a smear due to the different sizes of PAR polymers.
Clonogenic survival assays of neuroblastoma cell lines treated with MK-4827 and radiation. Surviving fractions were normalized to plating efficiency for each cell line and MK-4827 doses were the half maximal inhibitory concentration (IC50) values for each cell line. ▪ Radiation; ▴ MK-4827 plus radiation. All experiments were performed in quadruplicates. Surviving fractions are shown on a log scale as mean values and bars represent the standard error of the mean; where error bars are not visualized, they are smaller than the size of the symbol.
PARP inhibition with MK-4827 increases γ-H2AX expression in neuroblastoma cells. DNA damage caused by ionizing radiation induces rapid phosphorylation of γ-H2AX. Prolonged higher levels of γ-H2AX suggest that DNA repair mechanisms are impaired. To explore the effects of MK-4827 and radiation on DNA repair, we quantified levels of γ-H2AX in NB1691luc cells. Figure 4 demonstrates that combining MK-4827 with radiation led to increased expression levels of γ-H2AX in NB1691luc compared to cells treated with radiation or MK-4827 alone. The levels of γ-H2AX were significantly elevated 24 hours after treatment in cells treated with MK-4827 plus radiation compared to control cells or cells treated with radiation or MK-4927 alone (Student's t-test p<0.001). In these cell lines, we also assessed RAD51, another key player in the HR repair pathway but did not find any differences in RAD51 expression among the different treatment arms (data not shown).
Radiosensitization by MK-4827 in an in vivo model of metastatic neuroblastoma. To establish the effects of MK-4827 and radiation in vivo, we used a model of metastatic neuroblastoma (7). As shown in Figure 5A, combination of MK-4827 and radiation significantly prolonged survival compared to either treatment modality alone. To document that MK-4827 inhibited PARP activity in vivo, we evaluated levels of PAR in tumors removed from NB1691luc-bearing mice following treatment. Both the MK-4827 and the combination group had approximately 200,000 pg/ml PAR, while the radiation group expressed approximately 450,000 pg/ml PAR (Figure 5B). These results indicate that MK-4827 at the dose used in the in vivo study, inhibited PARP activity in vivo and resulted in the expected biochemical outcome.
Expression analysis of poly (ADP-ribose) polymerase (PARP)-1 and poly (ADP-ribose) polymerase (PARP)-2 in V-myc myelocytomatosis viral related gene, neuroblastoma derived (MYCN) amplified and non-amplified tumors. Expression levels of PARP-1 and PARP-2 were analyzed in a published gene expression data set (Gene Expression Omnibus 3960; GSE3960). The analysis started with raw data (CEL files) and applied pre-processing procedure with Robust Multiarray Average. Boxplot graphs for PARP-1 and PARP-2 expression in fetal brain (n=1), MYCN amplified (n=20) and MYCN non-amplified (n=81) neuroblastoma tumors are shown. Y scale is a log2 transformation of the expression measured and each unit corresponds to a two-fold change in expression. A significant difference in expression levels for PARP-1 expression (two-tailed t-test: p<0.002) but not for PARP-2 (two-tailed t-test: p=0.159) was found.
Our in vitro studies demonstrated that MK-4827 plus radiation increased γ-H2AX levels in neuroblastoma cells (Figure 4). As shown in Figure 6, our in vivo results parallel our in vitro findings, demonstrating significant increases in γ-H2AX levels in tumors from mice treated with MK-4827 plus radiation compared to those treated with MK-4827 (Wilcoxon rank sum test p=0.008) or radiation (Wilcoxon rank sum test p=0.03) alone.
To further elucidate the mechanisms of cell death in our in vivo model, we performed immunohistochemical staining for cleaved caspase-3 (CC3), a marker of apoptosis. CC3 was significantly increased in tumors from the combination group compared to the MK-4827 (Wilcoxon rank sum p=0.0078) or radiation (Wilcoxon rank sum p=0.0156) treatment arms (see Figure 6C). In addition, Ki 67, a marker of proliferation, was significantly reduced in tumors following treatment with MK-4827 plus radiation compared to treatment with MK-4827 (Wilcoxon rank sum p=0.0010) or radiation (Wilcoxon Rank Sum p=0.0026) alone.
Discussion
Clinical trials are currently testing PARP inhibitors for the treatment of adult tumors; however, this class of anti cancer agents has been slow to enter the clinic for childhood cancer (10, 11). The favorable toxicity profile of PARP inhibitors and their radiosensitizing effects render them ideal candidates for combination with external beam radiation for a variety of childhood cancer type, or with a targeted radiopharmaceutical, such as 131I-Metaiodobenzylguanidine (131I-MIBG), for neuroblastoma. In this study, we investigated the radiosensitizing effects of the PARP-1/2 inhibitor MK-4827 in vitro and in a murine xenograft model of metastatic neuroblastoma. In vitro, we demonstrated that combining MK-4827 with radiation increased the efficacy of radiotherapy in a variety of neuroblastoma cell lines independent of their MYCN status. While both MK-4827 and radiation had some anti proliferative effects individually, the combination regimen displayed a dose-dependent additive effect. To evaluate any potential correlations between MYCN status and response to a PARP1/2 inhibitor, we assessed PARP-1 and PARP-2 expression levels in a published gene expression data set from 20 MYCN amplified and 81 MYCN non-amplified tumors (Gene Expression Omnibus 3960). We found a significant difference in PARP-1 but not PARP-2 expression between MYCN amplified and non-amplified tumors. Additional studies are required to assess whether and how MYCN status affects response to PARP inhibition. Prior studies have shown that inhibition of PARP enhanced the activity of the combination of temozolomide with topotecan in neuroblastoma cells, but to our knowledge the present is the first report of combinations of external beam radiation and PARP inhibition in in vitro and in vivo models of neuroblastoma (12). Our data are consistent with previous reports of the radio sensitizing effects of PARP inhibitors in other tumor models and support the use of MK-4827 in combination with radiation for the treatment of neuroblastoma (13, 14).
Western blot analysis of poly-ADP-ribose (PAR) in Kelly and NB1691luc cells following treatment with radiation, MK-4827, or combination of MK-4827 plus radiation. NB1691luc and Kelly cells were treated with increasing radiation doses, and 600 nM of MK-4827. In samples undergoing combined treatment, radiation (RT) was given 30 minutes after MK-4827 administration. PAR levels were determined 3 and 24 hours after radiation. β-Actin is shown as loading control.
Prior studies have shown that PARP-1 and PARP-2 play roles in a variety of DNA repair pathways such as BER, NHEJ and HR repair (15-18). To assess the effects of MK-4827 on DNA repair in neuroblastoma, we assessed γ-H2AX, a marker of DNA DSBs. Our in vitro experiments demonstrated that MK-4827 plus radiation combinations significantly increased γ-H2AX expression and our in vivo results paralleled these in vitro findings and documented augmented γ-H2AX expression after treatment with MK-4827 plus radiation compared to single modality cohorts.
Prior reports have indicated that PARP-1 inhibition down-regulates RAD51, a key factor in the HR repair pathway (18). Contrary to published reports, we did not find a change in RAD51 expression in two independent neuroblastoma cell lines treated with radiation or a combination of radiation plus MK-4827 (data not shown).
γ-H2AX expression after treatment of NB1691luc cells with radiation, MK-4827 or combination of MK-4827 plus radiation. Immunofluorescent quantitation of γ-H2AX levels 3, 6, or 24 hours after treatment with MK-4827 (600 nM), radiation (RT; 2 Gy), or MK-4827 followed three hours later by RT. Average immunofluorescence is shown on the y-axis. Data are presented as mean values of triplicate samples and standard deviation. Student's t-test was used to assess statistical significance between γ-H2AX levels at 24 hours in cells treated with combination of MK-4827 plus radiation compared to radiation (p<0.001) or MK-4827 alone (p<0.001).
In vivo efficacy and target inhibition of combined MK-4827and radiation (RT) in metastatic neuroblastoma. A: In vivo NB1691luc metastases were treated with MK-4827 (50 mg/kg) every day for 10 days; RT (0.5 Gy) every day for 5 days (2.5 Gy total dose); or MK-4827 (50 mg/kg) followed by RT (0.5 Gy delivered one hour after MK-4827); control mice were treated with Ora-Plus only. Kaplan–Meier method was used to generate survival curves, and survival differences between cohorts were calculated using a log-rank test. B: In vivo poly-ADP-ribose (PAR) levels in tumors 24 hours after treatment with MK-4827 (50 mg/kg), RT (0.5 Gy) or combination therapy [0.5 Gy delivered one hour after MK-4827 (50 mg/kg)]; for each condition tumor samples n=2. The mean and standard deviation for one tumor sample are depicted.
In vivo, the combination of MK-4827 and radiation improved survival of mice in a model of metastatic neuroblastoma compared to either single treatment modality. In this in vivo model, we were able to assess mechanisms of cooperative anti-neoplastic activity, mode of cell death, and pharmacodynamic effects. The mechanism of combined MK-4827 and radiation activity included augmented inhibition of DNA repair; the mode of cell death involved apoptotic cell death based on increased CC3 levels, and pharmacodynamic results documented significant down-regulation of PAR in tumors from mice treated with MK-4827 (with or without radiation).
The critical role of radiation in the treatment of high-risk neuroblastoma is well established. In this study, we provide preclinical evidence to support introduction of the PARP-1/2 inhibitor MK-4827 in combination with radiation as therapy for patients with metastatic neuroblastoma. Radiation therapy is one of the mainstays of advanced neuroblastoma treatment, and combining a radio sensitizing agent such as MK-4827 with radiation should increase the efficacy of radiotherapy and potentially improve long-term clinical outcomes.
In vivo immunohistochemical analyses of γ-H2AX, cleaved caspase-3 (CC3) and Ki 67 in human metastatic neuroblastoma xenografts. A: Representative stains of NB1691luc liver metastases resected 24 hours after MK-4827 (50 mg/kg), RT (0.5 Gy), or MK-4827 (50 mg/kg) followed by RT (0.5 Gy). Arrows indicate positively stained cells. Image-based quantification of γ-H2AX (B), CC3 (C), and Ki 67 (D) positive tumor cells that were averaged for each tumor and treatment group and normalized to the control (set at 100%). Error bars represent the standard error. Treatment group differences were assessed by Kruskal-Wallis test followed by Wilcoxon rank sum test pairwise comparisons. γH2AX: Combination therapy versus control (p<0.0001), RT (p=0.0353), and MK-4827 (p=0.0008). CC3: Combination therapy versus control (p=0.0078), RT (p=0.0156); and MK-4827 (p=0.0078). Ki 67: Combination therapy versus control (p=0.0156), RT (p=0.0026), and MK-4827 (p=0.0010).
Acknowledgements
This research was supported in part by the National Institute of Neurological Disorders and Stroke grant K12NS001692 (SM), HHMI (SB), NIH Brain Tumor SPORE grant P50 CA097257 (DHK), Nancy and Stephen Grand Philanthropic Fund (DHK), The V Foundation (KKM, DHK), Merck Inc. (DHK), P01 81403 (KKM), Dougherty Foundation (KKM), Campini Foundation (SM, KKM) and Alex Lemonade Foundation (KKM).
Footnotes
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Conflicts of Interest
Dr. Haas-Kogan has received funding from Merck Oncology to perform laboratory investigations. The study sponsors had no involvement in the study design, collection of the data, analysis or interpretation of the data.
- Received December 12, 2012.
- Revision received February 1, 2013.
- Accepted February 1, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved