Preclinical Efficacy of a PARP-1 Targeted Auger-Emitting Radionuclide in Prostate Cancer

There is an unmet need for better therapeutic strategies for advanced prostate cancer. Poly (ADP-ribose) polymerase-1 (PARP-1) is a chromatin-binding DNA repair enzyme overexpressed in prostate cancer. This study evaluates whether PARP-1, on account of its proximity to the cell’s DNA, would be a good target for delivering high-linear energy transfer Auger radiation to induce lethal DNA damage in prostate cancer cells. We analyzed the correlation between PARP-1 expression and Gleason score in a prostate cancer tissue microarray. A radio-brominated Auger emitting inhibitor ([77Br]Br-WC-DZ) targeting PARP-1 was synthesized. The ability of [77Br]Br-WC-DZ to induce cytotoxicity and DNA damage was assessed in vitro. The antitumor efficacy of [77Br]Br-WC-DZ was investigated in prostate cancer xenograft models. PARP-1 expression was found to be positively correlated with the Gleason score, thus making it an attractive target for Auger therapy in advanced diseases. The Auger emitter, [77Br]Br-WC-DZ, induced DNA damage, G2-M cell cycle phase arrest, and cytotoxicity in PC-3 and IGR-CaP1 prostate cancer cells. A single dose of [77Br]Br-WC-DZ inhibited the growth of prostate cancer xenografts and improved the survival of tumor-bearing mice. Our studies establish the fact that PARP-1 targeting Auger emitters could have therapeutic implications in advanced prostate cancer and provides a strong rationale for future clinical investigation.


Introduction
Prostate cancer is one of the most diagnosed cancers in men worldwide and the second leading cause of cancer deaths in the USA [1,2]. Although patients with nonmetastatic prostate cancer at initial diagnosis have a high five-year survival rate, patients who present with or progress to castration resistance and metastases have a poor prognosis with a survival rate nearing 30% [3,4]. Despite the recent therapeutic advances to improve overall survival, including second-generation anti-androgen therapy, immunotherapy, chemotherapy, and radiopharmaceuticals targeting bone, a castration-resistant disease usually represents the lethal stage of advanced prostate cancer [5][6][7][8][9][10][11][12][13][14][15][16]. Hence, there remains a critical need for effective therapeutic strategies to improve the clinical outcomes of patients with advanced prostate cancer.
Targeting DNA damage repair pathways for cancer therapy has gained much attention over the past decades. Poly (ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme that binds DNA and facilitates single-strand break repair [17][18][19]. In response to DNA damage, PARP-1 gets activated and catalyzes the transfer of ADP-ribose from NAD+ onto PARP itself and other protein substrates [20]. Multiple ADP-ribose moieties are sequentially added by PARP-1 forming poly(ADP)ribose which facilitates the repair of damaged DNA [21]. PARP-1 inhibitors compete with NAD+ for the catalytically active site in vivo preclinical prostate cancer models. Our findings, for the first time, illustrated the potential of Auger emitters in prostate cancer therapy and provided the incentive for additional studies.

PARP-1 Expression in Prostate Cancer and Correlation with Gleason Score
Gleason score is the most widely used prostate cancer grading system and is an effective prognostic predictor in prostate cancer patients [52,53]. We evaluated the expression of PARP-1 in a prostate cancer TMA with normal prostate tissue (n = 6), prostate hyperplasia (n = 7), and prostatic adenocarcinoma (n = 94) graded based on Gleason score ranging from 6 to 10. PARP-1 expression was found to be elevated in prostate adenocarcinoma compared to normal prostate tissue (normal vs. Gleason score ≤ 6: p = 0.0011; normal vs. Gleason score 7: p < 0.0001; normal vs. Gleason score 8-10: p = <0.0001) and prostatic hyperplasia (hyperplasia vs. Gleason score ≤ 6: p = 0.0152; hyperplasia vs. Gleason score 7: p = 0.0016; hyperplasia vs. Gleason score 8-10: p ≤ 0.0001) ( Figure 1A,B). The Spearman correlation coefficient was computed to identify the association between PARP-1 expression and Gleason grades. PARP-1 exhibited a statistically significant correlation with the Gleason score (r = 0.35, p < 0.0006), displaying an increase in PARP-1 expression with corresponding increases in Gleason score (Gleason score ≤ 6 vs. 8-10: p = 0.0312; Gleason score 7 vs. 8-10: p = 0.0251). Our data indicate that patients with highly aggressive tumors with high Gleason scores and those with a predicted poor prognosis have elevated PARP-1, making them ideal candidates for PARP-1-targeted radiotherapeutics.

Synthesis, Purification, and PARP Binding Affinity of [ 77 Br]Br-WC-DZ
The radiochemical purity of the synthesized [ 77 Br]Br-WC-DZ was greater than 99%, and the molar activity was 21,000 ± 4900 mCi/µmol (n = 12). This high specific activity is ideal for targeted radiotherapy, especially for a saturable target, such as PARP-1, resulting in highly efficient radiation delivery [54]. In this study, we included PC-3, the commonly used in vitro model of CRPC derived from bone metastasis, and IGR-CaP1, a more recently developed cell line that produces completely penetrant bone, liver, and brain metastasis in mice emulating aggressive end-stage prostate cancer [55][56][57]. Both cell lines are androgenreceptor-negative [58]. We have confirmed the PARP-1 expression in PC-3 and IGR-CaP1 cells by immunoblot analysis ( Figure S3A). The [ 77 Br]Br-WC-DZ displayed high binding affinity (K d = 0.58 ± 0.25 nM) for PARP-1 and low non-specific binding, as determined by saturation binding studies in PC-3 tumor xenografts ( Figure S3B). To confirm whether the uptake of [ 77 Br]Br-WC-DZ in PC-3 and IGR-CaP1 is PARP-1 dependent, the radioligand uptake in these cells in the presence of molar excess of a non-radiolabeled PARP-1 inhibitor olaparib (50 µM) was evaluated. The uptake of [ 77 Br]Br-WC-DZ was blocked to background levels in the presence of olaparib in both cell lines, confirming that it is PARP-1-dependent ( Figure S3C).

[ 77 Br]Br-WC-DZ Induce PARP-1 Dependent DNA Damage
To elucidate the mechanism of cytotoxicity induced by [ 77 Br]Br-WC-DZ, we performed immunofluorescence analysis of phosphorylated histone 2A family member X (γH2AX) and tumor suppressor p53 binding protein 1 (53BP1) DNA repair nuclear foci, which are markers of the radiation-induced DNA damage [59]. In both PC-3 and IGR-CaP1 cells, [ 77 Br]Br-WC-DZ caused a significant (p < 0.0001) increase in γH2AX foci formation at both 1 and 5 nM concentrations ( Figure 3A,B). The increase was timedependent in both cell lines and dose-dependent (p < 0.0001) in IGR-CaP1 ( Figures 3A,B and S5A,B). There was also an increase in PARP-1 expression after treatment with [ 77 Br]Br-WC-DZ for 24h ( Figure 3A). In blocking experiments when cells were co-treated with [ 77 Br]Br-WC-DZ and an excess of PARP inhibitor olaparib, γH2AX foci formation was significantly decreased (p < 0.0001), suggesting that the DNA damage is specific to [ 77 Br]Br-WC-DZ binding to PARP-1 ( Figure 3A,B). The γH2AX foci were found to be colocalizing with 53BP1, which is an index of radiation-induced DNA damage ( Figure 3C). To further analyze the residual DNA damage after treatment with [ 77 Br]Br-WC-DZ, cells were treated with the Auger emitter for 1 h, media was washed out, and γH2AX foci formation was assessed. There was a significant increase in γH2AX foci formation in both PC-3 and IGR-CaP1 (p < 0.0001) cells compared to the control ( Figure S5C-D).

[ 77 Br]Br-WC-DZ Induce PARP-1 Dependent DNA Damage
To elucidate the mechanism of cytotoxicity induced by [ 77 Br]Br-WC-DZ, we performed immunofluorescence analysis of phosphorylated histone 2A family member X (γH2AX) and tumor suppressor p53 binding protein 1 (53BP1) DNA repair nuclear foci, which are markers of the radiation-induced DNA damage [59]. In both PC-3 and IGR-CaP1 cells, [ 77 Br]Br-WC-DZ caused a significant (p < 0.0001) increase in γH2AX foci formation at both 1 and 5 nM concentrations ( Figure 3A,B). The increase was time-dependent in both cell lines and dose-dependent (p < 0.0001) in IGR-CaP1 ( Figure 3A,B and Figure S5A,B). There was also an increase in PARP-1 expression after treatment with [ 77 Br]Br-WC-DZ for 24 h ( Figure 3A). In blocking experiments when cells were co-treated with [ 77 Br]Br-WC-DZ and an excess of PARP inhibitor olaparib, γH2AX foci formation was significantly decreased (p < 0.0001), suggesting that the DNA damage is specific to [ 77 Br]Br-WC-DZ binding to PARP-1 ( Figure 3A,B). The γH2AX foci were found to be co-localizing with 53BP1, which is an index of radiation-induced DNA damage ( Figure 3C). To further analyze the residual DNA damage after treatment with [ 77 Br]Br-WC-DZ, cells were treated with the Auger emitter for 1 h, media was washed out, and γH2AX foci formation was assessed. There was a significant increase in γH2AX foci formation in both PC-3 and IGR-CaP1 (p < 0.0001) cells compared to the control ( Figure S5C,D).

Biodistribution of [ 77 Br]Br-WC-DZ in Vivo
Biodistribution studies of [ 77 Br]Br-WC-DZ in the presence and absence of a blocking agent, olaparib, were performed in mice bearing PC-3 and IGR-CaP1 xenografts to confirm selective tumor localization and absence of specific uptake in normal tissues. Both PC-3 and IGR-CaP1 xenograft-bearing mice showed tumor uptake (PC-3, 3.58 ± 0.61 %ID/g; IGR-CaP1, 1.76 ± 0.32 %ID/g) of [ 77 Br]Br-WC-DZ at 4 h ( Figure 4A). Olaparib significantly reduced the radioactive uptake of both PC-3 (3.58 ± 0.61 vs. 0.31 ± 0.07 %ID/g p, 0.0001) and IGR-CaP1 (1.76 ± 0.32 vs. 0.52 ± 0.13 %ID/g p, 0.0001) tumors ( Figure 4A). Assessment of radioactive distribution patterns in blood and normal organs showed negligible uptake in blood, lung, muscle, bone, prostate, and pancreas (<0.5 %ID/g), slight uptake in the kidney and spleen (<1.2 %ID/g), and highest uptake in the liver (6-7 %ID/g) ( Figure S6). High tumor-to-tissue ratios (>2) were observed in both tumor models for all tissues except for the liver ( Figure 4B). Olaparib blocking did not significantly reduce the radioactivity uptake in any of the normal organs confirming that its uptake is PARP-1-independent ( Figure 4A). Taken together, these findings confirm the in vivo binding specificity and selectivity of [ 77 Br]Br-WC-DZ toward PARP-1-expressing tumors.  [ 77 Br]Br-WC-DZ more efficiently and showed more dramatic effects in terms of tumor growth restriction and survival advantage. This finding correlates with the increased uptake of the radioligand in PC-3 tumors, as evidenced in the biodistribution studies ( Figure 4). Overall, this study, for the first time, provides evidence that a PARP-1-targeting Auger emitter can offer antitumor effects in in vivo prostate cancer models with minimal toxicity.

Discussion
While there are cures available for localized prostate cancer, the advanced disease poses a therapeutic challenge because of castration or chemotherapy resistance, demanding effective curative strategies. Targeted radiotherapy is emerging as a promising treatment modality that efficiently delivers ionizing radiation to cancer cells while minimizing radiation exposure to untargeted cells. Despite the efficacy of the currently used radioligands, including the beta-emitter 177 Lu-PSMA-617 for mCRPC, there is still a need for more specific targets and radionuclides with improved treatment outcomes and minimal side effects [60,61]. In this study, we are reporting the preclinical

Discussion
While there are cures available for localized prostate cancer, the advanced disease poses a therapeutic challenge because of castration or chemotherapy resistance, demanding effective curative strategies. Targeted radiotherapy is emerging as a promising treatment modality that efficiently delivers ionizing radiation to cancer cells while minimizing radiation exposure to untargeted cells. Despite the efficacy of the currently used radioligands, including the beta-emitter 177 Lu-PSMA-617 for mCRPC, there is still a need for more specific targets and radionuclides with improved treatment outcomes and minimal side effects [60,61]. In this study, we are reporting the preclinical evaluation of an Auger-emitting radio-brominated PARP-1 inhibitor, [ 77 Br]Br-WC-DZ, in prostate cancer cell lines and in vivo models.
Our TMA analysis corroborated the previous studies that PARP-1 expression is higher in prostate cancer than in normal prostate tissues [24,25,62,63]. Most importantly, our study is the first to markedly correlate the PARP-1 expression with the Gleason score. We have previously reported the utility of PARP-1 radioligands, specifically [ 18 F]WC-DZ-F, for PET imaging in the prostate cancer [64]. The Auger emitter [ 77 Br]Br-WC-DZ synthesized for this study is an analog of [ 18 F] WC-DZ-F, except that fluorine is replaced with bromine. Although Auger therapy has been predominantly studied with 125 I, the advantages of 77 Br include its high radiotoxicity, longer half-life, higher theoretical specific activity, and lack of thyroid uptake [65,66]. As far as we know, this is the first study exploring the effectiveness of a 77 Br linked PARP-1 inhibitor in in vivo xenograft tumor models.
Cytotoxicity studies confirmed that, on a molar level, [ 77 Br]Br-WC-DZ is more potent than non-radiolabeled WC-DZ-Br displaying more than a 10,000-fold difference in IC 50 values. DNA repair foci analysis in the presence of blocking agents, such as olaparib, revealed that [ 77 Br]Br-WC-DZ-induced DNA damage is brought about by its specific binding to PARP-1. Furthermore, we observed an increase in PARP-1 expression with [ 77 Br]Br-WC-DZ because of the DNA-damage-induced upregulation of PARP-1. Considering the intact homologous recombination repair genes such as BRCA1 and 2 in the cell lines tested, the cytotoxic effect is brought about by DNA damage induced by ionizing radiation rather than synthetic lethality by PARP-1 inhibition. Additionally, keeping in mind that patients' harboring BRCA1/2 or HRR mutations are benefitted from conventional PARP-1 inhibitors, PARP-1 Auger therapy has the advantage that its use can be extended beyond BRCA1/2 defective cancers.
According to some of the earlier works, despite showing potent in vitro cytotoxic activity, PARP-1 inhibitor monotherapy showed limited clinical activity in certain tumor types [67]. Lee et al. have reported that, though a PARP-1-targeted Auger emitter 125 I-KX1 was highly cytotoxic in vitro, it was predicted to display limited therapeutic efficacy in solid tumor models of neuroblastoma [39]. When administered intratumorally, 131 I-PARPi elicited a significant reduction in tumor growth and improvement in median survival of the subcutaneous mouse model of the glioblastoma [38]. Similarly, Auger-emitting PARP inhibitor 123 I-MAPi displayed therapeutic efficacy in glioblastoma models employing a complex convection-enhanced drug delivery system [46]. In the present study, the systemically administered brominated Auger emitter [ 77 Br]Br-WC-DZ was effective in suppressing the growth of subcutaneous prostate cancer tumors and offered a survival advantage in tumor-bearing mice. These results could be improved by optimizing the radiation dose administered and by investigating multi-dose fractions. We would be expanding the in vivo studies to micro-metastatic models of prostate cancer.
Though PARP-1 expression levels were similar between the PC-3 and IGR-CaP1 cell lines, PC-3 cells showed increased sensitivity to [ 77 Br]Br-WC-DZ in in vitro assays in terms of cytotoxicity, clonogenic survival, and in in vivo xenograft tumor suppression. The differential sensitivity could be attributed to several factors, including cell type, DNA repair capacity, cell cycle phase at the time of exposure, and the microenvironment [68]. PC-3 cells have a homozygous deletion of the DNA repair-associated gene PTEN [69]. This could also be contributing to the increased sensitivity of PC-3 cells to [ 77 Br]Br-WC-DZ. Further studies are warranted to identify the contributing factors leading to the sensitivity and/or resistance of the cells to [ 77 Br]Br-WC-DZ to better understand the clinical outcome.
In conclusion, this study identifies the utility of PARP-1 inhibition as a means for delivering lethal high-LET Auger radiation to the cancer cell's DNA resulting in tumorspecific DNA damage and cytotoxicity. Radio-brominated Auger emitting PARP-1 inhibitor [ 77 Br]Br-WC-DZ could have the potential for clinical translation in advanced prostate cancer and warrants further investigation. Since PARP-1 is overexpressed in a multitude of cancers, this therapeutic approach has the prospective to be extrapolated to other cancers as well.

Cell Lines and Culture Conditions
Human prostate cancer cell line PC-3 was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in high-glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA). The IGR-CaP1 cell line, derived from primary prostate cancer, was kindly provided by Dr. Anne Chauchereau (Prostate Cancer Group, Institut Gustave Roussy, Villejuif, France) [57]. IGR-CaP1 cells were cultured according to the culture conditions described previously [56]. The cell lines were maintained at 37 • C, 5% CO 2 in a humidified incubator. The cell lines were authenticated by short-tandem repeat (STR) profiling (Arizona Genetics Core, Tucson, AZ, USA).

Chemistry and Radiochemistry
Br-WC-DZ and the labeling precursor were synthesized as reported (Patent No WO/2002/044183 [70,71]. The radiosynthesis, purification, and dose preparation of [ 77 Br]Br-WC-DZ are detailed in the supplementary methods. The [ 77 Br]Bromide was produced in the cyclotron facility of Washington University in Saint Louis [72]. The [ 77 Br]Br-WC-DZ was prepared via copper-mediated nucleophilic radiobromination of a boron precursor with [ 77 Br]Bromide, a strategy that has been reported previously by us [66]. The [ 77 Br]Br-WC-DZ was purified by HPLC ( Figures S1 and S2). Cellular uptake of [ 77 Br]Br-WC-DZ in PC-3 and IGR-CaP1 cell lines in the presence and absence of molar excess of PARP inhibitor olaparib (50 µM) was studied as reported earlier by us [64].

In Vitro Cytotoxicity
The cytotoxic effect of [ 77 Br]Br-WC-DZ on PC-3 and IGR-CaP1 prostate cancer cells was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation kit (Promega, Madison, WI, USA) following the manufacturer's instructions. Briefly, the cells were seeded at a density of 1000-2000 cells/well in 96-well plates. Approximately 24 h after cell seeding, the cells were treated with varying doses (10 −11.5 to 10 −8 M/1.8 KBq/mL-5.7 MBq/mL) of [ 77 Br]Br-WC-DZ. After incubating for 120 h, the medium was aspirated, and 100 µL of serum-free media and 20 µL of MTS solution (5 mg/mL solution in PBS) were added into each well; the cells were then incubated for 2 h at 37 • C. The absorbance was measured at 490 nm using VersaMax Microplate Reader (Molecular Devices LLC, San Jose, CA, USA). Experiments were completed in six replicates and were repeated three times. The absorbance of vehicle (0.1% ethanol)-treated control cells was considered 100% survival. The half-maximal inhibitory concentration (IC 50 ) values were determined using GraphPad Prism 7.0 software (GraphPad Software, Inc., San Diego, CA, USA). Cytotoxicity experiments with non-radioactive WC-DZ-Br and other PARP inhibitors (10 −10 to 10 −4 M) were carried out in a similar manner.

Clonogenic Assay
Prostate cancer cells were plated in 6-well plates at a density of 500 cells/well in triplicates, allowed to attach overnight, and treated with [ 77 Br]Br-WC-DZ (0.5, 1.0, and 5 nM/0.29, and 0.57 and 2.9 MBq/mL). Vehicle (0.01% ethanol)-treated cells served as control. After incubating for 3 h, the cells were washed twice with medium and cultured for 14 days, with fresh medium added every three days. The colonies were fixed (Acetic acid/methanol; 1:7) and stained with 0.5% crystal violet solution. The colonies with more than 50 cells in each well were counted. Plating efficiency (PE = the number of colonies/the number of seeded cells × 100%) and surviving fraction (SF = the number of colonies formed after treatment/number of cells seeded × PE) were calculated.

DNA Damage Assessment by Comet Assay
The ability of [ 77 Br]Br-WC-DZ to induce DNA damage was measured by comet assay. PC-3 and IGR-CaP1 cells were treated with 5 nM [ 77 Br]Br-WC-DZ for 4 h. Cells were harvested, suspended in PBS, mixed with low-melting agarose, and spread on a Trevigen's CometSlide (Gaithersburg, MD, USA). The slides were placed in lysis buffer, followed by single-cell electrophoresis, and stained with 1X SyberGreen (Thermo Fisher Scientific, Rockford, IL, USA) at RT for 30 min in the dark. The CometSlides were then washed and allowed to dry overnight at RT. Images were taken using Zeiss Axioplan 2 microscope using a 20× objective, and the comet tail length was measured using CometScore software (TriTek Corporation, Sumerduck, VA, USA), and results were expressed as the percent of DNA in the tail (tail intensity).

In vivo Biodistribution and Antitumor Activity Studies
The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Washington University School of Medicine (St Louis, MO, USA). Six-week-old athymic female/male nude mice (nu/nu) were purchased from Charles River Laboratories (Wilmington, MA, USA), and animals were acclimated for 1 week before the experiments. In vivo biodistribution studies were performed at the Washington University Small Animal Imaging Facility. PC-3 or IGR-CaP1 cells (1 × 10 7 cells in serum-free DMEM) were injected into the right flank of female and male mice, respectively. Once the tumors reached a volume of 100 mm 3 , 370 kBq of [ 77 Br]Br-WC-DZ was injected intravenously, and the biodistribution was evaluated at 2 h and 4 h time points (n = 5). In the blocking group, mice were pre-injected with olaparib (5 mg/kg bwt), and biodistribution was evaluated at 4 h. The animals were sacrificed at each time point, their organs were harvested, and radioactivity was measured on a Beckman Gamma-8000 counter. Tumor and organ uptake were analyzed and calculated as a percentage of injected dose per weight of tissue in grams (%ID/g). Tumor-to-organ ratios were calculated for each mouse based on %ID/g. For the in vivo efficiency study, once the tumors reached a mean volume of 175 mm 3 , the mice were divided into two groups. One group received saline, and the other received 56 MBq of [ 77 Br]Br-WC-DZ by intravenous injection. Tumor volumes were measured using calipers and calculated using the formula: Volume (mm 3 ) = (Length × Width 2 )/2. Tumor volume and body weight were monitored twice a week.

Statistical Analysis
Statistical analyses were performed using Graph Pad Prism 7 (San Diego, CA, USA) software. Comparisons between groups were made using an unpaired t-test (two groups) or one-way/two-way ANOVA (three or more groups) with Bonferroni multiple comparison test. In vivo efficacy studies were evaluated using Kaplan-Meier survival curves compared with the log-rank (Mantel-Cox) test. p values < 0.05 were considered significant and are indicated by asterisks in figures (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05).