Nanoformulation of Olaparib Amplifies PARP Inhibition and Sensitizes PTEN/TP53-Deficient Prostate Cancer to Radiation

Published OnlineFirst May 12, 2017; DOI: 10.1158/1535-7163.MCT-16-0740

Small Molecule Therapeutics Molecular Cancer Therapeutics Nanoformulation of Olaparib Amplifies PARP Inhibition and Sensitizes PTEN/TP53-Deficient Prostate Cancer to Radiation Anne L. van de Ven1,2, Shifalika Tangutoori2,3, Paige Baldwin2,4, Ju Qiao5, Codi Gharagouzloo4, Nina Seitzer6, John G. Clohessy6, G. Mike Makrigiorgos3, Robert Cormack3, Pier Paolo Pandolfi6, and Srinivas Sridhar1,2,3

Abstract

The use of PARP inhibitors in combination with radiotherapy is compared to radiation only controls. Half of mice treated with a promising strategy to locally enhance DNA damage in tumors. NanoOlaparib þ radiation achieved a complete response over the Here we show that radiation-resistant cells and tumors derived 13-week study duration. Using ferumoxytol as a surrogate nano- from a Pten/Trp53-deficient mouse model of advanced prostate particle, MRI studies revealed that NanoOlaparib enhances cancer are rendered radiation sensitive following treatment with the intratumoral accumulation of systemically administered NanoOlaparib, a lipid-based injectable nanoformulation of ola- nanoparticles. NanoOlaparib-treated tumors showed up to parib. This enhancement in radiosensitivity is accompanied by 19-fold higher nanoparticle accumulation compared to radiation dose-dependent changes in g-H2AX expression and is untreated and radiation-only controls, suggesting that the specific to NanoOlaparib alone. In animals, twice-weekly intra- in vivo efficacy of NanoOlaparib may be potentiated by its venous administration of NanoOlaparib results in significant ability to enhance its own accumulation. Together, these data tumor growth inhibition, whereas previous studies of oral ola- suggest that NanoOlaparib may be a promising new strategy for parib as monotherapy have shown no therapeutic efficacy. When enhancing the radiosensitivity of radiation-resistant tumors NanoOlaparib is administered prior to radiation, a single dose of lacking BRCA mutations, such as those with PTEN and TP53 radiation is sufficient to triple the median mouse survival time deletions. Mol Cancer Ther; 16(7); 1279–89. 2017 AACR.

Introduction tors have also been shown to trap PARP-1 and PARP-2 on DNA (4), thereby forming PARP–DNA complexes that are thought to PARP inhibitors are an emerging class of drugs that inhibit be responsible for the synergism seen with PARP inhibition and PARP-1 and PARP-2, proteins that play a critical role in base alkylating agents. There is also growing evidence that PARP excision repair (1). When PARP function is impaired, double- inhibitors may provide clinical benefit for subsets of patients stranded DNA (dsDNA) breaks accumulate with time. Cells proficient in homologous repair (5); however, it is not yet clear deficient in homologous recombination cannot accurately repair how to best select patients for treatment with PARP inhibitors. these breaks, leading to genetic instability, chromosome rear- PARP inhibitor olaparib (Lynparza, AstraZeneca Pharmaceu- rangement, and cell death (2). This synthetic lethality has proven ticals LP) has been approved by the FDA as a monotherapy for effective for improving progression-free survival in patients with advanced ovarian cancer in patients with germline BRCA muta- homologous repair-deficient BRCA mutations (3). PARP inhibi- tions (5, 6). Patient eligibility is determined using the BRACA- nalysis CDx in vitro companion diagnostic (Myriad Genetics) to identify BRCA1 and BRCA2 gene variants. Olaparib has also 1Department of Physics, Northeastern University, Boston, Massachusetts. been granted Breakthrough Therapy status by the FDA for 2Nanomedicine Science & Technology Center, Northeastern University, Boston, metastatic castration-resistant prostate cancer (mCRPC) follow- Massachusetts. 3Department of Radiation Oncology, Dana-Farber Cancer Insti- ing the phase II TOPARP-A clinical trial in which men with 4 tute, Boston, Massachusetts. Department of Bioengineering, Northeastern defective DNA damage repair mechanisms responded to ola- University, Boston, Massachusetts. 5Department of Mechanical and Industrial 6 parib (7). Importantly, this trial demonstrated that mutations Engineering, Northeastern University, Boston, Massachusetts. Cancer Research BRCA Institute, Beth Israel Deaconess Cancer Center, Boston, Massachusetts. in other DNA damage repair genes, not just ,caninduce sensitivity to PARP inhibition. Olaparib monotherapy has Note: Supplementary data for this article are available at Molecular Cancer fi fi Therapeutics Online (http://mct.aacrjournals.org/). shown ef cacy across a variety of advanced tumors classi ed by mutation status (5, 7–10), and thus olaparib continues to be A.L. van de Ven and S. Tangutoori contributed equally to this article. investigated in more than 40 active phase II and III clinical trials Corresponding Author: Srinivas Sridhar, Northeastern University, 360 Hunting- (11). Notably, 47% of these trials seek to determine whether ton Avenue, Boston, MA 02115. Phone: 617-373-2930; Fax: 617-373-2823; E-mail: the therapeutic efficacy of olaparib can be enhanced in com- [email protected] bination with other small molecules, including DNA-damaging doi: 10.1158/1535-7163.MCT-16-0740 agents, cytotoxic chemotherapy, anti-angiogenic agents, and 2017 American Association for Cancer Research. molecular-specificinhibitors.

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Olaparib has been demonstrated to enhance the cytotoxicity of ually dissolved in chloroform and combined at a molar ratio of therapies that induce DNA damage. Oral olaparib capsules, 13.6:0.43:1.29:0.717:5.75. Following overnight solvent evapora- administered in combination with paclitaxel for advanced gastric tion under rotary vacuum, the lipid-drug melt was hydrated using cancer, improved overall survival for patients with known DNA PBS (pH 7.4, final concentration 2.5 mg/mL olaparib) at 50C, damage repair impairments (12). Olaparib in combination with emulsified by heating and cooling five times with agitation, and paclitaxel and/or platinum therapy enhanced progression-free then sonicated at 25C for 10 minutes to produce highly con- survival in advanced ovarian cancer, breast cancer, and other densed lipid nanoparticles containing hydrophobic olaparib. solid tumors (13, 14). Unfortunately, these drug combinations Unbound drug was removed by dialysis for 4 to 6 hours. For produced significant off-target toxicity, necessitating a reduction animal studies, nanoparticles were concentrated by centrifugation in olaparib dose compared with monotherapy to avoid life- at 3,200 rcf for 45 minutes and resuspension to yield a final drug threatening side effects such neutropenia, leukopenia, anemia, concentration of 5.0 to 10.0 mg/mL. Conventional olaparib was and thrombocytopenia (14). Similarly, dose-escalation studies of dissolved in DMSO for use as a control. olaparib in combination gemcitabine (15), doxorubicin (16), and paclitaxel (17) found that the monotherapy daily dose (400 Nanoparticle characterization mg b.i.d.) could not be reached with an acceptable tolerability The mean nanoparticle diameter, zeta potential, and concen- fi pro le. In contrast, daily olaparib maintenance therapy after tration were measured by laser scattering microscopy (Zetaview, combination therapy was well tolerated (18). ParticleMetrix) using PBS (pH 7.4 or 6.0) as solvent. Nanoparti- Combining olaparib with radiotherapy is a promising strategy cles were visualized using transmission electron microscopy to selectively enhance DNA-damage at the primary site of drug (TEM; JEM-1010, JEOL) after being dried on 300-mesh copper- action. Focused-beam X-ray radiation produces localized dsDNA coated carbon grids (Electron Microscopy Sciences) and negative- breaks and thus would be expected to render cells more sensitive ly stained with a 1.5% uranyl acetate solution (Sigma-Aldrich). to PARP inhibition (19, 20). Olaparib has been shown to enhance Drug encapsulation and release was measured using high-perfor- fi fi the effect of radiation in both BRCA2-pro cient and de cient mance liquid chromatography (HPLC; Agilent 1100 series). prostate cancer cells (21). Several PARP inhibitors have been Olaparib was detected at 207 nm on a reverse phase C18 column in vivo – shown to have radiosensitizer activity (20, 22 25); how- (SUPELCO) with a mobile phase of methanol: water (64 : 36). The ever, these agents have not yet been demonstrated to provide nanoparticle loading efficiency was determined by dissolving radiosensitivity to radiation-resistant tumors. Clinically, olaparib nanoparticles in methanol prior to HPLC. Drug release was has been demonstrated to have antitumor activity in a subset of determined by dialyzing nanoparticles against PBS and measuring prostate cancer patients with mutated DNA damage repair genes, the supernatant with HPLC. notably BRCA2 and ATM (7), but has not been tested in combination with radiation. Here we describe NanoOlaparib, a lipid-based nanoformula- Cell culture PTEN fi tion of olaparib with demonstrated efficacy in radiation-resistant Three -de cient prostate cancer cell lines were evaluated mice. Three PTEN-deficient prostate cancer cell lines were tested for their response to olaparib, including human PC3 (ATCC), Ptenpc/;Trp53pc-/ for their sensitivity to olaparib and NanoOlaparib. The most human LNCaP (ATCC), and mouse FKO1 ( , fi clinically relevant line, radiation-resistant FKO1 cells derived provided by Dr. Pandol ; refs. 27, 28). All cell lines were tested fi from a Pten/Trp53-deficient mouse model of advanced prostate and authenticated using short tandem repeat (STR) pro ling. All cancer (26), was further investigated and found to selectively cell lines were passaged for fewer than 6 months after receipt. Cells increase g-H2AX expression in a radiation-dose dependent man- were grown in F12-K (PC3), RPMI (LNCaP), or DMEM media ner using immunocytochemistry and Western blotting. A subcu- (FKO1) supplemented with 10% FBS (HyClone). The FKO1 taneous model of prostate cancer, generated in mice, was used to media was further supplemented with 25 mg/mL bovine pituitary assess the therapeutic efficacy of NanoOlaparib alone and in extract (Life Technologies), 5 mg/mL insulin (Sigma Aldrich), and 6 ng/mL epidermal growth factor (Life Technologies). All combination with a single dose of focused-beam X-ray radiation. To determine the potential mechanism by which NanoOlaparib cells were maintained in a 5% CO2 atmosphere at 37 C. To works in vivo, ultra-short time-to-echo MRI (UTE-MRI) was uti- determine the optimal seeding densities for extended cell assays, lized to quantify ferumoxytol accumulation in tumors following the doubling rate of each line was measured by MTS assay for NanoOlaparib administration. The results of these studies suggest metabolic activity (Promega). The cell doubling times were found that NanoOlaparib may be a promising strategy for enhancing to be 36.5 hours (PC3), 64.9 hours (LNCAP), and 19.5 hours radiosensitivity of advanced prostate tumors with PTEN and TP53 (FKO1). deletions. IC50, cell viability, and clonogenic survival assays For IC50 determination, exponentially growing cells were trea- Materials and Methods ted continuously with 0 to 100 mmol/L olaparib or NanoOlaparib Nanoparticle synthesis for four population doublings. The final cell count was deter- sn NanoOlaparib was synthesized using 1, 2-dipalmitoyl- -gly- mined using an MTS assay and the IC50 was calculated as the drug cero-3-phosphocholine (DPPC), 1,2-dioleoyl-3-tri methyl- concentration required to inhibit growth of 50% of cells. For the ammonium-propane (chloride salt) (DOTAP), cholesterol, and short-term cell viability assay, cells in exponential growth phase 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[methoxy were pretreated with 1 mmol/L olaparib or NanoOlaparib (polyethyleneglycol)-2000 (DSPE-PEG2000) purchased from for 24 hours, irradiated (0, 2, or 4 Gy), replated at low density Avanti Polar Lipids and olaparib (Selleck Chemicals). DPPC, (300–600 cells/cm2) 4 hours after irradiation, and then cultured DOTAP, cholesterol, DSPE-PEG2000, and olaparib were individ- in complete media without drug for nine additional doubling

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cycles (6–24 days). The colonies were fixed, stained using crystal the human oral daily dose (800 mg) and then reduced by 40% to violet (BioPioneer), and dissolved in acetic acid for quantification compensate for the full bioavailability of intravenously admin- of absorbance at 590 nm. The percent cell viability was deter- istered drug. Mice treated with radiation received a one-time X-ray mined relative to an untreated control. Drug uptake by subcon- dose of 10 Gy when tumors reached 250 50 mm3 using a fluent monolayers was measured using HPLC following 24-hour Precision Small Animal Radiation Research Platform (SARRP, drug treatment and normalized for protein content using a XRad). Tumor size was measured twice weekly using calipers, Bradford assay. For the long-term clonogenic cell survival assay, and all animals were monitored and weighed at least twice per cells in exponential growth phase were pretreated with 1 mmol/L week. Mice were removed from the therapeutic study when olaparib or NanoOlaparib for 24 hours, irradiated (0–10 Gy), tumors reached 1,500 mm3, imaged using MRI, and sacrificed replated at low density (300–600 cells/cm2) 4 hours after immediately thereafter. Harvested tissues were fixed in 10% irradiation, and then incubated continuously with drug for a formalin. All animals were maintained in accordance with insti- minimum of 14 days (29, 30). Colonies stained with crystal tutional rules and ethical guidelines for animal care. violet were manually counted using a pen and used to estimate the surviving fraction as a function of the plating efficiency (31). MRI of ferumoxytol accumulation To generate the radiation dose–response curves, the data were Animal imaging was performed at the Center for Translational fitted to a linear quadratic model. The sensitizer enhancement Neuroimaging (CTNI) at Northeastern University in accordance ratio (SER) at 10% cell survival was calculated according to the with the Division of Laboratory Animal Medicine and Institu- equation: tional Animal Care and Use Committee. MRI images were obtained at ambient temperature (25C) using a Bruker Biospec D ðÞ 7.0T/20-cm USR horizontal magnet (Bruker) equipped with a ¼ 10 no treatment SER10 fi ¼ D10ðÞdrug treatment 20-G/cm magnetic eld gradient insert (ID 12 cm, Bruker) and a 300 MHz, 30 mm mouse coil (Animal Imaging Research, LLC). – For all radiation studies, cells received a single dose of 0 10 Gy T1-weighted, T2-weighted, and ultra-short time-to-echo (UTE) X-rays using a 220 kVp beam delivered at 13 mA (dose rate of images were acquired at 150 mm resolution within a 3 cm 5.45 Gy/min) via a 0.15 mm copper filter using a Precision Small field-of-view as previously described (33). The UTE pulse- Animal Radiation Research Platform (SARRP, XRad). sequence used a 200 kHz fixed trajectory with acquisition para- meters TE ¼ 13 microseconds, TR ¼ 4 milliseconds, and u ¼ 20. Confocal microscopy Mice were randomized into one of three treatment groups: Subconfluent monolayers of FKO1 cells (5 104 cells/cm2) untreated (no olaparib), 4 hours pretreated (NanoOlaparib, were pretreated for 24 hours with 0 to 5 mmol/L olaparib, 1 40 mg/kg), or chronically treated (NanoOlaparib, 40 mg/kg NanoOlaparib, or a vehicle control and then irradiated with 2 twice weekly for 2 weeks). Ferumoxytol accumulation in to 10 Gy. Cells were fixed 30 minutes after radiation treatment, the vasculature and tumor was monitored 0 and 24 hours respec- permeabilized with 0.05% Triton-X100 for 10 minutes, tively after a one-time intravenous bolus injection of 14 mg/kg blocked with a 1% BSA/2% goat serum solution for 10 minutes, ferumoxytol. Precontrast images were acquired immediately prior and then labeled with anti-g-H2AX 1:1,500 (Cell Signaling to ferumoxytol injection. To assess how NanoOlaparib treatment Technology), anti-Rad51 1:500 (AbCam), and/or anti-DNA changes ferumoxytol accumulation, vehicle-treated mice were polymerase q (polq) 1:500 for 1 hour. Following three washes, given one injection of NanoOlaparib (40 mg/kg) following the cells were probed with Alexa Fluor 488 and 647 IgG 1:500 ferumoxytol clearance and then re-injected with a new bolus of (Invitrogen) for 1 hour, washed, and counterstained with DAPI ferumoxytol (14 mg/kg) 4 hours later. T1,T2, and UTE signal (Life Technologies). Images were acquired using a Zeiss LSM intensity images were transformed and rendered using 3DSlicer 710 laser-scanning confocal microscope (DAPI Ex: 350 nm, Em: v4.4 (34). To quantify ferumoxytol accumulation, the UTE signal 410–480 nm; Rad51 Ex: 488 nm, Em: 505–530 nm; Polq Ex: intensity was measured for each voxel within the tumor volume. 488 nm, Em: 505–530 nm; gH2AX Ex: 633 nm, Em: 660–710 The fold-increase in voxel number as a function of signal intensity nm) using a 40 or 63 objective. The mean g-H2AX was determined by subtracting the precontrast signal histogram fluorescent signal intensity was analyzed using 200 nuclei per from the postcontrast histogram. The percentage of tumor volume treatment group from a minimum of 10 random fields, using with ferumoxytol accumulation was calculated from the fold- ImageJ v4.1. The damage enhancement ratio was calculated increase histogram by counting the total number of voxels above a from the mean g-H2AX fluorescence intensity at each radiation threshold intensity of 750 and normalizing by the total number of dose as described in ref. 32. tumor voxels. Histology Animal models Harvested tissues were embedded in paraffin, cut, and stained A subcutaneous mouse model of radiation-resistant prostate by the Dana-Farber/Harvard Cancer Center Research Pathology cancer was generated by a one-time subcutaneous implantation Core. To assess tissue pathology, tumor sections were stained with of 106 (in 200 mL PBS) FKO1 cells (derived from Ptenpc / ; H&E according to manufacturer's recommendations. Trp53pc / mice) in the right flank of nude (nu/nu) mice at Northeastern University. Mice were randomized into four treat- Statistical analysis ment groups (n ¼ 6–7) when tumors reached 180 20 mm3 Statistical significances were analyzed by Student t test or one- in volume. NanoOlaparib (40 mg/kg drug) was administered way analysis of variance with P 0.05 considered statistically twice weekly via intravenous injection for up to 12 weeks. The significant. All data were expressed as mean SD (in vitro data) or NanoOlaparib dose was selected based on allometric scaling of mean SEM (in vivo data). Survival fraction curve was analyzed by

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KaleigaGraph 4.0, then fitted to the data using a linear–quadratic physiological pH occurred slowly (Fig. 1B), characterized by first- model. The median survival time was estimated from the Kaplan– order drug release with a half-life of 4.3 days (R2 ¼ 0.99; Fig. 1B, Meier curve and a log-rank test was used pairwise to test for inset). statistically significant differences (35). PARP inhibition sensitizes prostate cancer cell lines to Results radiotherapy Loss of the PTEN or TP53 tumor suppressor genes is commonly Nanoformulation of lipid nanoparticles containing olaparib observed in prostate cancer, whereas their combined loss is often Poorly soluble olaparib was encapsulated into lipid nanopar- observed in advanced prostate cancer. The therapeutic efficacy of ticles using bulk synthesis techniques. Lipid films containing olaparib and NanoOlaparib treatment was assessed in three PTEN- cholesterol, DPPC, DOTAP, DSPE-PEG , and olaparib were 2000 deficient prostate cancer cell lines with differing radiation resistance hydrated with aqueous solvent and sonicated to produce mono- including LNCaP (ARpos,p53wt), PC-3 (ARneg,p53null), and FKO1 disperse, unilamellar nanoparticles loaded with clinically relevant (ARinsensitive,p53null; Fig. 2A). Nanoparticles were diluted to concentrations of inhibitor. Nanoparticles were characterized contain the same number of drug molecules as the free olaparib, using a laser scattering video microscope to obtain nanoparticle with mmol/L NanoOlaparib denoting the amount of drug contained count, size, and zeta potential information. The mean nanopar- (not the concentration of nanoparticles). Cell viability was deter- ticle size was measured to be 71 5 mm (PDI ¼ 0.07) with less mined by the counting of cells transiently treated (24 hours) with than 7% batch-to-batch variation at time of synthesis. The rep- olaparib or NanoOlaparib and then allowed to proliferate in the resentative size distribution at synthesis and following 3 months absence of drug for nine population doublings. Here, radiation was of storage is shown in Fig. 1A. Nanoparticles had an overall surface performed 24 hours after drug addition. Both LNCaP and PC-3 cells charge of 24 7 mV, indicative of effective charge shielding of displayed a dose-dependent response to radiation alone, whereas the cationic lipids by PEG. Nanoparticles had a rounded or the FKO1 cells were radiation-resistant. All cell lines demonstrated partially concave appearance under TEM when counterstained increased sensitivity to radiation following pretreatment with 1 uranyl acetate (Fig. 1A, inset). Using HPLC, the average nanopar- mmol/L olaparib or NanoOlaparib. The relative change in sensitivity ticle loading was estimated to be 5.0 mg/mL for solutions contain- was greatest for the FKO1 cells, which demonstrated approximately ing approximately 4 107 nanoparticles/mL. Olaparib release at 70% a decrease in cell viability with PARP inhibition compared to radiation alone. Cumulative drug uptake did not significantly differ for FKO1 cells treated 24 hours with olaparib or NanoOlaparib, as measured by solid-phase extraction followed by HPLC. The IC50 for continuously treated FKO1 cells was determined to be 2.2 mmol/L (olaparib) and 3.0 mmol/L (NanoOlaparib) in the absence of radiation, demonstrating that these cells are relatively insensitive to PARP-1 inhibition as a monotherapy. Reported values for prostate cancer cell lines considered olaparib-sensitive are on the order of 0.59 mmol/L (LNCaP) and 0.79 mmol/L (PC-3), whereas cells with BRCA1/BRCA2 mutations are 0.02 to 0.2 mmol/L (29). A long- term clonogenic assay was performed to determine the fraction of FKO1 cells surviving radiation treatment alone and in combination with continuous PARP inhibition (Fig. 2B and C). Both olaparib and NanoOlaparib were found to significantly enhance the effect of radiation at doses of 6 Gyand above (P < 0.05). At 10% cell survival, the SER (SER10) was measured to be 1.28 (olaparib) and 1.81 (NanoOlaparib). The relative benefit of NanoOlaparib over olaparib was observed to be greatest at 10 Gy.

NanoOlaparib pretreatment enhances DNA damage in vitro FKO1 cell monolayers treated with radiation were examined for expression of phosphorylated histone H2AX (g-H2AX), a bio- marker of cellular response to dsDNA breaks (36). Compared to untreated controls, NanoOlaparib pretreatment with concentrations of 0.2 mmol/L and above produced a statistically significant increase in mean nuclear fluorescence intensity that persisted for 4 hours following irradiation (Supplementary Fig. S1A and S1B). The nuclei of cells pretreated with 1 mmol/L olaparib or Nano- Olaparib showed a significant increase in both g-H2AX foci Figure 1. In vitro characterization of nanoformulated olaparib. A, Representative number and foci intensity as early as 0.5 hours after irradiation fl fi nanoparticle size distribution following synthesis and 3 months of storage, as (Fig. 3A). The mean nuclear uorescence was quanti ed across a measured by individual nanoparticle tracking. Inset, Representative range of different radiation doses (Fig. 3B). Olaparib alone was transmission electron micrograph of nanoparticles counterstained with uranyl found to increase nuclear g-H2AX expression at all radiation acetate. B, Cumulative olaparib release in phosphate buffered saline at pH 7.4. doses; however, this damage paralleled that caused by radiation fi fi Inset, Linear tof rst-order release kinetics. alone, suggesting that free drug increases the basal level of g-H2AX

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Figure 2. In vitro therapeutic efficacy of olaparib and NanoOlaparib, alone, and in combination with radiation. A, Cell viability of three PTEN-deficient prostate cancer cell lines with differing radiation resistance including LNCaP (ARpos, p53wt), PC-3 (ARneg, p53neg), and FKO1 (ARinsensitive, p53neg), as measured by cell counting nine population doublings after 24-hour pretreatment with olaparib or NanoOlaparib. B, Representative images of FKO1 colony formation after one-time irradiation followed by 14 days of continuous drug treatment with free olaparib or NanoOlaparib containing 1 mmol/L drug. Cells treated with 2 to 10 Gy radiation þ drug were seeded at 3,000 cells/well, all others were seeded at 6,000 cells/well. Cells were pretreated with drug 24 hours prior to irradiation. C, Quantitative analysis of the colony formation assay. Mean values SD. expression in a radiation-insensitive manner. In contrast, cells cancer with PTEN (40) or BRCA (41) mutations, and may treated with NanoOlaparib displayed a clear radiation dose- reflect impaired nuclear transport of Rad51 (42). dependent enhancement in g-H2AX expression. At 10 Gy, NanoOlaparib pretreatment resulted in a damage enhancement NanoOlaparib in combination with radiation enhances tumor factor of 4.2 relative to vehicle-treated controls. growth inhibition Rad51, a recombinase that assists in the repair of dsDNA The ability of NanoOlaparib to overcome radiation-resistance breaks (37), and polq, a DNA polymerase that can promote in vivo was assessed using FKO1 cells (derived from Ptenpc-/ ; both homologous recombination (38) and microhomology- Trp53pc-/ mice; ref. 26) implanted subcutaneously in nude mice. mediated end-joining (39) in response to dsDNA breaks, were Given the large number of mice enrolled in the study and the need also examined for changes in protein expression. Both Rad51 for regular tumor size monitoring, a subcutaneous model was and polq foci were observed in the nuclei of cells following selected over the spontaneous prostate cancer model. Mice NanoOlaparib treatment (Supplementary Fig. S2); however, received one of four treatments: untreated, 10 Gy radiation, neither the number of foci nor the fraction of cells expressing 40 mg/kg NanoOlaparib (i.v. twice-weekly), or 40 mg/kg Nano- foci increased following combination therapy (NanoOlaparib Olaparib (i.v. twice-weekly) þ 1 10 Gy radiation. Tumors þ 6 Gy radiation). Only Rad51 expression in the nucleus was receiving combination therapy were pretreated three times (on found to colocalize with g-H2AX staining. Cytoplasmic Rad51 days 0, 3, 7) with NanoOlaparib and then irradiated on day 9, staining was found to increase transiently in a drug-dose when the tumor size matched that of the radiation only controls dependent manner following irradiation, with peak staining (irradiated on day 1). For both irradiated and non-irradiated observed 30 minutes after radiation (Supplementary Fig. S1A mice, NanoOlaparib treatment was continued twice weekly for and S1C). Cytoplasmic Rad51 staining correlated linearly with the duration of the study. The individual tumor response is shown increasing NanoOlaparib concentration, resulting in a 4.7-fold in Fig. 4A and averaged group response is shown in Fig. 4B. increase in Rad51 staining compared to non-irradiated cells Irradiated FKO1 tumors demonstrated radiation-resistance in vivo, treated with an equivalent dose of NanoOlaparib (Supplemen- growing at the same average rate as untreated controls. Nano- tary Fig. S1D). This pattern of staining has previously been Olaparib-treated tumors demonstrated a slower rate tumor shown to be a clinical hallmark of highly aggressive prostate growth compared to mice receiving radiation and untreated

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different treatment groups showed clear cytological differences. Untreated and irradiated tumors showed densely packed, viable cells throughout the tumor volume. NanoOlaparib-treated tumors showed regional variations in tumor cell density but no necrosis. Relapsed tumors (NanoOlaparib þ 10 Gy radiation) were characterized by large areas of necrosis and swollen, vacuolated cells surrounded by a dense border of cells only at the tumor margin (Fig. 5B).

NanoOlaparib increases nanoparticle accumulation in tumors Given the apparent long-term benefit of administering Nano- Olaparib before and after a single dose of radiation, we sought to determine whether NanoOlaparib administration changes drug delivery to the tumor, using ferumoxytol as a surrogate nanoparticle (Fig. 6). Ferumoxytol accumulation was imaged 24 hours after nanoparticle administration using ultra-short time-to-echo (UTE) MRI, a technique that produces positive-contrast images of ferumoxytol (33). In longitudinal experiments, multiple ferumoxytol boluses were administered sufficiently apart (Fig. 6A) to allow for blood pool clearance (33). Figure 6B shows representative T1,T2, and UTE signal intensity images from a single longitudinal experiment, collected in a mouse treated with a lipid vehicle followed by NanoOlaparib. Here, darkening of the T1 and T2 images, and brightening of the UTE images, indicate the presence of ferumoxytol. A small amount of ferumoxytol accumulation was observed following vehicle pre-treatment; however, this accumulation was visibly enhanced following NanoOlaparib pretreatment. The UTE signal intensity was measured for each voxel in the tumor volume and plotted as a histogram (Fig. 6C). Ferumoxytol accumulation appeared as an extended tail that diverged from the precontrast histogram (no ferumoxytol) at a UTE signal intensity of approximately 750. To determine the Figure 3. relative signal enhancement with NanoOlaparib treatment, the Immunostaining for biomarkers of DNA damage and repair. A, Representative confocal microscopy images of nuclear g-H2AX (red) and DAPI (blue) staining in fold-increase in voxel number was plotted as a function of signal FKO1 cells 30 minutes following irradiation. Cells were pretreated for 24 hours intensity (Fig. 6D). At any given UTE signal intensity, up to a four- with 1 mmol/L NanoOlaparib, olaparib, or a vehicle control before irradiation. fold increase in the number of voxels was observed, demonstrat- B, Quantification of mean g-H2AX immunostaining per nucleus as a function of ing that NanoOlaparib-enhanced ferumoxytol accumulation is radiation dose. higher than would be expected for a second bolus of ferumoxytol alone. To assess the reproducibility of NanoOlaparib-enhanced accu- controls. All tumors treated with combination therapy showed a mulation, the percentage of signal-enhanced voxels with feru- decrease in tumor size following irradiation, and in three of six moxytol accumulation (defined here as a UTE signal above 750) mice, the tumors continued to shrink until completely gone. The was quantified across 11 different tumors treated NanoOlaparib, average rate of tumor growth following combination therapy 1 10 Gy radiation, or no treatment (Fig. 6E). Tumors treated showed increased variability with time as a result of three tumors with a single dose of NanoOlaparib demonstrated ferumoxytol growing while three were shrinking. accumulation in 18.5 8% of voxels, an overall 5- and 19-fold Enhanced survival was observed for mice treated with enhancement compared to irradiated tumors and untreated NanoOlaparib and combination therapy (Fig. 4C), with a tumors, respectively. The overall distribution of ferumoxytol median survival time of 33 days (NanoOlaparib) and 66 days accumulation was observed to vary widely from tumor-to-tumor (combination) compared to 22 days (controls and radiation when rendered as maximum intensity projection images, gener- only). In mice receiving combination therapy, the average time ally appearing as one or more foci of high accumulation sur- to reach 1,500 mm3 was found to be 48 days for those with rounded by large areas of diffuse accumulation (Fig. 6F). relapsing tumors (n ¼ 3; Fig. 4D) whereas the remaining mice showed no tumor relapse over the 90-day observation period (n ¼ 3). No significant weight loss was observed for all treat- Discussion ment groups (Fig. 4E). For mice treated with NanoOlaparib, no There is a compelling need for developing an intravenous adverse correlates of hematopoietic damage were found in the formulation of olaparib. Like chemotherapy, olaparib is organs of mononuclear phagocyte system, including the liver, administered at the maximum tolerated dose (400 mg b.i.d.) spleen, kidney, lung, and heart (Fig. 5A). Minor necrosis and because only a small fraction of consumed drug reaches the inflammation appeared in the heart of only one mouse that tumor. Oral olaparib pharmacokinetics is far from ideal: the received both radiation and NanoOlaparib. The tumors of drug is metabolized rapidly into nonfunctional metabolites

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Figure 4. Effect of NanoOlaparib alone or in combination with radiation on a FKO1 allograft. A, Individual growth curves of mice randomized into three treatment groups: 1 10 Gy radiation, twice-weekly i.v. NanoOlaparib (40 mg/kg), or twice-weekly NanoOlaparib (40 mg/kg) þ 1 10 Gy radiation. NanoOlaparib treatment was continued for the duration of the study. B, Average rate of tumor growth (n ¼ 6–7) to treatment, shown as mean SE. C, Overall survival, with mice sacrificed when tumors reached 1,500 mm3. D, Time required for tumors to reach 1,500 mm3, shown as mean SE. , three mice omitted due to complete response as of day 90. E, Average group weight, measured on Day 0 and the day of sacrifice. and thus patients must swallow up to 16 capsules per day to tant cells in vitro at both high and low doses of radiation. maintain effective drug concentrations. Up to 80% of patients Considering the poor bioavailability of oral olaparib, we would can expect to experience one or more adverse events including expect this difference to be further magnified when a bioequiva- nausea, fatigue, or diarrhea (43). In contrast, nanoformulation lent dose is administered in vivo. Second, NanoOlaparib does allows poorly soluble drugs such as olaparib to be administered not raise the basal level of g-H2AX expression but instead directly into the bloodstream and circulate longer. This enhances g-H2AX expression in a radiation-dose dependent man- increased bioavailability is expected to enhance drug's thera- ner. Given that g-H2AX expression generally correlates with peutic efficacy at clinically equivalent doses or below, opening dsDNA damage, this suggests NanoOlaparib may be less toxic possibilities to reduce the olaparib dose and thereby ameliorate than the free drug in tissues not receiving radiation. Third, the systemic side effects. damage enhancement factor of NanoOlaparib was significantly The nanoformulation described here has several unique advan- higher (P < 0.05) than that of olaparib for all radiation doses tages that are not observed with the free drug. First, the nano- above 2 Gy. In stereotactic body radiation therapy (SBRT) for formulation has enhanced therapeutic efficacy in radiation-resis- prostate cancer ablation, patients can safely receive five fractions

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Figure 5. Tissue histology. A, Representative H&E staining of the liver, spleen, kidney, heart, and lung of mice that received 40 mg/kg twice-weekly NanoOlaparib or no treatment. B, Representative H&E staining of FKO1 tumors from mice receiving no treatment, 1 10 Gy radiation, and i.v. NanoOlaparib (50 days) þ 10 Gy radiation.

consisting of 9 to 10 Gy each (44), thus it is realistic to envision that NanoOlaparib can act synergistically with radiation to over- combining NanoOlaparib with high doses of radiation. For these come radiation-resistant tumors. NanoOlaparib alone did show reasons, we chose to study the therapeutic benefit of combining statistically significant growth inhibition and enhanced overall NanoOlaparib and radiation in cells derived from Ptenpc / ; survival. Given that previous reports of Olaparib monotherapy in Trp53pc / mice that have previously been reported as unrespon- Ptenpc-/ ;Trp53pc-/- mice demonstrated no tumor growth inhibi- sive to olaparib monotherapy (26). tion (26), we believe this difference is due to improved delivery Cancers lacking homologous recombination (HR) capacity are and/or uptake of olaparib. generally sensitive to PARP inhibition. Here we looked at polq,a Several PARP inhibitors exhibit vasoactive properties, allowing protein whose expression is related to HR. Polq expression is for increased tumor oxygenation and/or improved drug delivery frequently elevated in cancers with HR deficiency (45), likely due (20, 21, 48). Olaparib has been reported to transiently enhance its secondary capacity as a promoter of microhomology-mediated tumor vessel perfusion in non–small cell lung cancer xenografts end-joining (39). The restoration of normal HR function in these (20), which may explain the enhanced sensitivity of these tumors cells reduces polq expression to normal levels (38), thus polq to olaparib in combination with fractionated radiation. Here, expression is believed to inversely correlate with HR activity. In changes in nanoparticle accumulation were measured after a our studies, polq expression remained constant with treatment single dose of PARP inhibitor. We found that NanoOlaparib and no colocalization of polq was observed, suggesting that actively enhances nanoparticle accumulation in tumors by as alternative nonhomologous end-joining is not a dominant mech- much as 19-fold. Given the robust enhancement in median anism of dsDNA repair in FKO1 cells. Given the normal expres- survival time following NanoOlaparib þ radiation treatment, we sion of Polq, it is unsurprising that FKO1 cells are relatively speculate that the in vivo efficacy of NanoOlaparib may be poten- insensitive olaparib and NanoOlaparib monotherapy. tiated by its ability to enhance its own accumulation in tumors. NanoOlaparib clearly displayed radiosensitizer activity in vivo. Whether this behavior is mediated by changes in tumor perfusion, Because the half-life of peglyated lipid nanoparticles is generally or some other mechanism, remains to be determined. several days (46), mice were pretreated three times (days 0, 3, 7) Ferumoxytol accumulation has recently been shown to pre- with NanoOlaparib to allow drug accumulation. Mice then dict the localization of therapeutic nanoparticles in tumor received a single 10 Gy dose of radiation, selected to maximize microvasculature (49). Here, the use of ferumoxytol as a the radiation dose-enhancement factor of NanoOlaparib while surrogate provides interesting new insights into the treatment allowing rapid recovery of body weight after irradiation. Given efficacy data. We observed that ferumoxytol accumulation that radiation-induced DNA damage and genomic instability following NanoOlaparib treatment varied tumor by tumor, requires days to weeks to result in widespread cell death (47), with one tumor showing significantly less accumulation. This NanoOlaparib treatment was continued twice-weekly for the heterogeneity may account for some of the observed differences duration of the study. The single dose of radiation, when com- in radiosensitization. Interestingly, the three relapsed Nano- bined with NanoOlaparib pretreatment and maintenance, was Olaparib þ radiation tumors all showed a greater rate of sufficient to produce a complete response in half of the mice. This tumor growth during the NanoOlaparib pretreatment phase statistically significant response (P < 0.01) was well beyond that (days 0–9) than those with a complete response, suggesting observed by adding the effects of each monotherapy, indicating that these relapsed tumors were less responsive to NanoOlaparib

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Figure 6. Ferumoxytol delivery and accumulation in FKO1 tumors. A, Timeline for longitudinal MRI experiments involving multiple boluses of ferumoxytol. B, Representative negative- and positive-contrast MRI image slices of a single tumor before ferumoxytol injection (left), 24 hours after ferumoxytol injection with 4 hours vehicle pretreatment (middle), and 24 hours after ferumoxytol injection with 4 hours NanoOlaparib pretreatment (right). Ferumoxytol accumulation appeared as a darkening in the T1 and T2 images and a brightening of the UTE image. This tumor received 1 10 Gy radiation 3 weeks prior to MRI study. C, Histogram of the UTE signal intensity for all voxels within the tumor volume following each treatment. D, The fold-increase in voxel number as a function of signal intensity between the first and second rounds of ferumoxytol accumulation. E, The percentage of tumor volume with significant nanoparticle accumulation (as measured from the UTE signal intensity) following a single ferumoxytol injection in 11 different tumors pretreated with NanoOlaparib, radiation, or no treatment. Statistically significant differences (P < 0.01) were observed between the average of the indicated groups (). F, Two-dimensional maximum intensity projections of ferumoxytol accumulation in all 11 different tumors. treatment alone. Given that radiosensitization is determined by Development of methodology: A.L. van de Ven, S. Tangutoori, J. Qiao, the quantity of radiosensitizer present, as well as the distribution C. Gharagouzloo, R. Cormack, S. Sridhar of the radiosensitizer throughout the tumor (50), we would expect Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.L. van de Ven, S. Tangutoori, P. Baldwin, J. Qiao, that tumors with poor NanoOlaparib uptake would display a less C. Gharagouzloo, N. Seitzer, J.G. Clohessy, R. Cormack, S. Sridhar robust response to radiation. It may be that some FKO1 tumors are Analysis and interpretation of data (e.g., statistical analysis, biostatistics, intrinsically less conducive to nanoparticle accumulation, or computational analysis): A.L. van de Ven, S. Tangutoori, P. Baldwin, J. Qiao, alternatively, less amenable to NanoOlaparib-induced enhance- C. Gharagouzloo, N. Seitzer, R. Cormack, S. Sridhar ments in nanoparticle accumulation. Writing, review, and/or revision of the manuscript: A.L. van de Ven, In conclusion, we have shown that NanoOlaparib renders S. Tangutoori, C. Gharagouzloo, N. Seitzer, G.M. Makrigiorgos, R. Cormack, PTEN/TP53 fi S. Sridhar -de cient radiation-resistant tumors sensitive to radi- Administrative, technical, or material support (i.e., reporting or organizing ation. NanoOlaparib treatment is sufficient to trigger a significant data, constructing databases): S. Tangutoori, C. Gharagouzloo, P.P. Pandolfi, tumor response, and when administered in combination with S. Sridhar radiation, can lead to a complete response. The mechanism by Study supervision: S. Sridhar which nanoformulated olaparib sensitizes tumors to radiation is complex and likely extends beyond a simple enhancement in drug Acknowledgments accumulation, as NanoOlaparib displays several unique beha- FK01 cells were kindly donated by the Pandolfi group (BIDMC/Harvard viors in vitro that are not observed using an equivalent concen- Medical School). The authors thank Benjamin Geilich for his assistance with the tration of free drug. Additionally, NanoOlaparib appears to have TEM. We thank Dana-Farber/Harvard Cancer Center for the use of the Rodent Histopathology Core. the capacity to enhance nanoparticle accumulation in tumors and this may help potentiate its own accumulation. The results of this study show that NanoOlaparib may be a promising strategy for Grant Support enhancing the sensitivity of radiation-resistant tumors lacking This work was supported by the grants NSF-DGE-0965843, NIH HHS BRCA mutations, such as those with PTEN and TP53 deletions. U54CA151881, and CIMIT 13-1807 (to S. Sridhar), the Mazzone Foundation (to S. Sridhar and R. Cormack), and NIH NCIR01CA082328 (to P.P. Pandolfi). fl The costs of publication of this article were defrayed in part by the Disclosure of Potential Con icts of Interest payment of page charges. This article must therefore be hereby marked No potential conflicts of interest were disclosed. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: A.L. van de Ven, S. Tangutoori, C. Gharagouzloo, Received November 3, 2016; revised March 9, 2017; accepted April 28, 2017; R. Cormack, S. Sridhar published OnlineFirst May 12, 2017.

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Nanoformulation of Olaparib Amplifies PARP Inhibition and Sensitizes PTEN/TP53-Deficient Prostate Cancer to Radiation

Anne L. van de Ven, Shifalika Tangutoori, Paige Baldwin, et al.

Mol Cancer Ther 2017;16:1279-1289. Published OnlineFirst May 12, 2017.

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