[18F]-Labeled PARP-1 PET Imaging of PSMA Targeted Alpha Particle Radiotherapy Response

Motivation The growing interest and clinical translation of alpha particle (α) therapies brings with it new challenges to assess target cell engagement and to monitor therapeutic effect. Noninvasive imaging has great potential to guide α-treatment and to harness the potential of these agents in the complex environment of disseminated disease. Poly(ADP) ribose polymerase 1 (PARP-1) is among the most abundantly expressed DNA repair enzymes with key roles in multiple repair pathways - such as those induced by irradiation. Materials and Methods Here, we used a third-generation PARP1-specific radiotracer, [18F]-PARPZ, to delineate castrate resistant prostate cancer xenografts. Following treatment with the clinically applied [225Ac]-PSMA-617, positron emission tomography was performed and analyzed, along with and correlative autoradiography and histology acquired. Results [18F]-PARPZ was able to distinguish treated from control (saline) xenografts by uptake. Kinetic analysis of tracer accumulation also suggests that the localization of the agent to sites of increased PARP-1 expression is a consequence of DNA damage response. Conclusions Together, these data support expanded investigation of [18F]-PARPZ to facilitate clinical translation in the α-therapy space.

The double strand DNA breaks and severe genotoxic damage incurred by -traversal of the nucleus are difficult to repair. In controlled in vitro study, a single can kill a target cell 8 . However, the short path length of these emission means that many cells are spared any exposure; as evinced in part by the fact that these are not curative treatments 3,9 . Furthermore, we have shown previously that genomic factors such as DNA damage repair (DDR) status may influence sensitivity to -therapy 10 . We and others posit that detection of DDR, ideally through non-invasive methods, can be used to personalize treatment approaches [11][12][13][14] .
The multipathway mechanisms of eukaryotic DDR are intricate, orchestrated and tightly controlled processes. Summarily, these involve sensing of damage, signal transduction and recruitment of enzymatic complexes to remodel and repair DNA. Poly (ADP-ribose) polymerase 1 (PARP-1) is among the most well studied DDR proteins [15][16][17] and has critical roles in multiple single and double strand repair processes 18 . As such, inhibition of catalytic activity of this enzyme, and in particular the PARP-1 isoform, has been exploited to induce synthetic lethality in combination with loss of BRCA1 or BRCA2 function. The capacity to assess the PARP-1 status in a target cancer cell is emerging as a means to select patients for and to monitor PARP-1 inhibitor therapy 19,20 .
[ 18 F]PARPZ, also known as [ 18 F]WC-DZ-F, was developed as an analogue of [ 18 F]FluorThanatrace (FTT) and [ 125 I] KX1 which are well-established PARP-1 ligands for measuring PARP-1 expression. Our previous work has shown that this third-generation radiolabeled PARP-1 inhibitor has highly specific targeted uptake and favorable pharmacokinetic properties for use as a non-invasive imaging agent and targeted therapy 21 . In this study, we have evaluated the application of [ 18 F]PARPZ in a model of BRCA1 null castrate resistant prostate cancer (22Rv1) to detect DDR to in vivo -irradiation using [ 225 Ac]-PSMA-617 ( Figure 1). This chelate-conjugated peptide targets prostate specific membrane antigen (PSMA, also known as Glutamate carboxypeptidase II [GCPII]), a cell surface receptor of considerable clinical and research interest in prostate cancer 4,22,23 .

MATERIALS AND METHODS: Synthesis and Radiosynthesis of [ 18 F]PARPZ:
[ 18 F]PARPZ was synthesized and prepared for injection as previously described 21  F]-PARPZ: At room temperature, the above reaction mixture passed through a Waters MCX cartridge (100 mg in 1 mL cartridge, pretreated with 3 mL acetonitrile), following by acetonitrile (0.5 mL) for rinsing, and all were eluted into a 10 mL Pyrex tube containing precursor 1 (2.6 mg, 14.7 µmol)) and 10% Pd/C (6 mg) in methanol (0.5 mL). The tube was flushed with argon, and then capped firmly and heated at 120 °C in a heating block. After 20 min, the reaction was completed according to HPLC. The reaction mixture passed through a pad of Celite (pre-treated with acetonitrile), and the reaction tube and Celite were rinsed with acetonitrile (2 mL). All the eluted solution was collected in a tube, and solvents were removed under a flow of argon at 108 °C. The residue was dissolved in HPLC mobile phase/water 1:1 (4 mL), and injected onto HPLC via a nylon filter. After HPLC purification (Agilent SB-C18 250×9.4 mm, 17% acetonitrile/83% water/0.1% TFA, 4 mL/min, 250 nm), [ 18 F]-PARPZ (0.44 GBq/12 mCi) was collected at 11 min and diluted in water (40 mL). [ 18 F]-PARPZ was extracted from the solution by a standard solid phase extract procedure using a Waters HLB light and was formulated in 10% ethanol/saline.

Animal Studies:
All animal studies were performed under the Guide for the Care and Use of Laboratory Animals through the Washington University Animal Studies Committee. The tumor model used for these studies were 22Rv1-Luc cells (a gift of Dr. Ken Pienta of Johns Hopkins University) grown in conditions specified by the American Type Culture Collection. Four million cells were implanted in a 1:1 mixture of cells in DPBS (ThermoFisher) and matrigel (Corning) into NCI Athymic NCR-nu/nu male mice (8wk; Charles River/NCI).
Tumors were monitored by bioluminescence and caliper measurements, and used for treatment and imaging upon reaching approximately 500 mm 3 . A larger tumor volume than for traditional tumor control studies was chosen for the present investigations to preclude full local tumor control in the therapy group; reducing the variable of tumor size between groups.
Tumor bearing mice were anesthetized with isoflurane (2% mixture in 2L/min) and radioligand were delivered by injection into the retro-orbital sinus 24 . 300 nCi of [ 225 Ac]-PSMA-617 was administered to each of the animals in the treatment group. For dynamic imaging, an animal from each treatment and control group were simultaneously injected with approximately 200 µCi on the bed of the dedicated small animal tomograph (microPET R4, Concorde Microsystems), in triplicate, and imaged for 30 minutes. All static images were acquired for 10 minutes at between 110-130 minutes post-administration. Acquisitions were recorded using an energy window of 350-700 keV and coincidence-timing window of 6 ns. The 30 minute duration datasets were histogrammed to 5 minute frames and were reconstructed using a maximum a priori MAP and 3D filtered back projection using a ramp filter with a cut-off frequency equal to the Nyquist frequency into a 128 × 128 × 63 matrix. Region of interest analysis was conducted in ASIPro (v6.3.3.0, Concorde Microsystems) and statistical analyses were performed in Prism (v8.0, GraphPad Software).

Autoradiography and Immunohistochemistry:
Fresh frozen sections of [ 225 Ac]-PSMA-617 treated tumors were prepared and exposed, as previously 25,26 . Briefly, 8 µm thick sections in O.C.T. Compound (TissuePlus, Fisher Healthcare) were fixed opposite a storage phosphor screen (MS, PerkinElmer) at -20˚ C for 3 d. Imaging and analysis were performed on the CyclonePlus and Optiquant (v4.1, PerkinElmer). Contrast limited enhanced histogram equalization was performed on the lossless image and displayed with an optimized colormap (Viridis 27 ) using FIJI 28 .
Sectioned tissue slides were fixed briefly in 4% paraformaldehyde (Affymetrix), dried and stored. Slides were rehydrated before steaming in EDTA pH 8 (Invitrogen) for 40 min. Endogenous peroxidases were quenched with BLOXALL (Vector Labs), and the slides were blocked for 1 h with Serum Free Protein Block (Dako). Slides were incubated with antibodies directed against PARP-1 (Abcam ab194586). Staining was visualized with ImmPRESS Polymer detection kit and ImmPACT DAB (Vector Labs).

RESULTS: Radiochemistry:
[ 18 F]-PARPZ was synthesized from a 4fluorobenzaldehyde precursor using an improved procedure for greater yeild 21 . We achieved a radiochemical yield of 52% and radiochemical purity of 99.9%. at the end of synthesis. Specific activity of the tracer at preparation was 76 GBq/µmol (≥2000 mCi/µmol per synthesis). Baseline PARP-1 imaging was performed at 2 h post administration of [ 18 F]-PARPZ one day following the delivery [ 225 Ac]-PSMA-617 was administered 2 h prior to baseline PARP-1 imaging. We chose the 6 day treatment date for followup imaging as we would not expect significant differences between saline control and [ 225 Ac]-PSMA-617 treated 22Rv1 tumors at this interim time. The halflife of Actinium-225 is 10 days, and the energy deposited from the initially localized radiotherapy at day 6 will be roughly 35% of the total dose. Indeed tumor volumes between the groups were not significantly different at either imaging dates.

[ 18 F]-PARPZ PET:
We performed PET imaging of treated and control groups at day 1 and day 6 post-administration. Figure 2 shows coronal slices (left) and maximum intensity projection (right) images for representative control untreated (top) and [ 225 Ac]-PSMA-617 treated (bottom) animals from the two imaging time points. As expected, hepatobiliary clearance resulted in a majority of PARP tracer activity in the intestine at the imaging timepoints indicated in both baseline and treatment PET acquisitions. Distinction of the tumor can be made in all subjects using [ 18 F]-PARPZ (as annotated with an arrow, Figure 2A-D). Qualitatively, there are no notable differences in tracer distribution or tumor delineation between the control and treated subjects at day 1 or the untreated animals at day 6. Prominent uptake in the treated tumor in the latter (day 6) imaging session ( Figure 2D) is observed, and more clearly resolved in the projection imaging data ( Figure 2H).
Quantitation of [ 18 F]-PAPRZ PET uptake in tumors was assessed noninvasively and compared across control and treatment groups between the two imaging timepoints (Figures 3A and 3C). No significant difference in the tracer localization was noted between control and [ 225 Ac]PSMA-617 groups at the initial baseline (1 day post-administration) scan. Likewise, no difference in mean or maximum tumor uptake was noted in the untreated group between day 1 and day 6 timepoints. In contrast, there was both a significant difference in PARP-1 tracer localization (mean percent injected activity per gram) for the treated group between the initial and latter scan dates (P<0.01); as well as a measurable difference between the control and treated tumor uptake at day 6 (P<0.05). Maximum tumor uptake as percent injected activity per gram was also significant between the treated animals across the two imaging timepoints (P=0.05).
The change in mean and maximum [ 18 F]-PAPRZ uptake on an individual subject basis is shown in Figures 3B and 3D. The maximum intensity values increase in nearly all tumors investigated. We surmise that the increase in this metric is a function of both therapy induced DNA A subset of animals were imaged on-camera upon tracer administration at the 6 day post-treatment imaging session in order to investigate the kinetics of tumor localization. The (mean) percent injected activity per gram accumulation in the tumor was recorded across 180 second frames out to 30 minutes. Two representative animals from each saline control and treated groups were imaged together on the imaging bed, and are plotted in Figure 4. These kinetic scans show more rapid uptake at an increased magnitude for the [ 225 Ac]-PSMA-617 treated subjects.

Autoradiography and Immunohistochemistry:
The dissected tumor tissue were sectioned and evaluated for [ 225 Ac]-PSMA-617 signal by autoradiography in the treatment group and both groups were immunostained for PARP-1 ( Figure 5). These tumors expressed PARP-1 either throughout, or in defined, non-stromal areas. The areas for which PARP-1 staining was present generally correlated with [ 225 Ac]-PSMA-617 localization by phosphor autoradiography. No significant difference in staining intensity was observed between the control and treated groups. In the control group there was a trend towards more mild PARP-1 staining.

DISCUSSION:
We have evaluated the utility of a PARP-1 tracer to delineate responses to targeted alpha particle radiotherapy in a mouse model of prostate cancer. This work shows that we are able to quantitatively distinguish [ 18 F]-PARPZ uptake in tumors treated with an alpha particle emitting targeted radiotherapy. The general focus of extant PARP-1 positron emitting radiotracer work has been to evaluate and validate PARP-1 expression for eventual clinical use 19,20,29 . This may provide the capacity to characterize PARP-1 expression in patients which may help to guide selection and dosing of PARP inhibitors, of which several are clinically approved for various indications 30 .
Previous investigations of positron emitting PARP-1 tracers to noninvasively measure the activation of DNA repair cascades invoked in tumors by external beam X-ray irradiation have been published using [ 18 F]-  FluorThanatrace and [ 18 F]-Olaparib 11,29 . A direct comparison between the various PARP-1 imaging agents published to date is difficult, as various oncologic models and therapies have been utilized 11,21,[31][32][33][34] . What distinguishes this work is the use of [ 18 F]-PARPZ, with minimal degradation or metabolic products 21 , comparatively very high tumor localization (at roughly 10% Mean IA/g and 20% Maximum IA/g; Figures 2,3), sustained uptake ( Figure 4) and the use of potent alpha particle radiotherapy directed to a widely investigated prostate cancer radioligand target. While it is difficult to draw significant conclusions regarding the differences in PARP-1 expression following treatment, we were able to generally correlated PARP-1 expression IHC with areas of radiotherapeutic localization by autoradiography.
The differences in tracer kinetics in the tumors between treated and control groups (

CONCLUSIONS:
We are able to measure significant increases in PARP imaging tracer localization to targeted -therapy treated tumors in a model system of prostate cancer. The improved imaging characteristics and stability of [ 18 F]-PARPZ coupled with our institution's successful translation and implementation of a previousgeneration agent, [ 18 F]FTT, lay the ground-work for the initiation of clinical investigation of DNA damage response imaging in patients treated with both approved (Radium-223 dichloride) and investigational alpha particle radiotherapy.

ACKNOWLEDGEMENTS:
This work was supported by R01-CA201035, AND R01-CA229893. The Siteman Cancer Center is funded by NCI Cancer Center Support Grant #P30 CA091842.