Beyond Average: α-Particle Distribution and Dose Heterogeneity in Bone Metastatic Prostate Cancer.

α-particle emitters are emerging as a potent modality for disseminated cancer therapy because of their high linear energy transfer and localized absorbed dose profile. Despite great interest and pharmaceutical development, there is scant information on the distribution of these agents at the scale of the α-particle pathlength. We sought to determine the distribution of clinically approved [223Ra]RaCl2 in bone metastatic castration-resistant prostate cancer at this resolution, for the first time to our knowledge, to inform activity distribution and dose at the near-cell scale. Methods: Biopsy specimens and blood were collected from 7 patients 24 h after administration. 223Ra activity in each sample was recorded, and the microstructure of biopsy specimens was analyzed by micro-CT. Quantitative autoradiography and histopathology were segmented and registered with an automated procedure. Activity distributions by tissue compartment and dosimetry calculations based on the MIRD formalism were performed. Results: We revealed the activity distribution differences across and within patient samples at the macro- and microscopic scales. Microdistribution analysis confirmed localized high-activity regions in a background of low-activity tissue. We evaluated heterogeneous α-particle emission distribution concentrated at bone-tissue interfaces and calculated spatially nonuniform absorbed-dose profiles. Conclusion: Primary patient data of radiopharmaceutical therapy distribution at the small scale revealed that 223Ra uptake is nonuniform. Dose estimates present both opportunities and challenges to enhance patient outcomes and are a first step toward personalized treatment approaches and improved understanding of α-particle radiopharmaceutical therapies.

Evaluation of Candidate Theranostics for 227Th/89Zr Paired Radioimmunotherapy of Lymphoma.

227Th is a promising radioisotope for targeted α-particle therapy. It produces 5 α-particles through its decay, with the clinically approved 223Ra as its first daughter. There is an ample supply of 227Th, allowing for clinical use; however, the chemical challenges of chelating this large tetravalent f-block cation are considerable. Using the CD20-targeting antibody ofatumumab, we evaluated chelation of 227Th4+ for α-particle-emitting and radiotheranostic applications. Methods: We compared 4 bifunctional chelators for thorium radiopharmaceutical preparation: S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2-(4-isothicyanatobenzyl)-1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid (p-SCN-Bn-HEHA), p-isothiacyanatophenyl-1-hydroxy-2-oxopiperidine-desferrioxamine (DFOcyclo*-p-Phe-NCS), and macrocyclic 1,2-HOPO N-hydroxysuccinimide (L804-NHS). Immunoconstructs were evaluated for yield, purity, and stability in vitro and in vivo. Tumor targeting of the lead 227Th-labeled compound in vivo was performed in CD20-expressing models and compared with a companion 89Zr-labeled PET agent. Results: 227Th-labeled ofatumumab-chelator constructs were synthesized to a radiochemical purity of more than 95%, excepting HEHA. 227Th-HEHA-ofatumumab showed moderate in vitro stability. 227Th-DFOcyclo*-ofatumumab presented excellent 227Th labeling efficiency; however, high liver and spleen uptake was revealed in vivo, indicative of aggregation. 227Th-DOTA-ofatumumab labeled poorly, yielding no more than 5%, with low specific activity (0.08 GBq/g) and modest long-term in vitro stability (<80%). 227Th-L804-ofatumumab coordinated 227Th rapidly and efficiently at high yields, purity, and specific activity (8 GBq/g) and demonstrated extended stability. In vivo tumor targeting confirmed the utility of this chelator, and the diagnostic analog, 89Zr-L804-ofatumumab, showed organ distribution matching that of 227Th to delineate SU-DHL-6 tumors. Conclusion: Commercially available and novel chelators for 227Th showed a range of performances. The L804 chelator can be used with potent radiotheranostic capabilities for 89Zr/227Th quantitative imaging and α-particle therapy.

Quantitative In Vivo Imaging of the Androgen Receptor Axis Reveals Degree of Prostate Cancer Radiotherapy Response.

Noninvasive biomarkers for androgen receptor (AR) pathway activation are urgently needed to better monitor patient response to prostate cancer therapies. AR is a critical driver and mediator of resistance of prostate cancer but currently available noninvasive prostate cancer biomarkers to monitor AR activity are discordant with downstream AR pathway activity. External beam radiotherapy (EBRT) remains a common treatment for all stages of prostate cancer, and DNA damage induced by EBRT upregulates AR pathway activity to promote therapeutic resistance. [89Zr]11B6-PET is a novel modality targeting prostate-specific protein human kallikrein 2 (hK2), which is a surrogate biomarker for AR activity. Here, we studied whether [89Zr]11B6-PET can accurately assess EBRT-induced AR activity.Genetic and human prostate cancer mouse models received EBRT (2-50 Gy) and treatment response was monitored by [89Zr]11B6-PET/CT. Radiotracer uptake and expression of AR and AR target genes was quantified in resected tissue.EBRT increased AR pathway activity and [89Zr]11B6 uptake in LNCaP-AR and 22RV1 tumors. EBRT increased prostate-specific [89Zr]11B6 uptake in prostate cancer-bearing mice (Hi-Myc x Pb_KLK2) with no significant changes in uptake in healthy (Pb_KLK2) mice, and this correlated with hK2 protein levels. IMPLICATIONS: hK2 expression in prostate cancer tissue is a proxy of EBRT-induced AR activity that can noninvasively be detected using [89Zr]11B6-PET; further clinical evaluation of hK2-PET for monitoring response and development of resistance to EBRT in real time is warranted.

[18F]-Labeled PARP-1 PET imaging of PSMA targeted alpha particle radiotherapy response.

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. 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 correlative autoradiography and histology acquired. [18F]-PARPZ was able to distinguish treated from control (saline) xenografts by increased 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. Together, these data support expanded investigation of [18F]-PARPZ to facilitate clinical translation in the ⍺-therapy space.

Prostate cancer polygenic risk score and prediction of lethal prostate cancer.

Polygenic risk scores (PRS) for prostate cancer incidence have been proposed to optimize prostate cancer screening. Prediction of lethal prostate cancer is key to any stratified screening program to avoid excessive overdiagnosis. Herein, PRS for incident prostate cancer was evaluated in two population-based cohorts of unscreened middle-aged men linked to cancer and death registries: the Västerbotten Intervention Project (VIP) and the Malmö Diet and Cancer study (MDC). SNP genotypes were measured by genome-wide SNP genotyping by array followed by imputation or genotyping of selected SNPs using mass spectrometry. The ability of PRS to predict lethal prostate cancer was compared to PSA and a commercialized pre-specified model based on four kallikrein markers. The PRS was associated with incident prostate cancer, replicating previously reported relative risks, and was also associated with prostate cancer death. However, unlike PSA, the PRS did not show stronger association with lethal disease: the hazard ratio for prostate cancer incidence vs. prostate cancer metastasis and death was 1.69 vs. 1.65 in VIP and 1.25 vs. 1.25 in MDC. PSA was a much stronger predictor of prostate cancer metastasis or death with an area-under-the-curve of 0.78 versus 0.63 for the PRS. Importantly, addition of PRS to PSA did not contribute additional risk stratification for lethal prostate cancer. We have shown that a PRS that predicts prostate cancer incidence does not have utility above and beyond that of PSA measured at baseline when applied to the clinically relevant endpoint of prostate cancer death. These findings have implications for public health policies for delivery of prostate cancer screening. Focusing polygenic risk scores on clinically significant endpoints such as prostate cancer metastasis or death would likely improve clinical utility.

Improved Radium-223 Therapy with Combination Epithelial Sodium Channel Blockade.

Background: Radium-223 dichloride ([223Ra]RaCl2) is the first approved alpha particle-emitting therapy and is indicated for treatment of bone metastatic castrate resistant prostate cancer. Approximately half of the dose is absorbed into the gastrointestinal (GI) tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigate the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Utilizing primary human duodenal organoids (enteroids) as in vitro models of the functional GI epithelium, we found that Amiloride (ENaC blocker) and NS-1619 (K+ channel activator) presented significant effects in 223Ra membranal transport. The radioactive drug distribution was evaluated for lead combinations in vivo, and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223Ra uptake in vivo from the gut, to nearly double the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent and X-ray imaging was significantly greater than single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may be readily implemented as a clinical approach to improve outcomes of bone metastatic cancer patients with the benefit of ameliorated tolerability.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

PSA-Targeted Alpha-, Beta-, and Positron-Emitting Immunotheranostics in Murine Prostate Cancer Models and Nonhuman Primates.

PURPOSE: Most patients with prostate cancer treated with androgen receptor (AR) signaling inhibitors develop therapeutic resistance due to restoration of AR functionality. Thus, there is a critical need for novel treatment approaches. Here we investigate the theranostic potential of hu5A10, a humanized mAb specifically targeting free PSA (KLK3). EXPERIMENTAL DESIGN: LNCaP-AR (LNCaP with overexpression of wildtype AR) xenografts (NSG mice) and KLK3_Hi-Myc transgenic mice were imaged with 89Zr- or treated with 90Y- or 225Ac-labeled hu5A10; biodistribution and subcellular localization were analyzed by gamma counting, PET, autoradiography, and microscopy. Therapeutic efficacy of [225Ac]hu5A10 and [90Y]hu5A10 in LNCaP-AR tumors was assessed by tumor volume measurements, time to nadir (TTN), time to progression (TTP), and survival. Pharmacokinetics of [89Zr]hu5A10 in nonhuman primates (NHP) were determined using PET. RESULTS: Biodistribution of radiolabeled hu5A10 constructs was comparable in different mouse models. Specific tumor uptake increased over time and correlated with PSA expression. Treatment with [90Y]/[225Ac]hu5A10 effectively reduced tumor burden and prolonged survival (P ≤ 0.0054). Effects of [90Y]hu5A10 were more immediate than [225Ac]hu5A10 (TTN, P < 0.0001) but less sustained (TTP, P < 0.0001). Complete responses were observed in 7 of 18 [225Ac]hu5A10 and 1 of 9 mice [90Y]hu5A10. Pharmacokinetics of [89Zr]hu5A10 were consistent between NHPs and comparable with those in mice. [89Zr]hu5A10-PET visualized the NHP-prostate over the 2-week observation period. CONCLUSIONS: We present a complete preclinical evaluation of radiolabeled hu5A10 in mouse prostate cancer models and NHPs, and establish hu5A10 as a new theranostic agent that allows highly specific and effective downstream targeting of AR in PSA-expressing tissue. Our data support the clinical translation of radiolabeled hu5A10 for treating prostate cancer.

Fibrillar pharmacology of functionalized nanocellulose.

Cellulose nanocrystals (CNC) are linear organic nanomaterials derived from an abundant naturally occurring biopolymer resource. Strategic modification of the primary and secondary hydroxyl groups on the CNC introduces amine and iodine group substitution, respectively. The amine groups (0.285 mmol of amine per gram of functionalized CNC (fCNC)) are further reacted with radiometal loaded-chelates or fluorescent dyes as tracers to evaluate the pharmacokinetic profile of the fCNC in vivo. In this way, these nanoscale macromolecules can be covalently functionalized and yield water-soluble and biocompatible fibrillar nanoplatforms for gene, drug and radionuclide delivery in vivo. Transmission electron microscopy of fCNC reveals a length of 162.4 ± 16.3 nm, diameter of 11.2 ± 1.52 nm and aspect ratio of 16.4 ± 1.94 per particle (mean ± SEM) and is confirmed using atomic force microscopy. Size exclusion chromatography of macromolecular fCNC describes a fibrillar molecular behavior as evidenced by retention times typical of late eluting small molecules and functionalized carbon nanotubes. In vivo, greater than 50% of intravenously injected radiolabeled fCNC is excreted in the urine within 1 h post administration and is consistent with the pharmacological profile observed for other rigid, high aspect ratio macromolecules. Tissue distribution of fCNC shows accumulation in kidneys, liver, and spleen (14.6 ± 6.0; 6.1 ± 2.6; and 7.7 ± 1.4% of the injected activity per gram of tissue, respectively) at 72 h post-administration. Confocal fluorescence microscopy reveals cell-specific accumulation in these target tissue sinks. In summary, our findings suggest that functionalized nanocellulose can be used as a potential drug delivery platform for the kidneys.

Expression and clinical implications of leucine-rich repeat containing 15 (LRRC15) in osteosarcoma.

Leucine-rich repeat containing 15 (LRRC15) is a member of the leucine-rich repeat superfamily that is overexpressed in various cancers and associated with higher tumor grade and aggression. Despite its known tumorigenicity, its roles within osteosarcoma are unknown, prompting us to evaluate its expression and clinical significance within this rare yet aggressive cancer. Western blots showed differential expression of LRRC15 in the osteosarcoma cell lines MNNG/HOS, KHOS, 143B, MG63, Saos-2, and U2OS. We additionally validated this positive expression, as well as sublocalization to the cell membrane, with immunofluorescence. A tissue microarray constructed from 69 osteosarcoma patient tissues was immunohistochemically stained for LRRC15 expression, stratified, and used for clinicopathological analysis. Publicly available databases on LRRC15 expression, including RNA sequencing data from the Therapeutically Applicable Research to Generate Effective Treatments on Osteosarcoma (TARGET-OS) and the Gene Expression database of Normal and Tumor tissues 2 (GENT2) were also analyzed. We found 63 of the 69 (91.3%) patient tissues exhibited some degree of LRRC15 immunostaining, including no staining (6 of 69, 8.7%), 1+ staining (12 of 69, 17.4%), 2+ staining (25 of 69, 36.2%), and 3+ staining (26 of 69, 37.7%). The patients with osteosarcomas having elevated LRRC15 expression demonstrated comparatively increased metastasis, chemoresistance, and shorter 5-year survival rates. Our analysis of the TARGET-OS and GENT2 databases also showed increased LRRC15 gene expression in osteosarcoma. Taken together, our study supports LRRC15 as a prognostic biomarker and emerging therapeutic target in osteosarcoma.

Genome-wide association study identifies novel single nucleotide polymorphisms having age-specific effect on prostate-specific antigen levels.

BACKGROUND: Testing for prostate-specific antigen (PSA) levels in blood are widely used and associated with prostate cancer risk and outcome. After puberty, PSA levels increase by age and multiple single nucleotide polymorphisms (SNPs) have been found to be associated with PSA levels. However, the relationship between the effects of SNPs and age on PSA remains unknown. METHODS: To test for SNP × age interaction, we conducted a genome-wide association study using 2394 men without prostate cancer diagnosis from Malmö, Sweden as a discovery set and 2137 men from the eMERGE study (USA) for validation. Linear regression was used to identify significant interactions between SNP and age (p < 1 × 10-4 for discovery, p < .05 for validation). RESULTS: The 15 SNPs from three different loci (8p11.22, 8p12, 3q25.31) are found to have age-specific effect on PSA levels. Expression quantitative trait loci (eQTLs) analysis shows that 12 SNPs from 3q25.31 locus affect the expression level of three genes: KCNAB1, SLC33A1, PLCH1. CONCLUSIONS: Our results suggest that SNPs may have age-specific effect on PSA levels, which provides new direction to study genetic markers for PSA.

Genetic signature of prostate cancer mouse models resistant to optimized hK2 targeted α-particle therapy.

Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.

Preclinical Single Photon Emission Computed Tomography of Alpha Particle-Emitting Radium-223.

Objective: Dose optimization and pharmacokinetic evaluation of α-particle emitting radium-223 dichloride (223RaCl2) by planar γ-camera or single photon emission computed tomography (SPECT) imaging are hampered by the low photon abundance and injected activities. In this study, we demonstrate SPECT of 223Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223Ra were imaged using a dedicated small animal SPECT by detecting the low-energy photon emissions from 223Ra. Localization of the therapeutic agent was verified by whole-body and whole-limb autoradiography and its radiobiological effect confirmed by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction at reasonable administered activities and acquisition times. Line sources of 223Ra in both air and in a water scattering phantom gave a line spread function with a full-width-at-half-maximum of 1.45 mm. Early and late-phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling was correlated with DNA damage from the α particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223RaCl2, a recently approved α-particle-emitting radionuclide. This approach allows quantitative assessment of 223Ra distribution and may assist radiation-dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning.

Preclinical efficacy of hK2 targeted [177Lu]hu11B6 for prostate cancer theranostics.

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops. In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression. Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease. The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway. In a previous study, hu11B6 conjugated with Actinium-225 (225Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy. We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 (177Lu) and investigated whether similar tumor killing and AR-enhancement is produced. Moreover, single-photon emission computed tomography (SPECT) imaging of 177Lu is quantitatively accurate and can be used to perform treatment planning. [177Lu]hu11B6 therefore has significant potential as a theranostic agent. Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice. Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied. Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [177Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging. Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom. Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-ß-gal activity (senescence) and analyzed by digital autoradiography (DAR). Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg). Tumor accumulation of [177Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen. However, SPECT imaging with therapeutic amounts of [177Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177Lu and decreasing tumor volumes. For [177Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection. Tumor volume reduction correlated with injected activity and the absorbed dose. IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA- ß-gal activity (14 d). Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake. AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017). Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects. The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level. Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225Ac labeled hu11B6, however emerging at a later timepoint.

PSMA expression in the Hi-Myc model; extended utility of a representative model of prostate adenocarcinoma for biological insight and as a drug discovery tool.

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is highly overexpressed in primary and metastatic prostate cancer (PCa). This has led to the development of radiopharmaceuticals for targeted imaging and therapy under current clinical evaluation. Despite this progress, the exact biological role of the protein in prostate cancer development and progression has not been fully elucidated. This is in part because the human PSMA and mouse PSMA (mPSMA) have different patterns of anatomical expression which confound study in the most widely utilized model organisms. Most notably, mPSMA is not expressed in the healthy murine prostate. Here, we reveal that mPSMA is highly upregulated in the prostate adenocarcinoma of the spontaneous Hi-Myc mouse model, a highly accurate and well characterized mouse model of prostate cancer development. Antibody detection and molecular imaging tools are used to confirm that mPSMA is expressed from early prostatic intraepithelial neoplasia (PIN) through adenocarcinoma.

Harnessing Androgen Receptor Pathway Activation for Targeted Alpha Particle Radioimmunotherapy of Breast Cancer.

PURPOSE: The impact of androgen receptor (AR) activity in breast cancer biology is unclear. We characterized and tested a novel therapy to an AR-governed target in breast cancer.Experimental Design: We evaluated the expression of prototypical AR gene products human kallikrein 2 (hK2) and PSA in breast cancer models. We screened 13 well-characterized breast cancer cell lines for hK2 and PSA production upon in vitro hormone stimulation by testosterone [dihydrotestosterone (DHT)]. AR-positive lines were further evaluated by exposure to estrogen (17ß-Estradiol) and the synthetic progestin D-Norgestrel. We then evaluated an anti-hK2-targeted radiotherapy platform (hu11B6), labeled with alpha (α)-particle emitting Actinium-225, to specifically treat AR-expressing breast cancer xenografts under hormone stimulation. RESULTS: D-Norgestrel and DHT activated the AR pathway, while 17ß-Estradiol did not. Competitive binding for AR protein showed similar affinity between DHT and D-Norgestrel, indicating direct AR-ligand interaction. In vivo production of hK2 was sufficient to achieve site-specific delivery of therapeutic radionuclide to tumor tissue at >20-fold over background muscle uptake; effecting long-term local tumor control. CONCLUSIONS: [225Ac]hu11B6 targeted radiotherapy was potentiated by DHT and by D-Norgestrel in murine xenograft models of breast cancer. AR activity in breast cancer correlates with kallikrein-related peptidase-2 and can be activated by D-Norgestrel, a common contraceptive, and AR induction can be harnessed for hK2-targeted breast cancer α-emitter radiotherapy.

Genome-wide Scan Identifies Role for AOX1 in Prostate Cancer Survival.

BACKGROUND: Most men diagnosed with prostate cancer have low-risk cancers. How to predict prostate cancer progression at the time of diagnosis remains challenging. OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with death from prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Blood samples from 11 506 men in Sweden were collected during 1991-1996. Of these, 1053 men were diagnosed with prostate cancer and 245 died from the disease. Stage and grade at diagnosis and outcome information were obtained, and DNA from all cases was genotyped. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A total of 6 126 633 SNPs were tested for association with prostate-cancer-specific survival time using a Cox proportional hazard model, adjusted for age, stage, and grade at diagnosis. A value of 1×10-6 was used as suggestive significance threshold. Positive candidate SNPs were tested for association with gene expression using expression quantitative trait locus analysis. RESULTS AND LIMITATIONS: We found 12 SNPs at seven independent loci associated with prostate-cancer-specific survival time. One of 6 126 633 SNPs tested reached genome-wide significance (p<5×10-8) and replicated in an independent cohort: rs73055188 (p=5.27×10-9, per-allele hazard ratio [HR]=2.27, 95% confidence interval [CI] 1.72-2.98) in the AOX1 gene. A second SNP reached a suggestive level of significance (p<1×10-6) and replicated in an independent cohort: rs2702185 (p=7.1×10-7, per-allele HR=2.55, 95% CI=1.76-3.69) in the SMG7 gene. The SNP rs73055188 is correlated with AOX1 expression levels, which is associated with biochemical recurrence of prostate cancer in independent cohorts. This association is yet to be validated in other ethnic groups. CONCLUSIONS: The SNP rs73055188 at the AOX1 locus is associated with prostate-cancer-specific survival time, and AOX1 gene expression level is correlated with biochemical recurrence of prostate cancer. PATIENT SUMMARY: We identify two genetic markers that are associated with prostate-cancer-specific survival time.

Elective Nodal Irradiation Attenuates the Combinatorial Efficacy of Stereotactic Radiation Therapy and Immunotherapy.

Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB).Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN.Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8+ effector T cells as well as FoxP3+ regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8+ T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1.Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8+ T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058-71. ©2018 AACR.

The impact of age on radium-223 distribution and an evaluation of molecular imaging surrogates.

INTRODUCTION: Radium-223 dichloride is the first alpha-particle emitting therapeutic agent approved by FDA and EMA for bone metastatic castration-resistant prostate cancer. We studied its age-dependent biodistribution in mice, and compared it with [99mTc]Tc-MDP and [18F]NaF aiming to identify a potential imaging surrogate to predict [223Ra]RaCl2 whole-body localization. METHODS: Male C57Bl/6 mice dosed with [223Ra]RaCl2 were sacrificed at different time points to explore [223Ra]RaCl2 whole-body distribution. In another experiment, mice at different ages were dosed with [223Ra]RaCl2 to evaluate the aging impact. Finally, [99mTc]Tc-MDP and [18F]NaF were administered to mice, and we compared their biodistributions with [223Ra]RaCl2. Detailed micro-localization of each tracer was visualized using autoradiography and histochemical staining. RESULTS: [223Ra]RaCl2 uptake in bone was rapid and stable. We observed persistent localization at bone epiphyses, as well as the red pulp of the spleen, while its uptake in most soft tissues cleared within 24 h. [223Ra]RaCl2 distribution in soft tissues is similar in all age groups tested, while bone activity significantly decreased with aging. Although the diagnostic tracers cleared much faster from soft tissues than the therapeutic radionuclide, [99mTc]Tc-MDP and [18F]NaF both co-localized with [223Ra]RaCl2 in the skeletal compartment. CONCLUSIONS: Radium-223 localization to the bone is dependent on age-varying factors, which implies that radium-223 dosimetry should take patient age into account. [99mTc]Tc-MDP shows a different biodistribution from [223Ra]RaCl2, both in soft tissues and in bone. [18F]NaF presents a high similarity with [223Ra]RaCl2 in skeletal uptake, which validates the potential of [18F]NaF as an imaging surrogate to predict radium-223 radiotherapeutic distribution in bone.

Feed-forward alpha particle radiotherapy ablates androgen receptor-addicted prostate cancer.

Human kallikrein peptidase 2 (hK2) is a prostate specific enzyme whose expression is governed by the androgen receptor (AR). AR is the central oncogenic driver of prostate cancer (PCa) and is also a key regulator of DNA repair in cancer. We report an innovative therapeutic strategy that exploits the hormone-DNA repair circuit to enable molecularly-specific alpha particle irradiation of PCa. Alpha-particle irradiation of PCa is prompted by molecularly specific-targeting and internalization of the humanized monoclonal antibody hu11B6 targeting hK2 and further accelerated by inherent DNA-repair that up-regulate hK2 (KLK2) expression in vivo. hu11B6 demonstrates exquisite targeting specificity for KLK2. A single administration of actinium-225 labeled hu11B6 eradicates disease and significantly prolongs survival in animal models. DNA damage arising from alpha particle irradiation induces AR and subsequently KLK2, generating a unique feed-forward mechanism, which increases binding of hu11B6. Imaging data in nonhuman primates support the possibility of utilizing hu11B6 in man.