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Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) is effective against prostate cancer (PCa), but all patients relapse eventually. Poor understanding of the underlying resistance mechanisms represents a key barrier to development of more effective RLT. We investigate the proteome and phosphoproteome in a mouse model of PCa to identify signaling adaptations triggered by PSMA RLT. Methods: Therapeutic efficacy of PSMA RLT was assessed by tumor volume measurements, time to progression, and survival in C4-2 or C4-2 TP53-/- tumor-bearing nonobese diabetic scid γ-mice. Two days after RLT, the proteome and phosphoproteome were analyzed by mass spectrometry. Results: PSMA RLT significantly improved disease control in a dose-dependent manner. Proteome and phosphoproteome datasets revealed activation of genotoxic stress response pathways, including deregulation of DNA damage/replication stress response, TP53, androgen receptor, phosphatidylinositol-3-kinase/AKT, and MYC signaling. C4-2 TP53-/- tumors were less sensitive to PSMA RLT than were parental counterparts, supporting a role for TP53 in mediating RLT responsiveness. Conclusion: We identified signaling alterations that may mediate resistance to PSMA RLT in a PCa mouse model. Our data enable the development of rational synergistic RLT-combination therapies to improve outcomes for PCa patients.

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.

225Ac-PSMA-617 targeted-therapy has demonstrated efficacy in 75-85% of patients; however, responses are not durable. We aimed to establish translatable mouse models of disseminated prostate cancer (PCa) to evaluate effectiveness of 225Ac-PSMA-617 at various disease stages. Methods: C4-2, C4-2B, or 22Rv1 cells were injected into the left ventricle of male NSG mice. Disease progression was monitored using bioluminescence imaging (BLI). For treatment, mice were injected with 40 kBq 225Ac-PSMA-617 at one (early treatment cohort) or three weeks (late treatment cohort) post-inoculation. Treatment efficacy was monitored by BLI of whole-body tumor burden. Mice were sacrificed based on body conditioning score. Results: C4-2 cells yielded metastases in liver, lungs, spleen, stomach, bones, and brain - achieving a clinically relevant model of widespread metastatic disease. The disease burden in the early treatment cohort was stable over 27 weeks in 5/9 mice and progressive in 4/9 mice. These mice were sacrificed due to brain metastases. Median survival of the late treatment cohort was superior to controls (13 vs. 7 weeks; p

BackgroundProstate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) is a promising yet not curative approach in castration-resistant (CR) prostate cancer (PC). Rational combination therapies may improve treatment efficacy. Here, we explored the effect of androgen receptor blockade (ARB) on PSMA expression visualized by PET and its potential additive effect when combined with 177Lu-PSMA RLT in a mouse model of prostate cancer.MethodsMice bearing human CRPC (C4-2 cells) xenografts were treated with 10 mg/kg enzalutamide (ENZ), with 50 mg/kg bicalutamide (BIC), or vehicle (control) for 21 days. PSMA expression was evaluated by 68Ga-PSMA11 PET/CT and quantified by flow cytometry of tumor fine needle aspirations before treatment and on days 23, 29, 34, and 39 post-therapy induction. For the RLT combination approach, mice bearing C4-2 tumors were treated with 10 mg/kg ENZ or vehicle for 21 days before receiving either 15 MBq (84 GBq/μmol) 177Lu-PSMA617 or vehicle. DNA damage was assessed as phospho-γH2A.X foci in tumor biopsies. Reduction of tumor volume on CT and survival were used as study endpoints.ResultsTumor growth was delayed by ARB while 68Ga-PSMA11 uptake increased up to 2.3-fold over time when compared to controls. ABR-induced upregulation of PSMA expression was confirmed by flow cytometry. Phospho-γH2A.X levels increased 1.8- and 3.4-fold at 48 h in response to single treatment ENZ or RLT and ENZ+RLT, respectively. Despite significantly greater DNA damage and persistent increase of PSMA expression at the time of RLT, no additional tumor growth retardation was observed in the ENZ+RLT group (vs. RLT only, p = 0.372 at day 81). Median survival did not improve significantly when ENZ was combined with RLT.ConclusionARB-mediated increases in PSMA expression in PC xenografts were evident by 68Ga-PSMA11 PET imaging and flow cytometry. 177Lu-PSMA617 effectively decreased C4-2 tumor size. However, while pre-treatment with ARB increased DNA damage significantly, it did not result in synergistic effects when combined with RLT.

To improve prostate-specific membrane antigen (PSMA)-targeted theranostic approaches, robust murine models of prostate cancer are needed. However, important characteristics of preclinical PSMA imaging-that is, the reproducibility of the imaging signal and the relationship between quantitative cell surface PSMA expression and lesion detectability with small-animal PET/CT-have not been defined yet. Methods: Murine prostate cancer RM1 sublines (ras myc transformed cells of C57BL/6 prostate origin) expressing varying levels of human PSMA were injected into the shoulder of C57BL/6 mice on day 0. 68Ga-PSMA11 PET/CT was performed on days 7 and 8 and interpreted by 2 masked readers to determine interday and interreader reproducibility. PSMA expression was quantified on days 7 and 8 by flow cytometry of fine-needle aspiration tumor biopsy samples. Cell surface PSMA expression was correlated with PET signal. The threshold for PET positivity was based on the clinical Prostate Cancer Molecular Imaging Standardized Evaluation (PROMISE) criteria. Results: The maximum and average percentages of injected 68Ga-PSMA11 activity per gram of tissue (%IA/g) correlated nearly perfectly as determined by 2 independent readers and on 2 separate days (intraclass correlation coefficient, 1.00/0.89 and 0.95/0.88, respectively). The number of PSMA molecules per cell increased from the RM1-yellow fluorescent protein subline (PSMA-; 2,000/cell) to the RM1-low subline (PSMA+; 17,000/cell), the RM1-medium subline (PSMA++; 22,000/cell), and the RM1-PGLS subline (PSMA-positive, green fluorescent protein-positive, and luciferase-positive; PSMA+++; 45,000/cell). Expression levels correlated with the visual positivity rate on 68Ga-PSMA11 PET and with the PSMA PET %IA/g. The PSMA threshold for PET positivity was approximately 20,000 per cell. Signal correlation was close at lower PSMA levels (RM1-low to RM1-medium; 10-23 %IA/g) but was lost at higher PSMA levels (RM1-medium to RM1-PGLS; 23-27 %IA/g). Conclusion: The in vivo relationship between 68Ga-PSMA11 PET/CT and PSMA expression level in a murine model of prostate cancer was robust for lower cell surface PSMA expression levels (≤22,000/cell). Thus, preclinical 68Ga-PSMA11 PET/CT can be used as an imaging biomarker to test PSMA-targeted interventions in murine models.

Clinical 177Lu-PSMA-617 radioligand therapy (RLT) is applied in advanced-stage prostate cancer. However, to the best of our knowledge murine models to study the biologic effects of various activity levels have not been established. The aim of this study was to optimize specific and total activity for 177Lu-PSMA-617 RLT in a syngeneic model of murine prostate cancer. Methods: Murine-reconstituted, oncogene-driven prostate cancer cells (0.1 × 106) (RM1), transduced to express human prostate-specific membrane antigen (PSMA), were injected into the left flank of C57Bl6 immunocompetent mice. RLT was performed by administering a single tail vein injection of 177Lu-PSMA-617 at different formulations for specific (60 MBq at high, 62 MBq/nmol; intermediate, 31 MBq/nmol; or low 15 MBq/nmol specific activity) or total activity (30, 60, or 120 MBq). Organ distribution was determined by ex vivo γ-counter measurement. DNA double-strand breaks were measured using anti-gamma-H2A.X (phospho S139) immunohistochemistry. Efficacy was assessed by serial CT tumor volumetry and 18F-FDG PET metabolic volume. Toxicity was evaluated 4 wk after the start of RLT. Results: Mean tumor-to-kidney ratios ± SEM were 19 ± 5, 10 ± 5, and 2 ± 0 for high, intermediate, and low (each n = 3) specific activity, respectively. Four of 6 (67%) mice treated with intermediate or high specific activity and none of 6 (0%) mice treated with low specific activity or formulation demonstrated significant DNA double-strand breaks (≥5% γ-H2A.X-positive cells). High when compared with intermediate or low specific activity resulted in a lower mean ± SEM tumor load by histopathology (vital tissue, 4 ± 2 vs. 8 ± 3 mm2; n = 3 vs. 6), day-4 18F-FDG PET (metabolic volume, 87 ± 23 vs. 118 ± 14 mm3; n = 6 vs. 12), and day-7 CT (volume, 323 ± 122 vs. 590 ± 46 mm3; n = 3 vs. 6; P = 0.039). 177Lu-PSMA-617 (120 MBq) with high specific activity induced superior tumor growth inhibition (P = 0.021, n = 5/group) without subacute hematologic toxicity (n = 3/group). Conclusion:177Lu-PSMA-617 (120 MBq) and high specific activity resulted in the highest efficacy in a syngeneic model of murine prostate cancer. The model will be useful for studying the effects of PSMA-directed RLT combined with potentially synergistic pharmacologic approaches.