LNCaP Xenografts Research Articles

Identification of Oral Bioavailable Coumarin Derivatives as Potential AR Antagonists Targeting Prostate Cancer.

Androgen receptor (AR) is a crucial driver of prostate cancer (PCa), but acquired resistance to AR antagonists significantly undermines their clinical efficacy. We previously discovered coumarin derivative 1, which is capable of disrupting AR ligand-binding domain dimers, offering the potential for overcoming resistance. However, its poor oral bioavailability limited further development. In this study, comprehensive structure optimizations led to compound 4a (IC50 = 0.051 μM), which exhibited comparable AR antagonistic activity to enzalutamide (IC50 = 0.060 μM) and demonstrated excellent selectivity over other nuclear receptors in vitro. Especially, 4a showed superior efficacy against ARF876L/T877A and ARW741C mutants compared to darolutamide and enzalutamide. Moreover, 4a exhibited favorable pharmacokinetic profiles (F = 66.24%) in vivo and significant tumor growth inhibition in an LNCaP xenograft mouse model upon oral administration. These results highlight the potential of 4a as a promising oral AR antagonist for overcoming drug resistance in PCa.

Discovery of Thiadiazoleamide Derivatives as Potent, Selective, and Orally Available Antagonists Disrupting Androgen Receptor Homodimer.

Androgen receptor (AR) is an important therapeutic target for prostate cancer (PCa) treatment, but prolonged use of AR antagonists has led to variant drug-resistant mutations. Since all marketed AR antagonists target the ligand binding pocket (LBP) of AR, to mitigate cross-resistance, a new drug pocket named Dimer Interface Pocket was discovered and a novel AR antagonist M17-B15 was identified. M17-B15 showed strong in vitro efficacy against PCa but had poor pharmacokinetic properties in vivo. In this study, through rational design and structure-activity relationship exploration, a series of thiadiazoleamide derivatives represented by N29 (IC50 = 0.018 μM) were identified with dominant AR antagonistic activity and remarkable anti-PCa activity in vitro. Furthermore, N29 effectively inhibited a series of typical drug-resistant AR mutants. The improved oral bioavailability of N29 facilitated its efficacy via oral administration, significantly inhibiting LNCaP xenograft tumor in vivo, presenting a promising therapeutic application for PCa.

61Cu-PSMA-Targeted PET for Prostate Cancer: From Radiotracer Development to First-in-Human Imaging.

The demand for PET tracers that target prostate-specific membrane antigen (PSMA) continues to increase. Meeting this demand with approved 68Ga- and 18F-labeled PSMA tracers is challenging outside of major urban centers. This is because the short physical half-life of these radionuclides makes it necessary to produce them near their sites of usage. To overcome this challenge, we propose cyclotron-produced 61Cu for labeling PSMA PET tracers. 61Cu can be produced on a large scale, and its 3.33-h half-life allows shipping over considerably longer distances than possible for 68Ga and 18F. Production of true theranostic twins using 61Cu and the β--emitter 67Cu is also feasible. Methods: PSMA-I&T (DOTAGA-(l-y)fk(sub-KuE)) and its derivative in which the DOTAGA chelator was replaced by NODAGA (NODAGA-(l-y)fk(sub-KuE)), herein reported as DOTAGA-PSMA-I&T and NODAGA-PSMA-I&T, respectively, were labeled with 61Cu and compared with [68Ga]Ga-DOTAGA-PSMA-I&T, [68Ga]Ga-NODAGA-PSMA-I&T, [68Ga]Ga-PSMA-11, and [18F]PSMA-1007. In vitro (lipophilicity, affinity, cellular uptake, and distribution) and invivo (PET/CT, biodistribution, and stability) studies were performed in LNCaP cells and xenografts. Human dosimetry estimates were calculated for [61Cu]Cu-NODAGA-PSMA-I&T. First-in-human imaging with [61Cu]Cu-NODAGA-PSMA-I&T was performed in a patient with metastatic prostate cancer. Results: [61Cu]Cu-DOTAGA-PSMA-I&T and [61Cu]Cu-NODAGA-PSMA-I&T were synthesized with radiochemical purity of more than 97%, at an apparent molar activity of 24 MBq/nmol, without purification after labeling. In vitro, natural Cu (natCu)-DOTAGA-PSMA-I&T and natCu-NODAGA-PSMA-I&T showed high affinity for PSMA (inhibitory concentration of 50%, 11.2 ± 2.3 and 9.3 ± 1.8 nM, respectively), although lower than the reference natGa-PSMA-11 (inhibitory concentration of 50%, 2.4 ± 0.4 nM). Their cellular uptake and distribution were comparable to those of [68Ga]Ga-PSMA-11. In vivo, [61Cu]Cu-NODAGA-PSMA-I&T showed significantly lower uptake in nontargeted tissues than [61Cu]Cu-DOTAGA-PSMA-I&T and higher tumor uptake (14.0 ± 5.0 percentage injected activity per gram of tissue [%IA/g]) than [61Cu]Cu-DOTAGA-PSMA-I&T (6.06 ± 0.25 %IA/g, P = 0.0059), [68Ga]Ga-PSMA-11 (10.2 ± 1.5 %IA/g, P = 0.0972), and [18F]PSMA-1007 (9.70 ± 2.57 %IA/g, P = 0.080) at 1 h after injection. Tumor uptake was also higher for [61Cu]Cu-NODAGA-PSMA-I&T at 4 h after injection (10.7 ± 3.3 %IA/g) than for [61Cu]Cu-DOTAGA-PSMA-I&T (4.88 ± 0.63 %IA/g, P = 0.0014) and [18F]PSMA-1007 (6.28 ± 2.19 %IA/g, P = 0.0145). Tumor-to-nontumor ratios of [61Cu]Cu-NODAGA-PSMA-I&T were superior to those of [61Cu]Cu-DOTAGA-PSMA-I&T and comparable to those of [68Ga]Ga-PSMA-11 and [18F]PSMA-1007 at 1 h after injection and increased significantly between 1 and 4 h after injection in most cases. Human dosimetry estimates for [61Cu]Cu-NODAGA-PSMA-I&T were similar to the ones reported for 18F-PSMA ligands. First-in-human imaging demonstrated multifocal osseous and hepatic metastases. Conclusion: [61Cu]Cu-NODAGA-PSMA-I&T is a promising PSMA radiotracer that compares favorably with [68Ga]Ga-PSMA-11 and [18F]PSMA-1007, while allowing delayed imaging.

Abstract 6037: Copper-67 as a next-generation radioconjugate warhead for cancer therapy

Abstract Background and Purpose: Antibody drug conjugates (ADCs) have shown promise as cancer treatments. However, the efficacy of ADCs has been limited in solid tumors and can come with significant toxicities. Radioconjugates utilize various radionuclides that are chelated and can be conjugated to small molecules, peptides and antibodies. We propose Targeted Copper Theranostics (TCTs) which use copper-64 to image tumors and beta-emitting copper-67 to irradiate and kill cancer cells using various molecular targets of oncological relevance. We have evaluated the efficacy of copper-67 based therapy in three formats; peptides, antibodies, and pre-targeting of antibody conjugates in relevant tumor mouse models. Benefits of copper-67 [half-life, 61.8 h; 100% β- energy in the range of 0.18-0.56 MeV] include high purity, high specific activity, manufacturing in a centralized distribution model at high yields on electron accelerators, and half-life suitable to various targeting formats. Copper-64 and copper-67 provide multiple logistical benefits relative to other radionuclides including Ga-68, F-18 and Lu-177. Methods: We evaluated three tumor-targeting moieties conjugated to a sarcophagine bifunctional chelator (SAR), including: a peptide dimer targeting prostate specific membrane antigen (SAR-bisPSMA); an antibody 5B1, targeting the PDAC-specific biomarker carbohydrate antigen-19-9 (CA19-9); and a Tz-TCO IDEEA huA33 antibody/sarcophagine tetrazine conjugate targeting transmembrane glycoprotein A33. Various doses were administered of [67Cu]Cu-SAR-bisPSMA in LNCAP xenografts; [67Cu]Cu-SAR-5B1 (~10 μg) in FC1245 Ft2Ab PDAC tumor-bearing C57BL/6 mice; and [67Cu]Cu-MeCOSAR-Tz in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts 72 hours after administration of huA33-TCO. Results: Administration of either 7.5, 15 or 30 MBq of [67Cu]Cu-SAR-bisPSMA increased overall survival relative to the control group. Similarly, after administration of 4, 11, 18 MBq of [67Cu]Cu-5B1, treated groups enhanced overall survival to 32 days compared to control (14 days). Pre-targeting using ~18.5, 37.0 or 55.5 MBq or fractionated dosing of two 27.8 MBq doses of [67Cu]Cu-MeCOSAR-Tz showed efficient tumor growth reduction with enhanced overall survival for treated mice pre-administered huA33-TCO, from 68 days to >200 days (dose dependent) compared to control (21 days). Conclusion: We report the inhibition of tumor growth with minimal radiotoxicity and each format demonstrated significantly improved overall survival compared to controls. Collectively our results demonstrated that TCTs using the sarcophagine chelator with copper-67 are effective radioconjugates across multiple formats with an acceptable safety profile. Copper-67-based TCTs are being translated in multiple theranostic clinical trials in the USA and Australia. Citation Format: Matt Harris, Kurt Gehlsen, Lachlan McInnes, Jason Lewis. Copper-67 as a next-generation radioconjugate warhead for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6037.

Abstract 4168: Pre-clinical demonstration of Raman spectroscopy-assisted photothermal therapy for prostate cancer

Abstract Prostate cancer (PCa) ranks among the most prevalent malignancies in men. The combination of ultrasound and fine-needle aspiration biopsy has greatly facilitated early diagnosis and expanded the range of treatment options. Traditional treatments primarily involve anti-androgens and androgen deprivation therapy; however, a recurrence occurs in approximately 30% of cases following these interventions. Our current focus is on the exploration of photothermal therapies using nanoparticles for precise tumor ablation. Among these nanoparticles, multiwalled carbon nanotubes (MWCNTs) exhibit unique structural, optical, and thermal properties that make them exceptionally appealing for biomedical applications, especially as a foundation for photothermal therapy. Our platform employs the copper (I)-catalyzed azide alkyne cycloaddition of prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys) ligand to the MWCNTs. We used confocal microscopy to observe a significantly enhanced binding of the targeted Glu-urea-Lys-MWCNTs to the surfaces of PSMA-expressing LNCaP cells compared to IgG-MWCNTs and to PSMA-null PC3 cells. Raman microscopy further confirmed the increased presence of Glu-urea-Lys-MWCNTs on LNCaP spheroids compared to non-targeting MWCNTs. We also confirmed enhanced photothermal cell ablation in vitro using the targeted MWCNTs. Based on in vitro evidence, we established LNCaP and PC3 xenografts in nu/nu mice and evaluated the therapeutic efficacy. Using fluorescence-based imaging, we observed the enhanced localization of Glu-urea-Lys-MWCNTs within the tumors. Furthermore, we collected the tumors and conducted ex vivo analysis using Raman microscopy for a label-free detection of the targeted MWCNTs, which complemented the fluorescence imaging and laying the foundation for real-time Raman imaging in vivo. Subsequently, we applied photothermal ablation of the tumor xenografts based on a pre-optimized treatment protocol established from COMSOL simulations and prior experiments. This treatment led to complete tumor ablation with minimal recurrence and minimal non-specific damage. In conclusion, we have assessed the effectiveness of our targeted nano-photothermal platform in a pre-clinical model of PCa. Further studies will aid in the transition of photothermal therapy to clinical trials. Moreover, we aim to investigate the role of photothermal therapy in the PCa tumor microenvironment and whether such an approach can trigger post-therapeutic activation of immune responses against the tumor. Ultimately, we anticipate that our nanotherapeutic platform will contribute to a focused therapeutic approach where targeted MWCNT-laser treatment exploits overexpressed tumor markers in conjunction with various irradiation strategies to enhance ablation while minimizing damage to surrounding tissue. Citation Format: Seung Soo Lee, Gabriel Beaudoin, Jun Hui Yeoh, Frederic Leblond, Mark Trifiro, Miltiadis Paliouras. Pre-clinical demonstration of Raman spectroscopy-assisted photothermal therapy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4168.

Abstract 6023: 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity

Abstract Despite significant developments over the last few decades, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Prostate specific membrane antigen (PSMA), a prostate cancer-specific membrane marker which directly correlates with androgen independence, metastasis, and disease progression, has been well-established as a radioligand target for the diagnosis and treatment of mCRPC. Lutetium-177 (177Lu)-PSMA-617 (PLUVICTOTM) was approved in 2022 for the treatment of progressive PSMA-positive mCRPC. However, only 30% of patients showed a radiological response in the registrational trial. Meanwhile, recent clinical experience has shown very limited efficacy in low PSMA expressing patients. These data collectively request for further improvement in the clinical benefit of PSMA-targeted radiotherapy. Actinium-225 (225Ac), an alpha emitter, is significantly more potent on cancer cell killing and has a shorter range in tissue penetration when compared to the beta emitter 177Lu. Such a profile supports the development of 225Ac based radiotherapies. Using our proprietary Clear-XTM technology platform, we developed 225Ac-FL-020, a novel 225Ac-based PSMA radioligand therapy candidate which has demonstrated prominent pre-clinical properties. We have assessed the binding affinity against PSMA of the non-labeled vector FL-020 in vitro. The in vivo biodistribution profile of FL-020 was characterized by SPECT/CT imaging and biodistribution using Indium-111 (111In)-FL-020 in PSMA high LNCaP tumor-bearing nude mice. In addition, anti-tumor activities of 225Ac-FL-020 were evaluated in the LNCaP xenograft model and directly compared to 225Ac-PSMA-617. FL-020 was found bound to LNCaP cells with an IC50 value of 51.55 nM. Meanwhile, the off-target screening showed that less than 50% inhibition of binding or activity was observed by FL-020 at 10 µM against 85 targets including receptors, ion channels, enzymes, and transporters, indicating the high selectivity of FL-020. Moreover, 111In-FL-020 displayed a very promising in vivo distribution profile with high and sustained tumor uptake and fast systemic clearance. Furthermore, 225Ac-FL-020 exhibited superior anti-tumor activity compared to 225Ac-PSMA-617 at the same dose level (10 KBq/mouse) in the LNCaP xenograft model with a favorable safety profile as indicated by body weight and hematological parameters. A mechanistic study was also conducted in 225Ac-FL-020-treated LNCaP tumor samples where DNA double-strand breaks and tumor cell apoptosis were observed, confirming the MOA of alpha emitters. Taken together, these results collectively demonstrate that 225Ac-FL-020 is a potent and selective PSMA-targeting radioligand therapy candidate with superior anti-tumor activity and a favorable safety profile warranting further clinical development. Citation Format: Fa Liu, Jiangwei Zhang, Junyu Yang, Kirstine T. Thrane, Mathias W. Hallund, Rikke V. Grønlund, Nicholas C. Wong. 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6023.

64Cu production via the 68Zn(p,nα)64Cu nuclear reaction: An untapped, cost-effective and high energy production route

IntroductionCopper-64 (64Cu, t1/2 = 12.7 h) is a positron emitter well suited for theranostic applications with beta-emitting 67Cu for targeted molecular imaging and radionuclide therapy. The present work aims to evaluate the radionuclidic purity and radiochemistry of 64Cu produced via the 68Zn(p,nα)64Cu nuclear reaction. Macrocyclic chelators DOTA, NOTA, TETA, and prostate-specific membrane antigen ligand PSMA I&T were radiolabeled with purified 64Cu and tested for in vitro stability. [64Cu]Cu-PSMA I&T was used to demonstrate in vivo PET imaging using 64Cu synthesized via the 68Zn(p,nα)64Cu production route and its suitability as a theranostic imaging partner alongside 67Cu therapy. Methods64Cu was produced on a 24 MeV TR-24 cyclotron at a beam energy of 23.5 MeV and currents up to 70 μA using 200 mg 68Zn encapsulated within an aluminum‑indium-graphite sealed solid target assembly. 64Cu semi-automated purification was performed using a NEPTIS Mosaic-LC synthesis unit employing CU, TBP, and TK201 (TrisKem) resins. Radionuclidic purity was measured by HPGe gamma spectroscopy, while ICP-OES assessed elemental purity. Radiolabeling was performed with NOTA at room temperature and DOTA, TETA, and PSMA I&T at 95 °C. 64Cu incorporation was studied by radio-TLC. 64Cu in vitro stability of [64Cu]Cu-NOTA, [64Cu]Cu-DOTA, [64Cu]Cu-TETA, and [64Cu]Cu-PSMA I&T was assessed at 37 °C from 0 to 72 h in human blood serum. Preclinical PET imaging was performed at 1, 24, and 48 h post-injection with [64Cu]Cu-PSMA I&T in LNCaP tumor-bearing mice and compared with [68Ga]Ga-PSMA I&T. ResultsMaximum purified activity of 4.9 GBq [64Cu]CuCl2 was obtained in 5 mL of pH 2–3 solution, with 2.9 GBq 64Cu concentrated in 0.5 mL. HPGe gamma spectroscopy of purified 64Cu detected <0.3 % co-produced 67Cu at EOB with no other radionuclidic impurities. ICP-OES elemental analysis determined <1 ppm Al, Zn, In, Fe, and Cu in the [64Cu]CuCl2 product. NOTA, DOTA, TETA, and PSMA I&T were radiolabeled with 64Cu, resulting in maximum molar activities of 164 ± 6 GBq/μmol, 155 ± 31 GBq/μmol, 266 ± 34 GBq/μmol, and 117 ± 2 GBq/μmol, respectively. PET imaging in PSMA-expressing LNCaP xenografts resulted in high tumor uptake (SUVmean = 1.65 ± 0.1) using [64Cu]Cu-PSMA I&T, while [68Ga]Ga-PSMA I&T yielded an SUVmean of 0.76 ± 0.14 after 60 min post-injection. Conclusions64Cu was purified in a small volume amenable for radiolabeling, with yields suitable for preclinical and clinical application. The 64Cu production and purification process and the favourable PET imaging properties confirm the 68Zn(p,nα)64Cu nuclear reaction as a viable 64Cu production route for facilities with access to a higher energy proton cyclotron, compared to using expensive 64Ni target material and the 64Ni(p,n)64Cu nuclear reaction. Advances in knowledge and implications for patient careOur 64Cu production technique provides an alternative production route with the potential to improve 64Cu availability for preclinical and clinical studies alongside 67Cu therapy.

Discovery of BWA-522, a First-in-Class and Orally Bioavailable PROTAC Degrader of the Androgen Receptor Targeting N-Terminal Domain for the Treatment of Prostate Cancer.

We report small molecular PROTAC compounds targeting the androgen receptor N-terminal domain (AR-NTD), which were obtained by tethering AR-NTD antagonists and different classes of E3 ligase ligands through chemical linkers. A representative compound, BWA-522, effectively induces degradation of both AR-FL and AR-V7 and is more potent than the corresponding antagonist against prostate cancer (PC) cells in vitro. We have shown that the degradation of AR-FL and AR-V7 proteins by BWA-522 can suppress the expression of AR downstream proteins and induce PC cell apoptosis. BWA-522 achieves 40.5% oral bioavailability in mice and 69.3% in beagle dogs. In a LNCaP xenograft model study, BWA-522 was also proved to be an efficacious PROTAC degrader, resulting in 76% tumor growth inhibition after oral administration of a dose of 60 mg/kg. This study indicates that BWA-522 is a promising AR-NTD PROTAC for the treatment of AR-FL- and AR-V7-dependent tumors.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti-Hook Effect for Tumor Therapy.

Proteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post-translational protein degradation capabilities. However, off-target induced unintended tissue effects and intrinsic "hook effect" hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano-PROTACs) modality with a center-spoke degradation network for achieving efficient dose-dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self-assemble into Nano-PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano-PROTACs can form effective polynary complexes and E3 ligases degradation network with multi-binding sites, achieving dose-dependent protein degradation with "anti-hook effect". The generality and efficacy of Nano-PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide-range dose-dependent manner with a 95 % degradation rate and long-lasting potency up to 72 h in vitro. Significantly, Nano-PROTACs achieve in vivo dose-dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self-assembly strategy, the Nano-PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

Nano Proteolysis Targeting Chimeras (PROTACs) with Anti‐Hook Effect for Tumor Therapy

AbstractProteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post‐translational protein degradation capabilities. However, off‐target induced unintended tissue effects and intrinsic “hook effect” hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano‐PROTACs) modality with a center‐spoke degradation network for achieving efficient dose‐dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self‐assemble into Nano‐PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano‐PROTACs can form effective polynary complexes and E3 ligases degradation network with multi‐binding sites, achieving dose‐dependent protein degradation with “anti‐hook effect”. The generality and efficacy of Nano‐PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide‐range dose‐dependent manner with a 95 % degradation rate and long‐lasting potency up to 72 h in vitro. Significantly, Nano‐PROTACs achieve in vivo dose‐dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self‐assembly strategy, the Nano‐PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.

A GATA2-CDC6 axis modulates androgen receptor blockade-induced senescence in prostate cancer

BackgroundProstate cancer is a major cause of cancer morbidity and mortality in men worldwide. Androgen deprivation therapy (ADT) has proven effective in early-stage androgen-sensitive disease, but prostate cancer gradually develops into an androgen-resistant metastatic state in the vast majority of patients. According to our oncogene-induced model for cancer development, senescence is a major tumor progression barrier. However, whether senescence is implicated in the progression of early-stage androgen-sensitive to highly aggressive castration-resistant prostate cancer (CRPC) remains poorly addressed.MethodsAndrogen-dependent (LNCaP) and –independent (C4-2B and PC-3) cells were treated or not with enzalutamide, an Androgen Receptor (AR) inhibitor. RNA sequencing and pathway analyses were carried out in LNCaP cells to identify potential senescence regulators upon treatment. Assessment of the invasive potential of cells and senescence status following enzalutamide treatment and/or RNAi-mediated silencing of selected targets was performed in all cell lines, complemented by bioinformatics analyses on a wide range of in vitro and in vivo datasets. Key observations were validated in LNCaP and C4-2B mouse xenografts. Senescence induction was assessed by state-of-the-art GL13 staining by immunocytochemistry and confocal microscopy.ResultsWe demonstrate that enzalutamide treatment induces senescence in androgen-sensitive cells via reduction of the replication licensing factor CDC6. Mechanistically, we show that CDC6 downregulation is mediated through endogenous activation of the GATA2 transcription factor functioning as a CDC6 repressor. Intriguingly, GATA2 levels decrease in enzalutamide-resistant cells, leading to CDC6 stabilization accompanied by activation of Epithelial-To-Mesenchymal Transition (EMT) markers and absence of senescence. We show that CDC6 loss is sufficient to reverse oncogenic features and induce senescence regardless of treatment responsiveness, thereby identifying CDC6 as a critical determinant of prostate cancer progression.ConclusionsWe identify a key GATA2-CDC6 signaling axis which is reciprocally regulated in enzalutamide-sensitive and -resistant prostate cancer environments. Upon acquired resistance, GATA2 repression leads to CDC6 stabilization, with detrimental effects in disease progression through exacerbation of EMT and abrogation of senescence. However, bypassing the GATA2-CDC6 axis by direct inhibition of CDC6 reverses oncogenic features and establishes senescence, thereby offering a therapeutic window even after acquiring resistance to therapy.

Abstract ND03: Discovery of ARV-766, an androgen receptor degrading PROTAC® for the treatment of men with metastatic castration resistant prostate cancer

Abstract Prostate cancer is the second leading cause of cancer death in men in the United States. The androgen receptor (AR) plays critical roles in both early disease and advanced prostate cancer. Current therapeutic approaches targeting the androgen/AR axis are initially effective, but castration resistant prostate cancer (CRPC) inevitably develops. CRPC is linked with increased AR activity via gene overexpression, amplification, and gain-of-function mutations. To address these mechanisms of AR-dependent prostate tumor growth, we have developed a novel therapeutic agent, ARV-766, a proteolysis targeting chimera (PROTAC®) that induces a protein-protein interaction between the AR and specific E3 ubiquitin ligase complexes, leading to the ubiquitination of AR and its subsequent degradation via the proteasome. In vitro, ARV-766 degrades AR in various prostate cancer cell lines, including those harboring resistance-conferring, clinically relevant point mutations, with a half-maximal degradation concentration (DC50) of &amp;lt;1 nM in wild type VCaP. Importantly ARV-766 also maintains potency against the AR L702H mutant, which has been associated with resistance to some AR antagonists. In vivo, ARV-766 is orally bioavailable and robustly degrades AR with a &amp;gt;90% observed maximum degradation (Dmax) at efficacious doses. ARV-766 significantly and dose-dependently inhibits tumor growth in murine LNCaP and VCaP xenograft models, including an enzalutamide-insensitive non-castrated VCaP model. These preclinical data supported the clinical development of ARV-766 for the treatment of men with metastatic CRPC. Selected pre-clinical data along with the chemical structure of ARV-766 will be presented. Citation Format: Larry Snyder, S H. Lee, T K. Neklesa, X Chen, H Dong, C Ferraro, D A. Gordon, J Macaluso, J Pizzano, J Wang, R R. Willard, N Vitale, R Peck, M D. Moore, C M. Crews, J Houston, A P. Crew, I Taylor. Discovery of ARV-766, an androgen receptor degrading PROTAC® for the treatment of men with metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND03.

Discovery of thiohydantoin based antagonists of androgen receptor with efficient degradation for the treatment of prostate cancer

Prostate cancer (PC) is one of the most prevalent cancers in men worldwide, and androgen receptor (AR) is a well-validated drug target for the treatment of PC. However, PC often exhibits resistance to AR antagonists over time. Thus, it is urgent to identify novel and effective drugs for PC treatment. A series of novel thiohydantoin based AR antagonists with efficient degradation against AR were designed, synthesized, and evaluated. Based on our previous SAR and further structural optimization, a tool molecule 26h was discovered with dual mechanisms including improved antagonistic activity and potent degradation (AR-fl and AR-V7). Moreover, 26h can also effectively block AR nuclear translocation and inhibit AR/AR-V7 heterodimerization, thereby inhibiting downstream gene transcription. Importantly, 26h displayed potent robust efficacy in LNCaP (TGI: 70.70%) and 22Rv1 (TGI: 78.89%) xenograft models. This provides new design strategies and advantageous potential compounds for the treatment of prostate cancer.

Old Drug, New Delivery Strategy: MMAE Repackaged.

Targeting therapy is a concept that has gained significant importance in recent years, especially in oncology. The severe dose-limiting side effects of chemotherapy necessitate the development of novel, efficient and tolerable therapy approaches. In this regard, the prostate specific membrane antigene (PSMA) has been well established as a molecular target for diagnosis of, as well as therapy for, prostate cancer. Although most PSMA-targeting ligands are radiopharmaceuticals used in imaging or radioligand therapy, this article evaluates a PSMA-targeting small molecule-drug conjugate, and, thus, addresses a hitherto little-explored field. PSMA binding affinity and cytotoxicity were determined in vitro using cell-based assays. Enzyme-specific cleavage of the active drug was quantified via an enzyme-based assay. Efficacy and tolerability in vivo were assessed using an LNCaP xenograft model. Histopathological characterization of the tumor in terms of apoptotic status and proliferation rate was carried out using caspase-3 and Ki67 staining. The binding affinity of the Monomethyl auristatin E (MMAE) conjugate was moderate, compared to the drug-free PSMA ligand. Cytotoxicity in vitro was in the nanomolar range. Both binding and cytotoxicity were found to be PSMA-specific. Additionally, complete MMAE release could be reached after incubation with cathepsin B. In vivo, the MMAE conjugate displayed good tolerability and dose-dependent inhibition of tumor growth. Immunohistochemical and histological studies revealed the antitumor effect of MMAE.VC.SA.617, resulting in the inhibition of proliferation and the enhancement of apoptosis. The developed MMAE conjugate showed good properties in vitro, as well as in vivo, and should, therefore, be considered a promising candidate for a translational approach.

Molecular Design of 68Ga- and 89Zr-Labeled Anticalin Radioligands for PET-Imaging of PSMA-Positive Tumors.

Anticalin proteins directed against the prostate-specific membrane antigen (PSMA), optionally having tailored plasma half-life using PASylation technology, show promise as radioligands for PET-imaging of xenograft tumors in mice. To investigate their suitability, the short-circulating unmodified Anticalin was labeled with 68Ga (τ1/2 = 68 min), using the NODAGA chelator, whereas the half-life extended PASylated Anticalin was labeled with 89Zr (τ1/2 = 78 h), using either the linear chelator deferoxamine (Dfo) or a cyclic derivative, fusarinine C (FsC). Different PSMA targeting Anticalin versions (optionally carrying the PASylation sequence) were produced carrying a single exposed N- or C-terminal Cys residue and site-specifically conjugated with the different radiochelators via maleimide chemistry. These protein conjugates were labeled with radioisotopes having distinct physical half-lives and, subsequently, applied for PET-imaging of subcutaneous LNCaP xenograft tumors in CB17 SCID mice. Uptake of the protein tracers into tumor versus healthy tissues was assessed by segmentation of PET data as well as biodistribution analyses. PET-imaging with both the 68Ga-labeled plain Anticalin and the 89Zr-labeled PASylated Anticalin allowed clear delineation of the xenograft tumor. The radioligand A3A5.1-PAS(200)-FsC·89Zr, having an extended plasma half-life, led to a higher tumor uptake 24 h p.i. compared to the 68Ga·NODAGA-Anticalin imaged 60 min p.i. (2.5% ID/g vs 1.2% ID/g). Pronounced demetallation was observed for the 89Zr·Dfo-labeled PASylated Anticalin, which was ∼50% lower in the case of the cyclic radiochelator FsC (p < 0.0001). Adjusting the plasma half-life of Anticalin radioligands using PASylation technology is a viable approach to increase radioisotope accumulation within the tumor. Furthermore, 89Zr-ImmunoPET-imaging using the FsC radiochelator is superior to that using Dfo. Our strategy for the half-life adjustment of a tumor-targeting Anticalin to match the physical half-life of the applied radioisotope illustrates the potential of small binding proteins as an alternative to antibodies for PET-imaging.

Abstract 5043: Radium-223 demonstrates increased antitumor activity in combination with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model of bone metastatic prostate cancer

Abstract Radium-223, a bone-specific alpha particle therapy, increased survival of patients with metastatic castration-resistant prostate cancer (mCRPC) and bone metastases (Parker et al., NEJM 2013) 177Lu-PSMA-617 is a small molecule radioligand that delivers beta-particle radiation to cancer cells expressing prostate-specific membrane antigen (PSMA). Despite improving overall survival of patients with heavily pretreated mCRPC (Sator et al., NEJM 2021), responses to 177Lu-PSMA-617 are often not durable and many patients develop progressive disease presumably due to microscopic osseous lesions. Compared to beta-particle radiation, alpha particles have a shorter range and higher linear energy transfer, making them more suitable for treating microscopic lesions. In this study, we evaluated preclinical antitumor efficacy and potential beneficial combination effects of radium-223 combined with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model mimicking progressive prostate cancer micrometastases in bone. LNCaP prostate cancer cells were inoculated into the right tibiae of male NOD.scid mice. Six weeks after inoculation, the mice were randomized to treatment groups based on serum prostate-specific antigen (PSA) and treated with two injections (Q4Wx2, i.v.) of vehicle, radium-223 (330 kBq/kg), 177Lu-PSMA-617 (10 MBq) or their combination 177Lu-PSMA-617 was given 24 hours after radium-223. PSA and bone formation marker procollagen type I intact N-terminal propeptide (PINP) were measured from blood samples and tumor-induced abnormal bone growth was determined by radiography. Terminal blood samples were analyzed for hematologic status at sacrifice. The combination treatment of radium-223 and 177Lu-PSMA-617 demonstrated robust antitumor efficacy, as indicated by lower serum PSA (mean change relative to pretreatment levels: 63.9 % vs 631 % (vehicle control), p=0.026), decreased tumor-induced abnormal bone area in tumor-bearing tibiae (p&amp;lt;0.001) and suppression of abnormal bone metabolic activity as evidenced by a reduced level of the bone formation marker PINP (p&amp;lt;0.001), compared with vehicle. Tolerability of the combination treatment was evidenced by the lack of significant body weight loss and the absence of cumulative hematological toxicity. In conclusion, these preclinical results show that the combination of radium-223 and 177Lu-PSMA-617 is well-tolerated and efficacious in inhibiting osseous tumor growth and improving response. The combination of radium-223 and 177Lu-PSMA-I&amp;T is currently being investigated in patients with mCRPC in a phase 1/2 trial (AlphaBet, NCT05383079). Citation Format: Arne Scholz, Matias Knuuttila, Justyna Zdrojewska, Mari I. Suominen, Esa Alhoniemi, Christoph A. Schatz, Sabine Zitzmann-Kolbe, Sanna-Maria Käkönen, Urs B. Hagemann. Radium-223 demonstrates increased antitumor activity in combination with 177Lu-PSMA-617 in the intratibial LNCaP xenograft model of bone metastatic prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5043.

The ethanol extract of Cyperus exaltatus var. iwasakii exhibits cell cycle dysregulation, ERK1/2/p38 MAPK/AKT phosphorylation, and reduced MMP-9-mediated metastatic capacity in prostate cancer models in vitro and in vivo

BackgroundProstate cancer is the second most common cause of cancer death worldwide in men. The development of novel and highly efficient therapeutic strategies is strongly recommended to treat prostate cancer. Cyperaceae are an ecologically and economically important family of plants with several pharmacological effects. However, the biological efficacy of Cyperus exaltatus var. iwasakii (CE) is unknown. PurposeThis study aimed to investigate the antitumor effect of the ethanol extract of CE against prostate cancer. MethodsIn vitro antitumor efficacy of CE was explored by the MTT assay, cell counting assay, FACS analysis, immunoblot, wound-healing migration, invasion assay, zymographic assay, and EMSA in prostate cancer cells, DU145 and LNCaP. For in vivo experiments, xenograft mice were injected with LNCaP cells. Histology (H&E and Ki-67) and biochemical enzyme assay were then performed. The toxicity test was evaluated by an acute toxicity assay. The phytochemical constituents of CE were identified by spectrometric and chromatographic analyses. ResultsCE exerted a significant antiproliferative effect against prostate cancer cells. CE-induced antiproliferative cells were associated with cell cycle arrest at G0/G1 (cyclin D1/CDK4, cyclin E/CDK2, p21Waf1) in DU145 cells, but G2/M (ATR, CHK1, Cdc2, Cdc25c, p21Waf1, and p53) in LNCaP cells. CE stimulated the phosphorylation of ERK1/2, p38 MAPK, and AKT in DU145 cells, but only p38 MAPK phosphorylation was increased in LNCaP cells. CE treatment suppressed migration and invasion in the two types of prostate cancer cells by inhibiting MMP-9 activity through the regulation of transcription factors, such as AP-1 and NF-κB. In vivo experiments showed a reduction in tumor weight and size following oral CE administration. Histochemistry confirmed that CE inhibited tumor growth in the mouse LNCaP xenograft model. The administration of CE had no adverse effects on body weight, behavioral patterns, blood biochemistry, and histopathology findings of vital organs in mice. Finally, a total of 13 phytochemical constituents were identified and quantified in CE. The most abundant secondary metabolites in CE were astragalin, tricin, and p-coumaric acid. ConclusionOur results demonstrated the antitumor efficacy of CE against prostate cancer. These findings suggest that CE might be a potential candidate for prostate cancer prevention or treatment.

Therapeutic targeting of histone lysine demethylase KDM4B blocks the growth of castration-resistant prostate cancer

Epigenetics is an emerging mechanism for tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated that upregulation of histone lysine demethylase KDM4B correlated with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we further investigated the role of KDM4B in promoting PCa progression and tested the efficacy of B3 using clinically relevant PCa models including PCa cell line LNCaP and 22Rv1 and xenografts derived from these cell lines. In loss and gain-functional studies of KDM4B in PCa cells, we found that overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of 22Rv1 xenografts and sensitized tumor to anti-androgen receptor (AR) antagonist enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin, leading to cell apoptosis. Comparative transcriptomic analysis performed on KDM4B knockdown and B3-treated 22Rv1 cells revealed that B3 inhibited both H3K9me3 and H3K27me3 demethylase activities. Our studies establish KDM4B as a target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMs for CRPC patients.

Targeted α-therapy using astatine (211At)-labeled PSMA1, 5, and 6: a preclinical evaluation as a novel compound

PurposeTargeted α-therapy (TAT) for prostate-specific membrane antigen (PSMA) is a promising treatment for metastatic castration-resistant prostate cancer (CRPC). Astatine is an α-emitter (half-life=7.2 h) that can be produced by a 30-MeV cyclotron. This study evaluated the treatment effect of 211At-labeled PSMA compounds in mouse xenograft models.MethodsTumor xenograft models were established by subcutaneous transplantation of human prostate cancer cells (LNCaP) in NOD/SCID mouse. [211At]PSMA1, [211At]PSMA5, or [211At]PSMA6 was administered to LNCaP xenograft mice to evaluate biodistribution at 3 and 24 h. The treatment effect was evaluated by administering [211At]PSMA1 (0.40 ± 0.07 MBq), [211At]PSMA5 (0.39 ± 0.03 MBq), or saline. Histopathological evaluation was performed for the at-risk organs at 3 and 6 weeks after administration.Results[211At]PSMA5 resulted in higher tumor retention compared to [211At]PSMA1 and [211At]PSMA6 (30.6 ± 17.8, 12.4 ± 4.8, and 19.1 ± 4.5 %ID/g at 3 h versus 40.7 ± 2.6, 8.7 ± 3.5, and 18.1 ± 2.2%ID/g at 24 h, respectively), whereas kidney excretion was superior in [211At]PSMA1 compared to [211At]PSMA5 and [211At]PSMA6. An excellent treatment effect on tumor growth was observed after [211At]PSMA5 administration. [211At]PSMA1 also showed a substantial treatment effect; however, the tumor size was relatively larger compared to that with [211At]PSMA5. In the histopathological evaluation, regenerated tubules were detected in the kidneys at 3 and 6 weeks after the administration of [211At]PSMA5.ConclusionTAT using [211At]PSMA5 resulted in excellent tumor growth suppression with minimal side effects in the normal organs. [211At]PSMA5 should be considered a new possible TAT for metastatic CRPC, and translational prospective trials are warranted.

Efficacy of PACE4 pharmacotherapy in JHU-LNCaP-SM preclinical model of androgen independent prostate cancer

Prostate cancer (PCa) is a complex disease progressing from in situ to invasive or metastatic tumors while also being capable of modulating its androgen dependence. Understanding how novel therapies are working across the different stages of the disease is critical for their proper positioning in the spectrum of PCa treatments. The targeting of proprotein convertase PACE4 (Paired basic Amino Acid-Cleaving Enzyme 4) has been proposed as a novel approach to treat PCa. Animal studies performed on LNCaP xenografts, an androgen-dependent model, already yielded positive results. In this study, we tested PACE4 inhibition on JHU-LNCaP-SM, a newly described androgen-independent model, in cell-based and xenograft assays. Like LNCaP, JHU-LNCaP-SM cells express PACE4 and its oncogenic isoform PACE4-altCT. Using isoform-specific siRNAs, downregulation of PACE4-altCT resulted in JHU-LNCaP-SM growth inhibition. Furthermore, JHU-LNCaP-SM responded to the PACE4 pharmacological inhibitor known as C23 in cell-based assays as well as in athymic nude mice xenografts. These data support the efficacy of PACE4 inhibitors against androgen independent PCa thereby demonstrating that PACE4 is a key target in PCa. The JHU-LNCaP-SM cell line represents a model featuring important aspects of androgen-independent PCa, but it also represents a very convenient model as opposed to LNCaP cells for in vivo studies, as it allows rapid screening due to its high implantation rate and growth characteristics as xenografts.