22Rv1 Xenografts Research Articles

Targeted Delivery of AR-V7 siRNA with Bivalent PSMA Aptamers Effectively Suppresses the Growth of Enzalutamide-Resistant Prostate Cancer.

Androgen deprivation therapy has been the primary treatment strategy for advanced prostate cancer (PCa). But most patients develop castration resistance over time. For FDA-approved second-generation androgen receptor (AR) antagonists, including enzalutamide (ENZ) and abiraterone (AA), patients who initially respond to them eventually develop resistance. The key mechanism for resistance to ENZ/AA involves AR splice variants (AR-Vs) and specifically AR-V7. Current AR antagonists cannot target AR-V7 due to its lack of the C-terminal ligand-binding domain (LBD) but keeping the AR N-terminal domain (NTD) which still can activate androgen-responsive genes. Therefore, targeting the AR NTD and AR-V7 is critically important to overcome ENZ resistance. Unfortunately, AR NTD has been considered an "undruggable" target due to the difficulty in defining its three-dimensional (3D) structure. In this context, siRNA is highly suitable to address this undruggable target. However, siRNA cannot freely diffuse into cells, and a carrier is needed. In this regard, nucleic acid-based aptamers are highly suitable for cell type-specific delivery of siRNA in vivo. In this study, we have developed a serum-stable bivalent prostate-specific membrane antigen (PSMA) aptamer-AR-V7 siRNA chimera (PAP). The results show that PAP can knock down both AR-full length and AR-V7 in PSMA-expressing castration-resistant cells. It can resensitize ENZ in cell lines and PCa xenografts. ENZ combined with PAP can significantly inhibit 22Rv1 xenograft growth in mice without experiencing castration. Owing to the low toxicity, PAP has potential to offer a new antiandrogen treatment for current ENZ-resistant PCa.

Metformin in Overcoming Enzalutamide Resistance in Castration-Resistant Prostate Cancer.

Androgen deprivation is the standard treatment for prostate cancer (PCa) patients. However, the disease eventually progresses as castration-resistant PCa (CRPC). Enzalutamide, an AR inhibitor, is a typical drug to treating CRPC and due to continuous reliance on the drug, can lead to Enzalutamide-resistance (ENZ-r). This highlights the necessity for developing novel therapeutic targets to combat the gain of resistance. Metformin has been recently investigated for its potential anti-tumorigenic effects in many cancer types. In this study, we used enzalutamide and metformin in combination to explore the possible rescued efficacy of enzalutamide in the treatment of ENZ-r CRPC. We first tested the effects of this combination treatment on cell viability, drug synergy, and cell proliferation in ENZ-r CRPC cell lines. After combination treatment, we observed a decrease in cell proliferation and viability as well as a synergistic effect of both enzalutamide and metformin in vitro Following these results, we sought to explore how combination treatment effected mitochondrial fitness utilizing mitochondrial stress test analysis and mitochondrial membrane potential (MMP) shifts due to metformin's action in inhibiting Complex I of oxidative phosphorylation. We employed 2 different strategies of in vivo testing using 22Rv1 and LuCaP35CR xenograft models. Finally, RNA sequencing revealed a potential link in the downregulation of Ras/MAPK signaling following combination treatment. Significance Statement Increasing evidence suggests that oxidative phosphorylation might play a critical role in the development of resistance to cancer therapy. We showed that targeting oxidative phosphorylation with metformin can enhance the efficacy of enzalutamide in castration-resistant prostate cancer in vitro.

Design, synthesis and biological evaluation of dual inhibitors targeting AR/AR-Vs and PARP1 in castration resistant prostate cancer therapy

The combination of androgen signaling inhibitors and PARP inhibitors has shown promising results in clinical trials for the treatment of castration-resistant prostate cancer (CRPC). Multi-target inhibitors can inhibit tumors through different pathways, addressing the limitations of traditional single target inhibitors. We designed and synthesized dual inhibitors targeting AR/AR-Vs and PARP1 using a pharmacophore hybridization strategy. The most potent compound, II-3, inhibits AR/AR-Vs signaling and induces DNA damage by inhibiting PARP1. The IC50 values of II-3 in the castration-resistant prostate cancer cell lines 22RV1 and C4–2 are 4.38 ± 0.56 µM, and 3.44 ± 0.63 µM, respectively. II-3 not only suppresses the proliferation and migration of 22RV1 and C4–2 cells, but also promotes their apoptosis. Intraperitoneal injection of II-3 effectively inhibits tumor growth in 22RV1 xenograft nude mice without evident drug-induced toxicity. Overall, a series of novel dual inhibitors targeting AR/AR-Vs and PARP1 were designed and synthesized, and meanwhile the in vivo and in vitro effects were comprehensively explored, which provided a potential new therapeutic strategy for CRPC.

Design, Synthesis, and Biological Evaluation of Potent EZH2/LSD1 Dual Inhibitors for Prostate Cancer.

As histone modification enzymes, EZH2 mediates H3K27 trimethylation (H3K27me3), whereas LSD1 removes methyl groups from H3K4me1/2 and H3K9me1/2. Synergistic anticancer effects of combining inhibitors of these two enzymes are observed in leukemia and prostate cancer. Thus, a series of EZH2/LSD1 dual inhibitors are designed and synthesized to evaluate their anticancer activity. After the structure-activity study, one of the best compounds, ML234, displayed excellent antiproliferative capacity against prostate cancer cell lines LNCAP, PC3, and 22RV1. Enzymatic assays ascertained that the anticancer potency of ML234 was mediated through coinhibition of EZH2 and LSD1. Moreover, the accumulation of H3K4me2 and H3K9me2 and the decrease of H3K27me3 induced by ML234 were verified by Western blot analysis. More importantly, the compound remarkably suppressed the tumor growth and enhanced the therapeutic efficacy of clinical drug enzalutamide in the 22RV1 xenograft mouse model, indicating that it may have potential as an anticancer agent in prostate cancer.

Enzalutamide Sensitizes Castration-Resistant Prostate Cancer to Copper-Mediated Cell Death.

Despite the initial efficacy of enzalutamide in castration-resistant prostate cancer (CRPC), inevitable resistance remains a significant challenge. Here, the synergistic induction of copper-dependent cell death (cuproptosis) in CRPC cells is reported by enzalutamide and copper ionophores (elesclomol/disulfiram). Mechanistically, enzalutamide treatment increases mitochondrial dependence in CRPC cells, rendering them susceptible to cuproptosis, as evidenced by specific reversal with the copper chelator tetrathiomolybdate. This susceptibility is characterized by hallmarks of cuproptosis, including lipoylated protein aggregation and iron-sulfur cluster protein instability. Interestingly, the mitochondrial matrix reductase, FDX1, specifically correlates with elesclomol sensitivity, suggesting a potential mechanistic divergence between the two copper ionophores. Notably, this synergistic effect extends beyond in vitro models, demonstrating efficacy in 22Rv1 xenografts, mouse Pten p53 knockout organoids. Importantly, enzalutamide significantly enhances copper ionophore-mediated cytotoxicity in enzalutamide-resistant cells. Collectively, these findings indicate that enzalutamide and copper ionophores synergistically induce cuproptosis, offering a promising therapeutic avenue for CRPC, potentially including enzalutamide-resistant cases.

Caffeic acid phenethyl ester suppresses the expression of androgen receptor variant 7 via inhibition of CDK1 and AKT.

Androgen receptor (AR) splice variant 7 (AR-V7) is capable to enter nucleus and activate downstream signaling without ligand. AR-V7 assists the tumor growth, cancer metastasis, cancer stemness, and the evolvement of therapy-resistant prostate cancer (PCa). We discovered that caffeic acid phenethyl ester (CAPE) can repress the expression and downstream signaling of AR-V7 in PCa cells. CAPE blocked the gene transcription, nuclear localization, and protein abundance of AR-V7. CAPE inhibited the expression of U2AF65, SF2 and hnRNPF, which were splicing factors for AR-V7 intron. Additionally, CAPE decreased protein stability of AR-V7 and enhanced the proteosome-degradation of AR-V7. We observed that CDK1 and AKT regulated the expression and stability of AR-V7 via phosphorylation of Ser81 and Ser213, respectively. CAPE decreased the expression of CDK1 and AKT. Overexpression of CDK1 restored the abundance of AR-V7 in CAPE-treated PCa cells. Overexpression of AR-V7, AKT or CDK1 rescued the proliferation of PCa cells under CAPE treatment. Intraperitoneal injection of 10 mg/kg CAPE retarded the growth of 22Rv1 xenografts in nude mice and suppressed the protein levels of AR-V7, CDK1 and AKT in 22Rv1 xenografts. Our study provided the rationale of applying CAPE for inhibition of AR-V7 in prostate tumors.

Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer.

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Design, synthesis and evaluation of novel prostate-specific membrane antigen-targeted aryl [18F]fluorosulfate PET tracers

The expression of prostate-specific membrane antigen (PSMA) in prostate cancer is 100–1000 times higher than that in normal tissues, and it has shown great advantages in the diagnosis and treatment of prostate cancer. The combination of PSMA and PET imaging technology based on the principle of metabolic imaging can achieve high sensitivity and high specificity for diagnosis. Due to its suitable half-life (109 min) and good positron abundance (97%), as well as its cyclotron accelerated generation, 18F has the potential to be commercialize, which has attracted much attention. In this article, we synthesized a series of fluorosulfate PET tracers targeting PSMA. All four analogues have shown high affinity to PSMA (IC50 = 1.85–5.15 nM). After the radioisotope exchange labeling, [18F]L9 and [18F]L10 have PSMA specific cellular uptake (0.65 ± 0.04% AD and 1.19 ± 0.03% AD) and effectively accumulated in 22Rv1 xenograft mice model. This study demonstrates that PSMA-1007-based PSMA-targeted aryl [18F]fluorosulfate novel tracers have the potential for PET imaging in tumor tissues.

Novel selective agents for the degradation of AR/AR-V7 to treat advanced prostate cancer

The androgen receptor AR antagonists, such as enzalutamide and apalutamide, are efficient therapeutics for the treatment of prostate cancer (PCa). Even though they are effective at first, resistance to both drugs occurs frequently. Resistance is mainly driven by aberrations of the AR signaling pathway including AR gene amplification and the expression of AR splice variants (e.g. AR-V7). This highlights the urgent need for alternative therapeutic strategies. Here, a total of 24 compounds were synthesized and biologically evaluated to disclose compound 20i, exhibiting potent AR antagonistic activities (IC50 = 172.85 ± 21.33 nM), promising AR/AR-V7 protein degradation potency, and dual targeting site of probably AR (ligand-binding domain, LBD and N-terminal domain, NTD). It potently inhibits cell growth with IC50 values of 4.87 ± 0.52 and 2.07 ± 0.34 μM in the LNCaP and 22RV1 cell lines, respectively, and exhibited effective tumor growth inhibition (TGI = 50.9 %) in the 22RV1 xenograft study. These data suggest that 20i has the potential for development as an AR/AR-V7 inhibitor with degradation ability to treat advanced prostate cancer.

Abstract 694: Evaluation of a synergistic drug combination with 177Lu-rhPSMA-10.1 for prostate cancer: Results of an in vitro screen and in vivo proof of concept study

Abstract Purpose: Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) has been shown to extend survival in men with advanced prostate cancer (PCa). Novel radiohybrid (rh) PSMA-targeted 177Lu-rhPSMA-10.1 has shown promising preclinical efficacy and advantageous radiation dosimetry in humans. We conducted an in vitro screen to identify known anticancer drugs with potential for synergistic interaction with 177Lu-rhPSMA-10.1. Here we present key screening data and a subsequent in vivo efficacy analysis of the lead novel drug combination in PSMA-expressing 22Rv1 PCa xenografts. Methods: Over 150 FDA-approved anticancer drugs were screened in a clonogenic survival assay of 22Rv1 cells using the test drug alone, at a range of concentrations <20 µM to determine the IC50, and results compared to incubations of the drug + 15 MBq/mL 177Lu-rhPSMA-10.1 after 10 d. A focused screen of 5 lead candidates was then conducted to determine the impact of 177Lu-rhPSMA-10.1 (0-25 MBq/mL) on the drug IC50. A synergy score was determined using the zero interaction potency (ZIP) reference model and the multi-dimensional synergy of combinations (MuSyc) platform. Subsequently, to evaluate the efficacy of the lead combination, 177Lu-rhPSMA-10.1 (single 30 MBq iv dose) and Cobimetinib (0.25 mg orally per day for 21 d) alone and in combination were administered to 22Rv1 tumor-bearing NMRI nude mice (n = 8 per group plus untreated controls). Tumor volume was measured 2x week for 69 d. Two-way ANOVA and Tukey’s multiple comparisons test (data analyzed until n = 3 remained per group) and Kaplan-Meier Log-rank survival analyses were performed. Results: The in vitro screen identified MEK inhibitor Cobimetinib as a lead candidate for synergistic combination with 177Lu-rhPSMA-10.1 across a wide concentration range, with a ZIP synergy score of 13.25% (95% CI ± 2.17) and promising results on MuSyc analysis. The 177Lu-rhPSMA-10.1 + Cobimetinib combination significantly suppressed tumor growth in vivo vs untreated controls (from day 13-30; p<0.01) and 177Lu-rhPSMA-10.1 alone (from day 17-30; p<0.001). The median survival in the combination group (49 d) was significantly longer vs the untreated group (23 d; p=0.001) and the group treated with 177Lu-rhPSMA-10.1 alone (36 d; p=0.002). Conclusions: Through an extensive in vitro screen, we identified Cobimetinib to have potential for a synergistic anti-tumor effect in combination with 177Lu-rhPSMA-10.1. This may be due to inhibition of the MEK-MAPK pathway by Cobimetinib during DNA damage response, resulting in radiosensitization of cancer cells to 177Lu-labeled RLT agents such as 177Lu-rhPSMA-10.1. This novel combination showed an enhanced therapeutic efficacy vs the single agents in 22Rv1 xenografts and the lack of overlapping monotherapy toxicity reported in the clinic supports clinical investigation in men with PCa. Citation Format: Caroline Foxton, Bart Cornelissen, Edward O'Neill, Bradley Waldron, Freja Pretzmann, Rikke Veggerby Grønlund, Mathias Wikke Hallund, Daniel J. Stevens. Evaluation of a synergistic drug combination with 177Lu-rhPSMA-10.1 for prostate cancer: Results of an in vitro screen and in vivo proof of concept study [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 694.

Abstract 3223: Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa)

Abstract Advanced PCa, especially castration-resistant PCa, represents an urgent unmet treatment need. Dysregulation of the polycomb repressive complex 2 (PRC2), which mediates trimethylation of histone H3 at lysine 27 (H3K27me3) and leads to gene silencing, is common in PCa and correlates with poor prognosis. EED, a core PRC2 component, is crucial for histone methyltransferase activity. MDM2, a negative regulator of p53, is often amplified or overexpressed in PCa and associated with metastasis and poor clinical outcomes. We evaluated antitumor activity and molecular mechanisms of EED inhibitor APG-5918/EEDi-5273 and MDM2 selective inhibitor alrizomadlin in preclinical PCa models. Human castration-resistant (22Rv1) and androgen-dependent (LNCaP) PCa cell lines were used in antiproliferation assays. Cell viability was determined by CellTiter-Glo luminescent assays. Western blotting was used to explore mechanisms of action. Subcutaneous 22Rv1 and LNCaP cell line-derived xenograft (CDX) models were established to evaluate antitumor effects of APG-5918 and alrizomadlin in vivo. Single-agent APG-5918 or alrizomadlin had limited inhibition on PCa cell growth; combined, they demonstrated synergistic antitumor activity. In the 22Rv1 CDX model, single-agent alrizomadlin (100 mg/kg) or APG-5918 (100 mg/kg) administered for 26 days exerted no/limited antitumor activity, with treatment-to-control (T/C) values of 93.39% and 49.89%, respectively. In contrast, the doublet enhanced antitumor activity (T/C value, 33.22%), with a synergistic index of 1.40. In the LNCaP CDX model, APG-5918 (100 mg/kg) administered for 28 days exhibited minimal antitumor effects (T/C value on last day of dosing, 80.32%). Alrizomadlin (100 or 50 mg/kg) showed limited antitumor activity (T/C value, 47.61%). Combined treatment significantly enhanced antitumor activity (T/C value, 15.69%), with a synergistic index of 2.44. Regarding putative mechanisms, western blot analyses showed that APG-5918 downregulated oncogenic drivers and DNA methylation factors (UHRF1, DNMT1) and on-target-related pharmacodynamic markers (H3K27me3, EED, EZH2). Alrizomadlin markedly downregulated UHRF1 and DNMT1, and upregulated p53 and p21 expression. Combined treatment further enhanced downregulation of DNMT1, UHRF1, cell cycle pathway proteins (pRb, CDK6), antiapoptotic protein MCL-1, and synergistically increased cleavage of PARP-1, a marker of apoptosis. In summary, we show that targeting EED and MDM2 synergistically enhances antitumor activity in PCa cell lines and CDX models. Mechanistically, this combination likely modulated pathways related to DNA methylation, cell cycle, and apoptosis. The findings provide a scientific rationale for future clinical development of APG-5918 and alrizomadlin for PCa patients. Citation Format: Yan Yin, Yun Yang, Eric Liang, Baisong Li, Hengbang Wang, Dajun Yang, Yifan Zhai. Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa) [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 3223.

Abstract 3145: HRA00242-C004, a novel anti-PSMA ADC with high DAR reveals potent in vitro and in vivo anti-tumor efficacy

Abstract Prostate cancer (PCa) is the second most common cancer, and it is the fifth leading cause of cancer-related death among men. The incidence rates of PCa are 37.5 per 100,000 in developed countries and 11.3 per 100,000 in developing countries, while mortality rates are 8.1 per 100,000 in developed countries and 5.9 per 100,000 in developing countries1. As androgen receptor (AR) signaling plays an essential role in PCa initiation and disease progression, androgen deprivation therapy (ADT) has been a backbone of treatment for patients with advanced disease2. However, patient responses to ADT vary, and most patients eventually develop castration-resistant prostate cancer (CRPC) which remains large unmet clinical need3. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein located on the cell membrane, is specifically and highly expressed in PCa with an extremely low expression level in non-prostatic tissues4, which makes it an ideal target for the treatment of PCa5.Here, we presented a PSMA-directed ADC, HRA00242-C004, which features a differentiated topoisomerase I inhibitor payload DXh conjugated via a cleavable linker to a humanized anti-PSMA IgG1 antibody (HRP04732). HRP04732 showed good binding affinity in both protein and cell-level assays, and a better cell internalization profile when compared with the reference antibody (AB-PG1-XG1-006, published sequences from US8114965B2). HRA00242-C004 exerted strong inhibition effect on cell lines with different PSMA expression levels. In vivo efficacy study, HRA00242-C004 showed potent anti-tumor activity in PSMA medium expression human prostate cancer 22RV1 xenograft model without significant weight loss during the experiments. Moreover, HRA00242-C004 demonstrated satisfactory PK profiles in SD rats and cynomolgus monkeys without any toxicity observed in PK assays. Compared with AB-PG1-XG1-006-DXh (the reference ADC), HRA00242-C004 exhibited a longer elimination half-life as well as higher serum exposure level of both the total antibody and the intact ADC. Meanwhile, as employing the same linker-payload as SHR-A1811, HRA00242-C004 has potential good plasma stability which was consistent with the results of PK study. In summary, with a highly permeable payload, high DAR, potent in vitro and in vivo anti-tumor efficacy, HRA00242-C004 demonstrated the best-in-class potential. 1.CA Cancer J. Clin. 2021, 71, 209-249.2.Nat. Rev. Urol. 2019, 16, 645-654.3.J. Clin. Oncol. 2015, 33, 1151-1156.4.World J Surg. 2006; 30: 628-36.5.Front Oncol. 2018; 8: 653-63. Citation Format: Guang Lin, Haoying Zhang, Zhibin Xu, Lingfeng You, Zhendong Xue, Yuchang Mao, Xiaoying Yang, Bing Hu, Sophie Lin, Jun Feng, Zhe Zhang, Xin Ye, Min Hu, Feng He. HRA00242-C004, a novel anti-PSMA ADC with high DAR reveals potent in vitro and in vivo anti-tumor efficacy [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 3145.

Abstract 6059: Discovery of ETS-007, a first-in-class degrader targeting N-terminal domain of androgen receptor derived from AR liquid-liquid phase separation inhibitor

Abstract Patients with advanced prostate cancer inevitably acquire resistance to anti-androgen therapies, and progress to lethal castration-resistant prostate cancer (CRPC). The resistance mechanisms involve AR gene amplification and overexpression, constitutively active ligand-independent splice variants such as AR-V7, and the mutations in the AR ligand-binding domain (LBD). Our previous study has shown that the AR N-terminal domain (NTD) is responsible for AR liquid-liquid phase separation (LLPS) capability, which is essential for AR/AR-V7 transcriptional activity. We conducted a compound library screening using an AR LLPS phenotypic assay and identified ET0516 as a hit to specifically inhibit AR LLPS and its downstream gene expression, indicating that inhibition of AR. LLPS through targeting its NTD is a feasible approach to overcome the resistance to current anti-androgens therapy. Here we report the identification of a novel AR degrader, ETS-007 which is originated and evolved from internal LLPS phenotypic screening, that effectively degrades both AR and AR-V7 proteins with DC50 of single-digit nM by inducing a protein-protein interaction between AR NTD and an E3 ligase complex. ETS-007 induced AR and AR-V7 polyubiquitination, followed by degradation which could be blocked by the protease inhibitor MG132. ETS-007 significantly inhibited cell proliferation in AR and/or AR-V7 positive cell lines, e.g. LNCaP and 22RV1, and downregulated the androgen responsive genes such as PSA and TMPRSS2. Compared with enzalutamide and ARV-110, ETS-007 could block transcriptional activities of AR-V7, and AR mutants in luciferase report assay. Oral administration of ETS-007 in an enzalutamide-resistant 22RV1 xenograft model led to intratumoral AR-V7 degradation and concomitant decrease in PSA protein levels, resulting in a remarkable tumor regression. Taken together, our findings provide preclinical evidence that ETS-007 as a potent AR-NTD degrader derived from our LLPS platform can effectively block the transcriptional activities of both AR and AR-V7 for the treatment of metastatic CRPC. Citation Format: Yingjie Li, Mingyue Fei, Yingke Miao, Lijian Feng, Xiaofei Fan, Jinping Li, Yaozhong Chen, Ming Xu, Wenqi Cui, Qiangang Zheng, Jidong Zhu. Discovery of ETS-007, a first-in-class degrader targeting N-terminal domain of androgen receptor derived from AR liquid-liquid phase separation inhibitor [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 6059.

Abstract 4504: Discovery of CBPD-409 and CBPD-268 as highly potent and orally efficacious CBP/p300 PROTAC degraders for the treatment of castration-resistant prostate cancer

Abstract Prostate cancer remains the leading cause of cancer-related death globally in men. While current AR-targeted therapies are effective for the treatment of prostate cancer and improve patient survival, resistance in patients typically develops within 18 months. CBP/p300 are AR transcriptional co-activators and are promising therapeutic targets for the treatment of castration-resistant prostate cancer (CRPC). We developed a series of highly potent, selective, and orally efficacious CBP/p300 PROTAC degraders, with CBPD-409 being the best compound. CBPD-409 induces robust CBP/p300 degradation with DC50 0.2-0.4 nM and displays strong anti-proliferative effects with IC50 1.2-2.0 nM in VCaP, LNCaP and 22Rv1 AR+ cell lines. It has a favorable oral pharmacokinetic profile and achieves 50% of oral bioavailability in mice. A single oral administration of CBPD-409 at 1 mg/kg achieves >95% depletion of CBP/p300 proteins in the VCaP tumor tissue at 3 h and 24 h time points. CBPD-409 exhibits strong and dose/schedule-dependent tumor growth inhibition and is more potent and efficacious than two CBP/p300 inhibitors CCS1477 and GNE-049 and the AR antagonist Enzalutamide. To further improve the degradation potency and the oral bioavailability in rats, we developed a new class of CBP/p300 degraders based on our optimized CRBN ligand TX-16. Our efforts led to the discovery of CBPD-268 as an exceptionally potent, effective, and orally efficacious degrader of CBP/p300 proteins. In the VCaP, LNCaP and 22Rv1 cell lines, CBPD-268 induces consistent and robust CBP/p300 degradation with DC50 of ≤0.03 nM and Dmax >95%, leading to potent cell growth inhibition. CBPD-268 has excellent oral bioavailability in both mice and rats and has a good ADME profile. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent depletion of CBP and p300 proteins in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taken together, CBPD-409 and CBPD-268 are highly promising CBP/p300 degraders for further extensive evaluations for the treatment of CRPC and other types of human cancers. Citation Format: Zhixiang Chen, Mi Wang, Dimin Wu, Lijie Zhao, Tianfeng Xu, Hoda Metwally, Yu Wang, Donna McEachern, Wei Jiang, Longchuan Bai, Jie Luo, Meilin Wang, Ruiting Li, John Takyi-Williams, Lu Wang, Qiuxia Li, Bo Wen, Duxin Sun, Arul M. Chinnaiyan, Shaomeng Wang. Discovery of CBPD-409 and CBPD-268 as highly potent and orally efficacious CBP/p300 PROTAC degraders for the treatment of castration-resistant 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 4504.

Structure-Activity Relationship (SAR) Studies of Novel Monovalent AR/AR-V7 Dual Degraders with Potent Efficacy against Advanced Prostate Cancer.

Androgen receptor (AR) has been extensively established as a potential therapeutic target for nearly all stages of prostate cancer (PCa). However, acquired resistance to AR-targeted drugs inevitably develops and severely limits their clinical efficacy. Particularly, there currently exists no efficient treatment for patients expressing the constitutively active AR splice variants, such as AR-V7. Herein, we report the structure-activity relationship studies of 55 N-heterocycle-substituted hydantoins, which identified the structural motifs required for AR/AR-V7 degradation. Among them, the most potent compound 27c exhibited selective AR/AR-V7 degradation over other hormone receptors and excellent antiproliferative activities in LNCaP and 22RV1 cells. RNA sequence analysis confirmed that 27c effectively suppressed transcriptional activity of the AR signaling pathway. Importantly, 27c demonstrated potent antitumor efficacy in an enzalutamide-resistant 22RV1 xenograft model. These results highlight the potential of 27c as a promising dual AR/AR-V7 degrader for overcoming drug resistance in advanced PCa expressing AR splice variants.

Discovery of CBPD-268 as an Exceptionally Potent and Orally Efficacious CBP/p300 PROTAC Degrader Capable of Achieving Tumor Regression.

CBP/p300 proteins are key epigenetic regulators and promising targets for the treatment of castration-resistant prostate cancer and other types of human cancers. Herein, we report the discovery and characterization of CBPD-268 as an exceptionally potent, effective, and orally efficacious PROTAC degrader of CBP/p300 proteins. CBPD-268 induces CBP/p300 degradation in three androgen receptor-positive prostate cancer cell lines, with DC50 ≤ 0.03 nM and Dmax > 95%, leading to potent cell growth inhibition. It has an excellent oral bioavailability in mice and rats. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent CBP/p300 depletion in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taking these results together, CBPD-268 is a highly promising CBP/p300 degrader as a potential new cancer therapy.

Development of CD46 targeted alpha theranostics in prostate cancer using 134Ce/225Ac-Macropa-PEG4-YS5.

Rationale: 225Ac, a long-lived α-emitter with a half-life of 9.92 days, has garnered significant attention as a therapeutic radionuclide when coupled with monoclonal antibodies and other targeting vectors. Nevertheless, its clinical utility has been hampered by potential off-target toxicity, a lack of optimized chelators for 225Ac, and limitations in radiolabeling methods. In a prior study evaluating the effectiveness of CD46-targeted radioimmunotherapy, we found great therapeutic efficacy but also significant toxicity at higher doses. To address these challenges, we have developed a radioimmunoconjugate called 225Ac-Macropa-PEG4-YS5, incorporating a stable PEGylated linker to maximize tumoral uptake and increase tumor-to-background ratios. Our research demonstrates that this conjugate exhibits greater anti-tumor efficacy while minimizing toxicity in prostate cancer 22Rv1 tumors. Methods: We synthesized Macropa.NCS and Macropa-PEG4/8-TFP esters and prepared Macropa-PEG0/4/8-YS5 (with nearly ~1:1 ratio of macropa chelator to antibody YS5) as well as DOTA-YS5 conjugates. These conjugates were then radiolabeled with 225Ac in a 2 M NH4OAc solution at 30 °C, followed by purification using YM30K centrifugal purification. Subsequently, we conducted biodistribution studies and evaluated antitumor activity in nude mice (nu/nu) bearing prostate 22Rv1 xenografts in both single-dose and fractionated dosing studies. Micro-PET imaging studies were performed with 134Ce-Macropa-PEG0/4/8-YS5 in 22Rv1 xenografts for 7 days. Toxicity studies were also performed in healthy athymic nude mice. Results: As expected, we achieved a >95% radiochemical yield when labeling Macropa-PEG0/4/8-YS5 with 225Ac, regardless of the chelator ratios (ranging from 1 to 7.76 per YS5 antibody). The isolated yield exceeded 60% after purification. Such high conversions were not observed with the DOTA-YS5 conjugate, even at a higher ratio of 8.5 chelators per antibody (RCY of 83%, an isolated yield of 40%). Biodistribution analysis at 7 days post-injection revealed higher tumor uptake for the 225Ac-Macropa-PEG4-YS5 (82.82 ± 38.27 %ID/g) compared to other conjugates, namely 225Ac-Macropa-PEG0/8-YS5 (38.2 ± 14.4/36.39 ± 12.4 %ID/g) and 225Ac-DOTA-YS5 (29.35 ± 7.76 %ID/g). The PET Imaging of 134Ce-Macropa-PEG0/4/8-YS5 conjugates resulted in a high tumor uptake, and tumor to background ratios. In terms of antitumor activity, 225Ac-Macropa-PEG4-YS5 exhibited a substantial response, leading to prolonged survival compared to 225Ac-DOTA-YS5, particularly when administered at 4.625 kBq doses, in single or fractionated dose regimens. Chronic toxicity studies observed mild to moderate renal toxicity at 4.625 and 9.25 kBq doses. Conclusions: Our study highlights the promise of 225Ac-Macropa-PEG4-YS5 for targeted alpha particle therapy. The 225Ac-Macropa-PEG4-YS5 conjugate demonstrates improved biodistribution, reduced off-target binding, and enhanced therapeutic efficacy, particularly at lower doses, compared to 225Ac-DOTA-YS5. Incorporating theranostic 134Ce PET imaging further enhances the versatility of macropa-PEG conjugates, offering a more effective and safer approach to cancer treatment. Overall, this methodology has a high potential for broader clinical applications.

Доклиническое изучение терапевтической эффективности радиофармацевтического препарата [Lu-177]Lu-PSMA I&T

The appearance of radiopharmaceuticals based on prostate-specific membrane antigen (PSMA) ligands labeled with beta- or alpha-emitting radionuclides led to a breakthrough on the development of targeted therapy for metastatic castration-resistant prostate cancer. For the completeness of the preclinical evaluation of therapeutic radiopharmaceuticals based on PSMA, the study of their effectiveness should be supplemented by an assessment of absorbed doses of internal radiation in experimental tumor foci. The objectives of this study were: 1) Examine the dose-effect relationship to assess the therapeutic efficacy of [Lu-177]Lu-PSMA I&T with activities comparable to those of humans; 2) Assessing the expected absorbed dose levels created in 22RV1 xenografts after the therapeutic effects have been introduced, with the potential for both minimal and maximum radiopharmaceutical accumulation. The studies were conducted on male nu/nu mice with subcutaneous 22RV1 xenografts. The dynamic of accumulation was examined after 2.2 MBq of a radiopharmaceutical was introduced. The main findings were obtained when [Lu-177]Lu-PSMA was introduced at 9.2 MBq (EDmin) and 36.6 MBq (EDmax). A statistically significant dose-dependent inhibition of xenograft growth was found compared with the control. The expected absorbed doses for EDmin in xenografts were between 2.7 Gy for minimal accumulation and 3.9 Gy for maximum accumulation. For EDmax in a similar scenario, this range was 10.7 to 15.5 Gy. The results support the preclinical assessment's informationality in determining the range of doses absorbed in tumor foci that cause the observed therapeutic effects.

Design, synthesis and biological evaluation of a novel PSMA-PI3K small molecule drug conjugate.

Small molecule drug conjugates are an emerging targeted therapy for cancer treatment. Building upon the overexpressed prostate-specific membrane antigen (PSMA) in prostate cancer, we herein report the design and synthesis of a novel PSMA-PI3K small molecule drug conjugate 1. Conjugate 1 demonstrates potent inhibition against PI3K with an IC50 value of 0.40 nM and simultaneously targets PSMA, giving rise to selective growth inhibition activity for PSMA-positive cancer cells. Conjugate 1 also potently inhibits the phosphorylation of PI3K main downstream effectors and arrests the cell cycle in the G0/G1 phase in PSMA-positive 22Rv1 prostate cancer cells. Further in vivo evaluation shows that conjugate 1 has favorable efficacy and tolerability in a 22Rv1 xenograft model, demonstrating its potential application in targeted cancer therapy.

Antiproliferative activity of the novel CYP17A1 inhibitor alsevirone

BACKGROUND: Prostate cancer is the most frequently diagnosed type of cancer in men in developed countries. It is dependent upon androgens and could be effectively combated by androgen deprivation therapy. Reduction of androgen synthesis can be accomplished through the inhibition of the enzyme 17α-hydroxylase/17.20-lyase (CYP17A1), which catalyzes two sequential reactions in the production of androgens. Steroid derivatives modified with nitrogen-containing heterocycles attract attention as antineoplastic agents for prostate cancer treatment due to their inhibitory potential against CYP17A1.
 AIM: Evaluate cytotoxic activity and antitumor effects of the synthesized alsevirone in comparison with abiraterone.
 METHODS: Cytotoxicity was evaluated using MTT test. Anticancer effect was researched in vivo in prostate cancer xenograft models 22Rv1 and DU145 in Balb/c nude mice. Testosterone concentration was determined using an enzyme-linked immunosorbent assay in blood serum of BDF1 mice.
 RESULTS: Alsevirone demonstrated cytotoxic activity in prostate cancer cells: DU145 (23.8±1.2 µM vs 151.4±23.7 µM for abiraterone), 22Rv1 (35.9±5.6 µM vs 109.9±35.2 µM for abiraterone) and LNCaP (22.9±0.5 µM vs 28.8±1.6 µM for abiraterone). Testosterone concentration in blood serum of BDF1 mice reduced by 80% after 10-day treatment. Inhibition of the tumors’ growth in 22Rv1 xenograft model was statistically significant when using alsevirone in comparison with the control group: average tumor volume was 171.6±50.1 mm3 (р=0.022) vs 424.2±70.3 mm3 in control, with tumor growth inhibition index of 59%.
 CONCLUSIONS: Alsevirone has a higher cytotoxic potential against prostate cancer cells (DU145, LNCaP and 22Rv1) compared to abiraterone. Alsevirone demonstrated the ability to reduce the concentration of testosterone in the blood serum of BDF1 mice, and statistically significant antitumor activity in 22Rv1 xenograft models.