Androgen Receptor Splice Variants Research Articles

Reproducible preclinical models of androgen receptor driven human prostate cancer bone metastasis.

Preclinical models recapitulating the metastatic phenotypes are essential for developing the next-generation therapies for metastatic prostate cancer (mPC). We aimed to establish a cohort of clinically relevant mPC models, particularly androgen receptor positive (AR+) bone metastasis models, from LuCaP patient-derived xenografts (PDX) that reflect the heterogeneity and complexity of mPC. PDX tumors were dissociated into single cells, modified to express luciferase, and were inoculated into NSG mice via intracardiac injection. The progression of metastases was monitored by bioluminescent imaging. Histological phenotypes of metastases were characterized by immunohistochemistry and immunofluorescence staining. Castration responses were further investigated in two AR-positive models. Our PDX-derived metastasis (PDM) model collection comprises three AR+ adenocarcinomas (ARPC) and one AR- neuroendocrine carcinoma (NEPC). All ARPC models developed bone metastases with either an osteoblastic, osteolytic, or mixed phenotype, while the NEPC model mainly developed brain metastasis. Different mechanisms of castration resistance were observed in two AR+ PDM models with distinct genotypes, such as combined loss of TP53 and RB1 in one model and expression of AR splice variant 7 (AR-V7) expression in another model. Intriguingly, the castration-resistant tumors displayed inter- and intra-tumor as well as organ-specific heterogeneity in lineage specification. Genetically diverse PDM models provide a clinically relevant system for biomarker identification and personalized medicine in metastatic castration-resistant prostate cancer.

Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs.

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared to the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remains unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

Discovery of an Aldo-Keto reductase 1C3 (AKR1C3) degrader.

Aldo-keto reductase 1C3 (AKR1C3) is a protein upregulated in prostate cancer, hematological malignancies, and other cancers where it contributes to proliferation and chemotherapeutic resistance. Androgen receptor splice variant 7 (ARv7) is the most common mutation of the AR receptor that confers resistance to clinical androgen receptor signalling inhibitors in castration-resistant prostate cancer. AKR1C3 interacts with ARv7 promoting stabilization. Herein we report the discovery of the first-in-class AKR1C3 Proteolysis-Targeting Chimera (PROTAC) degrader. This first-generation degrader potently reduced AKR1C3 expression in 22Rv1 prostate cancer cells with a half-maximal degradation concentration (DC50) of 52 nM. Gratifyingly, concomitant degradation of ARv7 was observed with a DC50 = 70 nM, along with degradation of the AKR1C3 isoforms AKR1C1 and AKR1C2 to a lesser extent. This compound represents a highly useful chemical tool and a promising strategy for prostate cancer intervention.

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest

BackgroundTherapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported.MethodsTo characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay.ResultsAR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA.ConclusionVTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

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.

Manzamine A reduces androgen receptor transcription and synthesis by blocking E2F8-DNA interactions and effectively inhibits prostate tumor growth in mice.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

TAS3681, an androgen receptor antagonist, prevents drug resistance driven by aberrant androgen receptor signaling in prostate cancer.

Second-generation androgen receptor (AR) signaling inhibitors (ARSIs), such as abiraterone and enzalutamide, prolong the life of patients with castration-resistant prostate cancer (CRPC). However, patients receiving ARSIs ultimately develop resistance through various complex mechanisms, including AR mutations, constitutively active AR-splice variants (AR-Vs), and AR overexpression. Here, we characterized a novel AR pure antagonist, TAS3681, which inhibits AR transcriptional activity and downregulates AR-full length (AR-FL) and AR-Vs. TAS3681 reduced the protein levels of AR-FL and AR-Vs including AR-V7 in enzalutamide-resistant cells (SAS MDV No. 3-14), in vitro and in vivo, showing strong antitumor efficacy in an AR-V7-positive xenograft model. In AR-overexpressing VCaP (prostate cancer) cells, conversely to enzalutamide, TAS3681 effectively suppressed cell proliferation and downregulated AR expression. Importantly, TAS3681 blocked the transcriptional activity of various mutant ARs, including mutations F877L/T878A and H875Y/T878A, which confer resistance to enzalutamide, and V716M and H875Y mutations, which confer resistance to darolutamide. Our results demonstrate that TAS3681 suppresses the reactivation of AR signaling, which causes resistance to ARSIs, via a newly identified mechanism of action. Therefore, TAS3681 could be a new therapeutic option for CRPC treatment.

Abstract LB177: Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC

Abstract Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men worldwide. Androgens play an important role in the development and progression of prostate cancers and targeting androgen receptor (AR) signaling axis over decades has remained a mainstay of prostate cancer therapy. Next generation hormonal agents (NHAs) enzalutamide and abiraterone have brought clinical benefits for metastatic castrate-resistant prostate cancer (mCRPC) patients, AR axis-targeted therapy resistance is inevitable due to AR gene amplification or mutations. AR-V7 is the most clinically relevant AR splice variant associated with endocrine resistance and poor prognosis. AR-V7 constitutively activates AR signaling and lacks the ligand binding domain (LBD) where hormones and AR antagonists interact, resulting in resistance to AR signaling inhibitors (ARSi). Thus, AR-V7 is a promising therapeutic target to address ARSi resistance in mCRPC. We have discovered a highly potent AR-V7 proteolysis targeting chimera (PROTAC), HC-4955 that degrades both the wild-type AR (WT-AR) and AR-V7 mutant. HC-4955 degrades AR-V7 with DC50 < 5 nM. In silico molecular docking analysis predicts the binding of HC-4955 to the N-terminal domain of AR and then the essential amino acids contributed to HC-4955 binding are validated by mutational analysis studies. HC-4955 also potently degrades mutants of AR-LBD mutations including L702H and T878A and these mutations are also associated with ARSi resistance. HC-4955 strongly inhibits proliferation of ARSi-resistant prostate cancer cell lines with IC50 < 10 nM. HC-4955 shows a robust anti-tumor activity in the AR-V7-bearing 22Rv1 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models, with majority of the tumors completely regressed and > 90% AR-V7 expression decreased at 1-5 mg/kg (QD, PO) in these models. The pre-clinical results support the clinical development of HC-4955 for the treatment of mCRPC. Selected pre-clinical data along with the chemical structure of HC-4955 will be presented. Citation Format: Jing Li, Zhilin Tu, Wu Du, Xi Xiang, Zeyu Wen, Yang You, Lijuan Zhao, Yin Chen, Chengcheng Yang, Hongwei Yuan, Xinghai Li. Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB177.

EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells

The androgen receptor (AR) is a primary target for treating prostate cancer (PCa), forming the bedrock of its clinical management. Despite their efficacy, resistance often hampers AR-targeted therapies, necessitating new strategies against therapy-resistant PCa. These resistances involve various mechanisms, including AR splice variant overexpression and altered activities of transcription factors like the glucocorticoid receptor (GR) and FOXA1. These factors rely on common coregulators, such as EP300/CREBBP, suggesting a rationale for coregulator-targeted therapies. Our study explores EP300/CREBBP acetyltransferase inhibition’s impact on steroid receptor and FOXA1 signaling in PCa cells using genome-wide techniques. Results reveal that EP300/CREBBP inhibition significantly disrupts the AR-regulated transcriptome and receptor chromatin binding by reducing the AR-gene expression. Similarly, GR’s regulated transcriptome and receptor binding were hindered, not linked to reduced GR expression but to diminished FOXA1 chromatin binding, restricting GR signaling. Overall, our findings highlight how EP300/CREBBP inhibition distinctively curtails oncogenic transcription factors’ signaling, suggesting the potential of coregulatory-targeted therapies in PCa.

Developing a Novel Enzalutamide-Resistant Prostate Cancer Model via AR F877L Mutation in LNCaP Cells.

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol: Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo.

Abstract 7505: Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays

Abstract Introduction: Androgen-receptor splice variant 7 (AR-V7) is constitutively activated isoform of AR and has been associated with resistance towards AR targeting therapies and development into mCRPC. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the drug, but the detection of AR-V7 with tissues has limitations such as clinical challenge to poorly accessible tumor tissues before and after treatment. Here, we developed the method of AR-V7 detection in circulating tumor cells (CTC) after isolation with CytoGen’s Smart BiopsyTM CTC isolator by in vitro assays of qPCR and immunofluorescence (IF) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Method: qPCR and IF methods were developed for high sensitivity and specificity of AR-V7 detection. Sensitivity and specificity were tested using AR-V7 positive cells (VCaP and 22Rv1) and negative cells (PC-3 and Raji). Limit of detection (LOD) test of AR-V7 expression was performed with AR-V7 positive cells (200 cells to 50 cells, 3 points) mixed with Raji cells (1 × 10^5cells). Then, AR-V7 positive cells (200 cells to 50 cells, 3 points) were spiked into peripheral blood mononuclear cells (PBMC) of blood sample (5mL, healthy donor) to mimic CTC after isolation by CytoGen’s Smart BiopsyTM CTC isolator, subsequently AR-V7 expression was tested with qPCR and IF assay. Results: AR-V7 expression was clearly detected by qPCR and IF in the LOD test of AR-V7 positive cells mixed with Raji cells and was not detected at all in the negative controls. The similar clear results were also observed in the spiking test of AR-V7 positive cells into PBMC isolated by CytoGen’s Smart BiopsyTM CTC isolator, suggesting that clinical samples of prostate cancer could be applied with excellent sensitivity and specificity for AR-V7. Conclusions: AR-V7 expresses only a part of cancer tissue, so it is difficult to accurately diagnose with traditional histological assays. Detection of AR-V7 in intact live CTC isolated by CytoGen’s Smart BiopsyTM CTC isolator might overcome limitations of tissue biopsy. In this study, we developed the method to detect AR-V7 in CTC with high sensitivity and specificity with qPCR and IF assay, and presented the results of verifying AR-V7 expression by double check using two kinds of assays. We plan to conduct clinical validation of the developed method through additional study using patient samples. Citation Format: Hoin Kang, Jihyun Lee, Min Hyung Kim, Soee Kim, Hyoyong Kim, Jubi Lee, Hae Ung Lee, Hai-Chon Lee, Jung Won Kim. Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays [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 7505.

Abstract 2266: Detection of androgen receptor splice variants from clinical sequencing

Abstract Background: Androgen receptor splice variant-7 (AR-v7) has been widely studied as a biomarker of resistance to AR-targeted therapy in castration-resistant prostate cancer (CRPC). Various methods have been used to detect AR-v7 in CRPC specimens, including next generation sequencing (NGS) technology. However, NGS technologies applied in clinical sequencing have focused on using blood or fresh-frozen CRPC tissue specimens. There is not yet an AR-v7 detection system tailored to formalin-fixed paraffin-embedded (FFPE) tissue specimens commonly used for clinical sequencing in hospitals. Methods: In this respect, we propose a novel approach to identify AR-v7 in targeted RNA sequencing (RNA-seq) data derived from FFPE specimens. This approach can identify, in addition to AR-v7, all constitutively active AR splice variants (AR-Vs) that are co-expressed with AR-v7. In short, our two-step approach first gathers soft-clipped or divided reads that are adjacent to the splicing sites of AR-Vs and then yields the number of splitting reads that support the existence of AR-Vs. Next, the algorithm selects the paired-end reads whose one side and the other side are mapped to the preceding and following exons, respectively. The final number of spanning reads are calculated following to the removal of low-quality reads. Results: We validated the proposed approach using two large-scale, independent RNA-seq datasets of prostate cancer (PC) samples: 111 samples from Seoul National University Hospital (SNUH) and 558 samples from The Cancer Genome Atlas (TCGA) dataset. The majority of both datasets were localized PC tumors, especially in the SNUH dataset, where localized PC accounted for 91% of the total. Overall, our approach successfully identified samples with putative AR-Vs, including AR-v7, AR-v3, and AR-v9. Statistical analyses of 111 SNUH PC patients suggested potential detection threshold in clinical sequencing settings (at least 200 split reads). In the SNUH dataset, the AR-v7 positive group, which included 5 patients (5%) with values above the threshold, shared the following characteristics: higher AR-v7/full length AR expression levels, AR amplification and co-expression with AR-v3/AR-v9. AR-v7 positivity was experimentally validated by Sanger sequencing. The similar pattern was also found in the analysis of TCGA dataset. Conclusions: These results demonstrate that the detection system can successfully identify AR-v7 positivity not only for metastatic PC but also for patients with localized PC through clinical sequencing. We expect that the further in-depth analyses including larger samples and clinical outcomes can discover clinical applicability of AR-v7. Citation Format: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun. Detection of androgen receptor splice variants from clinical sequencing [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 2266.

Abstract 4102: Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models

Abstract Background: Androgen receptor (AR) signaling inhibition is part of the mechanism behind many effective prostate cancer therapies, including androgen deprivation and next generation anti-androgens. However in castration resistant prostate cancer up to 50% of treatment resistance has been linked to AR amplification or expression of the constitutively active RNA splice variant, AR-V7. Although the use of immune checkpoint blockade has failed to demonstrate clinical success, immunotherapeutic interventions remain attractive as prostate cancer’s relatively slow progression provides time for anti-tumor immune responses to develop. Cancer specific vaccines have the potential to enable and enhance tumor specific immune responses by stimulating and directing T cell immunity to important oncologic targets. We hypothesized that a cancer vaccine designed to induce a T cell response against cells expressing high levels of AR would result in an anti-tumor effect and potentially prevent or delay the development of therapeutic resistance. Methods: Adenoviral vaccines against AR (Ad-AR) or AR splice variant 7 (Ad-AR-V7) were used to evaluate the potential of AR targeting vaccination in mouse models. We evaluated AR and AR variant specific adaptive responses produced by our vaccines with naïve and tumor-bearing mice by ex vivo peptide restimulation. We challenged vaccinated mice with multiple syngeneic subcutaneous tumor models that we engineered to express AR-V7, including a PTEN-/- p53-/- prostate tumor cell line. Finally we tested our vaccines in a therapeutic regimen, vaccinating mice after implanting our subcutaneous AR-V7 expressing models. Results: Across multiple strains of mice, AR or AR-V7 targeting adenoviral vaccination induced robust adaptive immune responses against the N-terminal domain of AR. Vaccination with either Ad-AR or Ad-AR-V7 resulted in significant anti-tumor responses, providing a significant overall survival benefit and complete tumor rejection by the majority of mice vaccinated against AR or AR-V7. Similarly tumor bearing mice treated with Ad-AR or Ad-AR-V7 vaccination displayed tumor growth inhibition or complete tumor regression resulting in an overall survival benefit. Additionally, we found that vaccine responses were unaffected by the use of the approved antiandrogen drug enzalutamide, suggesting that vaccinations could be deployed in tandem with standard-of-care therapies. Conclusions: Vaccination against AR and AR splice variants can induce AR specific adaptive immune responses capable of inhibiting the growth of AR-V7 expressing tumor models, illustrating the potential of AR and its splice variants as immunologic targets in prostate cancer. Citation Format: Robert D. Marek, Junping Wei, Tao Wang, Xiao-Yi Yang, Gangjun Lei, Zachary C. Hartman. Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models [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 4102.

Abstract 4750: The MNK inhibitor EB1 sensitizes AR-V7 positive castration-resistant prostate cancer to enzalutamide

Abstract Introduction: Resistance to androgen receptor signaling inhibitors (ARSI) like Enzalutamide is frequent in castration resistant prostate cancer (CRPC). The mechanisms driving drug resistance include the overexpression of androgen receptor (AR) and the presence of splice variants like androgen receptor splice variant 7 (AR-V7). Clinical studies (PROPHECY) have demonstrated that AR-V7 positive CRPC patients treated with Enzalutamide show poorer overall survival and thus lack an effective targeted line of treatment. Recently, AR signaling and the regulation of protein translation via the translation initiation factor 4E (eIF4E) and MAPK-interacting kinases (MNKs) have been shown to depend on each other and targeting eIF4E has been proposed to increase the efficacy of ARSI. Objectives: We have recently described EB1 as the first in class non-ATP competitive inhibitor of MNK1/2 (Bou-Petit et al. JMC, 2022) and have now tested its applicability in CRPC. In this work we describe the impact of EB1 on the efficacy of Enzalutamide treatment in CRPC. Methodology: Cell lines 22RV1 and LNCaP-95 were used as AR-V7 positive models of CRPC. Effects of EB1 and Enzalutamide alone and in combination on cell growth were studied through crystal violet assay. Drug combination Index was calculated following Chou-Talalay method using CompuSyn Software. Cell death was studied through propidium iodide (PI) staining. AR transcriptional activity was assessed through RT-qPCR of AR canonical target genes like PSA. A head-to-head study with eFT-508 (Tomivosertib, eFFECTOR Therapeutics, Inc.) as the most advanced Type I MNK inhibitor was performed. Lastly, spheroids obtained through centrifugation in ultra-low attachment plates were used for validating drug interaction in 3D growth and viability studies. Spheroid viability was assessed through PI and Calcein AM staining as well as MTT assay. Results: In vitro results demonstrate strong to moderate synergistic inhibition of cell growth through the induction of cell death by the combination of Enzalutamide and EB1 in AR-V7 positive CRPC cell lines, as well as reduction of AR transcriptional activity (PSA). Drug combination also reduced growth and viability of spheroids, indicating good tumor penetrating properties in 3D models and giving in vitro proof-of-concept for in vivo applications. Furthermore, drug synergy with Enzalutamide could only be observed with EB1 and not with Type-I MNK inhibitors like eFT-508, indicating that MNK1/2 functions beyond its kinase activity might be relevant for cancer progression. Conclusion: Here we demonstrate that the novel MNK inhibitor EB1 shows promising in vitro results, which cannot be obtained with Type-I MNK inhibitors. The combination of EB1 with the standard of care Enzalutamide provide a novel therapeutic opportunity for the treatment of CRPC, particularly in AR-V7 positive patients. Citation Format: Sara García-Ortega, Leticia Suárez-Cabrera, Joan Morote, Olga Méndez, Marta Cano-Galietero, Elena Muro-Blanc, Jose I. Borrell, Anna Santamaria, Santiago Ramón y Cajal, Stefan Hümmer. The MNK inhibitor EB1 sensitizes AR-V7 positive castration-resistant prostate cancer to enzalutamide [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 4750.

Abstract 6045: Development and characterization of ATC-324: An AUTOTAC-based androgen receptor degrader for prostate cancer treatment

Abstract Prostate cancer (PCa) progression is largely driven by the androgen receptor (AR), making it a prime target for therapy. However, therapy resistance often arises due to AR mutations and splice variants, such as AR-v7. AUTOTAC (AUTOphagy-TArgeting Chimera) is a novel protein degradation platform that utilizes the autophagy-lysosomal pathway to selectively degrade disease-causing proteins. In this study, we characterize ATC-324, an AUTOTAC-based AR degrader designed for the treatment of PCa. ATC-324 comprises enzalutamide linked to YT 6-2, a small molecule activator of the autophagy receptor p62/SQSTM1. ATC-324 induces the formation of the AR/p62 complex while activating autophagic flux, leading to the autophagy-lysosomal degradation of AR. To characterize ATC-324, we employed various AR-dependent, AR-null, and enzalutamide-resistant PCa cell lines. Given that bone metastasis is a major clinical complication of castrate-resistant PCa associated with greater morbidity and mortality, we utilized the bone-in-culture array (BICA) assay, an ex vivo bones culture model, to investigate the impact of ATC-324 on PCa cell survival and growth in the bone microenvironment. We demonstrated that ATC-324 effectively degrades wild-type, mutant, and splice variants of AR. In addition, ATC-324 induces AR-dependent apoptosis and cytotoxicity. Importantly, ATC-324 maintains its potency in enzalutamide-resistant PCa cells. Utilizing the BICA assay, we showed that ATC-324 exhibits cytotoxicity against 22Rv1 bone micrometastases at even lower concentrations than in 2D cell culture. In conclusion, our study demonstrates the successful development and characterization of ATC-324 as a potent and selective degrader of various forms of AR. Furthermore, its effectiveness against enzalutamide-resistant cells indicates its capability to overcome therapy resistance. The observed cytotoxicity against bone micrometastases suggests a therapeutic potential for combating bone metastasis, a major complication of castration-resistant PCa. Our study underscores the potential of the AUTOTAC platform to selectively degrade disease-causing proteins Citation Format: Tri Pham, Tae Hyun Bae, Ki Woon Sung, Abdo Najy, Alaleh Zamiri, Hyejeong Jang, Su Ran Mun, Seongho Kim, Dongping Shi, Steven Kregel, Elizabeth Heath, Michael Cher, Yong Tae Kwon, Hyeong-Reh Kim. Development and characterization of ATC-324: An AUTOTAC-based androgen receptor degrader for prostate cancer treatment [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 6045.

Abstract 7222: Antiandrogen equipped with HDAC inhibitors activates autophagy-mediated growth inhibition in castrate-resistant prostate cancer

Abstract Targeting androgen receptor (AR) by pharmacologic intervention is one of the practical approaches for the treatment of castration-resistant prostate cancer (CRPC). Hence, this study aimed to develop novel molecules that employ multiple mechanisms to inhibit AR expression and curtail the growth of CRPC. The target antiandrogen-equipped histone deacetylase inhibitors were synthesized, adapting the convergent chemistry we used to synthesize the first-generation compounds. The final compounds were characterized using 1H-NMR, 13C-NMR, and high-resolution mass spectrometry. We synthesized and analyzed several compounds, in which KK62 emerged as the lead compound. KK62 significantly inhibits a panel of CRPC cell lines in nanomolar concentrations without causing toxicity to healthy prostate epithelial cells. The molecular analysis suggested that inhibition of AR and AR-splice variants was evident, followed by downregulation of PSA expression in AR-positive CRPC cell lines. Further analysis confirmed that KK62 overcomes dihydrotestosterone (DHT) induced AR signaling and enzalutamide-resistant CRPC cells by downregulating AR and AR-splice variants in CRPC cells. Subsequent analysis confirmed that KK62 induces autophagy signaling by upregulating autophagosome (LC3B) and lysosome (LAMP1) markers expression and facilitating the autophagosome and lysosome fusion, which resulted in the induction of apoptosis in all AR-positive CRPC cell lines. The pro-apoptotic markers, such as BAX and cleaved caspase, were upregulated, whereas the downregulation of pro-survival markers was evident in KK62-treated CRPC cells. Our ongoing castrated and non-castrated xenotransplanted CRPC tumors and Patient-derived xenograft in vivo studies would validate our in vitro findings and confirm that KK62 is a potent compound that may improve therapeutic advances in treating CRPC. Citation Format: Balaji Chandrasekaran, Kiran Kumar Yalla, Ashish Tyagi, Vaibhav Shukla, Neha Tyagi, Bhawna Tyagi, Mohit Vashishta, Adegboyega K Oyelere, Chendil Damodaran. Antiandrogen equipped with HDAC inhibitors activates autophagy-mediated growth inhibition in castrate-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 7222.

Abstract 6071: Identification of a molecular glue degrader targeting the full-length AR and AR-V7 splice variant

Abstract Prostate cancer is the most common cancer in males and the fifth leading cause of cancer mortality worldwide. A variety of therapeutic approaches exist including steroid synthesis inhibitors and next-generation ligand binding domain (LBD) antagonists [e.g., enzalutamide (Enza)]. However, these approaches are only effective against tumors bearing the full-length androgen receptor (AR-FL). In the course of their disease, many patients acquire androgen receptor splice variant 7 (AR-V7), a mutant form of the receptor which lacks the LBD and is constitutively activated. To date, no effective therapy has been demonstrated for patients with metastatic prostate cancer bearing the AR-V7 signature. We have recently developed a high throughput platform that allows for the identification of ligands that can bind to a wide variety of protein targets. Using this approach, we screened a diverse chemical library of 100,000 small molecules to identify compounds that are bound to AR-V7. One such molecule, AR-600, directly bound to AR-V7 and moreover, induced proteasomal-mediated targeted protein degradation of both AR-V7 and AR-FL. In prostate cancer cell lines expressing AR-FL, AR-600 induced a gene expression profile similar to cells treated with Enza including a marked reduction in KLK3 (PSA) expression. Moreover, AR-600 inhibited the growth of 22RV1 cells (IC50=23 nM), a cell line that predominantly expresses AR-V7, as well as C4-2 cells that express predominantly full-length AR (IC50=51 nM). In contrast, the compound did not affect the growth of a wide variety of non-tumorigenic cells, non-prostate tumor cell lines or PC3 cells, a prostate cell line that exhibits AR-independent cell growth (IC50>100uM). Preliminary exploration of the mechanism of action of AR-600 (M.W. ~300 Daltons) demonstrates the compound works as a molecular glue. These results thereby identify a small molecule glue degrader that can specifically induce the targeted protein degradation of the AR-V7 splice variant. Given the strong association of this isoform with treatment resistance, this approach would appear to provide an attractive strategy for patients with metastatic castration resistant prostate cancer. Citation Format: Yuan Liu, Mads B. Larsen, Bo Lin, Irene A. Cardenal, Jason Kennerdell, Toren Finkel, Bill B. Chen. Identification of a molecular glue degrader targeting the full-length AR and AR-V7 splice variant [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 6071.

Abstract 2105: Targeting the “Achilles Heel” of androgen receptor activity in castration resistant prostate cancer

Abstract Background: Each year, millions of men are diagnosed with prostate cancer (CaP). The unchecked activation of the Androgen Receptor (AR) spurs CaP development and progression. Mutations or the presence of AR splice variants can add complexity to tumor ecology, leading to chemoresistance. This study aims to develop novel inhibitors targeting the “Achilles Heel” of AR activity: the N-terminal domain (NTD). This would circumvent the limitations of LBD (Ligand Binding Domain) therapies and offer a superior treatment option for patients. Though the AR-NTD is an intrinsically disordered protein, which complicates structure-based drug design, we have developed a unique small molecule inhibitor, ASR600, which specifically targets AR-NTD and promotes AR and AR- variants degradation by ubiquitination at previously unknown sites. Methods: Mass-spec analysis was performed to identify distinct ubiquitination sites in AR's NTD. To perform spatial transcriptome analysis, CytAssist Visium (10X genomics) was used. Furthermore, we used western blotting, immunofluorescence, immunoprecipitation, and PDX mice studies in CRPC mouse models to examine the impact of ASR600 on CRPC. Results: Four AR ubiquitination sites: K845, K847, and K913 (LBD) and K311 (NTD) are already identified. Interestingly, ASR600 continued to inhibit AR expression even with mutated ubiquitination sites. Mass spectrometry analysis of ASR-mediated mono-ubiquitinated N-terminal AR identified K16 as a unique ubiquitin acceptor. Altering this ubiquitination site rescues AR expression from ASR600-mediated degradation, emphasizing AR N-terminus as an ASR600 target. To discern the role of the K16 ubiquitination site in AR protein turnover, we evaluated AR protein stability in LNCaP cells that stably expressed ARWT or AR-K16R constructs. Our data indicates the pivotal importance of K16 for AR transcriptional activity. Invivo, ASR600, when used as a sole agent, exhibited inhibitory responses in PDX of clinically aggressive, AR-expressing tumors. Spatial gene expression analysis revealed marked expression differences in AR signature genes between control and ASR600-treated PDX tissues. Crucially, AR signaling, and related markers mainly aligned with epithelial markers rather than stromal ones. In contrast, the ASR600-treated PDX tumor showcased reduced AR signaling within epithelial cells. Conclusion: Targeting AR-NTD has received a lot of attention, but its innately disordered structure has hindered progress. ASR600 targets AR-NTD specifically by ubiquitinating the protein at a novel location, K16. Our in vivo, PDX studies, combined with the spatial analysis, lay a strong foundation for initiating IND-enabling TOX studies, followed by a phase-I clinical trial for ASR-600 in CRPC patients. Citation Format: Ashish Tyagi, Balaji Chandrasekaran, Arun K. Sharma, Chendil Damoadaran. Targeting the “Achilles Heel” of androgen receptor activity in 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 2105.

Abstract 1231: First-in-class AR-V7/AR-fl small molecule molecular glue degrader for prostate cancer treatment

Abstract Metastatic castration resistance prostate cancer (mCRPC) is a lethal disease due to the development of treatment to androgen receptor (AR) signaling inhibitors (ARSi) and taxane chemotherapy. Treatment resistance occurs partly due to the expression of AR splice variants that lack the ligand binding domain (LBD) and are constitutively active in the nucleus. AR-V7 is the most prevalent variant conferring clinical resistance to both ARSi and taxanes. Currently, there is no selective AR-V7 inhibitor leaving patients with limited therapeutic options. Thus, the development of selective AR-V7 inhibitors is a high priority, clinically unmet need. To identify AR-V7 pharmacologic inhibitors, we performed a high throughput small molecule phenotypic screen using enzymatic complementation and nuclear AR-V7 as the assay endpoint. Our primary screen of ~170K compounds (z score = 0.8), followed by a cell-toxicity counter screen and a secondary GFP screen identified hit compound 7907, as a dual AR-V7/AR-fl protein degrader, with unique chemotype compared to all known AR modulators. Hit to lead optimization by medicinal chemistry/SAR studies identified lead compound 15, with increased potency compared to 7907. Mechanistically, we showed that compound 15 shortened AR-V7 protein half-life by activating the ubiquitin-proteasome pathway and inducing proteasomal degradation of both AR-V7/AR-fl, without affecting their transcription. Importantly, compound 15 induced degradation occurred within 3hr of treatment and was blocked by the clinically approved proteasome inhibitor, bortezomib. TurboID proximity ligation assay identified distinct E3 ligases, uniquely interacting with AR-V7 or AR-fl. Using AR-V7/AR-fl deletion mutants we further showed that compound 15 activity is mediated by the N-terminal domain of AR, present in both proteins. Remarkably, compound 15 sensitized LNCaP95 cells (endogenous AR-V7/AR-fl expression) to enzalutamide suggesting potential therapeutic synergism and ability to reverse enzalutamide resistance. Ongoing studies aim to narrow down the binding site of compound 15 as well as identify the E3 ligases mediating its activity. Together, these data support a molecular glue degrader mechanism of action, consistent with published studies showing that molecular glue degraders are ideal for targeting classically “undruggable” proteins lacking an LBD or containing intrinsically disordered domains, as is the case for AR-V7. Currently, all AR-directed therapies target the LBD of AR-fl, inhibiting AR signaling. AR-V7 expression is a direct outcome of this inhibition, leading to AR-fl and AR-V7 co-expression in patient tumors. We posit that our drug candidate, by offering dual AR-V7/AR-fl inhibition in a single treatment, has the potential to not only benefit patients with mCRPC but also patients with hormone-sensitive disease, and delay AR-V7 expression. Citation Format: CheukMan C. Au, Catrina Estrella, Prerna Vatsa, Michelle Naidoo, Michael Miller, Peter T. Meinke, David M. Nanus, Paraskevi Giannakakou. First-in-class AR-V7/AR-fl small molecule molecular glue degrader for prostate cancer treatment [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 1231.

Abstract 6271: Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer

Abstract Prostate cancer (PC) stands as the most frequently diagnosed and second leading cause of cancer death among American men. PC mortality is predominantly attributed to castration-resistant prostate cancer (CRPC), often emerging within 2-3 years following the initiation of androgen deprivation therapy. This resistance primarily arises from sustained androgen receptor (AR) signaling, ultimately resulting in a compromised response to AR-directed therapies. AR splice variant expression is a key contributor to the persistent AR signaling in CRPC. AR-variant 7 (AR-V7) is the most clinically prevalent variant, expressed in about 75% of patients with CRPC and confers resistance to standard of care (SoC) treatments. AR-V7 differs from the full-length AR (AR-fl) because it lacks the ligand binding and hinge domains, rendering it resistant to SoC treatments. Currently, there are no selective inhibitors for AR-V7. Therefore, we sought to identify unique biological features of AR-V7, distinct from AR-fl, to exploit therapeutic options for targeting AR-V7. Previous studies have demonstrated high sequence homology, largely overlapping cistromes, and gene transactivation profiles between AR-V7 and AR-FL. Our published mechanistic studies revealed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl, allowing for constitutive import and activation of target genes in the nucleus. Herein, we report an additional novel subnuclear phenotype of AR-V7 in LNCAP95 and 22RV1 cells modeling CRPC. Staining with AR-V7- and AR-fl- specific antibodies revealed that unlike AR-fl, AR-V7 exhibited prominent nucleolar localization. To validate AR-V7 nucleolar localization, CRPC cells stained with fibrillarin (nucleolar marker), and subsequent fluorescence intensity analysis revealed that AR-V7 was predominantly localized in fibrillarin positive nucleoli, whereas AR-fl protein was solely localized in the nucleoplasm. This suggests a novel functional role of AR-V7 in the nucleus. To further confirm AR-V7 nucleolar localization, Actinomycin D (ActD) was used as a tool compound to disrupt nucleolar integrity. Treatment with ActD for 4 hours in CRPC cells significantly reduced the area of fibrillarin positive nucleoli and prompted AR-V7 re-distribution out of the nucleoli and into the nucleoplasm. Ongoing efforts to further investigate AR-V7 nucleolar function in CRPC cells include mutating the predicted nucleolar localization signal located in AR-V7 and identifying nucleolar-interacting proteins through proximity-labeling assays. In conclusion, we unveiled a novel nuclear compartmentalization of AR-V7 within the nucleolus, the primary hub for ribosomal biogenesis. These observations suggest that AR-V7 could regulate the high levels of protein synthesis occurring in CRPC cells, creating a window of a potential therapeutic opportunity for selective inhibition. Citation Format: Michelle K. Naidoo, Paraskevi Giannakakou. Novel insights into androgen receptor-variant 7 subnuclear localization and function in 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 6271.