Androgen Receptor Splice Variants Research Articles

Prognostic role of Androgen Receptor splice variant 7 (AR-V7) in the pathogenesis of breast cancer

Prognostic role of Androgen Receptor splice variant 7 (AR-V7) in the pathogenesis of breast cancer

Vaccination Against Androgen Receptor Splice Variants to Immunologically Target Prostate Cancer

Background/Objectives: Androgen receptor (AR) expression and signaling are critical for the progression of prostate cancer and have been the therapeutic focus of prostate cancer for over 50 years. While a variety of agents have been developed to target this axis, many of these fail due to the emergent expression of AR RNA splice variants, such as AR-V7, that can signal independently of ligand binding. Other therapies, such as vaccination against prostate-specific antigens, have achieved FDA approvals but have fallen short of being incorporated as standard-of-care therapies for advanced prostate cancer. This may be due to the elevated level of immunosuppression observed in prostate cancer, which remains largely refractory to immune checkpoint blockade. Methods: We developed a vaccine targeting AR-V7, a common isoform associated with treatment resistance, and demonstrated its ability to elicit AR-V7-specific immunity and enable anti-tumor responses against AR-V7+ cancers in subcutaneous tumor models. Results: Our studies also revealed that AR-V7 expression conferred an immune suppressive phenotype that was significant in a non-AR-dependent prostate cancer model. Notably, in this model, we found that vaccination in combination with enzalutamide, an AR antagonist, suppressed these aggressive immune suppressive cancers and resulted in enhanced survival in comparison to control vaccinated and enzalutamide-treated mice. While anti-PD-1 immune checkpoint inhibition (ICI) alone slowed tumor growth, the majority of vaccinated mice that received anti-PD-1 therapy showed complete tumor elimination. Conclusions: Collectively, these results validate the importance of AR signaling in prostate cancer immune suppression and suggest the potential of AR-V7-specific vaccines as therapeutic strategies against prostate cancer, offering significant protective and therapeutic anti-tumor responses, even in the presence of androgen signaling inhibitors.

An Autophagy-Targeting Chimera Induces Degradation of Androgen Receptor Mutants and AR-v7 in Castration-Resistant Prostate Cancer

Abstract Genetic alterations play a pivotal role in various human diseases, particularly cancer. The androgen receptor (AR) is a crucial transcription factor driving prostate cancer (PCa) progression across all stages. Current AR-targeting therapies utilize competitive AR antagonists or pathway suppressors. However, therapy resistance often emerges due to AR mutations and AR splice variants, such as AR-v7. To overcome this, we developed ATC-324, an AR degrader using the innovative protein degradation technology platform AUTOphagy-TArgeting Chimera (AUTOTAC). ATC-324 was designed to comprise enzalutamide, an AR inhibitor, as a target-binding ligand and YT 6-2, a ligand of the autophagy receptor p62/SQSTM1, as an autophagy-targeting ligand. ATC-324 induces the formation of the AR/p62 complex, leading to autophagy-lysosomal degradation of AR. Importantly, ATC-324 effectively degrades AR mutants frequently detected in PCa and co-degrades AR-v7 as a heterodimer with full-length AR. ATC-324 reduces nuclear AR levels and downregulates the target gene expression of AR and AR-v7, leading to cytotoxicity in AR-positive PCa cells. We also provide evidence of the therapeutic potential of ATC-324 in vivo as well as ex vivo bone organ culture. Moreover, ATC-324 remains potent in enzalutamide-resistant PCa cells. These results demonstrate the potential of the AUTOTAC platform to target previously considered undruggable proteins and overcome certain drug resistance mechanisms.

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.

B-255 Identification of the protein expression of Androgen Receptor variants using Targeted proteomics in clinical Castration Resistant Prostate Cancer models

Abstract Background Castration Resistant Prostate Cancer (CRPC) is a treatment resistant form of prostate cancer (PCa). Currently, there is not a way to identify which patients will develop this resistance before full blown CRPC develops. Therefore, all PCa treatment approaches are similar yet this results in tumor regression in some cases and progression in others. Targeting the androgen receptor (AR) is still the main focus of current therapies even in CRPC. Emergence of AR splice variants (AR-Vs) after initial treatment is thought to be one of the primary mechanisms of resistance. AR-Vs lack the ligand binding domain rendering Androgen Deprivation Therapy (ADT) ineffective in tumors expressing these variants. Recent work has identified the DNA and RNA species of ARVs in CRPC but investigation into whether the protein is translated are unknown. One exception is the approval of an antibody test that detects a specific AR-v, AR-v7, from the blood of PC patients and predicts ADT response. However, there are instances where a patient may not express ARv7 and are still resistant to ADT. This may suggest that other AR-Vs, not currently detected at the protein level, are important predictors for ADT response. Methods The primary goal of this work is to expand the protein identification of known and unknown AR-Vs that may predict response to ADT using targeted liquid chromatography tandem mass spectrometry (LC-MS/MS). We developed the AR-V targeted LC-MS/MS assay by first designing AR splice variants from unique splice regions via RNA-guided sequences translated to amino acid sequences. The AR-variants full amino acid sequences were then in-silico trypsin digested using Expasy software tool that generated tryptic peptides. Blast analysis of the AR-V peptides was performed where no overlap with other human proteins was observed. These AR-V peptide sequences were commercially synthesized along with heavy isotopically analogs to be used as internal standards in the LC-MS/MS method. The method examined 9 peptides and included 2 to 3 MRM transitions per peptide and calibration curves were constructed using unlabeled peptides. Specimens examined consisted of a panel of PCa cell lines (n= 6), CRPC patient derived xenografts tumors (PDX) (n=4) and a negative control cell line (no androgen receptor expression), Cos-1, which served with a dual function and was used as matrix for peptide MRM optimization experiments. Linearity and sensitivity for each peptide included in the method was examined using calibration curves. Results The targeted MRM approached was successfully achieved for the quantification of 9 peptides from 8 proteins in 6 PCa cell lines and 4 PDX tumor specimens. Out of the 8 proteins evaluated, we observed 3 known targets (AR-exon1, AR-V7 and AR-V12) and 4 novel AR-Vs in 22Rv1 PCa cell lines. Additionally, CRPC PDX were assessed where 3 novel AR-Vs were detected. Conclusions These preliminary results using a LC-MS/MS platform show promising identification and quantification of novel AR-Vs that have not been measured before at the protein level. Subsequent analysis of larger cohorts will further elucidate AR-Vs role in PCa treatment resistance mechanisms and possibly assist in future treatment approaches.

Targeting the CLK2/SRSF9 splicing axis in prostate cancer leads to decreased ARV7 expression.

In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant-7 (ARV7) is clinically the most relevant and has a distinct 3' untranslated region (3'UTR) compared to the AR full-length variant, suggesting a unique post-transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3'UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine-rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2-like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti-proliferative effects in enzalutamide-treated or -naive PC models. Thus, targeting aberrant alternative splicing at the 3'UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI-resistant PC.

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Synergistic Strategies for Castration-Resistant Prostate Cancer: Targeting AR-V7, Exploring Natural Compounds, and Optimizing FDA-Approved Therapies.

Castration-resistant prostate cancer (CRPC) remains a significant therapeutic challenge due to its resistance to standard androgen deprivation therapy (ADT). The emergence of androgen receptor splice variant 7 (AR-V7) has been implicated in CRPC progression, contributing to treatment resistance. Current treatments, including first-generation chemotherapy, androgen receptor blockers, radiation therapy, immune therapy, and PARP inhibitors, often come with substantial side effects and limited efficacy. Natural compounds, particularly those derived from herbal medicine, have garnered increasing interest as adjunctive therapeutic agents against CRPC. This review explores the role of AR-V7 in CRPC and highlights the promising benefits of natural compounds as complementary treatments to conventional drugs in reducing CRPC and overcoming therapeutic resistance. We delve into the mechanisms of action underlying the anti-CRPC effects of natural compounds, showcasing their potential to enhance therapeutic outcomes while mitigating the side effects associated with conventional therapies. The exploration of natural compounds offers promising avenues for developing novel treatment strategies that enhance therapeutic outcomes and reduce the adverse effects of conventional CRPC therapies. These compounds provide a safer, more effective approach to managing CRPC, representing a significant advancement in improving patient care.

Increased chromatin accessibility mediated by nuclear factor I drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer.

Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

Simultaneous detection of tumor-derived mutations in DNA and RNA extracted from a single circulating tumor cell based on droplet digital polymerase chain reaction.

e15053 Background: Androgen receptor splice variant 7 (AR-V7) is a constitutively activated isoform of AR and has been associated with resistance to AR-targeted therapies and development of metastatic castration-resistant prostate cancer. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the appropriate drug, however, the detection of AR-V7 with tissues has limitations, such as the clinical challenge to poorly accessible tumor tissues before and after therapy. In addition, KRAS mutations serve as a prognostic biomarker for the administration of appropriate anti-cancer drugs in patients with various cancers, including non-small cell lung cancer (NSCLC). Here, we developed the process for the simultaneous detection of AR-V7 and KRAS mutation in CTCs using isolation with CytoGen’s Smart Biopsy™ System with droplet digital polymerase chain reaction (ddPCR) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Methods: ddPCR assays were performed to detect the KRAS G12C mutation using CTC gDNA and to evaluate the absolute levels of AR-V7 using CTC mRNA. Analytical validation was performed using NSCLC NCI-H358 and prostate cancer 22Rv1 cells (10 and 5 cells each), which harbor KRAS G12C and AR-V7, respectively, spiked into 5 mL of healthy blood to mimic CTC. CTCs were isolated using CytoGen's Smart Biopsy™ System. Oligo-dT magnetic beads were used to isolate mRNA from CTCs, and gDNA was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/mRNA Nano Kit. PCR and droplet reading for each assay were performed on the QX200 AutoDG Droplet Digital PCR System. Results: As a result of CTC KRAS G12C detection using the ddPCR™ KRAS G12/G13 Screening Kit, the mutated KRAS gene was successfully detected in as few as 5 NCI-H358 cells. In the results of CTC AR-V7 detection using the custom TaqMan ddPCR assay, transcripts (AR-V7 and HPRT1) were successfully detected even with only 5 22Rv1 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to the simultaneous detection of DNA/RNA mutated genes in clinical NSCLC and prostate cancer patients with detection technology requiring high sensitivity and specificity. Conclusions: Accurately detecting multiple genetic mutations in CTCs is a challenging because CTCs are very rare in the blood. In this study, we developed the process to simultaneously extract and detect DNA mutations and RNA splicing variants in CTCs using CytoGen's Smart Biopsy™ System. This process, along with the results from our ongoing clinical trials, could provide information for the diagnosis and appropriate treatment of patients with various cancers, including NSCLC and prostate cancer.

Androgen Receptor Splice Variant 7 in Asian Patients With Metastatic Castration-Resistant Prostate Cancer.

Androgen receptor splice variant 7 (ARV-7) is a resistance mechanism to hormonal therapy in metastatic castrate-resistant prostate cancer (mCRPC). It has been associated with poor outcomes. On progression to castrate resistance, ARV-7 positivity has been identified in global populations at an incidence of 17.8%-28.8%. Here, we characterize the incidence of ARV-7 positivity in Asian patients with mCRPC in a prospective fashion and evaluate its implications on treatment outcomes. Patients with mCRPC from multiple centers in Southeast and East Asia were enrolled in a prospective manner before initiation of androgen receptor signaling inhibitors or docetaxel. ARV-7 status was evaluated at baseline with three commercially available assays: AdnaTest Prostate Cancer platform, Clearbridge method, and IBN method. Clinical outcomes at progression were assessed. The primary end point of this study was prevalence of ARV-7 positivity; secondary end points were incidence of ARV-7 positivity, prostate specific antigen (PSA) response rate, PSA progression-free survival (PFS), and overall survival (OS). A total of 102 patients with a median age of 72 years at enrollment participated. Overall, an incidence of ARV-7 positivity of between 14.3% and 33.7% in Asian patients with mCRPC was demonstrated depending on the assay used. Patients found to have ARV-7 positivity at enrollment had a numerically worse PSA PFS compared with ARV-7 negative patients. In this study, the incidence of ARV-7 positivity in Asian patients with mCRPC was shown to be similar to the global population. Patients with ARV-7 positivity appear to have more aggressive disease with numerically worse PSA PFS and OS. Further prospective studies are needed to fully characterize the relationship that ARV-7 positivity has on prognosis of Asian patients with mCRPC.

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.

A phase 1/2 study of ONCT-534, a dual-action androgen receptor inhibitor (DAARI), in patients with metastatic castration-resistant prostate cancer.

TPS5111 Background: Current therapeutic strategies for metastatic castration-resistant prostate cancer (mCRPC) include treatment with next-generation androgen receptor pathway inhibitors (ARPIs), which target the ligand-binding domain (LBD) of the AR. Approximately 5% to 10% of patients will have primary resistance to ARPIs and most initial responders will develop resistance within 1 to 3 years. Eventually, nearly all men with prostate cancer treated with ARPIs develop resistance via several mechanisms including AR genomic alterations, epigenetic alterations, and expression of truncated constitutively active AR splice variants. Hence, there is substantial unmet need for treatment of men with mCRPC resistant to at least one next-generation ARPI. ONCT-534 is a DAARI with a novel mechanism of action that combines inhibition of AR function with degradation of the AR protein. Importantly, this activity includes interaction with both LBD and the N-terminal domain (NTD) of the AR, rendering it effective against splice variants and LBD mutants, unlike existing ARPIs. ONCT-534 has demonstrated preclinical activity in prostate cancer models against unmutated AR and multiple forms of AR alteration, including amplification, mutations in the LBD, and splice variants with loss of LBD. Methods: ONCT-534-101 is a phase 1/2, multi-center study to evaluate the safety, tolerability, antitumor activity, and pharmacokinetics of ONCT-534 in subjects with histologically confirmed mCRPC without neuroendocrine differentiation or small cell features who have relapsed or are refractory (R/R) to at least one next-generation ARPI. The study is separated into Phase 1 Dose Escalation and a Phase 2 Dose Expansion portions. The Phase 1 portion will evaluate approximately 27 subjects in 5 dose levels using an adaptive Bayesian Optimal Interval (BOIN) design to assess safety, tolerability and dose limiting toxicities (DLTs) at escalating doses and to determine the maximum tolerated dose and inform the 2 dose levels or schedules to be tested in Phase 2. DLTs will be assessed during the first 28 days of treatment. AR phenotype and AR levels will be evaluated for each subject pre- and post-treatment. The Phase 2 portion will evaluate approximately 32 subjects in 2 randomized cohorts to assess safety and tolerability of ONCT-534, compare the 2 different dose levels or schedules to select the optimal dose for further study, and assess the preliminary antitumor activity of ONCT-534. In both phases, after a screening period, eligible subjects with mCRPC will receive their assigned dose regimen of ONCT-534, which will be administered orally daily for 2 years or until disease progression and no longer clinically benefiting from treatment or development of unacceptable toxicity. Cohorts 1 and 2 have been completed without DLT. Enrollment into Cohort 3 began in January 2024 (NCT 05917470). Clinical trial information: NCT05917470 .

Novel Androgen Receptor Splice Variant 7 in Gynecologic Tumors.

Androgen receptor splicing variant 7 (AR-V7) is a truncated variant of the AR mRNA that may be a predictive biomarker for AR-targeted therapy. AR-V7 has been described in prostate, breast, salivary duct, and hepatocellular carcinomas as well as mammary and extra-mammary Paget disease. We report 2 gynecologic cancers occurring in the lower uterine segment and ovary and both harboring AR-V7 by targeted RNA sequencing. The uterine tumor was an undifferentiated carcinoma consisting of epithelioid cells and focally spindled cells arranged in sheets, nests, and cords associated with brisk mitotic activity and tumor necrosis. The ovarian tumor consisted of glands with cribriform and solid architecture and uniform cytologic atypia. ER and PR were positive in the ovarian tumor and negative in the uterine tumor. Both were positive for AR and negative for HER2, GATA3, and NKX3.1. DNA methylation profiling showed epigenetic similarity of the AR-V7-positive gynecologic cancers to AR-V7-positive breast cancers rather than to prostate cancers. AR-V7 may underpin rare gynecologic carcinomas with undifferentiated histology or cribriform growth reminiscent of prostatic adenocarcinoma and breast invasive ductal carcinoma.

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 with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain 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.