Androgen Receptor Variants Research Articles

Targeting Androgen Receptor Variants in Castration-Resistant Prostate Cancer (CRPC)

Castration-resistant prostate cancer (CRPC) remains a lethal stage of prostate cancer, characterized by resistance to androgen deprivation therapy (ADT) and poor clinical prognosis. One of the key drivers in CRPC progression is the androgen receptor (AR), specifically the emergence of androgen receptor variants (AR-Vs) that sustain AR signaling even in the absence of androgens. AR-Vs, such as AR-V7, promote androgen-independent activation of oncogenic pathways, leading to uncontrolled cell proliferation and therapeutic resistance. Targeting AR-Vs offers a promising therapeutic strategy to manage CRPC effectively. This review explores the molecular mechanisms underpinning AR-V expression, examines the roles of major AR variants in CRPC progression, and evaluates current and emerging therapies targeting AR-Vs. By summarizing recent advancements and the limitations of current approaches, we highlight potential future directions to improve therapeutic outcomes for CRPC patients. Keywords: Androgen receptor variants, Castration-resistant prostate cancer, AR-V7, Androgen deprivation therapy, Targeted therapy, Oncogenic pathways

Targeting Androgen Receptor Variants in Castration-Resistant Prostate Cancer (CRPC): A Comprehensive Review

Targeting Androgen Receptor Variants in Castration-Resistant Prostate Cancer (CRPC): A Comprehensive Review

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

BackgroundThe Androgen Receptor (AR) has emerged as an endocrine therapy target in Breast Cancer, exhibiting up to 80% expression in clinical cases. AR-V7, a constitutively activated splice variant of AR with a truncated ligand-binding domain (LBD), demonstrates ligand-independent transcriptional activity and resistance to nonsteroidal antiandrogens like Bicalutamide or Enzalutamide, targeting the LBD. In metastatic prostate cancer, elevated AR-V7 levels lead to therapeutic resistance and increased metastasis.MethodsIn this study, we evaluated the expression of AR and AR-V7 in cell lines and a cohort of 89 patients undergoing surgical intervention for treatment-naïve breast cancer. Further clinicopathological correlations and survival analysis were performed to evaluate the relationship between the AR and AR-V7 expression and clinical outcomes.ResultsAR-V7/AR-FL ratio was elevated in the TNBC cell line and downregulation of AR-FL upon AR antagonists’ treatment led to a compensatory increase in AR-V7. Clinical samples showed significantly elevated expression of AR and AR-V7 in tumors compared to control cases. Further clinicopathological correlation revealed aggressive clinical traits, higher pathological grades, and poor survival with AR-V7 expression.ConclusionsOur study unravels AR-V7 as a marker for poor clinical outcomes, predicting breast cancer aggressiveness, and encourages consideration of AR-V7 as a probable target for therapeutic intervention.

Discovery of 5-Nitro-N-(3-(trifluoromethyl)phenyl) Pyridin-2-amine as a Novel Pure Androgen Receptor Antagonist against Antiandrogen Resistance.

The transformation of clinical androgen receptor (AR) antagonists into agonists driven by AR mutations poses a significant challenge in treating prostate cancer (PCa). Novel anti-AR therapeutics combating mutation-induced resistance are required. Herein, by combining structure-based virtual screening and biological evaluation, a high-affinity agonist E10 was first discovered. Then guided by the representative conformation of State 1 at the free energy landscape, the structural optimization of E10 was performed, and pure AR antagonists EL15 (IC50 = 0.94 μM) and EF2 (IC50 = 0.30 μM) were successfully identified. Both can antagonize wild-type and variant drug-resistant ARs. Therein, EF2 demonstrated potent inhibition of the AR pathway and effectively suppressed tumor growth in a C4-2B xenograft mouse model following oral administration. Further molecular dynamics simulation and mutagenesis studies revealed atomic insights into the mode of action of EF2 which may serve as a novel lead compound for developing therapeutics against AR-driven PCa.

A study of the role of androgen receptor and androgen receptor variant 7 in TNBC patients and the effect of their targeting by Enzalutamide and EPI-001 in MDA-MB-231

The lack of targeted therapy for triple-negative breast cancer (TNBC) is among the mainsprings of its poor prognosis. This study aimed to elucidate the role of the androgen receptor (AR) and its splice variant 7 (ARv7) in TNBC patients. Further, the molecular impact of their blockers, Enzalutamide and EPI-001, on the TNBC cell line MDA-MB-231 was investigated. Thereby, immunohistochemical expression of AR/ARv7 was assessed for TNBC Egyptian patients. Moreover, bioinformatics analysis of AR/ARv7 RNA status was carried out on TNBC patients from The Cancer Genome Atlas Breast Carcinoma project (TCGA-BRCA). Data from both groups was correlated with patients’ clinicopathological features. Besides, scratch wound healing assay and ELISA were employed to assess the effect of AR/ARv7 blockers on several metastasis markers in MDA-MB-231 cell line. In the Egyptian-TNBC patients, AR expression was associated with worse 7-year DFS (40.6±18.6%). In addition, ARv7 showed cytoplasmic and nuclear patterns, and both cytoplasmic and nuclear ARv7+ patients demonstrated a worse 7-year DFS (22.7±17.7% and 20±17.9%) and overall survival (63.6±14.5% and 40±21.8%). Importantly, 80% of the nuclear ARv7+ patients developed distant metastasis. The data of the TCGA-TNBC patients showed a tendency for poor outcomes in the high ARv7-expressing patients. Molecularly, in MDA-MB-231, both inhibitors modulated metastasis and epithelial to mesenchymal transition (EMT) markers ROCK1, ROCK2, c-Myc, E-cadherin and N-cadherin, with EPI-001 downregulating NF-ĸB level as well. We concluded that ARv7 indicated poor prognosis in the studied cohorts and that blocking of AR/ARv7 abated metastasis and key regulators of EMT in MDA-MB-231, at least in part by targeting ROCK/NF-ĸB/c-Myc axis.

Diverse phenotypes and fertility outcomes of patients with androgen insensitivity syndrome in a Chinese family harboring identical AR gene variant

BackgroundAndrogen insensitivity syndrome (AIS) is a rare genetic disorder characterized by resistance to androgens, mainly due to mutations in the androgen receptor (AR) gene. It can manifest as complete AIS, partial AIS and mild AIS. While there have been studies linking specific AR gene mutations to AIS phenotypes, different clinical AIS phenotypes are also reported in patients with the same AR gene mutation. So far, the precise correlations between phenotypes and genotypes remain incompletely understood.MethodsWe conducted a thorough investigation involving four patients diagnosed with different types of AIS from a single Chinese family. Clinical manifestations, laboratory examinations, and fertility outcomes were well-documented. Furthermore, we performed genetic sequencing to detect possible pathogenetic variants.ResultsWhole exome sequencing identified a hemizygous missense variant (c.2263T > C; p.Phe755Leu) of AR gene in all four affected patients with different degrees of undermasculinisation and heterogeneous spermatogenesis. The proband, diagnosed with partial AIS, opted for treatment with donated sperm due to non-obstructive azoospermia, while their older sibling, diagnosed with complete AIS, was raised as a girl. His two maternal uncles were both diagnosed with mild AIS, the older uncle fathered two girls naturally, whereas the younger uncle utilized assisted reproductive technology to conceive a boy because of severe oligoasthenozoospermia.ConclusionOur study first identified the same AR variant (c.2263T > C;p.Phe755Leu) in four affected patients displaying highly diverse phenotypes of AIS and fertility outcomes, thereby significantly expanding the phenotypic spectrum of AIS. Notably, we presented a clear insight into different fertility outcomes of AIS patients with identical AR (c.2263T > C;p.Phe755Leu) variant, which provided reliable evidence that males harboring this variant may obtain biological offspring naturally or in combination with assisted reproductive technology. Furthermore, our study underscored the potential role of androgen concentration in shaping the phenotypic diversity of AIS, warranting further investigation.

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.

Complete Androgen Insensitivity Syndrome in a Young Girl with Primary Amenorrhea and Suspected Delayed Puberty: A Case-Based Review of Clinical Management, Surgical Follow-Up, and Oncological Risk.

Background: Complete androgen insensitivity syndrome (CAIS) is a rare disorder of sex development characterized by 46,XY karyotype and testes, yet presenting with a complete female phenotype, which is related to mutations in the androgen receptor (AR) gene. Case presentation: We herein present the case of a 14-year-old adolescent with primary amenorrhea and suspected delayed puberty whose diagnostic journey led to the identification of CAIS through the demonstration of a novel AR variant (c.159_207del). Case-based review: Our report encompasses the complexity of CAIS management, focusing on the risk of malignancy, surveillance options, hormone replacement therapy, timing of an eventual gonadectomy, and the psychosocial impact of such a diagnosis. An algorithm has been formulated for the management of CAIS starting in adolescence, highlighting the conservative approach for those patients unwilling to undergo gonadectomy. Conclusions: Primary amenorrhea and delay in puberty development may provide clues, ultimately leading to a diagnosis of CAIS. This review emphasizes the cruciality of a multidisciplinary approach in managing patients with CAIS, needing for an individualized care to optimize the overall outcome.

LX1 Dual Targets AR Variants and AKR1C3 in Advanced Prostate Cancer Therapy.

The development of resistance to current standard-of-care treatments, such as androgen receptor (AR) targeting therapies, remains a major challenge in the management of advanced prostate cancer. There is an urgent need for new therapeutic strategies targeting key resistant drivers, such as AR variants like AR-V7, and steroidogenic enzymes, such as aldo-keto reductase 1C3 (AKR1C3), to overcome drug resistance and improve outcomes for patients with advanced prostate cancer. Here, we have designed, synthesized, and characterized a novel class of LX compounds targeting both the AR/AR variants and AKR1C3 pathways. Molecular docking and in vitro studies demonstrated that LX compounds bind to the AKR1C3 active sites and inhibit AKR1C3 enzymatic activity. LX compounds were also shown to reduce AR/AR-V7 expression and to inhibit their target gene signaling. LX1 inhibited the conversion of androstenedione into testosterone in tumor-based ex vivo enzyme assays. In addition, LX1 inhibited the growth of cells resistant to antiandrogens including enzalutamide (Enza), abiraterone, apalutamide, and darolutamide in vitro. A synergistic effect was observed when LX1 was combined with antiandrogens and taxanes, indicating the potential for this combination in treating resistant prostate cancer. Treatment with LX1 significantly decreased tumor volume, serum PSA levels, as well as reduced intratumoral testosterone levels, without affecting mouse body weight. Furthermore, LX1 was found to overcome resistance to Enza treatment, and its combination with Enza further suppressed tumor growth in both the CWR22Rv1 xenograft and LuCaP35CR patient-derived xenograft models. Collectively, the dual effect of LX1 in reducing AR signaling and intratumoral testosterone, along with its synergy with standard therapies in resistant models, underscores its potential as a valuable treatment option for advanced prostate cancer. Significance: LX1 simultaneously targets androgen receptor variants and the steroidogenic enzyme AKR1C3, offering a promising approach to combat drug resistance and enhancing therapeutic efficacy in conjunction with standard treatments for advanced prostate cancer.

Analysis of genetic and clinical characteristics of androgen insensitivity syndrome: a cohort study including 12 families.

Androgen insensitivity syndrome (AIS) manifests itself as variable symptoms of under-virilization in patients with 46,XY disorders caused by androgen receptor (AR) gene variants. This large-sample study aimed to correlate the genotypes and phenotypes to the fertility of individuals. This was a cohort study that analyzed the genetic and clinical characteristics of patients with AIS from a single center in China. The 117 patients were divided into 53 with complete AIS (CAIS) and 64 with partial AIS (PAIS). At their first visit, the median age was 1.83 years (0.92-4.17), and the external masculinization score was 3.0 (2.0-6.0). At the last follow-up, 92% (49/53) of patients with CAIS maintained their female gender, and 94% (60/64) of patients with PAIS were raised as males. No gender anxiety was observed in this study. Eighty-eight AR variants were identified, with 31 (35%) being unreported. Moreover, 24% (21/88) occurred more than once. The variants that appeared most frequently were located at amino acid 841, including p.R841H (n = 5) and p.R841C (n = 2). Variants p.N706S, p.R856H, and p.A871V were each observed 4 times. In terms of inheritance, 83% of patients with parental verification inherited variants from their mothers. We also observed that the variants from 1 case were inherited from his maternal grandfather who had hypospadias. Most children with PAIS were raised as males. The abundance of maternally inheritable variants and the presence of case of preserved fertility indicate the fertility potential in patients with AIS. Hence, we recommend a careful evaluation of gonadectomy when fertility preservation is being considered.

Hydropathic AF-2 variants in the androgen receptor gene among androgen insensitivity patients.

Androgen insensitivity syndrome (AIS) is a common condition among individuals with differences of sexual development (DSD) and results from germline allelic variants in the androgen receptor (AR) gene. Understanding the phenotypic consequences of AR allelic variants that disrupt the activation function 2 (AF2) region is essential to grasping its clinical significance. This study aims to provide insights into the phenotypic characteristics and clinical impact of AR mutations affecting the AF2 region in AIS patients. We achieve this by reviewing reported AR variants in the AF2 region among individuals with AIS, including identifying a new phenotype associated with the c.2138T>C variant (p.Leu713Pro) in the AR gene. We comprehensively reviewed AR variants within the AF2 region reported in AIS and applied molecular dynamics simulations to assess the impact of the p.Leu713Pro variant on protein dynamics. Our review of reported AR variants in the AF2 region revealed a spectrum of phenotypic outcomes in AIS patients. Molecular dynamics simulations indicated that the p.Leu713Pro variant significantly alters the local dynamics of the AR protein and disrupts the correlation and covariance between variables. The diverse phenotypic presentations observed among individuals with AR variants in the AF2 region highlight the complexity of AIS. The altered protein dynamics resulting from the p.Leu713Pro variant further emphasize the importance of the AF2 region in AR function. Our study provides valuable insights into AR mutations' phenotypic characteristics and clinical impact on the AF2 region in AIS. Moreover, the disruption of protein dynamics underscores the significance of the AF2 region in AR function and its role in the pathogenesis of AIS.

TBCRC 058: A randomized phase II study of enzalutamide, enzalutamide with mifepristone, and treatment of physician’s choice in patients with androgen receptor-positive metastatic triple-negative or estrogen receptor-low breast cancer (NCT06099769).

TPS1138 Background: Triple-negative breast cancer (TNBC) refers to a heterogenous group of breast cancers that lack expression of ER, PR, and HER2. Despite recent advances with immunotherapy (IO) and antibody-drug conjugates (ADCs), TNBC remains the most aggressive subtype, characterized by a high risk of recurrence and a short overall survival in the metastatic setting. Breast tumors with low levels of ER and PR expression (1-10%) clinically behave like TNBC, and clinical management follows the TNBC treatment (tx) paradigm. We and others have identified a subset of ER/PR/HER2 negative breast cancers (BC) that express the androgen receptor (AR). Enzalutamide (enza), an AR-antagonist, has demonstrated activity in AR+ metastatic TNBC (Traina et al, JCO 2018). Activation of the glucocorticoid receptor (GR) has been implicated as a mechanism of resistance to AR inhibition in prostate and BCs (Kach et al, Sci Transl Med 2015). Effective therapies for advanced TNBC remain an unmet need, particularly in patients who are ineligible for or progress following a checkpoint inhibitor. This randomized study will evaluate the efficacy of enzalutamide or enzalutamide plus the GR antagonist mifepristone (mif) as compared to physician’s choice chemotherapy (TPC). Methods: This is a randomized phase II trial; 201 patients (pts) will be randomized in a 1:1:1 fashion to enza, enza with mif, or TPC (carboplatin, paclitaxel, eribulin, or capecitabine). The primary endpoint (endpt) is progression free survival (PFS), and the trial is designed to test the hypothesis that PFS in the pooled enzalutamide arms is superior to TPC; there is 80% power to detect a hazard ratio (HR) of 0.70, corresponding to increase in PFS from 3.5 months (mos) with TPC to 5.0 mos with enza-based tx. Secondary endpts include comparisons of PFS among 3 arms and evaluation of response rate, clinical benefit rate, duration of response, overall survival, safety/toxicity, and patient-reported outcomes by arm. Exploratory endpts include correlation of tumor and circulating markers (constitutively active AR variants in circulating tumor cells and circulating tumor cell DNA) with tx response. Eligible pts must have: ECOG 0-2, metastatic ER/PR low or negative, HER2 negative breast cancer (BC), measurable or evaluable disease (dz), < 2 prior lines of chemotx, any # prior endocrine txs, no prior anti-AR tx, no prior mif, concurrent CYP17 inhibitors prohibited. Pts with PD-L1+ BC must have received prior IO if not contraindicated. Tumors must have AR ≥10%, normal organ function, no history of brain mets. As of 1/15/2024, 2 of 201 pts have begun protocol-specified tx. Clinical trial information: NCT06099769 .

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.

Androgen receptor and estrogen receptor variants in prostate and breast cancers

The androgen receptor (AR) and estrogen receptor alpha (ERα) are steroid receptor transcription factors with critical roles in the development and progression of prostate and breast cancers. Advances in the understanding of mechanisms underlying the ligand-dependent activation of these transcription factors have contributed to the development of small molecule inhibitors that block AR and ERα actions. These inhibitors include competitive antagonists and degraders that directly bind the ligand binding domains of these receptors, luteinizing hormone releasing hormone (LHRH) analogs that suppress gonadal synthesis of testosterone or estrogen, and drugs that block specific enzymes required for biosynthesis of testosterone or estrogen. However, resistance to these therapies is frequent, and is often driven by selection for tumor cells with alterations in the AR or ESR1 genes and/or alternatively spliced AR or ESR1 mRNAs that encode variant forms AR or ERα. While most investigations involving AR have been within the context of prostate cancer, and the majority of investigations involving ERα have been within the context of breast cancer, important roles for AR have been elucidated in breast cancer, and important roles for ERα have been elucidated in prostate cancer. Here, we will discuss the roles of AR and ERα in breast and prostate cancers, outline the effects of gene- and mRNA-level alterations in AR and ESR1 on progression of these diseases, and identify strategies that are being developed to target these alterations therapeutically.

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.

Abstract LB027: Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository

Abstract Second-generation androgen ablation therapies (ADT), enzalutamide, and abiraterone are initially effective on metastatic castration-resistant prostate cancer (mCRPC). However, nearly 30-40% of patients eventually become resistant to these treatments due to the presence of androgen-receptor variants (AR-V), which lack a ligand-binding domain. The grim reality that metastatic CRPC is virtually incurable for most patients poses a significant challenge for clinicians striving to enhance these individuals' quality of life and lifespan. Considering drug discovery efforts, we partnered with the National Cancer Institute's (NCI) Natural Product Repository, which possesses a diverse collection of natural product extracts derived from plant, marine, and microbial organisms. We developed high-throughput phenotypic screening assays to screen the crude natural product extract libraries on AR and AR-variants CRPC cell lines (C4-2B and 22Rv1). Several extracts inhibited the growth of both CRPC cell lines and were further confirmed for dose-dependent cell viability inhibition assays. Once we determined the IC50 dose, we performed a Western blot analysis that confirmed the inhibition of AR and AR-V7 expression in CRPC cells.In subsequent studies, we narrowed down by fractionating the most potent extracts and confirmed their ability to inhibit AR expression and signaling on CRPC cell lines. Currently, we are characterizing the potent compounds and their inhibitory activity on AR and AR-variants, in vitro and in vivo CRPC models. Many nutraceuticals have been shown to target AR signaling, synthesis, or degradation which effectively inhibited the growth of CRPC. Our collaborative efforts with NCI will permit us find N-terminal AR degrading molecules (AF-1), which are essential for the AR transcriptional activities of CRPC. In sum, discovery of ovel small molecules from our drug development program have the potential to translate these effective natural products into drugs with clinical benefits to treat CRPC. Citation Format: Neha Tyagi, Sepideh Hosseiniporgham, Ashish Tyagi, Balaji Chandrasekaran, Katherine Bonilla, Inna Kriger, James Saccchettini, Chendil Damodaran. Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository [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 LB027.

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.

Abstract 4364: Molecular insight of non-canonical Gsk3β-Gli-AR axis: Therapeutic implications of truncated-Gli3 in castration-resistant prostate cancer

Abstract The aberrant activation of the hedgehog (Hh) signaling pathway causes the progression of therapy-resistant castration-resistant prostate cancer (CRPC). In this study, we aimed to unravel the biological significance of Gli-family proteins and their functional involvement of Gsk3β-mediated regulatory mechanism(s) in CRPC. We have discovered a novel function of the truncated form of Gli (t-Gli3) in advancing CRPC. This was accomplished through in silico analysis, in vitro experiments, PCa patient clinical data, and in vivo PCa MYC overexpression mouse models. Our investigation into the Gli3 regulation, typically influenced by receptor-mediated ciliary localization, uncovered a Smo-independent mechanism. Surprisingly, despite the lack of a transactivation domain in t-Gli3, we found a specific requirement for an androgen receptor variant 7 (AR-V7) for its action, as evidenced by co-immunoprecipitation and chromatin immunoprecipitation assays. Mechanistically, Gsk3β activation leads to the t-Gli3 generation, and inhibition of Gsk3β supported the accumulation of full-length Gli3 through a non-canonical mechanism. Knockdown of Gsk3β (Gsk3β KD) reduces CRPC cell proliferation in 2-D and 3-D culture models, inhibiting key proliferative signaling pathways and inducing apoptosis via mitochondrial fragmentation. These findings were supported using an orthotropic PCa mouse model. RNA-seq analysis also showed upregulated phenotype enrichment of the apoptosis, P53, and glycolysis pathway genes in 22Rv1Gsk3β KD cells. Compared to scramble, a lower OCR/mitochondrial respiratory rate is evident in 22Rv1Gsk3β KD cells. Next, our exploration of potential therapies for PCa showed that individually targeting Smo or Gli1 is insufficient to inhibit cell viability. Interestingly, we uncovered that solely targeting Gli3 was sufficient to inhibit the growth of CRPC cells. In conclusion, our study provides the significance of Gli-family proteins and novel insights into the interplay of Gsk3β, t-Gli3, and AR, offering therapeutic implications in CRPC. Citation Format: Jyoti Bala Kaushal, Pratima Raut, Sarathy Seshacharyulu, Zahraa W. Alsafwani, Gunjan Sharma, Abdullah M. Khan, Sushanta Halder, Satyanarayana Rachagani, Surinder K. Batra, Jawed A. Siddiqui. Molecular insight of non-canonical Gsk3β-Gli-AR axis: Therapeutic implications of truncated-Gli3 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 4364.

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 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.