Androgen Receptor Transcriptional Activity Research Articles

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 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 7592: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)

Abstract The LAR subtype of TNBC is enriched for targetable biomarkers such as androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combination of CDK4/6, AR, and PI3K-AKT inhibitors in preclinical models of LAR TNBC. The MDAMB453 and MFM223 (AR positive/Rb intact/PIK3CAmutant) LAR TNBC cells were treated with each inhibitor alone or in combination. Drug sensitivity was assessed using colony formation, cell viability, and live-cell analysis. The Loewe additivity equation was used to measure the additive drug effects. Immunoblot analysis was used to evaluate cell cycle and PI3K-AKT signaling and ARE luciferase assay was used to assess AR transcriptional activity. MDAMB453 and MFM223 cells were sensitive to the single agents palbociclib, alpelisib, and capivasertib (IC50, ~500 nM), which inhibited CDK4/6, PI3Kα, and AKT, respectively. Enzalutamide, an AR antagonist, exerted no growth inhibitory activity (IC50, 15-25 μM). Treatment with palbociclib, alpelisib, or capivasertib did not alter the AR protein levels or transcriptional activity. The combination of palbociclib+capivasertib synergistically inhibited the proliferation of MDAMB453 and MFM223 cells (synergistic scores 7.34, p=5.81-11, and 4.78, p=0.012, respectively) better than palbociclib+alpelisib. Enzalutamide did not enhance either combination. Palbociclib+capivasertib inhibited PI3KCA-mutant and Rb-intact LAR TNBC PDX growth more potently than palbociclib+alpelisib (p=0.046). Treatment of LAR TNBC cells with palbociclib suppressed Rb phosphorylation and resulted in adaptive activation of P-AKTS473/T308 and the AKT substrates GSK3β, PRAS40, and FoxO3a after 24 h. Capivasertib inhibited the phosphorylation of AKT and AKT substrates, following CDK4/6 blockade with palbociclib, more potently than alpelisib. PI3Kβ inhibitors (TGX221, GSK2636771) did not block AKT substrate upregulation, suggesting PI3Kβ does not mediate adaptive responses. Rictor knockdown failed to reduce AKT activation following palbociclib treatment, suggesting an mTORC2-independent mechanism. Human Phospho-kinase Array of MDA-MB-453 cells treated with palbociclib showed time dependent upregulation of GSK3βS9, STAT3Y727 and PDFGRβY751. PDGFRβ activation after palbociclib treatment was confirmed in MFM223 cells. PDGFRβ siRNAs in MDAMB453 cells blocked P-AKTS473 upregulation after palbociclib treatment. Treatment with palbociclib and the PDGFRβ small-molecule antagonist CP673451 arrested LAR TNBC growth similar to palbociclib+capivasertib, suggesting an adaptive response involving PDGFRβ signaling. In conclusion, this study supports the clinical testing of the palbociclib and capivasertib combination for LAR TNBC patients and reveals adaptive responses after palbociclib treatment through PDGFRβ signaling. Citation Format: Rosario Chica-Parrado, Gun Min Kim, Yasuaki Uemoto, Dan Ye, Fabiana Napolitano, Chang-Ching Lin, Emmanuel Bikorimana, Kyung-min Lee, Saurabh Mendiratta, Ariella B. Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC) [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 7592.

Abstract 5343: Depletion of gut microbiota with broad spectrum antibiotics drives Pten-null prostate cancer growth in mice

Abstract Gut microbes have the potential to be either promoters or inhibitors of cancer. Use of antibiotics during cancer therapy can lead to dysbiosis, which may affect treatment outcomes. Here, we examine the effects of broad-spectrum antibiotics on cancer progression in mice with Pten-null castration resistant prostate cancer (CRPC). After five or eight weeks, orchiectomized conditional Pten-null knockout (KO) mice aged 50 and 90 weeks were given two biweekly cycles of a broad-spectrum antibody cocktail (BSAbx), respectively. The effects of BSAbx on tumor burden were compared, and molecular and immunological responses were analyzed by quantitative IHC (qIHC) and qRt-PCR at week four after antibiotic treatment. BSAbx treatment resulted in tumor burdens of 1.2 and 2.4-fold higher than in control mice in mice aged 50 and 90 weeks, respectively. qIHC analysis of KI67 showed increased proliferation in stromal and cancer cells. Additionally, androgen receptor (AR) protein and gene expression levels of Ar and Ar target genes (Fkbp5, Tmprss2, and Timp4) were elevated in tumors of BSAbx-treated mice. Restoring gut microbiota by fecal microbiota transplantation (FMT) from KO mice CRPC after BSAbx restored AR transcriptional activity to basal levels and lessened tumor growth. FMT from non-castrated KO with castration-naïve prostate cancer or heathy wildtype mice further reduced AR Transcriptional activity. Moreover, altering the gut microbiota of CRPC mice with FMT from heathy mice resulted in a tendency for lower tumor burden, 21% reduction versus control mice. Immunologically, BSAbx treatment resulted in decreased CD11b+/GR1+ cells in tumors and were associated with decreased interferon-gamma (IFN-gamma), dendritic cell functions and T cell-mediated cytotoxicity gene signatures. Mice that received FMT form health mice showed enhanced T cell mediated immunity as demonstrated by higher IFN-gamma signatures and tumor abundances of CD8+ T cells expression IFN-gamma, TNF-alpha and the cytotoxic granule granzyme B. Our study shows that broad spectrum antibiotics caused dysbiosis and promoted prostate cancer growth in mice with Pten-null prostate cancer. The effects were partly mediated through the upregulation of AR signaling and the impairment of neutrophil and T cell-mediated immune responses in tumors. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Mitsuhisa Nishimoto, Yasunori Mori, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Depletion of gut microbiota with broad spectrum antibiotics drives Pten-null prostate cancer growth in mice [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 5343.

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 940: Integrated analysis of cfDNA fragmentomics, DNA methylation and cfRNA transcription in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background Liquid biopsies based on cell-free DNA (cfDNA) analysis provide non-invasive clinical diagnostic insights. Recently, cfDNA fragmentomics has emerged as a tool for inferring epigenomic and transcriptional information from tumor-derived cfDNA. We conducted a comprehensive profiling of cfDNA fragmentomics, whole transcriptome sequencing (WTS), and genome-wide DNA methylation on two metastatic castration-resistant prostate cancer (mCRPC) samples to systematically investigate molecular aberrations in mCRPC. Methods Two mCRPC patient plasma samples were extensively profiled using high-depth 150x whole-genome sequencing (WGS), 30x whole-genome methylation, and cfRNA WTS. CfDNA fragmentomics analysis was performed to assess androgen receptor (AR) transcriptional activity. We calculated fragment coverage around AR binding sites (ARBS) and quantified ARBS nucleosome profiling abnormality scores (ARBS scores) by comparing centric fragment coverage with normal plasma background. Additionally, we calculated fragment size entropy around AR promoter and enhancer regions to reflect AR gene expression activity. Fragmentomics-inferred gene activity was validated through promoter-targeted panels and WTS profiling of matched mCRPC samples. Results Both mCRPC plasma samples exhibited significant ARBS nucleosome depletion. Sample A showed a higher ARBS score compared to Sample B. Interestingly, AR enhancer activity inferred from high-depth WGS and targeted panels was high in both samples, while AR promoter activity was high only in Sample A. Genome-wide DNA methylation revealed hypo-methylation in AR enhancer and ARBS regions in both samples, with Sample A showing more pronounced hypo-methylation. Copy number variation analysis indicated AR amplification in Sample A and RB1 loss in Sample B. Collectively, these findings suggest that Sample A may represent androgen receptor-dependent prostate cancer (ARPC), while Sample B may have developed resistance to androgen therapy, possibly indicating neuroendocrine prostate cancer (NEPC). Conclusions This study, to our knowledge, is the first to combine high-depth WGS, promoter-targeted panels, WTS, and genome-wide DNA methylation to systematically study epigenomic and transcriptional dysregulation in mCRPC. The integration of mutation, copy number variation, fragmentomics, and DNA methylation profiling holds potential clinical utility for inferring prostate cancer subtypes, facilitating patient stratification, and guiding treatment selection. Citation Format: Chao Dai, Lisha Zhu, Yong Huang, Giancarlo Bonora, Kemin Zhou, Shidong Jia, Pan Du. Integrated analysis of cfDNA fragmentomics, DNA methylation and cfRNA transcription in metastatic castration-resistant prostate cancer (mCRPC) [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 940.

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.

Regulating Androgen Receptor Function in Prostate Cancer: Exploring the Diversity of Post-Translational Modifications.

Androgen receptor (AR) transcriptional activity significantly influences prostate cancer (PCa) progression. In addition to ligand stimulation, AR transcriptional activity is also influenced by a variety of post-translational modifications (PTMs). A number of oncogenes and tumor suppressors have been observed leveraging PTMs to influence AR activity. Subjectively targeting these post-translational modifiers based on their impact on PCa cell proliferation is a rapidly developing area of research. This review elucidates the modifiers, contextualizes the effects of these PTMs on AR activity, and connects these cellular interactions to the progression of PCa.

Regulation of androgen receptor stability by the β1 Pix/STUB1 complex.

The androgen receptor (AR) is essential in the development and differentiation of testes and male genitalia. AR expression is tightly regulated at the translational and posttranslational levels. AR posttranscriptional regulation is a major determinant of AR availability since AR is a direct target of E3 ubiquitin ligase STUB1. Our work indicated that the Rac/Cdc42 guanosine triphosphatase guanine nucleotide exchange factor, β1 Pix, enhanced AR levels after AR stimulation in HEK293 and HeLa cells. AR stimulation decreased AR ubiquitination which is accompanied by increased β1 Pix binding to AR. Ectopic expression of β1 Pix decreased AR ubiquitination in Tm4 and HEK293 cells. We demonstrated that the formation of a multimolecular complex comprised of AR/β1 Pix/STUB1 responded in a time-dependent manner to AR stimulation. β1 Pix binding dissociated STUB1 from AR and thus prevented STUB1 from catalyzing receptor ubiquitination. β1 Pix enhanced AR transcriptional activity and increased AR target gene expression. Irrespective of treatment, immunofluorescence analysis showed a strong nuclear colocalization of endogenous AR and endogenous βPix in Tm4 cells. However, using Tm4 cell fractionation, AR stimulation decreased βPix/AR association in the cytosolic fraction and increased binding of AR to βPix in the nuclear fraction. To support the role of β1 Pix in androgen regulation, we found that individuals lacking this gene have a significant increase in genitourinary malformations associated with androgen dysfunction. Our data indicate that β1 Pix is an important modulator of AR stability and ligand-dependent AR transcriptional activity. We propose that β1 Pix could serve as a promising therapeutic target to modulate AR signaling.

A carboxy-terminal ubiquitylation site regulates androgen receptor activity

Degradation of unliganded androgen receptor (AR) in prostate cancer cells can be prevented by proteasome inhibition, but this is associated with only modest increases in polyubiquitylated AR. An inhibitor (VLX1570) of the deubiquitylases associated with the proteasome did not increase ubiquitylation of unliganded AR, indicating that AR is not targeted by these deubiquitylases. We then identified a series of AR ubiquitylation sites, including a not previously identified site at K911, as well as methylation sites and previously identified phosphorylation sites. Mutagenesis of K911 increases AR stability, chromatin binding, and transcriptional activity. We further found that K313, a previously reported ubiquitylation site, could also be methylated and acetylated. Mutagenesis of K313, in combination with K318, increases AR transcriptional activity, indicating that distinct posttranslational modifications at K313 differentially regulate AR activity. Together these studies expand the spectrum of AR posttranslational modifications, and indicate that the K911 site may regulate AR turnover on chromatin.

Inhibition of N-myristoyltransferase activity promotes androgen receptor degradation in prostate cancer.

Even though prostate cancer (PCa) patients initially respond to androgen deprivation therapy, some will eventually develop castration resistant prostate cancer (CRPC). Androgen receptor (AR) mediated cell signaling is a major driver in the progression of CRPC while only a fraction of PCa becomes AR negative. This study aimed to understand the regulation of AR levels by N-myristoyltransferase in PCa cells. Two enantiomers, (1S,2S)- d-NMAPPD and (1R,2R)- d-NMAPPD (LCL4), were characterized by various methods (1 H and 13 C NMR, UHPLC, high-resolution mass spectra, circular dichroism) and evaluated for the ability to bind to N-myristoyltransferase 1 (NMT1) using computational docking analysis. structure-activity relationship analysis of these compounds led to the synthesis of (1R,2R)-LCL204 and evaluation as a potential NMT1 inhibitor utilizing the purified full length NMT1 enzyme. The NMT inhibitory activity wase determined by Click chemistry and immunoblotting. Regulation of NMT1 on tumor growth was evaluated in a xenograft tumor model. (1R,2R)- d-NMAPPD, but not its enantiomer (1S,2S)- d-NMAPPD, inhibited NMT1 activity and reduced AR protein levels. (1R,2R)-LCL204, a derivative of (1R,2R)- d-NMAPPD, inhibited global protein myristoylation. It also suppressed protein levels, nuclear translocation, and transcriptional activity of AR full-length or variants in PCa cells. This was due to enhanced ubiquitin and proteasome-mediated degradation of AR. Knockdown of NMT1 levels inhibited tumor growth and proliferation of cancer cells. Inhibitory efficacy on N-myristoyltransferase activity by d-NMAPPD is stereospecific. (1R,2R)-LCL204 reduced global N-myristoylation and androgen receptor protein levels at low micromolar concentrations in prostate cancer cells. pharmacological inhibition of NMT1 enhances ubiquitin-mediated proteasome degradation of AR. This study illustrates a novel function of N-myristoyltransferase and provides a potential strategy for treatment of CRPC.

SAT023 The Role Of Cysteine Residues In The Binding Of Inhibitors Of The N-Terminal Domain Of The Androgen Receptor

Abstract Disclosure: L. Deneault: None. A. Tien: None. A. Banuelos: Stock Owner; Self; ESSA. T. Tam: None. R. Andersen: Stock Owner; Self; ESSA. M.D. Sadar: Stock Owner; Self; ESSA. Background: Therapies for prostate cancer (PC) involve pharmaceutical castration by targeting the androgen receptor (AR) ligand-binding domain (LBD). These therapies are not curative with the malignancy progressing to castration-resistant PC (CRPC). Most CRPC remains driven by AR through multiple resistances, e.g. constitutively active AR splice variants that lack LBD (AR-Vs), or gain-of-function mutations in LBD. This has led to clinical development of EPI analogues (“EPI”) which target the AR N-terminal domain (NTD) to block the transcriptional activities of, AR-Vs and mutated ARs. The EPI-binding pocket involves region 341-446 amino acid residues of the NTD. AR NTD is enriched in cysteine residues with C404 suggested to be important in the binding mechanism of EPI. Free cysteine residues are able interact forming disulfide bonds, which are important in intra- and intermolecular protein-protein interactions. Here we begin to elucidate the role of cysteines in the inhibition of AR activity by EPI. Methods: Binding assays assess the role of cysteines in the covalent binding mechanism used a fluorescein-labelled EPI with fragments of recombinant AR-NTD (rNTD) with or without the addition of iodoacetamide (caps cysteines). Protein-protein interactions requiring disulfide bonds with AR examined by non-reducing SDS-PAGE and diagonal SDS-PAGE of cells treated with inhibitors. A role of specific cysteines in the activity of EPI to block AR transcriptional activity was assessed by cysteine mutants using a reporter gene construct driven by AR. Results: Capping cysteine residues on rNTD with iodoacetamide reduced the amount of covalent binding of EPI. This data suggests that cysteines are essential for covalent binding of EPI to AR-NTD. EPI bound preferentially to the oxidized form of rNTD suggesting the importance of disulfide bridges. Diagonal SDS-PAGE revealed differential alterations in intramolecular and intermolecular disulfide bonds in response to EPI compared to AR LBD inhibitors. These gels also revealed unique differences in protein-protein interactions with AR depending on the inhibitor. AR-driven reporter gene assays revealed mutations of cysteine residues in the AR- NTD did not adversely affect AR transcriptional activity in response to androgen. Conclusions: Although cysteines residing in the AR-NTD were required for covalent binding of EPI, mutation of C404 in the EPI-binding site was not essential for the efficacy of EPI to block AR transcriptional activity. There were differential patterns in protein-protein interactions with AR depending upon which domain the inhibitor targeted. Our future goal is to identify these unique proteins to provide insight into the mechanisms and develop more potent and more specific therapeutics for the treatment of CRPC. Presentation: Saturday, June 17, 2023

SAT022 A Mechanism Of Resistance To Novel Inhibitors Of The N-Terminal Domain Of Androgen Receptor

Abstract Disclosure: J. Setiawan: None. N.R. Mawji: None. A.H. Tien: None. R.J. Andersen: Stock Owner; Self; ESSA Pharma. M.D. Sadar: Stock Owner; Self; ESSA Pharma. Background: Androgen receptor (AR) is a steroid hormone receptor and a major therapeutic target in prostate cancer. Antiandrogen therapies directed at the AR C-terminal ligand-binding domain (LBD) provide initial benefit to patients with advanced prostate cancer, but they eventually progress to lethal metastatic castration-resistant prostate cancer (CRPC). AR N-terminal domain (NTD) inhibitors are a novel class of antagonists that were developed to overcome resistance mechanisms related to the AR-LBD, including splice variants lacking the LBD (AR-V7). While mutations in the AR-LBD have been well-characterized, the impact of NTD mutations on AR transcriptional activity and drug efficacy remains unclear. The mutation W435L was discovered from clinical samples of prostate cancer from patients treated with antiandrogens. This mutation stabilizes interaction between the AR’s NTD and LBD (N/C interaction) to increase AR transcriptional activity. Tryptophan residues W435 and W397 in AR NTD are essential in the binding site for the AR-NTD inhibitor EPI-7386 that is currently in clinical trials. We hypothesize that tryptophan mutations within the EPI-7386 binding site of AR are a potential mechanism of resistance. Methods: Site-directed mutagenesis was used to create expression vectors with W435 and W397 point mutations in AR. Plasmids were transfected into AR-negative CV-1 cells to compare the transcriptional activity of wild-type (WT) versus mutant ARs. IC50s for each inhibitor were generated for androgen-induced reporter gene constructs. A mammalian two-hybrid system assay was used to assess the impact of inhibitors and AR mutations on N/C interaction. Results: The AR-W435L mutation led to higher IC50s to inhibit androgen-induced MMTV-luciferase activity by NTD inhibitors and antiandrogens. Differences in N/C interaction were observed between WT and mutant ARs treated with EPI-7386. Importantly, the ability of another EPI analog, EPI-7170, to decrease AR-N/C interaction was not significantly impacted by W435L. These data support that loss of efficacy with EPI-7386 is not across the entire class of EPI analogs and may be specific to EPI-7386. Conclusion: Tryptophan mutations in the EPI-7386 binding site are predictive of resistance to this specific antagonist and do not broadly apply to the entire class of EPI analogs. Key differences were revealed between AR-LBD and AR-NTD inhibitors that support the recruitment of different tau regions in the NTD. Presentation: Saturday, June 17, 2023

Number of CAG repeats and mortality in middle aged and older men.

The androgen receptor (AR) mediates peripheral effects of testosterone. Previous data suggests an association between the number of CAG repeats in exon-1 of the AR gene and AR transcriptional activity. The aim of this analysis was to determine the association between the number of AR CAG repeats and all-cause mortality in men and the influence of testosterone level on the association. Follow-up data to 27 January 2018 were available for men aged 40-79 years recruited across six countries of the European Male Aging Study between 2003 and 2005. Cox proportional hazards modelling was used to determine the association between CAG repeat number/mortality. Results were expressed as hazard ratios (HR)/95% confidence intervals (CI). One thousand nine hundred and seventy-seven men were followed up. Mean baseline age was 60 ± 11.1 years. Mean duration of follow-up was 12.2 years. At follow up 25.1% of men had died. CAG repeat length ranged from 6 to 39, with the highest proportion of CAG repeat number at 21 repeats (16.4%). In a multivariable model, compared to men with 22-23 AR CAG repeats: for men with <22 and >23 AR CAG HR, 95%CIfor mortality were, <22 CAG repeats 1.17 (0.93-1.49) and >23 CAG repeats 1.14 (0.88-1.47). In a post-hoc analysis, the association was significant for men in the lowest tertile of baseline testosterone (<14.2 nmol/L) with >23 CAG repeats: in the adjusted model for <22 and >23 CAG repeats, respectively, 1.49 (0.97-2.27) and 1.68 (1.06-2.67) versus 22-23 repeats. Our European-wide cohort data overall found no association of androgen receptor CAG repeat number and mortality in men. However, post hoc analysis suggested that an association might be present in men with lower baseline testosterone concentrations, which merits further investigation.

Novel progestogenic androgens for male contraception: design, synthesis, and activity of C7 α-substituted testosterone†.

Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for suppression of testicular T production. Expanding upon the vast amount of data accumulated from nortestosterone (NT), NT analogs, and their prodrugs, a new series of PA, the C7 methyl, and ethyl α-substituted T analogs 7α-Methyltestosterone (7α-MT) and 7α-Ethyltestosterone (7α-ET), respectively, were hypothesized and designed to have superior androgenic and progestogenic activities when compared with parent T. Results from androgen receptor and progesterone receptor competitive binding and transcriptional activation assays showed favorable activities for these T analogs. Additionally, 7α-MT and 7α-ET were shown to be active substrates for aromatase in vitro, mitigating a potential negative impact on bone mineral density with long-term use. In conjunction with this observation, the diminished metabolism of these T analogs by 5α-reductase may reduce potential concerns for prostatic growth. In the Hershberger in vivo rat bioassay, 7α-MT and 7α-ET showed superior androgenic and anabolic activities as compared with T. These C7 α-substituted T analogs also showed clear progestogenic activity in the McPhail bioassay which evaluated endometrial glandular arborization in a rabbit model. The discovery of aromatizable molecules with reduced metabolism by 5α-reductase that have androgenic, anabolic, and progestogenic properties indicates that the core and/or prodrugs of 7α-MT and 7α-ET are promising molecules for further development as male contraceptive PAs.

Bromodomain-containing protein 4 activates androgen receptor transcription and promotes ovarian fibrosis in PCOS

Polycystic ovary syndrome (PCOS) is an endocrine disorder and the main cause of anovulatory infertility, in which persistent activation of androgen receptor (AR) due to aberrant acetylation modifications of transcription is a potential trigger; however, the precise mechanisms of AR activation are poorly understood. In this study, AR activation in dehydroepiandrosterone- and letrozole-induced rat PCOS ovaries coincided with a marked increase of a chromatin acetylation "reader" BRD4. Further bioinformatic analysis showed that the AR promoter contained highly conserved binding motifs of BRD4 and HIF-1α. BRD4 and HIF-1α inducibly bound to the histone 3/4 acetylation-modified AR promoter, while administration of a BRD4-selective inhibitor JQ1 reduced the binding and AR transcription and improved the adverse expression of the core fibrotic mediators in PCOS ovaries and DHT-treated granulosa cells. Our data indicate that BRD4 upregulation and the resultant AR transcriptional activation constitute an important regulatory pathway that promotes ovarian fibrosis in PCOS.

A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Androgen receptor transcriptional activity is required for heregulin-1β–mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while invivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. Invitro treatment with the ErbB3 ligand heregulin-1β (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Tricyclic Diterpenoids Selectively Suppress Androgen Receptor-Positive Prostate Cancer Cells.

Androgen receptor (AR) is a viable therapeutic target for lethal castration-resistant prostate cancer (CRPC), because the continued progression of CRPC is mainly driven by the reactivation of AR transcriptional activity. The current FDA-approved AR antagonists binding to ligand binding domain (LBD) become ineffective in CRPC with AR gene amplification, LBD mutation, and the evolution of LBD-truncated AR splice variants. Encouraged by the fact that tricyclic aromatic diterpenoid QW07 has recently been established as a potential N-terminal AR antagonist, this study aims to explore the structure-activity relationship of tricyclic diterpenoids and their potential to suppress AR-positive cell proliferation. Dehydroabietylamine, abietic acid, dehydroabietic acid, and their derivatives were selected, since they have a similar core structure as QW07. Twenty diterpenoids were prepared for the evaluation of their antiproliferative potency on AR-positive prostate cancer cell models (LNCaP and 22Rv1) using AR-null cell models (PC-3 and DU145) as comparisons. Our data indicated that six tricyclic diterpenoids possess greater potency than enzalutamide (FDA-approved AR antagonist) towards LNCaP and 22Rv1 AR-positive cells, and four diterpenoids are more potent than enzalutamide against 22Rv1 AR-positive cells. The optimal derivative possesses greater potency (IC50 = 0.27 µM) and selectivity than QW07 towards AR-positive 22Rv1 cells.

Abstract A019: Enzalutamide resistance is driven by adaptations that enhance AR splice variant and FOXA1 activities

Abstract Prostate cancer (PCa) that progresses on therapy with an androgen receptor (AR) inhibitor such as enzalutamide (Enz) generally continues to express high levels of transcriptionally active AR, but mechanisms driving this persistent AR activity remain to be established. These tumors also generally express increased levels of AR splice variants that lack the ligand binding domain and hence have androgen-independent activity (with ARv7 being the most common), but the extent to which these variants contribute to AR activity, and their dependence on the full length AR (ARfl), also remain unclear. We found Enz treatment initially suppressed AR activity in VCaP cells, despite an increase in ARv7 splice variant, while Enz-resistant VCaP cells (VCaPEnzR) generated by prolonged culture in Enz had restoration of AR transcriptional activity. Notably, this restored AR activity was not associated with further increases in ARv7 expression. However, by ChIP-seq we found it was associated with increased ARv7 chromatin binding, with ARv7 binding at a subset of the sites occupied by ARfl, but not at any unique sites. Moreover, approaches using RNAi and an AR degrader targeting ARfl showed that ARv7 was the major driver of AR transcriptional activity in the VCaPEnzR cells, despite much higher levels of ARfl expression, and was not dependent on ARfl. ATAC-seq showed there was a global increase in chromatin accessibility in the VCaPEnzR cells, with the greatest increase being at ARv7 sites. This suggested these sites had decreased nucleosome occupancy, which was confirmed by MNase-seq. As FOXA1 can act as a pioneer factor to open chromatin, we next carried out FOXA1 ChIP-seq. This showed increased FOXA1 binding across all FOXA1 sites in the VCaPEnzR cells versus parental VCaP, with the greatest increases being at ARv7 binding sites and at sites that showed the greatest increases in accessibility by ATAC-seq. Notably, frameshift mutations in the C-terminus of FOXA1 are found at increased frequency in castration-resistant prostate cancer (CRPC) and may enhance FOXA1 activity, but FOXA1 was not mutated in the VCaPEnzR versus parental VCaP cells. Mass spectrometry then identified posttranslational modifications (PTMs) in FOXA1 that have been previously reported in PCa, including K270 methylation that can impair FOXA1 chromatin binding, but this was not consistently decreased in VCaPEnzR cells. In contrast, we found consistent increases in phosphorylation at C-terminal sites including T334 and Y429. Notably, the Y429 phosphorylation was reported previously to enhance FOXA1 chromatin binding and estrogen receptor activity. Together these findings indicate that progression to Enz-resistance is dependent on adaptations that enhance ARv7 activity. These adaptations include increasing chromatin accessibility at ARv7 binding sites, which may be driven by PTMs in FOXA1. Citation Format: Betul Ersoy-Fazlioglu, Larysa Poluben, Mannan Nouri, Henry W. Long, Joshua W. Russo, Steven P. Balk. Enzalutamide resistance is driven by adaptations that enhance AR splice variant and FOXA1 activities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A019.