DNA-binding Domain Of Androgen Receptor Research Articles

Structural mechanism underlying variations in DNA binding by the androgen receptor

The androgen receptor (AR) is a steroid activated transcription factor which recognizes DNA motifs resembling inverted repeats of a conserved 5’-AGAACA-3’-like hexanucleotides separated by a three-nucleotide spacer from a similar, but less conserved hexanucleotide. Here, we report the structures of the human AR DNA binding domain (DBD) bound to two natural AREs (C3 and MTV) in head-to-head dimer conformations, diffracting at 2.05 Å and 2.25 Å, respectively. These structures help to explain the impact of androgen insensitivity mutations on the structure integrity, DNA binding and DBD dimerization. The binding affinity of the AR DBD to different DNA motifs were measured by the BioLayer Interferometry (BLI) and further validated by Molecular Dynamics (MD) simulations. This shows that the high binding affinity of the first DBD to the upstream 5’-AGAACA-3’ motif induces the cooperative binding of the second DBD to the second hexanucleotide. Our data indicate identical interaction of the DBDs to the upstream hexanucleotides, while forming an induced closer contact of the second DBD on the non-canonical hexanucleotides. The variation in binding between the DBD monomers are the result of differences in DNA occupancy, protein-protein interactions, DNA binding affinity, and DNA binding energy profiles. We propose this has functional consequences.

Novel Inhibitors of androgen receptor's DNA binding domain identified using an ultra-large virtual screening.

Androgen receptor (AR) inhibition remains the primary strategy to combat the progression of prostate cancer (PC). However, all clinically used AR inhibitors target the ligand-binding domain (LBD), which is highly susceptible to truncations through splicing or mutations that confer drug resistance. Thus, there exists an urgent need for AR inhibitors with novel modes of action. We thus launched a virtual screening of an ultra-large chemical library to find novel inhibitors of the AR DNA-binding domain (DBD) at two sites: protein-DNA interface (P-box) and dimerization site (D-box). The compounds selected through vigorous computational filtering were then experimentally validated. We identified several novel chemotypes that effectively suppress transcriptional activity of AR and its splice variant V7. The identified compounds represent previously unexplored chemical scaffolds with a mechanism of action that evades the conventional drug resistance manifested through LBD mutations. Additionally, we describe the binding features required to inhibit AR DBD at both P-box and D-box target sites.

The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.

Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain and Modulation via Direct Interactions with DNA Abasic Sites: Understanding the Mechanisms Involved in Castration-Resistant Prostate Cancer.

The androgen receptor (AR) is an important drug target in prostate cancer and a driver of castration-resistant prostate cancer (CRPC). A significant challenge in designing effective drugs lies in targeting constitutively active AR variants and, most importantly, nearly all AR variants lacking the ligand-binding domain (LBD). Recent findings show that an AR's constitutive activity may occur in the presence of somatic DNA mutations within non-coding regions, but the role of these mutations remains elusive. The discovery of new drugs targeting CRPC is hampered by the limited molecular understanding of how AR binds mutated DNA sequences, frequently observed in prostate cancer, and how mutations within the protein and DNA regulate AR-DNA interactions. Using atomistic molecular dynamics (MD) simulations and quantum mechanical calculations, we focused our efforts on (i) rationalising the role of several activating DBD mutations linked to prostate cancer, and (ii) DBD interactions in the presence of abasic DNA lesions, which frequently occur in CRPC. Our results elucidate the role of mutations within DBD through their modulation of the intrinsic dynamics of the DBD-DNA ternary complex. Furthermore, our results indicate that the DNA apurinic lesions occurring in the androgen-responsive element (ARE) enhance direct AR-DNA interactions and stabilise the DBD homodimerisation interface. Moreover, our results strongly suggest that those abasic lesions may form reversible covalent crosslinks between DNA and lysine residues of an AR via a Schiff base. In addition to providing an atomistic model explaining how protein mutations within the AR DNA-binding domain affect AR dimerisation and AR-DNA interactions, our findings provide insight into how somatic mutations occurring in DNA non-coding regions may activate ARs. These mutations are frequently observed in prostate cancer and may contribute to disease progression by enhancing direct AR-DNA interactions.

De Novo Design of an Androgen Receptor DNA Binding Domain-Targeted peptide PROTAC for Prostate Cancer Therapy.

Androgen receptor splice variant‐7 (AR‐V7), one of the major driving factors, is the most attractive drug target in castration‐resistant prostate cancer (CRPC). Currently, no available drugs efficiently target AR‐V7 in clinical practice. The DNA binding domain (DBD) is indispensable for the transcriptional activity of AR full length and AR splice variants, including AR‐V7. Based on the homodimerization structure of the AR DBD, a novel peptide‐based proteolysis‐targeting chimera (PROTAC) drug is designed to induce AR and AR‐V7 degradation in a DBD and MDM2‐dependent manner, without showing any activity on other hormone receptors. To overcome the short half‐life and poor cell penetrability of peptide PROTAC drugs, an ultrasmall gold (Au)‐peptide complex platform to deliver the AR DBD PROTAC in vivo is developed. The obtained Au‐AR pep‐PROTAC effectively degrades AR and AR‐V7 in prostate cancer cell lines, particularly in CWR22Rv1 cells with DC50 values 48.8 and 79.2 nM, respectively. Au‐AR pep‐PROTAC results in suppression of AR levels and induces tumor regression in both enzalutamide sensitive and resistant prostate cancer animal models. Further optimization of the Au‐AR pep‐PROTAC can ultimately lead to a new therapy for AR‐V7‐positive CRPC.

Abstract 2650: Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer

Abstract Background: The androgen receptor (AR), which plays a major role in prostate cancer (PCa), consists of a C-terminal ligand-binding domain (LBD), a hinge domain, a DNA-binding domain (DBD), and an N-terminal domain (NTD). Current AR antagonists and AR signaling inhibitors (ASIs), used in androgen deprivation therapy (ADT) and to prevent disease progression, often fail due to production of alternatively spliced AR variants (AR-Vs) that lack the LBD but retain the DBD, which is essential to AR transcriptional activity. This project examines a series of novel compounds designed to inhibit AR-DNA interaction and study how these compounds affect castration-resistant PCa (CRPC). Methods: Compounds potentially targeting the DBD were used to predict direction and position of compounds when docked to the AR. MTT assays, flow cytometry, immunoblots, qRT-PCR, luciferase assay, and immunofluorescence were used to test compounds on AR and PCa. Drug Affinity Responsive Target Stability (DARTS) tested potential binding of the compounds to the AR. Studies in intact mice were used to determine toxicity of the compounds and the maximum tolerated dose. Results: Of 48 compounds synthesized, C15 had the highest and most consistent efficacy overall. C15 decreased metabolic activity in 22Rv1 and CWR-R1 cells that express AR-Vs and in C4 and C4-2 that do not express AR-Vs. Further, C15 had a reduced effect in AR-null PC3 cells and minimal effect in normal human dermal fibroblast (NHDF) cells. In C4-2 and 22Rv1, C15 significantly reduced PSA mRNA and AR transcriptional activity, and in C4-2, C15 significantly reduced TMPRSS2 and Nrdp1 mRNA levels. C08 and C15 reduced pronase-induced AR proteolysis only when the AR DBD is present. Furthermore, C15 reduces AR binding at the PSA enhancer, but DHT stimulation may weaken this binding inhibition and reduction in AR transcriptional activity. However, combining C15 with an AR inhibitor targeting the LBD (enzalutamide, darolutamide, apalutamide) restores the full inhibitory potential and even reduces metabolic activity and AR transcriptional activity further than either drug alone in both C4-2 and 22Rv1s. No toxicity in intact mice was observed with C15 with the highest dose tested being 50 mg/kg. Conclusion: Our preliminary data demonstrate that C15 directly binds to the DBD of the AR and potently inhibits CRPC cells, including those that express AR-Vs missing the LBD. Although increased DHT levels may weaken C15’s inhibition of the AR DBD, addition of a LBD-targeting AR inhibitor improves the inhibitory potential of both drugs. Based on these results, we conclude that C15 may hold great clinical potential as it successfully inhibits CRPC by targeting the AR DBD with minimal toxicity, and it can improve the effects of standard-of-care LBD-targeting AR inhibitors by reducing AR-V-induced resistance. Citation Format: Thomas M. Steele, Elisabeth A. Messner, Maria Malvina Tsamouri, Salma Siddiqui, Brian Bennion, Ruiwu Liu, Paramita Ghosh. Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2650.

Chronologically modified androgen receptor in recurrent castration-resistant prostate cancer and its therapeutic targeting.

Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys609 in the zinc finger DNA binding domain of AR (acK609-AR) that not only allowed AR translocation but also galvanized a distinct global transcription program, conferring enzalutamide insensitivity. Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase-mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, (R)-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.

Design, synthesis, and biological evaluation of phenyl thiazole-based AR-V7 degraders

Multiple Splice variants of AR have been reported in the past few years. These splice variants are upregulated in most cases of CRPC resulting in poor prognosis. Most of these variants lack the ligand binding domain (LBD) but still bind to DNA resulting in constitutive activation of downstream targets. The AR-V7 splice variant has been characterized extensively and current clinical trials in CRPC are exploring the use of AR-V7 as a biomarker. New therapeutic molecules that selectively target AR-V7 are also being explored. However, there is a dearth of information available on the selectivity, phenotypic responses in AR-V7 dependent cell lines and pharmacokinetic properties of such molecules. Using our proprietary computational algorithms and rational SAR optimization, we have developed a potent and selective AR-V7 degrader from a known AR DNA binding domain (DBD) binder. This molecule effectively degraded AR-V7 in a CRPC cell line and demonstrated good oral bioavailability in mouse PK studies. This tool compound can be used to evaluate the pharmacological effects of AR-V7 degraders. Further exploration of SAR can be pursued to develop more optimized lead compounds.

Discovery of novel antagonists targeting the DNA binding domain of androgen receptor by integrated docking-based virtual screening and bioassays.

Androgen receptor (AR), a ligand-activated transcription factor, is a master regulator in the development and progress of prostate cancer (PCa). A major challenge for the clinically used AR antagonists is the rapid emergence of resistance induced by the mutations at AR ligand binding domain (LBD), and therefore the discovery of novel anti-AR therapeutics that can combat mutation-induced resistance is quite demanding. Therein, blocking the interaction between AR and DNA represents an innovative strategy. However, the hits confirmed targeting on it so far are all structurally based on a sole chemical scaffold. In this study, an integrated docking-based virtual screening (VS) strategy based on the crystal structure of the DNA binding domain (DBD) of AR was conducted to search for novel AR antagonists with new scaffolds and 2-(2-butyl-1,3-dioxoisoindoline-5-carboxamido)-4,5-dimethoxybenzoicacid (Cpd39) was identified as a potential hit, which was competent to block the binding of AR DBD to DNA and showed decent potency against AR transcriptional activity. Furthermore, Cpd39 was safe and capable of effectively inhibiting the proliferation of PCa cell lines (i.e., LNCaP, PC3, DU145, and 22RV1) and reducing the expression of the genes regulated by not only the full-length AR but also the splice variant AR-V7. The novel AR DBD-ARE blocker Cpd39 could serve as a starting point for the development of new therapeutics for castration-resistant PCa.

Development of Novel Inhibitors Targeting the D-Box of the DNA Binding Domain of Androgen Receptor.

The inhibition of the androgen receptor (AR) is an established strategy in prostate cancer (PCa) treatment until drug resistance develops either through mutations in the ligand-binding domain (LBD) portion of the receptor or its deletion. We previously identified a druggable pocket on the DNA binding domain (DBD) dimerization surface of the AR and reported several potent inhibitors that effectively disrupted DBD-DBD interactions and consequently demonstrated certain antineoplastic activity. Here we describe further development of small molecule inhibitors of AR DBD dimerization and provide their broad biological characterization. The developed compounds demonstrate improved activity in the mammalian two-hybrid assay, enhanced inhibition of AR-V7 transcriptional activity, and improved microsomal stability. These findings position us for the development of AR inhibitors with entirely novel mechanisms of action that would bypass most forms of PCa treatment resistance, including the truncation of the LBD of the AR.

Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy

Although second-line antiandrogen therapy (SAT) is the standard of care in men with castration-resistant prostate cancer (CRPC), resistance inevitably occurs. One major proposed mechanism of resistance to SAT involves the emergence of androgen receptor (AR) splice variant-7, AR-V7. Recently, we developed MTX-23 using the principle of proteolysis targeting chimera (PROTAC) to target both AR-V7 and AR-full length (AR-FL). MTX-23 has been designed to simultaneously bind AR's DNA binding domain (DBD) and the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. Immunoblots demonstrated that MTX-23's degradation concentration 50% (DC50) for AR-V7 and AR-FL was 0.37 and 2 μmol/L, respectively. Further studies revealed that MTX-23 inhibited prostate cancer cellular proliferation and increased apoptosis only in androgen-responsive prostate cancer cells. The antiproliferative effect of MTX-23 was partially reversed when either AR-V7 or AR-FL was overexpressed and was completely abrogated when both were overexpressed. To assess the potential therapeutic value of MTX-23, we next generated 12 human prostate cancer cell lines that are resistant to the four FDA-approved SAT agents-abiraterone, enzalutamide, apalutamide, and darolutamide. When resistant cells were treated with MTX-23, decreased cellular proliferation and reduced tumor growth were observed both in vitro and in mice. These results collectively suggest that MTX-23 is a novel PROTAC small molecule that may be effective against SAT-resistant CRPC by degrading both AR-V7 and AR-FL.

Selective targeting of the androgen receptor-DNA binding domain by the novel antiandrogen SBF-1 and inhibition of the growth of prostate cancer cells.

Prostate cancers are reliant on androgens for growth and survival. Clinicians and researchers are looking for potent treatments for the resistant forms of prostate cancer; however, a handful of small molecules used in the treatment of castration-resistant prostate cancer have not shown potent effects owing to the mutations in the AR (Androgen Receptor). We used SBF-1, a well-characterized antitumor agent with potent cytotoxic effects against different kinds of cancers and investigated its effect on human prostate cancer. SBF-1 substantially inhibited the proliferation, induced apoptosis, and caused cell cycle arrest in LNCaP and PC3/AR+ prostate cancer cell lines. SBF-1 inhibited the activation of the IGF-1-PNCA pathway, as demonstrated by decreased expression of IGF-1 (insulin-like growth factor 1), proliferating cell nuclear antigen (PCNA), and its downstream Bcl-2 protein. Using microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) assays, we observed a direct binding of SBF-1 to the AR. SBF-1 binds to the AR-DBD (DNA-binding domain) and blocks the transcription of its target gene. SBF-1 demonstrated a potent antitumor effect in vivo; it inhibited AR signaling and suppressed tumor growth in animals. Our study suggests that SBF-1 is an inhibitor of the AR and might be used in the treatment of prostate cancer.

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a.

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood-testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3'-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.

A Mechanistic High-Content Analysis Assay Using a Chimeric Androgen Receptor That Rapidly Characterizes Androgenic Chemicals.

Human health is at risk from environmental exposures to a wide range of chemical toxicants and endocrine-disrupting chemicals (EDCs). As part of understanding this risk, the U.S. Environmental Protection Agency (EPA) has been pursuing new high-throughput in vitro assays and computational models to characterize EDCs. EPA models have incorporated our high-content analysis-based green fluorescent protein estrogen receptor (GFP-ER): PRL-HeLa assay, which allows direct visualization of ER binding to DNA regulatory elements. Here, we characterize a modified functional assay based on the stable expression of a chimeric androgen receptor (ARER), wherein a region containing the native AR DNA-binding domain (DBD) was replaced with the ERα DBD (amino acids 183-254). We demonstrate that the AR agonist dihydrotestosterone induces GFP-ARER nuclear translocation, PRL promoter binding, and transcriptional activity at physiologically relevant concentrations (<1 nM). In contrast, the AR antagonist bicalutamide induces only nuclear translocation of the GFP-ARER receptor (at μM concentrations). Estradiol also fails to induce visible chromatin binding, indicating androgen specificity. In a screen of reference chemicals from the EPA and the Agency for Toxic Substances and Disease Registry, the GFP-ARER cell model identified and mechanistically grouped activity by known (anti-)androgens based on the ability to induce nuclear translocation and/or chromatin binding. Finally, the cell model was used to identify potential (anti-)androgens in environmental samples in collaboration with the Houston Ship Channel/Galveston Bay Texas A&M University EPA Superfund Research Program. Based on these data, the chromatin-binding, in vitro assay-based GFP-ARER model represents a selective tool for rapidly identifying androgenic activity associated with drugs, chemicals, and environmental samples.

SUN-LB136 A Comparison of Androgen Receptor Splice Variant, AR-V7, and Glucocorticoid Receptor Activity in Prostate Cancer

Prostate Cancer (PCa) is an androgen dependent disease and patients with metastatic PCa are treated with androgen deprivation therapy (ADT). Although most tumors respond initially, tumors become resistant and are termed castration resistant prostate cancer (CRPC). There is compelling evidence that most of these tumors retain androgen receptor (AR) dependence and some data to suggest that, in some cases, the glucocorticoid receptor (GR) substitutes for AR. AR, itself, is re-activated through a variety of mechanisms including the expression of constitutively active AR splice variants that lack the ligand binding domain (LBD) of AR. Expression of one variant, AR-V7, which contains the amino-terminal domain and DNA binding domain of AR and 16 unique amino-acids, has been correlated with resistance to second line ADT. Although there has been some debate regarding the role of AR-V7, whether it is only a partial substitute for AR or has unique activities, our studies of engineered cell lines treated to express levels of AR-V7 equivalent to AR, clearly show that while the AR isoforms have common targets, they each also have unique targets. Consistent with this, the cistromes of the two show many unique sites as well as common sites. AR-V7 binding is enriched near the transcription start site (TSS) and we have identified a novel de novo binding motif. These findings suggest the possibility of developing a gene signature unique to AR-V7.Because GR activity in PCa has also been suggested as an escape mechanism in response to ADT, and GR binds to the same consensus response elements, we sought to identify a GR signature in PCa, to compare it with the AR and AR-V7 signatures, and to ask whether the AR-V7 and/or GR signatures are enriched in CRPC. Because much of the gene signatures are cell line dependent, we sought to compare GR and AR-V7 action in cells that express both. LN-95 cells express both AR-V7 and GR. MDA-PCa-2b cells, a cell line derived from an African American patient, expresses GR, but not AR-V7. The parental line was infected with a lentivirus that expresses AR-V7 in response to doxycycline. Transcriptomes were determined for AR, GR, and AR-V7 in these lines using RNA-Seq. Overall the magnitude of regulation of gene expression was generally lower than in the LNCaP AR-V7 and VCaP-AR-V7 lines, but there was good overlap of the MDA-PCa-2b AR-V7 regulated with the LNCaP AR-V7 regulated genes. Genes induced by GR overlapped with AR and isoform common genes, but did not overlap with AR-V7 specific genes. A comparison of AR-V7 specific genes common to the LNCaP and VCaP models as well as to publicly available data sets for LN-95 and 22RV1 AR-V7 signatures, show a strong correlation with CRPC compared to primary tumors when analyzed in the Grasso data set.

Comparing the DNA‐Binding Specificity of Glucocorticoid and Androgen Receptors

Prostate cancer is the second most common cancer among men, with an estimated 175,000 diagnoses expected in 2019. Androgen hormones fuel prostate tumor growth through the actions of the androgen receptor (AR), a member of the nuclear hormone family of transcription factors. Therapies for prostate cancer include surgery and inhibition of AR with antagonists. Patients, however, often relapse with castration‐resistant prostate cancer (CRPC) in which the cancer grows in the absence of AR ligand action. In some cases, the need for androgens is subverted by overexpression of the AR‐related glucocorticoid receptor (GR). GR effectively replaces AR by regulating genes that drive prostate growth and survival. We have previously used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method along with the SelexGLM computational algorithm to demonstrate that GR and AR have similar, but distinct DNA sequence specificities. Upon overexpression, GR is able to bind genomic loci once preferentially bound by AR and regulate associated genes. To better understand differences in AR and GR DNA recognition, we are now measuring DNA sequence preferences of both AR and GR using the No Read Left Behind (NRLB) method. NRLB yields high resolution models of specificity after just one round of selection, allowing rapid screening of both wildtype and hybrid AR and GR DNA binding domains. We have over‐expressed and purified both the AR and GR DNA binding domains, and are designing hybrid mutants. Exploring the similarities and differences between AR, GR and the hybrid molecules should help us understand how GR might be playing a role in CRPC.Support or Funding Information• Grant: NSF:CAREER (MCB‐1552862) to M.A. Pufall

Probing the Effects of Androgen and Glucocorticoid Receptor Sequences on DNA Binding Preferences

Prostate cancer is the second leading cause of cancer among men and although survival rates are high for regional tumors, it is estimated that almost 30,000 men will succumb to their diagnosis in 2018. Androgen hormones fuel prostate tumor growth by signaling through the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. Blocking androgen signaling through administration of antagonists (e.g bicalutamide, enzalutamide) or halting production of androgens through chemical or surgical castration arrests progression of the disease. Patients often relapse with castration resistant prostate cancer (CRPC) that continues to grow despite the lack of androgens. One mechanism of resistance is due to upregulation of a related nuclear receptor, the glucocorticoid receptor (GR). Because AR and GR have overlapping DNA binding specificities, GR is able to functionally substitute for AR at key disease genes. We have shown previously, using SELEX (Selective Evolution of Ligands by Exponential Enrichment) coupled with a novel computational algorithm (SelexGLM), that although AR and GR bind similar sequences, they recognize DNA differently. GR is less specific than AR, allowing it to accommodate predominantly AR sequences. To better understand the selectivity of AR and GR, and to identify the determinants of their specificity, we are measuring the sequence preference of AR and GR mutants using the No Read Left Behind (NRLB) method. As opposed to traditional SELEX, NRLB yields high resolution models of specificity with a single round of selection, which allows rapid screening of our mutants. We have over‐expressed and purified both the AR and GR DNA binding domains, and have designed hybrid mutants. Comparison of the DNA binding preferences of AR, GR and the hybrid molecules by NRLB should help uncover how GR might be playing a role in CRPC.Support or Funding InformationThis work was supported in part by the FUTURE program of the University of Iowa Carver College of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain Inhibitor Pyrvinium.

Pyrvinium was identified as the first small molecule inhibitor of the androgen receptor (AR) DNA-binding domain (DBD). It was also among the first small molecules shown to directly inhibit the activity of AR splice variants (ARVs), which has important clinical implications in the treatment of castration-resistant prostate cancer. Important questions about pyrvinium’s mechanism of action remain. Here, we demonstrate through mutational analysis that amino acids 609 and 612 are important for pyrvinium action. Nuclear magnetic resonance demonstrates a specific interaction between a soluble pyrvinium derivative and the AR DBD homodimer–DNA complex. Chromatin immunoprecipitation and electrophoretic mobility shift assay experiments demonstrate that, despite an interaction with this complex, pyrvinium does not alter the DNA-binding kinetics in either assay. AR immunoprecipitation followed by mass spectrometry was used to identify proteins whose interaction with AR is altered by pyrvinium. Several splicing factors, including DDX17, had reduced interactions with AR in the presence of pyrvinium. RNA sequencing of prostate cancer cells treated with pyrvinium demonstrated changes in splicing, as well as in several other pathways. However, pyrvinium did not alter the levels of ARVs in several prostate cancer cell lines. Taken together, our new data pinpoint the direct interaction between pyrvinium and AR DBD and shed light on the mechanism by which it inhibits AR transcriptional activity.

Abstract 3741: Deciphering the regulome of androgen receptor variants in prostate cancer

Abstract Prostate cancer is an androgen-dependent disease. Androgen function is controlled by the androgen receptor (AR), and treatments targeting AR are effective at preventing disease progression. Despite this, resistance and emergence of castration resistant prostate cancer (CRPC) is responsible for virtually all prostate cancer-related death. Constitutively active AR variants (AR-Vs) have been implicated as drivers of CRPC. AR-Vs consist of the unstructured AR NH2-terminal domain (NTD), and the AR DNA binding domain, but lack the ligand binding domain (LBD), which is targeted by all current therapies. While our understanding of proteins that regulate the AR LBD is mature, there is less known about the molecular regulation of the NTD. This represents a gap in knowledge given that the AR NTD is essential for AR/AR-V transcriptional activity. To address this, we used a technique termed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) with a pair of isogenic prostate cancer cell lines that were manipulated via genome engineering to express either full-length AR or AR-V. RIME identified several candidate proteins that interact with the AR NTD including BCL9, a β-catenin transcriptional coactivator. Interrogation of public databases revealed that BCL9 mRNA is overexpressed in metastatic prostate cancer compared with localized disease. Additionally, knockdown of BCL9 in cell lines expressing full-length AR inhibited expression of AR target genes as well as β-catenin target genes. Interestingly, knockdown of BCL9 did not affect these same genes in cell lines expressing AR-Vs, indicating that AR truncation could represent a mechanism of escape from BCL9 regulation. Collectively, these findings identify a new mechanism of AR regulation by BCL9 in prostate cancer. Citation Format: Mark L. Daniel, Theresa E. Hickey, Jason S. Carroll, Wayne D. Tilley, Luke A. Selth, Scott M. Dehm. Deciphering the regulome of androgen receptor variants in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3741.

Androgen signaling regulates the transcription of anti-Müllerian hormone via synergy with SRY-related protein SOX9A

Anti-Müllerian hormone (amh) is one of the earliest functional genes expressed during testicular differentiation. It has been suggested that androgen signaling regulates critical genes for the differentiation and development of the testis. To elucidate the exact regulatory mechanisms involved in amh transcription mediated by androgen signaling, androgen signaling was manipulated in zebrafish by cytochrome P450 17a1 (cyp17a1) knockout and Flutamide treatment. In cyp17a1-deficient and Flutamide-treated testes, up-regulated sry-box9a (sox9a) and down-regulated amh were observed. Moreover, a physical association of the zebrafish androgen receptor (AR) and SOX9A was found. The interaction between AR and SOX9A was mediated via the DNA binding domain (DBD) of AR and the transactivation domain (TA) of SOX9A, and was further enhanced by 5-alpha dihydrotestosterone (DHT), one of the most potent androgens. Intriguingly, together with SOX9A, androgen signaling synergistically promoted amh transcription, mainly through the proximal 1kb of the amh promoter region. Taken together, our data demonstrate a critical mechanism underlying the direct synergy of androgen signaling and SOX9A in the regulation of amh transcription.