Mutant Androgen Receptor Research Articles

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

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

CRISPR-Cas9 guided RNA-based model for the silencing of spinal bulbar muscular atrophy: A functional genetic disorder.

This study explores a novel therapeutic approach for spinal bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a mutation in the Androgen Receptor (AR) gene. The aim is to investigate the potential of CRISPR-Cas9 technology in targeting the mutant AR gene to inhibit its production. The objectives include assessing the accuracy and efficacy of CRISPR-Cas9 guided RNAs in silencing the mutant gene and evaluating the feasibility of this approach as a treatment for SBMA. Computational and in-silico approaches are used to evaluate the feasibility of using CRISPR-Cas9 technology for treating SBMA. Computational analysis is used to design CRISPR-Cas9 guided RNAs targeting the mutant AR gene, assessing their on-target and off-target scores, GC content, and structural accuracy. In-silico simulations predict the potential therapeutic outcomes of the CRISPR-Cas9 approach in an artificial environment. Three guided RNA (gRNA) sequences were designed using the CHOPCHOP tool, targeting specific regions of the AR gene with high efficiency and 100% match. These gRNAs demonstrated effective targeting with minimal off-target scores and optimal GC content. Additionally, lentiCRISPR v2 plasmids were designed for the delivery of CRISPR materials, enabling high-efficiency multiplex genome editing of the AR gene. Thermodynamic ensemble predictions indicated favorable secondary structure stability of the designed gRNAs, further supporting their suitability for gene editing. The evaluation of designed gRNAs confirmed their strong binding ability to the target sequences, validating their potential as effective tools for genome editing. The study highlights the potential of CRISPR-Cas9 technology for targeting the Androgen Receptor gene associated with spinal bulbar muscular atrophy (SBMA). The findings support the feasibility of this approach for gene editing and suggest further exploration in preclinical and clinical settings. Recommendations include continued research to optimize CRISPR-Cas9 delivery methods and enhance specificity for therapeutic applications in SBMA.

Discovery of Thiadiazoleamide Derivatives as Potent, Selective, and Orally Available Antagonists Disrupting Androgen Receptor Homodimer.

Androgen receptor (AR) is an important therapeutic target for prostate cancer (PCa) treatment, but prolonged use of AR antagonists has led to variant drug-resistant mutations. Since all marketed AR antagonists target the ligand binding pocket (LBP) of AR, to mitigate cross-resistance, a new drug pocket named Dimer Interface Pocket was discovered and a novel AR antagonist M17-B15 was identified. M17-B15 showed strong in vitro efficacy against PCa but had poor pharmacokinetic properties in vivo. In this study, through rational design and structure-activity relationship exploration, a series of thiadiazoleamide derivatives represented by N29 (IC50 = 0.018 μM) were identified with dominant AR antagonistic activity and remarkable anti-PCa activity in vitro. Furthermore, N29 effectively inhibited a series of typical drug-resistant AR mutants. The improved oral bioavailability of N29 facilitated its efficacy via oral administration, significantly inhibiting LNCaP xenograft tumor in vivo, presenting a promising therapeutic application for PCa.

Discovery of a Series of Orally Bioavailable Androgen Receptor Degraders for the Treatment of Prostate Cancer.

Androgen receptor (AR) signaling plays a key role in the progression of prostate cancer. This study describes the discovery and optimization of a novel series of AR PROTAC degraders that recruit the Cereblon (CRBN) E3 ligase. Having identified a series of AR ligands based on 4-(4-phenyl-1-piperidyl)-2-(trifluoromethyl)benzonitrile, our PROTAC optimization strategy focused on linker connectivity and CRBN ligand SAR to deliver potent degradation of AR in LNCaP cells. This work culminated in compounds 11 and 16 which demonstrated good rodent oral bioavailability. Subsequent SAR around the AR binding region brought in an additional desirable feature, degradation of the important treatment resistance mutation L702H. Compound 22 (AZ'3137) possessed an attractive profile showing degradation of AR and L702H mutant AR with good oral bioavailability across species. The compound also inhibited AR signaling in vitro and tumor growth in vivo in a mouse prostate cancer xenograft model.

Mutant androgen receptor induces neurite loss and senescence independently of ARE binding in a neuronal model of SBMA

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite outgrowth, induced neurite retraction, and inhibited neurite regrowth. This dysfunction was independent of polyQ-expanded AR transcriptional activity at androgen response elements (ARE). We further showed that the formation of polyQ-expanded AR intranuclear inclusions promoted neurite retraction, which coincided with reduced expression of the neuronal differentiation marker β-III-Tubulin. Finally, we revealed that cell death is not the primary outcome for cells undergoing neurite retraction; rather, these cells become senescent. Our findings reveal that mechanisms independent of AR canonical transcriptional activity underly neurite defects in a cell model of SBMA and identify senescence as a pathway implicated in this pathology. These findings suggest that in the absence of a role for AR canonical transcriptional activity in the SBMA pathologies described here, the development of SBMA therapeutics that preserve this activity may be desirable. This approach may be broadly applicable to other polyglutamine diseases such as Huntington's disease and spinocerebellar ataxias.

UBX-390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer.

The androgen receptor (AR) is an attractive target for treating prostate cancer, considering its role in the development and progression of localized and metastatic prostate cancer. The high global mortality burden of prostate cancer, despite medical treatments such as androgen deprivation or AR antagonist therapy, highlights the need to explore alternative strategies. One strategy involves the use of heterobifunctional degraders, also known as proteolysis-targeting chimeras, which are novel small-molecule therapeutics that inhibit amplified or mutated targets. Here, the study reports a novel cereblon-based AR degrader, UBX-390, and demonstrates its superior activity over established AR degraders, such as ARV-110 or ARCC-4, in prostate cancer cells under short- and long-term treatment conditions. UBX-390 suppresses chromatin binding and gene expression of AR and demonstrates substantial efficacy in the degradation of AR mutants in patients with treatment-resistant prostate cancer. UBX-390 is presented as an optimized AR degrader with remarkable potential for treating castration-resistant prostate cancer.

Reduced mesencephalic astrocyte-derived neurotrophic factor expression by mutant androgen receptor contributes to neurodegeneration in a model of spinal and bulbar muscular atrophy pathology.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by extended CAG trinucleotide repeats in the androgen receptor (AR) gene, which encodes a ligand-dependent transcription factor. The mutant AR protein, characterized by polyglutamine expansion, is prone to misfolding and forms aggregates in both the nucleus and cytoplasm in the brain in SBMA patients. These aggregates alter protein-protein interactions and compromise transcriptional activity. In this study, we reported that in both cultured N2a cells and mouse brain, mutant AR with polyglutamine expansion causes reduced expression of mesencephalic astrocyte-derived neurotrophic factor (MANF). Overexpression of MANF ameliorated the neurotoxicity of mutant AR through the inhibition of mutant AR aggregation. Conversely, knocking down endogenous MANF in the mouse brain exacerbated neuronal damage and mutant AR aggregation. Our findings suggest that inhibition of MANF expression by mutant AR is a potential mechanism underlying neurodegeneration in SBMA.

Female cichlids mate with novel androgen receptor mutant males that lack coloration

A key challenge in animal behavior is disentangling the social stimuli that drive conspecific behaviors. For some species, like teleost fish, putative sexual signaling cues are inextricably linked to others, making it difficult to parse the precise roles distinct signals play in driving conspecific behaviors. In the African cichlid Astatotilapia burtoni, males are either dominant or subordinate, wherein bright coloration, territoriality, and courtship behavior inextricably correlate positively with rank. Here, we leveraged androgen receptor (AR) mutant male A. burtoni that lack dominance-typical coloration but not behavior to isolate the role of male coloration in driving female mating behaviors in this species. We found in independent behavioral assays that females behave aggressively towards AR mutant but not WT males, yet still mated with both types of males. Females showed enhanced activation of esr2b + cells in the hypothalamus when housed with either mutant or WT males and this activation scaled with spawning activities. Therefore, there is not a simple relationship between male coloration and female mating behaviors in A. burtoni, suggesting independent sensory mechanisms converge on hypothalamic esr2b cells to coordinate behavioral output.

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 LB178: HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of all breast carcinomas and has poor prognosis. Chemotherapy is the mainstay treatment for TNBC patients, yet with limited clinical benefit. Androgen receptor (AR) is expressed in approximately 50% of TNBC tumors and thus the anti-androgen therapy could be a promising solution for the treatment of AR+ TNBC. Nevertheless, AR inhibitors including enzalutamide and bicalutamide or the androgen biosynthesis inhibitor abiraterone have failed to demonstrate satisfying anti-tumor activity in clinical trials. We have discovered HP518, an AR-targeting PROTAC (proteolysis targeting chimera) that induces potent degradation of both the wild-type AR and AR mutants. HP518 is currently under the clinical development for the treatment of mCRPC. Here we report the preclinical data on HP518 for the treatment of AR+ TNBC. HP518 robustly degrades AR in multiple TNBC cell lines with a half-maximal degradation concentration (DC50) < 1 nM. HP518 strongly inhibits AR+ TNBC cell growth, whereas the AR inhibitor enzalutamide does not show significant anti-proliferative effect on these cells in the presence or absence of androgen. Consistent with the in vitro results, HP518 significantly and dose-dependently inhibits tumor growth in MDA-MB-453 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models. PI3KCA activating mutations are frequently expressed in AR+ TNBC. HP518 in combination with alpelisib, a PI3Kα inhibitor shows a synergistic anti-tumor activity. Mechanistically, HP518, but not AR inhibitor enzalutamide downregulates the mRNA expression levels of a set of DNA replication-related genes in AR+ TNBC cells, indicating a novel and ligand-independent role for AR in regulating growth of AR+ TNBC cells. Therefore, HP518, an AR-targeting PROTAC degrader holds great potential to fulfill the unmet clinical needs for the AR+ TNBC therapy. Selected pre-clinical data will be presented. HP518 structure will not be disclosed. Citation Format: Jing Li, Hongwei Yuan, Kefan Chen, Yongxu Huo, Guoqing Liu, Wu Du, Xinghai Li. HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action [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 LB178.

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

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

Abstract 4487: Simple modifications of 3-HPT for improved in-vivo activtiy

Abstract One of the key targets in the treatment of castration resistant prostate cancer is HDAC6. HDAC6 removes acetyl groups from Heat shock protein 90 (HSP90), which is a protein chaperone that folds the androgen receptor. By inhibiting HDAC6 it is possible to prevent the expression of mutated androgen receptors, that are no longer responsive to androgen deprivation therapy, treating castration resistant prostate cancer. The Oyelere lab has previously demonstrated that 3-Hydroxypyridin-2-thione (3-HPT) can serve as a scaffold for HDAC6 inhibitors. While the unaltered 3-HPT has been shown on its own to inhibit HDAC6 in enzyme specific assays. The unaltered 3-HPT had no effect in whole cell assays. This work investigated simple modification of the 3-HPT scaffold to improve the in-vivo activity of 3-HPT. This work is an extension of work previously done in the Oyelere lab. The aim of this work is to apply simple modifications to the 3-HPT scaffold to improve the in-vitro activity. Previously 3-HPT was shown on its own to inhibit HDAC6 with an IC50 of 681nM but have no effect on cell assays. The hypothesis was that by converting the secondary amine into a tertiary amine the pharmacokinetic-properties of 3-HPT allowing can be improved focusing on cellular entry. The specific modification was the addition of alkanes and fluorinated alkanes at the secondary amine. This addition should improve the charge of the compounds improving the invitro activity. Based on molecular docking studies using Autodock vina, the introduction of fluorines improves the binding to HDAC6 by interacting with a hydrophilic pocket near the zinc ion. The preliminary in-vivo results in LnCAP and DU145 cells demonstrate that the addition of fluorinated alkanes has substantially improved the IC50 compared to the base 3-HPT compound. The base 3-HPT compound has an IC50 of greater than 100µM while the compounds with fluorinated alkanes have IC50 values in the range of 2-15µM. this data indicates improved drug likeness. Preliminary IC50 values of compounds in prostate cancer cell lines Compound Name LNCaPIC50 (µM) DU145IC50 (µM) Vero IC50 (µM) SH-1-78 >100 >100 >100 SH-1-94 2.50 19.29 22.53 RK-1-187 96.95 95.45 88.57 SH-1-110 1.27 21.47 17.41 RK-2-16 2.09 62.86 18.76 RK-2-17 >100 >100 >100 RK-2-64 78.36 >100 76.45 RK-2-67 14.19 26.95 11.38 Citation Format: Ryan E. Kern, Susanna Huang, Uche Arunsi, Adegboyega Oyelere. Simple modifications of 3-HPT for improved in-vivo activtiy [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 4487.

Discovery of Novel Anti-Resistance AR Antagonists Guided by Funnel Metadynamics Simulation.

Androgen receptor (AR) antagonists are widely used for the treatment of prostate cancer (PCa), but their therapeutic efficacy is usually compromised by the rapid emergence of drug resistance. However, the lack of the detailed interaction between AR and its antagonists poses a major obstacle to the design of novel AR antagonists. Here, funnel metadynamics is employed to elucidate the inherent regulation mechanisms of three AR antagonists (hydroxyflutamide, enzalutamide, and darolutamide) on AR. For the first time it is observed that the binding of antagonists significantly disturbed the C-terminus of AR helix-11, thereby disrupting the specific internal hydrophobic contacts of AR-LBD and correspondingly the communication between AR ligand binding pocket (AR-LBP), activation function 2 (AF2), and binding function 3 (BF3). The subsequent bioassays verified the necessity of the hydrophobic contacts for AR function. Furthermore, it is found that darolutamide, a newly approved AR antagonist capable of fighting almost all reported drug resistant AR mutants, can induce antagonistic binding structure. Subsequently, docking-based virtual screening toward the dominant binding conformation of AR for darolutamide is conducted, and three novel AR antagonists with favorable binding affinity and strong capability to combat drug resistance are identified by in vitro bioassays. This work provides a novel rational strategy for the development of anti-resistant AR antagonists.

Differentially disrupted spinal cord and muscle energy metabolism in spinal and bulbar muscular atrophy.

Prior studies showed that polyglutamine-expanded androgen receptor (AR) is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of nicotinamide adenine dinucleotide-dependent (NAD+-dependent) sirtuin 1 is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating postsymptomatic transgenic SBMA mice with the NAD+ precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of NAD+ in the spinal cords of SBMA mice. Metabolomic and proteomic profiles of SBMA quadriceps muscles indicated alterations in several important energy-related pathways that use NAD+, in addition to the NAD+ salvage pathway, which is critical for NAD+ regeneration for use in cellular energy production. We also observed decreased mRNA levels of nicotinamide riboside kinase 2 (Nmrk2), which encodes a key kinase responsible for NR phosphorylation, allowing its use by the NAD+ salvage pathway. Together, these data suggest a model in which NAD+ levels are significantly decreased in muscles of an SBMA mouse model and intransigent to NR supplementation because of decreased levels of Nmrk2.

Structural Development of Androgen Receptor Antagonists Using Phenylferrocene Framework as a Hydrophobic Pharmacophore.

We previously identified nitrophenylferrocenes and cyanophenylferrocenes as promising lead structures of novel androgen receptor (AR) antagonists, based on the structural similarity between ferrocene and the steroidal skeleton. In the present research, we explored the structure-activity relationship (SAR) of phenylferrocene derivatives. Introduction of a hydrophobic substituent such as a chlorine atom at the 2-position or 3-position of phenylferrocene derivatives significantly increased the antagonistic activity toward wild-type AR, and among the synthesized compounds, 3-chloro-4-cyanophenylferrocene (29) exhibited the most potent anti-proliferative activity toward the androgen-dependent growth of SC-3 cells expressing wild-type AR (IC50 14 nM). Like conventional antiandrogens such as hydroxyflutamide, the major active metabolite of flutamide, compound 29 exhibited agonistic activity toward T877A-AR, a mutant AR expressed in human prostate cancer cell line LNCaP. Notably, however, the 2-chloro isomer 27 showed potent antagonistic activity toward wild-type AR (IC50 49 nM) and also exhibited antagonistic activity toward T877A-AR. Our SAR data should prove helpful for the development of new-generation AR antagonists based on phenylferrocene as candidate agents to treat drug-resistant prostate cancer.

First-in-human phase 1 study of CC-94676, a first-in-class androgen receptor (AR) ligand-directed degrader (LDD), in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC).

134 Background: Androgen receptor (AR) signaling is the principal driver of PC at all stages. CC-94676 (BMS-986365) is a heterobifunctional, first-in-class, orally bioavailable AR LDD designed to induce rapid, sustained, and highly selective AR degradation in pts who progressed on standard of care therapies. We report initial results from an open-label, multicenter study, NCT04428788, evaluating CC-94676 in pts with progressive mCRPC. Methods: Pts with mCRPC who progressed on androgen deprivation therapy, ≥ 1 second generation hormonal therapy (eg, enzalutamide [enza], abiraterone [abi], darolutamide, and apalutamide) and taxane chemotherapy (chemo) (unless refused or not indicated) were enrolled to evaluate the safety, tolerability, PK/PD, and preliminary efficacy of CC-94676. Escalation doses evaluated were 100–1200 mg QD and 600–900 mg BID; expansion doses were 600 mg QD and 400–900 mg BID. Results: As of Aug 21, 2023, 95 pts received CC-94676 (median age 71 yrs) with a median of 5 (range 2–12) prior therapies, including enza (80%), abi (72%), both enza & abi (56%), and chemo (56%) (docetaxel 55%; cabazitaxel 20%). There were no ≥ Grade (G) 4 treatment-related adverse events (TRAEs) or discontinuations due to TRAEs. Of 27 pts treated in escalation, treatment (Tx) was well tolerated; 2 pts had non-serious G3 TRAEs at doses ≥ 800 mg QD, which were manageable with dose modifications. One dose-limiting toxicity of asymptomatic QTc prolongation was observed at 900 mg BID and resolved with dose interruption. The maximum tolerated dose was not reached. Of 68 pts treated in expansion, the most common TRAEs were dose-dependent: QTc prolongation (asymptomatic) (43%; G3: 9%), bradycardia (31%; ≥G3: none), and fatigue (24%; ≥G3: none). G3 TRAEs were manageable with dose modifications. The rate of pts with confirmed PSA reductions ≥ 30% (PSA30) increased dose-dependently from 400 to 900 mg BID. 23/68 (34%) pts across all dose levels achieved ≥ PSA30. At 900 mg BID, 11/20 (55%) pts showed PSA30; 7/20 (35%) and 2/20 (10%) had confirmed PSA declines of ≥50% and ≥90%, respectively. PSA responses and radiographic tumor shrinkage occurred across all dose levels, including in pts with AR wildtype (WT) , amplifications, and mutations (by cfDNA), and in heavily pretreated pts who progressed on abi, enza, and chemo. At 900 mg BID, the median duration of Tx was 182 days (range 21-448) and 9/20 (45%) pts remained free of radiographic progression with treatment ongoing at 6 months. Conclusions: CC-94676 is well tolerated with a manageable safety profile. CC-94676 shows promising and prolonged clinical activity in heavily pretreated mCRPC pts who progressed on abi, enza, and chemo with activity seen in pts with tumors expressing WT and mutant ARs. Selection of the recommended phase 2 dose is ongoing. Clinical trial information: NCT04428788 .

In silico comparison between the mutated and wild-type androgen receptors and their influence on the selection of optimum androgenic receptor blockers for the treatment of prostate cancer

Background: Prostate cancer is a disease that occurs in men aged more than 50 years. In Iraq, 8.89 men per 100,000 population suffer from prostate cancer, with the incidence being 14,016 cases and mortality being 6,367 cases. Despite advances in treatment against prostate cancer, it can become resistant to drugs. Therefore, the aim of current study was to search and identify binding sites for the repositioning of drugs by computational methods (docking). Methods: Based on the protein structure of the wild androgen receptor, the analysis parameters (22x22x22 on the X, Y, and Z axes) were established. Results: The interactions of the natural ligands with androgen receptor were 10.0 (testosterone) and 10.8 (dihydrotestosterone) while mutated androgen receptor (T877A) had a low affinity with testosterone and dihydrotestosterone (-5.3 and -6.7, respectively). In the interactions of both receptors with the reported inhibitors (antagonists), a decrease with Bicalutamide (-8.3 and -4.3, respectively) and an increase in affinity with Flutamide and Nilutamide (-7.7 and 8.6, wild AR; -8.7 and -9.3 AR T877A) were observed. As for Enzalutamide and Apalutamide (second-generation antagonists), the change was minimal between wild androgen receptor and T877A (-7.6 and -7.7; -7.3 and -7.3, respectively). The change in the affinity of the ligands with androgen receptor and androgen receptor T877A shows how a mutation alters the bonds between these molecules. Conclusion: The identification of key sites and potent inhibitors against abnormal androgen receptor functions will enrich prostate cancer treatments.

Low androgen signaling rescues genome integrity with innate immune response by reducing fertility in humans

Development of the gonads under complex androgen regulation is critical for germ cells specification. In this work we addressed the relationship between androgens and genomic integrity determining human fertility. We used different study groups: individuals with Differences of Sex Development (DSD), including Complete Androgen Insensitivity Syndrome (CAIS) due to mutated androgen receptor (AR), and men with idiopathic nonobstructive azoospermia. Both showed genome integrity status influenced by androgen signaling via innate immune response activation in blood and gonads. Whole proteome analysis connected low AR to interleukin-specific gene expression, while compromised genome stability and tumorigenesis were also supported by interferons. AR expression was associated with predominant DNA damage phenotype, that eliminated AR-positive Sertoli cells as the degeneration of gonads increased. Low AR contributed to resistance from the inhibition of DNA repair in primary leukocytes. Downregulation of androgen promoted apoptosis and specific innate immune response with higher susceptibility in cells carrying genomic instability.

Calcium Activation of the Androgen Receptor in Prostate Cells.

Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor. The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured. To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels. The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

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