Antiandrogen Research Articles

Abstract PS11-07: High prevalence of HER2-Low and AR expression in breast cancer brain metastases provide novel therapeutic options

Abstract Background: Although survival rates for patients with advanced breast cancer (BC) have improved overall, rates of BC brain metastases (BCBM) have increased over the past two decades, occurring in up to 15% to 30% of patients. BCBM greatly reduce quality of life and overall survival (OS) and are now the leading cause of BC patient mortality. Patients with primary HER2+ and triple negative BC (TNBC) are at highest risk for BM. Locoregional therapy with surgery and radiation are currently standard of care (SOC), but these treatments have significant morbidity and BCBM progressing despite these interventions are hard to treat. Systemic therapies to reduce cognitive impairment and increase survival are desperately needed, particularly in HER2-negative patients. Currently, immune checkpoint inhibitors (ICIs) and small molecules such as PARP inhibitors are among the limited systemic options that have BCBM activity in HER2-negative BC. However, the benefit of these therapies is limited to a select group of patients. For example, androgen receptor (AR) is expressed in 60-80% of all BC and, in our recently completed clinical trial (NCT03206203), patients with AR+ metastatic TNBC treated with carboplatin +/- atezolizumab received no clinical benefit from the addition of atezolizumab. Alternatives to ICIs are needed for patients with AR+ TNBC. HER2-low (H2L) BC is a newly defined subset of HER2-negative BC with a HER2 immunohistochemical (IHC) score of 1+ ( >10%) or 2+/in situ hybridization (ISH) negative phenotype. Recent clinical trials have demonstrated significant clinical benefit from novel HER2 antibody-drug conjugates (ADCs) in treating H2L BC. Trastuzumab-deruxtecan (T-Dxd), a HER2-directing ADC (topoisomerase I inhibitor conjugated to a humanized HER2-targeted antibody) was recently FDA approved as the first targeted therapy to treat H2L BC and can penetrate the blood–brain barrier. DESTINY-Breast04 demonstrated notable PFS and OS benefit of T-DXd compared to SOC in patients with H2L metastatic BC and included 61 patients with BCBM (results anticipated shortly). The overall rates of AR and H2L expression in patients with BCBM is unknown. The goal of this study is to investigate the prevalence of AR and HER2-low expression in a large cohort of BCBM treated at our institution. Methods: We retrospectively assessed rates of H2L and AR positivity (AR+) among patients with BCBM whose treatment included surgery at our institution between 2005-2023. Results were correlated with clinical receptor status (HR+/HER2-, HER2+/HR+, HER2+/HR- and TNBC) and clinical characteristics. Results of ER, PR, and HER2 testing performed for clinical purposes on the brain metastases and primary tumors were obtained from pathology reports. We retrieved formalin-fixed paraffin-embedded tissue blocks of the brain metastases and performed IHC for AR (SP107) and prognostic markers for cases with missing data. Results: We identified 101 cases of BCBM. Based on clinical prognostic markers, BCBM subtypes were HR+/HER2- (22%), HR+/HER2+ (14%), HER2+/HR- (21%) and TNBC (43%). Forty-five percent of the BCBM (n=45) were AR+ ( >10%) and 24% (n=24) demonstrated > 50% AR+. Among tumors with >50% AR+, 42% (n=10) are H2L (7 HR+[30%] and 3 TNBC [13%]). Forty-two percent of BCBM were H2L. Among the HER2-negative BCBM (n= 66), 64% (n=42) were H2L, including 23 of 44 TNBC (52%) and 19 of 22 HR+ (86)%. Fifteen percent of BMBC changed clinical subtype from the primary, including conversion to TNBC and H2L. Conclusion: AR+ positive (24% > 50% expression) and HER2-Low expressing (42%) tumors represent a significant proportion of our BCBM cohort providing exciting new options for treatment. Trials re-evaluating AR antagonists in combination with SOC utilizing high AR+ thresholds may benefit patients with BCBM. At least 10% of BCBM are both H2L and demonstrate >50% AR positivity providing the rationale for the combination of ADCs such as T-DXd and AR-targeted therapies for BCBM. Citation Format: Guadalupe Garcia, Paula Gonzalez-Ericsson, Brian Lehmann, Bret Mobley, Jennifer Pietenpol, Laura Kennedy, Ben Park, Melinda Sanders. High prevalence of HER2-Low and AR expression in breast cancer brain metastases provide novel therapeutic options [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-07.

Abstract PO5-05-09: Testing AI-Predicted Protein Motifs that Direct Constitutive Genomic AR Activity in Endocrine Resistant Breast Cancer

Abstract Testing AI-Predicted Protein Motifs that Direct Constitutive Genomic AR Activity in Endocrine-Resistant Breast Cancer Ashfia F. Khan 1,2, Anthony S. Peidl 1,2, Shaymaa Bahnassy 3, Henry Vo2, Micah B. Castillo 2, Sarah K Herzog4,5, Suzanne AW Fuqua4,6, Preethi Gunaratne 2, Xiaolian Gao 2, Subash C. Pakhrin7, Tasneem Bawa-Khalfe1,2 1 Center for Nuclear Receptors & Cell Signaling, University of Houston, Houston, TX 2 Department of Biology & Biochemistry, University of Houston, Houston, TX 3 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 4 Lester & Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 5 Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 6 Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 7 Department of Computer Science & Engineering Technology, University of Houston-Downtown, Houston, TX Background: Endocrine therapy (ET) remains the first-line treatment for hormone-receptor positive (HR+) breast cancer (BCa). Approximately 15–20% of HR+ BCa are intrinsically resistant to ET, and 30–40% of patients acquire resistance. Resistance to ET (ET-R) supports cancer progression with reduced disease-free survival and greater incidence of metastatic disease. Hence, therapeutic strategies for ET-R HR+ BCa remain an overarching challenge. The androgen receptor (AR) is emerging as an attractive alternative target for BCa subtypes, and elevated AR levels drive HR+ BCa progression. Targeting AR in HR+ BCa is proving difficult with preclinical studies showing conflicting results for AR antagonists. Yet clinical trials with several AR-targeting drugs are ongoing. Our recent report highlights a unique constitutively active modified AR population that drives HR+ BCa metastatic properties and is insensitive to AR inhibitors. Our current objectives are to 1) use a novel machine-learning model to predict AR modifications and 2) establish a strategy to identify patients with high modified AR levels. Methods: An advanced artificial intelligence (AI) tool and mid-throughput microfluidic peptide array technology were used to map modification domains on AR. SUMO post-translational modification of AR (SUMO-AR) was eliminated in HR+ BCa using CRISPR-Cas9 technology. RNA-seq was employed to identify a unique gene signature for SUMO-AR, and comparative bioinformatic analysis stratified patients with high versus low SUMO-AR. Results: A novel deep-learning AI platform SumoPred-PLM is trained to identify consensus, non-consensus, and SUMO2/3-specific motifs on AR. We verified SUMO2/3-specific sites on AR with a mid-throughput microfluidic peptide array. The identified SUMO2/3-acceptor site of AR is important for HR+ BCa cell pathophysiology; loss of this SUMO2/3-acceptor site impacts endogenous AR SUMOylation, cell morphology, and proliferation/apoptosis. Using both high and low SUMO-AR BCa lines, a unique SUMO-AR gene profile was established. Our SUMO-AR gene signature identifies HR+ BCa patients with greater susceptibility to metastatic progression. Conclusion: Our studies present a unique pipeline that incorporates deep-learning AI technology to identify vulnerable motifs in AR for future drug discovery. Drug screens are currently ongoing. In addition, we establish a SUMO-AR gene signature that stratifies HR+ BCa patients with high/low SUMO-AR and predicts disease progression. We expect the results could be utilized to identify responders to AR inhibitors in ongoing clinical trials. Citation Format: Ashfia Khan, Anthony Peidl, Shaymaa Bahnassy, Henry Vo, Micah Castillo, Sarah Herzog, Suzanne Fuqua, Preethi Gunaratne, Xiaolian Gao, Subash Pakhrin, Tasneem Bawa-Khalfe. Testing AI-Predicted Protein Motifs that Direct Constitutive Genomic AR Activity in Endocrine Resistant Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-09.

Abstract PS15-07: Proxalutamide plus Endocrine Therapies in Women with HR+/HER2-/AR+ Metastatic Breast Cancer: A Phase Ic Study

Abstract Abstract Background: Resistance to endocrine therapy (ET) and cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) is a major obstacle to the management of hormone-receptor (HR)+/human epidermal growth factor receptor 2 (HER2)- metastatic breast cancer (mBC). Targeting the AR signaling pathway has been demonstrated with promising results in this population. This phase Ic study aimed to assess the safety, efficacy, pharmacokinetic (PK), and pharmacodynamic (PD) characteristics of the combination of proxalutamide, a high-affinity AR antagonist, with ETs in androgen receptor (AR)+/HR+/HER2- mBC. Methods: Patients in part 1 regimen-finding phase received proxalutamide plus specific ETs (letrozole [cohort A], exemestane [cohort B], or fulvestrant [cohort C]) and were assessed for dose-limiting toxicity (DLT), PK, PD, and anti-tumor activity. Part 2 expansion phase evaluated the safety and efficacy of proxalutamide plus fulvestrant. The primary endpoint was safety and tolerability. Results: Between June 18, 2019, and Sep 15, 2022, 38 (18 in part 1 and 20 in part 2) patients were treated. Efficacy analysis indicated that the ORR was 16.7% (2/12, 95% CI, 2.1%-48.4%) for cohort C and 15.0% (3/20; 95% CI, 3.2%-37.9%) for part 2. Patients achieved a median progression-free survival (PFS) of 6.4 months (95% CI, 2.7-19.3) in cohort C while an mPFS of 11.0 months (95% CI, 5.5- not estimable) in part 2. In part 1, no DLTs were reported. Grade 3 or more treatment-emergent adverse events (TEAEs) were observed in 11 (29.7%) patients (7 [18.9%] in part 1 and 4 [10.8%] in part 2) and mainly included neutrophil count decreased (8.1%) and platelet count decreased (5.4%). Seven (18.9%) had serious AEs (4 [10.8%] in part 1 and 3 [8.1%] in part 2)[1]. PD results showed a greater reduction of estradiol in cohort C compared with that in cohort B. In addition, proxalutamide was absorbed rapidly into the body with the plasma concentrations of proxalutamide and metabolites reaching a nearly steady state on day 29. Patients with AR/ER of ≤1 seemed to achieve longer PFS over those of >1 (8.4 months vs. 4.1 months). Conclusions: These findings suggested favorable clinical outcomes and safety profiles of the combination of proxalutamide and fulvestrant in AR+/HR+/HER2- mBC patients who have progressed on the first-line therapy, and maybe with better efficacy in patients with lower AR/ER ratio. Trial registration: NCT20191063 Citation Format: Hui-Ping Li, Guohong Song, Hanfang Jiang, Xu Liang, Xiaojia Wang, Hua Yang, Lili Zhang, Youzhi Tong. Proxalutamide plus Endocrine Therapies in Women with HR+/HER2-/AR+ Metastatic Breast Cancer: A Phase Ic Study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS15-07.

Low Contact Strategy to Measure Flutamide’s Effects on Territorial Aggression in C57BL/6J Male Mice

While numerous factors contribute to mouse behavior, biological drivers are paramount to many regulatory processes that undergird behavior. In particular, the sex hormone testosterone plays a key role in the regulation of aggression, physical activity, mating, and other behaviors known to be influenced by the presence of an experimenter. Due to this experimenter bias, strategies that minimize direct and frequent contact with research animals are needed to reduce any undesirable effects. The purpose of this research was to explore the effects of an androgen receptor antagonist (Flutamide) on territorial aggression in male C57BL/6J mice utilizing a low contact technique for chemical delivery. It was hypothesized that the introduction of an androgen receptor inhibiting substance under a low contact delivery strategy would result in effective reduction of the territorial aggression response in mice introduced to an unknown male mouse in the home cage environment, mimicking similar blunting responses characterized in previous work evaluating aggression in rodents. Silastic (Dow Corning, Midland, MI) implants were used as the lower contact delivery strategy for drug administration. This strategy allowed minimal contact with the experimental technician and eliminated the need for daily contact required for the commonly used injection strategies employed elsewhere. A series of aggression trials before, 24, 96, and 192 hours after Flutamide implantation were conducted in three groups of mice. Control mice (n=4) received 100% Cholesterol-containing implants, low exposure mice (n=5) received 80% Cholesterol/20% Flutamide-containing implants, and high exposure mice (n=5) received 100% Flutamide-containing implants. Each aggression trial consisted of introducing a foreign mouse to an experimental mouse’s home cage for an 8-minute period. The time to initial aggression and the total number of aggressions over the 8 minutes was quantified. The time to initial aggression was unaffected by flutamide administration throughout the study [F(26,91) =0.37, p=0.864]. The total number of aggressions were significantly affected by flutamide exposure [F(28,82) = 2.90, p=0.029]. Follow-up post hoc testing indicated a significant reduction in aggression in both the low and high dose groups 24 (low: p = 0.001; high = 0.001) and 96 (low: p = 0.009; high = 0.012) hours after implantation compared to the control at the same time points. The use of Silastic implants to deliver the anti-androgenic compound was a valuable technique that reduced research animal contact with experimental technicians. This technique can be modified for prolonged exposure by lengthening the implant or increasing the diameter of the Silastic tubing. This anti-androgen exposure methodology requiring less interaction reduces the confounding variables that impact mouse behavior and could prove beneficial to observe and study other androgen-related behaviors including physical activity and mating. All funding for this research was provided by the Union University Department of Biology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

ACSM1 and ACSM3 Regulate Fatty Acid Metabolism to Support Prostate Cancer Growth and Constrain Ferroptosis.

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared with nonmalignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, whereas silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation, and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function of medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance. Significance: Androgen receptor-induced ACSM1 and ACSM3 mediate a metabolic pathway in prostate cancer that enables the utilization of medium chain fatty acids for energy production, blocks ferroptosis, and drives resistance to clinically approved antiandrogens.

Amino Terminal Acetylation of HOXB13 Regulates the DNA Damage Response in Prostate Cancer.

Advanced localized prostate cancers (PC) recur despite chemotherapy, radiotherapy and/or androgen deprivation therapy. We recently reported HOXB13 lysine (K)13 acetylation as a gain-of-function modification that regulates interaction with the SWI/SNF chromatin remodeling complex and is critical for anti-androgen resistance. However, whether acetylated HOXB13 promotes PC cell survival following treatment with genotoxic agents is not known. Herein, we show that K13-acetylated HOXB13 is induced rapidly in PC cells in response to DNA damage induced by irradiation (IR). It colocalizes with the histone variant γH2AX at sites of double strand breaks (DSBs). Treatment of PCs with the Androgen Receptor (AR) antagonist Enzalutamide (ENZ) did not suppress DNA-damage-induced HOXB13 acetylation. In contrast, HOXB13 depletion or loss of acetylation overcame resistance of PC cells to ENZ and synergized with IR. HOXB13K13A mutants show diminished replication fork progression, impaired G2/M arrest with significant cell death following DNA damage. Mechanistically, we found that amino terminus regulates HOXB13 nuclear puncta formation that is essential for proper DNA damage response. Therefore, targeting HOXB13 acetylation with CBP/p300 inhibitors in combination with DNA damaging therapy may be an effective strategy to overcome anti-androgen resistance of PCs.

Novel selective agents for the degradation of AR/AR-V7 to treat advanced prostate cancer

The androgen receptor AR antagonists, such as enzalutamide and apalutamide, are efficient therapeutics for the treatment of prostate cancer (PCa). Even though they are effective at first, resistance to both drugs occurs frequently. Resistance is mainly driven by aberrations of the AR signaling pathway including AR gene amplification and the expression of AR splice variants (e.g. AR-V7). This highlights the urgent need for alternative therapeutic strategies. Here, a total of 24 compounds were synthesized and biologically evaluated to disclose compound 20i, exhibiting potent AR antagonistic activities (IC50 = 172.85 ± 21.33 nM), promising AR/AR-V7 protein degradation potency, and dual targeting site of probably AR (ligand-binding domain, LBD and N-terminal domain, NTD). It potently inhibits cell growth with IC50 values of 4.87 ± 0.52 and 2.07 ± 0.34 μM in the LNCaP and 22RV1 cell lines, respectively, and exhibited effective tumor growth inhibition (TGI = 50.9 %) in the 22RV1 xenograft study. These data suggest that 20i has the potential for development as an AR/AR-V7 inhibitor with degradation ability to treat advanced prostate cancer.

Comparative Analysis of CDK4/6 Inhibitors (Ribociclib and Palbociclib) combined with Enzalutamide in Triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is recognized as a challenging subtype due to its poor prognosis. Recent molecular profiling studies have unveiled a significant subset expressing the androgen receptor (AR) subset which may respond to AR-blocking agents, offering a potentially effective treatment strategy. This study aims to investigate the potential synergistic cytotoxic and apoptotic effects of the AR antagonist enzalutamide (ENZA) in combination with CDK4/6 inhibitors palbociclib (PB) or ribociclib (RB) and compare the effectiveness of these combinations in TNBC cells. Results revaled that ENZA in combination with PB or RB induced synergistic cytotoxic cytotoxicity in all tested TNBC cell lines. While synergistic cytotoxic combinations of ENZA with PB did not induce apoptosis in any TNBC cell line, ENZA+RB combinations exhibited a synergistic apoptotic effect. This study suggests that the ENZA+RB combination may be more favorable due to its apoptosis-inducing effect. However, these data need to be further supported by detailed in vivo and clinical studies

Manzamine A reduces androgen receptor transcription and synthesis by blocking E2F8-DNA interactions and effectively inhibits prostate tumor growth in mice.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

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.

Enhancing therapeutic efficacy in luminal androgen receptor triple-negative breast cancer: exploring chidamide and enzalutamide as a promising combination strategy

Extensive exploration of the molecular subtypes of triple-negative breast cancer (TNBC) is critical for advancing precision medicine. Notably, the luminal androgen receptor (LAR) subtype has attracted attention for targeted treatment combining androgen receptor antagonists and CDK4/6 inhibitors. Unfortunately, this strategy has proven to be of limited efficacy, highlighting the need for further optimization. Using our center’s comprehensive multiomics dataset (n = 465), we identified novel therapeutic targets and evaluated their efficacy through multiple models, including in vitro LAR cell lines, in vivo cell-derived allograft models and ex vivo patient-derived organoids. Moreover, we conducted flow cytometry and RNA-seq analysis to unveil potential mechanisms underlying the regulation of tumor progression by these therapeutic strategies. LAR breast cancer cells exhibited sensitivity to chidamide and enzalutamide individually, with a drug combination assay revealing their synergistic effect. Crucially, this synergistic effect was verified through in vivo allograft models and patient-derived organoids. Furthermore, transcriptomic analysis demonstrated that the combination therapeutic strategy could inhibit tumor progression by regulating metabolism and autophagy. This study confirmed that the combination of histone deacetylase (HDAC) inhibitors and androgen receptor (AR) antagonists possessed greater therapeutic efficacy than monotherapy in LAR TNBC. This finding significantly bolsters the theoretical basis for the clinical translation of this combination therapy and provides an innovative strategy for the targeted treatment of LAR TNBC.

The protein composition of exosomes released by prostate cancer cells is distinctly regulated by androgen receptor-antagonists and -agonist to stimulate growth of target cells.

Prostate cancer (PCa) is a prevalent malignancy in men worldwide, ranking as the second leading cause of cancer-related death in Western countries. Various PCa hormone therapies, such as androgen receptor (AR)-antagonists or supraphysiological androgen level (SAL) reduce cancer cell proliferation. However, treated cells may influence the growth of neighboring cells through secreted exosomes in the tumor microenvironment (TME). Here, the change of protein content of exosomes secreted from PCa cells through treatment with different AR-antagonists or SAL has been analyzed. Isolation of exosomes via ultracentrifugation of treated human PCa LNCaP cells with AR-agonist and various AR-antagonists; analysis of cellular senescence by detection of senescence associated beta galactosidase activity (SA β-Gal); Western blotting and immunofluorescence staining; Mass spectrometry (MS-spec) of exosomes and bioinformatic analyses to identify ligand-specific exosomal proteins. Growth assays to analyze influence of exosomes on non-treated cells. MS-spec analysis identified ligand-specific proteins in exosomes. One thousand seventy proteins were up- and 52 proteins downregulated by SAL whereas enzalutamide upregulated 151 proteins and downregulated 42 exosomal proteins. The bioinformatic prediction indicates an up-regulation of pro-proliferative pathways. AR ligands augment hub factors in exosomes that include AKT1, CALM1, PAK2 and CTNND1. Accordingly, functional assays confirmed that the isolated exosomes from AR-ligand treated cells promote growth of untreated PCa cells. The data suggest that the cargo of exosomes is controlled by AR-agonist and -antagonists and distinct among the AR-antagonists. Further, exosomes promote growth that might influence the TME. This finding sheds light into the complex interplay between AR signaling and exosome-mediated communication between PCa cells.

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.

Enzalutamide induces cytotoxicity in desmoplastic small round cell tumor independent of the androgen receptor

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, pediatric cancer caused by the EWSR1::WT1 fusion protein. DSRCT predominantly occurs in males, which comprise 80-90% of the patient population. While the reason for this male predominance remains unknown, one hypothesis is that the androgen receptor (AR) plays a critical role in DSRCT and elevated testosterone levels in males help drive tumor growth. Here, we demonstrate that AR is highly expressed in DSRCT relative to other fusion-driven sarcomas and that the AR antagonists enzalutamide and flutamide reduce DSRCT growth. However, despite these findings, which suggest an important role for AR in DSRCT, we show that DSRCT cell lines form xenografts in female mice at the same rate as male mice and AR depletion does not significantly alter DSRCT growth in vitro. Further, we find that AR antagonists reduce DSRCT growth in cells depleted of AR, establishing an AR-independent mechanism of action. These findings suggest that AR dependence is not the reason for male predominance in DSRCT and that AR-targeted therapies may provide therapeutic benefit primarily through an AR-independent mechanism that requires further elucidation.

Exploration of vitamin D metabolic activity-related biological effects and corresponding therapeutic targets in prostate cancer

BackgroundPrevious studies have unequivocally demonstrated that the vitamin D (VD) metabolism pathway significantly influences prognosis and sensitivity to hormone therapy in prostate cancer (PCa). However, the precise underlying mechanism remains unclear.MethodsWe performed molecular profiling of 1045 PCa patients, leveraging genes linked to VD synthesis and VD receptors. We then identified highly variable gene modules with substantial associations with patient stratification. Subsequently, we intersected these modules with differentially expressed genes between PCa and adjacent paracancerous tissues. Following a meticulous process involving single-factor regression and LASSO regression to eliminate extraneous variables and construct a prognostic model. Within the high-risk subgroup defined by the calculated risk score, we analyzed their differences in cell infiltration, immune status, mutation landscape, and drug sensitivity. Finally, we selected Apolipoprotein E (APOE), which featured prominently in this model for further experimental exploration to evaluate its potential as a therapeutic target.ResultsThe prognostic model established in this study had commendable predictive efficacy. We observed diminished infiltration of various T-cell subtypes and reduced expression of co-stimulatory signals from antigen-presenting cells. Mutation analysis revealed that the high-risk cohort harbored a higher frequency of mutations in the TP53 and FOXA genes. Notably, drug sensitivity analysis suggested the heightened responsiveness of high-risk patients to molecular inhibitors targeting the Bcl-2 and MAPK pathways. Finally, our investigation also confirmed that APOE upregulates the proliferative and invasive capacity of PCa cells and concurrently enhances resistance to androgen receptor antagonist therapy.ConclusionThis comprehensive study elucidated the potential mechanisms through which this metabolic pathway orchestrates the biological behavior of PCa and findings hold promise in advancing the development of combination therapies in PCa.

Anti-COVID-19 Potential of Withaferin-A and Caffeic Acid Phenethyl Ester.

The recent COVID-19 (coronavirus disease 2019) pandemic triggered research on the development of new vaccines/drugs, repurposing of clinically approved drugs, and assessment of natural anti-COVID-19 compounds. Based on the gender difference in the severity of the disease, such as a higher number of men hospitalized and in intense care units, variations in sex hormones have been predicted to play a role in disease susceptibility. Cell surface receptors (Angiotensin-Converting Enzyme 2; ACE2 and a connected transmembrane protease serine 2- TMPSS2) are upregulated by androgens. Conversely, androgen antagonists have also been shown to lower ACE2 levels, implying their usefulness in COVID-19 management. In this study, we performed computational and cell-based assays to investigate the anti- COVID-19 potential of Withaferin-A and Caffeic acid phenethyl ester, natural compounds from Withania somnifera and honeybee propolis, respectively. Structure-based computational approach was adopted to predict binding stability, interactions, and dynamics of the two test compounds to three target proteins (androgen receptor, ACE2, and TMPRSS2). Further, in vitro, cell-based experimental approaches were used to investigate the effect of compounds on target protein expression and SARS-CoV-2 replication. Computation and experimental analyses revealed that (i) CAPE, but not Wi-A, can act as androgen antagonist and hence inhibit the transcriptional activation function of androgen receptor, (ii) while both Wi-A and CAPE could interact with ACE2 and TMPRSS2, Wi-A showed higher binding affinity, and (iii) combination of Wi-A and CAPE (Wi-ACAPE) caused strong downregulation of ACE2 and TMPRSS2 expression and inhibition of virus infection. Wi-A and CAPE possess multimodal anti-COVID-19 potential, and their combination (Wi-ACAPE) is expected to provide better activity and hence warrant further attention in the laboratory and clinic.

Function-Oriented Synthesis of Pentacyclic Triterpenoids and Discovery of an ent-Estrane as a Natural Product-Inspired Androgen Receptor Antagonist.

While pentacyclic triterpenoids have a rich history in chemistry and biology, the challenges associated with their asymmetric synthesis contribute to the current reality that medicinal exploration in the area is largely constrained to natural product derivatization. To address this deficiency, a function-oriented synthesis of pentacyclic triterpenoids was pursued. Overall, we report a divergent synthesis of 26-norgermanicol and 26-norlupeol and we have identified a new class of androgen receptor antagonist that is ∼6× more potent than lupeol.

Developing a Novel Enzalutamide-Resistant Prostate Cancer Model via AR F877L Mutation in LNCaP Cells.

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival. However, scientific evidence suggests that sustained treatment with AR antagonists may induce acquired AR mutations or splice variants, such as AR F877L, T878A, and H875Y, leading to drug resistance and thereby diminishing the therapeutic efficacy of these agents. Thus, the establishment of prostate cancer models incorporating these particular mutations is essential for developing new therapeutic strategies to overcome such resistance and evaluate the efficacy of next-generation AR-targeting drugs. We have developed a CRISPR (clustered regularly interspaced short palindromic repeats)-based knock-in technology to introduce an additional F877L mutation in AR into the human prostate cell line LNCaP. This article provides comprehensive descriptions of the methodologies for cellular gene editing and establishment of an in vivo model. Using these methods, we successfully identified an enzalutamide-resistant phenotype in both in vitro and in vivo models. We also assessed the efficacy of target protein degraders (TPDs), such as ARV-110 and ARV-667, in both models, and the corresponding validation data are also included here. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Generation of AR F877L-mutated LNCaP cell line using CRISPR technology Basic Protocol 2: Validation of drug resistance in AR F877L-mutated LNCaP cell line using the 2D CTG assay Support Protocol: Testing of sgRNA efficiency in HEK 293 cells Basic Protocol 3: Validation of drug resistance in AR F877L-mutated LNCaP cell line in vivo.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.