AR-mediated Transcription Research Articles

Discovery of a Highly Potent PROTAC Degrader of p300/CBP Proteins for the Treatment of Enzalutamide-Resistant Prostate Cancer.

Prostate cancer therapies against androgen receptor (AR) eventually develop lethal resistance; thus, exploring new therapeutic approaches is urgent for prostate cancer treatment. Acetyltransferase p300/CBP are key coactivators for AR-mediated transcription and represent promising therapeutic targets to inhibit AR activity in prostate cancer. We describe the design synthesis and evaluation of a new class of p300/CBP PROTAC degraders. We identified an excellent p300/CBP degrader MJP6412, which effectively induced degradation of p300/CBP proteins, downregulated AR target genes, and inhibited cell growth of human prostate cancer cell lines and enzalutamide-resistant cells with IC50 even at nanomolar concentrations. Furthermore, MJP6412 demonstrated significant inhibition of tumor growth in a VCaP xenograft model. Collectively, MJP6412 is a promising lead compound for the treatment of prostate cancer, especially enzalutamide-resistant prostate cancer.

Examining the Effect of PARP-1 Inhibitors on Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells.

Since the early 1940s, androgen ablation has been the cornerstone of treatment for prostate cancer (PC). Importantly, androgen receptor (AR) signaling is vital not only for the initiation of PC, which is initially androgen-dependent, but also for castration-resistant disease. Recent studies demonstrated clear promise of the poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors for targeting prostate cancer cells harboring mutations in DNA damage-repair genes. In addition, it has been established that PARP-1 inhibition suppresses growth of AR-positive prostate cancer cells in cell and animal models. Thus, prostate cancer represents a particularly promising disease site for targeting PARP-1, given that both DNA repair and AR-mediated transcription depend on PARP-1 function. Here, we describe the development and use of cell-based assay to evaluate the impact of PARP-1 inhibitors on the AR signaling in prostate cancer cells.

Targeting the androgen receptor signaling pathway in advanced prostate cancer.

This article summarizes current androgen receptor (AR)-directed therapies that have received regulatory approval for the treatment of advanced prostate adenocarcinoma (herein referred to as prostate cancer, PC). PC is an androgen-dependent malignancy in which ligands including testosterone and dihydrotestosterone bind to AR, initiating androgen-AR complex translocation to the nucleus followed by AR-mediated transcription of target genes. Androgen deprivation therapy (ADT), including gonadotropin hormone-releasing hormone (GnRH) agonists with or without AR antagonists (antiandrogens), GnRH antagonists, or bilateral orchiectomy, forms the backbone of treatment for patients with metastatic castration-naive PC and/or castration-resistant PC (CRPC). ADT is also an option for high-risk, early-stage PC after prostatectomy and/or radiation. While ADT is often very effective as initial therapy, resistance ultimately develops despite suppression of gonadal and/or adrenal androgens, leading to CRPC, which is characterized by mechanisms such as reactivation of the AR signaling pathway, AR gene overexpression, and mutations in the ligand-binding domain of AR that lead to disease progression, resulting in increased symptom burden and ultimately death. However, disease in patients with CRPC is still dependent on androgen signaling, and these patients continue on ADT to maintain a castrate level of serum testosterone. Novel hormonal therapies including agents that target AR directly (eg, AR antagonists) are often added to ADT in this setting. Targeting the AR signaling pathway led to the development of second-generation AR antagonists, examples of which include enzalutamide, apalutamide, and darolutamide. These agents do not exhibit partial agonism, possess a higher affinity for AR, and are postulated to improve survival outcomes relative to their first-generation counterparts for patients with CRPC. Lastly, the emergence of ADT, including second-generation AR antagonists, has led to the development of supportive care for treatment-related adverse effects. Major advances have been made in targeting the AR signaling pathway in patients with advanced PC. Further studies are warranted to identify the optimal sequencing of therapies to maximize treatment benefit. Mitigation of treatment-related adverse effects presents new opportunities to advance clinical pharmacy practice.

Roles of Key Epigenetic Regulators in the Gene Transcription and Progression of Prostate Cancer.

Prostate cancer (PCa) is a top-incidence malignancy, and the second most common cause of death amongst American men and the fifth leading cause of cancer death in men around the world. Androgen receptor (AR), the key transcription factor, is critical for the progression of PCa by regulating a series of target genes by androgen stimulation. A number of co-regulators of AR, including co-activators or co-repressors, have been implicated in AR-mediated gene transcription and PCa progression. Epigenetic regulators, by modifying chromatin integrity and accessibility for transcription regulation without altering DNA sequences, influence the transcriptional activity of AR and further regulate the gene expression of AR target genes in determining cell fate, PCa progression and therapeutic response. In this review, we summarized the structural interaction of AR and epigenetic regulators including histone or DNA methylation, histone acetylation or non-coding RNA, and functional synergy in PCa progression. Importantly, epigenetic regulators have been validated as diagnostic markers and therapeutic targets. A series of epigenetic target drugs have been developed, and have demonstrated the potential to treat PCa alone or in combination with antiandrogens.

Inflammation induced by lipopolysaccharide advanced androgen receptor expression and epithelial-mesenchymal transition progress in prostatitis and prostate cancer.

BackgroundTo explore the mechanism of prostatic inflammation on prostate cancer (PCa) by comparing the changes of prostate epithelial cells and PCa cells in an inflammatory environment.MethodsFirst, immunohistochemistry (IHC) was used to compare the level of expression of TNF-α, IL-1β, IL-6, and TGF-β between benign prostatic hyperplasia (BPH), prostatitis, and PCa. Then primary prostate epithelial cells were sampled from patients who were suspected of PCa and had histological prostatitis (HP) confirmed by pathological biopsy. Lipopolysaccharide (LPS) or BAY11-7082 were used to investigate the change of androgen receptor (AR) and AR-mediated transcription, epithelial-mesenchymal transition (EMT) in primary prostate epithelial cells, and lymph node carcinoma of the prostate (LNCap) cells.ResultsTNF-α, IL-1β, IL-6, and TGF-β were significantly increased in HP and PCa compared with those in BPH patients. The proliferation of primary prostate epithelial cells and LNCap cells got the inflection point at LPS 10 µg/mL. In an inflammatory environment with 10 µg/mL LPS, both primary prostate epithelial cell and LNCap cell viability increased, and AR, AR-mediated transcription, and EMT processes were significantly increased. Inhibitors of NF-κB with 10 nM BAY11-7082 decreased AR, AR-mediated transcription, and EMT processes.ConclusionsNF-κB regulates AR expression and EMT in prostatitis and PCa, and NF-κB inhibitors may have potential therapeutic value.

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer.

Simple SummaryIn almost all patients, the proliferation and growth of prostate cancer is driven by the androgen receptor. Given this critical role, inhibiting the androgen receptor is the standard of care following surgery or radiotherapy. However, the effectiveness of current anti-androgen therapeutics is almost always temporary, with the cancer eventually developing drug resistance. There are no curative treatment options for resistant prostate cancer and these patients have an average life-expectancy of only 20 months. Currently, all clinical inhibitors of the androgen receptor act by direct binding to the hormone binding site. These drugs are therefore vulnerable to mutations which frequently arise at this region or to the emergence of truncated forms of the androgen receptor that are constitutively active without any need of hormone binding. In this study, we aim to develop small molecules inhibitors that attack the molecular functions of the androgen receptor at the N-terminal domain, distinct from steroid recruitment to evade resistance to conventional treatments.Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

The Courage study: A first-in-human phase 1 study of the CBP/p300 inhibitor FT-7051 in men with metastatic castration-resistant prostate cancer.

TPS5085 Background: Prostate cancer is the second leading cause of cancer-related death among men in the U.S., largely due to metastatic disease that progresses despite hormonal therapy (tx). The role for androgen receptor (AR) signaling in prostate cancer and hormone tx resistance is well-established. CBP/p300 are essential co-activators of AR-mediated transcription. FT-7051 is an oral, potent, and selective inhibitor of CBP/p300 with activity in preclinical models of prostate cancer including models resistant to currently used AR inhibitors like enzalutamide. The Courage Study (NCT04575766) is a first-in-human, multicenter, phase 1, open-label study examining the safety, pharmacokinetics (PK), preliminary anti-tumor activity, and pharmacodynamics (PD) of FT-7051 for the treatment of men with metastatic castration-resistant prostate cancer (mCRPC) who have progressed despite prior tx and have been treated with at least one approved androgen receptor pathway inhibitor. The study will enroll up to 45 men with mCRPC at ̃8-15 US sites. Methods: The study employs a Bayesian optimal interval (BOIN) design with an accelerated titration. Patients (pts) will initially be enrolled in a dose level cohort size of 1 until a Grade 2 or higher toxicity occurs that is considered related to FT-7051 or the highest dose level is reached. Upon completion of the accelerated titration phase, subsequent cohorts will enroll 3-5 pts. Treatment: FT-7051 capsules will be administered on a 28 d cycle (21 d on / 7 d off) with Dose Levels -1 to 7 assigned per protocol using the BOIN design. Key inclusion criteria: Diagnosis of mCRPC with either adenocarcinoma or mixed histology AND rising PSA; previously failed at least one approved androgen receptor pathway inhibitor; ≥ 18 yrs of age; prior taxane chemotherapy permitted. Key exclusion criteria: Previous solid organ transplant, prior anticancer tx including prior tx with small molecules within 4 wks of first dose of study treatment, prior radiation tx within 4 wks prior to initiation of study treatment, prior androgen antagonist tx within 2 wks, prior radium-223 tx within 6 wks. Endpoints: Primary endpoints are to define the recommended phase 2 monotherapy dose of FT-7051 through assessments of DLTs, SAEs, clinically relevant AEs, and clinically relevant safety laboratory values. Key secondary endpoints include: PSA at 12 wks, time to PSA progression, time to radiographic progression, overall response rate, and plasma PK parameters. PD assessments of CBP/p300 inhibition in surrogate tissue, biomarker assessments in CTCs (AR, AR-v7), and peripheral blood are included. Duration: Pts will remain on study treatment until they are deemed to be no longer clinically benefiting (NLCB) by the treating Investigator or until unacceptable toxicity. Pt may be followed for survival for up to 24 months from last dose of study treatment. The first pt was dosed January 2021. Clinical trial information: NCT04575766.

Functional mapping of androgen receptor enhancer activity

BackgroundAndrogen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10–100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription.ResultsTo characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity.ConclusionsUsing a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.

COMPARATIVE ANALYSIS OF FK506-BINDING PROTEINS 51 (FKBP51) AND 52 (FKBP52) IN UTERINE FIBROIDS COMPARED TO NORMAL MYOMETRIUM

FK506-binding protein 51 (FKBP51) and 52 (FKBP52) are immunophilins and common co-regulators of transcription meditated by the glucocorticoid receptor (GR), progesterone receptor (PR) and androgen receptor (AR). While FKBP51-PR or -GR binding inhibits transcriptional activity, FKBP51-AR binding induces AR-mediated transcription. FKBP52 has the opposite effect on PR and GR transcriptional activity, enhancing transcription. Although all three steroid receptors are expressed in both uterine myometrial and fibroid cells, the steroid-induced proliferative response of fibroid cells differ from that in adjacent myometrial cells, indicating aberrant steroid signaling in fibroids.

Abstract 1262: Targeting transcriptional addiction in anti-androgen refractory castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related mortality in men of the western world. Androgen receptor (AR) is the primary driver alteration in metastatic castration-resistant prostate cancer (mCRPC). Advanced PCa treated with first line androgen-deprivation therapy (ADT) eventually relapse in a hormone refractory or castration-resistant (CR) form. Relapsed disease is highly aggressive and poses an increased risk of morbidity and death. This refractory state of CRPC is characterized by a strong addiction to AR-mediated transcription driven by the synergy between a variety of genomic alterations and epigenetic remodeling. Despite the success of recently approved therapies targeting AR signaling, such as abiraterone and second-generation anti-androgens including enzalutamide, durable responses are limited, owing to acquired resistance. Epigenetic therapies targeting coactivators within the AR transcription machinery have shown promise as effective options for the treatment of CRPC in both its naive and Enzalutamide Resistant state (Enza-Res). Using basic proteomic and next generation genomic approaches, we have identified CDK7, as an indispensable component of the AR-transcription machinery and a novel target in CRPC. The phosphorylation of MED1 (T1457) by CDK7 leads to the formation of the chromatin bound AR-MED1 complex and is essential for its stability and onset of AR-mediated transcription. Loss of CDK7 activity either through genetic silencing or through treatment with the CDK7 inhibitor THZ1 led to de-recruitment of chromatin bound MED1, loss of AR-target gene expressions, increased apoptosis, and a drastic reduction in cell proliferation. Here, we present evidence for the efficacy of targeting CDK7 in curtailing AR signaling in the Enza-Res state. Further, we observed hyper-phosphorylation of MED1 and AR, and a concomitant decrease in the catalytic-subunit (alpha-isoform) of the protein phosphatase 2A (PP2ACα) in enzalutamide resistant cell lines and metastatic biopsies. This hyper-phosphorylated state was strongly dependent on the catalytic activity of CDK7, wherein treatment of Enza-Res cells with THZ1 led to complete loss of phosphorylation of MED1 and AR, and death in Enza-res cells. These findings point to the indispensable role of CDK7 in driving the AR oncogenic transcription program in anti-androgen refractory CRPC and its viability as a potential therapeutic target. Citation Format: Reyaz ur Rasool, Ramakrishnan Natesan, Qu Deng, Shweta Aras, Samuel Sander Effron, Samuel Sander Effron, Erick Mitchell Velasquez, Jessica M. Posimo, Priti Lal, Donita C. Brady, Irfan A. Asangani. Targeting transcriptional addiction in anti-androgen refractory castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1262.

MiR-30a inhibits androgen-independent growth of prostate cancer via targeting MYBL2, FOXD1, and SOX4.

Castrate-resistant prostate cancer (CRPC) is an aggressive and lethal disease. The pathogenesis of CRPC is not fully understood and novel therapeutic targets need to be identified to improve the patients' prognosis. MicroRNA-30a (miR-30a) has been demonstrated to be a tumor suppressor in many types of solid malignancies. However, its role in androgen-independent (AI) growth of prostate cancer (PCa) received limited attention as yet. The clinical association of miR-30a and its potential targets with AI growth was characterized by bioinformatics analyses. Regulation of cell proliferation and colony formation rates by miR-30a were tested using PCa cell models. Xenograft models were used to measure the regulation of prostate tumor growth by miR-30a. The real-time quantitative polymerase chain reaction was used to validate whether miR-30a and its targets regulate cell cycle control genes and androgen receptor (AR)-dependent transcription. Bioinformatics tools, Western blot, and luciferase reporter assays were utilized to identify miR-30a targets. Bioinformatic analysis showed that low expression of miR-30a is associated with castration resistance of PCa patients and poor outcomes. Transfection of miR-30a mimics inhibited the AI growth of PCa cells in vitro and in vivo. Upregulation of miR-30a in 22RV1 cells altered the expression of cell cycle control genes and AR-mediated transcription, while downregulation of miR-30a in LNCaP cells had the opposite effects to AR-mediated transcription. MYBL2, FOXD1, and SOX4 were identified as miR-30a targets. Downregulation of MYBL2, FOXD1, and SOX4 affected the expression of cell cycle control genes and AR-mediated transcription and suppressed the AI growth of 22RV1 cells. Our results suggest that miR-30a inhibits AI growth of PCa by targeting MYBL2, FOXD1, and SOX4. They provide novel insights into developing new treatment strategies for CRPC.

Androgen receptor-binding sites are highly mutated in prostate cancer

Androgen receptor (AR) signalling is essential in nearly all prostate cancers. Any alterations to AR-mediated transcription can have a profound effect on carcinogenesis and tumor growth. While mutations of the AR protein have been extensively studied, little is known about those somatic mutations that occur at the non-coding regions where AR binds DNA. Using clinical whole genome sequencing, we show that AR binding sites have a dramatically increased rate of mutations that is greater than any other transcription factor and specific to only prostate cancer. Demonstrating this may be common to lineage-specific transcription factors, estrogen receptor binding sites were also found to have elevated rate of mutations in breast cancer. We provide evidence that these mutations at AR binding sites, and likely other related transcription factors, are caused by faulty repair of abasic sites. Overall, this work demonstrates that non-coding AR binding sites are frequently mutated in prostate cancer and can impact enhancer activity.

Oxidative stress and redox signaling in CRPC progression: therapeutic potential of clinically-tested Nrf2-activators.

Androgen deprivation therapy (ADT) is the mainstay regimen in patients with androgen-dependent prostate cancer (PCa). However, the selection of androgen-independent cancer cells leads to castrate resistant prostate cancer (CRPC). The aggressive phenotype of CRPC cells underscores the need to elucidate mechanisms and therapeutic strategies to suppress CRPC outgrowth. Despite ADT, the activation of androgen receptor (AR) transcription factor continues via crosstalk with parallel signaling pathways. Understanding of how these signaling cascades are initiated and amplified post-ADT is lacking. Hormone deprivation can increase oxidative stress and the resultant reactive oxygen species (ROS) may activate both AR and non-AR signaling. Moreover, ROS-induced inflammatory cytokines may further amplify these redox signaling pathways to augment AR function. However, clinical trials using ROS quenching small molecule antioxidants have not suppressed CRPC progression, suggesting that more potent and persistent suppression of redox signaling in CRPC cells will be needed. The transcription factor Nrf2 increases the expression of numerous antioxidant enzymes and downregulates the function of inflammatory transcription factors, e.g., nuclear factor kappa B. We documented that Nrf2 overexpression can suppress AR-mediated transcription in CRPC cell lines. Furthermore, two Nrf2 activating agents, sulforaphane (a phytochemical) and bardoxolone-methyl (a drug in clinical trial) suppress AR levels and sensitize CRPC cells to anti-androgens. These observations implicate the benefits of potent Nrf2-activators to suppress the lethal signaling cascades that lead to CRPC outgrowth. This review article will address the redox signaling networks that augment AR signaling during PCa progression to CRPC, and the possible utility of Nrf2-activating agents as an adjunct to ADT.

Abstract B016: CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation

Abstract Metastatic castration-resistant prostate cancer (CRPC) is an aggressive disease with high mortality rate, primarily resulting from the transcriptional addiction driven by Androgen Receptor (AR). First-line CRPC treatments typically target AR-signaling, but are rapidly bypassed, resulting in only a modest survival benefit with the FDA approved anti-androgen therapies such abiraterone or enzalutamide. Therefore, molecular approaches that more effectively block the AR-transcriptional program are urgently needed. Here, we present evidence demonstrating that AR transcriptional signaling is activated through a “phosphoswitch” catalyzed by cyclin-dependent-kinase 7 (CDK7). Specifically, CDK7 phosphorylates (in vitro and in vivo) the transcriptional co-activator MED1, a key subunit of Mediator complex, at Threonine 1457 to promote the formation of the AR-transcriptional complex at enhancers and super-enhancer (SE) sites. Furthermore, knockdown or inhibition of CDK7 with the recently developed covalent inhibitor THZ1 abolished T1457 phosphorylation that led to MED1 decruitment from the chromatin, attenuated AR-signaling and eliminated AR-addicted naïve or enzalutamide refractory prostate cancer cells. Interestingly, the reduced AR transcriptional output and cellular phenotypes associated with CDK7 knockdown or inhibition was reversed by T1457D phosphomimic suggesting MED1 as a major effector substrate of CDK7 transcription kinase. Finally, THZ1 demonstrated tumor regression in CRPC xenograft models in vivo. In summary, our identification of MED1 T1457 as a novel CDK7 substrate, which is essential for driving AR-mediated transcription, makes CDK7 a potential “non-oncogene dependency” in AR addicted advanced prostate cancer. Taken together, we will present data that strongly support the clinical evaluation of CDK7 specific inhibitors as a monotherapy or in combination with second generation anti-androgens in refractory castration-resistant prostate cancer. Conflict of Interest: None (P.S. This work is undergoing final revision for publication in Cancer Discovery) Citation Format: Reyaz Rasool, Ramakrishnan Natesan, qu deng, Shweta Aras, Priti Lal, Jessica Posimo, Nallasivam Palanisamy, Goutham Narla, Robert Den, Matthew Freedman, donita brady, irfan a asangani. CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B016. doi:10.1158/1535-7163.TARG-19-B016

CDK7 Inhibition Suppresses Castration-Resistant Prostate Cancer through MED1 Inactivation.

Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by androgen receptor (AR). First-line CRPC treatments typically target AR signaling, but are rapidly bypassed, resulting in only a modest survival benefit with antiandrogens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional coactivator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at superenhancer sites, and is essential for AR-mediated transcription. In addition, a CDK7-specific inhibitor, THZ1, blunts AR-dependent neoplastic growth by blocking AR/MED1 corecruitment genome-wide, as well as reverses the hyperphosphorylated MED1-associated enzalutamide-resistant phenotype. In vivo, THZ1 induces tumor regression of AR-amplified human CRPC in a xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC. SIGNIFICANCE: Potent inhibition of AR signaling is critical to treat CRPC. This study uncovers a driver role for CDK7 in regulating AR-mediated transcription through phosphorylation of MED1, thus revealing a therapeutically targetable potential vulnerability in AR-addicted CRPC.See related commentary by Russo et al., p. 1490.This article is highlighted in the In This Issue feature, p. 1469.

Amyloid precursor protein, an androgen-regulated gene, is targeted by RNA-binding protein PSF/SFPQ in neuronal cells.

Amyloid precursor protein (APP) is a representative gene related to Alzheimer's disease (AD). Androgens function by binding to the androgen receptor (AR). Both androgen and RNA-binding protein PSF play a role in the pathology of AD. However, the involvement of AR and PSF in APP regulation in neuron has not been investigated. Here, we explored the regulatory mechanism of APP expression by AR and PSF using neuron-derived cells. We demonstrated that androgen up-regulates the production of APP at the mRNA and protein levels. This induction is enhanced by AR over-expression and inhibited by its silencing. One candidate AR-binding region was identified in the intron region of APP and validated its activity as AR-dependent enhancer by the luciferase assay. Furthermore, the public transcriptome data of brain tissues of mice indicated that APP is regulated by PSF post-transcriptionally. We observed a decreased expression of APP after PSF knockdown and interaction of PSF with the APP transcript. Moreover, we revealed that silencing of PSF inhibited the stability of the APP mRNA. Thus, these results presented a new regulatory mechanism of APP expression by androgen through AR-mediated transcription and PSF at the post-transcriptional level that might be associated with the occurrence of AD.

Abstract 4718: SR-4370, a potent and selective inhibitor of class I HDACs, suppresses AR signaling and in vivo prostate tumor growth

Abstract Androgen receptor (AR) is an androgen-activated transcription factor and drives prostate cancer (PCa) progression. Class I HDACs 1-3 are critical for activating AR-mediated transcription. Thus, targeting these HDACs is a promising strategy for treating PCa. Notably, along with significant adverse effects, several FDA-approved HDAC inhibitors broadly inhibiting different HDACs were ineffective for treating castration-resistant prostate cancer in clinical trials. Significantly, entinostat, an aminobenzamide analog specific to HDACs 1-3, extended overall survival for patients with breast cancer resistant to endocrine therapy. These observations suggest that HDACi selective to HDACs 1-3 may be effective for treating solid tumors including PCa. We have recently discovered the novel benzoylhydrazide class of HDAC inhibitors highly specific to HDACs 1-3. An optimized analog, SR-4370, exhibited low µM to nM potency against HDACs 1-3. SR-4370 markedly suppressed AR signaling, PCa cell proliferation in vitro, and prostate tumor growth in vivo. Gene expression profiling experiments revealed that SR-4370 downregulated AR, AR-Vs and AR target genes as well as the MYC oncogenic network. Chromatin accessibility assay using ATAC-seq showed that SR-4370 altered chromatin states in PCa cells. The chromatins with AR-binding sites became inaccessible on SR-4730 treatment, indicating that altered chromatin accessibility may contribute to the inhibition of AR signaling. Interestingly, SR-4370 sensitized C4-2 cells to enzalutamide. In PCa xenograft models, SR-4370 was effective to suppress tumor growth in vivo. Importantly, SR-4370 was well tolerated and did not cause observable adverse effects as judged by body weight and blood chemistry tests of treated mice. Our data suggest that SR-4370 may be a safe and clinically applicable treatment for advanced PCa refractory to current frontline treatments. (Supported by UFHealth Cancer Center, Florida Breast Cancer Foundation, James and Esther King Biomedical Research Program, and Bankhead-Coley Cancer Research Program, Florida Department of Health) Citation Format: Iqbal Mahmud, Guimei Tian, Jia Wang, Ryan Stowe, Zhiguang Huo, Yushan Zhang, Hamsa Thayek Purayil, Eric Helm, Theodore Drashansky, Dorina Avram, Yehia Daaka, William R. Roush, Daiqing Liao. SR-4370, a potent and selective inhibitor of class I HDACs, suppresses AR signaling and in vivo prostate tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4718.

Abstract 5208: Genome-wide AR enhancer activity in prostate cancer

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed forms of cancer with one out of every nine North American men developing the disease in their lifetime. Despite robust early detection methods, new therapeutics, and advancements in surgical operations, PCa is still second leading cause of cancer death in men. An increasing number of studies have shown that non-coding mutations plays a critical role in the development and progression of PCa. These somatic mutations can disrupt regulatory elements such as enhancers and alter the transcriptional landscape of the cancer. Discovering and interpreting the non-coding elements is crucial to understand the nature of this disease. At all stages of PCa, androgen receptor (AR) signaling is essential to the cancer. Recent work from our laboratory demonstrated that there is a significant increase in somatic mutations at AR binding sites (ARBS). Given the importance of AR-mediated transcription, as well as the recurrent nature of these mutations, we proposed that these somatic mutations could play an important role in PCa development. To stratify the mutations, we conducted a massively multi-parallel enhancer assay to identify those ARBS sites with enhancer activity. With STARR-seq we found that 8% of the ARBS had strong inducible AR enhancer activity (341/4139). Interestingly, the majority of these sites did not contain a canonical androgen response element. With these results we incorporated high-throughput chromosome conformation capture (HiC) to formally define the genes regulated by each of these AR enhancers. Based on these results we have selected and characterized several non-coding ARBS mutations. We believe that the importance of the regulatory role of these regions will play a tremendous role in understanding of the prostate cancer and its progression. Citation Format: Dogancan Ozturan, Flora Huang, Tunc Morova, Mohammadali Saffarzadeh, Nathan A. Lack. Genome-wide AR enhancer activity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5208.

BAF53A regulates androgen receptor-mediated gene expression and proliferation in LNCaP cells

The actin-like protein of the SWI/SNF complex, BAF53A, regulates gene expression by the gene-specific chromatin remodeling of target genes. However, the function of BAF53A in the androgen receptor pathway in prostate cancer cells remains unclear. Here, we demonstrated that BAF53A positively regulates the expression of endogenous AR target genes (e.g. PSA, TMPRSS2, FKBP5, and KLK2) in LNCaP cells. It functions as a coactivator in AR-mediated transcription by interacting with other nuclear receptor coactivators, such as p300 and FLII, and is associated with AR in the presence of dihydrotestosterone (DHT). The DHT-induced recruitment of BAF53A to the proximal and distal androgen response elements (AREs) of the PSA gene in the presence of BRG1 (but not BRM) was inhibited by an AR antagonist, suggesting the coactivator function of BAF53A in the SWI/SNF complex. Depletion of BAF53A in LNCaP cells resulted in a significant decrease in growth rate. Furthermore, the expression of BAF53A in prostate cancer tissue was significantly elevated, compared to that in normal prostate tissue, and correlated with the expression of AR, and BRG1, but not BRM. Therefore, our results suggested that BAF53A plays an important role in the expression of AR target genes in prostate cancer, and can be used clinically for the treatment of prostate cancer.