Direct Androgen Receptor Antagonist Research Articles

Phosphorylation of the androgen receptor at Ser81 is co-sustained by CDK1 and CDK9 and leads to AR-mediated transactivation in prostate cancer.

Androgen receptor (AR) is the principal molecule in prostate cancer (PCa) etiology and therapy. AR re‐activation still remains a major challenge during treatment of castration‐resistant prostate cancer (CRPC) tumors that relapse after castration therapies. Recent reports have indicated the enrichment of Ser81‐phosphorylated AR (pS81) in the nucleus of CRPC cells, and CDK1 and CDK9 as the kinases phosphorylating AR at S81. In the current study we showed that pS81 is preferentially localized in the nucleus in both rapid biopsy metastatic CRPC samples and PCa xenografts, and nuclear pS81 localization is correlated with AR transactivation in tumor xenografts. Chromatin immunoprecipitation (ChIP) analysis demonstrated an alignment of S81 phosphorylation and AR‐mediated transactivation with the chromatin locus openness. Moreover, pS81‐specific ChIP‐Seq showed a disproportional occupancy of pS81 on AR‐activated promoters, while 3C‐ChIP assays further indicated an enrichment of pS81 at the PSA enhancer‐promoter loop, a known AR activating hub. In the latter, CDK9 was shown to modulate the transactivation of the AR and RNA Pol II. Indeed, ChIP and re‐ChIP assays also confirmed that AR‐dependent activation of the PSA enhancer and promoter mediated by pS81 was coupled with activation of Pol II and the pTEFb complex. Mechanistically, we determined that CDK1 and CDK9 sustained the pS81 AR modification in the soluble and chromatin‐bound fractions of PCa cells, respectively. Finally, we demonstrated that CDK1 activity was maintained throughout the cell cycle, and that CDK1 inhibitors restored androgen sensitivity in CRPC tumor cells. Based on these findings, CDK1 and CDK9 could be targeted as pS81 kinases in patients with CRPC, either alone or in conjunction with direct AR antagonists.

A 3-(4-nitronaphthen-1-yl) amino-benzoate analog as a bifunctional AKR1C3 inhibitor and AR antagonist: Head to head comparison with other advanced AKR1C3 targeted therapeutics

Drugs used for the treatment of castration resistant prostate cancer (CRPC) include Abiraterone acetate (Zytiga®) and Enzalutamide (XTANDI®). However, these drugs provide clinical benefit in metastatic disease for only a brief period before drug resistance emerges. One mechanism of drug resistance involves the overexpression of type 5 17-β-hydroxysteroid dehydrogenase (aldo-keto reductase 1C3 or AKR1C3), a major enzyme responsible for the formation of intratumoral androgens that activate the androgen receptor (AR). 3-((4-Nitronaphthalen-1-yl)amino)benzoic acid 1 is a “first-in-class” AKR1C3 competitive inhibitor and AR antagonist. Compound 1 was compared in a battery of in vitro studies with structurally related N-naphthyl-aminobenzoates, and AKR1C3 targeted therapeutics e.g. GTx-560 and ASP9521, as well as with R-bicalutamide, enzalutamide and abiraterone acetate. Compound 1 was the only naphthyl derivative that was a selective AKR1C3 inhibitor and AR antagonist in direct competitive binding assays and in AR driven reporter gene assays. GTx-560 displayed weak activity as a direct AR antagonist but had high potency in the AR reporter gene assay consistent with its ability to inhibit the co-activator function of AKR1C3. By contrast ASP9521 did not act as either an AR antagonist or block AR reporter gene activity. Compound 1 was the only compound that showed comparable potency to inhibit AKR1C3 and act as a direct AR antagonist. Compound 1 blocked the formation of testosterone in LNCaP-AKR1C3 cells, and the expression of PSA driven by the AKR1C3 substrate (4-androstene-3,17-dione) and by an AR agonist, 5α-dihydrotestosterone consistent with its bifunctional role. Compound 1 blocked the nuclear translocation of the AR at similar concentrations to enzalutamide and caused disappearance of the AR from cell lysates. R-biaclutamide and enzalutamide inhibited AKR1C3 at concentrations 200x greater than compound 1, suggesting that its bifunctionality can be explained by a shared pharmacophore that can be optimized.

Abstract IA24: Targeting DNA repair dysfunction in advanced prostate cancer

Abstract Prostatic adenocarcinoma (PCa) is the 2nd leading cause of cancer death in US men. Organ-confined PCa can be effectively managed, but there is no durable treatment for advanced disease. Advanced PCa is treated through androgen deprivation therapy, often coupled with direct AR antagonists, as PCa is exquisitely dependent on androgen receptor (AR) activity for survival. Furthermore, recent studies identified AR as a major effector of DNA repair, manifest through the ability of the receptor to regulate DNAPK expression and activity. While AR-directed therapeutics effectively suppress the proproliferative, prosurvival, and pro-DNA repair functions of AR and result in tumor remission, relapse is common. Recurrent disease arises largely due to resurgent AR activity with 2-3 years, and there is no cure for this castration-resistant phase (CRPC, castration-resistant PCa). Thus, there is a significant need to develop new means for targeting recurrent AR activity or develop adjuvant therapies in advanced PCa. Emerging data from our laboratory and others strongly support the concept that alterations in DNA damage repair (DDR) pathways are more common than previously thought in sporadic PCa, and that alterations in these pathways may accord new, more effective means of therapeutic intervention. New studies to be discussed will address underlying mechanisms of action with regard to the pathway, and identify clinically actionable ramifications of DNA repair dysfunction. Major concepts to be considered include new discoveries regarding PARP1 function and activity in lethal disease, and that a newly identified gene signature of PARP1-regulated networks is associated with poor outcome. Further, mechanistic investigation revealed new insight into the means by which PARP1 inhibitors likely function as single agents in advanced prostate cancer, manifest through both DNA repair and transcriptional regulatory functions. Other major concepts to be considered include the differential roles of DNAPK in controlling PCa progression. Findings to be discussed strongly support a model wherein selected DDR pathways can be developed as therapeutic targets to tailor treatment for prostate cancer and improve outcome for advanced disease. Citation Format: Karen E. Knudsen. Targeting DNA repair dysfunction in advanced prostate cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA24.

Sigma1 Targeting to Suppress Aberrant Androgen Receptor Signaling in Prostate Cancer.

Suppression of androgen receptor (AR) activity in prostate cancer by androgen depletion or direct AR antagonist treatment, although initially effective, leads to incurable castration-resistant prostate cancer (CRPC) via compensatory mechanisms including resurgence of AR and AR splice variant (ARV) signaling. Emerging evidence suggests that Sigma1 (also known as sigma-1 receptor) is a unique chaperone or scaffolding protein that contributes to cellular protein homeostasis. We reported previously that some Sigma1-selective small molecules can be used to pharmacologically modulate protein homeostasis pathways. We hypothesized that these Sigma1-mediated responses could be exploited to suppress AR protein levels and activity. Here we demonstrate that treatment with a small-molecule Sigma1 inhibitor prevented 5α- dihydrotestosterone-mediated nuclear translocation of AR and induced proteasomal degradation of AR and ARV, suppressing the transcriptional activity and protein levels of both full-length and splice-variant AR. Consistent with these data, RNAi knockdown of Sigma1 resulted in decreased AR levels and transcriptional activity. Furthermore, Sigma1 physically associated with ARV7 and ARv567es as well as full-length AR. Treatment of mice xenografted with ARV-driven CRPC tumors with a drug-like small-molecule Sigma1 inhibitor significantly inhibited tumor growth associated with elimination of AR and ARV7 in responsive tumors. Together, our data show that Sigma1 modulators can be used to suppress AR/ARV-driven prostate cancer cells via regulation of pharmacologically responsive Sigma1-AR/ARV interactions, both in vitro and in vivoCancer Res; 77(9); 2439-52. ©2017 AACR.

Abstract IA26: Dual functions of PARP1 in prostate cancer: mechanisms and implications for therapeutic intervention

Abstract Prostatic adenocarcinoma (PCa) is the 2nd leading cause of cancer death in US men. Organ-confined PCa can be effectively managed, but there is no durable treatment for advanced disease. Advanced PCa is treated through androgen deprivation therapy, often coupled with direct AR antagonists, as PCa is exquisitely dependent on androgen receptor (AR) activity for survival. Furthermore, recent studies identified AR as a major effector of DNA repair, manifest through the ability of the receptor to regulate DNAPK expression and activity. While AR directed therapeutics effectively suppress the pro-proliferative, pro-survival, and pro-DNA repair functions of AR and result in tumor remission, relapse is common. Recurrent disease arises largely due to resurgent AR activity with 2-3 years, and there is no cure for this castration-resistant phase (CRPC, castration-resistant PCa). Thus, there is a significant need to develop new means for targeting recurrent AR activity or develop adjuvant therapies in advanced PCa. The function of PARP1 in DNA repair has been cultivated as a therapeutic target for tumors incurring alterations of specific DNA repair pathways. However, it is now clear that factors beyond DNA repair alterations play a role in the response to PARP1 inhibitors-- as noted in the TO-PARP trial, not all patients with DNA repair alterations responded to PARP1 inhibitors as single agents; conversely, a significant number tumors lacking BRCA1/2 or other DNA repair alterations show objective responses to PARP1 inhibitors in PCa and other cancers. PARP1 is known to harbor a second major cellular function on chromatin as a transcriptional co-regulator, capable of modulating chromatin structure and selected transcription factor activity. Notably, previous studies demonstrated that PARP1 regulates AR residence on chromatin, and downstream AR-dependent tumor progression. Recent unpublished studies to be discussed demonstrate that PARP1 activity is significantly enriched in lethal disease, and that a newly identified gene signature of PARP1-regulated networks is associated with poor outcome. Further, mechanistic investigation revealed new insight into the means by which PARP1 inhibitors likely act function as single agents in advanced prostate cancer, manifest through both DNA repair and transcriptional regulatory functions. Citation Format: Karen E. Knudsen. Dual functions of PARP1 in prostate cancer: mechanisms and implications for therapeutic intervention [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA26.

Abstract P3-14-04: Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo

Abstract VT-464 is a lyase-selective inhibitor of the dual-activity CYP17A1 enzyme that is required for the synthesis of androgens and estrogens in the gonads, adrenals, and tumors. In addition to its role as a CYP17A1 lyase inhibitor, we previously showed that VT-464 also functions as a direct and effective AR antagonist in prostate cancer models. In our current study, we evaluated the therapeutic potential of VT-464 in breast cancer by analyzing its effectiveness in several AR-positive breast cancer cell lines, including those that are ER-positive and ER-negative. Through in vitro assays and xenograft analysis, we compared the activity of VT-464 to enzalutamide, a second generation AR antagonist that is approved for the treatment of castration resistant prostate cancer and is in multiple Phase 2 breast cancer studies. Our results showed that VT-464 was highly effective in preventing proliferation of both ER-positive and ER-negative breast cancer cell lines in vitro. Importantly, significant inhibition was also observed in soft agar assays that assesses anchorage-independent growth. In mechanistic studies, VT-464 and enzalutamide both inhibited induction of AR target genes and recruitment of AR to target promoters, verifying direct AR antagonistic activity observed previously in prostate cancer cells. Furthermore, we evaluated the ability of VT-464 and enzalutamide to inhibit tumor formation in a tamoxifen-resistant model of breast cancer. Oral administration of either enzalutamide or VT-464 significantly decreased tumor growth in mice, with VT-464 achieving greater growth inhibition than enzalutamide. Together, these data provide further rationale for the future study of the AR as a viable therapeutic target in breast cancer, and importantly, suggest that AR inhibition can impact tamoxifen-resistant tumor growth. The dual effects of CYP17A1 lyase inhibition and AR antagonism that are achieved with VT-464 further supports its development as an effective oral therapy option for AR-positive breast cancer. A phase 1 / 2 clinical study of oral VT-464 in women with AR+ triple-negative breast cancer or ER+ cancer resistant to aromatase inhibitors will commence in 2015. Citation Format: Ellison SJ, Norris JD, Wardell S, Eisner JR, Hoekstra WJ, Stagg DB, Alley HM, Moore WR, McDonnell DP. Effects of the dual selective CYP17 lyase inhibitor and androgen receptor (AR) antagonist, VT-464, on AR+ and ER+ tumor models in vitro and in vivo. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-04.

The association of D4, 3-keto-abi (D4A) and response to abiraterone in castration-resistant prostate cancer (CRPC).

245 Background: Abiraterone (Abi), a potent inhibitor of 17α-hydroxylase/17,20-lyase (CYP17A1), is a standard treatment for men with metastatic CRPC. Abi is converted to D4A by 3β-hydroxysteroid dehydrogenase (3βHSD). D4A inhibits CYP17A1, 3βHSD, and steroid-5α-reductase (SRD5A) and has direct androgen receptor antagonist activity, which together make it a more potent agent than Abi in xenograft models. It is not known if conversion to D4A in patients (pts) correlates with response or resistance to Abi. Methods: Blood was collected (single time point on Abi) from CRPC pts who started Abi during 2011-2015. Abi and D4A were extracted from serum and analyzed by mass spectrometry. The purpose of this ongoing study is to assess the potential correlation between D4A and response to treatment. Results: 32 patients with CRPC had blood collected. 4 pts (12.5%) received ketoconazole and 6 (18.8%) chemotherapy prior to Abi. Median pre-Abi prostate-specific antigen (PSA) was 14.3 ng/ml (0.6-646.1). Median time from initiation of androgen deprivation therapy (ADT) to CRPC was 34.3 months (m) (6-129.6) and median time from CRPC to Abi initiation was 4.8 m (0-14.9). PSA decline > 50% (PSA50) on Abi was seen in 68% (12/31) and 73% (19/26) of pts at 3 and 6 m, respectively. Treatment with Abi was ongoing in 23/32 pts (74%), and discontinued in 8 pts (26%) due to disease progression (no follow up data on 1 pt) with median duration of Abi treatment 14.6 m (2.9-44.4 m) at last follow up. As the absolute concentration of detected levels of Abi and D4A varied significantly among pts, the percentage of the total extracted metabolites was used to assess correlation with response. Abi and D4A comprised 94.3% (73.8-97.4%) and 5.7% (2.6-26.2%), respectively, of total levels. Conclusions: Abi absorption and metabolism significantly vary among pts. Most of these pts had prolonged duration of response to Abi (74% ongoing treatment and median treatment duration 13.3 m in 8 pts that came off treatment). Longer follow up, accrual of pts with shorter duration of response, and sampling at multiple time points on Abi and at progression is ongoing to further evaluate the impact of D4A on treatment response.

Abstract 3316: Characterizing mechanisms of resistance to androgen deprivation in prostate cancer

Abstract Despite significant advances in the treatment of prostate cancer (PCa), this malignancy remains a leading cause of cancer death among men. PCa cells require androgen receptor (AR) signaling for their growth and survival. Androgen deprivation therapy (ADT) constitutes the main therapeutic option for patients with advanced PCa. However, the major cause of death in men with metastatic prostate cancer involves progression to castration-resistant prostate cancer (CRPC). Characterizing mechanisms of resistance to ADT could enable the development of more effective therapeutic strategies. To identify genes whose silencing drives resistance to ADT in androgen-sensitive PCa cells, we performed a systematic genome-wide suppressor RNAi screen in the androgen-sensitive LNCaP cells, using a 98,000-shRNA library, targeting approximately 16,000 human genes. After infection, cells were cultured in absence of androgens for several weeks. Cells showing the ability to proliferate upon androgen deprivation were harvested at different time points and shRNA sequences were identified and quantitated through nextgen sequencing. Hit genes were those with at least 2 shRNAs enriched >4 fold, and whose silencing exerted the most robust effects on cell proliferation. To validate the identified top-tier genes in vitro, we infected LNCaP cells with lentiviruses carrying shRNAs (2 shRNAs/candidate gene) targeting the top ∼30 candidate genes, in parallel with 15 negative control shRNAs. We monitored the ability of each shRNA to increase proliferation under androgen-deprived conditions, using cell viability assays. To determine whether any shRNAs drive castration resistance by reactivating the AR-signaling pathway, we analyzed the expression of known AR effector genes in the presence of candidate shRNAs and also tested their effect on cell proliferation in the presence of the direct AR antagonist MDV3100. In future, we will also validate candidate genes in vivo, using a LNCaP xenograft model. This study has identified several genes whose silencing may modulate resistance to ADT in prostate cancer. Additional functional studies should determine whether a subset of these may promote new therapeutic strategies for men with CRPC. Citation Format: Ginevra Botta, Judit Jane-Valbuena, Terence Wong, John Doench, Levi A. Garraway. Characterizing mechanisms of resistance to androgen deprivation in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2014-3316

Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer.

Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling1-3. Despite the success of recently approved therapies targeting AR signaling such as abiraterone4-6 and second generation anti-androgens MDV3100 (enzalutamide)7,8, durable responses are limited, presumably due to acquired resistance. Recently JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit anti-proliferative effects in a range of malignancies9-12. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ111,13. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR-mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer.

Outsmarting androgen receptor: creative approaches for targeting aberrant androgen signaling in advanced prostate cancer

Prostatic adenocarcinomas are reliant on androgen receptor (AR) activity for survival and progression. Therefore, first-line therapeutic intervention for disseminated disease entails the use of AR-directed therapeutics, achieved through androgen deprivation and direct AR antagonists. While initially effective, recurrent, ‘castrate-resistant’ prostate cancers arise, for which there is no durable means of treatment. An abundance of clinical study and preclinical modeling has led to the revelation that restored AR activity is a major driver of therapeutic failure and castrate-resistant prostate cancer development. The mechanisms underpinning AR reactivation have been identified, providing the foundation for a new era of drug discovery and rapid translation into the clinic. As will be reviewed in this article, these creative new ways of suppressing AR show early promise.