Androgen Receptor N-terminal Domain Research Articles

A phase 1 study to assess the safety, pharmacokinetics, and anti-tumor activity of the androgen receptor n-terminal domain inhibitor epi-506 in patients with metastatic castration-resistant prostate cancer

EPI-506 is the first of a new class of drugs targeting the N-terminal domain (NTD) of the androgen receptor (AR), potentially overcoming known resistance mechanisms to androgen receptor pathway inhibitors (ARPIs) among men with metastatic castration resistant prostate cancer (mCRPC). Patients with mCRPC who had progressed on prior ARPI were enrolled in this phase 1 open-label, adaptive 3 + 3 dose escalation study. The primary outcome was safety and tolerability of oral EPI-506. Secondary objectives included determination of the maximal tolerated dose (MTD), pharmacokinetic profile, and antitumor efficacy. 28 mCRPC patients were enrolled into 7 dose cohorts of EPI-506 ranging from 80-3600mg given once daily and 1800mg given twice daily. Six DLTs occurred in 4 patients; Grade 4 elevated amylase; Grade 3 abdominal pain; Grade 3 elevated ALT and Grade 3 elevated AST; Grade 2 nausea and Grade 1 vomiting which resulted in study drug intake of < 75% of the expected dose during the DLT assessment period. The most common drug-related adverse events included diarrhea, nausea and fatigue. Six patients had a PSA decline not meeting PSA response criteria. The study was terminated prior to reaching the MTD due to poor oral bioavailability. This phase 1 trial established the safety of EPI-506 and provides proof of concept for targeting the AR NTD. Next generation compounds with improved bioavailability and potency are in clinical development.

Development of a Benzothiazole Scaffold-Based Androgen Receptor N-Terminal Inhibitor for Treating Androgen-Responsive Prostate Cancer.

All current clinically approved androgen deprivation therapies for prostate cancer target the C-terminal ligand-binding domain of the androgen receptor (AR). However, the main transactivation function of the receptor is localized at the AR N-terminal domain (NTD). Targeting the AR NTD directly is a challenge because of its intrinsically disordered structure and the lack of pockets for drugs to bind. Here, we have taken an alternative approach using the cochaperone BAG1L, which interacts with the NTD, to develop a novel AR inhibitor. We describe the identification of 2-(4-fluorophenyl)-5-(trifluoromethyl)-1,3-benzothiazole (A4B17), a small molecule that inhibits BAG1L-AR NTD interaction, attenuates BAG1L-mediated AR NTD activity, downregulates AR target gene expression, and inhibits proliferation of AR-positive prostate cancer cells. This compound represents a prototype of AR antagonists that could be key in the development of future prostate cancer 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.

Abstract 1209: Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Abstract The androgen receptor (AR) is a key driver in the growth of prostate cancer and remains a crucial target for therapeutic intervention even in late stages of the disease. While current anti-androgen therapies targeting directly or indirectly the AR ligand binding domain (LBD) are initially effective, resistance ultimately develops. The selective targeting of the N-terminal domain (NTD) of the AR represents a novel method of blocking AR signaling to by-pass LBD-related resistance. EPI-7386 is a potent and metabolically stable NTD inhibitor (aniten) currently in a phase 1 dose-escalation study in mCRPC patients. Here we report the results of a comprehensive in vitro characterization of its mechanism of action. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. Target engagement was measured by a Cellular Thermal Shift Assay (CETSA). Both Nanostring and RNAseq were used to explore the activity of EPI-7386 on the AR transcriptome. Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR were carried out to determine the effect of EPI-7386 on AR genomic occupancy. EPI-7386 exhibited potent activity in inhibiting full-length AR (AR-FL) driven transcriptional activity and also strongly impaired the transcriptional activity and the viability of cellular models driven exclusively by truncated AR protein. Using CETSA, we confirmed that EPI-7386 induced a thermal shift of both AR-FL and AR-V7 (lacking the AR LBD) in LNCaP and LNCaP95, respectively, which is an indication of AR target engagement by EPI-7386. ChIP analyses allowed a deeper understanding of epigenetic and transcriptional regulation driven by EPI-7386. It showed EPI-7386 inhibits androgen-activated AR binding to target gene loci such as KLK3. Additionally, EPI-7386 suppresses AR-regulated target gene expression in a comparable manner as lutamides in three human prostate cancer cell lines, LNCaP, 22Rv1, and VCaP, with a few notable exceptions. As a consequence, the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-associated transcriptional activity in both LNCaP and VCaP cells. In AR-V7 driven cell lines, LNCaP95 and 22Rv1, EPI-7386 showed superior activity to enzalutamide in inhibiting AR-regulated genes expression. In conclusion, EPI-7386 is a potent AR NTD inhibitor that has the capacity to by-pass AR LBD resistance mechanisms to current anti-androgen therapies by uniquely inhibiting AR-mediated signaling. The agent has the potential for providing clinical benefit as a single agent in patients whose tumors are progressing on anti-androgens or in combination with current anti-androgens in earlier line patients. The Phase I dose escalation first in human clinical trial of EPI-7386 single agent (NCT04421222) is currently enrolling. Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Han-Jie Zhou, Ronan Le Moigne. Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1209.

Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes

Steroid hormone receptors activate gene transcription by binding specific DNA sequences and recruiting coactivators to initiate transcription of their target genes. For most nuclear hormone receptors (NRs), the ligand-dependent activation function domain-2 (AF-2), residing in the C-terminal ligand binding domain (LBD), is a primary contributor to the NR transcriptional activity. In contrast to other steroid receptors such as estrogen receptor-α (ERα), the transcriptional activation function of androgen receptor (AR) is thought to be largely dependent on its ligand-independent activation function domain-1 (AF-1) located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different activation function domain from other steroid receptors despite the fact that NRs share similar domain organizations and have a highly conserved DNA binding domain (DBD) and LBD. Here we present cryo-electron microscopic structures of DNA-bound full-length AR and its functional complex structure with its key coactivators, steroid receptor coactivator-3 (SRC-3) and the histone acetyltransferase p300. The structures reveal that androgen-bound full-length AR dimerization follows a unique head-to-head and tail-to-tail manner with all of the domains involved. The DBD and LBD are located at the center of the dimer interface. The NTDs wrap around the LBDs through their unique intra- and inter-molecular N- and C-terminal interactions and connect to each other. We observe that unlike ERα, AR binds a single SRC-3 molecule along with a single p300 molecule. The AR NTD appears to be the primary site for recruitment of both coactivators. The N-terminal region of SRC-3, rather than its receptor interaction domain, is important for this interaction. The structures presented here highlight the importance of the AR NTD for its transcriptional functions and provide a structural basis for understanding the assembly of the AR:coactivator complex and its domain contributions to transcriptional regulation.

Pin1 inhibition improves the efficacy of ralaniten compounds that bind to the N-terminal domain of androgen receptor

Therapies for lethal castration-resistant prostate cancer (CRPC) are an unmet medical need. One mechanism underlying CRPC and resistance to hormonal therapies is the expression of constitutively active splice variant(s) of androgen receptor (AR-Vs) that lack its C-terminus ligand-binding domain. Transcriptional activities of AR-Vs and full-length AR reside in its N-terminal domain (NTD). Ralaniten is the only drug proven to bind AR NTD, and it showed promise of efficacy in Phase 1 trials. The peptidyl-prolyl isomerase Pin1 is frequently overexpressed in prostate cancer. Here we show that Pin1 interacted with AR NTD. The inhibition of Pin1 expression or its activity selectively reduced the transcriptional activities of full-length AR and AR-V7. Combination of Pin1 inhibitor with ralaniten promoted cell cycle arrest and had improved antitumor activity against CRPC xenografts in vivo compared to individual monotherapies. These findings support the rationale for therapy that combines a Pin1 inhibitor with ralaniten for treating CRPC.

Preclinical and clinical pharmacology of EPI-7386, an androgen receptor N-terminal domain inhibitor for castration-resistant prostate cancer.

119 Background: EPI-7386 is the newest of the “anitens”, a new class of compounds designed to inhibit androgen receptor activity by binding to the N-terminal domain (NTD) of the AR. Through this novel method of AR inhibition, anitens can block AR transcription even in the presence of AR ligand-binding domain (LBD) resistance mechanisms including point mutations and splice variants. Compared to the first generation aniten, EPI-506, which showed poor pharmacokinetic properties in patients, EPI-7386 is metabolically stable in vitro and in vivo. A Phase 1 clinical trial of EPI-7386 in metastatic castration-resistant prostate cancer patients failing standard of care therapies is ongoing and the pharmacokinetic properties of the drug in preclinical models as well as in the initial cohort of patients are presented. Methods: The metabolic stability of EPI-7386 was evaluated in vitro in mouse, rat, dog, monkey, and human hepatocytes. Projected PK parameters in humans were estimated from in vitro and in vivo clearance correlation (IVIVC). Induction of CYP isoforms was evaluated in human hepatocyte cultures. In patients, plasma concentrations of EPI-7386 were determined by LC-MS-MS, and 4-beta-hydroxycholesterol levels in plasma were followed over time as an indirect indicator of CYP3A induction. Results: In vitro hepatocyte studies demonstrated good metabolic stability for EPI-7386 with an in vitro half-life &gt; 360 min. In animal PK studies, the terminal half-life of EPI-7386 was approximately 5.8 hours in mouse, 4.9 hours in rat, 13.4 hours in dog and the plasma clearance was low across species. The oral bioavailability of EPI-7386 ranged from 33–112% in mouse to &gt; 100% in rat and dog. Using IVIVC, a predicted human clearance of 0.16–0.39 mL/min/kg was calculated for EPI-7386, which was in line with allometric scaling from animal PK parameters. Human PK profiles of different doses of EPI-7386 were simulated using predicted oral bioavailability, clearance, and volume of distribution. Cmax and AUC0–24h for the Phase 1 first-in-human study (NCT04421222) starting dose of 200 mg dose were predicted to be 6,915 ng/mL and 137,278 ng•h/mL respectively. A comparison between estimated PK parameters and actual values observed in the first patient cohort will be presented. Human hepatocyte CYP induction studies showed that EPI-7386 is not an inducer of CYP1A2 but may have the potential to induce CYP2B6 and CYP3A4. A comparison of 4-beta-hydroxy cholesterol levels measured during the phase 1 will be presented along with a comparison drawn from in vitro models. Conclusions: Pre-clinical characterization predicts that EPI-7386 has the appropriate PK and metabolic properties to afford exposure in patients at potentially efficacious levels following once-daily oral administration. PK measurements in the initial cohort of patients treated in the Phase 1 study will be presented. Clinical trial information: NCT04421222.

Structure-Activity Relationships for the Marine Natural Product Sintokamides: Androgen Receptor N-Terminus Antagonists of Interest for Treatment of Metastatic Castration-Resistant Prostate Cancer.

Synthetic analogues of the marine natural product sintokamides have been prepared in order to investigate the structure-activity relationships for the androgen receptor N-terminal domain (AR NTD) antagonist activity of the sintokamide scaffold. An in vitro LNCaP cell-based transcriptional activity assay with an androgen-driven luciferase (Luc) reporter was used to monitor the potency of analogues. The data have shown that the chlorine atoms on the leucine side chains are essential for potent activity. Analogues missing the nonchlorinated methyl groups of the leucine side chains (C-1 and C-17) are just as active and in some cases more active than the natural products. Analogues with the natural R configuration at C-10 and the unnatural R configuration at C-4 are most potent. Replacing the natural propionamide N-terminus cap with the more sterically hindered pivaloylamide N-terminus cap leads to enhanced potency. The tetramic acid fragment and the methyl ether on the tetramic acid fragment are essential for activity. The SAR optimized analogue 76 is more selective, easier to synthesize, more potent, and presumed to be more resistant to proteolysis than the natural sintokamides.

Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

The androgen receptor (AR) is a validated therapeutic target for prostate cancer and has been a focus for drug development for more than six decades. Currently approved therapies that inhibit AR signaling, such as enzalutamide, rely solely on targeting the AR ligand-binding domain and, therefore, have limited efficacy on prostate cancer cells that express truncated, constitutively active AR splice variants (AR-Vs). The LNCaP95 cell line is a human prostate cancer cell line that expresses both functional full-length AR and AR-V7. LNCaP95 is a heterogeneous cell population that is resistant to enzalutamide, with its proliferation dependent on transcriptionally active AR-V7. The purpose of this study was to identify a LNCaP95 clone that would be useful for evaluating therapies for their effectiveness against enzalutamide-resistant prostate cancer cells. Seven clones from the LNCaP95 cell line were isolated and characterized using morphology, in vitro growth rate, and response to ralaniten (AR N-terminal domain inhibitor) and enzalutamide (antiandrogen). In vivo growth of the clones as subcutaneous xenografts was evaluated in castrated immunodeficient mice. All of the clones maintained the expression of full-length AR and AR-V7. Cell proliferation of the clones was insensitive to androgen and enzalutamide but importantly was inhibited by ralaniten, which is consistent with AR-Vs driving the proliferation of parental LNCaP95 cells. In castrated immunodeficient animals, the growth of subcutaneous xenografts of the D3 clone was the most reproducible compared to the parental cell line and other clones. These data support that the enzalutamide-resistant LNCaP95-D3 subline may be suitable as a xenograft tumor model for preclinical drug development with improved reproducibility.

545P Preclinical profile of EPI-7386, a second-generation N-terminal domain androgen receptor inhibitor for the treatment of prostate cancer

Disease progression in metastatic castration-resistant prostate cancer (mCRPC) patients largely remains androgen receptor (AR) dependent. Resistance inevitably arises to current anti-androgens through numerous ligand binding domain (LBD)-associated mechanisms. EPI-7386 is a second-generation aniten which inhibits AR transcriptional activity by binding to the AR N-terminal domain (NTD), thus bypassing these resistance mechanisms. A phase I trial in patients with anti-androgen resistant mCRPC is currently being initiated. The preclinical anti-tumor and safety profile of EPI-7386 will be presented. EPI-7386 potency and selectivity was assessed using reporter and viability assays in cellular models. Pharmacodynamic markers were measured by qPCR, NanoString and RNAseq. The pre-clinical safety profile was assessed both in in vitro assays and in rat and dog toxicology studies. EPI-7386 has an IC50 of 421 nM in LNCaP reporter assays, it inhibited both AR full-length and AR-V7 dependent gene expression and maintained antitumor activity in AR-V7-expressing tumor models, including in in vivo models resistant to enzalutamide (ENZ). Transcriptomic analysis of EPI-7386 revealed on-target anti-AR activity, with a profile overlapping but distinct from ENZ, while the combination of the two agents demonstrated broader and deeper AR pathway inhibition. IND-enabling studies in rats and dogs showed on-target activity with good tolerability at drug exposures several fold higher than those active in tumor xenograft models. PK simulations suggest predicted tolerated and active doses can be achieved during the phase I dose escalation. EPI-7386, a second-generation aniten, is a potent AR NTD inhibitor, with a favorable safety and ADME profile, good pharmaceutical characteristics, and a long predicted human half-life supporting once-daily oral dosing with tablets. In vivo, EPI-7386 demonstrated anti-tumor activity as a single agent in the setting of anti-androgen clinical resistance and enhanced efficacy in combination therapy. A phase I study is scheduled to begin Q2 2020.

Combination therapy with androgen receptor N-terminal domain antagonist EPI-7170 and enzalutamide yields synergistic activity in AR-V7-positive prostate cancer.

Resistance of castration-resistant prostate cancer (CRPC) to enzalutamide and abiraterone involves the expression of constitutively active, truncated androgen receptor (AR) splice variants (AR-Vs) that lack a C-terminal ligand-binding domain (LBD). Both full-length AR and truncated AR-Vs require a functional N-terminal domain (NTD) for transcriptional activity thereby providing rationale for the development of ralaniten (EPI-002) as a first-in-class antagonist of the AR-NTD. Here, we evaluated the antitumor effect of a next-generation analog of ralaniten (EPI-7170) as a monotherapy or in combination with enzalutamide in prostate cancer cells that express AR-V7 that were resistant to enzalutamide. EPI-7170 had 8-9 times improved potency compared to ralaniten. Enzalutamide increased levels of AR-V7 and expression of its target genes. Knockdown of AR-V7 restored sensitivity to enzalutamide, indicating a role for AR-V7 in the mechanism of resistance. EPI-7170 inhibited expression of genes transcriptionally regulated by full-length AR and AR-V7. A combination of EPI-7170 and enzalutamide resulted in synergistic inhibition of proliferation of enzalutamide-resistant cells that was consistent with results from cell cycle and clonogenic assays. In addition, this drug enhanced the antitumor effect of enzalutamide in enzalutamide-resistant CRPC preclinical models. Thus, a combination therapy targeting both the NTD and LBD of AR, and thereby blocking both full-length AR and AR-Vs, has potential for the treatment of enzalutamide-resistant CRPC.

Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes

Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2 (AF-2) is a primary contributor to the nuclear receptor (NR) transcriptional activity. In contrast to other steroid receptors, such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here, we present cryoelectron microscopy (cryo-EM) structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR directly contacts a single SRC-3 and p300. The AR NTD is the primary site for coactivator recruitment. The structures provide a basis for understanding assembly of the AR:coactivator complex and its domain contributions for coactivator assembly and transcriptional regulation.

Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes

Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2(AF-2) is a primary contributor to the NR transcriptional activity. In contrast to other steroid receptors such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here we present cryoEM structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR binds a single SRC-3 and p300. The AR NTD is the primary site for both coactivator recruitment. The structures provide a basis for understanding the assembly of the AR:coactivator complex and its domain contributions for transcriptional regulation.

IND candidate EPI-7386 as an N-terminal domain androgen receptor inhibitor in development for the treatment of prostate cancer.

142 Background: The androgen receptor (AR) pathway drives most metastatic castration-resistant prostate cancers (mCRPC) even in late stages of the disease. Anti-androgen resistance mechanisms include AR gene amplification, C-terminal ligand-binding domain (LBD) mutations and expression of constitutively active AR splice variants lacking the LBD (e.g. AR-V7). Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its’ transcriptional activity even in the presence of LBD-driven resistance. In a phase I trial, the first-generation AR NTD inhibitor (aniten) EPI-002, demonstrated minor PSA declines in mCRPC patients. The efficacy and safety profile of second generation aniten IND-candidate EPI-7386 will be presented. Methods: Novel aniten chemical structures were developed to increase molecule potency using a wide variety of CRPC models. Similarly, the stability and selectivity of the molecule were characterized with screening and functional assays. IND-enabling studies further support the compounds’ safety profile. Results: EPI-7386 demonstrated a 20-fold improvement in AR-driven cellular potency compared to EPI-002, while being highly stable in human hepatocytes and across animal species. In vitro proliferation assays demonstrated on-target activity across a panel of prostate cancer cell lines, with activity in AR-V7-driven cellular models. EPI-7386 was able to induce tumor regression in CRPC xenografts and show superiority to enzalutamide (ENZ) in ENZ resistant models. In addition, the combination of ENZ with EPI-7386 demonstrated a more robust antitumor response. IND-enabling studies demonstrate a safe and well-tolerated profile supporting an IND filing in 1Q 2020. Pharmacodynamic markers specific to anitens will be presented, in addition to early clinical development plans. Conclusions: The next generation aniten compound EPI-7386 is more active and metabolically stable than EPI-002. It demonstrated potential as a single agent in overcoming anti-androgen clinical resistance as well as in combination therapy in earlier stages of the disease. The clinical strategy supporting the development of this new generation of aniten will be discussed.

Regression of castration-resistant prostate cancer by a novel compound QW07 targeting androgen receptor N-terminal domain.

Androgen deprivation therapy (ADT) via surgical or chemical castration frequently fails to halt lethal castration-resistant prostate cancer (CRPC), which is induced by multiple mechanisms involving constitutive androgen receptor (AR) splice variants, AR mutation, and/or de novo androgen synthesis. The AR N-terminal domain (NTD) possesses most transcriptional activity and is proposed as a potential target for CRPC drug development. We constructed a screening system targeting AR-NTD transcription activity to screening a compound library and identified a novel small molecule compound named QW07. The function evaluation and mechanism investigation of QW07 were carried out in vitro and in vivo. QW07 bound to AR-NTD directly, blocked the transactivation of AR-NTD, blocked interactions between co-regulatory proteins and androgen response elements (AREs), inhibited the expression of genes downstream of AR, and inhibited prostate cancer growth in vitro and in vivo. QW07 was demonstrated as an AR-NTD-specific antagonist with the potential to inhibit both canonical and variant-mediated AR signaling to regress the CRPC xenografts and is proposed as a lead compound for a specific antagonist targeting AR-NTD.

Targeting the Oncogenic Long Non-coding RNA SLNCR1 by Blocking Its Sequence-Specific Binding to the Androgen Receptor

Long non-coding RNAs (lncRNAs) are critical regulators of numerous physiological processes and diseases, especially cancers. However, development of lncRNA-based therapies is limited because the mechanisms of many lncRNAs are obscure, and interactions with functional partners, including proteins, remain uncharacterized. The lncRNA SLNCR1 binds to and regulates the androgen receptor (AR) to mediate melanoma invasion and proliferation in an androgen-independent manner. Here, we use biochemical analyses coupled with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing to show that the N-terminal domain of AR binds a pyrimidine-rich motif in an unstructured region of SLNCR1. This motif is predictive of AR binding, as we identify an AR-binding motif in lncRNA HOXA11-AS-203. Oligonucleotides that bind either the AR N-terminal domain or the AR RNA motif block the SLNCR1-AR interaction and reduce SLNCR1-mediated melanoma invasion. Delivery of oligos that block SLNCR1-AR interaction thus represent a plausible therapeutic strategy.

Abstract B117: Treatment of castrated resistant prostate cancer with EPI-7386, a second generation N-terminal domain androgen receptor inhibitor

Abstract Background: The androgen receptor (AR) pathway drives most metastatic castration-resistant prostate cancers (mCRPC) even in late stages of the disease and resistance to ligand binding domain (LBD)-linked therapies inevitably emerges. Mechanisms of resistance to anti-androgens include AR gene amplification, C-terminal ligand-binding domain (LBD) mutations and expression of constitutively-active truncated AR splice variants lacking the LBD (e.g. AR-V7). Inhibition of the N-terminal domain (NTD) of the AR can inhibit transcriptional activity even in the presence of LBD-driven resistance. A Phase I clinical trial of the first-generation AR NTD inhibitor, EPI-506, demonstrated minor PSA declines in anti-androgen refractory mCRPC patients, revealing the need for more potent and metabolically stable NTD inhibitors. EPI-7386 represents a second generation of NTD inhibitors (Anitens) that are more active and more metabolically stable than EPI-506. Methods: Chemical structure activity relationships were explored to increase molecule potency while maintaining target specificity using a wide variety of CRPC models in vivo and in vitro. The stability and selectivity of the molecules were characterized with a panel of selective screening and functional assays. The mechanism of action and pathway engagement markers were followed by qPCR and RNAseq. Results: EPI-7386 was chosen as the IND candidate. The molecule demonstrated a 20-fold improvement in AR-driven cellular potency compared to EPI-002, while being highly stable in human and animal hepatocytes. In vitro proliferation assays demonstrated on-target activity across a panel of prostate cancer cell lines, with activity demonstrated in AR-V7-driven cellular models. The antiproliferative effect correlated with the inhibitory effect on AR driven genes, including specifically AR-V7 driven genes such as UBE2C. EPI-7386 controlled tumor growth and induced tumor regressions in several CRPC xenografts, including AR-V7-driven 22Rv1 and enzalutamide resistant LNCaP95 models, as well as the VCaP model. Importantly, the combination of enzalutamide with EPI-7386 demonstrated a more robust and consistent PSA and antitumor response in the VCaP model. EPI-7386 was well tolerated in animal models, and therapeutic levels could be achieved without displaying signs of toxicity in a 14-day rat study. Pharmacodynamic markers will be presented, in addition to their incorporation in the future clinical plan. Conclusions: The next generation aniten compound EPI-7386 is more active and metabolically stable than EPI-506. It shows on-target activity in AR full length and AR-V7 driven models, and demonstrates a high therapeutic index in preclinical models. As a single agent, EPI-7386 may overcome anti-androgen clinical resistance in advanced mCRPC as well as potentially in combination therapy with anti-androgens in earlier stages of the disease. The clinical strategy supporting the development of this Aniten N-terminal domain inhibitor of AR will be discussed. Citation Format: Ronan Le Moigne, C. Adriana Banuelos, Nasrin R Mawji, Teresa Tam, Jun Wang, Kunzhong Jian, Raymond J Andersen, Alessandra Cesano, Marianne D Sadar, Han-Jie Zhou, Peter Virsik. Treatment of castrated resistant prostate cancer with EPI-7386, a second generation N-terminal domain androgen receptor inhibitor [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 B117. doi:10.1158/1535-7163.TARG-19-B117

503P - EPI-7386 is a novel N-terminal domain androgen receptor inhibitor for the treatment of prostate cancer

BackgroundThe androgen receptor (AR) pathway drives most metastatic castration-resistant prostate cancers (mCRPC) even in late stages of the disease. Anti-androgen resistance mechanisms include AR gene amplification, C-terminal ligand-binding domain (LBD) mutations and expression of constitutively-active truncated AR splice variants lacking the LBD (eg. AR-V7). Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its’ transcriptional activity even in the presence of LBD-driven resistance. A Phase I clinical trial of the first-generation AR NTD inhibitor, EPI-506, demonstrated minor PSA declines in mCRPC patients. EPI-7386 is a more potent and stable NTD inhibitor (Anitens) that is advancing to clinical trials. The compounds’ characteristics and the initial clinical study plans including potential biomarkers will be reviewed. MethodsChemical structure activity relationships were developed to increase molecule potency using a wide variety of CRPC models in vivo and in vitro. Similarly, the stability and selectivity of the molecule were characterized with screening and functional assays. Biomarkers were also explored. ResultsEPI-7386 demonstrated a 20 fold improvement in AR-driven cellular potency compared to EPI-002, while being highly stable in human and animal hepatocytes. In vitro proliferation assays demonstrated on-target activity across a panel of prostante cancer cell lines, with activity in AR-V7-driven cellular models. EPI-7386 was able to control tumor growth and induce tumor regressions in several CRPC xenografts, including enzalutamide resistant models. In addition, the combination of enzalutamide with EPI-7386 demonstrated a more robust and more homogeneuous antitumor response. Pharmacodynamic markers specific to NTD inhibitors will be presented. ConclusionsThe next generation aniten compound EPI-7386 is more active and more metabolically stable than EPI-002. It demonstrated potential as single agent in overcoming anti-androgen clinical resistance as well as in combination therapy in earlier stages of the disease. The clinical strategy supporting the development of this new generation of Aniten will be discussed. Legal entity responsible for the studyESSA Pharmaceuticals. FundingESSA Pharmaceuticals. DisclosureR. Le Moigne: Full / Part-time employment: ESSA pharma. C.A. Banuelos: Shareholder / Stockholder / Stock options: ESSA pharma. N.R. Mawji: Shareholder / Stockholder / Stock options: ESSA pharma. R.J. Andersen: Research grant / Funding (institution), Shareholder / Stockholder / Stock options, Officer / Board of Directors: ESSA pharma. A. Cesano: Advisory / Consultancy: ESSA pharma; Full / Part-time employment: Nanostring. M.D. Sadar: Speaker Bureau / Expert testimony, Leadership role, Research grant / Funding (institution), Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Officer / Board of Directors: ESSA pharma; Speaker Bureau / Expert testimony: Pfizer. H. Zhou: Shareholder / Stockholder / Stock options, Full / Part-time employment: ESSA pharma. P. Virsik: Shareholder / Stockholder / Stock options, Full / Part-time employment: ESSA pharma. All other authors have declared no conflicts of interest.

Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.

Abstract 1023: Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant prostate cancer

Abstract Androgen receptor (AR) signaling plays an essential role in all stages of prostate cancer. Androgen-deprivation therapy is generally effective for advanced prostate cancer until progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Most CRPC continues to be driven by AR signaling. AR transcriptional activity requires a functional N-terminal domain (NTD). Transactivation of AR is mediated by ligand-independent activation by cross-talk with signal transduction pathways targeting the AR NTD, gain-of-function mutations in AR ligand-binding domain (LBD), or expression of truncated AR splice variants that lack LBD (e.g., AR-V7). The intrinsically disordered AR NTD is essential for its transcriptional activity. It harbors six putative binding sites for Pin1, a proline isomerase that regulates protein conformation at specific phosphorylated-Ser/Thr-Pro motifs. All-trans retinoic acid (ATRA) is a validated and potent Pin1 inhibitor. Since conformational changes within the AR NTD are required for transactivation, perturbation of its structure may be a promising approach to block its activity. The purpose of this study was to assess a therapy that combined ATRA with antagonists of AR NTD, ralaniten (EPI-002) and its analogues. We hypothesized that targeting Pin1 activity with ATRA would disrupt the AR NTD and enhance inhibition by EPI compounds which bind to Tau-5 in the NTD. Using reporter gene assays, proliferation assays, and cell cycle analysis by flow cytometry, we tested ATRA in isolation and in combination with EPI in androgen-sensitive (LNCaP) and androgen-independent (LN95) prostate cancer cell lines. We found that treatment with ATRA decreased the transcriptional activity of AR and androgen-induced expression of PSA. ATRA also attenuated the transcriptional activity of AR-V7 and androgen-independent growth of LN95 cells expressing both full-length AR and AR-V7. Co-immunoprecipitation studies confirmed interactions between Pin1 and specific regions of AR NTD. In combination, ATRA had synergistic interaction with EPI compounds and lowered the effective inhibitory concentrations for blocking AR transcriptional activity. Furthermore, combinations decreased cell cycle progression of LN95 cells through S-phase, leading to accumulation of cells in G1 and induction of senescence. These preclinical findings showed that ATRA enhanced the potency of AR NTD inhibitors and support a novel therapeutic strategy for CRPC. Future studies will determine the in vivo antitumor effect of ATRA in combination with EPI compounds on the growth of CRPC xenografts. Citation Format: Jacky K. Leung, Marianne D. Sadar. Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant 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 1023.