Truncated Androgen Receptor Variants Research Articles

Targeting androgen receptor and the variants by an orally bioavailable Proteolysis Targeting Chimeras compound in castration resistant prostate cancer.

Despite the advent of improved therapeutic options for advanced prostate cancer, the durability of clinical benefits is limited due to inevitable development of resistance. By constitutively sustaining androgen receptor (AR) signaling, expression of ligand-binding domain truncated AR variants (AR-V(ΔLBD)) accounts for the major mechanism underlying the resistance to anti-androgen drugs. Strategies to target AR and its LBD truncated variants are needed to prevent the emergence or overcome drug resistance. We utilize Proteolysis Targeting Chimeras (PROTAC) technology to achieve induced degradation of both full-length AR (AR-FL) and AR-V(ΔLBD) proteins. In the ITRI-PROTAC design, an AR N-terminal domain (NTD) binding moiety is appended to von-Hippel-Lindau (VHL) or Cereblon (CRBN) E3 ligase binding ligand with linker. Invitro studies demonstrate that ITRI-PROTAC compounds mechanistically degrade AR-FL and AR-V(ΔLBD) proteins via ubiquitin-proteasome system, leading to impaired AR transactivation on target gene expression, and inhibited cell proliferation accompanied by apoptosis activation. The compounds also significantly inhibit enzalutamide-resistant growth of castration resistant prostate cancer (CRPC) cells. In castration-, enzalutamide-resistant CWR22Rv1 xenograft model without hormone ablation, ITRI-90 displays a pharmacokinetic profile with decent oral bioavailability and strong antitumor efficacy. AR NTD that governs the transcriptional activities of all active variants has been considered attractive therapeutic target to block AR signaling in prostate cancer cells. We demonstrated that utilizing PROTAC for induced AR protein degradation via NTD represents an efficient alternative therapeutic strategy for CRPC to overcome anti-androgen resistance. The funding detail can be found in the Acknowledgements section.

Abstract 2686: Is ARV7 a regulator of prostate cancer stemness and plasticity

Abstract Therapy options for advanced and castration-resistant prostate cancer (CRPC) include the use of drugs targeting directly/indirectly the ligand binding domain (LBD) of androgen receptor (AR) such as enzalutamide (Enza) and abiraterone acetate. However, primary or acquired treatment failures are frequently observed and have been associated with the expression of truncated AR variants devoid of the LBD. In particular, the AR variant AR3, also known as ARV7, is detected in over 80% of CRPC specimen and is increasingly expressed in metastatic lesions. In PC models ARV7 expression causes treatment resistance to Enza. Moreover, ARV7 expression leads to increased expression of mesenchymal markers and stem cell signature genes. CDH2 (N-Cadherin) expression is increased by ARV7 binding to regulatory elements in intron 1 of CDH2, which is antagonized by full length (FL) AR. Based on these observations we hypothesize that ARV7 is a marker and regulator of PC stemness and/or plasticity. ARFL or ARV7 mRNA and protein was detected in cell lines and tissue samples by standard procedures including qRT-PCR, RNA in situ hybridization (RISH), immunofluorescence (IF) and western blotting using the monoclonal ARV7 antibody RM7. ARFL or ARV7 stably expressing LNCaP were obtained by lentiviral transduction. LNCaP cells resistant to Enza (LNCaP-Enza) were generated by exposure to increasing concentrations (up to 5 µM) of the drug. Transcriptome analysis in LNCaP-Enza vs. LNCaP was done by RNAseq and Gene Set Enrichment Analysis. ARV7 mRNA was detectable in LNCaP, albeit gene expression was 2.3% - 2.6% of the known ARV7 positive cell lines 22Rv1 and VCaP, respectively. RISH and IF demonstrated that only a fraction (~10%) of LNCaP cells expressed ARV7 mRNA and nuclear localized ARV7 protein. ARV7 mRNA was also detected in isolated epithelial and stromal areas in treatment-naïve prostate cancer specimen by RISH. In contrast, all LNCaP-Enza cells were positive for nuclear ARV7 protein. Transcriptome analysis of LNCaP-Enza cells showed enrichments for published ARV7 gene expression signatures. Insular spots of ARV7 mRNA were detected in treatment-naïve tissue and in isolated cells of the castration-sensitive cell line LNCaP suggesting that these cells could be intrinsically resistant against LBD-targeting drugs and responsible for the failure of various endocrine therapies. This project will further analyze whether this subpopulation possesses stem cell (-like)/plasticity characteristics and whether these are regulated by ARV7. Citation Format: Tobias Furlan, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Is ARV7 a regulator of prostate cancer stemness and plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2686.

Abstract P192: Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Abstract Background: Androgen receptor (AR) signaling is a main driver of prostate cancer progression 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, and new methods of inhibiting the AR pathway are needed. 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 (NCT04421222). Here we further characterized the binding to AR NTD and the mechanism of action of EPI-7386. Methods: Target engagement was measured by Cellular Thermal Shift Assay (CETSA) and two-dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. qPCR, NanoString, and RNA sequencing were used to explore the activity of EPI-7386 on the AR transcriptome. To determine the effect of EPI-7386 on AR genomic occupancy, Chromatin immunoprecipitation sequencing (ChIP-seq) was performed. Results: We confirmed target engagement of EPI-7386 with an LBD truncated AR variant by CETSA using a cell line which expresses only AR-V567es, suggesting the interaction of EPI-7386 with AR NTD. In the same cell line, AR antagonist enzalutamide that binds to AR LBD showed no target engagement with AR-V567es. Furthermore, 2D NMR study results demonstrate an interaction of EPI-7386 with amino acid residues located in the transcription activation unit 5 (Tau-5) region of the AR NTD, a region which has been described to be involved in interactions with transcriptional cofactors such as CBP/p300. EPI-7386 strongly impaired the transcriptional activity and gene expression driven exclusively by LBD truncated AR variants including AR-V567es and AR-V7 and decreased cell viability. EPI-7386 has been shown to suppress the AR regulated transcriptome and the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-regulated gene expression. The analysis of the AR cistrome by ChIP-seq showed that EPI-7386 displaces genome-wide androgen induced AR binding and the combination with enzalutamide completely abrogated AR binding. 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. Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Berenger Biannic, Han-Jie Zhou, Ronan Le Moigne. Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P192.

Diverse AR Gene Rearrangements Mediate Resistance to Androgen Receptor Inhibitors in Metastatic Prostate Cancer.

Prostate cancer is the second leading cause of male cancer deaths. Castration-resistant prostate cancer (CRPC) is a lethal stage of the disease that emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. The purpose of this study was to identify genomic mechanisms that contribute to the development and progression of CRPC. We used whole-genome and targeted DNA-sequencing approaches to identify mechanisms underlying CRPC in an aggregate cohort of 272 prostate cancer patients. We analyzed structural rearrangements at the genome-wide level and carried out a detailed structural rearrangement analysis of the AR locus. We used genome engineering to perform experimental modeling of AR gene rearrangements and long-read RNA sequencing to analyze effects on expression of AR and truncated AR variants (AR-V). AR was among the most frequently rearranged genes in CRPC tumors. AR gene rearrangements promoted expression of diverse AR-V species. AR gene rearrangements occurring in the context of AR amplification correlated with AR overexpression. Cell lines with experimentally derived AR gene rearrangements displayed high expression of tumor-specific AR-Vs and were resistant to endocrine therapies, including the AR antagonist enzalutamide. AR gene rearrangements are an important mechanism of resistance to endocrine therapies in CRPC.

Modulation of androgen receptor DNA binding activity through direct interaction with the ETS transcription factor ERG

The androgen receptor (AR) is a type I nuclear hormone receptor and the primary drug target in prostate cancer due to its role as a lineage survival factor in prostate luminal epithelium. In prostate cancer, the AR cistrome is reprogrammed relative to normal prostate epithelium and particularly in cancers driven by oncogenic ETS fusion genes. The molecular basis for this change has remained elusive. Using purified proteins, we report a minimal cell-free system that demonstrates interdomain cooperativity between the ligand (LBD) and DNA binding domains (DBD) of AR, and its autoinhibition by the N terminus of AR. Furthermore, we identify ERG as a cofactor that activates AR's ability to bind DNA in both high and lower affinity contexts through direct interaction within a newly identified AR-interacting motif (AIM) in the ETS domain, independent of ERG's own DNA binding ability. Finally, we present evidence that this interaction is conserved among ETS factors whose expression is altered in prostate cancer. Our work highlights, at a biochemical level, how tumor-initiating ETS translocations result in reprogramming of the AR cistrome.

SAT-330 Preclinical Screen of Patient CRPC Tumors Shows Sensitivity to Bromodomain Inhibitors Includes AR-null and Neuroendocrine Disease

Castration-resistant prostate cancer (CRPC) remains a lethal disease despite the introduction of second generation androgen receptor (AR) directed therapies. Resistance to AR directed therapies, such as abiraterone and enzalutamide, emerges through diverse alterations in the AR pathway or transition to aggressive AR-null phenotypes. Although bromodomain and extra-terminal (BET) inhibitor therapies are in current clinical trials for CRPC, understanding of the full spectrum of patient responders is limited by a lack of models for preclincial testing. Thus, the goal of this study was to examine the efficacy of BET inhibitors in a panel of patient-derived models of CRPC, including AR-null and neuroendocrine differentiated tumors that are inherently resistant to AR-directed therapy. To rapidly screen the sensitivity of tumors to BET inhibitors, we used ex vivo cultures of patient-derived xenograft tissue from the MURAL (Melbourne Urological Research Alliance) cohort of patient tumors and treated them with JQ1. Changes in proliferation and apoptosis were measured using histology for Ki67 and cleaved caspase 3. Consistent with previous studies, our results showed that all AR-positive tumors were sensitive to JQ1, with decreases in proliferation and increases in apoptosis. Importantly, this includes tumors from patients who failed abiraterone and/or enzalutamide and have diverse alterations of the AR, including mutations, genomic amplifications and expression of truncated AR variants. Surprisingly, 50% of AR null tumors were just as sensitive to BET inhibitors and AR-positive tumors. This includes tumors with large cell and small cell neuroendocrine histopathology, which are inherently resistant to AR-directed therapies. We also assessed intra-patient heterogeneity by examining tumors from different metastases of the same patient. We observed consistent responses in one patient, but divergent responses in another, likely due to differences in AR status. Collectively, our data show that BET inhibitors are promising treatments for tumors with diverse mechanisms of resistance to AR-directed therapies; however, the responses of AR-null tumors are more heterogeneous. Therefore, further studies to identify biomarkers of responsiveness to BET inhibitors in AR-null and neuroendocrine tumors are warranted.

Contribution of Adrenal Glands to Intratumor Androgens and Growth of Castration-Resistant Prostate Cancer.

Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo. We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients. Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3). Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true "nonsurgical ADX" are warranted.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Highlights of This Issue

The EWS/FLI (EF) fusion protein is well known as an aberrant transcription factor that drives oncogenesis in Ewing sarcoma by inducing widespread transcriptional reprogramming. In this issue, Tanner and colleagues explore the previously unstudied role of EF in driving metabolic misregulation. Silencing of EF resulted in significant changes in the global metabolic profile and glucose utilization. Additionally, it was demonstrated that EF is required for high expression of de novo serine synthesis and 1-carbon metabolism pathways, which correlated with worse patient survival. These findings expand the understanding of Ewing sarcoma biology and offer potential new avenues of therapeutic development.Incurable castration-resistant prostate cancer (CRPC) is often accompanied by unrestrained androgen receptor (AR) signaling driven by (currently undruggable) constitutively active, C-terminally truncated AR variants (e.g., AR-V7); which retain the potent transactivating N-terminal domain of AR. Transcriptional activities of AR and variants are enhanced by select coactivators (e.g., Vav3) with elevated expression in CRPC. Magani and colleagues demonstrate that Vav3 (via its DH domain) interacts with the N-termini of AR and AR-V7. Vav3 DH domain overexpression disrupts coactivator interactions with and transcriptional activities of AR-V7 and AR, decreasing CRPC aggressiveness. This study identifies AR N-terminal coactivator interactions as exploitable vulnerabilities in CRPC.Spontaneous nuclear envelope (NE) rupture has been observed in several cancer cell lines but does not occur in untransformed human cells. NE rupture can lead to mixing of nuclear and cytoplasmic factors and may compromise genome integrity. Yang and colleagues have identified the tumor suppressor proteins Rb and p53 as suppressors of NE rupture. Depletion of either Rb or p53 increased the frequency of NE rupture in primary cells. This finding reveals novel protective functions of Rb and p53 and provides a mechanistic explanation for the occurrence of NE rupture in cancer cells.Glioblastoma (GBM) is an invasive and malignant brain cancer. Heparan sulfate proteoglycans (HSPG), present on cell surfaces and in the extracellular matrix (ECM), regulate cell signaling and cell-microenvironment interactions. Heparan sulfate (HS) expression and enzymes that modulate their function are altered in GBM, but the actual HS content and structure are unknown. Using liquid chromatography-mass spectrometry (LC/MS), Tran and colleagues define the patterns of HS structure and their modification in patient-derived tumorspheres. Using this approach, a novel role is uncovered for the HS-modifying enzyme heparanase in GBM invasion. These results suggest that HS-interacting factors are potential therapeutic targets in GBM.

Novel Androgen Receptor Coregulator GRHL2 Exerts Both Oncogenic and Antimetastatic Functions in Prostate Cancer.

Alteration to the expression and activity of androgen receptor (AR) coregulators in prostate cancer is an important mechanism driving disease progression and therapy resistance. Using a novel proteomic technique, we identified a new AR coregulator, the transcription factor Grainyhead-like 2 (GRHL2), and demonstrated its essential role in the oncogenic AR signaling axis. GRHL2 colocalized with AR in prostate tumors and was frequently amplified and upregulated in prostate cancer. Importantly, GRHL2 maintained AR expression in multiple prostate cancer model systems, was required for cell proliferation, enhanced AR's transcriptional activity, and colocated with AR at specific sites on chromatin to regulate genes relevant to disease progression. GRHL2 is itself an AR-regulated gene, creating a positive feedback loop between the two factors. The link between GRHL2 and AR also applied to constitutively active truncated AR variants (ARV), as GRHL2 interacted with and regulated ARVs and vice versa. These oncogenic functions of GRHL2 were counterbalanced by its ability to suppress epithelial-mesenchymal transition and cell invasion. Mechanistic evidence suggested that AR assisted GRHL2 in maintaining the epithelial phenotype. In summary, this study has identified a new AR coregulator with a multifaceted role in prostate cancer, functioning as an enhancer of the oncogenic AR signaling pathway but also as a suppressor of metastasis-related phenotypes. Cancer Res; 77(13); 3417-30. ©2017 AACR.

Abstract 3522: Structural characterization of androgen receptor variant 7 in prostate cancer

Abstract Prostate cancer is an extremely common disease that affects one in every seven men in their lifetime. The standard care for late-stage cancer is designed to inhibit the activation of androgen receptor (AR). While this therapeutic approach is initially efficient, the cancer almost always develops resistance and gives rise to castration-resistant prostate cancer (CRPC). When therapy fails, the median survival of patients who suffer from CRPC is 12-16 months. It is critical to understand the mechanism of CRPC better to ameliorate the outcome of late-stage prostate cancer patients. AR is a transcription factor that consists of an N-terminal domain (NTD), a DNA binding domain (DBD) and a ligand binding domain (LBD). Upon activation by androgen, an intramolecular interaction between NTD and LBD occurs. The AR translocates into the nucleus where it forms a homodimer. It binds to the promoter and enhancer elements of the AR target genes and triggers gene transcription. It has been demonstrated in many clinical studies that AR is important for the progression of prostate cancer including CRPC. In CRPC, although androgen production and binding is inhibited, how the AR can still be activated is a fundamental question to be answered. One of the several different mechanisms proposed is the alternative splicing of the AR generating truncated AR variants that lack LBD. These variants do not require androgens to be activated and they are intrinsically resistant to clinically approved therapeutics. Despite the fact that the AR variants have been shown to be critical in late-stage prostate cancer, the mechanism of gene transcription initiation by the AR variants is not well-known. We propose to characterize the mechanism of the AR variant activation. Since ARV7 is the most commonly observed variant, we utilize ARV7. We develop a FRET based methodology to identify how ARV7 dimers form and to test if full-length AR is necessary for transcriptional activation mediated by ARV7. We are able to demonstrate possible intermolecular interaction between the full length AR and ARV7 measuring the FRET intensity between the fluorophores attached to the N and C terminus of two proteins. FRET measurements are analyzed by tracking single cells and recording the FRET intensities during the process. Our preliminary data suggest that the full-length AR and ARV7 may interact in the presence of androgen. ARV7 is always localized in the nucleus whereas the AR requires androgen to be activated and translocates into the nucleus. Being in close proximity, putative interaction between the AR and ARV7 occurs in the nucleus. Additionally, we use NTD and DBD point mutants to determine critical positions of intermolecular interaction between them. Supporting the significance of ARV7 in prostate cancer, our results clarify the dimerization process. It gives an opportunity to test clinical drugs currently in development to effectively inhibit ARV7 activation. Citation Format: Fatma Özgün, Zeynep Kaya, Halil Bayraktar, Selen Manioğlu, Doğancan Özturan, Nathan Lack. Structural characterization of androgen receptor variant 7 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3522. doi:10.1158/1538-7445.AM2017-3522

Androgen Receptor Rearrangement and Splicing Variants in Resistance to Endocrine Therapies in Prostate Cancer.

In the last few years, the survival of patients with castration-resistant prostate cancer (CRPC) has significantly improved as a result of the development of second-generation androgen deprivation therapies such as abiraterone and second-generation antagonists such as enzalutamide. However, CRPC patients rapidly develop resistance to these drugs, in many cases because of reactivation of the therapeutic target, the androgen receptor (AR) transcription factor. Several mechanisms responsible for AR transcriptional reactivation have been demonstrated, including mutation, amplification, and rearrangement of the AR gene, transcriptional compensation by alternative steroid receptors, and mutation or copy number alteration of genes encoding AR coregulators. In addition, CRPC tumors display elevated expression of truncated AR variants (AR-Vs) that can arise from alternative splicing or underlying AR gene rearrangements. In this review, we discuss general mechanisms of resistance to androgen/AR-targeted therapies, with a focus on the role of AR-Vs in conferring resistance to abiraterone or enzalutamide in CRPC patients.

MP84-15 PREVENT METASTASIS BY DOUBLE CHIMERIC ANTIGEN RECEPTOR T CELLS TARGETING CIRCULATING CANCER CELLS FOR PROSTATE CANCER TREATMENT

INTRODUCTION AND OBJECTIVES: We and others have demonstrated that the androgen receptor (AR) is essential for the development and progression of prostate cancer. Although androgendeprivation therapy (ADT) is effective initially, unfortunately, metastatic prostate cancer often recurs as so-called castration resistant prostate cancer (CRPC). Overexpression of AR, AR hypersensitivity, c-terminal truncated AR variants, as well as altered steroid metabolism are considered the main mechanisms for CRPC. Current anti-AR treatments (Abiraterone and Enzalutamide) for CRPC only extend patient survival for approximately 5 months, indicating novel therapeutic agents are urgently needed. It has been shown that AR variants possess an intact and functional N-terminal domain that contains several key motifs and tyrosine residues involved in AR transactivation. METHODS: We recently designed a 14-amino acid peptide spanning the 355-368 residues (ARi14 peptide) on AR N-terminal, consisting of the partial transactivation activation unit-1 and three key tyrosine residues involved in CRPC progression. Multiple prostate cancer cell lines were used in the experiments. Trypan blue and Alamar Blue assays were used to assess cell proliferation. Western blot and anti-AR immunoprecipitation were conducted to assess AR protein levels and tyrosine phosphorylation. Quantitative RT-PCR was performed to assess PSA mRNA expression. RESULTS: Treatment of multiple AR-positive prostate cancer cells with this short peptide induced a significant reduction of AR protein, as well as AR transactivation in a luciferase reporter assay in a time-dependent manner. However, AR gene expression at the mRNA level was not altered. Further analysis revealed that ARi14 peptide abolished androgen-stimulated AR tyrosine phosphorylation in LNCaP cells, indicating a disruption of AR with its tyrosine kinase. In 22RV1 cells that harbor AR variants the ARi14 peptide dramatically reduced the protein levels of full length AR and AR variants, abolished AR-target PSA gene expression and also suppressed 22RV1 cell proliferation that Enzalutamide failed to do so. Sequence mutation analysis demonstrated that the C-terminal 8 residue portion (361-368aa) of the ARi14 is the key element for its anti-AR activities. CONCLUSIONS: Our data demonstrated that the novel short peptide ARi14 exerts a potent anti-AR activity by inducing AR protein degradation possibly through reduced tyrosine phosphorylation in Enzalutamide-resistant prostate cancer cells.

MP84-14 OVERCOMING ENZALUTAMIDE-RESISTANT WITH A NOVEL ANTI-AR PEPTIDE IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-14 OVERCOMING ENZALUTAMIDE-RESISTANT WITH A NOVEL ANTI-AR PEPTIDE IN PROSTATE CANCER CELLS Daniel Zainfeld, Ruibao Chen, J Brantley Thrasher, and Benyi Li Daniel ZainfeldDaniel Zainfeld More articles by this author , Ruibao ChenRuibao Chen More articles by this author , J Brantley ThrasherJ Brantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2238AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We and others have demonstrated that the androgen receptor (AR) is essential for the development and progression of prostate cancer. Although androgen-deprivation therapy (ADT) is effective initially, unfortunately, metastatic prostate cancer often recurs as so-called castration resistant prostate cancer (CRPC). Overexpression of AR, AR hypersensitivity, c-terminal truncated AR variants, as well as altered steroid metabolism are considered the main mechanisms for CRPC. Current anti-AR treatments (Abiraterone and Enzalutamide) for CRPC only extend patient survival for approximately 5 months, indicating novel therapeutic agents are urgently needed. It has been shown that AR variants possess an intact and functional N-terminal domain that contains several key motifs and tyrosine residues involved in AR transactivation. METHODS We recently designed a 14-amino acid peptide spanning the 355-368 residues (ARi14 peptide) on AR N-terminal, consisting of the partial transactivation activation unit-1 and three key tyrosine residues involved in CRPC progression. Multiple prostate cancer cell lines were used in the experiments. Trypan blue and Alamar Blue assays were used to assess cell proliferation. Western blot and anti-AR immunoprecipitation were conducted to assess AR protein levels and tyrosine phosphorylation. Quantitative RT-PCR was performed to assess PSA mRNA expression. RESULTS Treatment of multiple AR-positive prostate cancer cells with this short peptide induced a significant reduction of AR protein, as well as AR transactivation in a luciferase reporter assay in a time-dependent manner. However, AR gene expression at the mRNA level was not altered. Further analysis revealed that ARi14 peptide abolished androgen-stimulated AR tyrosine phosphorylation in LNCaP cells, indicating a disruption of AR with its tyrosine kinase. In 22RV1 cells that harbor AR variants the ARi14 peptide dramatically reduced the protein levels of full length AR and AR variants, abolished AR-target PSA gene expression and also suppressed 22RV1 cell proliferation that Enzalutamide failed to do so. Sequence mutation analysis demonstrated that the C-terminal 8 residue portion (361-368aa) of the ARi14 is the key element for its anti-AR activities. CONCLUSIONS Our data demonstrated that the novel short peptide ARi14 exerts a potent anti-AR activity by inducing AR protein degradation possibly through reduced tyrosine phosphorylation in Enzalutamide-resistant prostate cancer cells. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1095 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Daniel Zainfeld More articles by this author Ruibao Chen More articles by this author J Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.

Molecular Pathways: Targeting Resistance in the Androgen Receptor for Therapeutic Benefit

Androgen receptor signaling is critical in the development and progression of prostate cancer, leading to intensive efforts to elucidate all potential points of inflection for therapeutic intervention. These efforts have revealed new mechanisms of resistance and raise the possibility that known mechanisms may become even more relevant in the context of effective androgen receptor suppression. These mechanisms include tumoral appropriation of alternative androgen sources, alterations in androgen receptor expression, androgen receptor mutations, truncated androgen receptor variants, alterations and cross-talk in recruitment of cofactors to androgen receptor binding sites in the genome, and androgen receptor-driven oncogenic gene fusions. New agents such as enzalutamide, EPI-001, androgen receptor-specific peptidomimetics, novel HSP90 inhibitors, and PARP inhibitors, as well as new approaches to cotargeting the androgen receptor pathway, point to the potential for more complete and durable control of androgen receptor-mediated growth.

Constitutively-active androgen receptor variants function independently of the HSP90 chaperone but do not confer resistance to HSP90 inhibitors

The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer.

Androgen Receptor Gene Rearrangements: New Perspectives on Prostate Cancer Progression

The androgen receptor (AR) is a master regulator transcription factor in normal and cancerous prostate cells. Canonical AR activation requires binding of androgen ligand to the AR ligand binding domain, translocation to the nucleus, and transcriptional activation of AR target genes. This regulatory axis is targeted for systemic therapy of advanced prostate cancer. However, a new paradigm for AR activation in castration-resistant prostate cancer (CRPC) has emerged wherein alternative splicing of AR mRNA promotes synthesis of constitutively active AR variants that lack the AR ligand binding domain (LBD). Recent work has indicated that structural alteration of the AR gene locus represents a key mechanism by which alterations in AR mRNA splicing arise. In this review, we examine the role of truncated AR variants (ARVs) and their corresponding genomic origins in models of prostate cancer progression, as well as the challenges they pose to the current standard of prostate cancer therapies targeting the AR ligand binding domain. Since ARVs lack the COOH-terminal LBD, the genesis of these AR gene rearrangements and their resulting ARVs provides strong rationale for the pursuit of new avenues of therapeutic intervention targeted at the AR NH2-terminal domain. We further suggest that genomic events leading to ARV expression could act as novel biomarkers of disease progression that may guide the optimal use of current and next-generation AR-targeted therapy.

Effects of Sorafenib on C-Terminally Truncated Androgen Receptor Variants in Human Prostate Cancer Cells

Recent evidence suggests that the development of castration resistant prostate cancer (CRPCa) is commonly associated with an aberrant, ligand-independent activation of the androgen receptor (AR). A putative mechanism allowing prostate cancer (PCa) cells to grow under low levels of androgens, is the expression of constitutively active, C-terminally truncated AR lacking the AR-ligand binding domain (LBD). Due to the absence of a LBD, these receptors, termed ARΔLBD, are unable to respond to any form of anti-hormonal therapies. In this study we demonstrate that the multikinase inhibitor sorafenib inhibits AR as well as ARΔLBD-signalling in CRPCa cells. This inhibition was paralleled by proteasomal degradation of the AR- and ARΔLBD-molecules. In line with these observations, maximal antiproliferative effects of sorafenib were achieved in AR and ARΔLBD-positive PCa cells. The present findings warrant further investigations on sorafenib as an option for the treatment of advanced AR-positive PCa.

Androgen Receptor Splice Variants Activate Androgen Receptor Target Genes and Support Aberrant Prostate Cancer Cell Growth Independent of Canonical Androgen Receptor Nuclear Localization Signal

Synthesis of truncated androgen receptor (AR) splice variants has emerged as an important mechanism of prostate cancer (PCa) resistance to AR-targeted therapy and progression to a lethal castration-resistant phenotype. However, the precise role of these factors at this stage of the disease is not clear due to loss of multiple COOH-terminal AR protein domains, including the canonical nuclear localization signal (NLS) in the AR hinge region. Despite loss of this NLS, we show that diverse truncated AR variant species have a basal level of nuclear localization sufficient for ligand-independent transcriptional activity. Whereas full-length AR requires Hsp90 and importin-β for active nuclear translocation, basal nuclear localization of truncated AR variants is independent of these classical signals. For a subset of truncated AR variants, this basal level of nuclear import can be augmented by unique COOH-terminal sequences that reconstitute classical AR NLS activity. However, this property is separable from ligand-independent transcriptional activity. Therefore, the AR splice variant core consisting of the AR NH(2)-terminal domain and DNA binding domain is sufficient for nuclear localization and androgen-independent transcriptional activation of endogenous AR target genes. Indeed, we show that truncated AR variants with nuclear as well as nuclear/cytoplasmic localization patterns can drive androgen-independent growth of PCa cells. Together, our data demonstrate that diverse truncated AR species with varying efficiencies of nuclear localization can contribute to castration-resistant PCa pathology by driving persistent ligand-independent AR transcriptional activity.