Androgen Receptor Variants Research Articles

Abstract 5508: Artesunate increases enzalutamide efficacy in advanced prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed worldwide. PCa development and progression require androgen receptor (AR) signaling, which stimulates its downstream gene expressions and cancer progression. While second-generation anti-androgen drugs plus androgen deprivation therapy (ADT) remains the first-line treatment for advanced prostate cancer patients, around one-third of patients will relapse in a short period. Advanced prostate cancer results in more mortalities than primary PCa patients. Evidence shows that the recurrence is caused by AR overexpression, AR variants, AR mutations, and signaling crosstalk. Thus, it is urgently needed to discover a novel therapeutic strategy for treating advanced prostate cancer. The heat shock protein family (HSP), including HSP90 and HSP70, play important roles in refolding aggregated protein for cancer cell proteomic equilibrium. HSP is induced primarily by heat shock factor (HSF1). As AR’s chaperone protein, HSP70 and HSP90 increase AR transcription activity. Inhibiting HSP70 and HSP90 promotes STUB1, an E3 ligase, binding to AR and AR - V7, and ubiquitination. Artesunate (ART) is a semi-synthetic ingredient from Artemisia annua and is the most common treatment for malaria throughout the world. It was approved for medical use by the FDA (Food and Drug Administration) (Food and Drug Administration). Recently ART has been unveiled for its anticancer properties. However, the efficacy of ART treatment in advanced prostate cancer and the direct target of ART have not been investigated yet. Herein, we have examined the efficacy of combining Enzalutamide (Enza) and ART in advanced prostate cancer cell lines. We also performed unbiased bioinformatics analysis using RNA seq results in enzalutamide-resistant cell line C4-2R cells and 22RV1 cells to investigate the cell response toward ART treatment. We identified ART could downregulate of AR signaling pathway. Moreover, we determined that ART treatment induces AR degradation in proteasome dependent manner. Interestingly, we found HSP70 and HSP90 are also decreased in RNA seq results. Taking these together suggests that ART may target HSF1 directly. Our results suggest ART induced AR degradation could be a promising clinical strategy for advanced prostate cancer. Citation Format: Xinyi Wang, Fengyi Mao, Jinghui Liu, Yifan Kong, Dang he, Chi Wang, Zhiguo Li, Xiaoqi Liu. Artesunate increases enzalutamide efficacy in advanced prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5508.

Abstract 6542: Detection of androgen receptor splice variants from clinical sequencing

Abstract Androgen receptor (AR) splice variants (AR-Vs) have been widely studied on its important role in prostate cancer (PC) progression. In particular, AR-Vs lacking ligand binding domains, such as AR splice variant 7 (AR-v7) that links exon 3 and cryptic exon 3 or AR variant that losses exons 5 to 7 (ARv567es), have been demonstrated as one of the resistant mechanisms to androgen deprivation therapy in PC. Despite its clinical importance, the current algorithms to detect fusions such as TopHat-Fusion have limitation to detect intra-chromosomal rearrangements including AR-Vs. In this respect, we propose a novel approach to identify AR-Vs from targeted RNA sequencing (RNA-seq) dataset. In short, our two-step approach first gathers soft-clipped or divided reads that are adjacent to the splicing sites of AR-Vs and then yields the number of splitting reads that support the existence of AR-Vs. Next, the algorithm selects the paired-end reads whose one side and the other side are mapped to the preceding and following exons, respectively. The final number of spanning reads are calculated following to the removal of low-quality reads. We validated the proposed approach using two large-scale, independent RNA-seq datasets of PC samples: 34 samples from Seoul National University Hospital (SNUH) and 558 samples from The Cancer Genome Atlas (TCGA) dataset. Overall, our approach successfully identified samples with putative AR-Vs, including AR-v7 and ARv567es. In addition, our approach successfully detected AR-v7 with reliable number of supporting reads, from the RNA-seq dataset of AR-v7-expressing cell line. Finally, statistical analyses of 34 SNUH PC patients suggested potential detection threshold in clinical sequencing settings (at least 10 split reads and 1% proportion). In the SNUH dataset, The AR-v7 positive group, which included 6 patients (17.1%) with values above the threshold, had higher AR-v7 expression levels than the negative group (p=4.8E-04). The similar pattern was also found in the analysis of TCGA dataset. Owing to its flexibility, we also confirmed that the developed approach can be used to detection of other AR-Vs, including AR-v3 and ARv567es. We expect that the further in-depth analyses including larger samples and clinical outcomes can discover clinical applicability of AR-Vs. Citation Format: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun. Detection of androgen receptor splice variants from clinical sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6542.

Abstract 4802: Identification of critical hypoxia induced factors in castrate resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the second most diagnosed cancer in men and the second cause of cancer-related death amongst men worldwide. PCa is a heterogeneous disease, and the outcome is worse for patients when the disease progresses from localized PCa to a castrate-resistant disease. Androgen receptor (AR) signaling is crucial for PCa development and has been a major therapeutic target for decades. Despite the success made with hormone therapy to block AR signaling, castration resistant disease can arise through mutations and amplifications of the androgen receptor (AR) and AR splice variants (AR-Vs). Amongst them, AR-V7 and AR-V12 can promote AR signaling independent of androgen. Furthermore, prostate tumors are highly hypoxic due to abnormal tumor vasculature. Hypoxia influences the efficacy of local and systemic treatment strategies, such as radiotherapy, hormone therapy, and chemotherapy, resulting in poorer clinical outcomes for PCa patients. Therefore, a complete understanding of AR and AR variant function, as well as how they are modulated by hypoxia and therapy is warranted for developing new strategies to treat PCa. Methods: To identify the transcriptional apparatus associated with the AR and its variants under normal and hypoxic conditions, we carried out Bio-ID proximity labeling mass spectrometry (BioID-MS). We generated stable androgen-independent human prostate cancer cell lines, PC-3 and DU145, expressing AR and the AR-V7 and AR-V12 variants fused to a MiniTurboID (MTID) enzyme. To delineate the AR proximal interaction network, we treated cells with androgen or vehicle for 3 hours and affinity-purified biotinylated proteins after the addition of biotin for the final 2 hours. Cells were grown in normoxia (20% O2) or treated with hypoxia (95% N2,5% CO2 and 0.5% O2) for 24 h. Nonspecific interactions were filtered using controls and statistical methods to identify high-confidence interactomes. Results: BioID proximity labeling in PC3 cells identified several AR-associated proteins, including many knowninteractors, validating the approach. Hypoxic conditions led to a striking alteration in proximal interactions and many metabolic proteins were highly enriched with AR upon exposure of cells to hypoxia for 24h. Among these, phosphoglycerate kinase 1 (PGK1) was a top hit. PGK1 has been reported to interact with AR to mediate the expression of genes that regulates cell proliferation and apoptosis. Additionally, PGK1 has an essential role in metabolic reprogramming induced by c-MYC and HIF-1α, leading to enhanced tumorigenesis. Conclusion: Bio-ID mass spectrometry was used to map the proximal interactome of AR and its variants, identifying novel interactions partners for further analysis. We found that hypoxia strongly alters interactors with the AR, and we identified PGK1 as a hypoxia induced AR interaction in PCA. Current work to functionally validate this interaction will be presented. Citation Format: Oloruntoba I. Osagie, Jordann Smakk, Deborah E. Citrin, Travis H. Stracker. Identification of critical hypoxia induced factors in castrate resistant prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4802.

α-Mangostin Promotes In Vitro and In Vivo Degradation of Androgen Receptor and AR-V7 Splice Variant in Prostate Cancer Cells.

A major limitation of current prostate cancer pharmacotherapy approaches is the inability of these compounds to target androgen receptor variants or mutants that develop during prostate cancer progression. The demand for novel therapeutics to prevent, slow, and treat prostate cancer is significant because FDA approved anti-androgens are associated with adverse events and can eventually drive drug-resistant prostate cancer. This study evaluated α-mangostin for its novel ability to degrade the androgen receptor and androgen receptor variants. α-Mangostin is one of more than 70 isoprenylated xanthones isolated from Garcinia mangostana that we have been evaluating for their anticancer potential. Prostate cancer cells treated with α-mangostin exhibited decreased levels of wild-type and mutated androgen receptors. Immunoblot, immunoprecipitation, and transfection experiments demonstrated that the androgen receptor was ubiquitinated and subsequently degraded via the proteasome, which we hypothesize occurs with the assistance of BiP, an ER chaperone protein that we have shown to associate with the androgen receptor. We also evaluated α-mangostin for its antitumor activity and promotion of androgen receptor degradation in vivo. In summary, our study demonstrates that androgen receptor degradation occurs through the novel activation of BiP and suggests a new therapeutic approach for prostate cancer.

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.

A compendium of Androgen Receptor Variant 7 target genes and their role in Castration Resistant Prostate Cancer.

Persistent androgen receptor (AR) signalling is the main driver of prostate cancer (PCa). Truncated isoforms of the AR called androgen receptor variants (AR-Vs) lacking the ligand binding domain often emerge during treatment resistance against AR pathway inhibitors such as Enzalutamide. This review discusses how AR-Vs drive a more aggressive form of PCa through the regulation of some of their target genes involved in oncogenic pathways, enabling disease progression. There is a pressing need for the development of a new generation of AR inhibitors which can repress the activity of both the full-length AR and AR-Vs, for which the knowledge of differentially expressed target genes will allow evaluation of inhibition efficacy. This review provides a detailed account of the most common variant, AR-V7, the AR-V7 regulated genes which have been experimentally validated, endeavours to understand their relevance in aggressive AR-V driven PCa and discusses the utility of the downstream protein products as potential drug targets for PCa treatment.

Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4–2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

Androgen receptor variant 7 exacerbates hepatocarcinogenesis in a c-MYC-driven mouse HCC model

Androgen receptor variant 7 (AR-V7), an AR isoform with a truncated ligand-binding domain, functions as a transcription factor in an androgen-independent manner. AR-V7 is expressed in a subpopulation of hepatocellular carcinoma (HCC), however, its role(s) in this cancer is undefined. In this study, we investigated the potential roles of AR-V7 in hepatocarcinogenesis in vivo in a c-MYC-driven mouse HCC model generated by the hydrodynamic tail-vein injection system. The impacts of AR-V7 on gene expression in mouse HCC were elucidated by RNA-seq transcriptome and ontology analyses. The results showed that AR-V7 significantly exacerbated the c-MYC-mediated oncogenesis in the livers of both sexes. The transcriptome and bioinformatics analyses revealed that AR-V7 and c-MYC synergistically altered the gene sets involved in various cancer-related biological processes, particularly in lipid and steroid/sterol metabolisms. Importantly, AR-V7 suppressed a tumor suppressor Claudin 7 expression, upregulated by c-MYC overexpression via the p53 signaling pathway. Claudin 7 overexpression significantly suppressed the c-MYC-driven HCC development under p53-deficient conditions. Our results suggest that the AR-V7 exacerbates the c-MYC-driven hepatocarcinogenesis by potentiating the oncogenic roles and minimizing the anti-oncogenic functions of c-MYC. Since AR-V7 is expressed in a subpopulation of HCC cases, it could contribute to the inter- and intra-heterogeneity of HCC.

Targeted Next-Generation Sequencing for the Diagnosis of Gene Variants in Patients with 46,XY Disorder of Sex Development

Introduction: Disorders of sex development (DSDs) are congenital abnormalities in which chromosomal, gonadal, and anatomical sex development are atypical. One of these disorders, 46,XY DSD, is particularly difficult to diagnose and manage because its etiology and clinical phenotypes are highly heterogeneous. Methods: We used a gene panel containing 141 genes implicated in DSDs to perform targeted next-generation sequencing (NGS) in 50 patients with 46,XY DSD. Results: Gene variants were detected in 23 patients (46%). Among them, 13 patients had previously reported pathogenic or likely pathogenic variants, 9 patients had novel variants, and 1 patient had a previously reported variant of uncertain significance. Three of the novel variants were pathogenic, and the remaining were variants of uncertain significance; therefore, 16 patients had pathogenic or likely pathogenic variants according to ACMG guidelines, and the overall diagnostic rate of 46,XY DSD was 32%. The most common gene variants were SRD5A2 variants, followed by the AR variant. In addition, we analyzed the association between gene variants and clinical phenotypes. Most patients presented with multiple DSD phenotypes (i.e., two or more DSD phenotypes were observed, such as micropenis, hypospadias, and cryptorchidism), but the phenotype with the highest diagnostic rate was micropenis. Conclusion: Our results indicate that targeted NGS can effectively detect pathogenic gene variants in patients with 46,XY DSD.

Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain and Modulation via Direct Interactions with DNA Abasic Sites: Understanding the Mechanisms Involved in Castration-Resistant Prostate Cancer.

The androgen receptor (AR) is an important drug target in prostate cancer and a driver of castration-resistant prostate cancer (CRPC). A significant challenge in designing effective drugs lies in targeting constitutively active AR variants and, most importantly, nearly all AR variants lacking the ligand-binding domain (LBD). Recent findings show that an AR's constitutive activity may occur in the presence of somatic DNA mutations within non-coding regions, but the role of these mutations remains elusive. The discovery of new drugs targeting CRPC is hampered by the limited molecular understanding of how AR binds mutated DNA sequences, frequently observed in prostate cancer, and how mutations within the protein and DNA regulate AR-DNA interactions. Using atomistic molecular dynamics (MD) simulations and quantum mechanical calculations, we focused our efforts on (i) rationalising the role of several activating DBD mutations linked to prostate cancer, and (ii) DBD interactions in the presence of abasic DNA lesions, which frequently occur in CRPC. Our results elucidate the role of mutations within DBD through their modulation of the intrinsic dynamics of the DBD-DNA ternary complex. Furthermore, our results indicate that the DNA apurinic lesions occurring in the androgen-responsive element (ARE) enhance direct AR-DNA interactions and stabilise the DBD homodimerisation interface. Moreover, our results strongly suggest that those abasic lesions may form reversible covalent crosslinks between DNA and lysine residues of an AR via a Schiff base. In addition to providing an atomistic model explaining how protein mutations within the AR DNA-binding domain affect AR dimerisation and AR-DNA interactions, our findings provide insight into how somatic mutations occurring in DNA non-coding regions may activate ARs. These mutations are frequently observed in prostate cancer and may contribute to disease progression by enhancing direct AR-DNA interactions.

Novel inhibition of AKR1C3 and androgen receptor axis by PTUPB synergizes enzalutamide treatment in advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is the main driving force of mortality in prostate cancer patients. Among the parameters contributing to the progression of CRPC and treatment failure, elevation of the steroidogenic enzyme AKR1C3 and androgen receptor variant 7 (AR-V7) are frequently reported. The AKR1C3/AR-V7 complex has been recognized as a major driver for drug resistance in advanced prostate cancer. Herein we report that the level of AKR1C3 is reciprocally regulated by the full-length androgen receptor (AR-FL) through binding to the distal enhancer region of the AKR1C3 gene. A novel function of PTUPB in AKR1C3 inhibition was discovered and PTUPB showed more effectiveness than indomethacin and celecoxib in suppressing AKR1C3 activity and CRPC cell growth. PTUPB synergizes with enzalutamide treatment in tumor suppression and gene signature regulation. Combination treatments with PTUPB and enzalutamide provide benefits by blocking AR/AR-V7 signaling, which inhibits the growth of castration relapsed VCaP xenograft tumors and patient-derived xenograft organoids. Targeting of the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer.

YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth.

Hippo signaling restricts tumor growth by inhibiting the oncogenic potential of YAP/TAZ-TEAD transcriptional complex. Here, we uncover a context-dependent tumor suppressor function of YAP in androgen receptor (AR) positive prostate cancer (PCa) and show that YAP impedes AR+ PCa growth by antagonizing TEAD-mediated AR signaling. TEAD forms a complex with AR to enhance its promoter/enhancer occupancy and transcriptional activity. YAP and AR compete for TEAD binding and consequently, elevated YAP in the nucleus disrupts AR-TEAD interaction and prevents TEAD from promoting AR signaling. Pharmacological inhibition of MST1/2 or LATS1/2, or transgenic activation of YAP suppressed the growth of PCa expressing therapy resistant AR splicing variants. Our study uncovers an unanticipated crosstalk between Hippo and AR signaling pathways, reveals an antagonistic relationship between YAP and TEAD in AR+ PCa, and suggests that targeting the Hippo signaling pathway may provide a therapeutical opportunity to treat PCa driven by therapy resistant AR variants.

Plasma progastrin-releasing peptide level shows different predictive profiles for treatment response by androgen receptor axis-targeted agents in patients with metastatic castration-resistant prostate cancer.

The neuroendocrine (NE) pathway cannot be ignored as a mechanism for castration-resistant prostate cancer (CRPC) progression. The neuromediator, gastrin-releasing peptide (GRP) may be involved in the aberrant activation of the normal androgen receptor (AR) and increased AR variants. This study focused on plasma levels of progastrin-releasing peptide (ProGRP) and examined the treatment outcomes with androgen receptor axis-targeted (ARAT) agents. One hundred patients with metastatic CRPC were enrolled. Enzalutamide (ENZ) or abiraterone acetate/prednisone (AA/P) were administered to 50 patients each in a nonrandomized manner as a first-line or later choice. Plasma ProGRP levels were determined using a chemiluminescent enzyme immunoassay, and data were collected prospectively. The study endpoints were prostate-specific antigen (PSA) response and survival estimates. In the ENZ series, ProGRP levels correlated with the maximum PSA change from baseline (high ProGRP: -34.5% vs. low ProGRP: -85.7% p= .033). PSA progression-free survival (PFS), radiographic/symptomatic (r/s) PFS, and overall survival (OS) in patients with high ProGRP were significantly worse than those in patients with low ProGRP (median PSA-PFS: 3.3 vs. 10.0months, p =.001, r/s PFS: 5.0 vs. 15.0months, p< 0.001, and OS 17.5 vs. 49.0months, p< .001, respectively). In addition, ProGRP showed an independent predictive value for all survival estimates in multivariate analyses. In the AA/P series, ProGRP levels did not correlate with the PSA change or predict PSA-PFS and r/s PFS, but they maintained a significant difference in OS (19.0 vs. 48.0months, p= .003). Plasma ProGRP provides a consistent predictive value for OS in metastatic CRPC patients who underwent therapy with ARAT agents. Meanwhile, ProGRP showed different predictive profiles for PSA- and r/s PFS between ENZ and AA/P. These findings clinically suggest a mechanism for CRPC progression involving the NE pathway via the GRP. The underlying mechanism of different predictive profiles by the ARAT agent should be explored in future research.

Mutational Screening of Androgen Receptor Gene in 8224 Men of Infertile Couples.

Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity. The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males. A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses. We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic. This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.

OR26-1 Alternative Polyadenylation as a Therapeutic Vulnerability in Prostate Cancer

Abstract Prostate cancer is the second leading cause of male cancer death in the United States. While localized disease can be cured by radiation or surgery, metastatic prostate cancer presents a clinical challenge. Metastatic prostate cancer can initially be controlled by endocrine therapies that target the androgen receptor (AR), however, these tumors will inevitably develop resistance. This stage of the disease, termed castration-resistant prostate cancer (CRPC), is responsible for the majority of prostate cancer-specific deaths. Truncated AR variant (AR-V) proteins are broadly enriched in CRPC cell lines and clinical samples, and can function as ligand-independent, constitutively active transcription factors. We found that blocking an alternative poly(A) site located in AR intron 3 reduced expression of multiple AR-V mRNA and protein species and increased expression of full-length (FL) AR mRNA and protein in 22Rv1 and LNCaP95 CRPC cells. We found the cleavage and polyadenylation specificity factor (CPSF) component, CPSF1, regulates selection of this alternative poly(A) site based on the finding that knockdown of CPSF1 also reduced expression of AR-Vs and increased expression of FL-AR in 22Rv1 and LNCaP95 cells. Further, knockdown of CPSF1 inhibited growth of these CRPC cell lines, as well as androgen-dependent LNCaP cells. To define how CPSF1 regulates alternative polyadenylation of AR and to determine novel pathways that are manipulated by CPSF1 in prostate cancer, we identified CPSF1-dependent gene expression using RNA-seq and CPSF1-dependent poly(A) site usage using poly(A)-ClickSeq (PAC-Seq). Gene set enrichment analysis of RNA-seq data revealed the Glycolysis Hallmark gene was positively regulated by CPSF1, and nominated multiple differentially-expressed genes encoding regulators of glycolysis as CPSF1 targets. These results highlight AR independent pathways that are also regulated by CPSF1 in prostate cancer. We are currently integrating RNA-Seq and PAC-seq datasets to define novel CPSF1 regulated pathways that can be targeted therapeutically in prostate cancer. This research will determine trans-acting factors and cis-regulatory elements that drive alternative polyadenylation of AR mRNA transcripts to promote CRPC progression. This research will also identify AR independent targets of CPSF1 that could be exploited for development of new therapies for CRPC. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

A cryptic transactivation domain of EZH2 binds AR and AR's splice variant, promoting oncogene activation and tumorous transformation.

Enhancer of Zeste Homolog 2 (EZH2) and androgen receptor (AR) are crucial chromatin/gene regulators involved in the development and/or progression of prostate cancer, including advanced castration-resistant prostate cancer (CRPC). To sustain prostate tumorigenicity, EZH2 establishes non-canonical biochemical interaction with AR for mediating oncogene activation, in addition to its canonical role as a transcriptional repressor and enzymatic subunit of Polycomb Repressive Complex 2 (PRC2). However, the molecular basis underlying non-canonical activities of EZH2 in prostate cancer remains elusive, and a therapeutic strategy for targeting EZH2:AR-mediated oncogene activation is also lacking. Here, we report that a cryptic transactivation domain of EZH2 (EZH2TAD) binds both AR and AR spliced variant 7 (AR-V7), a constitutively active AR variant enriched in CRPC, mediating assembly and/or recruitment of transactivation-related machineries at genomic sites that lack PRC2 binding. Such non-canonical targets of EZH2:AR/AR-V7:(co-)activators are enriched for the clinically relevant oncogenes. We also show that EZH2TAD is required for the chromatin recruitment of EZH2 to oncogenes, for EZH2-mediated oncogene activation and for CRPC growth in vitro and in vivo. To completely block EZH2's multifaceted oncogenic activities in prostate cancer, we employed MS177, a recently developed proteolysis-targeting chimera (PROTAC) of EZH2. Strikingly, MS177 achieved on-target depletion of bothEZH2's canonical (EZH2:PRC2) and non-canonical (EZH2TAD:AR/AR-V7:co-activators) complexes in prostate cancer cells, eliciting far more potent antitumor effects than the catalytic inhibitors of EZH2. Overall, this study reports a previously unappreciated requirement for EZH2TAD for mediating EZH2's non-canonical (co-)activator recruitment and gene activation functions in prostate cancer and suggests EZH2-targeting PROTACs as a potentially attractive therapeutic for the treatment of aggressive prostate cancerthat rely on the circuits wired by EZH2 and AR.

The evolution and polymorphism of mono-amino acid repeats in androgen receptor and their regulatory role in health and disease.

Androgen receptor (AR) is a key member of nuclear hormone receptors with the longest intrinsically disordered N-terminal domain (NTD) in its protein family. There are four mono-amino acid repeats (polyQ1, polyQ2, polyG, and polyP) located within its NTD, of which two are polymorphic (polyQ1 and polyG). The length of both polymorphic repeats shows clinically important correlations with disease, especially with cancer and neurodegenerative diseases, as shorter and longer alleles exhibit significant differences in expression, activity and solubility. Importantly, AR has also been shown to undergo condensation in the nucleus by liquid-liquid phase separation, a process highly sensitive to protein solubility and concentration. Nonetheless, in prostate cancer cells, AR variants also partition into transcriptional condensates, which have been shown to alter the expression of target gene products. In this review, we summarize current knowledge on the link between AR repeat polymorphisms and cancer types, including mechanistic explanations and models comprising the relationship between condensate formation, polyQ1 length and transcriptional activity. Moreover, we outline the evolutionary paths of these recently evolved amino acid repeats across mammalian species, and discuss new research directions with potential breakthroughs and controversies in the literature.

Androgen receptor mutations for precision medicine in prostate cancer

Hormonal therapies including androgen deprivation therapy and androgen receptor (AR) pathway inhibitors such as abiraterone and enzalutamide have been widely used to treat advanced prostate cancer. However, treatment resistance emerges after hormonal manipulation in most prostate cancers, and it is attributable to a number of mechanisms, including AR amplification and overexpression, AR mutations, the expression of constitutively active AR variants, intra-tumor androgen synthesis, and promiscuous AR activation by other factors. Although various AR mutations have been reported in prostate cancer, specific AR mutations (L702H, W742L/C, H875Y, F877L, and T878A/S) were frequently identified after treatment resistance emerged. Intriguingly, these hot spot mutations were also revealed to change the binding affinity of ligands including steroids and antiandrogens and potentially result in altered responses to AR pathway inhibitors. Currently, precision medicine utilizing genetic and genomic data to choose suitable treatment for the patient is becoming to play an increasingly important role in clinical practice for prostate cancer management. Since clinical data between AR mutations and the efficacy of AR pathway inhibitors are accumulating, monitoring the AR mutation status is a promising approach for providing precision medicine in prostate cancer, which would be implemented through the development of clinically available testing modalities for AR mutations using liquid biopsy. However, there are few reviews on clinical significance of AR hot spot mutations in prostate cancer. Then, this review summarized the clinical landscape of AR mutations and discussed their potential implication for clinical utilization.

Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes.

CYCLIN K down-regulation induces androgen receptor gene intronic polyadenylation, variant expression and PARP inhibitor vulnerability in castration-resistant prostate cancer

Androgen receptor (AR) messenger RNA (mRNA) alternative splicing variants (AR-Vs) are implicated in castration-resistant progression of prostate cancer (PCa), although the molecular mechanism underlying the genesis of AR-Vs remains poorly understood. The CDK12 gene is often deleted or mutated in PCa and CDK12 deficiency is known to cause homologous recombination repair gene alteration or BRCAness via alternative polyadenylation (APA). Here, we demonstrate that pharmacological inhibition or genetic inactivation of CDK12 induces AR gene intronic (intron 3) polyadenylation (IPA) usage, AR-V expression, and PCa cell resistance to the antiandrogen enzalutamide (ENZ). We further show that AR binds to the CCNK gene promoter and up-regulates CYCLIN K expression. In contrast, ENZ decreases AR occupancy at the CCNK gene promoter and suppresses CYCLIN K expression. Similar to the effect of the CDK12 inhibitor, CYCLIN K degrader or ENZ treatment promotes AR gene IPA usage, AR-V expression, and ENZ-resistant growth of PCa cells. Importantly, we show that targeting BRCAness induced by CYCLIN K down-regulation with the PARP inhibitor overcomes ENZ resistance. Our findings identify CYCLIN K down-regulation as a key driver of IPA usage, hormonal therapy-induced AR-V expression, and castration resistance in PCa. These results suggest that hormonal therapy-induced AR-V expression and therapy resistance are vulnerable to PARP inhibitor treatment.