Expression Of Androgen Receptor Splice Variants Research Articles

Abstract 1231: First-in-class AR-V7/AR-fl small molecule molecular glue degrader for prostate cancer treatment

Abstract Metastatic castration resistance prostate cancer (mCRPC) is a lethal disease due to the development of treatment to androgen receptor (AR) signaling inhibitors (ARSi) and taxane chemotherapy. Treatment resistance occurs partly due to the expression of AR splice variants that lack the ligand binding domain (LBD) and are constitutively active in the nucleus. AR-V7 is the most prevalent variant conferring clinical resistance to both ARSi and taxanes. Currently, there is no selective AR-V7 inhibitor leaving patients with limited therapeutic options. Thus, the development of selective AR-V7 inhibitors is a high priority, clinically unmet need. To identify AR-V7 pharmacologic inhibitors, we performed a high throughput small molecule phenotypic screen using enzymatic complementation and nuclear AR-V7 as the assay endpoint. Our primary screen of ~170K compounds (z score = 0.8), followed by a cell-toxicity counter screen and a secondary GFP screen identified hit compound 7907, as a dual AR-V7/AR-fl protein degrader, with unique chemotype compared to all known AR modulators. Hit to lead optimization by medicinal chemistry/SAR studies identified lead compound 15, with increased potency compared to 7907. Mechanistically, we showed that compound 15 shortened AR-V7 protein half-life by activating the ubiquitin-proteasome pathway and inducing proteasomal degradation of both AR-V7/AR-fl, without affecting their transcription. Importantly, compound 15 induced degradation occurred within 3hr of treatment and was blocked by the clinically approved proteasome inhibitor, bortezomib. TurboID proximity ligation assay identified distinct E3 ligases, uniquely interacting with AR-V7 or AR-fl. Using AR-V7/AR-fl deletion mutants we further showed that compound 15 activity is mediated by the N-terminal domain of AR, present in both proteins. Remarkably, compound 15 sensitized LNCaP95 cells (endogenous AR-V7/AR-fl expression) to enzalutamide suggesting potential therapeutic synergism and ability to reverse enzalutamide resistance. Ongoing studies aim to narrow down the binding site of compound 15 as well as identify the E3 ligases mediating its activity. Together, these data support a molecular glue degrader mechanism of action, consistent with published studies showing that molecular glue degraders are ideal for targeting classically “undruggable” proteins lacking an LBD or containing intrinsically disordered domains, as is the case for AR-V7. Currently, all AR-directed therapies target the LBD of AR-fl, inhibiting AR signaling. AR-V7 expression is a direct outcome of this inhibition, leading to AR-fl and AR-V7 co-expression in patient tumors. We posit that our drug candidate, by offering dual AR-V7/AR-fl inhibition in a single treatment, has the potential to not only benefit patients with mCRPC but also patients with hormone-sensitive disease, and delay AR-V7 expression. Citation Format: CheukMan C. Au, Catrina Estrella, Prerna Vatsa, Michelle Naidoo, Michael Miller, Peter T. Meinke, David M. Nanus, Paraskevi Giannakakou. First-in-class AR-V7/AR-fl small molecule molecular glue degrader for prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1231.

Abstract 6271: Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer

Abstract Prostate cancer (PC) stands as the most frequently diagnosed and second leading cause of cancer death among American men. PC mortality is predominantly attributed to castration-resistant prostate cancer (CRPC), often emerging within 2-3 years following the initiation of androgen deprivation therapy. This resistance primarily arises from sustained androgen receptor (AR) signaling, ultimately resulting in a compromised response to AR-directed therapies. AR splice variant expression is a key contributor to the persistent AR signaling in CRPC. AR-variant 7 (AR-V7) is the most clinically prevalent variant, expressed in about 75% of patients with CRPC and confers resistance to standard of care (SoC) treatments. AR-V7 differs from the full-length AR (AR-fl) because it lacks the ligand binding and hinge domains, rendering it resistant to SoC treatments. Currently, there are no selective inhibitors for AR-V7. Therefore, we sought to identify unique biological features of AR-V7, distinct from AR-fl, to exploit therapeutic options for targeting AR-V7. Previous studies have demonstrated high sequence homology, largely overlapping cistromes, and gene transactivation profiles between AR-V7 and AR-FL. Our published mechanistic studies revealed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl, allowing for constitutive import and activation of target genes in the nucleus. Herein, we report an additional novel subnuclear phenotype of AR-V7 in LNCAP95 and 22RV1 cells modeling CRPC. Staining with AR-V7- and AR-fl- specific antibodies revealed that unlike AR-fl, AR-V7 exhibited prominent nucleolar localization. To validate AR-V7 nucleolar localization, CRPC cells stained with fibrillarin (nucleolar marker), and subsequent fluorescence intensity analysis revealed that AR-V7 was predominantly localized in fibrillarin positive nucleoli, whereas AR-fl protein was solely localized in the nucleoplasm. This suggests a novel functional role of AR-V7 in the nucleus. To further confirm AR-V7 nucleolar localization, Actinomycin D (ActD) was used as a tool compound to disrupt nucleolar integrity. Treatment with ActD for 4 hours in CRPC cells significantly reduced the area of fibrillarin positive nucleoli and prompted AR-V7 re-distribution out of the nucleoli and into the nucleoplasm. Ongoing efforts to further investigate AR-V7 nucleolar function in CRPC cells include mutating the predicted nucleolar localization signal located in AR-V7 and identifying nucleolar-interacting proteins through proximity-labeling assays. In conclusion, we unveiled a novel nuclear compartmentalization of AR-V7 within the nucleolus, the primary hub for ribosomal biogenesis. These observations suggest that AR-V7 could regulate the high levels of protein synthesis occurring in CRPC cells, creating a window of a potential therapeutic opportunity for selective inhibition. Citation Format: Michelle K. Naidoo, Paraskevi Giannakakou. Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6271.

Identification of a targetable JAK-STAT enriched androgen receptor and androgen receptor splice variant positive triple-negative breast cancer subtype

Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.

Beyond Prostate Cancer: An Androgen Receptor Splice Variant Expression in Multiple Malignancies, Non-Cancer Pathologies, and Development.

Multiple studies have demonstrated the importance of androgen receptor (AR) splice variants (SVs) in the progression of prostate cancer to the castration-resistant phenotype and their utility as a diagnostic. However, studies on AR expression in non-prostatic malignancies uncovered that AR-SVs are expressed in glioblastoma, breast, salivary, bladder, kidney, and liver cancers, where they have diverse roles in tumorigenesis. AR-SVs also have roles in non-cancer pathologies. In granulosa cells from women with polycystic ovarian syndrome, unique AR-SVs lead to an increase in androgen production. In patients with nonobstructive azoospermia, testicular Sertoli cells exhibit differential expression of AR-SVs, which is associated with impaired spermatogenesis. Moreover, AR-SVs have been identified in normal cells, including blood mononuclear cells, neuronal lipid rafts, and the placenta. The detection and characterization of AR-SVs in mammalian and non-mammalian species argue that AR-SV expression is evolutionarily conserved and that AR-SV-dependent signaling is a fundamental regulatory feature in multiple cellular contexts. These discoveries argue that alternative splicing of the AR transcript is a commonly used mechanism that leads to an expansion in the repertoire of signaling molecules needed in certain tissues. Various malignancies appropriate this mechanism of alternative AR splicing to acquire a proliferative and survival advantage.

Lineage plasticity and treatment resistance in prostate cancer: the intersection of genetics, epigenetics, and evolution.

Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, and the development of castrate-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-related mortality. While CRPC typically develops through a gain in androgen receptor (AR) signaling, a subset of CRPC will lose reliance on the AR. This process involves genetic, epigenetic, and hormonal changes that promote cellular plasticity, leading to AR-indifferent disease, with neuroendocrine prostate cancer (NEPC) being the quintessential example. NEPC is enriched following treatment with second-generation anti-androgens and exhibits resistance to endocrine therapy. Loss of RB1, TP53, and PTEN expression and MYCN and AURKA amplification appear to be key drivers for NEPC differentiation. Epigenetic modifications also play an important role in the transition to a neuroendocrine phenotype. DNA methylation of specific gene promoters can regulate lineage commitment and differentiation. Histone methylation can suppress AR expression and promote neuroendocrine-specific gene expression. Emerging data suggest that EZH2 is a key regulator of this epigenetic rewiring. Several mechanisms drive AR-dependent castration resistance, notably AR splice variant expression, expression of the adrenal-permissive 3βHSD1 allele, and glucocorticoid receptor expression. Aberrant epigenetic regulation also promotes radioresistance by altering the expression of DNA repair- and cell cycle-related genes. Novel therapies are currently being developed to target these diverse genetic, epigenetic, and hormonal mechanisms promoting lineage plasticity-driven NEPC.

Abstract P1-13-14: Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, and despite recent treatment advances, clinical outcomes remain poor, particularly in the metastatic setting. TNBC is a biologically heterogeneous entity, and up to 50% of TNBC express the androgen receptor (AR). Gene expression subtyping has identified a subset of AR-TNBC with a luminal AR-driven (LAR) phenotype. Consequently, there has been great interest in translating androgen receptor signaling inhibitors (ARSIs) approved for use in prostate cancer such as enzalutamide and bicalutamide into the management of LAR-TNBC. However, early phase clinical trials of anti-androgens in metastatic AR-TNBC have had modest results. While multiple mechanisms of anti-androgen resistance have been identified in prostate cancer, including AR amplification and over-expression, emergence of AR splice variants (AR-Vs), and transition to AR independent growth, molecular determinants of anti-androgen resistance in TNBC remain poorly understood, and pre-clinical models to study acquired ARSI resistance in LAR-TNBC have been limited. Methods: A novel pre-clincial model of acquired ARSI resistance was derived from the LAR subtype MDA-MB-453 cell line through serial passaging with increasing concentrations of enzalutamide. Celltiter Blue viability assays were used to determine enzalutamide IC50 of parental and enzalutamide-resistant (EnzR) cell lines. RNA was extracted from the parental and resistant cells, reverse transcribed and expression of AR, splice-variants and canonical AR target genes were evaluated using RT-qPCR. Immunofluorescence was used to investigate the amount and nuclear localization of the AR protein within the parental and enzalutamide resistant cells. Results: EnzR MDA-MB-453 cells were derived through culture in increasing concentrations of enzalutamide for >6 months, and found to have a significant increase in enzalutamide IC50 compared to the parental cell line. EnzR MDA-MB-453 cells had a 4.5 fold increase in full-length AR expression at the transcriptional level compared to the parental cell line. AR protein expression and nuclear localization were also increased in EnzR cells compared to the parental cell line. Canonical AR targets including NKX3.1 were downregulated in response to enzalutamide in parental but not EnzR cells. While pathogenic AR splice variants (AR-Vs) implicated in ARSI resistance in prostate cancer were detected at low levels in MDA-MB-453 cells, no increase in AR-V expression was observed in the EnzR cell line. Conclusions: We have developed a novel pre-clinical model of anti-androgen/ARSI resistance in LAR-TNBC, and identified AR overexpression and persistent AR signaling associated with acquired enzalutamide resistance in LAR-TNBC. While pathogenic AR splice variants have been implicated in ARSI resistance in metastatic prostate cancer and are detected in the MDA-MB-453 cell line model, enzalutamide resistance was not associated with increased AR splice variant expression in this model. Future work will investigate mechanisms leading to AR overexpression as well as combination therapeutic strategies to overcome acquired ARSI resistance. Citation Format: Hannah Krause, Marina N. Sharifi, Serena K. Wolfe, Jamie M. Sperger, Kari B. Wisinski, Ruth O’Regan, Joshua Lang. Increased androgen receptor expression as a mechanism of acquired anti-androgen resistance in androgen receptor-positive triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-14.

Racial Differences in Androgen Receptor (AR) and AR Splice Variants (AR-SVs) Expression in Treatment-Naïve Androgen-Dependent Prostate Cancer.

Androgen receptor splice variants (AR-SVs) contribute to the aggressive growth of castration-resistant prostate cancer (CRPC). AR-SVs, including AR-V7, are expressed in ~30% of CRPC, but minimally in treatment-naïve primary prostate cancer (PCa). Compared to Caucasian American (CA) men, African American (AA) men are more likely to be diagnosed with aggressive/potentially lethal PCa and have shorter disease-free survival. Expression of a truncated AR in an aggressively growing patient-derived xenograft developed with a primary PCa specimen from an AA patient led us to hypothesize that the expression of AR-SVs could be an indicator of aggressive growth both in PCa progression and at the CRPC stage in AA men. Tissue microarrays (TMAs) were created from formalin-fixed paraffin-embedded (FFPE) prostatectomy tumor blocks from 118 AA and 115 CA treatment-naïve PCa patients. TMAs were stained with AR-V7-speicifc antibody and with antibodies binding to the N-terminus domain (NTD) and ligand-binding domain (LBD) of the AR. Since over 20 AR-SVs have been identified, and most AR-SVs do not as yet have a specific antibody, we considered a 2.0-fold or greater difference in the NTD vs. LBD staining as indication of potential AR-SV expression. Two AA, but no CA, patient tumors stained positively for AR-V7. AR staining with NTD and LBD antibodies was robust in most patients, with 21% of patients staining at least 2-fold more for NTD than LBD, indicating that AR-SVs other than AR-V7 are expressed in primary treatment-naïve PCa. About 24% of the patients were AR-negative, and race differences in AR expression were not statistically significant. These results indicate that AR-SVs are not restricted to CRPC, but also are expressed in primary PCa at higher rate than previously reported. Future investigation of the relative expression of NTD vs. LBD AR-SVs could guide the use of newly developed treatments targeting the NTD earlier in the treatment paradigm.

Prostate fibroblasts enhance androgen receptor splice variant 7 expression in prostate cancer cells

Androgen receptor splice variant (AR-V) expression has been associated with prostate cancer (PCa) progression to castration-resistant PCaduring androgen deprivation therapy, which reduces androgen production and inhibits androgen action in PCa cells. However, the mechanisms whereby aberrant AR-V expression is increased in PCa are still largely unknown. Fibroblasts in tumor stroma influence PCa initiation and aggressiveness, and which may play a crucial role in eliciting genetic changes during malignant transformation in human prostate epithelium. Here, our aim was to determine whether prostate fibroblasts in tumor stroma induce aberrant AR-V7 expression in PCa cells under low androgen concentration. We performed in vitro experiments using androgen-sensitive, AR-positive PCa cell lines (LNCaP and 22Rv1 cells), commercially available prostate stromal cells (PrSC), and primary cultured prostate fibroblasts (pcPrF) from PCa specimens collected from biopsies of patients with advanced PCa. PCa cells were cocultured with each of the three fibroblast lines (PrSC, pcPrF-M37, and pcPrF-M48). The proliferation under low androgen concentration of LNCaP and 22Rv1 cells cocultured with PrSC, pcPrF-M37, or pcPrF-M48 was significantly increased compared to that of PCa cells cultured alone. Androgen receptor-full length (AR-FL) protein expression was increased in LNCaP and 22Rv1 cells cocultured with PrSC, pcPrF-M37, or pcPrF-M48. AR-V7 protein expression was increased in 22Rv1 cells cocultured with PrSC, pcPrF-M37, or pcPrF-M48. Under low androgen concentration, AR-V7 protein expression was slightly detected in LNCaP cells cocultured with PrSC or pcPrF-M37. Cytokine array analysis revealed that monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) levels in the conditioned medium of 22Rv1 cells cocultured with PrSC, pcPrF-M37, or pcPrF-M48 were increased under low androgen concentration. High IL-8 concentration (30 ng/ml) resulted in significantly increased protein expression of AR-FL, AR-V7, and phospho-NF-κB p65 in 22Rv1 cells. In contrast, IL-8 antibody (1 µg/ml) decreased AR-V7 protein expression in 22Rv1 cells cocultured with PrSC, pcPrF-M37, or pcPrF-M48. pcPrF from PCa specimens increase the expression of aberrant AR-V7 in PCa cells. IL-8 may be a target for preventing the expression of aberrant AR-Vs in PCa.

Abstract 1030: IX2X2

Abstract Introduction: Prostate cancer (PCa) is the second leading cause of cancer-related mortality in men. More than 85% of PCa and advanced aggressive castration-resistant prostate cancer (CRPC) depend on the androgen receptor (AR) for growth. AR-targeted therapeutics are the mainstay treatment option for PCa. Majority of PCa and CRPC respond to AR-LBD-binding antagonists such as enzalutamide and become refractory after a brief period of response. One of the main resistance mechanisms is the expression of AR splice variants (AR-SVs). AR-SVs lack LBD and are constitutively active, causing aggressive phenotype. Description: Rationale-based drug design was employed to synthesize selective AR irreversible covalent antagonists (SARICA). SARICAs were evaluated in AR transactivation to determine the antagonistic activity. SARICAs were incubated with purified recombinant AR activation function-1 (AF-1) protein and mass spectrometry was performed to determine their covalent binding. SARICAs were evaluated in AR-SV transactivation assay and in a Schild’s plot to determine the nature of inhibition (reversible Vs irreversible). SARICAs were tested in PCa cell line gene expression and proliferation assays and in tumor xenograft experiments. Summary: SARICAs inhibited AR transactivation in submicromolar range. Mass spectrometry with purified AF-1 protein demonstrated covalent binding of SARICAs and identified C406 and C327 as SARICA-interacting nucleophiles. Both amino acids are important for AR-V7 function and stability, making them appropriate drug targets. SARICAs inhibited the activity of AR-V7 and irreversibly inhibited AR transactivation, indicating that these covalent binders are irreversible antagonists. SARICAs selectively inhibit the proliferation and growth of AR-positive prostate cancer cells and tumors, but not AR-negative cells and tumors. Conclusion: These first-in-class AR-selective covalent molecules thus serve as chemical probes to advance our understanding of the AR-AF-1 and they potentially offer an opportunity to treat refractory cancers. Disclosure: This work was supported by an NCI grant (CA229164 and CA229164S1), a DOD grant (W81XWH2110055), and by Muirhead endowment. The SARICAs are licensed to Oncternal therapeutics, San Diego, CA. RN is a consultant to Oncternal therapeutics. Citation Format: Thamarai U. Ponnusamy, Thirumagal T. Thiyagarajan, Ramesh Narayanan. IX2X2 [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 1030.

Impact of AR-V7 and other androgen receptor splice variant expression on outcomes of post-prostatectomy salvage therapy.

274 Background: Radiotherapy (RT) with or without androgen deprivation therapy (ADT) plays a key role in salvage therapy of prostate cancer recurrent after prostatectomy. However, not all patients benefit from salvage therapy, and there is an unmet need for biomarkers to distinguish responders from non-responders. Prostate cancer depends on androgen receptor (AR) signaling, and expression of AR splice variants (ARVs) that enable androgen-independent AR signaling is associated with resistance to ADT in the metastatic setting. Recent in vitro data suggest that ARVs also mediate DNA repair after irradiation, suggesting that ARV expression may also be a biomarker of RT resistance. However, the landscape of ARVs in primary prostate cancer and its effect on treatment outcomes remain unexplored. Here we hypothesize that ARVs are detectable in primary prostate cancer and may modulate response to salvage RT + ADT. Methods: We retrospectively identified 46 prostate cancer patients treated with prostatectomy followed by salvage RT+ ADT at a single institution from 1995 to 2012. The indication for salvage therapy was biochemical failure after undetectable post-operative PSA in 72%, gross local recurrence in 17%, and persistently elevated PSA after surgery in 11%. Median RT dose was 64.8 Gy, and all patients received concurrent ADT. We comprehensively interrogated the landscape of ARVs by performing ultra-deep targeted RNA-seq of archival formalin-fixed paraffin-embedded prostatectomy samples. Using a custom library of > 3000 primers spanning all AR exons and introns, we evaluated 21 native splice junction sites and 20 splice variants with a mean depth of coverage of > 5000x. Decipher score was also evaluated. We tested for association between splice variant expression and clinical outcomes using the log-rank test and Cox proportional hazards model. Results: In total, 76% of patients had one or more detectable AR splice variants. The most commonly detected variants were AR-45 in 41%, AR-V9 in 20%, and AR-V7 in 13%. At a median follow-up of 33.8 months, biochemical progression-free survival (BPFS) at 3 years was 60%, distant metastasis-free survival was 90%, and overall survival was 100%. Among detected splice variants, only AR-V7 was associated with differential clinical outcomes, with a median BPFS of 10.9 months in AR-V7 positive vs 73.4 months in AR-V7 negative patients ( p = 0.0020, HR 5.23, 95% CI 1.62-16.87). Conclusions: Using ultra-deep targeted RNA-Seq, we provide among the first comprehensive descriptions of the ARV landscape in primary prostate cancer. Moreover, we show that detectable AR-V7 in prostatectomy specimens was associated with inferior outcomes following salvage RT+ADT, suggesting for the first time that AR-V7 may modulate outcomes for localized as well as metastatic disease. Ongoing work includes comparison with Decipher score and validation in independent cohorts.

Abstract LBA016: Androgen Receptor (AR) N-Terminus-Domain-Binding Small Molecule Degraders for the Treatment of AR Splice Variant-Positive Castration-Resistant Prostate Cancer

Abstract Prostate cancer is the second leading cause of cancer-related deaths in men in the United States. Over 30,000 men die of prostate cancer each year in the U.S. and the deaths predominantly occur due to late-stage castration-resistant prostate cancer (CRPC). Androgen receptor (AR) is the primary therapeutic target in castration-sensitive prostate cancer and CRPC, with over 85% of the cases expressing AR. About 30% of CRPC fail to respond to ligand-binding domain (LBD)-binding AR-signaling inhibitors, while the remaining develop resistance after an initial period of response of 12-24 months. Expression of AR splice variants (AR-SVs) that lack LBD has been identified as one of the primary causes for resistance development to AR-signaling inhibitors. AR-SVs also promote aggressive CRPC growth, resulting in shorter overall survival. We discovered novel AR N-terminus domain (NTD)-binding small molecule, GTx-534 that induce degradation of AR and AR-SV proteins, resulting in loss of AR signaling. Surface Plasmon Resonance (SPR), intrinsic fluorescence biophysical studies and thermal stability analysis with purified AR activation function-1 (AF-1) domain in the NTD showed GTX-534 binding. Notably, GTx-534 significantly inhibited AR and AR-V7-positive 22RV1 cell line xenograft in castrated immune compromised rodents with a tumor growth inhibition (TGI) of over 50% (p<0.005), while enzalutamide failed to inhibit the 22RV1 tumor growth. Serum PSA concentrations of 22RV1-bearing animals were reduced to background levels by GTx-534 (p<0.01), but not by enzalutamide. Genome-wide expression studies demonstrated a distinct mechanism of action for GTx-534 in AR-SV-positive tumors. The results from 22RV1 xenograft studies were validated in LuCaP 86.2 patient-derived xenograft (PDX) that express AR v567es, another AR variant that lacks the LBD widely expressed in CRPC. GTx-534 completely inhibited LuCaP 86.2 tumor growth with a TGI of greater than 90%, while enzalutamide failed to alter the tumor growth. These results provide preclinical proof-of-concept for the efficacy of GTx-534 in AR and AR-SV-positive CRPC. Disclosure. The work presented in here was funded by a Sponsored Research Agreement from Oncternal Therapeutics, Inc. to RN and DDM and by National Cancer Institute grant R01CA229164 to RN. RN is a consultant to Oncternal Therapeutics Inc. GFK and JBB are employees of Oncternal Therapeutics Inc. Citation Format: Ramesh Narayanan. Androgen Receptor (AR) N-Terminus-Domain-Binding Small Molecule Degraders for the Treatment of AR Splice Variant-Positive Castration-Resistant Prostate Cancer [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 LBA016.

Impact of AR-V7 and Other Androgen Receptor Splice Variant Expression on Outcomes of Post-Prostatectomy Salvage Therapy

Radiotherapy (RT) with or without androgen deprivation therapy (ADT) plays a key role in the salvage treatment of prostate cancer recurrent after prostatectomy. However, not all patients benefit from salvage therapy, and there is an unmet need for biomarkers to distinguish responders from non-responders. Prostate cancer depends on androgen receptor (AR) signaling, and expression of AR splice variants (ARVs) that enable androgen-independent AR signaling is associated with resistance to ADT in the metastatic setting. Recent in vitro data suggest that ARVs also mediate DNA repair after irradiation, suggesting that ARV expression may also be a biomarker of resistance to RT. However, the landscape of ARV expression in primary prostate cancer and its effect on treatment outcomes remain unexplored. Here, we hypothesized that ARVs may be detectable in primary prostate cancer and furthermore may modulate response to salvage RT+ADT.We retrospectively identified 46 prostate cancer patients treated with prostatectomy followed by salvage RT+ ADT at a single institution from 1995 to 2012. Median age at salvage was 64.5 years. The indication for salvage therapy was biochemical failure after an undetectable post-operative PSA in 72%, gross local recurrence in 17%, and persistently elevated PSA after surgery in 11%. Median RT dose was 64.8 Gy, and all patients received concurrent ADT. We comprehensively interrogated the landscape of ARVs by performing ultra-deep targeted RNA-seq of archival formalin-fixed paraffin-embedded prostatectomy samples. Using a custom library of > 3000 primers spanning all AR exons and introns, we evaluated 21 native splice junction sites and 20 splice variants with a mean depth of coverage of > 5000x. We tested for association between splice variant expression and clinical outcomes using the log-rank test and Cox proportional hazards model.In total, 76% of patients had one or more detectable AR splice variants. The most commonly detected variants were AR-45 (exon 1b-2) in 41%, AR-V9 (exon 3-cryptic exon (CE) 5) in 20%, and AR-V7 (exon 3-CE3) in 13%. At a median follow-up of 33.8 months, biochemical progression-free survival (BPFS) at 3 years was 60%, distant metastasis-free survival was 90%, and overall survival was 100%. Among detected splice variants, only AR-V7 was associated with differential clinical outcomes, with a median BPFS of 10.9 months in AR-V7 positive vs 73.4 months in AR-V7 negative patients (P = 0.0020, HR 5.23, 95% CI 1.62-16.87).Using a custom ultra-deep targeted RNA-Seq approach, we provide among the first comprehensive descriptions of the AR splice variant landscape in primary prostate cancer. Moreover, we show that detectable AR-V7 expression in prostatectomy specimens was associated with inferior outcomes following subsequent salvage RT+ADT, suggesting for the first time that AR-V7 may modulate outcomes for localized as well as metastatic disease.

Blocking GRP/GRP-R signaling decreases expression of androgen receptor splice variants and inhibits tumor growth in castration-resistant prostate cancer

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.

Prospective Evaluation of Clinical Outcomes Using a Multiplex Liquid Biopsy Targeting Diverse Resistance Mechanisms in Metastatic Prostate Cancer.

PURPOSENearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.MATERIALS AND METHODSA novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board–approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings.RESULTSHierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]).CONCLUSIONWe demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.

Depletion of androgen receptor low molecular weight isoform reduces bladder tumor cell viability and induces apoptosis.

Bladder cancer (BlCa) exhibits a gender disparity where men are three times more likely to develop the malignancy than women suggesting a role for the androgen receptor (AR). Here we report that BlCa cells express low molecular weight (LMW) AR isoforms that are missing the ligand binding domain (LBD). Isoform expression was detected in most BlCa cells, while a few express the full-length AR. Immunofluorescence studies detect AR in the nucleus and cytoplasm, and localization is cell dependent. Cells with nuclear AR expression exhibit reduced viability and increased apoptosis on total AR depletion. A novel AR-LMW variant, AR-v19, that is missing the LBD and contains 15 additional amino acids encoded by intron 3 sequences was detected in most BlCa malignancies. AR-v19 localizes to the nucleus and can transactivate AR-dependent transcription in a dose dependent manner. AR-v19 depletion impairs cell viability and promotes apoptosis in cells that express this variant. Thus, AR splice variant expression is common in BlCa and instrumental in ensuring cell survival. This suggests that targeting AR or AR downstream effectors may be a therapeutic strategy for the treatment of this malignancy.

Identification of Genes Required for Enzalutamide Resistance in Castration-Resistant Prostate Cancer Cells In Vitro.

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Combined Cell-free DNA and RNA Profiling of the Androgen Receptor: Clinical Utility of a Novel Multianalyte Liquid Biopsy Assay for Metastatic Prostate Cancer

BackgroundThe androgen receptor (AR) remains a critical driver in metastatic castration-resistant prostate cancer (mCRPC). Profiling AR aberrations in both circulating DNA and RNA may identify key predictive and/or prognostic biomarkers in the context of contemporary systemic therapy. ObjectiveTo profile AR aberrations in circulating nucleic acids and correlate with clinical outcomes. Design, setting, and participantsWe prospectively enrolled 67 mCRPC patients commencing AR pathway inhibitors (ARPIs; n = 41) or taxane chemotherapy (n = 26). Using a first-in-class next-generation sequencing-based assay, we performed integrated cell-free DNA (cfDNA) and cell-free RNA (cfRNA) profiling from a single 10 ml blood tube. Outcome measurements and statistical analysisKaplan-Meier survival estimates and multivariable Cox regression analyses were used to assess associations between clinical outcomes and the following AR aberrations: copy number variation, splice variants (AR-V7 and AR-V9) and somatic mutations. Results and limitationsCell-free DNA and cfRNA were successfully sequenced in 67 (100%) and 59 (88%) patients, respectively. Thirty-six (54%) patients had one or more AR aberrations. AR gain and cumulative number of AR aberrations were independently associated with clinical/radiographic progression-free survival (PFS; hazard ratio [HR] 3.2, p = 0.01 and HR 3.0 for 0 vs ≥2, p = 0.04) and overall survival (HR 2.8, p = 0.04 and HR 2.9 for 0 vs ≥2, p = 0.03). Notably, concurrent AR gain and AR splice variant expression (AR gain/AR-V+) was associated with shorter prostate-specific antigen PFS on both ARPIs (HR 6.7, p = 0.009) and chemotherapy (HR 3.9, p = 0.04). Importantly, key findings were validated in an independent cohort of mCRPC patients (n = 40), including shorter OS in AR gain/AR-V+ disease (HR 3.3, p = 0.02). Limitations include sample size and follow-up period. ConclusionsWe demonstrate the utility of a novel, multianalyte liquid biopsy assay capable of simultaneously detecting AR alterations in cfDNA and cfRNA. Concurrent profiling of cfDNA and cfRNA may provide vital insights into disease biology and resistance mechanisms in mCRPC. Patient summaryIn this study of men with advanced prostate cancer, DNA and RNA abnormalities in the androgen receptor detected in blood were associated with poor outcomes on available drug treatments. This information could be used to better guide treatment of advanced prostate cancer.

Combinatorial expression of androgen receptor splice variants: No predictive value in castration-resistant prostate cancer patients treated with enzalutamide (enza) or abiraterone (abi).

e17547 Background: Playing an important role in prostate cancer, androgen receptor (AR) signaling is a common therapeutic target. Novel hormonal treatment (NHT) using enza or abi prolongs overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, biomarkers predicting therapy response are limited. AR-V7, as the most abundant AR splice variant, has gained clinical interest. Nonetheless, current discussions on its predictive power are diverse. Given that AR-V7 as a sole biomarker is not efficient in predicting response to NHT, we aimed to increase the predictive potential by analysis of combinatorial AR splice variant (AR-V) expression in mCRPC patients undergoing NHT. Methods: We prospectively enrolled 60 patients who started on either abi or enza. Presence of circulating tumor cells (CTC) as well as expression of AR-V3, -7 and -9 were assessed. Outcomes in CTC-, CTC+/AR-V- and CTC+/AR-V+ patients were analyzed considering PSA reduction, PSA-PFS, PFS and OS. Results: PSA reduction of 50% was predominantly found in CTC- patients (78.5%) compared to CTC+/AR-V- (55.5%) and CTC+/AR-V+ (39.3%) without statistical significance (P = 0.059). When taking co-expression of two or more AR-V into account there was no difference in PSA response either (one AR-V 42.9%, two AR-V 33.3%, three AR-V 41.6%, P = 0.154). Median PSA-PFS was 17 months (95%CI 15.7 – 18.3), 13 months (95%CI 6.8 – 19.2) and 5 months (95%CI 3.6 – 6.4) for CTC- pts, CTC+/AR-V- pts and CTC+/AR-V+ pts, respectively (P = 0.005). However, comparing CTC- and CTC+ pts, differences become even more apparent (P = 0.004), CTC+/AR-V- and AR-V+ pts showed less statistically significant differences (P = 0.029). Median PFS and OS were not reached for CTC- pts. PFS was 10 months (95%CI 6.2 – 13.8) for CTC+/AR-V- pts and 9 months (95%CI 1.1 – 16.9) for CTC+/AR-V+ pts (P = 0.004, only CTC- vs. CTC+ P = 0.002). OS was 28 months (95%CI 16.8 – 39.2) for CTC+/AR-V- pts and 15 months (95%CI 7.9 – 22.1) for CTC+/AR-V+ pts (P = 0.014, only CTC- vs. CTC+ P = 0.006). Regarding PFS and OS, there was no difference comparing only CTC+/AR-V- and AR-V+ pts (P = 0.356 and P = 0.244). Conclusions: AR splice variants have prognostic power in stratifying mCRPC patients suffering from a more advanced stage of disease. Nonetheless, our study clearly demonstrates the lack of predictive power of AR splice variants for response to NHT. Additionally, we prove the importance of CTC analysis rather than AR-V expression being more valuable in mCRPC.

IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.

Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

Owing to the marked sexual dimorphism of hepatocellular carcinoma (HCC), sex hormone receptor signaling has been implicated in numerous aspects of liver cancer pathogenesis. We sought to reconcile the clear contribution of androgen receptor (AR) activity that has been established in preclinical models of HCC with the clinical failure of AR antagonists in patients with advanced HCC by evaluating potential resistance mechanisms to AR-targeted therapy. The AR locus was interrogated for resistance-causing genomic modifications using publicly available primary HCC datasets (1,019 samples). Analysis of HCC tumor and cell line RNA-seq data revealed enriched expression of constitutively active, treatment-refractory AR splice variants (AR-SV). HCC cell lines expressed C-terminal-truncated AR-SV; 28 primary HCC samples abundantly expressed AR-SV. Low molecular weight AR species were nuclear localized and constitutively active. Furthermore, AR/AR-SV signaling promoted AR-mediated HCC cell progression and conferred resistance to AR antagonists. Ligand-dependent and -independent AR signaling mediated HCC epithelial-to-mesenchymal transition by regulating the transcription factor SLUG. These data suggest that AR-SV expression in HCC drives HCC progression and resistance to traditional AR antagonists. Novel therapeutic approaches that successfully target AR-SVs may be therapeutically beneficial for HCC. SIGNIFICANCE: Treatment-refractory, constitutively active androgen receptor splice variants promote hepatocellular carcinoma progression by regulating the epithelial-to-mesenchymal transition pathway.