Androgen Receptor Target Gene Expression Research Articles

GOLM1 promotes prostate cancer progression via interaction with PSMD1 and enhancing AR-driven transcriptional activation.

Aberrant transcriptional activation of the androgen receptor (AR) is a predominant cause of prostate cancer (PCa), including both in the initial and androgen-independent stages. Our study highlights Golgi membrane protein 1 (GOLM1) as a key regulator of AR-driven transcriptional activity in PCa progression. Utilizing local clinical data and TCGA data, we have established a robust association between GOLM1 and AR target genes, and further demonstrated that GOLM1 can enhance the expression of AR target genes. We discovered that GOLM1 interacts with PSMD1, a component of the 19S regulatory complex in the 26S proteasome, using mass spectrometry and Co-IP analysis. It is well known that ubiquitin-proteasome plays a vital role in AR expression and transcriptional regulation. Our findings demonstrate that GOLM1 enhances ubiquitin proteasome activity by binding to PSMD1, thereby facilitating AR-driven transcriptional activity and PCa progression. These results indicate that GOLM1 and its associated proteins may become potential therapeutic targets for PCa characterized by dysregulated AR-driven transcriptional activation.

Targeting proliferating cell nuclear antigen enhances ionizing radiation-induced cytotoxicity in prostate cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, cell growth, and survival. PCNA also enhances androgen receptor (AR) signaling in prostate cancer (PC) cells. We identified a PCNA interaction protein (PIP) box at the N-terminal domain of AR and developed a small peptide PCNA inhibitor R9-AR-PIP containing AR PIP-box. We also identified a series of small molecule PCNA inhibitors (PCNA-Is) that bind directly to PCNA and interrupt PCNA functions. The present study investigated the effects of the PCNA inhibitors on the sensitivity of PC cells to X-ray radiation. The effects of targeting PCNA on radio sensitivity of PC cells were investigated in four lines of castration-resistant PC (CRPC) cells with different AR expression statuses. The cells were treated with the PCNA inhibitors and X-ray radiation alone or in combination. The effects of the treatment on expression of AR target genes, DNA damage response, DNA damage, homologous recombination repair (HRR), and cytotoxicity were evaluated. We found that the androgen response element (ARE) occupancy of the DNA damage response gene PARP1 by AR is significantly attenuated by PCNA-I1S or R9-AR-PIP combined with X-ray radiation, while X-ray radiation alone does not enhance the ARE occupancy. PCNA-I1S or R9-AR-PIP alone significantly inhibits occupancy of the AR-occupied regions (AROR) in PRKDC and XRCC2 genes. R9-AR-PIP and PCNA-I1S inhibit expression of AR-Vs target gene cyclin A2 and show the additive effects with radiation in AR-positive CRPC cells. Targeting PCNA by PCNA-I1S and R9-AR-PIP downregulates expression of DNA damage response genes EXO1, Rad54L, Rad51, and/or PARP1 and shows the additive effects with radiation as compared with their respective controls in AR-positive CRPC LNCaP-AI, 22Rv1, and R1-D567 cells, but not in AR-negative PC-3 cells. R9-AR-PIP and PCNA-I1S elevate the levels of phospho-DNA-PKcs(S2056) and γH2AX, indicating DNA damage in response to radiation in AR-positive cells. The HRR is significantly attenuated by PCNA inhibitors PCNA-I1S, R9-AR-PIP, and T2AA in all four CRPC cells examined, and inhibited by Enzalutamide (Enz) only in 22RV1 cells. The cytotoxicity induced by X-ray radiation in androgen-dependent LNCaP cells is enhanced by Enz and a lower concentration of R9-AR-PIP in the colony formation assay. R9-AR-PIP at higher concentration reduces the colony formation and has an additive effect with X-ray radiation in all AR expressing cells, regardless of AR-FL and AR-Vs, but does not significantly alter the colony formation in AR-negative PC-3 cells. PCNA-I1S attenuates colony formation and has an additive effect with ionizing radiation in all four CRPC cells, regardless of AR expression status. These data provide a strong rationale for the therapy studies using PCNA-I1S or R9-AR-PIP in combination with X-ray radiation against CRPC tumors in preclinical models.

Abstract CT231: Identification of a novel agnostic predictive multiomic signature via Elastic Net/Machine Learning in TALAPRO-2 (TP-2), a phase 3 study of talazoparib (TALA) + enzalutamide (ENZA) vs placebo (PBO) + ENZA as first-line (1L) treatment in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: TP-2 (NCT03395197) demonstrated significantly improved radiologic progression-free survival (rPFS) regardless of homologous recombination repair (HRR) gene alteration status for pts with mCRPC who received 1L TALA + ENZA (n=402) vs PBO + ENZA (n=403; Agarwal et al. Lancet. 2023. PMID: 37285865). We explore potential associations of ctDNA genomics and tumor gene expression with efficacy in TP-2. Methods: The analysis was based on the safety population (n=799). Two data sets were used: a FoundationOne®Liquid CDx data set (Azad et al. ASCO 2023, #5056) of prospectively collected/retrospectively analyzed plasma samples (n=681); and a tumor transcriptomic data set generated via HTG’s Oncology Biomarker Panel (with 10 additional genes implicated in PARP inhibitor sensitivity; n=304). Correlations between baseline molecular biomarkers and rPFS were assessed via a Cox proportional hazards model. Multivariate elastic net regression analysis incorporated 151 pts in the TALA + ENZA arm with 2,868 molecular features (ctDNA: 309 genes [only short variant alterations incorporated]; HTG: 2,559 tumor transcripts). Results: Positively predictive candidate tumor biomarkers for TALA + ENZA included androgen response signature and individual androgen receptor (AR) target genes such as PSA, ALDH1A3, and CAMKK2. These expression signatures/transcripts had no/minimal predictive value for PBO + ENZA. A 33-feature (3 genes, 30 transcripts) elastic net signature was identified that was predictive of rPFS with TALA + ENZA: TP53 and AR short variant alteration status were each prognostic and associated with worse rPFS. Expression of multiple AR target genes, including ALDH1A3 (top selected feature) and CAMKK2, was positively associated with rPFS. For TALA + ENZA, this multiomic signature was predictive of rPFS in pts regardless of HRR gene alteration status, and to a lesser extent for PBO + ENZA. None of the 12 HRR genes used for stratification in TP-2 were included in the signature. Conclusions: Our exploratory analysis identified candidate gene expression signatures, including AR pathway elements, potentially associated with differential benefit from TALA + ENZA, reinforcing the potential for exploitable crosstalk between AR and DNA repair pathways. Though validation is necessary, a predictive multiomic signature for benefit from TALA + ENZA regardless of HRR alteration status was identified that included alterations in genes previously implicated in prognosis and expression of multiple AR target transcripts. Strikingly, it did not include any of the 12 HRR genes used in prospective stratification for TP-2, reinforcing the potential benefit for TALA + ENZA beyond HRR-deficient tumors. Citation Format: Glenn Liu, Xinmeng Jasmine Mu, Karim Fizazi, A. Douglas Laird, Nobuaki Matsubara, Steven M. Yip, Jie Pu, Joan Carles, Andre P. Fay, Robert J. Jones, Stefanie Zschäbitz, Josep M. Piulats, Jae Young Joung, Whijae Roh, Jijumon Chelliserry, Nicola Di Santo, Michelle Saul, Arun A. Azad, Neeraj Agarwal. Identification of a novel agnostic predictive multiomic signature via Elastic Net/Machine Learning in TALAPRO-2 (TP-2), a phase 3 study of talazoparib (TALA) + enzalutamide (ENZA) vs placebo (PBO) + ENZA as first-line (1L) treatment in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT231.

Regulation of androgen receptor stability by the β1 Pix/STUB1 complex.

The androgen receptor (AR) is essential in the development and differentiation of testes and male genitalia. AR expression is tightly regulated at the translational and posttranslational levels. AR posttranscriptional regulation is a major determinant of AR availability since AR is a direct target of E3 ubiquitin ligase STUB1. Our work indicated that the Rac/Cdc42 guanosine triphosphatase guanine nucleotide exchange factor, β1 Pix, enhanced AR levels after AR stimulation in HEK293 and HeLa cells. AR stimulation decreased AR ubiquitination which is accompanied by increased β1 Pix binding to AR. Ectopic expression of β1 Pix decreased AR ubiquitination in Tm4 and HEK293 cells. We demonstrated that the formation of a multimolecular complex comprised of AR/β1 Pix/STUB1 responded in a time-dependent manner to AR stimulation. β1 Pix binding dissociated STUB1 from AR and thus prevented STUB1 from catalyzing receptor ubiquitination. β1 Pix enhanced AR transcriptional activity and increased AR target gene expression. Irrespective of treatment, immunofluorescence analysis showed a strong nuclear colocalization of endogenous AR and endogenous βPix in Tm4 cells. However, using Tm4 cell fractionation, AR stimulation decreased βPix/AR association in the cytosolic fraction and increased binding of AR to βPix in the nuclear fraction. To support the role of β1 Pix in androgen regulation, we found that individuals lacking this gene have a significant increase in genitourinary malformations associated with androgen dysfunction. Our data indicate that β1 Pix is an important modulator of AR stability and ligand-dependent AR transcriptional activity. We propose that β1 Pix could serve as a promising therapeutic target to modulate AR signaling.

SAT634 The Androgen Receptor Does Not Directly Regulate The Transcription Of DNA Damage Response Genes

Abstract Disclosure: S. Hasterok: None. T.G. Scott: None. D.G. Roller: None. A. Spencer: None. A.B. Dutta: None. K. Sathyan: None. D.E. Frigo: None. M.J. Guertin: None. D. Gioeli: None. The clinical success of combined androgen deprivation therapy (ADT) and radiation therapy (RT) in prostate cancer (PCa) created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in PCa cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at steady state in asynchronously growing PCa cells. Our data indicate that the clinical benefit of ADT and RT is not due to the direct regulation of DDR gene transcription by AR and that the clinical benefit of this therapeutic combination is instead likely due to both treatments independently reducing tumor cell fitness. Presentation: Saturday, June 17, 2023

Abstract 3083: Targeting androgen receptor coregulators to enhance enzalutamide sensitivity in prostate cancer

Abstract Steroid hormone signaling axes drive proliferation and survival in prostate cancer (PCa), breast and other cancers. In late stage PCa, the androgen receptor (AR) is therapeutically antagonized with success, for example with Enzalutamide (Enza). Although initially successful, resistance emerges associated with tissue plasticity and patient mortality. Enza-resistance mechanisms include altered AR coregulator interactions. Identifying which coregulators change Enza sensitivity is attractive as they may represent new drug targets but challenging given the multitude of coregulators expressed in PCa and the diverse roles they play in transcription and translation.To meet this challenge, we designed a bioinformatic workflow to identify under-investigated yet clinically-impactful coregulators. We tested coactivators (CoA, n~750), corepressors (CoR, n ~600), and mixed function coregulators (Mixed, n~600) associations with patient outcome in TCGA PCa cohorts; applied partial correlation analyses to reveal coregulators that impacted AR target genes; utilized DepMap essentiality scores to rank importance; and examined interactions in publicly available AR RIME data. The impact of the top ranked coregulators (n=18) on Enza sensitivity was examined using shRNA or siRNA approaches, and with inhibitor drugs in Enza-resistant PCa cell lines (LNCaP C42B, 22Rv1). Finally, coregulator expression was examined in single cell RNA-Seq from PCa 3D organoids derived from the PBCre4:Ptenf/f:Rb1f/f (DKO) and the PBCre4:Ptenf/f:Rb1f/f:Trp53f/f (TKO) PCa mouse model, which recapitulate the loss of Enza sensitivity and lineage plasticity. ShRNA targeting coregulators was undertaken in organoids. The bioinformatics workflow identified 18 coregulators, including CoA (e.g., WDR5, KAT7, BLM, SMARCC1), CoR (e.g., SETDB1, PSPC1), and Mixed (e.g., KDM5A, BPTF, TCEGR1) to screen for impact on Enza. KAT7 shRNA modulated Enza sensitivity, regulated expression of AR target genes, and synergized with a KAT7 inhibitor (WM3835) to enhance Enza sensitivity. Similarly, a BLM inhibitor (ML216) synergized with Enza. Interestingly, although WDR5 siRNA alone did not alter Enza responses, the combination of KAT7 shRNA and WDR5 siRNA significantly potentiated Enza responses. Similarly, SMARCC1 shRNA cooperated with siRNA to several targets (TBX3, BPTF, or SETDB1) to enhance Enza responses. Smarcc1 and Tbx3 were upregulated in Enza-resistant neuroendocrine PCa, and the recently identified Pou2f3 Enza-resistant phenotypes, and Bptf was upregulated in the Tff3 Enza-resistant phenotype. We have shRNA targeted Blm, Smarcc1, Tbx3, and Bptf in the DKO and TKO organoids and are measuring the impact of Enza responses. Overall, we have identified potentially novel AR coregulators that associate with the development of therapy resistance and shown that targeting these coregulators may augment Enza sensitivity in PCa. Citation Format: Rayzel C. Fernandes, Debasis Nayak, Damien A. Leach, Meenalakshmi Chinnam, Wilbert Zwart, David W. Goodrich, Moray J. Campbell, Charlotte L. Bevan. Targeting androgen receptor coregulators to enhance enzalutamide sensitivity in 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 3083.

Abstract 6268: TT125-802 is a potent and highly selective CBP/p300 bromodomain inhibitor for the treatment of castration resistant prostate cancer and haematological malignancies

Abstract The paralogous lysine acetyltransferases CREB-binding protein (CBP) and p300 are key epigenetic regulators involved in diverse signaling pathways in cancer. The bromodomain (BRD) of CBP/p300 serves as an acetyl-lysine “reader” that allows CBP/p300 to bind chromatin at acetylated histone and non-histone proteins leading to the regulation of gene transcription. Indeed, CBP/p300 are critical co-activators of nuclear receptors, including the androgen receptor (AR) in castration resistant prostate cancer (CRPC). Thus, inhibition of CBP and p300 is an emerging therapeutic strategy to block the transactivation activity of the AR in CRPC. In addition, inhibition of the CBP/p300 BRD has been described as a potential therapeutic strategy to treat multiple myeloma (MM) through transcriptional suppression of interferon regulatory factor 4 (IRF4) and concomitant repression of its target genes MYC and MYB. TT125-802 is a highly selective and potent, oral small molecule inhibitor of the BRD of CBP/p300. In a BROMOscan assay against a panel of 40 BRD-containing proteins, TT125-802 revealed the unique selectivity to the BRD of CBP/p300 with a minimal off-target binding to all other BRDs, including BET proteins.TT125-802 shows selective anti-proliferative activity in AR-dependent prostate cancer cell lines (22Rv-1, C4-2, and LNCaP) and inhibits AR target gene expression (KLK2, KLK3, TMPRSS2, and MYC) in a dose-dependent manner (IC50 of 2 to 33 nM). AR-negative prostate cancer cell lines (DU-145 and PC-3) are insensitive to TT125-802 in vitro, pointing to an AR-selective mode of action. In an in vivo model of CRPC, TT125-802 inhibited tumor growth when administered orally to human C4-2 xenograft-bearing mice. Daily dosing of TT125-802 was well tolerated with stable bodyweights and platelet counts. In addition, preclinical studies in CRPC patient-derived xenograft (PDX)-bearing mice showed that the combination treatment of TT125-802 and enzalutamide had a synergistic effect on tumor growth inhibition compared to single agent treatments. TT125-802 reduced mRNA expression of the AR-target genes in tumor samples and decreased plasma PSA levels compared to enzalutamide alone, and the combination reduced levels even further. In a preclinical model of MM (OPM2), TT125-802 had a dose-dependent effect on tumor growth, inducing tumor regressions at the highest dose. Target genes such as MYC, MYB, and IRF4 were potently downregulated in tumors. We conclude that TT125-802 is a novel, highly selective inhibitor of the BRD of CBP/P300. It has therapeutic potential as monotherapy in prostate cancer and multiple myeloma and in combination with next-generation AR inhibitors for patients with lethal prostate cancer. A FIH study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Sara Laudato, Dorothea Gruber, Thomas Bohnacker, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. TT125-802 is a potent and highly selective CBP/p300 bromodomain inhibitor for the treatment of castration resistant prostate cancer and haematological malignancies [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 6268.

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.

FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

Association of emergent neuroendocrine prostate cancer detected by liquid biopsies with survival and treatment resistance.

247 Background: The mainstay of therapy in metastatic prostate cancer is androgen receptor (AR) signaling inhibition. However, the emergence of early castration resistance or neuroendocrine transformation is associated with poor prognosis. Reliable biomarkers are needed to identify these patients and guide selection of clinical therapy. Methods: mRNA was isolated from EpCAM-positive circulating tumor cells (CTCs) isolated from patients with CSPC, CRPC, or NEPC to measure expression of KLK2, KLK3 (PSA), TMPRSS2, FOLH1 (PSMA), synaptophysin ( SYP), and chromogranin ( CHGA). Post-hoc retrospective analysis of an institutional review board–approved prospective cohort (N = 98) was performed to identify patterns of gene expression. Samples were considered AR+ if 3 of 4 AR pathway genes ( TMPRSS2, KLK2, KLK3, and FOLH1) were positive, and were considered NE+ if either or both SYP or CHGA were positive. Blood samples from two prospective clinical trials of men with mCRPC treated with abiraterone and enzalutamide, respectively, were analyzed to confirm results. Longitudinal samples were collected from 17 patients (6 NEPC and 11 Adenocarcinoma) and cell free DNA was isolated and sequenced using a novel targeted exon panel. Results: AR and/or NE positive patients were found to have a median overall survival (OS) of 8.58 months as compared to a median OS of 29.6 months in the AR and NE negative population (p<0.0001; HR=2.75 [1.60-4.56]). In the subset of castrate resistant prostate cancer (CRPC) patients, AR+ and/or NE+ patients (n=31) were found to have a median OS of 6.74 months vs 18.79 months in the AR- and NE- group (n=39) (p= 0.0009; HR 2.38 [1.36-4.18]). We also tested samples from a phase II ARSI trials with abiraterone and enzalutamide, respectively. None of the baseline samples from these two trials met the above criteria for NEPC (AR- NE+) on their CTCs or histologically. Three of 48 total patients were identified with expression in their baseline blood samples without loss of AR target gene expression (AR+/NE+). Preliminary analysis of this small patient cohort in comparison to patients who were NE- shows that NE+ patients had worse OS (HR= 5.5906 [1.143-27.36), as would be expected by patients with emerging neuroendocrine differentiation. Integrated ctDNA sequencing identified mutations in genes associated with NEPC. Conclusions: The expression of NE genes in liquid biopsies while retaining AR target gene expression is associated with worse OS and may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype. Early identification of these patients may improve therapeutic decisions and improved patient outcomes. Pairing genomic alteration with changes in gene expression may additionally offer the basis for a new mechanism to assess efficacy of novel therapeutics in future clinical trials.

The Impact of Indoles Activating the Aryl Hydrocarbon Receptor on Androgen Receptor Activity in the 22Rv1 Prostate Cancer Cell Line.

The activation of the aryl hydrocarbon receptor (AhR) by xenobiotic compounds was demonstrated to result in the degradation of the androgen receptor (AR). Since prostate cancer is often dependent on AR, it has become a significant therapeutic target. As a result of the emerging concept of bacterial mimicry, we tested whether compounds with indole scaffolds capable of AhR activation have the potential to restrict AR activity in prostate cancer cells. Altogether, 22 indolic compounds were tested, and all of them activated AhR. However, only eight decreased DHT-induced AR luciferase activity. All indoles, which met the AhR-activating and AR-suppressing criteria, decreased the expression of DHT-inducible AR target genes, specifically KLK3 and FKBP5 mRNAs. The reduced AR binding to the KLK3 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay. In addition, some indoles significantly decreased AR protein and mRNA level. By using CRISPR/Cas9 AhR knockout technology, no relationship between AhR and AR, measured as target gene expression, was observed. In conclusion, some indoles that activate AhR possess AR-inhibiting activity, which seems to be related to the downregulation of AR expression rather than to AR degradation alone. Moreover, there does not seem to be a clear relationship that would connect AhR activation with AR activity suppression in 22Rv1 cells.

A clinical-grade liquid biomarker detects neuroendocrine differentiation in prostate cancer.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).

Androgen-Induced MIG6 Regulates Phosphorylation of Retinoblastoma Protein and AKT to Counteract Non-Genomic AR Signaling in Prostate Cancer Cells

The bipolar androgen therapy (BAT) includes the treatment of prostate cancer (PCa) patients with supraphysiological androgen level (SAL). Interestingly, SAL induces cell senescence in PCa cell lines as well as ex vivo in tumor samples of patients. The SAL-mediated cell senescence was shown to be androgen receptor (AR)-dependent and mediated in part by non-genomic AKT signaling. RNA-seq analyses compared with and without SAL treatment as well as by AKT inhibition (AKTi) revealed a specific transcriptome landscape. Comparing the top 100 genes similarly regulated by SAL in two human PCa cell lines that undergo cell senescence and being counteracted by AKTi revealed 33 commonly regulated genes. One gene, ERBB receptor feedback inhibitor 1 (ERRFI1), encodes the mitogen-inducible gene 6 (MIG6) that is potently upregulated by SAL, whereas the combinatory treatment of SAL with AKTi reverses the SAL-mediated upregulation. Functionally, knockdown of ERRFI1 enhances the pro-survival AKT pathway by enhancing phosphorylation of AKT and the downstream AKT target S6, whereas the phospho-retinoblastoma (pRb) protein levels were decreased. Further, the expression of the cell cycle inhibitor p15INK4b is enhanced by SAL and ERRFI1 knockdown. In line with this, cell senescence is induced by ERRFI1 knockdown and is enhanced slightly further by SAL. Treatment of SAL in the ERRFI1 knockdown background enhances phosphorylation of both AKT and S6 whereas pRb becomes hypophosphorylated. Interestingly, the ERRFI1 knockdown does not reduce AR protein levels or AR target gene expression, suggesting that MIG6 does not interfere with genomic signaling of AR but represses androgen-induced cell senescence and might therefore counteract SAL-induced signaling. The findings indicate that SAL treatment, used in BAT, upregulates MIG6, which inactivates both pRb and the pro-survival AKT signaling. This indicates a novel negative feedback loop integrating genomic and non-genomic AR signaling.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.

Abstract 2367: Characterizing the reciprocal regulation of an ABI1-dependent androgen receptor axis in prostate cancer

Abstract Background: Prostate cancer (PCa) affects nearly 50% of males over 60. Therapies for advanced PCa primarily target the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. Our previous research demonstrated that ABI1 acts as a tumor suppressor in PCa. ABI1 is a scaffold protein for the WAVE Regulatory Complex, positively regulates cell adhesion, and inhibits integrin activation through sequestering key kinases. We have observed a physical interaction between AR and ABI1. In this study, we aim to characterize the ABI1-dependent AR regulation in PCa. Method: We generated an ABI1 KO cell line model in LNCaP cells using CRISPR-Cas9. We then used mutagenesis to develop ABI1 binding mutants (W485N and ∆SH3) and generated ABI1 rescue cell lines in ABI1-KO. We performed qPCR, co-immunoprecipitation, subcellular fractionation, western blotting, in vitro assay, live-cell imaging, and PLA. We purified both AR and ABI1 proteins for in vitro analysis, performed turbidity and liquid-liquid droplet formation assays under DIC. Results: In cellulo binding assays indicate interaction of AR-NTD with the ABI1-SH3 domain. Expression of ABI1-W485N in KO cell line showed decreased binding, while ABI1-∆SH3 showed no observable binding to AR in co-IP assays compared to wildtype control. We then performed subcellular fractionation assays and saw decreased AR nuclear localization compared to ABI1-WT control. Decreased nuclear localization in ABI1-W485N was associated with decreased mRNA expression of hallmark AR target genes, KLK3 and FKBP5. In vitro turbidity assays indicated the conditions in which AR and ABI1 have propensity to phase separate. We then performed a liquid-liquid droplet assay. AR and ABI1 formed liquid-liquid droplets individually. We labeled ABI1 with Alexaflour 488 and saw that ABI1 was recruited to AR droplets indicating that AR and ABI1 can co-phase separate. PLA assays in cellulo and in vivo patient tumor samples showed positive interactions of AR-ABI1 in the nucleus. We then used live-cell observation and saw co-localization of biomolecular condensates for AR and ABI1. Conclusions: Our studies demonstrate that ABI1 plays a role in AR transcriptional pathway. Loss of ABI1 resulted in decreased nuclear AR and subsequent decrease in mRNA expression of AR target genes. Due to the intrinsically disordered structure of AR and ABI1, they were both able to phase separate individually as well as together. AR stimulation increased PLA interactions in nucleus of cells. The co-localization of AR and ABI1 in biomolecular condensates was changed upon androgen deprivation conditions. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and promote reactivation of AR pathway through ABI1-AR axis. Findings will allow for novel insights into the mechanisms underlying AR and ABI1 in prostate metastatic progression. Citation Format: Baylee A. Porter-Hansen, Alaji Bah, Alfonso Urbanucci, Konsta Kukkonen, Fan Zhang, Sonia Kung, Ladan Fazli, Martin E. Gleave, Gennady Bratslavsky, Leszek Kotula. Characterizing the reciprocal regulation of an ABI1-dependent androgen receptor axis in prostate cancer [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 2367.

Abstract 429: Androgen receptor (AR) N-terminal domain degraders can degrade AR full length and AR splice variants in CRPC preclinical models

Abstract Introduction: 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. Current antiandrogen therapies directly or indirectly target the AR ligand binding domain (LBD) and are initially effective in prostate cancer patients. However, 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 by small molecule anitens represents a novel method of blocking AR signaling that can bypass LBD-related resistance mechanisms. AR NTD is an intrinsically disordered region (IDR) which has no stable ordered structure and is generally regarded as an undruggable target due to the lack of a well-defined small-molecule binding pocket. However, we recently demonstrated that EPI-7386, an AR NTD small molecule inhibitor, inhibits AR activity by binding to Tau5 region of the NTD. By developing an aniten based bifunctional degrader (ANITAC for ANITen bAsed Chimera), our goal is to eliminate any forms of AR protein found in castration-resistant prostate cancer (CRPC), that can potentially drive disease progression, including LBD mutants and AR truncated variants. Methods: AR degradation was monitored by Western Blot or by using the HiBit assay. AR transcriptional activity was measured using different reporter assays following AR full length (FL) or truncated AR activity in in vitro models. Results: Here we report the first series of AR degraders targeting the NTD of AR, which lead to degradation of all forms of AR, including AR-V7 and AR-v567es. ANITACs eliminate AR in all cell lines tested through an E3 ligase dependent mechanism, with an observed 50% degradation concentration (DC50) < 20 nM in 22Rv1 cells for the most potent compounds. AR degradation mediated by ANITACs suppresses the expression of AR target genes and decreases the viability of prostate cancer cells. ANITACs also degrade clinically relevant AR mutants and AR splice variants and show inhibition of AR transcriptional activity in multiple cell lines expressing different forms of AR including AR FL (LNCaP, CWR-AD1), AR-V7 (22Rv1), AR-V567es (CWR-D567). In vitro and in vivo PK studies show the compounds are metabolically stable and exhibit excellent oral bioavailability in mice. Conclusion: In summary, we report preclinical data on the first generation of ANITAC molecules, that can be orally bioavailable and show activity against forms of AR expressed in late stage CRPC patients. Citation Format: Nan Hyung Hong, Berenger Biannic, Peter Virsik, Han-Jie Zhou, Ronan Le Moigne. Androgen receptor (AR) N-terminal domain degraders can degrade AR full length and AR splice variants in CRPC preclinical models [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 429.

The effect of PARP inhibition on androgen receptor localization and activity in castration resistant prostate cancer.

e17037 Background: The poly(ADP-ribose) polymerase inhibitors (PARPi) olaparib and rucaparib have been approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in the setting of homologous recombination deficiency (HRD). Additionally, PARPi have been shown to modulate androgen receptor (AR) signaling, with a recent report demonstrating mono-ADP-ribosylation of cysteine residues in AR by Parp7. Here, we further evaluated the effect of PARPi on AR activity and localization. Methods: The effect of PARPi on cell growth and survival of CRPC cell lines was evaluated in vitro and in vivo. AR ribosylation was assessed in CRPC cell lines by immunoprecipitation (IP) assays and proximity ligation assays (PLA). The subcellular localization of AR was determined by quantitative immunofluorescence microscopy in CRPC cell lines and xenograft models. Changes in AR activity with PARPi treatment were evaluated by a luciferase reporter assay and AR target gene expression in a PDX model. Finally, PARPi mediated alteration in the AR protein interactome was evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. Results: The PARPi olaparib and talazoparib, and to a lesser extent veliparib, inhibited CRPC cell growth. Evidence of AR ribosylation was seen by IP and PLA. PARPi treatment of multiple in vitro and in vivo prostate cancer models resulted in a shift of AR subcellular localization, from predominantly nuclear to cytoplasmic compartments. In luciferase reporter assays, AR transactivation activity was decreased after PARPi treatment in a dose dependent manner. In vivo, in prostate cancer xenograft models, decreased AR target gene expression was seen upon PARPi treatment. LC-MS/MS proteomic studies revealed that PARP inhibition resulted in significant changes in the composition of AR interaction partners, in particular of proteins related to intracellular trafficking and nuclear transport. This suggests a potential link between altered AR complex assembly and the observed changes in AR subcellular localization in the context of PARPi treatment. Conclusions: We describe a novel sequela of PARPi therapy to alter AR localization and activity in CRPC. Single agent PARP inhibition can alter prostate cancer cell growth in vitro and in vivo. With PARPi treatment, AR localization is shifted to the cytoplasm, the AR interactome is altered, and AR transcriptional activity decreased. These findings implicate a collateral mechanism of PARPi in preventing prostate cancer cell growth/survival that may augment previously described mechanisms related to HDR and synthetic lethality. It is essential to understand the role of PARPi beyond synthetic lethality in the context of HRD in order to better define the spectrum of response to PARPi across patients, and for development of biology-informed combination therapies.

Antagonistic action of a synthetic androgen ligand mediated by chromatin remodeling in a human prostate cancer cell line

The clinical use of androgen receptor (AR) antagonists has been successful in treating prostate cancer patients, inducing remission of androgen-dependent tumors. However, a couple of years after treatment, prostate tumors transition into an androgen-independent state with altered gene expression profiles, but the molecular basis is not understood. Since the AR antagonists trigger this transition, we assessed whether AR antagonists induce chromatin reorganization in an androgen-dependent prostate cancer cell line (LNCaP). Treatment of LNCaP cells with two clinically used AR antagonists (bicalutamide [Bic] and enzalutamide [Enz]) expectedly resulted in antagonistic effects on cell proliferation, AR transactivation, and dihydrotestosterone (DHT)-induced expression of AR target genes. Thus, the antagonists expectedly acted to antagonize the transactivation function of AR activated by androgen binding. By ChIP-qPCR assay, AR bound to Bic, but not Enz, was recruited to an endogenous consensus AR-binding site within the kallikrein-related peptidase 3 gene promoter after treatment with Bic, similar to the effect of DHT. By ATAC-seq analysis of the cells after long-term treatment for 5 days, Bic and dihydrotestosterone DHT induced different chromatin reorganization patterns and gene expression profiles, suggesting that Bic exhibited a distinct action from that by DHT. Thus, these results suggest that the action of a known AR antagonist is mediated by chromatin reorganization in a prostate cancer cell line.

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway

SummaryBAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17, that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth.

(+)-JJ-74-138 is a Novel Noncompetitive Androgen Receptor Antagonist.

Identification of novel androgen receptor (AR) antagonists may lead to urgently needed new treatments for patients with prostate cancer resistant to current AR antagonists. AR is presently the main target for treating prostate cancer. Clinically approved AR antagonists compete with dihydrotestosterone (DHT) for binding to the ligand-binding domain (LBD) of AR, and patients eventually develop resistance to these treatments. One approach to overcoming resistance is to discover compounds that inhibit AR in alternative ways. Our lab previously identified a small molecule, JJ-450, that is capable of inhibiting AR lacking LBD. To optimize the efficacy of this class of inhibitors, we developed structural analogues of JJ-450 and identified (+)-JJ-74-138 as a promising candidate. Here, we show that (+)-JJ-74-138 is more potent than JJ-450 in the inhibition of androgen-independent AR activity in enzalutamide-resistant LN95 cells. Further studies showed (+)-JJ-74-138 inhibition of castration-resistant PSA expression in all tested castration-resistant prostate cancer (CRPC) cells. (+)-JJ-74-138 inhibited mRNA expression of AR and ARv7 target genes and reduced AR level in the nucleus in the absence of androgens. Also, this analogue noncompetitively inhibited androgen-stimulated AR activity in C4-2, LN95, and 22Rv1 CRPC cells. At low dosages, (+)-JJ-74-138 inhibited the proliferation of enzalutamide-resistant AR-positive LN95 and 22Rv1 cells, but not AR-negative PC3 and DU145 cells. A surface plasmon resonance assay detected (+)-JJ-74-138 binding to AR and a chromatin immunoprecipitation assay indicated (+)-JJ-74-138 inhibited AR binding to androgen response elements. In addition, (+)-JJ-74-138 inhibited 22Rv1 xenograft tumor growth. Our observations suggest that (+)-JJ-74-138 is a novel noncompetitive AR antagonist capable of inhibiting enzalutamide-resistant CRPC.