Androgen Receptor Binding Sites Research Articles

Phytochemical identification and in silico elucidation of interactions of bioactive compounds from Citrullus lanatus with androgen receptor towards prostate cancer treatment.

Androgen receptor (AR) is known to play a crucial role in the development and progression of prostate cancer, and compounds that inhibit its activity are regarded as promising for the development of drugs to treat the disease. This study aimed to investigate the AR-inhibiting potential of Citrullus lanatus fruit compounds forprostate cancer drug development. Following HPLC identification, the binding energies, molecular interactions, and pharmacological potentials of the compounds against AR were elucidated using in silico techniques such as, molecular docking, induced-fit docking, molecular dynamics simulation, and ADMET prediction. Some of the compounds found to be present in Citrullus lanatus fruit included flavonoids such as proanthocyanin, naringin, flavan 3 ol, flavonones, naringenin, epicatechin, citrulline, and catechin. Naringenin exhibited the highest docking score in the molecular docking analysis, followed by resveratrol, ribalinidine, and epicatechin. These compoundsshare a common AR binding site with the standard ligand, dihydrotestosterone (DHT). Some of the compounds showed favorable ADMET profiles, while others showedat least one toxicity potential. The induced-fit docking of naringenin with AR yielded a higher docking score than the initial score obtained from standard docking while preserving stable molecular contacts with the interacting amino acids. Consistent hydrogen bond interactions of naringenin with PHE 764, ASN 705, and THR 877 of AR, including a persistent pi-pi stacking contact with PHE 764, were observed from the molecular dynamic simulation. The Citrullus lanatus compounds, particularly naringenin, may therefore be considered for further research towards the development of drugs for prostate cancer therapy.

Abstract 3505: Classification of cancer subtypes by cfDNA fragmentomics analysis

Abstract Introduction Fragmentation patterns of cell-free DNA (cfDNA) from whole genome sequencing offer insights into nucleosome occupancy, providing a tool to infer epigenomic and transcriptomic information. The nucleosome complex, the primary protector of cfDNA, is reflected in the size distribution of cfDNA fragments, with a mode fragment size of 167 bp corresponding to the wrapping of DNA around a single nucleosome. Nucleosome occupancy patterns at transcription factor binding sites (TFBS) exhibit differences between circulating tumor DNA in cancer patients and normal plasma controls, as well as among different tumor subtypes. Therefore, cfDNA fragmentomics can be employed for cancer subtyping analysis. Methods We utilized the Griffin framework to classify tumor subtypes based on nucleosome profiling of cancer-specific TFBS and tumor subtype-specific chromatin accessibility regions from low-pass whole genome sequencing data of cfDNA. For prostate cancer subtyping, AR and ASCL1 binding sites were used to distinguish between androgen receptor-dependent prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). ER and ERBB2 were employed for breast cancer subtyping to differentiate between ER-positive and ER-negative tumor subtypes. Results Nucleosome profiling patterns at AR binding sites (ARBS) were compared among over 500 prostate cancer plasma samples and 42 normal plasma backgrounds. The ARBS nucleosome profiling abnormality score (ARBS score) was quantified by comparing its GC-corrected fragment central coverage with the normal background using a standard Z-score.Majority of prostate cancer samples with a high ARBS score could be classified as ARPC. For 12 samples with high tumor fraction but low ARBS score and no AR copy number amplification, we found that 8 had a high ASCL1 nucleosome profiling abnormality score. All these samples also exhibited RB1 copy number loss, suggesting that these 8 samples likely belong to NEPC. Nucleosome profiling of 171 breast cancer plasma samples at breast cancer-specific ER binding sites revealed that majority of samples had a high ERBS abnormality score, indicating likely ER-positive breast cancer. Conclusion Our results demonstrate that nucleosome profiling of tumor cfDNA exhibits distinct patterns across different tumor types. Integrating nucleosome profiling at cancer-specific transcription factor binding sites can facilitate cancer subtyping, highlighting its potential clinical utility for patient stratification. Citation Format: Lisha Zhu, Chao Dai, Shuang Gan, Shidong Jia, Pan Du. Classification of cancer subtypes by cfDNA fragmentomics analysis [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 3505.

Nucleosome profiling at AR binding sites (ARBS) to provide a prognostic tool for metastatic castration-resistant prostate cancer (mCRPC).

42 Background: Prostate cancer is the second leading cause of cancer death for men in the U.S., and metastatic castration-resistant prostate cancer (mCRPC) is the most advanced form. In previous work, we have reported prognostic significance of PTEN-PI3K-AKT pathway and DNA damage response aberrations detected in cell-free DNA (cfDNA) of mCRPC. Here we utilized nucleosome profiling pattern at androgen receptor (AR) binding sites to further investigate AR signaling pathway dysregulation of mCRPC. Methods: Using the next-generation sequencing PredicineScore liquid biopsy assay (low-pass whole genome sequencing, LP-WGS), we profiled cfDNA fragment coverage centered around AR binding sites (ARBS). For 118 mCRPC patient samples, we quantified sample-level ARBS nucleosome profiling abnormality score (ARBS score) by comparing its centric fragment coverage with normal plasma background using standard Z-score. ARBS score was correlated with clinical outcomes including PSA response rate (PSA RR), progression-free survival and overall survival (OS). Results: By comparing ARBS nucleosome profiling and normal plasma background, we found that mCRPC plasma samples had significant ARBS nucleosome depletion, reflective of transcription factor binding and persistent AR signaling. Notably, ARBS nucleosome depletion was prostate cancer specific, and did not occur in other examined cancer types. ARBS score was highly correlated with tumor fraction (R=0.82), indicating that high tumor fraction mCRPC sample had higher AR binding. Importantly, we found that high ARBS score (>2) in pre-treatment samples (n=86) was associated with significantly shorter OS and lower PSA RR compared with low ARBS (≤2) group for patients receiving AR pathway inhibitor (ARPI) or taxane chemotherapy (13.4 vs. 19.4 months, p=7.3×10-3, 45.7% vs. 77.5%, p=5.2×10-3). By integrating genome-wide DNA methylation profiling, we further found that ARBS was hypo-methylated in mCRPC patients, and identified a strong negative correlation between DNA methylation beta value and ARBS score (R=-0.83). Conclusions: ARBS nucleosome profiling abnormality inferred from LP-WGS can serve as a key prognostic tool for mCRPC patients. Patients with high ARBS score had worse clinical outcomes, highlighting the potential clinical utility of cfDNA fragmentomics analysis for mCRPC patient stratification. Integration of mutation, copy number variation and fragmentomics profiling of ARBS may further enhance our understanding of molecular aberrations and treatment outcomes in mCRPC.

Dissecting transcription of the 8q24-MYC locus in prostate cancer recognizes the equilibration between androgen receptor direct and indirect dual-functions

BackgroundAndrogen receptor (AR) activation and repression dual-functionality only became known recently and still remains intriguing in prostate cancer (PCa). MYC is a prominent oncogene that functionally entangles with AR signaling in PCa. Further exploration of AR regulatory mechanisms on MYC gene transcription bears clinical and translation significance.MethodsBioinformatics analysis of PCa cell line and clinical RNA-Seq and ChIP-Seq (chromatin immunoprecipitation-sequencing) datasets to anchor interactions of AR and MYC transcriptional networks. ChIP-qPCR and 3C (chromosome conformation capture) analyses to probe MYC distal regulation by AR binding sites (ABSs). CRISPR/Cas9-mediated genome-editing to specify functions of ABS within the 8q24-MYC locus on androgen-mediated MYC transcription. Global FoxA1 and HoxB13 distribution profiling to advance AR transcriptional mechanisms.ResultsHere we recognize AR bi-directional transcription mechanisms by exploiting the prominent 8q24-MYC locus conferring androgen hyper-sensitivity. At ~ 25 Kb downstream of the MYC gene, we identified an undefined ABS, P10. By chromatin analyses, we validated androgen-dependent spatial interaction between P10 and MYC-Promoter (MYC-Pro) and temporal epigenetic repression of these MYC-proximal elements. We next designed a CRISPR/Cas9-mediated double genomic knock-out (KO) strategy to show that P10-KO slightly lessened androgen-elicited MYC transrepression in LNCaP-AR cells. In similar genomic editing assays, androgen-mediated MYC repression became slightly deepened upon KO of P11, an ABS in the PVT1 gene locus highly enriched in AR-binding motifs and peaks. We also investigated multiple ABSs in the established PCAT1 super-enhancer that distally interacts with MYC-Pro for transactivation, with each KO pool consistently shown to relieve androgen-elicited MYC repression. In the end, we systemically assessed androgen effects in the 8q24-MYC locus and along PCa genome to generalize H3K27ac and BRD4 re-distribution from pioneer factors (FoxA1 and HoxB13) to AR sites.ConclusionTogether, we reconciled these observations by unifying AR dual-functions that are mechanistically coupled to and equilibrated by co-factor redistribution.Graphical

Luteolin inhibits A549 cells proliferation and migration by down-regulating androgen receptors

BackgroundYi Fei Qing Hua Granules (YQG) is a traditional Chinese herbal medicine with the effects of inhibiting the proliferation of lung cancer cells. Luteolin is one of the active compounds of YQG. Luteolin is a common flavonoid extracted from natural herbs and it can promote cancer cells apoptosis has been reported. However, the underlying molecular mechanism and effects of luteolin on human lung cancer needs to be validated.MethodsMolecular docking, network pharmacology methods and quantitative structure–activity relationship (QSAR) model were used to identify the active components of YQG and their possible mechanisms of action. Western blot analysis was used to measure AR expression in A549 cells. Cell migration assays were used to detect A549 cells proliferation transfected by AR plasmid and AR mutation plasmid, respectively.ResultsTCMSP search results revealed that there are 182 active compounds in YQG, which correspond to 232 target genes. Sixty-one genes were overlapping genes in the 2 datasets of TCMSP and GeneCards. Through bioinformatics tagging of these overlapping genes, a total of 1,951 GO functional tagging analysis and 133 KEGG pathways were obtained. Through molecular docking technology and QSAR model verification, the multi-target active compound luteolin was screened out as one of the active components of YQG for in vitro verification. Androgen receptor (AR) was the hub protein with the highest docking score of luteolin. Western blot showed that luteolin could inhibit AR protein expression in lung cancer cell line A549. After the phosphorylation site of AR protein 877 was inactivated, the ability of luteolin to inhibit the proliferation of lung cancer cells was weakened. Luteolin significantly inhibited the growth of A549 xenogeneic tumors at day 25 and 28 and inhibited the expression of AR.ConclusionIn this study, we have explored luteolin as one of the active components of YQG, and may inhibit the proliferation and migration of A549 cells by decreasing the expression of AR and the regulation of phosphorylation at AR-binding sites.

Androgen receptor binding sites enabling genetic prediction of mortality due to prostate cancer in cancer-free subjects

Prostate cancer (PrCa) is the second most common cancer worldwide in males. While strongly warranted, the prediction of mortality risk due to PrCa, especially before its development, is challenging. Here, we address this issue by maximizing the statistical power of genetic data with multi-ancestry meta-analysis and focusing on binding sites of the androgen receptor (AR), which has a critical role in PrCa. Taking advantage of large Japanese samples ever, a multi-ancestry meta-analysis comprising more than 300,000 subjects in total identifies 9 unreported loci including ZFHX3, a tumor suppressor gene, and successfully narrows down the statistically finemapped variants compared to European-only studies, and these variants strongly enrich in AR binding sites. A polygenic risk scores (PRS) analysis restricting to statistically finemapped variants in AR binding sites shows among cancer-free subjects, individuals with a PRS in the top 10% have a strongly higher risk of the future death of PrCa (HR: 5.57, P = 4.2 × 10−10). Our findings demonstrate the potential utility of leveraging large-scale genetic data and advanced analytical methods in predicting the mortality of PrCa.

Androgen receptor and MYC transcriptomes are equilibrated in multilayer regulatory circuitries in prostate cancer.

The discovery of androgen receptor (AR) having transrepression effects completes the circle of its functionalities as a typical transcription factor, which intrinsically bears dual functions of activation and repression linked to co-factor competition and redistribution. Indeed, AR dual functions are exemplified by locus-wide regulation of the oncogenic 8q24-MYC region. RT-qPCR assay and public RNA-profiling datasets were used to assess MYC transcription in androgen-sensitive cell lines. Public ChIP-seq and RNA-Seq datasets were computed to evaluate AR-MYC direct and indirect signatures. Gene sets in typical MYC and AR pathways were monitored to validate their cross-talks. Bio-informatics and chromosome conformation capture (3C) assay were performed in the AR gene locus to examine androgen-elicited distal regulation. Finally, co-factor re-distribution were globally tracked between AR and MYC binding sites. In this report, we found MYC responded negatively to androgen with hypersensitivity, rivaling AR natural functions as an innate androgen effector. Furthermore, both direct and indirect AR and MYC transcriptional programs were actively in equilibration. With established androgen-mediated versus MYC-mediated gene subsets, we validated AR and MYC pathways were both bidirectional and extensively entangled. In addition, we determined that the AR gene locus resembled the MYC gene region and both loci were androgen-repressed via epigenetics and chromatin architectural alterations. Significantly, transcriptional factor profiling along the prostate cancer (PCa) genome exposed that PCa transcriptomes were dynamically equilibrated between AR-binding site and MYC-binding site. Together, our findings stratified AR-MYC interactions that are extensively wired and intricately organized to compensate for essential PCa transcriptional programs and neutralize excessive signaling.

Influence of ADT on B7-H3 expression during CRPC progression from hormone-naïve prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for advanced prostate cancer (PCa) patients. Unfortunately, although tumors respond well initially, they enter dormancy and eventually progress to fatal/incurable castration-resistant prostate cancer (CRPC). B7-H3 is a promising new target for PCa immunotherapy. CD276 (B7-H3) gene has a presumptive androgen receptor (AR) binding site, suggesting potential AR regulation. However, the relationship between B7-H3 and AR is controversial. Meanwhile, the expression pattern of B7-H3 following ADT and during CRPC progression is largely unknown, but critically important for identifying patients and determining the optimal timing of B7-H3 targeting immunotherapy. In this study, we performed a longitudinal study using our unique PCa patient-derived xenograft (PDX) models and assessed B7-H3 expression during post-ADT disease progression. We further validated our findings at the clinical level in PCa patient samples. We found that B7-H3 expression was negatively regulated by AR during the early phase of ADT treatment, but positively associated with PCa proliferation during the remainder of disease progression. Our findings suggest its use as a biomarker for diagnosis, prognosis, and ADT treatment response, and the potential of combining ADT and B7-H3 targeting immunotherapy for hormone-naïve PCa treatment to prevent fatal CRPC relapse.

Abstract A014: NFI-family transcriptional factors direct AR cistrome redistribution during prostate cancer tumorigenesis

Abstract Previous studies found AR cistrome reprogramming during transition from normal prostate to PCa, and progression to CRPC. AR binding sites (bs) in PCa were reported to be enriched for FOXA1 and HOXB13, in comparison to normal prostate where these are not enriched. However, mechanisms that may drive FOXA1 and HOXB13 to these sites and initiate this neoplastic transformation remain unclear. To address this, we first asked if there are other transcription factors (TFs) involved in tumor evolution. A motif enrichment analysis using patient data set (GSE130408) showed significant enrichment of NFI and NFI half motifs at AR bs in localized hormone sensitive PCa in comparison to normal prostate (0.07 vs 0.04, p=0.0001; and 0.27 vs 0.21, p=0.0044, respectively). Notably, NFI TFs were shown previously to be associated with AR bs in PCa cells, and to modulate AR-target gene expression and changes in chromatin accessibility. This suggested that NFI TFs may contribute to AR cistrome redistribution that drives PCa development. Next, we generated a CRPC cell model resistant to enzalutamide (ENZ), and found that AR activity in these cells was driven by the ARv7 splice variant. Notably, ATAC-seq identified in these cells a subset of sites with greatly increased chromatin accessibility, and ChIP-seq showed that these ATAC-up sites were associated with ARv7 binding and increased H3K27acetylation (H3K27ac, the mark of active enhancers). Significantly, these sites also were enriched for NFI and NFI half motifs (0.04, p<0.0001 and 0.1, p<0.0001, respectively). Notably, while AR is known as a transcriptional activator, it also has repressive functions. To study AR-direct repression, we examined the redistribution of H3K27ac mark in response to AR-inhibition (ENZ) or stimulation (DHT). We identified subsets of AR-bound regions with increased H3K27ac after ENZ that corresponded to regions with decreased H3K27ac after DHT. These regions were highly enriched for NFI and NFI-half motifs (0.04, p<0.0001 and 0.14, p<0.0001, respectively). As expected, these regions also were associated with DHT-repressed genes including UGT2B15, physiologically involved in steroid hormones metabolism. Notably, depletion of NFI-B and X (the most expressed NFI-family members in PCa) resulted in increased H3K27ac levels at UGT2B15, and impaired the suppressive effect of DHT on UGT2B15 expression. RNA-seq after NFIB and X knockdown then showed 212 up- and 119 downregulated genes, indicating NFI is a modulator of both AR-activated and repressed genes. Ingenuity Pathway Analysis demonstrated contribution of NFIs to androgen signaling and maintaining the activity of Sirtuin (SIRT) pathways. SIRTs possess deacetylase activity, including deacetylation of histones, and inhibition of SIRT1 resulted in dramatically increased UGT2B15 expression. Collectively, these data support a role for NFI TFs in directing the AR cistrome during PCa tumorigenesis, and in modulating its transcriptional activation versus repressive functions, with the latter being mediated by recruitment of SIRT1. Citation Format: Larysa Poluben, Joshua Russo, Olga Voznesensky, Mannan Nouri, Anastasia Stavridi, Xintao Qiu, Matthew Freedman, Henry W. Long, Steven P. Balk. NFI-family transcriptional factors direct AR cistrome redistribution during prostate cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A014.

Abstract B004: Genomic loss of TP53 impairs AR-targeted activity in metastatic prostate cancer

Abstract Background: Metastatic prostate cancer (mPC) is notable for a spectrum of recurrent genomic alterations, some associated with overall prognosis and others predicting response to specific therapeutics. Standard mPC interventions are designed to suppress the signaling program regulated by the androgen receptor (AR). These include androgen deprivation therapy (ADT) to reduce AR ligand levels and AR signaling inhibitors (ARSI) that directly impair AR function. While ADT and ARSI produce clinical responses in most patients, the duration and depth of response vary greatly. In addition, specific recurring genomic alterations are associated with shorter responses after ADT and ARSI therapy. Among these are mutations and/or genomic copy loss involving key tumor suppressor genes, including PTEN, RB1 and TP53. However, the mechanistic contribution of TP53 loss to AR signaling and resistance to AR pathway-directed therapy remains unresolved. Methods: Isogenic prostate cancer cell lines with intact and TP53 deletions were constructed using CRISPR/Cas9. The transcriptome profiles were determined by RNA-seq, and ChIP-seq was used to determine the chromatin landscapes of AR localization. In addition, we performed ATAC-seq to assess chromatin accessibility. Finally, to further validate the findings, we comprehensively analyzed AR-regulated gene expression and AR cistromes across a panel of patient-derived xenograft (PDX) models and clinical cohorts of mPC patient samples with respect to TP53 status. Results: Compared to TP53-wild-type (WT) cells, the transcriptional results of isogenic knockout TP53 lines showed impaired AR canonical activity. However, the downregulated AR response pathway is not caused by reduced levels of AR transcript or protein levels. Additionally, a similar trend was also found while comparing TP53-WT PDX models with TP53-loss models. We found that the loss of TP53 can shift the chromatin binding of AR, and the altered AR binding sites correlated with genes involved in neurogenesis pathways, which provide a potential mechanism of how the loss of TP53 might impact AR signaling and promote ADT and ARSI treatment resistance. We also identified additional AR chromatin binding sites that were enriched due to the loss of TP53. The targets potentially regulated by these sites are involved in multiple pathways, including cell morphogenesis involved in differentiation and response to the endogenous stimulus. Conclusions and Future Directions: Overall, the results indicate that in addition to altering DNA damage and repair pathways and contributing to cancer progression, genetic loss of TP53 in mPC may directly impair ADT and ARSI therapies, partially through alteration of AR chromatin activity. In addition, the AR cistrome reprogramming resulting from TP53 loss could also affect other pathways that could be exploited to overcome ARSI treatment resistance. Citation Format: Wanting Han, Michael D. Nyquist, Ilsa Coleman, Navonil De Sarkar, Lisa Ang, Eva Corey, Peter S. Nelson. Genomic loss of TP53 impairs AR-targeted activity in metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B004.

Abstract A047: Panorama of complex structural variants in primary localized prostate cancer

Abstract Prostate cancer (PCa) is a hormone-driven disease characterized by an abundance of structural variations (SV). Deconvolving the SV patterns can both inform on prior mutational processes leading to cancer, and be leveraged to identify patients with more aggressive disease. Under the auspices of the Pan Prostate Cancer Group (PPCG) consortium, we used whole genome sequencing of 812 treatment naive PCa patients to study both simple and complex SVs. In total, we classified 15,708 simple SVs and 1,448 complex SV events. Complex SVs such as chromothripsis, chromoplexy and templated insertion were found in more than half of the cohort. We also detected tandem duplicator phenotype (TDP) in a subset of the patients associated with CDK12 aberrations. Breakpoint recurrence analysis of driver genes revealed disruption of e.g. PTEN and formation of TMPRSS2-ERG fusion genes frequently coincide with occurrences of specific complex SV types, suggesting specific mutational processes driving these alterations of these cancer genes. Based on the simple and complex SV classifications we extracted six SV signatures, including two TDP-like signatures, distinct deletion-specific SV signatures and a signature characteristic of AR binding sites. We found signatures associated with disease markers, including Gleason score, risk scores, as well as age. Together, these findings provide insights into mechanisms driving SV formation and driver gene alterations in PCa, with potential for identifying markers of aggressive disease. Citation Format: André Olsen, Francesco Favero, Yilong Li, Etsehiwot Girma, Breon Feran, Tony Papenfuss, Kristian Helin, Jüri Reimand, Joachim Weischenfeldt. Panorama of complex structural variants in primary localized prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A047.

Organotin Antifouling Compounds and Sex-Steroid Nuclear Receptor Perturbation: Some Structural Insights.

Organotin compounds (OTCs) are a commercially important group of organometallic compounds of tin used globally as polyvinyl chloride stabilizers and marine antifouling biocides. Worldwide use of OTCs has resulted in their ubiquitous presence in ecosystems across all the continents. OTCs have metabolic and endocrine disrupting effects in marine and terrestrial organisms. Thus, harmful OTCs (tributyltin) have been banned by the International Convention on the Control of Harmful Antifouling Systems since 2008. However, continued manufacturing by non-member countries poses a substantial risk for animal and human health. In this study, structural binding of common commercial OTCs, tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), monophenyltin (MPT), and azocyclotin (ACT) against sex-steroid nuclear receptors, androgen receptor (AR), and estrogen receptors (ERα, ERβ) was performed using molecular docking and MD simulation. TBT, DBT, DPT, and MPT bound deep within the binding sites of AR, ERα, and Erβ, showing good dock score, binding energy and dissociation constants that were comparable to bound native ligands, testosterone and estradiol. The stability of docking complex was shown by MD simulation of organotin/receptor complex with RMSD, RMSF, Rg, and SASA plots showing stable interaction, low deviation, and compactness of the complex. A high commonality (50-100%) of interacting residues of ERα and ERβ for the docked ligands and bound native ligand (estradiol) indicated that the organotin compounds bound in the same binding site of the receptor as the native ligand. The results suggested that organotins may interfere with the natural steroid/receptor binding and perturb steroid signaling.

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in >90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

Genetic determinants of chromatin reveal prostate cancer risk mediated by context-dependent gene regulation.

Many genetic variants affect disease risk by altering context-dependent gene regulation. Such variants are difficult to study mechanistically using current methods that link genetic variation to steady-state gene expression levels, such as expression quantitative trait loci (eQTLs). To address this challenge, we developed the cistrome-wide association study (CWAS), a framework for identifying genotypic and allele-specific effects on chromatin that are also associated with disease. In prostate cancer, CWAS identified regulatory elements and androgen receptor-binding sites that explained the association at 52 of 98 known prostate cancer risk loci and discovered 17 additional risk loci. CWAS implicated key developmental transcription factors in prostate cancer risk that are overlooked by eQTL-based approaches due to context-dependent gene regulation. We experimentally validated associations and demonstrated the extensibility of CWAS to additional epigenomic datasets and phenotypes, including response to prostate cancer treatment. CWAS is a powerful and biologically interpretable paradigm for studying variants that influence traits by affecting transcriptional regulation.

Positive epigenetic regulation loop between AR and NSUN2 promotes prostate cancer progression.

BackgroundProstate cancer (PCa) is a major type of cancer in man worldwide. Androgen deprivation therapy (ADT) and the next‐generation androgen receptor (AR) pathway inhibitors have acquired great success in treating PCa. However, patients treated with ADT or AR targeted therapy are inevitably developing into castration‐resistant prostate cancer (CRPC) or becoming drug resistance. The role of mRNA 5‐methylcytosine (m5C) modification in cancers is largely unknown. This study aimed to explore the role of the m5C methyltransferase NSUN2 in Prostate cancer (PCa).MethodsThe expression of NSUN2 and its clinicopathological impact were evaluated in PCa cohorts. The effect of NSUN2 on the biological characteristics of PCa cells was investigated on the basis of gain‐offunction and loss‐of‐function analyses. Subcutaneous models further uncovered the role of NSUN2 in tumor growth. Epi‐transcriptome assays with RNA bisulfite sequencing (RNA‐BisSeq) analysis and in vitro enzyme reaction assays were performed to validate the targeted effect of NSUN2 on AR. AR‐binding sites in the NSUN2 promoter were investigated by ChIP and luciferase assays to uncover the interplay between NSUN2 and AR signaling. RIP‐qPCR and EMSA methods were performed to confirm that YBX1 binds to AR m5C sites.ResultsNSUN2 is highly expressed in PCa and predicts poor outcome. NSUN2 plays roles as a PCa oncogene both in vitro and in vivo. Depletion of NSUN2 results in decreased expression and activities of AR, including AR‐V7. Mechanistically, NSUN2 posttranscriptionally stabilized AR by cluster m5C modification in a m5CYBX1‐dependent manner. Strikingly, treatment with enzalutamide, an effective AR inhibitor, reduces NSUN2 expression and decreases the m5C modification level in prostate cancer cells. Finally, we found that AR transcriptionally regulates NSUN2.ConclusionNSUN2 stabilizes AR mRNA through cluster 5‐methylcytosine modification and activates a positive feedback loop to promote prostate cancer.

An allostatic epigenetic memory on chromatin footprints after double-hit acute stress

Stress induces allostatic responses, whose limits depend on genetic background and the nature of the challenges. Allostatic load reflects the cumulation of these reponses over the course of life. Acute stress is usually associated with adaptive responses, although, depending on the intensity of the stress and individual differences , some may experience maladaptive coping that persists through life and may influence subsequent responses to stressful events, as is the case of post-traumatic stress disorder. We investigated the behavioral traits and epigenetic signatures in a double-hit mouse model of acute stress in which heterotypic stressors (acute swim stress and acute restraint stress) were applied within a 7-day interval period. The ventral hippocampus was isolated to study the footprints of chromatin accessibility driven by exposure to double-hit stress. Using ATAC sequencing to determine regions of open chromatin, we showed that depending on the number of acute stressors, several gene sets related to development, immune function, cell starvation, translation, the cytoskeleton, and DNA modification were reprogrammed in both males and females. Chromatin accessibility for transcription factor binding sites showed that stress altered the accessibility for androgen, glucocorticoid, and mineralocorticoid receptor binding sites (AREs/GREs) at the genome-wide level, with double-hit stressed mice displaying a profile unique from either single hit of acute stress. The investigation of AREs/GREs adjacent to gene coding regions revealed several stress-related genes, including Fkbp5, Zbtb16, and Ddc, whose chromatin accessibility was affected by prior exposure to stress. These data demonstrate that acute stress is not truly acute because it induces allostatic signatures that persist in the epigenome and may manifest when a second challenge hits later in life.

Abstract 3625: Clonal architecture and evolution of treatment-resistant prostate cancer via deep whole-genome ctDNA sequencing

Abstract Background: Circulating tumor DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring. However, integration of ctDNA tests into clinical management is critically impeded by the poor understanding of the distinct somatic populations comprising bulk ctDNA—including their relationship to synchronous metastatic tissue, and their temporal dynamics during standard-of-care treatment. Prior approaches relying on targeted and/or low-resolution techniques (e.g. targeted exon sequencing, low-pass (shallow) whole-genome sequencing; WGS) do not permit comprehensive dissection of clonal architecture and unbiased analysis of putative resistance mechanisms. Methods: We performed deep WGS on serial plasma ctDNA (median depth: 185×) and synchronous metastatic tissue biopsies with high tumor purity from 35 patients with metastatic castration-resistant prostate cancer. We developed a subclonal reconstruction algorithm optimized for our data enabling resolution of per-patient evolutionary histories and ctDNA clonal composition. ctDNA nucleosome footprinting was used to infer mRNA abundance in synchronously biopsied metastases and androgen receptor (AR) transcription factor activity at 3224 AR binding sites (ARBS). Results: We comprehensively assess all classes of genomic alterations and demonstrate that ctDNA harbors greater populational heterogeneity than metastatic tissue (p<0.001). The evolutionary histories of ctDNA populations indicate frequent whole-genome doubling and attenuation of C>T aging-associated mutation signature during subclonal differentiation. Although driver alterations were largely concordant between tissue and ctDNA, each individual metastasis contributed only a minor share of total ctDNA (average ctDNA contribution: 17%). By comparing serial ctDNA before and after clinical progression on potent AR pathway inhibitors, we reveal population restructuring converging solely on AR copy augmentation as the dominant genomic driver of acquired treatment-resistance. Nucleosome depletion at transcription start-sites is highly correlated with same-patient metastatic tissue mRNA abundance, indicating that ctDNA fragmentomics can recapitulate transcriptomic patterns in metastatic lesions. Most ctDNA samples exhibited strong ARBS nucleosome depletion which correlated with AR gene copy number (R=0.36, p=0.003). Finally, serial ctDNA nucleosome profiling at ARBS revealed adaptive transcriptomic resistance to AR pathway inhibitors, including lineage switch to a neuroendocrine-like (AR-low) state. Conclusions: We show that the populations comprising ctDNA are typically complex and more heterogeneous than those found in bulk WGS of a synchronous metastasis. Our work advocates for liquid biopsy as a comprehensive multi-omic discovery tool for cancers with high ctDNA fractions. Citation Format: Cameron Herberts, Matti Annala, Joonatan Sipola, Sarah W. Ng, Xinyi E. Chen, Anssi Nurminen, Olga Korhonen, Aslı D. Munzur, Kevin Beja, Elena Schönlau, Cecily Q. Bernales, Elie Ritch, Jack V. Bacon, Nathan A. Lack, Matti Nykter, Rahul Aggarwal, Eric J. Small, Martin E. Gleave, David A. Quigley, Felix Y. Feng, Kim N. Chi, Alexander W. Wyatt. Clonal architecture and evolution of treatment-resistant prostate cancer via deep whole-genome ctDNA sequencing [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 3625.

Abstract 680: Topoisomerase 2 beta facilitates chromatin reorganization during Androgen Receptor induced transcription and contributes to chromoplexy in prostate cancer

Abstract Prostate cancer (PCa) is the most common malignancy and second leading cause of cancer death in American men. Androgen Receptor (AR) mediated transcriptional program is central to normal prostate homeostasis and drives PCa growth and survival. Chromoplexy, a highly complex genomic architecture with several intra- and inter-chromosomal segments joined in a chain, is among the most prominent genetic alterations that drive both prostate cancer initiation and progression, and often involves sites of AR transcription. Previous studies have shown that AR induced, topoisomerase 2 beta (TOP2B) mediated double strand breaks were recombinogenic and led to de novo formation of TMPRSS2-ERG fusion gene, shedding light on the potential role of TOP2B in chromoplexy formation. However, the precise role of TOP2B in AR transcription was not well understood. Here, we hypothesize that TOP2B is recruited to resolve topological constraints arising during induction of AR transcriptional programs, and its catalytic activity is required to facilitate or maintain chromosomal interactions optimal for transcriptional induction. We performed Chromosomal Conformation Capture related techniques (3C and HiC) on LNCaP cells before and after androgen stimulation and observed an increase in chromatin interactions within 15kb from promoters of AR target genes upon androgen induction. These interactions depended on TOP2B, as TOP2B catalytic inhibition or knockdown reduced them significantly. Furthermore, TOP2B Hi-CHIP revealed that TOP2B is involved in key enhancer-promoter looping and in several interactions among gene body, enhancers, promoters of AR target genes, and nearby topological associated domain borders. We went on to isolate which steps during AR transcription induction required TOP2B by examining chromatin localization of the key factors, including AR, cohesin (SMC1A), CTCF, histone 3 lysine 27 acetylation (H3K27ac), and total and phosphorylated RNA Polymerase II (RNAPII) using ChIP-seq. These experiments revealed that TOP2B was not required for AR binding nor for localization of H3K27ac marks. However, it was required for recruitment of cohesin to AR binding sites as well as to AR target gene promoters and gene bodies, for displacement of CTCF near AR target genes, and for localization and phosphorylation of RNAPII at AR target genes. These data nominate TOP2B as a key AR coactivator, assisting in the proper assembly of cohesin during transcription induction, and maintaining chromosomal interactions optimal for binding and activation of RNAPII. Intriguingly, sites of binding of TOP2B, as well as of cohesin, were highly associated with sites of chromoplexy complex rearrangements in human prostate cancers. Taken together, this work elucidates the role of TOP2B in AR-induced transcription, and implicates its involvement in chromoplexy formation in PCa. Citation Format: Minh-Tam N. Pham, Michael C. Haffner, Heather C. Wick, Jonathan B. Coulter, Anuj Gupta, Roshan V. Chikarmane, Harshath Gupta, Sarah Wheelan, William G. Nelson, Srinivasan Yegnasubramanian. Topoisomerase 2 beta facilitates chromatin reorganization during Androgen Receptor induced transcription and contributes to chromoplexy 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 680.

Targeting B7-H3 in prostate cancer: Phase 2 trial in localized prostate cancer using the anti-B7-H3 antibody enoblituzumab, with biomarker correlatives.

5015 Background: B7-H3/CD276, a member of the B7 superfamily, is highly expressed in prostate cancer (PCa) and is associated with rapid biochemical recurrence and early metastases. B7-H3 is the only checkpoint candidate to have a presumptive androgen receptor binding site, suggesting interaction with the androgen axis. Enoblituzumab (MacroGenics) is an investigational humanized Fc-optimized B7-H3–targeting antibody that induces antibody dependent cellular cytotoxicity (ADCC). Methods: In this phase 2 single-arm biomarker-rich neoadjuvant trial, men with operable intermediate- and high-risk localized prostate cancer (Grade Groups 3-5) were enrolled to evaluate the safety, anti-tumor efficacy, and immunogenicity of enoblituzumab when given prior to prostatectomy. Patients received enoblituzumab (15 mg/kg IV weekly x 6) prior to surgery. Prostate glands were harvested 2 weeks after the last dose, and were examined for pathologic and immunologic endpoints. The co-primary outcomes were safety and PSA0 at 1 year post-op. Pre-planned secondary outcomes were PSA and Gleason grade group change from biopsy to prostatectomy. Results: 32 men were enrolled. Grade 3/4 adverse events occurred in 12% of patients. One patient developed a grade-3 infusion reaction, and one had immune myocarditis that improved with steroids. Pre-prostatectomy PSA declines of >10% were observed in 31% of patients (95% CI: 16-50%). PSA0 at 1 year post-op was seen in 66% of men (95% CI: 47-81%). Median time to PSA recurrence was not reached, with a median follow-up of 30 months. Gleason group upgrade, no change, and downgrade was observed in 13%, 37%, and 50% of patients. Gleason grade group changes were significantly associated with enoblituzumab treatment compared to 1:1 matched historical controls (p=0.023). Tumor microenvironment profiling by NanoString GeoMx spatial proteomics and PanCancer IO 360 mRNA expression analysis revealed post-treatment upregulation of CD8+ T cells, PD-1/PD-L1 expression, and immune activation (granzyme B, IFN signaling, myeloid inflammation). There was a significant association between CD8+ T-cell increases and Gleason grade group declines. First-in-human antigen spread profiling revealed no safety concerns. TCR sequencing showed focused peripheral expansion of tumor associated T-cell clones that correlated with PSA0 at 1 year. Whole exome and RNAseq data, and clinical correlations, will be presented. Conclusions: In this neoadjuvant trial, inhibition of B7-H3 with enoblituzumab demonstrated favorable safety and encouraging activity in localized PCa patients. Data suggest robust intratumoral induction (adaptive upregulation) of immune checkpoints, T-cell activation, and myeloid inflammation. Enoblituzumab-induced peripheral expansion of tumor associated T-cell clones may be associated with tumor control. Clinical trial information: NCT02923180.

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.