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African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function. Significance: Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.

SF_6 RNA-Seq PCA

ST_12 ChIP-Seq to RNA-Seq

SF_8 RNA-Seq GSEA

SF_13 GSEA tumors

Supplementary Table _2 - RIME II

ST_3 ATAC-Seq

SF_9 overlap miRNA

African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function.Significance:Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.

SF_3 motifs in ATAC-Seq

ST_4 ChIP-Seq

ST_5 ChromHMM

ST_7 ATAC-Seq to RIME

SF_5 ChIP-Seq motif

SF_2 Ancestry cell lines

SF_4 VDR cistrome

ST_6 ChIP-Seq Motif

ST_8 ATAC/ChIP-Seq to splicing

SF_7 RNA-Seq volcanos

BackgroundAfrican American (AA) prostate cancer (PCa) appears uniquely sensitive to 1α,25(OH)2D3 signaling, compared to European American (EA) PCa, but the extent and impact of vitamin D receptor genomic functions remain poorly defined.ResultsA panel of EA and AA prostate epithelial cells (EA: HPr1-AR, LNCaP, AA: RC43N, RC43T, RC77N, RC77T) were analyzed with RIME to reveal the cell-specific composition of the VDR- complex. 1α,25(OH)2D3-dependent ATAC-Seq revealed the greatest impact on nucleosome positioning in RC43N and RC43T, with gain of nucleosome-free at enhancer regions. VDR ChIP-Seq identified stronger and more frequent VDR binding in RC43N and RC43T that was enriched for a larger and distinct motif repertoire, than EA cells. VDR binding significantly overlapped with core circadian rhythm transcription factors in AA cell line models. RNA-Seq also revealed significantly stronger 1α,25(OH)2D3 dependent VDR transcriptional responses enriched for circadian rhythm and inflammation networks in AA cells. Whilst RC43N was most responsive, RC43T displayed distorted responses. Significantly reduced BAZ1A/SMARCA5 in AA PCa samples was identified, and restored BAZ1A expression uniquely and significantly increased 1α,25(OH)2D3-regulated VDR targets in AA cells. These VDR- dependent cistrome-annotated genes were also uniquely and most significantly identified in three cohorts of AA PCa patients.ConclusionThese data suggest VDR transcriptional control in the prostate is more potent and dynamic in AA men, and primed to govern inflammatory and circadian pathways. Reduced BAZ1A/SMARCA5 expression and/or reduced environmentally-regulated serum vitamin D3 levels suppress these actions. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes including stress responses, access to quality early detection and treatment, social determinants and that collectively contribute to PCa health disparities.

The human cell requires ways to specify its transcriptome without altering the essential sequence of DNA; this is achieved through mechanisms which govern the epigenetic state of DNA and epitranscriptomic state of RNA. These alterations can be found as modified histone proteins, cytosine DNA methylation, non-coding RNAs, and mRNA modifications, such as N6-methyladenosine (m6A). The different aspects of epigenomic and epitranscriptomic modifications require protein complexes to write, read, and erase these chemical alterations. Reflecting these important roles, many of these reader/writer/eraser proteins are either frequently mutated or differentially expressed in cancer. The disruption of epigenetic regulation in the cell can both contribute to cancer initiation and progression, and increase the likelihood of developing resistance to chemotherapies. Development of therapeutics to target proteins involved in epigenomic/epitranscriptomic modifications has been intensive, but further refinement is necessary to achieve ideal treatment outcomes without too many off-target effects for cancer patients. Therefore, further integration of clinical outcomes combined with large-scale genomic analyses is imperative for furthering understanding of epigenomic mechanisms in cancer.

The biological drivers of prostate cancer (PCa) health disparities remain enigmatic. There are genomic differences, for example in the incidence of TMPRSS2 and ERG gene fusions which differ between European American (EA) and African American (AA) PCa. Accumulating epidemiological evidence suggests loss of vitamin D receptor VDR signaling contributes to AA PCa. Therefore, we investigated the VDR signaling in AA and EA prostate models. VDR protein levels were comparable between AA (RC43N, RC43T, RC77N, RC77T) and EA cells (HPr1AR, LNCaP) but RIME analyses identified significant AA-EA differences in VDR binding of coactivators (e.g. NCOA5), corepressors (e.g. HDAC2), and components of SWI/SNF and circadian rhythm complexes. ATAC-Seq identified 1α,25(OH)2D3-stimulated gain in nucleosome free (NF) regions was greatest in RC43N (non-malignant AA cells) and enriched for circadian rhythm transcription motifs (e.g. CLOCK). In RC43T AA PCa cells 1α,25(OH)2D3 led to a loss of NF free regions. Similarly, VDR ChIP-Seq revealed the cistrome was greater in AA cell models, and was dynamically altered in RC43N cells by 1α,25(OH)2D3 and overlapped with the SWI/SNF component SMARCA4, but 1α,25(OH)2D3 led to reduced VDR binding in RC43T cells. The 1α,25(OH)2D3-regulated transcriptome was largest in RC43N and RC43T but GSEA terms were frequently and oppositely enriched, for example Reactome S phase genes were negatively enriched in RC43N, but positively enriched in RC43T. Cistrome-transcriptome integration identified the most significant relationships between VDR binding and the magnitude of 1α,25(OH)2D3-regulated genes in RC43N. Global coregulator analyses identified significantly reduced BAZ1A/SMARCA5 in AA PCa in TCGA and a published cohort of ~1500 AA/EA tumors. Restoring BAZ1A expression in cells resulted in significantly enhanced 1α,25(OH)2D3-regulated transcriptome in RC43N and RC43T cells, and hypergeometric tests revealed the significant enrichment of direct VDR target genes. The footprint of altered AA VDR signaling was evident in three previously established clinical cohorts. Serum miRNA that predicted AA progression from HGPIN to PCa were enriched for 1α,25(OH)2D3-regulated VDR targets; AA tumors from a vitaminD3-treated chemotherapy trail were enriched for VDR-bound genes; and, tumors from men with low serum 25(OH)D3 levels were significantly enriched for VDR target genes identified in AA but not EA PCa. Together, these data suggest VDR transcriptional control is most potent and dynamic in AA prostate cells, and is primed to govern inflammatory and circadian pathways. Reduced BAZ1A/SMARCA5 expression and/or reduced environmental-regulated serum vitamin D3 levels most acutely suppress these actions in AA PCa. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes that contributes to PCa health disparities. Citation Format: Shahid Hussain, Manjunath Siddappa, Sajad A. Wani, Hedieh Jafari, Jaimie S. Gray, Hsu-Chang Wu, Hancong Tang, Mark D. Long, Isra Elhussin, Balasubramanyam Karanam, Honghe Wang, Rebecca Morgan, Gary Hardiman, Isaacson B. Adelani, Rotimi Oladapo, Adam R. Murphy, James R. Marshall, Chanita Highes Halbert, Lara E. Sucheston-Campbell, Clayton Yates, Moray J. Campbell. Vitamin D receptor function in African American prostate cancer is disrupted by reduced BAZ1A/SMARCA5 [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 3619.

Epigenetic mechanisms in prostate cancer (PCa) alter androgen receptor (AR) enhancer access resulting in re-wired AR signaling and cancer progression and/or therapy resistance. Previously, we established miR-96 targets RARγ and its coactivator TACC1, which in turn impacts AR signaling associated with PCa progression. In the current study we sought to define the RARγ complex, its interactions with the AR, and in turn how this controls gene expression. Increased RARγ levels through miR-96 treatments increased both ENZA antiproliferative sensitivity associated with an increase in G1 and G2/M. IMPACT-seq identified miR-96 recognition elements (MREs) in HPr1AR (~1950 at 24h) and LNCaP cells (~ 350) with ~ 150 shared. In parallel, m6A-Seq revealed that the epitranscriptome mark m6A differed significantly between cells. For example, m6A was more significantly enriched on TACC1 mRNA in LNCaP compared to HPr1AR and m6A-marked MRE resulted in genes being more signficantly down-regulated by miR96, including TACC1. RIME revealed that RARγ significantly interacts miR-96 differentially expressed transcripts (DETs) and proteins (DEPs) including m6A regulators (METTL3) and QSER1 complex that protects against enhancer methylation. Reflecting this, restoring RARγ levels in 22Rv1 cells, increased AR genomic binding 12-fold to greater than 9,500 sites and further by TACC1. These gained AR sites were enriched for GATA and Homeobox motifs, significantly overlapped with H3K27ac and super-enhancers, and also ChromHMM-defined Poised Enhancers (logPV = 163) and Transcribed regions (logPV = 179). RNA-Seq experiments revealed that RARγ/TACC1 significantly altered the frequency and fold change of DHT-regulated genes. Finally, miR-96 antagomir/ENZA treatments drove a unique transcriptome that triggered cell cycle arrest and significantly associated with worse survival in the SU2C cohort. To summarize these data suggest that miR-96 is directed to control the RARγ/TACC1 complex in part by m6A marks. Restoring the RARγ/TACC1 complex increased association with potential bookmarking factor like QSER1 and redirects AR to active enhancers that promote ENA-regulated genes and increase propensity for cells to undergo cell arrest. Citation Format: Sajad A. Wani, Jaimie S. Gray, Moray J. Campbell. RAR gamma interactome and cistrome guide the androgen receptor enhancer usage in prosate 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 6243.

Background: Historically, estrogens were used therapeutically in prostate cancer (PCa) but discontinued due to serious side effects. Given estrogen receptor alpha (ERα) and beta (ERβ) exert opposing actions, it is unclear how estrogen actions control PCa growth. The OSU Drug Development Institute developed a novel, highly selective, ERβ ligand (OSU-ERB-12 (ERB12)) which has excellent in vivo PK/PD properties. Methods: A combination robotic high-throughput drug viability screen was undertaken in androgen deprivation therapy (ADT)-sensitive and resistant PCa cell models that combined multiple concentrations of ERB12 with 30 compounds including Enzalutamide (ENZA), and epigenomic regulators; potent combinations were examined by clonogenic assays. The ERB12 and ENZA combination was examined in 22RV1 by RNA-Seq and ATAC-Seq and CRISPR-editing undertaken to delete ERα or ERb. Result: All cell lines (LNCaP, 22RV1, PC-3, and DU145) were sensitive to high doses of ERB12 alone (1-10μM) whereas each cell line displayed unique responses to ERB12 drug interactions. 22RV1 cells responded with the greatest additive effects to combinations with ENZA and HDAC or DNMT inhibitors. Clonogenic assays also demonstrated significant suppression of colony formation by ERB12 combined with all three classes of drug. Also, 22RV1 cells with CRISPR-deleted ERα grew slower whereas faster with ERβ deletion. ATAC-Seq in 22RV1 cells revealed that the combination of ENZA (1μM) with ERB12 (100nM), but not 17b-estradiol (E2) + ENZA), led to unique gain of nucleosome free (NF) regions enriched for nuclear receptor motifs (e.g. AR, RARa, PPARa) and loss of CTCF motifs. These regions also significantly overlapped with ChromHMM-defined Active and Poised Enhancers. Parallel RNA-Seq also identified a unique ERB12/ENZA-dependent transcriptome significantly associated with repression of androgen and MYC signaling. MYC repression was confirmed at the protein level. Integration of ATAC-Seq and RNA-Seq data revealed antagonism between ERB12 and E2 treatments notably with ERB12/ENZA leading to gain of NF regions and increased expression of coregulators (e.g. RUNX1 and NCOA3) that were repressed by E2/ENZA. Finally, transcript-aware analyses identified unique and common ERB12/ENZA driven splice variation including coregulators NCOR2 and PRAME. Conclusion: Our data suggest that there is a cooperative action between ERB12 and ENZA in limiting cell proliferation and altering nucleosome positioning and leading to a unique transcriptome. ERB12, is an orally-available and well-tolerated drug that appears to antagonize the AR driven signaling pathways that lead to lethal prostate cancer. We propose that ERB12 could allow patients to take a lower, and more well-tolerated dose of ADT drugs, which at high dose can be very harsh on patients. Citation Format: Jaimie S. Gray. Cooperative actions of a novel ERβ ligand, OSU-ERb-12, and Enzalutamide in advanced 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 884.