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Supplementary Table 2: List of genes defined as RA target genes on basis of RARα binding within 20 kb of the Gene Symbol

Neuroblastoma cell lines can differentiate upon treatment with retinoic acid (RA), a finding that provided the basis for the clinical use of RA to treat neuroblastoma. However, resistance to RA is often observed, which limits its clinical utility. Using a gain-of-function genetic screen, we identified an unexpected link between RA signaling and mastermind-like 3 (MAML3), a known transcriptional coactivator for NOTCH. Our findings indicate that MAML3 expression leads to the loss of activation of a subset of RA target genes, which hampers RA-induced differentiation and promotes resistance to RA. The regulatory DNA elements of this subset of RA target genes show overlap in binding of MAML3 and the RA receptor, suggesting a direct role for MAML3 in the regulation of these genes. In addition, MAML3 has RA-independent functions, including the activation of IGF1R and downstream AKT signaling via upregulation of IGF2, resulting in increased proliferation. These results demonstrate an important mechanistic role for MAML3 in proliferation and RA-mediated differentiation.Implications: MAML3 coordinates transcription regulation with receptor tyrosine kinase pathway activation, shedding new light on why this gene is mutated in multiple cancers. Mol Cancer Res; 14(5); 411–22. ©2016 AACR.

Supplementary method, Supplementary figures and supplementary figure legends: Supplementary Figure 1. Viral VBIM integration site in MAML3 gene and equal sensitivity to cisplatin of parental and SD3.23 cells. Supplementary Figure 2. Overexpression of MAML3, but not MAML1 and MAML2, mediates resistance to retinoic acid in multiple neuroblastoma cell lines. Supplementary Figure 3. Induction of neuronal markers upon RA treatment is decreased in MAML3 overexpressing cells. Supplementary Figure 4. MAML3 binds to RXR. Supplementary Figure 5. IGF2 is upregulated in MAML3 overexpressing cells and results in hyperproliferation, but not RA resistance.

S1: Image of full size blot for figure 1A; S2: Image of full size blot for figure 1B; S3: SRC3-pS543 and SRC3 chromatin binding patterns in MCF7 cells; S4: Venn diagram of SRC1, SRC2 and SRC3-pS543 enriched binding sites; S5: A. ChIP-QPCR validation of differential enriched chromatin binding sites for total SRC3 and SRC3-pS543; S6: Hormone dependence and phospho-specificity of SRC3-pS543 at sites either or not shared with total SRC3; S7: Overlap of ERïƒ¡ï€ and SRC3 with other publically available datasets; S8: Fulvestrant decreases SRC3-pS543/chromatin interactions at sites shared with ER; S9: Venn diagram, showing the overlap of GO pathways enriched for genes proximal to chromatin binding sites for SRC3-pS543 unique, SRC3 unique or shared regions; S10: 2D graph of normalized ChIP-seq data, illustrating signal for ER, total SRC3, H3K4me1, H3K4me2, H3K4me3 and H3K27Ac binding events at genomic regions occupied by SRC3-pS543; S11: Histological analysis of SRC3 and SRC3-pS543 expression in representative tumour sections; S12: Scatterplot analysis for total SRC3 versus SRC3-pS543 signal in the Nottingham Tenovus Primary Breast Carcinoma Series; S13: Disease Free Survival and breast cancer specific survival in breast cancer patients based on SRC3 protein expression; S14: Disease Free Survival and breast cancer specific survival in patients with HER2 positive and SRC3 high breast cancers compared with all other patients.

QPCR primer sequences for ChIP validations.

Figure and table legends for supplementary data

Purpose: The steroid receptor coactivator SRC3 is essential for the transcriptional activity of estrogen receptor α (ERα). SRC3 is sufficient to cause mammary tumorigenesis, and has also been implicated in endocrine resistance. SRC3 is posttranslationally modified by phosphorylation, but these events have not been investigated with regard to functionality or disease association. Here, we investigate the spatial selectivity of SRC3-pS543/DNA binding over the human genome and its expression in primary human breast cancer in relation with outcome.Experimental Design: Chromatin immunoprecipitation, coupled with sequencing, was used to determine the chromatin binding patterns of SRC3-pS543 in the breast cancer cell line MCF7 and two untreated primary breast cancers. IHC was used to assess the expression of SRC3 and SRC3-pS543 in 1,650 primary breast cancers. The relationship between the expression of SRC3 and SRC3-pS543, disease-free survival (DFS), and breast cancer specific survival (BCSS) was assessed.Results: Although total SRC3 is selectively found at enhancer regions, SRC3-pS543 is recruited to promoters of ERα responsive genes, both in the MCF7 cell line and primary breast tumor specimens. SRC3-pS543 was associated with both improved DFS (P = 0.003) and BCSS (P = 0.001) in tamoxifen untreated high-risk patients, such a correlation was not seen in tamoxifen-treated cases, the interaction was statistically significant (P = 0.001). Multivariate analysis showed SRC3-pS543 to be an independent prognostic factor.Conclusions: Phosphorylation of SRC3 at S543 affects its genomic interactions on a genome-wide level, where SRC3-pS543 is selectively recruited to promoters of ERα-responsive genes. SRC3-pS543 is a prognostic marker, and a predictive marker of response to endocrine therapy. Clin Cancer Res; 22(2); 479–91. ©2015 AACR.

Supplementary Figure S2. Exogenous overexpression of RIP140 does not affect RIP140 chromatin interactions.

Primer sequences corresponding with supplemental figure S5.

Kaplan-Meier survival plot of endometrial cancer patients who were not treated with tamoxifen for their breast cancer, categorized as ERalpha+/FOXA1+, ERalpha+/FOXA1-, ERalpha-/FOXA1+ or ERalpha-/FOXA1-.

Supplementary Table S3. Motif analyses for ERα and RIP140 binding sites.

supplementary methods

Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERα) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERα transcriptional action in breast and endometrial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERα binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERα binding sites proximal to genes involved in classical ERα target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERα and FOXA1 together with the enhancer-enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERα expression was associated with a longer interval between breast cancer and the emergence of endometrial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERα are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773–84. ©2016 AACR.

Supplementary figure and Table Legends. Legends to supplementary tables and figures

legends to supplementary tables and figures

Supplementary Methods Section

Contains fulltext : 244306.pdf (Publisher’s version ) (Open Access) Patients are the stewards of their own care and hence their voice is important when designing and implementing research. Patients should be involved not only as participants in research that impacts their care, as the recipients of that care and any associated harms, but also as research collaborators in prioritising important questions from the patient perspective and designing the research and the ways in which is it most appropriate to involve patients. The PIONEER Consortium, an international multistakeholder collaboration lead by the European Association of Urology, has developed a core outcome set (COS) for localised and metastatic prostate cancer relevant to all stakeholders in particular patients. Throughout the work of PIONEER, patient representatives were involved as collaborators in setting the research agenda, and a wider group of patients was involved as participants in developing COSs, for instance in consensus meetings on choosing important outcomes and appropriate definitions. This publication showcases the process for COS development and highlights the most important recommendations to ultimately inform future research projects co-created between patients and other stakeholders. PATIENT SUMMARY: An important step in involving patients in the selection of outcomes for clinical trials, clinical audits, and real-world evidence is the development of a core outcome set (COS) that is relevant to all stakeholders. This report highlights the patient participation throughout our PIONEER COS development. TAKE HOME MESSAGE: An important step in involving patients in the selection of outcomes for clinical trials, clinical audits, and real-world evidence is to develop a core outcome set (COS) that is relevant to all stakeholders. As part of the work of the PIONEER Consortium, we aim to highlight the patient participation throughout our PIONEER COS development.

206 Background: In prostate cancer, homologous recombination deficiency (HRD) is associated with poor prognosis, and sensitivity to DNA damaging agents and DNA damage repair (DDR) inhibitors. As new classes of DDR inhibitors become available, identifying patients with HRD will be critical for treatment selection. Here, we present machine learning (ML)-based models trained to predict HRD status directly from hematoxylin and eosin (H&E) whole slide images (WSI). Methods: ML models were trained to predict and segment cells and tissue regions within the tumor microenvironment (TME) using annotated (N=91,021 annotations) WSI of H&E-stained resections from the cancer genome atlas prostate adenocarcinoma (TCGA PRAD) dataset (N=401) and needle core biopsies from a proprietary dataset (N=1,000). Quantified Human Interpretable Features (HIFs) that describe the TME composition were extracted. Three models were trained to predict HRD status using 373 WSI with known HRD score (TCGA PRAD; train N=259, validation N=76, and test N=38). Two models used input from the TME model: An HIF multivariate logistic regression model, and a graph neural network (GNN) where predictions are based on the complex spatial relationships within the TME. An end-to-end (E2E) multiple instance learning model predicted directly from the WSI. Two cutoffs for HRD were defined using Gaussian Mixture Models, resulting in 99 WSI (train N=72, validation N=18, and test N=9) positive for the Genomic Instability (>16 events) cutoff, and 58 WSI (train N=44, validation N=10, test N=4) positive for the Genomic Instability (>22 events) cutoff. An independent validation set of 45 biopsies and 16 resections from a biobank of metastatic castration resistant prostate cancer with HRD status determined by whole-exome sequencing was compared to ML model H&E-based HRD prediction. Results: In the TCGA test set of resection samples, all three models moderately or strongly predicted HRD status, with the HIF model showing the best performance (AUROC 0.87, sensitivity 0.88, specificity 0.62). The same HIF model performed equally well (AUROC 0.85, Sensitivity 0.93, specificity 0.67) in the resection samples from the independent validation set. However, the model performance went down (AUROC 0.69, sensitivity 0.91, specificity: 0.3) when both resection and needle biopsy samples were included, highlighting the importance of a representative training set to achieve robust performance in a real world setting. Further model training and validation with a more diverse dataset is required to accurately assess the performance of the model on needle biopsies. Conclusions: ML models trained on resection prostate cancer samples performed well in predicting HRD status when applied to the same sample type, demonstrating the potential of ML models to predict genomic biomarkers status in surgical specimens for treatment decision.

5′ adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor. High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation. We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux. The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.

Prostate cancer (PCa) is one of the most frequent tumor types in the male Western population. Early‐stage PCa and late‐stage PCa are dependent on androgen signaling, and inhibitors of the androgen receptor (AR) axis represent the standard therapy. Here, we studied in detail the global impact of darolutamide, a newly approved AR antagonist, on the transcriptome and AR‐bound cistrome in two PCa cell models. Darolutamide strongly depleted the AR from gene regulatory regions and abolished AR‐driven transcriptional signaling. Enhancer activation was blocked at the chromatin level as evaluated by H3K27 acetylation (H3K27ac), H3K4 monomethylation (H3K4me1), and FOXA1, MED1, and BRD4 binding. We identified genomic regions with high affinities for the AR in androgen‐stimulated, but also in androgen‐depleted conditions. A similar AR affinity pattern was observed in healthy and PCa tissue samples. High FOXA1, BRD4, H3K27ac, and H3K4me1 levels were found to mark regions showing AR binding in the hormone‐depleted setting. Conversely, low FOXA1, BRD4, and H3K27ac levels were observed at regulatory sites that responded strongly to androgen stimulation, and AR interactions at these sites were blocked by darolutamide. Beside marked loss of AR occupancy, FOXA1 recruitment to chromatin was also clearly reduced after darolutamide treatment. We furthermore identified numerous androgen‐regulated super‐enhancers (SEs) that were associated with hallmark androgen and cell proliferation‐associated gene sets. Importantly, these SEs are also active in PCa tissues and sensitive to darolutamide treatment in our models. Our findings demonstrate that darolutamide is a potent AR antagonist blocking genome‐wide AR enhancer and SE activation, and downstream transcription. We also show the existence of a dynamic AR cistrome that depends on the androgen levels and on high AR affinity regions present in PCa cell lines and also in tissue samples., Schematic model showing how darolutamide blocks androgen (R1881)‐mediated AR signaling by inhibiting the function of normal enhancers and super‐enhancers, and impairing downstream gene transcription.

Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease that often recurs despite aggressive treatment with neoadjuvant chemotherapy and (radical) cystectomy. Basal and luminal molecular subtypes have been identified that are linked to clinical characteristics and have differential sensitivities to chemotherapy. While it has been suggested that epigenetic mechanisms play a role in defining these subtypes, a thorough understanding of the biological mechanisms is lacking. This report details the first genome-wide analysis of histone methylation patterns of human primary bladder tumours by chromatin immunoprecipitations and next-generation sequencing (ChIP-seq). We profiled multiple histone marks: H3K27me3, a marker for repressed genes, and H3K4me1 and H3K4me3, which are indicators of active enhancers and active promoters. Integrated analysis of ChIP-seq data and RNA sequencing revealed that H3K4 mono-methylation demarcates MIBC subtypes, while no association was found for the other two histone modifications in relation to basal and luminal subtypes. Additionally, we identified differentially methylated H3K4me1 peaks in basal and luminal tumour samples, suggesting that active enhancers play a role in defining subtypes. Our study is the first analysis of histone modifications in primary bladder cancer tissue and provides an important resource for the bladder cancer community.

Prostate cancer development and progression is largely dependent on androgen receptor (AR) signaling. AR is a hormone-dependent transcription factor, which binds to thousands of sites throughout the human genome to regulate expression of directly responsive genes, including pro-survival genes that enable tumor cells to cope with increased cellular stress. ERN1 and XBP1 – two key players of the unfolded protein response (UPR) – are among such stress-associated genes. Here, we show that XBP1 levels in primary prostate cancer are associated with biochemical recurrence in five independent cohorts. Patients who received AR-targeted therapies had significantly lower XBP1 expression, whereas expression of the active form of XBP1 (XBP1s) was elevated. In vitro results show that AR-induced ERN1 expression led to increased XBP1s mRNA and protein levels. Furthermore, ChIP-seq analysis revealed that XBP1s binds enhancers upon stress stimuli regulating genes involved in UPR processes, eIF2 signaling and protein ubiquitination. We further demonstrate genomic overlap of AR- and XBP1s-binding sites, suggesting genomic conversion of the two signaling cascades. Transcriptomic effects of XBP1 were further studied by knockdown experiments, which lead to decreased expression of androgen-responsive genes and UPR genes. These results suggest a two-step mechanism of gene regulation, which involves androgen-induced expression of ERN1, thereby enhancing XBP1 splicing and transcriptional activity. This signaling cascade may prepare the cells for the increased protein folding, mRNA decay and translation that accompanies AR-regulated tumor cell proliferation.

Darolutamide is a novel androgen receptor (AR) antagonist with a distinct chemical structure compared to other AR antagonists and currently in clinical Phase 3 trials for prostate cancer. Using cell‐based transactivation assays, we demonstrate that darolutamide, its diastereomers and its main metabolite keto‐darolutamide are strong, competitive antagonists for AR wild type, and also for several mutants identified in prostate cancer patients for which other AR antagonists show reduced antagonism or even agonism. Darolutamide, its two diastereomers and main metabolite are also strong antagonists in assays measuring AR N/C interaction and homodimerization. Molecular modeling suggests that the flexibility of darolutamide allows accommodation in the W742C/L mutated AR ligand‐binding pocket while for enzalutamide the loss of the important hydrophobic interaction with W742 leads to reduced AR interaction. This correlates with an antagonistic pattern profile of coregulator recruitment for darolutamide. In vitro efficacy studies performed with androgen‐dependent prostate cancer cell lines show that darolutamide strongly reduces cell viability and potently inhibits spheroid formation. Also, a marked down‐regulation of androgen target genes paralleled by decreased AR binding to gene regulatory regions is seen. In vivo studies reveal that oral dosing of darolutamide markedly reduces growth of the LAPC‐4 cell line‐derived xenograft and of the KuCaP‐1 patient‐derived xenograft. Altogether, these results substantiate a unique antagonistic profile of darolutamide and support further development as a prostate cancer drug., What's new? Comparison of genomic landscapes from primary prostate cancer and metastatic tumor shows that resistance mechanisms are centered on androgen signaling and increased synthesis. Here, the novel androgen receptor (AR) antagonist darolutamide shows strong in vitro and in vivo efficacy in different prostate cancer models. Darolutamide retains its antagonistic properties at elevated androgen levels and for several AR mutants identified in therapy‐resistant patients. A unique binding profile inside the AR ligand‐binding domain linked to the flexibility of darolutamide is proposed. Altogether, these results substantiate a unique antagonistic profile of darolutamide and support further development as a prostate cancer drug.

Prostate cancer is mainly driven by androgen receptor (AR) signaling so that drugs directly inhibiting this pathway such as GnRH ligands and AR inhibitors are now established as mainstay treatments. Unfortunately, treatment resistance often develops and combination therapies may represent potential strategies to prolong disease remission. The PI3K/Akt/mTOR signaling pathway plays an essential role in prostate cancer and is upregulated in about 30-50% of patients. Loss of the PI3K/Akt/mTOR pathway inhibitor PTEN is observed in up to 40% of late-stage prostate cancer patients. Compounds inhibiting PI3K or Akt have shown promising efficacy in preclinical prostate cancer models, and advanced clinical trials with these inhibitors are currently ongoing in metastatic castration-resistant prostate cancer, often in combination with AR inhibitors. Here we examined the impact of combining the AR inhibitor darolutamide with the pan class I PI3K inhibitor copanlisib on preclinical models in vitro and in vivo. We found that treating different androgen-dependent prostate cancer cell lines with darolutamide and copanlisib had a very strong synergistic inhibitory effect. Induction of apoptosis was observed in the PARP cleavage and caspase 3/7 activation assays after treatment with copanlisib and more so after treatment with copanlisib plus darolutamide. Expression analysis showed that darolutamide strongly down-regulated AR target genes such as KLK3, FKBP5 and TMPRSS2, but copanlisib had no additional impact. However, when examining genes involved in the apoptotic pathway we found that the down-regulation of pro-apoptotic genes that follows androgen treatment was strongly reversed by the combined darolutamide and copanlisib treatment. Finally, in vivo efficacy studies showed that the LuCaP 96 prostate cancer patient-derived (PDX) xenograft, which has a non-functional PTEN gene, strongly responded to darolutamide, but additional treatment with copanlisib did not increase anti-tumor efficacy. In the LuCaP 35 PDX model, which expresses PTEN at low level, darolutamide or copanlisib treatment alone showed only limited efficacy, while the combination exhibited tumor inhibitory effects. In conclusion, we found that combining darolutamide with copanlisib strongly inhibited the proliferation of prostate cancer models in vitro. This was linked to strong induction of cell apoptosis and prevention of pro-apoptotic gene down-regulation. Superior efficacy was also observed in the LuCaP 35 PDX xenograft model in vivo. Additional data are needed to explore the underlying molecular mechanisms. Together these results further support the exploration of the impact of combining AR and PI3K signaling inhibitors in late-stage prostate cancer patients. Citation Format: Tatsuo Sugawara, Holly NGuyen, Eva Corey, Ekaterina Nevedomskaya, Oliver Politz, Dominik Mumberg, Bernard Haendler. Combination of the androgen receptor inhibitor darolutamide and the PI3K inhibitor copanlisib leads to improved anti-tumor efficacy and apoptosis in prostate cancer 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 651.

The Androgen Receptor (AR) is the key-driving transcription factor in prostate cancer, tightly controlled by epigenetic regulation. To date, most epigenetic profiling has been performed in cell lines or limited tissue samples. Here, to comprehensively study the epigenetic landscape, we perform RNA-seq with ChIP-seq for AR and histone modification marks (H3K27ac, H3K4me3, H3K27me3) in 100 primary prostate carcinomas. Integrative molecular subtyping of the five data streams revealed three major subtypes of which two were clearly TMPRSS2-ERG dictated. Importantly, we identify a third subtype with low chromatin binding and activity of AR, but with high activity of FGF and WNT signaling. While positive for neuroendocrine-hallmark genes, these tumors were copy number-neutral with low mutational burden, significantly depleted for genes characteristic of poor-outcome associated luminal B-subtype. We present a unique resource on transcriptional and epigenetic control in prostate cancer, revealing tight control of gene regulation differentially dictated by AR over three subtypes., The Androgen Receptor (AR) is the main driver of prostate cancer and functions in conjunction with chromatin modifications. Here, the authors comprehensively profile 100 primary prostate carcinomas by sequencing RNA transcripts in combination with ChIP-sequencing for AR and the active histone marks H3K27ac, H3K4me3 and repressive mark H3K27me3.

Background: 5´ adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis which has been associated with different pathologies, including cancer. Precisely defining the role of AMPK in these processes necessitates the availability of a potent and selective inhibitor.Methods: High-throughput screening and subsequent chemical optimization led to the identification of the selective inhibitor BAY-3827. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact of BAY-3827 and the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation.Results: BAY-3827 prevented phosphorylation of acetyl-CoA carboxylase 1 and showed strongest anti-proliferative activity in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive prostate cancer cell lines. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified androgen receptor (AR) binding peaks in these genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in the responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux.Conclusions: The availability of the potent and selective inhibitor BAY-3827 will contribute to a better understanding of the biological role of AMPK signaling in cancer, especially in prostate adenocarcinoma.

Fibroblasts are abundantly present in the prostate tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) which play a key role in cancer development. Androgen receptor (AR) signaling is the main driver of prostate cancer (PCa) progression, and stromal cells in the TME also express AR. High-grade tumor and poor clinical outcome are associated with low AR expression in the TME, which suggests a protective role of AR signaling in the stroma against PCa development. However, the mechanism of this relation is not clear. In this study, we isolated AR-expressing CAF-like cells. Testosterone (R1881) exposure did not affect CAF-like cell morphology, proliferation, or motility. PCa cell growth was not affected by culturing in medium from R1881-exposed CAF-like cells; however, migration of PCa cells was inhibited. AR chromatin immune precipitation sequencing (ChIP-seq) was performed and motif search suggested that AR in CAF-like cells bound the chromatin through AP-1-elements upon R1881 exposure, inducing enhancer-mediated AR chromatin interactions. The vast majority of chromatin binding sites in CAF-like cells were unique and not shared with AR sites observed in PCa cell lines or tumors. AR signaling in CAF-like cells decreased expression of multiple cytokines; most notably CCL2 and CXCL8 and both cytokines increased migration of PCa cells. These results suggest direct paracrine regulation of PCa cell migration by CAFs through AR signaling.

Androgen receptor (AR) signaling is the major driver in prostate cancer. Darolutamide is a high-affinity AR inhibitor recently approved for non-metastatic castration-resistant prostate cancer. The impact of the AR on gene transcription has been extensively examined but the downstream effects on protein levels are still poorly understood. Here we generated for the first time comprehensive quantitative proteomic and transcriptomic data from the androgen-sensitive, prostate cancer cell line VCaP treated with the androgen R1881 and darolutamide. For the proteomic analysis, a comprehensive and quantitative label-free protein expression profiling was performed using nanoscale liquid chromatography coupled to tandem mass spectrometry. For the transcriptomic analysis, sequencing was performed via single-end, 50 base-pair reads with an average depth of 21 million reads per sample. FASTQ reads were mapped with STAR aligner to the human genome GRCh38 and quantified with RSEM. Further analysis was performed in R statistical software. We investigated the expression levels of 12900 protein-coding genes and 8721 translated proteins. Androgen treatment resulted in increased expression of known androgen response genes, as well as of genes from the mTORC1 pathway, cell cycle and other pathways previously shown to be affected by androgens. This was paralleled by upregulation of the corresponding protein levels in the majority of cases. Importantly, a detailed comparative analysis of transcriptomic and proteomic data revealed a few notable differences in regulation. In androgen-treated prostate cancer cells, there was a strong increase of FOLH1, AMACR and FKBP5 transcript levels (10 to 40-fold) but only a moderate increase of the corresponding protein levels (2 to 6-fold). Additional darolutamide treatment led to strong down-regulation of androgen-induced gene expression and of the corresponding protein levels. Here also there were a few instances where protein and transcript levels were differentially regulated. The discrepancy in transcript and protein levels seen after androgen and anti-androgen treatment suggests differential post-transcriptional regulation of protein abundance to occur in some instances. In conclusion, we found that darolutamide strongly reversed androgen-induced activation of gene expression programs related to cell proliferation and survival, both at the transcript and protein levels. We furthermore identified for the first time androgen-activated genes whose function is additionally regulated at the post-transcription level. Citation Format: Ekaterina Nevedomskaya, Simon J. Baumgart, Tatsuo Sugawara, Ralf Lesche, Dominik Mumberg, Bernard Haendler. Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1008.

Recent advances in whole-genome and transcriptome sequencing of prostate cancer at different stages indicate that a large number of mutations found in tumors are present in non-protein coding regions of the genome and lead to dysregulated gene expression. Single nucleotide variations and small mutations affecting the recruitment of transcription factor complexes to DNA regulatory elements are observed in an increasing number of cases. Genomic rearrangements may position coding regions under the novel control of regulatory elements, as exemplified by the TMPRSS2-ERG fusion and the amplified enhancer identified upstream of the androgen receptor (AR) gene. Super-enhancers are increasingly found to play important roles in aberrant oncogenic transcription. Several players involved in these processes are currently being evaluated as drug targets and may represent new vulnerabilities that can be exploited for prostate cancer treatment. They include factors involved in enhancer and super-enhancer function such as bromodomain proteins and cyclin-dependent kinases. In addition, non-coding RNAs with an important gene regulatory role are being explored. The rapid progress made in understanding the influence of the non-coding part of the genome and of transcription dysregulation in prostate cancer could pave the way for the identification of novel treatment paradigms for the benefit of patients.

This study presents an optimized ChIP-seq protocol to enhance transcription factor profiling in human tumours, enabling the analysis of highly challenging samples, including core needle biopsies., Chromatin immunoprecipitation (ChIP)-seq analyses of transcription factors in clinical specimens are challenging due to the technical limitations and low quantities of starting material, often resulting in low enrichments and poor signal-to-noise ratio. Here, we present an optimized protocol for transcription factor ChIP-seq analyses in human tissue, yielding an ∼100% success rate for all transcription factors analyzed. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. To illustrate the sensitivity of the optimized protocol, we provide high-quality ChIP-seq data for three independent factors (AR, FOXA1, and H3K27ac) from a single core needle prostate cancer biopsy specimen. In summary, double-cross-linking strongly improved transcription factor ChIP-seq quality on human tumor samples, further facilitating and enhancing translational research on limited amounts of tissue.

Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERα) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERα transcriptional action in breast and endometrial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERα binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERα binding sites proximal to genes involved in classical ERα target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERα and FOXA1 together with the enhancer-enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERα expression was associated with a longer interval between breast cancer and the emergence of endometrial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERα are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773–84. ©2016 AACR.

Neuroblastoma cell lines can differentiate upon treatment with retinoic acid (RA), a finding that provided the basis for the clinical use of RA to treat neuroblastoma. However, resistance to RA is often observed, which limits its clinical utility. Using a gain-of-function genetic screen, we identified an unexpected link between RA signaling and mastermind-like 3 (MAML3), a known transcriptional coactivator for NOTCH. Our findings indicate that MAML3 expression leads to the loss of activation of a subset of RA target genes, which hampers RA-induced differentiation and promotes resistance to RA. The regulatory DNA elements of this subset of RA target genes show overlap in binding of MAML3 and the RA receptor, suggesting a direct role for MAML3 in the regulation of these genes. In addition, MAML3 has RA-independent functions, including the activation of IGF1R and downstream AKT signaling via upregulation of IGF2, resulting in increased proliferation. These results demonstrate an important mechanistic role for MAML3 in proliferation and RA-mediated differentiation. Implications: MAML3 coordinates transcription regulation with receptor tyrosine kinase pathway activation, shedding new light on why this gene is mutated in multiple cancers. Mol Cancer Res; 14(5); 411–22. ©2016 AACR.

Systematic identification of noncoding regulatory elements has, to date, mainly relied on large-scale reporter assays that do not reproduce endogenous conditions. We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERα (ESR1) gene regulation. Moreover, we show that a genomic CRISPR-Cas9 tiling screen can precisely map functional domains within enhancer elements. Our approach expands the utility of CRISPR-Cas9 to elucidate the functions of the noncoding genome.

Aberrant activation of the Hippo pathway effectors YAP1/TAZ promotes cell proliferation and tumorigenesis. To identify novel regulators of YAP1/TAZ in cancer, we established a FACS-based screening system monitoring YAP1/TAZ activity in MDA-MB-231 breast cancer cells. Using these cells, we performed pooled genome-wide CRISPR/Cas9 knockout and CRISPR activation/interference (a/i) screens. The list of hits included previously known YAP1/TAZ modulators such as LATS2, AJUBA, and TAZ itself, demonstrating the robustness of the screen. Moreover, we identified about 30 novel candidate genes with potential inhibitory activity on YAP1/TAZ and about 50 candidate genes that may play a role in YAP1/TAZ activation. These genes represent diverse cellular functions such as regulation of actin cytoskeleton, integrin signaling, and ER protein processing, among others. Modulation of endogenous YAP1/TAZ target genes was assessed by individual gene knockout using crRNAs. Functional characterization of the novel potential YAP1/TAZ modulators will aid to the further understanding of YAP1/TAZ biology in health and disease. Citation Format: Jan Naujoks, Lisette Potze, Julia Kuehnlenz, Atanas Kamburov, Ekaterina Nevedomskaya, Andreas Steffen, Claudia Luther, Anna Anurin, Anne Buttgereit, Stefan Prechtl, Benjamin Bader, Ralf Lesche, Peter Staller, Martin Lange, Barbara Nicke. Genome-wide CRISPR/Cas9 screens for the identification of novel YAP1/TAZ modulators [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A29.

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed.