Androgen Receptor Coactivator Research Articles

Abstract B009: Targeting the AR co-activator CBP/p300 axis in castration resistant prostate cancer Impacts DNA damage repair function

Abstract Prostate cancer (PCa) is the second leading cause of cancer related deaths in US men. PCa is an androgen dependent disease and the transcription factor, androgen receptor (AR), is crucial for the overall outcome. Currently, androgen-deprivation therapy (ADT) is the standard of care first-line therapy for metastatic PCa. Resistance to ADT almost uniformly leads to lethal disease, termed castration-resistant prostate cancer (CRPC). Thus, there is a significant unmet clinical need to identify and develop novel strategies to treat CRPC. CBP/p300 are potent co-activators for AR. High expression of CBP/p300 are associated with locally advanced disease and castration resistant function of AR; thus, resulting in poor patient outcome and further highlighting the need to discern the role of CBP/p300 to potentially develop novel therapeutic targets for precision medicine. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300 and its associated transcriptional machinery. In this study, CBP/p300 mediated bromodomain activity is targeted by CCS1477 (Inobrodib), a first-in-class bromodomain inhibitor developed by Cell Centric. CCS1477 was shown to demonstrate effective inhibition in growth and clonogenicity assays. Inhibition of the CBP/p300 bromodomain with CCS1477 resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression in addition to inhibition of associated factors such as c-MYC and its downstream targets in PCa cell lines as well as patient derived xenograft (PDX) models. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC patient samples. The clinical significance of CBP/p300 expression in PCa has been investigated via elucidation of CBP/p300 transcriptional reprogramming and role in DNA damage response (DDR) pathways. Specifically, findings revealed that CBP/p300 bromodomain suppression sensitizes to AR-dependent DNA-repair. Transcriptional mapping identified CBP/p300 as regulators of cell proliferation and DNA repair processes, which were functionally confirmed across several PCa model systems. To assess relevance, exogenous challenge with radiation revealed that CBP/p300 bromodomain is required for AR-mediated DNA repair, and CBP/p300 expression is linked to DNA repair capacity in the clinical setting. Molecular analyses revealed that CBP/p300 facilitate double-strand break (DSB) repair efficiency via homologous recombination (HR) mediated DDR. Congruently, CBP/p300 strongly correlated with HR gene expression in PCa patient tissue. These collective findings reveal that CBP/p300 govern repair of DNA DSBs by regulating HR, thus modulating genome integrity, and promoting CRPC growth. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Citation Format: Sumaira Sardar. Targeting the AR co-activator CBP/p300 axis in castration resistant prostate cancer Impacts DNA damage repair function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B009.

Abstract A052: Discovery and characterization of a p300-selective degrader demonstrates potent anti-tumor activity in preclinical models of prostate cancer

Abstract Strategies targeting androgen receptor (AR) in the treatment of prostate cancer (PrCa) have shown clear clinical benefit. However, a variety of AR-signaling bypass mechanisms ultimately result in progression to metastatic castrate resistant prostate cancer (mCRPC), and therapies for these patients remain an urgent unmet need. The histone acetyltransferase paralogs p300 and CBP, key coactivators of AR, are currently under investigation as attractive therapeutic targets to abrogate aberrant AR signaling but face a challenging clinical toxicity profile in hematopoietic cells. We postulate that p300 serves as a key regulator of AR signaling, and by sparing CBP we should retain potency against AR-driven PrCa while providing a larger safety margin. We discovered potent heterobifunctional degraders with demonstrated selectivity for p300 over CBP. Our tool compound exhibits degradation of p300 within 2 hours (DC50 = 7.5nM, Dmax = 79%) with minimal impact on CBP at 48 h (DC50 >1uM, Dmax = 5%). The AR/ARv7 driven VCaP PrCa cell line demonstrated exceptional sensitivity to this p300 degrader in proliferation assays (gIC50 = 0.8 nM) comparable to the dual p300/CBP inhibitor, CCS-1477, or the AR degrader, ARV-110 and undergoes a cytotoxic response. Profiled against known AR target genes, our degrader was able to suppress KLK2, KLK3 and FKBP5 transcript levels akin to CCS-1477 in both LNCaP and VCaP PrCa cell lines, supporting the hypothesis that p300, not CBP, acts as the primary coactivator of AR signaling. In addition, our compound led to greater downregulation of full-length AR, ARv7 and cMyc compared to CCS-1477. Subsequent assessment in an in vivo model (VCaP CDX) demonstrated tumor growth inhibition at the highest dose tested (88%; p=2.6e-4). Lastly, we profiled our p300 degrader in human bone marrow derived hematopoietic progenitor colony-forming assays where we observed a significant reduction in toxicity (IC50 = 3.9uM) compared to either CCS-1477 (IC50 = 121nM) or a dual degrader tool compound (IC50 = 16nM), supporting the hypothesis that a p300-specific mechanism will offer an improved therapeutic index. Treatment with our p300-specific degrader resulted in a potent anti-tumor effect on PrCa cells both in vitro and in vivo, comparing favorably to both dual p300/CBP inhibitor or AR degrader strategies. Furthermore, selective degradation of p300 has the potential to minimize toxicity to hematopoietic cells commonly observed with dual targeting of CBP and p300. Taken together, these results support the role p300 may play in driving AR-mediated signaling in PrCa and demonstrate that a p300-specific degrader may serve as an effective therapeutic modality in this disease setting. Citation Format: Mike Russell, Cassandra L Lowenstein, Xuqing Zhang, Jeremy Roach, Jianing Song, Rakesh Nagilla, Peter Orth, Matt Tudor, Qiaolin Deng, Zhihua Sui, Corey Strickland, Larry J Jolivette, E Scott Priestley, Helai P Mohammad. Discovery and characterization of a p300-selective degrader demonstrates potent anti-tumor activity in preclinical models of prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A052.

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

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

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

Abstract 4729: LSD1 activates oncogenic super-enhancers in castration-resistant prostate cancer by forming nuclear condensates with BRD4

Abstract Lysine-specific demethylase (LSD1) was initially identified as a transcriptional repressor through demethylation of mono/di-methylated histone 3 lysine 4 (H3K4me1/2). Subsequent work suggests that LSD1 may trigger gene activation via demethylating H3K9me1/2 in the context of steroid receptors. Our previous studies have confirmed that LSD1 functions broadly as a coactivator of androgen receptor (AR)-regulated enhancers but retains its H3K4 demethylation function at these sites. Importantly, our recent work revealed that LSD1 regulates the accessibility of active enhancers through the interaction with FOXA1, which functions as a pioneer transcription factor by facilitating AR access to chromatin LSD1 inhibition disrupts the global binding of FOXA1 by blocking the demethylation of K270 of FOXA1, leading to inhibition of AR binding and transcriptional activity. However, in addition to AR signaling, it remains largely unknown whether LSD1 regulates other critical oncogenic programs during prostate cancer (PCa) progression. In this study, we aimed to fully understand the functional targets of LSD1 in castration-resistant PCa (CRPC) models. We performed a large transcriptomic profiling of CRPC patient-derived xenograft (PDX) models that are sensitive to LSD1 inhibitors. Our analyses indicated that LSD1 inhibition targeted multiples oncogenic programs, including previously known pathways such as AR, FOXA1, and E2F, and a previously undefined pathway, MYC signaling. Through single-cell RNA-seq analysis in a CRPC PDX model, we confirmed that MYC signaling was decreased in the responsive tumor cells and identified a previously unknown molecular subtype, AR+/FOXA1+/LSD1low/RB1−, which was resistant to LSD1 inhibition. It is well known that MYC is driven by super-enhancers (SEs), which are regulated by BRD4 (bromodomain and extraterminal (BET) protein). We further evaluated LSD1, FOXA1 and BRD4 ChIP-seq and defined SEs by using H3K27ac ChIP-seq followed by ROSE analysis. We found that LSD1, FOXA1, and BRD4 co-occupied at MYC SEs and BRD4 was recruited by unmethylated FOXA1 but not K270-methylated FOXA1. In addition, we observed the puncta-like formation of LSD1/BRD4 marked nuclear condensates under confocal immunofluorescence microscopy. By examining the public datasets of H3K27ac ChIP-seq of samples with different stages of PCa development, we identified a subset of SEs which were distinctly activated in CRPC. Using multiple CRPC PDXs, we revealed that combining LSD1 inhibitors with BET inhibitors exhibited strong synergy possibly via disrupting the CRPC-specific SEs. In summary, this study reveals a novel oncogenic function of LSD1 in driving PCa progression by activating SE-driven oncogenic programs, such as MYC signaling. These results provide a strong therapeutic potential of combining inhibitors of LSD1 and BRD4 in treating CRPC. Citation Format: Mingyu Liu, Muqing Li, Dong Han, Zifeng Wang, Wanting Han, Housheng Hansen He, Shuai Gao, Changmeng Cai. LSD1 activates oncogenic super-enhancers in castration-resistant prostate cancer by forming nuclear condensates with BRD4. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4729.

LSD1 inhibition disrupts super-enhancer driven oncogenic transcriptional programs in castration-resistant prostate cancer.

The lysine demethylase LSD1 (also called KDM1A) plays important roles in promoting multiple malignancies including both hematologic cancers and solid tumors. LSD1 targets histone and non-histone proteins and can function as a transcriptional corepressor and coactivator. LSD1 has been reported to act as a coactivator of androgen receptor (AR) in prostate cancer (PCa) and to regulate the AR cistrome via demethylation of its pioneer factor FOXA1. A deeper understanding of the key oncogenic programs targeted by LSD1 could help stratify PCa patients for treatment with LSD1 inhibitors, which are currently under clinical investigation. In this study, we performed transcriptomic profiling in an array of castration-resistant PCa (CRPC) xenograft models that are sensitive to LSD1 inhibitor treatment. Impaired tumor growth by LSD1 inhibition was attributed to significantly decreased MYC signaling, and MYC was found to be a consistent target of LSD1. Moreover, LSD1 formed a network with BRD4 and FOXA1 and was enriched at super-enhancer (SE) regions exhibiting liquid-liquid phase separation. Combining LSD1 inhibitors with BET inhibitors exhibited strong synergy in disrupting the activities of multiple drivers in CRPC, thereby inducing significant growth repression of tumors. Importantly, the combination treatment showed superior effects than either inhibitor alone in disrupting a subset of newly identified CRPC-specific SEs. These results provide mechanistic and therapeutic insights for co-targeting two key epigenetic factors and could be rapidly translated in the clinic for CRPC patients.

Cholesterol Triggers Nuclear Co-Association of Androgen Receptor, p160 Steroid Coactivators, and p300/CBP-Associated Factor Leading to Androgenic Axis Transactivation in Castration-Resistant Prostate Cancer.

Cholesterol modulates intratumoral androgenic signaling in prostate cancer; however, the molecular mechanisms underlying these changes in castration-resistant prostate cancer (CRPC) are not fully elucidated. Herein, we investigated the effect of cholesterol on androgen receptor (AR) coactivators expression and tumorigenesis in vitro and in vivo. Herein, we monitored the expression of AR coactivators (SRC-1, 2, 3 and PCAF) genes in PC-3 cells exposed to 2µg/mL of cholesterol for 8 hours by qPCR. We also performed cell migration at 0, 8, 24, 48 and 72h and flow cytometry assays (viability, apoptosis, and cell cycle) after a 24h exposure. Immunofluorescence assay was performed to evaluate the protein expression of the AR coactivators. Additionally, in vivo experiments were conducted using 22 male NOD/SCID mice. Mice were fed a standard (Control) or hypercholesterolemic (HCOL) diet for 21 days and then subcutaneously implanted with PC-3 cells. The tumor volume was calculated every two days, and after four weeks, the tumors were resected, weighed, and the serum lipid profile was measured. We also measured the intratumoral lipid profile and AR coactivators gene and protein expression by qPCR and Western Blot, respectively. Intratumor testosterone and dihydrotestosterone (DHT) concentrations were determined using ELISA. Cholesterol up-regulated the gene expression of coactivators SRC-1, SRC-2, SRC-3 and PCAF, increasing AR expression in PC-3 cells. Next, cholesterol-supplemented PC-3 cells exhibited increased cell migration and altered cell cycle phases, leading to changes in proliferation and reduced apoptosis. We found that SRC-1, SRC-2, SRC-3 and PCAF proteins co-localized in the nucleus of cholesterol-supplemented cells and co-associate with AR. In the in vivo model, the hypercholesterolemic (HCOL) group displayed higher serum total and intratumoral cholesterol levels, increased testosterone and dihydrotestosterone concentrations, and up-regulated AR coactivator expression. The tumor volume of the HCOL group was significantly higher than the control group. Our findings revealed that increased nuclear translocation of the coactivators leads to up-regulated AR gene and protein expression, potentially influencing tumor progression. Studies targeting cholesterol-modulated changes in AR coactivator expression may provide insights into the molecular mechanisms associated with the CRPC phenotype.

TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation.

Androgen receptor (AR) is a master transcription factor that drives prostate cancer (PCa) development and progression. Alterations in the expression or activity of AR coregulators significantly impact the outcome of the disease. Using a proteomics approach, we identified the tripartite motif-containing 33 (TRIM33) as a novel transcriptional coactivator of AR. We demonstrate that TRIM33 facilitates AR chromatin binding to directly regulate a transcription program that promotes PCa progression. TRIM33 further stabilizes AR by protecting it from Skp2-mediated ubiquitination and proteasomal degradation. We also show that TRIM33 is essential for PCa tumor growth by avoiding cell-cycle arrest and apoptosis, and TRIM33 knockdown sensitizes PCa cells to AR antagonists. In clinical analyses, we find TRIM33 upregulated in multiple PCa patient cohorts. Finally, we uncover an AR-TRIM33-coactivated gene signature highly expressed in PCa tumors and predict disease recurrence. Overall, our results reveal that TRIM33 is an oncogenic AR coactivator in PCa and a potential therapeutic target for PCa treatment.

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.

Natural product myricetin is a pan-KDM4 inhibitor which with poly lactic-co-glycolic acid formulation effectively targets castration-resistant prostate cancer

BackgroundCastration-resistant prostate cancer (CRPC) with sustained androgen receptor (AR) signaling remains a critical clinical challenge, despite androgen depletion therapy. The Jumonji C-containing histone lysine demethylase family 4 (KDM4) members, KDM4A‒KDM4C, serve as critical coactivators of AR to promote tumor growth in prostate cancer and are candidate therapeutic targets to overcome AR mutations/alterations-mediated resistance in CRPC.MethodsIn this study, using a structure-based approach, we identified a natural product, myricetin, able to block the demethylation of histone 3 lysine 9 trimethylation by KDM4 members and evaluated its effects on CRPC. A structure-based screening was employed to search for a natural product that inhibited KDM4B. Inhibition kinetics of myricetin was determined. The cytotoxic effect of myricetin on various prostate cancer cells was evaluated. The combined effect of myricetin with enzalutamide, a second-generation AR inhibitor toward C4-2B, a CRPC cell line, was assessed. To improve bioavailability, myricetin encapsulated by poly lactic-co-glycolic acid (PLGA), the US food and drug administration (FDA)-approved material as drug carriers, was synthesized and its antitumor activity alone or with enzalutamide was evaluated using in vivo C4-2B xenografts.ResultsMyricetin was identified as a potent α-ketoglutarate-type inhibitor that blocks the demethylation activity by KDM4s and significantly reduced the proliferation of both androgen-dependent (LNCaP) and androgen-independent CRPC (CWR22Rv1 and C4-2B). A synergistic cytotoxic effect toward C4-2B was detected for the combination of myricetin and enzalutamide. PLGA-myricetin, enzalutamide, and the combined treatment showed significantly greater antitumor activity than that of the control group in the C4-2B xenograft model. Tumor growth was significantly lower for the combination treatment than for enzalutamide or myricetin treatment alone.ConclusionsThese results suggest that myricetin is a pan-KDM4 inhibitor and exhibited potent cell cytotoxicity toward CRPC cells. Importantly, the combination of PLGA-encapsulated myricetin with enzalutamide is potentially effective for CRPC.

Target Protein for Xklp2 Functions as Coactivator of Androgen Receptor and Promotes the Proliferation of Prostate Carcinoma Cells.

The activation of the androgen receptor (AR) pathway is crucial in the progression of human prostate cancer. Results of the present study indicated that the target protein xenopus kinesin-like protein (TPX2) enhanced the transcription activation of AR and promoted the proliferation of LNCaP (ligand-dependent prostate carcinoma) cells. The protein-protein interaction between AR and TPX2 was investigated using coimmunoprecipitation assays. Results of the present study further demonstrated that TPX2 enhanced the transcription factor activation of AR and enhanced the expression levels of the downstream gene prostate-specific antigen (PSA). TPX2 did this by promoting the accumulation of AR in the nucleus and also promoting the recruitment of AR to the androgen response element, located in the promoter region of the PSA gene. Overexpression of TPX2 enhanced both the in vitro and in vivo proliferation of LNCaP cells. By revealing a novel role of TPX2 in the AR signaling pathway, the present study indicated that TPX2 may be an activator of AR and thus exhibits potential as a novel target for prostate carcinoma treatment.

Abstract PD1-07: Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors

Abstract Background: Acquired ESR1 mutations are a dominant driver of distant metastasis in metastatic breast cancer, inducing a basal-like phenotype that is relatively resistant to ER antagonists with decreased progression free survival. The majority of ER+ patients also express the androgen receptor (AR), and although AR expression is associated with better outcomes, high AR expression has also been associated with resistance to endocrine therapy (ET). Thus, there is currently a conundrum on how best to target AR, and to define when it is participating as a proliferative/metastatic driver, or when it may be a potential tumor suppressor associated with good prognosis. Methods: We generated ESR1 Y537S or D538G homozygous mutations in MCF-7 cells using CRISPR Cas-9 technology. ChIP-Seq, transcriptome analyses, and quantitative analysis of ER ChIP-Seq profiles from patient biopsies were integrated. Nuclear fractionation and immunoblot analyses were performed. Results: Correlation and gene set enrichment analyses demonstrated that the androgen response pathway was significantly reduced in ESR1 mutant, compared to wild-type (WT) ER cells. A dramatic redistribution in AR binding sites to heterochromatin was observed in ESR1 mutant cells. AR and ER co-occupied DNA binding sites were redistributed only to ERE motifs present in a restricted set of gene enhancer regions. Quantitative analysis of ER DNA binding profiles from ER+ patients showed that the highest recruitment of ER to AR/ER binding sites were in patients with metastatic breast cancer. Integration of AR/ER co-bound sites with the differentially-expressed mutant transcriptome identified a gene signature that predicted poor disease-free and overall survival in ER+ primary breast cancer patients from the METABRIC database. Elevated gene expression of these AR/ER co-regulated genes, including NCOA3, BMP7, N4BP3, and FOXA1, were validated in a cohort of metastatic tumors (N=97), compared to primary tumors (N=276). AR protein levels were elevated in ESR1 mutant tumors. Mechanistic studies demonstrated that elevated AR phosphorylation at specific sites (pS515, pS650) and decreased phosphorylation (pS308) occurs in mutant cells. These sites are known to affect AR transcriptional activity, protein stability, and nuclear export. Decreased K48-AR ubiquitination was observed, suggesting that elevated AR protein levels result from altered post-transcriptional modification of AR in mutant cells. Conclusions: We propose a two-prong genomic activation mechanism in ESR1 mutant tumors with AR redistribution to heterochromatin, and genomic co-activation of AR and ER. We hypothesize that the loss of AR tumor suppressor function may help drive metastasis in ESR1 mutant tumors via AR’s collaboration with ESR1 mutant oncogenic activity. Efficacious targeting of AR may require disruption of this driver AR/ER-regulated transcriptional program. Citation Format: Sandra L. Grimm, Guowei Gu, Sarah K. Herzog, Thomas L. Gonzalez, Hangqing Lin, Amanda R. Beyer, Yassine Rechoum, Tasneem Bawa-Khalfe, Ashfia F. Khan, Lili Du, W. Fraser Symmans, Ralf Kittler, Cristian Coarfa, Suzanne A.W. Fuqua. Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-07.

Phytosterols in hull-less pumpkin seed oil, rich in ∆7-phytosterols, ameliorate benign prostatic hyperplasia by lowing 5α-reductase and regulating balance between cell proliferation and apoptosis in rats.

BackgroundPumpkin seed oil is widely used to treat benign prostatic hyperplasia (BPH), a common disease in elder men. However, its active components and mechanism have remained to be elucidated.ObjectiveThe objective of the present study was to investigate the active components of pumpkin seed oil and its mechanism against BPH.DesignTotal phytosterol (TPS) was isolated from hull-less pumpkin (Cucurbita pepo L. var. Styriaca) seed oil and analyzed by gas chromatography/mass spectrometry (GC/MS). Three phytosterols were purified by preparative HPLC (high performance liquid chromatography) and confirmed by NMR (nuclear magnetic resonance). TPS (3.3 mg/kg body weight, 1 mL/day/rat) was administered intragastrically to the testosterone propionate-induced BPH rats for 4 weeks. The structure changes of prostate tissues were assessed by hematoxylin & eosin (H&E) staining. The expression of androgen receptor (AR) and steroid receptor coactivator 1 (SRC-1) was analyzed by immunohistochemistry, while that of 5α-reductase (5AR), apoptosis, or proliferation-related growth factors/proteins was detected by real-time quantitative polymerase chain reaction or western blotting.ResultsThe ∆7-phytosterols in TPS reached up to 87.64%. Among them, 24β-ethylcholesta-7,22,25-trienol, 24β-ethylcholesta-7,25(27)-dien-3-ol, and ∆7-avenasterol were confirmed by NMR. TPS treatment significantly ameliorated the pathological prostate enlargement and restored histopathological alterations of prostate in BPH rats. It effectively suppressed the expressions of 5AR, AR, and coactivator SRC-1. TPS inhibited the expression of proliferation-related growth factor epidermal growth factor, whereas it increased the expressions of apoptosis-related growth factor/gene transforming growth factor-β1. The proliferation-inhibiting effect was achieved by decreasing the ERK (extracellular signal-regulated kinase) phosphorylation, while apoptosis was induced by Caspase 3 activation through JNK (c-Jun N-terminal kinase) and p38 phosphorylation.ConclusionTPS from hull-less pumpkin seed oil, with ∆7-phytosterols as its main ingredients, is a potential nutraceutical for BPH prevention.

Abstract P202: Initial findings from an ongoing first-in-human phase 1 study of the CBP/p300 inhibitor FT-7051 in men with metastatic castration-resistant prostate cancer

Abstract Background: Prostate cancer is the 2nd leading cause of cancer-related deaths among men in the US. CREB binding protein (CBP) and paralog p300 are co-activators of androgen receptor (AR) relevant to metastatic castration-resistant prostate cancer (mCRPC) progression and AR therapy resistance. FT-7051 is an oral, potent and selective inhibitor of CBP/p300 bromodomain with activity in preclinical prostate cancer models, including those resistant to enzalutamide. Methods: The Courage Study (NCT04575766) is a first-in-human, multicenter, phase 1, open-label study to examine the safety, PK/PD, and preliminary anti-tumor activity of FT-7051 in mCRPC patients (pts) who have progressed despite prior therapy, including at least one AR pathway inhibitor. The study uses a Bayesian optimal interval (BOIN) design with an accelerated titration phase. FT-7051 is dosed on a 28-d cycle (21-d on/7-d off). Following accelerated titration, dose escalation/de-escalation decisions are made by comparing the observed dose-limiting toxicity (DLT) rate at the current dose with pre-specified dose escalation/de-escalation boundaries. The primary objectives are to evaluate safety and tolerability of FT-7051 and determine the recommended phase 2 dose. Key secondary endpoints include PSA at 12 wks, time to PSA progression, time to radiographic progression, overall response rate, and PK parameters. Biomarker analyses include PD assessments of CBP/p300 inhibition in surrogate tissues and genetic analyses in circulating tumor cells (AR, AR-v7) and peripheral blood. Results: As of 18-June-2021, a total of 5 pts were enrolled with exposure data entered into the database, with 2 pts ongoing and 3 pts discontinued (disease progression, n=1; no longer clinically benefitting, n=1; subject withdrawal, n=1). Pts had a median age of 71 yrs (range: 66-82) with a median time since first mCRPC diagnosis of 2.3 yrs (0.4-4.7) and a median of 3 (1-5) prior lines of mCRPC therapy. Mutations reported by the investigator for 3 pts included MYC, p53, RB1, AR, and PTEN loss. Preliminary PK analyses indicated that FT-7051 exposure increased with dose in a greater than dose-proportional manner. Importantly, observed FT-7051 exposures were consistent with the predicted efficacious exposure threshold derived from PK/efficacy modeling in preclinical studies. TEAEs were reported in 4 (80%) pts. Most TEAEs were mild (Gr1) or moderate (Gr2), ≥Gr3 TEAEs included one event of possibly related Gr3 hyperglycemia and one event of unrelated Gr5 disease progression. Conclusions: Preliminary safety and PK data from the accelerated titration phase of this BOIN study support the continued investigation of FT-7051 in men with mCRPC. Initial PK data confirm that FT-7051 exposure is consistent with the predicted efficacious exposure threshold determined by PK/efficacy modeling. Additional analyses of PSA, PD assessments, and genetic analyses, including AR-v7 status, will be reported. Citation Format: Andrew J. Armstrong, Michael S. Gordon, Melissa A. Reimers, Arif Hussain, Vaibhav G. Patel, Elaine T. Lam, Alex Sedkov, Von Potter, Neal Shore. Initial findings from an ongoing first-in-human phase 1 study of the CBP/p300 inhibitor FT-7051 in men with metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P202.

Abstract P204: Targeting the p300/CBP epigenetic pathway to overcome hormone therapy resistance in advanced prostate cancer

Abstract Purpose/Objective(s): Despite improvements in the treatment of advanced prostate cancer, there remains a major unmet need for further treatment options in the setting of metastatic castration-resistant prostate cancer (mCRPC). Recent evidence has pinpointed androgen receptor (AR) co-activators, such as p300/CREB binding protein (CBP), as potential therapeutic targets. Here, we describe the impact of a novel small molecule inhibitor of the p300/CBP bromodomain, FT-6876, in preclinical models of mCRPC. Materials/Methods: FT-6876 is a potent inhibitor of the bromodomains of CBP and p300 with high selectivity against other bromodomain-containing proteins (Caligiuri et al. Proceedings: AACR Annual Meeting 2020; Cancer Research. DOI: 10.1158/1538-7445.AM2020-3079). FT-6876 was tested, both alone and in combination with enzalutamide, in a panel of enzalutamide-sensitive and enzalutamide-resistant human prostate cancer cell lines, including LNCaP, LN95, and CS2. A Snail-inducible system, which confers enzalutamide resistance, in both LNCaP and LN95 lines, was also used. To test the effect of FT-6876 in inhibiting prostate cancer growth in vitro, 2D colony forming assays and 3D spheroid assays were performed. RT-qPCR and multi-omics analysis were performed to assess changes in gene and protein expression. To test the effects of FT-6876 in physiologic settings, an ex vivo murine pulmonary metastasis assay (PuMA) as well as a patient-derived xenograft (PDX) model were used. Results: FT-6876 treatment resulted in concentration-dependent growth inhibition of all tested enzalutamide-sensitive and -resistant cell lines. Treated cells showed decreased expression of several key prostate cancer genes, including KLK3 (PSA), TMPRSS2, and MYC. A multi-omics approach using proteomics, acetylomics, and RNA-Seq illuminated the global landscape of alterations in epigenetic and downstream signaling pathways induced by FT-6876. Changes were observed in both enzalutamide-sensitive and -resistant models. Finally, FT-6876 reduced metastatic growth in the PuMA model and induced tumor stasis in a patient-derived xenograft model of prostate cancer resistant to enzalutamide. Conclusion: Overall, we demonstrate that inhibiting the p300/CBP axis with the novel bromodomain inhibitor FT-6876 could be an effective therapeutic approach for both enzalutamide-sensitive and enzalutamide-resistant prostate cancer models. The safety and tolerability of FT-7051, an oral p300/CBP inhibitor related to FT-6876, is currently being evaluated in the Courage Study (NCT04575766), a first-in-human phase 1 study in men with mCRPC. Citation Format: Emily L. Chen, Nathan Hawkey, Beatrice C. Thomas, Kathryn E. Ware, Daniella Runyambo, Maureen Caligiuri, Erik Wilker, Erik J. Soderblom, M. Arthur Moseley III, Sylvie M. Guichard, Andrew J. Armstrong, Jason A. Somarelli. Targeting the p300/CBP epigenetic pathway to overcome hormone therapy resistance in advanced prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P204.

AC016745.3 Regulates the Transcription of AR Target Genes by Antagonizing NONO.

The androgen receptor (AR) and its related signaling pathways play an important role in the development of prostate cancer (PCa). Long non-coding RNAs (lncRNAs) are involved in the regulation of tumorigenesis and development, but their specific mechanism of action remains unclear. This study examines the function and mechanisms of action of lncRNA AC016745.3 in the development of PCa. It shows that dihydrotestosterone (DHT) results in the AR-dependent suppression of AC016745.3 expression in the LNCaP androgen-sensitive human prostate adenocarcinoma cell line. In addition, overexpression of AC016745.3 inhibits the proliferation and migration of PCa cells, and suppresses the expression of AR target genes. This research also demonstrates that the protein NONO interacts with AR and functions as an AR co-activator, promoting AR transcriptional activity. Furthermore, using RNA immunoprecipitation (RIP)-PCR experiments, the study demonstrates that both NONO and AR can bind AC016745.3. Moreover, cell phenotypic experiments reveal that NONO can promote cellular proliferation and migration, and that AC016745.3 can partially antagonize the pro-oncogenic functions of NONO in PCa cells. In summary, the results indicate that AC016745.3 can bind NONO, suppressing its ability to promote AR-dependent transcriptional activity. Furthermore, DHT-dependent suppression of AC016745.3 expression can enhance NONO’s promotion effect on AR.

PIM1 phosphorylation of the androgen receptor and 14-3-3 \u03b6 regulates gene transcription in prostate cancer

PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.

A Novel Splice Variant of the Inhibitor of Growth 3 Lacks the Plant Homeodomain and Regulates Epithelial-Mesenchymal Transition in Prostate Cancer Cells.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial–mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

Abstract 2317: Targeting prostate cancer super enhancers by co-inhibiting LSD1 and BRD4

Abstract In prostate cancer (PCa) cells, “pioneer” factor FOXA1 resides at distal enhancers enriched with histone 3 lysine 4 methylation (H3K4me) and lysine 27 acetylation (H3K27ac). FOXA1 is capable of remodeling the compact chromatin to facilitate androgen receptor (AR) recruitment. Recent studies revealed overexpression of FOXA1 caused by frequent structural alterations in the lethal stage of PCa, namely castration-resistant PCa(CRPC), suggesting FOXA1 as a critical target in CRPC. However, a lack of targetable domain hinders the effective targeting of FOXA1. Our recent (epi)genomic profiling study revealed that lysine-specific demethylase (LSD1) and FOXA1 co-occupy at active enhancers in PCa cells and LSD1 demethylase activity is required for FOXA1 chromatin binding. LSD1 inhibition can quickly and drastically reduce FOXA1 global binding and concurrently impair the “openness” of enhancers, leading to disrupted AR cistrome. Mechanistically, we demonstrated that this activator function of LSD1 is mediated through directly demethylating K270 of FOXA1. By testing an array of adenocarcinoma CRPC in vivo models, we further showed that the efficacy of LSD1 inhibitors may be correlated with FOXA1 levels. Collectively, these results provide the molecular reasoning on co-targeting the epigenetic regulators that are important for promoting AR activity in treating CRPC. Furthermore, we have found that LSD1, FOXA1, and BRD4 (a Bromodomain and Extra-Terminal motif (BET) protein) co-occupy the super enhancers characterized by distinguishable high levels of H3K4me2 and H3K27ac in PCa cells. Super enhancers have been found to drive oncogenic transcription program in PCa. As an active enhancer marker and an important AR coactivator, BRD4 recognizes H3K27ac signal and promotes the initiation and elongation steps in gene transcription process. Herein, we focus on the potential synergism of combined targeting of LSD1 and BRD4. We found that FOXA1 interacted with BRD4 in different cell lines and patient-derived xenograft (PDX) tumor tissues. In line with this association, either LSD1 inhibitors or BET inhibitors could decrease FOXA1 and BRD4 chromatin binding. Characterization of the physical interaction is an ongoing study. We found that the combinational treatment lead to a more effective reduction of AR transcriptional activity than either single treatment. The PCa cell proliferation in vitro was also more profoundly inhibited by combined therapy. More importantly, we demonstrated this synergism in both 22RV1 xenograft and PDX LuCaP35CR in the castrated mice, with a lower dose of LSD1 inhibitor and BET inhibitor (both in clinical trials) which did not induce overt toxicity in the animals. Currently, we are performing RNA-seq studies to identify the convergent downstream pathways by this co-treatment. In summary, our study provides important pre-clinical insights for targeting the CRPC enhancers by combining inhibitors of LSD1 and BRD4. Citation Format: Shuai Gao, Muqing Li, Mingyu Liu, Dong Han, Zifeng Wang, Wanting Han, Changmeng Cai. Targeting prostate cancer super enhancers by co-inhibiting LSD1 and BRD4 [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 2317.

White Button Mushroom Disrupts AR Signaling in Prostate Cancer: The Scientific Basis for a Diet-Based Chemoprevention

Abstract White button mushroom (WBM) (Agaricus bisporus) is a potential prostate cancer (PCa) chemo-preventive and therapeutic agent. Our clinical phase trial of WBM powder in patients with biochemically recurrent PCa indicated that WBM intake reduced the circulating levels of prostate-specific antigen (PSA), with minimal side effects [1]. We hypothesized that WBM exerts its effects on PCa through the androgen receptor (AR) signaling axis. We thus conducted the reverse translational study. Androgen-sensitive PCa cell lines (LNCaP and VCaP) and patient-derived-xenografts (PDX), of a prostate tumor (TM00298) were used. In both LNCaP and VCaP cells, western blots and qRT-PCR assays indicated that WBM extract (6~30 mg/mL) suppressed DHT-induced PSA expression and cell proliferation in a dose-dependent manner. Immunofluorescence on AR revealed that the nuclear localization of AR was reduced upon WBM extract treatment, which agreed with the results of a PSA promotor-luciferase assay, suggesting that WBM extract inhibited DHT-induced luciferase activity. RNA-Seq on WBM-treated LNCaP cells confirmed that WBM treatment suppressed androgen response pathways and cell-cycle control pathways. Our prostate cancer PDX showed that oral intake of WBM extract (200 mg/kg/day) significantly suppressed tumor growth, as well as decreased PSA levels in both tumors and serum. Both in vitro and in vivo studies suggested that chemical(s) in WBM extract behave as AR antagonist(s). We previously identified a conjugated linoleic acid isomer (CLA-9Z11E) as an active component in WBM extract. In the present study, we extended these findings by performing LanthaScreen™ TR-FRET AR Coactivator Interaction Assays for a direct interaction of CLA-9Z11E with AR. We report here that CLA-9Z11E exerts a strong antagonist potency against the recruitment of an AR coactivator peptide towards AR. The inhibitory effect of CLA-9Z11E (IC50: 350 nM) was nearly two times stronger than the known AR antagonist, cyproterone acetate (IC50: 672 nM). The information gained from this study improves the overall understanding of how WBM may contribute to the prevention and treatment of PCa. It also serves as an important, scientific basis for developing diet-based chemoprevention and integrative therapeutic strategies for prostate cancer (supported by NIH R01 CA227230). Reference: [1] Twardowski P, et al. A phase I trial of mushroom powder in patients with biochemically recurrent prostate cancer: Roles of cytokines and myeloid-derived suppressor cells for Agaricus bisporus-induced prostate-specific antigen responses. Cancer. 2015.121(17):2942-50.