Androgen Receptor Target Genes Research Articles

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

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

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

R-loop resolution by ARIP4 helicase promotes androgen-mediated transcription induction.

Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.

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

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

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

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

Androgen receptor modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth.

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or "oncomiRs" and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271-5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271-5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271-5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271-5p may have therapeutic potential in prostate cancer.

Androgen Receptor/AP-1 Activates UGT2B15 Transcription to Promote Esophageal Squamous Cell Carcinoma Invasion.

Esophageal squamous cell carcinoma (ESCC) is an aggressive epithelial malignancy with poor prognosis. Interestingly, ESCC is strongly characterized by a male-predominant propensity. Our previous study showed that androgen receptor (AR) orchestrated a transcriptional repression program to promote ESCC growth, but it remains unclear whether AR can also activate oncogenic signaling during ESCC progression. In this study, by analyzing our previous AR cistromes and androgen-regulated transcriptomes, we identified uridine diphosphate glucuronosyltransferase family 2 member B15 (UGT2B15) as a bona fide target gene of AR. Mechanistically, AP-1 cofactors played important and collaborative roles in AR-mediated UGT2B15 upregulation. Functional studies have revealed that UGT2B15 promoted invasiveness in vitro and lymph node metastasis in vivo. UGT2B15 was partially responsible for the AR-induced invasive phenotype in ESCC cells. Importantly, simultaneous blocking of AP-1 and AR resulted in stronger inhibition of cell invasiveness compared to inhibiting AP-1 or AR alone. In conclusion, our study reveals the molecular mechanisms underlying the AR-driven ESCC invasion and suggests that the AR/AP1/UGT2B15 transcriptional axis can be potentially targeted in suppressing metastasis in male ESCC patients.

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.

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

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

Peptides disrupting TM4SF3 interaction with AR or AR-V7 block prostate cancer cell proliferation.

Androgen receptor (AR) plays a vital role in the development and progression of prostate cancer from the primary stage to the usually lethal stage known as castration-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain are partially responsible for the abnormal activation of AR and may be involved in resistance to AR-targeting drugs occurring in CRPC. There is increasing consensus on the potential of drugs targeting protein-protein interactions. Our lab has recently identified transmembrane 4 superfamily 3 (TM4SF3) as a critical interacting partner for AR and AR-V7 and mapped the minimal interaction regions. Thus, we hypothesized that these interaction domains can be used to design peptides that can disrupt the AR/TM4SF3 interaction and kill prostate cancer cells. Peptides TA1 and AT1 were designed based on the TM3SF3 or AR interaction domain, respectively. TA1 or AT1 was able to decrease AR/TM4SF3 protein interaction and protein stability. Peptide TA1 reduced the recruitment of AR and TM4SF3 to promoters of androgen-regulated genes and subsequent activation of these AR target genes. Peptides TA1 and AT1 were strongly cytotoxic to prostate cancer cells that express AR and/or AR-V7. Peptide TA1 inhibited the growth and induced apoptosis of both enzalutamide-sensitive and importantly enzalutamide-resistant prostate cancer cells. TA1 also blocked the migration and malignant transformation of prostate cancer cells. Our data clearly demonstrate that using peptides to target the important interaction AR has with TM4SF3 provides a novel method to kill enzalutamide-resistant prostate cancer cells that can potentially lead to new more effective therapy for CRPC.

IU1 and enzalutamide combination yields synergistic effects on castration-resistant prostate cancer.

Androgen deprivation therapy (ADT) is one of the main treatment modalities for prostate cancer (PCa); however, almost all patients treated with ADT eventually progress into castration-resistant PCa (CRPC). Although second-generation androgen receptor (AR) antagonists, such as enzalutamide, have been approved for CRPC treatment, AR signaling in CRPC cells is reactivated through multiple mechanisms, resulting in resistance to treatment and tumor progression with a very poor prognosis. The present study aimed to explore the anticancer effect of a treatment combining AR antagonist enzalutamide with AR degrader IU1 on PCa cells. The joint effects of enzalutamide and IU1 on PCa cell proliferation and apoptosis and associated cell signaling were evaluated in vitro. Mechanistically, the ubiquitination level and half-life of AR were examined under the combination treatment. The binding of IU1 and enzalutamide to AR was further verified using cellular thermal shift analysis and isothermal dose-response curve fingerprinting. The combination of IU1 and three AR antagonists showed synergistic effects in different prostate cell lines. IU1 and enzalutamide synergistically promoted the degradation of AR and AR-V7 proteins, as well as suppressed the expression levels of AR and AR-V7 downstream target genes at the transcriptional and protein levels. The combination also synergistically blocked the PCa cell cycle and promoted apoptosis in PCa cell lines. Mechanistically, the combination promoted increased levels of AR ubiquitination. In CRPC cell lines and in the presence of increased androgen concentrations, enzalutamide was still able to bind AR competitively with androgens, reducing the stability of AR and thus promoting the degradation effect of IU1 on AR, synergistically producing an inhibitory effect on PCa cells. Taken together, our findings suggest that the combination of AR degrader and enzalutamide potentially represents a new therapeutic strategy for CRPC.

Spermine is a natural suppressor of AR signaling in castration-resistant prostate cancer

In castration-resistant prostate cancer (CRPC), clinical response to androgen receptor (AR) antagonists is limited mainly due to AR-variants expression and restored AR signaling. The metabolite spermine is most abundant in prostate and it decreases as prostate cancer progresses, but its functions remain poorly understood. Here, we show spermine inhibits full-length androgen receptor (AR-FL) and androgen receptor splice variant 7 (AR-V7) signaling and suppresses CRPC cell proliferation by directly binding and inhibiting protein arginine methyltransferase PRMT1. Spermine reduces H4R3me2a modification at the AR locus and suppresses AR binding as well as H3K27ac modification levels at AR target genes. Spermine supplementation restrains CRPC growth invivo. PRMT1 inhibition also suppresses AR-FL and AR-V7 signaling and reduces CRPC growth. Collectively, we demonstrate spermine as an anticancer metabolite by inhibiting PRMT1 to transcriptionally inhibit AR-FL and AR-V7 signaling in CRPC, and we indicate spermine and PRMT1 inhibition as powerful strategies overcoming limitations of current AR-based therapies in CRPC.

Abstract A059: Epigenetic remodelling by class I HDAC inhibitors increases response to androgen signalling inhibitors in prostate cancer

Abstract Prostate cancer (PCa) is one of the most prevalent cancers in the male population with approximately 1 in 8 men diagnosed with the disease during their lifetime. The treatment options available varies depending on individual circumstances, with surgery or radiotherapy employed as first-line treatments for locally advanced PCa whilst hormone therapies are employed as the disease progresses toward castrate-resistant prostate cancer. Despite the advances in therapies and initial treatment effectiveness, tumours develop resistance and overcoming this resistance proves a major challenge for the treatment of the disease. Dysregulation of chromatin regulatory proteins and the epigenetic landscape are key factors contributing to the development, progression and persistence of PCa. Histone deacetylases (HDACs) are a family of chromatin-modifying proteins which assemble into various transcriptional repressor complexes and mediate gene repression through the removal of acetyl groups from histone tails, subsequently compacting the chromatin landscape impacting on cell proliferation, transcription, replication and DNA repair. Overexpression of HDACs in prostate cancer has been shown to shift the balance of acetylation, resulting in improper gene silencing. Therefore, we hypothesise that restoration of the acetylation homeostasis through HDAC inhibition is a potential therapy to overcome resistance in PCa. Here, we show that the class I HDAC inhibitor Entinostat as a single agent promotes anti-tumour effects in PCa by impairing cell proliferation and inducing apoptosis in a panel of PCa cell lines. Interestingly, we found that combination with the androgen signalling inhibitors (ASIs), Enzalutamide or Abiraterone, further enhances these anti-tumour effects. In vitro analysis of matched parental and Enzalutamide-/Abiraterone-resistant C4-2B and CWRR-1 cells further revealed an enhanced reduction in cell survival following combined Entinostat and ASIs treatments in the resistant models compared to parental. Genomic and transcriptomic analyses uncovered distinct molecular signatures of these resistant models. Treatment with Entinostat alone downregulated androgen receptor (AR) expression and combination with ASI further suppressed AR expression and downstream AR target genes. Taken together, these results demonstrate the potential for Entinostat to synergise with current standard-of-care therapies to target ASI resistance in PCa. In conclusion, this study provides strong evidence suggesting the effectiveness of class I HDAC inhibition by Entinostat in the treatment of castration-resistant prostate cancer and opens the door for further research into combinatorial therapies in targeting resistance. Citation Format: Rachel McCole, Ben McCullough, Judith M. Manley, Nicholas Forsythe, Simon S. McDade, Melissa LaBonte Wilson. Epigenetic remodelling by class I HDAC inhibitors increases response to androgen signalling inhibitors in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A059.

Abstract B008: Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer

Abstract Introduction and Objective Several studies have shown that cancer-associated fibroblasts (CAFs), the most abundant component in prostate cancer (PCa) microenvironment, promote resistance to androgen deprivation therapies, and metastatic development. The aim of this study is to elucidate the mechanisms of CAF-induced therapy resistance through the establishment of in vitro 3D co-cultures. Methods In this study, the androgen-dependent PNPCa patient-derived xenograft (PDX) model (derived from soft tissue metastasis), and the androgen-independent LAPC9 PDX model (bone metastasis) are used. Epithelial and stromal cells (human and mouse origin, respectively) are separated through magnetic-associated cell sorting to generate PDX-derived organoids and fibroblasts. Fibroblast RNA and protein expression is assessed through RT-qPCR, Western Blot, and Immunofluorescence. Direct 3D co-cultures are obtained by combining fluorescently labelled organoids and fibroblasts in ultra-low attachment plates in defined media. In indirect co-cultures, organoids are cultured in Spherical Plates (Kugelmeiers) with fibroblasts on transwell inserts. Organoid viability is measured after 9 days by CellTiter-Glo 3D Assay. Results PDX-derived fibroblasts express typical CAF genes (e.g. α-smooth muscle actin and Tenascin C), and androgen receptor (AR), both at RNA and protein level. AR protein expression (as well as RNA expression of the AR target gene Fkbp5) is increased in CAFs upon treatment with dyhydrotestosterone (DHT) 10nM, while co-treatment with the AR inhibitor Enzalutamide 10mM abrogates this effect, suggestive of functional AR signaling. These CAFs can organize into tumor/CAF organoid 3D structures upon combination with PDX-derived tumor epithelial cells, providing an in vitro functional model to study tumor-stromal interactions. In indirect (transwell) co-cultures, CAFs increase the viability of the androgen dependent PNPCa organoids, but not that of androgen independent LAPC9. This growth-promoting effect is observed even upon culturing of PNPCa organoids in non-optimal growth conditions, at low DHT concentrations (DHT 0.5nM and 0.25nM versus the standard DHT 1nM). The factors mediating this process will be identified by gene expression profiling of CAFs and organoids in transwell co-cultures and/or monoculture. Conclusions Our data indicate that PCa metastatic cells modify the properties of neighbor stromal cells to support tumorigenesis, since PDX-derived fibroblasts, originally part of the mouse dermal stroma, display CAF features and active AR signaling. 3D co-cultures represent a useful tool to study the tumor-stromal interactions. Our transwell co-culture experiments suggest that CAFs play an important role in supporting the viability and growth of early metastatic androgen dependent PCa cells (PNPCa) by secreting pro-tumorigenic factors. The possibility that CAFs exert different effects depending on the tumor stage setting (early vs advanced) will be subsequently investigated. Citation Format: Francesco Bonollo, Natalie Sampson, George Thalmann, Sofia Karkampouna, Marianna Kruithof-de Julio. Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B008.

Inhibition of Wnt/β-catenin pathway overcomes therapeutic resistance to abiraterone in castration-resistant prostate cancer.

Abiraterone acetate has been clinically approved for the treatment of patients with advanced-stage prostate cancer. It reduces testosterone production by blocking the enzyme cytochrome P450 17 alpha-hydroxylase. Despite improved survival outcomes with abiraterone, almost all patients develop therapeutic resistance and disease recurrence, progressing to a more aggressive and lethal phenotype. Bioinformatics analyses predicted activation of canonical Wnt/β-catenin and involvement of stem cell plasticity in abiraterone-resistant prostate cancer. Increased expression of androgen receptor (AR) and β-catenin and their crosstalk causes activation of AR target genes and regulatory networks for which overcoming acquired resistance remains a major challenge. Here we show that co-treatment with abiraterone and ICG001, a β-catenin inhibitor, overcomes therapeutic resistance and significantly inhibited markers of stem cell and cellular proliferation in abiraterone-resistant prostate cancer cells. Importantly, this combined treatment abrogated the association between AR and β-catenin; diminished SOX9 expression from the complex more prominently in abiraterone-resistant cells. In addition, combined treatment inhibited tumor growth in an in vivo abiraterone-resistant xenograft model, blocked stemness, migration, invasion, and colony formation ability of cancer cells. This study opens new therapeutic opportunity for advanced-stage castration-resistant prostate cancer patients.

Abstract 3859: Alterations in hormone receptors regulome following response to kinase inhibitors in lethal prostate cancer

Abstract The androgen receptor (AR) signaling axis is critical for prostate cancer (PCa) pathogenesis and all subsequent phases of disease progression. Although initial success with androgen deprivation therapy and AR-targeted agents such as enzalutamide or abiraterone, most patients inevitably relapse due to therapeutic resistance. Previous studies have shown that genomic AR is unchanged despite resistance to AR antagonists, and its gene expression is unaltered. Therapeutic resistance is broadly classified into two categories; restoration of AR activity and tumor progression despite AR blockade. Indeed, others have noted an extensive reprogramming of the AR cistrome in clinical and preclinical specimens, where a shift in canonical to non-canonical AR cistrome is associated with an increase in Gleason grade. In previous studies, we observed increases in known AR target genes following exposure to kinase inhibitors which was marked by a decrease in tumor growth. We hypothesized that restoration of canonical AR cistrome can be mediated through kinase inhibition and is associated with re-sensitization to AR-targeted therapy. In addition, we treated cell lines and patient-derived xenograft models of advanced prostate cancer with kinase signaling pathway inhibitors, AR signaling inhibitors, or a combination of both for 24 hours (in vitro) or five days (in vivo). In addition, samples were collected and processed for RNA-seq and ChIP-seq. Our transcriptomic analysis showed enrichment of previously published non-canonical AR target genes in cells resistant to AR-targeted agents. When resistant cells were exposed to kinase inhibitors, we observed an enrichment of canonical AR target genes. In addition, we identified unique AR-enriched peaks at active sites associated with resistance. Through GSEA of gene annotated peaks, we found unique regulatory transcription factor targets, some of which are associated with cell proliferation and differentiation and may play a functional role in AR cistrome adaptation to resistance and survival. Overall, our data suggest that AR transcription adapts to the therapeutic resistance of AR antagonists via the enrichment of non-canonical AR cistrome. Furthermore, restoration of canonical AR cistrome is achieved following kinase signaling pathway inhibition and is associated with re-sensitization to AR-targeted therapy. Citation Format: Remi M. Adelaiye-Ogala, Surendra Gulla, Jonathan E. Bard, David J. VanderWeele, Kathleen Kelly. Alterations in hormone receptors regulome following response to kinase inhibitors in lethal prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3859.

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

Abstract Steroid hormone signaling axes drive proliferation and survival in prostate cancer (PCa), breast and other cancers. In late stage PCa, the androgen receptor (AR) is therapeutically antagonized with success, for example with Enzalutamide (Enza). Although initially successful, resistance emerges associated with tissue plasticity and patient mortality. Enza-resistance mechanisms include altered AR coregulator interactions. Identifying which coregulators change Enza sensitivity is attractive as they may represent new drug targets but challenging given the multitude of coregulators expressed in PCa and the diverse roles they play in transcription and translation.To meet this challenge, we designed a bioinformatic workflow to identify under-investigated yet clinically-impactful coregulators. We tested coactivators (CoA, n~750), corepressors (CoR, n ~600), and mixed function coregulators (Mixed, n~600) associations with patient outcome in TCGA PCa cohorts; applied partial correlation analyses to reveal coregulators that impacted AR target genes; utilized DepMap essentiality scores to rank importance; and examined interactions in publicly available AR RIME data. The impact of the top ranked coregulators (n=18) on Enza sensitivity was examined using shRNA or siRNA approaches, and with inhibitor drugs in Enza-resistant PCa cell lines (LNCaP C42B, 22Rv1). Finally, coregulator expression was examined in single cell RNA-Seq from PCa 3D organoids derived from the PBCre4:Ptenf/f:Rb1f/f (DKO) and the PBCre4:Ptenf/f:Rb1f/f:Trp53f/f (TKO) PCa mouse model, which recapitulate the loss of Enza sensitivity and lineage plasticity. ShRNA targeting coregulators was undertaken in organoids. The bioinformatics workflow identified 18 coregulators, including CoA (e.g., WDR5, KAT7, BLM, SMARCC1), CoR (e.g., SETDB1, PSPC1), and Mixed (e.g., KDM5A, BPTF, TCEGR1) to screen for impact on Enza. KAT7 shRNA modulated Enza sensitivity, regulated expression of AR target genes, and synergized with a KAT7 inhibitor (WM3835) to enhance Enza sensitivity. Similarly, a BLM inhibitor (ML216) synergized with Enza. Interestingly, although WDR5 siRNA alone did not alter Enza responses, the combination of KAT7 shRNA and WDR5 siRNA significantly potentiated Enza responses. Similarly, SMARCC1 shRNA cooperated with siRNA to several targets (TBX3, BPTF, or SETDB1) to enhance Enza responses. Smarcc1 and Tbx3 were upregulated in Enza-resistant neuroendocrine PCa, and the recently identified Pou2f3 Enza-resistant phenotypes, and Bptf was upregulated in the Tff3 Enza-resistant phenotype. We have shRNA targeted Blm, Smarcc1, Tbx3, and Bptf in the DKO and TKO organoids and are measuring the impact of Enza responses. Overall, we have identified potentially novel AR coregulators that associate with the development of therapy resistance and shown that targeting these coregulators may augment Enza sensitivity in PCa. Citation Format: Rayzel C. Fernandes, Debasis Nayak, Damien A. Leach, Meenalakshmi Chinnam, Wilbert Zwart, David W. Goodrich, Moray J. Campbell, Charlotte L. Bevan. Targeting androgen receptor coregulators to enhance enzalutamide sensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3083.

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

Abstract The paralogous lysine acetyltransferases CREB-binding protein (CBP) and p300 are key epigenetic regulators involved in diverse signaling pathways in cancer. The bromodomain (BRD) of CBP/p300 serves as an acetyl-lysine “reader” that allows CBP/p300 to bind chromatin at acetylated histone and non-histone proteins leading to the regulation of gene transcription. Indeed, CBP/p300 are critical co-activators of nuclear receptors, including the androgen receptor (AR) in castration resistant prostate cancer (CRPC). Thus, inhibition of CBP and p300 is an emerging therapeutic strategy to block the transactivation activity of the AR in CRPC. In addition, inhibition of the CBP/p300 BRD has been described as a potential therapeutic strategy to treat multiple myeloma (MM) through transcriptional suppression of interferon regulatory factor 4 (IRF4) and concomitant repression of its target genes MYC and MYB. TT125-802 is a highly selective and potent, oral small molecule inhibitor of the BRD of CBP/p300. In a BROMOscan assay against a panel of 40 BRD-containing proteins, TT125-802 revealed the unique selectivity to the BRD of CBP/p300 with a minimal off-target binding to all other BRDs, including BET proteins.TT125-802 shows selective anti-proliferative activity in AR-dependent prostate cancer cell lines (22Rv-1, C4-2, and LNCaP) and inhibits AR target gene expression (KLK2, KLK3, TMPRSS2, and MYC) in a dose-dependent manner (IC50 of 2 to 33 nM). AR-negative prostate cancer cell lines (DU-145 and PC-3) are insensitive to TT125-802 in vitro, pointing to an AR-selective mode of action. In an in vivo model of CRPC, TT125-802 inhibited tumor growth when administered orally to human C4-2 xenograft-bearing mice. Daily dosing of TT125-802 was well tolerated with stable bodyweights and platelet counts. In addition, preclinical studies in CRPC patient-derived xenograft (PDX)-bearing mice showed that the combination treatment of TT125-802 and enzalutamide had a synergistic effect on tumor growth inhibition compared to single agent treatments. TT125-802 reduced mRNA expression of the AR-target genes in tumor samples and decreased plasma PSA levels compared to enzalutamide alone, and the combination reduced levels even further. In a preclinical model of MM (OPM2), TT125-802 had a dose-dependent effect on tumor growth, inducing tumor regressions at the highest dose. Target genes such as MYC, MYB, and IRF4 were potently downregulated in tumors. We conclude that TT125-802 is a novel, highly selective inhibitor of the BRD of CBP/P300. It has therapeutic potential as monotherapy in prostate cancer and multiple myeloma and in combination with next-generation AR inhibitors for patients with lethal prostate cancer. A FIH study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Sara Laudato, Dorothea Gruber, Thomas Bohnacker, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. TT125-802 is a potent and highly selective CBP/p300 bromodomain inhibitor for the treatment of castration resistant prostate cancer and haematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6268.

Abstract 5281: Role of TMEM97/Sigma-2 receptor in prostate cancer

Abstract Introduction: Prostate cancer is the most common cancer in men. About one in eight men will be diagnosed with prostate cancer in his lifetime, and one in forty-one men will die of it. Sigma 2 receptor (σ2R) has been implicated in carcinogenesis including prostate cancer. Its molecular identity remained elusive until 2017 that transmembrane protein 97 (TMEM97) was identified as the bona fide sigma 2 receptor. Past studies have shown that σ2R is highly expressed in various mammalian tumors and TMEM97 works as an oncogene in different cancers. In this study, we examined the expression of TMEM97/σ2 receptor in prostate cancer, evaluated its interaction with androgen receptor (AR), and determined its functions in tumor cell growth and progression. Methods: The expression of TMEM97 at mRNA levels and its association with clinical parameters were queried in the TCGA database using cBioportal. Its expression pattern in prostate tumor tissues was examined by IHC using a commercially validated antibody. A correlation analysis of TMEM97 with AR was conducted utilizing both PCTA and TCGA databases. To determine its roles in tumor proliferation, clonogenicity, and responses to hormone deprivation and chemo-therapy, TMEM97 expression was overexpressed and knocked down, and the subsequent effects were determined by cell proliferation MTS assay, colony formation assay with and without enzalutamide treatment. A xenograft model of nu/nu mice was used to assess the impact of TMEM97 overexpression on tumor formation and growth in vivo. The effects of TMEM97 overexpression and knockdown on AR transcriptional activity were examined. Results: A significant elevation of TMEM97 mRNA levels, with a Z-Score above 2.0, was found in 5% of the prostate cancer patients and was associated with significantly reduced survival. TMEM97 protein was detected in prostate tumor tissues of all stages and grades. Its expression is highly correlated with the expression of AR in prostate cancer. Prostate cancer cells with TMEM97 overexpression showed growth advantage over the control cells both in normal culture conditions and also under treatment of enzalutamide. TMEM97 knockdown weakened growth, and sensitized castration resistant 22Rv1 toward enzalutamide, which is linked to the decrease of AR-V7. TMEM97 overexpression in LNCaP cells also significantly increased tumor formation and growth when xenografted into nu/nu mice. In LNCaP cells, TMEM97 overexpression elevated AR target genes expression. Parallelly, these genes were all significantly downregulated in TMEM97 knockdown cells. A compound with a high affinity to TMEM97 was found to induce apoptosis of prostate cancer cells with preferential degradation of AR-V7. Conclusion: The observations suggest that TMEM97/sigma2 receptor plays a role in prostate tumor cell proliferation and the increased expression of TMEM97 can elevate AR activity and stimulate prostate tumor formation and growth. Citation Format: Xiangwei Fang, Yuanqin Zhang, Jiuhui Wang, Paul Cacioppo, Michael Crider, Daotai Nie. Role of TMEM97/Sigma-2 receptor in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5281.