Androgen Receptor Signaling Inhibitors Research Articles

A COP1-GATA2 axis suppresses AR signaling and prostate cancer

Androgen receptor (AR) signaling is crucial for driving prostate cancer (PCa), the most diagnosed and the second leading cause of death in male patients with cancer in the United States. Androgen deprivation therapy is initially effective in most instances of AR-positive advanced or metastatic PCa. However, patients inevitably develop lethal castration-resistant PCa (CRPC), which is also resistant to the next-generation AR signaling inhibitors. Most CRPCs maintain AR expression, and blocking AR signaling remains a main therapeutic approach. GATA2 is a pioneer transcription factor emerging as a key therapeutic target for PCa because it promotes AR expression and activation. While directly inhibiting GATA2 transcriptional activity remains challenging, enhancing GATA2 degradation is a plausible therapeutic strategy. How GATA2 protein stability is regulated in PCa remains unknown. Here, we show that constitutive photomorphogenesis protein 1 (COP1), an E3 ubiquitin ligase, drives GATA2 ubiquitination at K419/K424 for degradation. GATA2 lacks a conserved [D/E](x)xxVP[D/E] degron but uses alternate BR1/BR2 motifs to bind COP1. By promoting GATA2 degradation, COP1 inhibits AR expression and activation and represses PCa cell and xenograft growth and castration resistance. Accordingly, GATA2 overexpression or COP1 mutations that disrupt COP1-GATA2 binding block COP1 tumor-suppressing activities. We conclude that GATA2 is a major COP1 substrate in PCa and that COP1 promotion of GATA2 degradation is a direct mechanism for regulating AR expression and activation, PCa growth, and castration resistance.

Adverse events associated with androgen receptor signaling inhibitors in the treatment of prostate cancer: A systematic review and network meta-analysis.

317 Background: Androgen receptor signaling inhibitors (ARSi) are emerging as standard treatments for prostate cancer across the disease spectrum. With multiple available agents approved, comparing adverse effects between different therapies may guide clinical decision-making. The purpose of this meta-analysis was to compare adverse events (AE) and serious adverse events (sAE) between the ARSi drugs abiraterone, apalutamide, darolutamide, and enzalutamide. Methods: PubMed, Web of Science, Embase, and Clinicaltrials.gov were searched for double-blind, randomized controlled trials for ARSi therapy in the treatment of prostate cancer (both metastatic and non-metastatic settings). Two independent teams extracted AE and sAE data. Studies were synthesized using R with the “Netmeta” package. Results: Thirteen double-blinded, randomized trials of abiraterone, apalutamide, darolutamide or enzalutamide were included in this study. The relative risk ratio (RR) of the most common any-grade AE compared to placebo was highest in enzalutamide with a RR of 1.30 (95% confidence interval [CI], 1.13-1.50), followed by apalutamide (1.20, [0.95-1.53]), abiraterone (1.19, [0.98-1.44]), and darolutamide (1.17, [0.83-1.65]). The risk of any sAE was highest in darolutamide (1.80, [0.91-3.57]), followed by apalutamide (1.75, [1.07-2.87]), abiraterone (1.56, [1.08-2.25]), and enzalutamide (1.57, [1.18-2.09]). For overall ARSi analysis, special sAE of interest included acute coronary syndrome, arrhythmia, and seizure, for which ARSi therapy was associated with an odds ratio (OR) of 2.85 (1.65-4.91), 1.75 (1.25-2.44), and 1.31 (0.57-3.00) respectively. Amongst themselves, enzalutamide had the highest risk of cardiovascular AE compared to non-enzalutamide ARSi (RR = 1.77, [1.42-2.20], p < 0.05). Abiraterone had the lowest risk of musculoskeletal AEs compared to non-abiraterone ARSi (0.79, [0.70-0.88], p < 0.05). No significant differences existed in the other domains of interest. Conclusions: ARSi therapy was associated with a higher risk of AE and sAE in various domains compared to placebo. Our findings suggest that the side-effect profiles of ARSis do not significantly differ except in the case of increased cardiovascular risk with enzalutamide and decreased musculoskeletal risk with abiraterone. This data may aid clinicians in determining personalized therapeutic options for their patents based on side effect profiles.

296 (PB076) - Cellular hormone metabolism is critical for canonical androgen receptor antagonist activity

Background: Second-generation androgen receptor (AR) antagonists and an androgen synthesizing enzyme inhibitor have become the standard of care for advanced prostate cancer (PCa). Clinically-approved AR antagonists bind to the AR ligand binding domain (LBD) and competitively inhibit AR function. Dual action AR inhibitors (DAARIs), which bind to the N-terminal domain (NTD) of the AR inhibit AR signaling and lead AR protein degradation, have been identified by our group as potential therapeutics for PCa.

ARNEO: A Randomized Phase II Trial of Neoadjuvant Degarelix with or Without Apalutamide Prior to Radical Prostatectomy for High-risk Prostate Cancer

BackgroundHigh-risk prostate cancer (PCa) patients have a high risk of biochemical recurrence and metastatic progression following radical prostatectomy (RP). ObjectiveTo determine the efficacy of neoadjuvant degarelix plus apalutamide before RP compared with degarelix with a matching placebo. Design, setting, and participantsARNEO was a randomized, placebo-controlled, phase II neoadjuvant trial before RP performed between March 2019 and April 2021. Eligible patients had high-risk PCa and were amenable to RP. InterventionPatients were randomly assigned at a 1:1 ratio to degarelix (240–80–80 mg) + apalutamide (240 mg/d) versus degarelix + matching placebo for 3 mo followed by RP. Prior to and following neoadjuvant treatment, pelvic 18F-PSMA-1007 positron emission tomography (PET)/magnetic resonance imaging (MRI) was performed. Outcome measurements and statistical analysisThe primary endpoint was the difference in proportions of patients with minimal residual disease (MRD; = residual cancer burden (RCB) ≤0.25 cm3 at final pathology). Secondary endpoints included differences in prostate-specific antigen responses, pathological staging, and change in TNM stage on prostate-specific membrane antigen (PSMA) PET/MRI following hormonal treatment. Biomarkers (immunohistochemical staining on prostate biopsy [PTEN, ERG, Ki67, P53, GR, and PSMA] and PSMA PET/MRI-derived characteristics) associated with pathological response (MRD and RCB) were explored. Results and limitationsPatients were randomized to neoadjuvant degarelix + apalutamide (n = 45) or degarelix + matching placebo (n = 44) for 12 wk and underwent RP. Patients in the degarelix + apalutamide arm achieved a significantly higher rate of MRD than those in the control arm (38% vs 9.1%; relative risk [95% confidence interval] = 4.2 [1.5–11], p = 0.002). Patients with PTEN loss in baseline prostate biopsy attained significantly less MRD (11% vs 43%, p = 0.002) and had a higher RCB at final pathology (1.6 vs 0.40 cm3, p < 0.0001) than patients without PTEN loss. Following neoadjuvant hormonal therapy, PSMA PET–estimated tumor volumes (1.2 vs 2.5 ml, p = 0.01) and maximum standardized uptake value (SUVmax; 4.3 vs 5.7, p = 0.007) were lower in patients with MRD than in patients without MRD. PSMA PET–estimated volume and PSMA PET SUVmax following neoadjuvant treatment correlated significantly with RCB at final pathology (both p < 0.001). ConclusionsIn high-risk PCa patients, neoadjuvant degarelix plus apalutamide prior to RP results in a significantly improved pathological response (MRD and RCB) compared with degarelix alone. Our trial results provide a solid hypothesis-generating basis for neoadjuvant phase 3 trials, which are powered to detect differences in long-term oncological outcome following neoadjuvant androgen receptor signaling inhibitor therapy. Patient summaryIn this study, we looked at the difference in pathological responses in high-risk prostate cancer patients treated with degarelix plus apalutamide or degarelix plus matching placebo prior to radical prostatectomy. We demonstrated that patients treated with degarelix plus apalutamide achieved a significantly better tumor response than patients treated with degarelix plus matching placebo. Long-term follow-up is required to determine whether improved pathological outcome translates into better oncological outcomes.

Transcriptional profiling of matched patient biopsies clarifies molecular determinants of enzalutamide-induced lineage plasticity

The androgen receptor (AR) signaling inhibitor enzalutamide (enza) is one of the principal treatments for metastatic castration-resistant prostate cancer (CRPC). Several emergent enza clinical resistance mechanisms have been described, including lineage plasticity in which the tumors manifest reduced dependency on the AR. To improve our understanding of enza resistance, herein we analyze the transcriptomes of matched biopsies from men with metastatic CRPC obtained prior to treatment and at progression (n = 21). RNA-sequencing analysis demonstrates that enza does not induce marked, sustained changes in the tumor transcriptome in most patients. However, three patients’ progression biopsies show evidence of lineage plasticity. The transcription factor E2F1 and pathways linked to tumor stemness are highly activated in baseline biopsies from patients whose tumors undergo lineage plasticity. We find a gene signature enriched in these baseline biopsies that is strongly associated with poor survival in independent patient cohorts and with risk of castration-induced lineage plasticity in patient-derived xenograft models, suggesting that tumors harboring this gene expression program may be at particular risk for resistance mediated by lineage plasticity and poor outcomes.

1404P A phase II study of ZEN-3694 (ZEN), enzalutamide (ENZ), and pembrolizumab (P) in metastatic castration resistant prostate cancer (mCRPC): Interim safety results

ZEN is a potent and selective BET bromodomain inhibitor (BETi) with encouraging clinical activity in combination with ENZ in patients (pts) with second generation androgen receptor signaling inhibitor (ARSI)-resistant mCRPC. ZEN has synergy with immune checkpoint inhibition in pre-clinical assays. The safety and efficacy of ZEN in combination with ENZ plus P was evaluated (NCT04471974). Pts were required to have progressive mCRPC, prior resistance to ≥ 1 second generation ARSI, and no prior chemotherapy for mCRPC. A safety lead-in (N = 6) followed by dose expansion was utilized, with a starting dose of ZEN 72 mg/day, ENZ 160 mg/day, and P 200 mg IV every 3 weeks. The primary objective was to determine the recommended phase 2 dose (RP2D); key efficacy endpoints included PSA50 response. Twenty-two pts have been enrolled to date (n = 6 safety lead-in; n = 16 dose expansion). Median age and serum PSA at study entry was 71 (range 54 – 82) and 30.6 ng/mL (range 5.6 – 560.1), respectively. Ten pts (45%) had prior treatment with > 1 ARSI. Fourteen pts (64%) remain on treatment, 7 discontinued for progression, and one discontinued due to toxicity (Grade 3 LFTs). There were no dose-limiting toxicities during safety lead-in; ZEN 72 mg daily + ENZ 160 mg/day + P 200 mg IV q 3 weeks was the RP2D. Grade 3 treatment-related adverse events (AEs) for the study cohort included fatigue (n = 3), decreased appetite (n = 1), rash (n = 1), and elevated AST/ALT (n = 1). Immune-related AEs included: skin rash (Grade 1/2: n = 9; grade 3: n = 1), colitis (Grade 1/2: n = 6; grade 3: n = 0), pruritus (grade 1/2: n = 2; grade 3: n = 0), and autoimmune hepatitis (Grade 3, n = 1). The PSA50 response proportion was 27% (6/22 pts), including 2 pts who had previously progressed on ENZ. None of the responders were MSI-high by targeted next-gen sequencing. PSA responses were durable, including 2 pts who remain on treatment for 13.6+ and 12.7+ months, respectively. The combination of ZEN, ENZ, and P had an acceptable safety profile. Preliminary clinical activity was observed, including responses in ENZ-resistant microsatellite-stable mCRPC. Further investigation of the combination is ongoing including in an AR-independent cohort.

1406P Interim results of a phase II trial of sacituzumab govitecan (SG) in patients (Pts) with metastatic castration resistant prostate cancer (mCRPC) progressing on androgen receptor signaling inhibitors (ARSI)

Resistance to ARSIs (e.g. abiraterone, enzalutamide) is inevitable and there is critical need to develop new therapies for mCRPC. SG is an antibody-drug conjugate targeting Trop-2, currently FDA-approved for use in breast and bladder cancers.

Integrated analysis reveals FOXA1 and Ku70/Ku80 as targets of ivermectin in prostate cancer

Ivermectin is a widely used antiparasitic drug and shows promising anticancer activity in various cancer types. Although multiple signaling pathways modulated by ivermectin have been identified in tumor cells, few studies have focused on the exact target of ivermectin. Herein, we report the pharmacological effects and targets of ivermectin in prostate cancer. Ivermectin caused G0/G1 cell cycle arrest, induced cell apoptosis and DNA damage, and decreased androgen receptor (AR) signaling in prostate cancer cells. Further in vivo analysis showed ivermectin could suppress 22RV1 xenograft progression. Using integrated omics profiling, including RNA-seq and thermal proteome profiling, the forkhead box protein A1 (FOXA1) and non-homologous end joining (NHEJ) repair executer Ku70/Ku80 were strongly suggested as direct targets of ivermectin in prostate cancer. The interaction of ivermectin and FOXA1 reduced the chromatin accessibility of AR signaling and the G0/G1 cell cycle regulator E2F1, leading to cell proliferation inhibition. The interaction of ivermectin and Ku70/Ku80 impaired the NHEJ repair ability. Cooperating with the downregulation of homologous recombination repair ability after AR signaling inhibition, ivermectin increased intracellular DNA double-strand breaks and finally triggered cell death. Our findings demonstrate the anticancer effect of ivermectin in prostate cancer, indicating that its use may be a new therapeutic approach for prostate cancer.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells.

The novel selenium-aspirin compound AS-10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase-mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC-3 and DU145 androgen receptor (AR)-negative and P53-null/mutant aggressive human prostate cancer (PCa) cell lines in NCI-60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early-stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure. Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS-10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR-positive 22Rv1 human PCa cells were used to validate key findings. In addition to confirming AS-10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate-specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS-10 decreased AR and KLK3 mRNA levels without impacting pre-existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS-10 arrested cells predominantly in G1 , and induced caspase-mediated apoptosis without necrosis. The death induced by AS-10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N-terminal Kinase cellular stress signaling and was mitigated modestly by glutathione-boosting antioxidant N-acetylcysteine. AS-10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA-seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences. AS-10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell-cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR-axis targeting drugs.

Triplet therapy with androgen deprivation, docetaxel, and androgen receptor signalling inhibitors in metastatic castration-sensitive prostate cancer: A meta-analysis

BackgroundThe addition of either docetaxel or an androgen receptor signalling pathway inhibitor (ARSi) to androgen-deprivation therapy (ADT) has become the standard of care for metastatic castration-sensitive prostate cancer (mCSPC) patients. Recent phase III data support even greater survival impact of a triplet regimen with ADT plus docetaxel plus an ARSi (abiraterone or darolutamide) compared to ADT plus docetaxel. ObjectiveTo evaluate whether the addition of an ARSi to ADT improves outcomes of mCSPC patients treated with docetaxel. MethodsWe searched MEDLINE/PubMed, the Cochrane Library, and ASCO Meeting abstracts for randomised clinical trials (RCTs) testing the combination of ARSi + ADT in mCSPC men who received docetaxel. Data extraction was conducted according to the PRISMA statement. Summary hazard ratio (HR) was calculated using random- or fixed-effects models. The statistical analyses were performed with RevMan software (v.5.2.3). ResultsFive RCTs were selected. Triplet therapy improved overall survival (OS) compared to ADT + docetaxel in mCSPC patients (HR = 0.73; p < 0.00001). This intensified strategy maintained the OS benefit when the ARSi was administered concomitant to chemotherapy (HR = 0.72; p < 0.00001), but no statistical effect was detected if the ARSi was sequential to docetaxel (p = 0.44). Moreover, in the subgroup of men with de novo mCSPC, triplets significantly improved OS (HR = 0.72, p < 0.0001). The lack of access to raw data was the main limit of our analysis. ConclusionOur results support a clear survival advantage of adding an ARSi to ADT in mCSPC patients treated with docetaxel, mainly when the ARSi was administered concomitantly to chemotherapy and in the subgroup of de novo mCSPC.

Emerging Role of PARP Inhibitors in Metastatic Prostate Cancer.

We highlight the clinical development of Poly (ADP-Ribose) polymerase (PARP) inhibitors in prostate cancer. Approximately 10 to 30% of metastatic prostate cancer patients carry germline or somatic mutations in DNA repair pathways. BRCA2 is the most commonly mutated gene in DNA damage repair pathways. Because of its critical function in homologous recombination repair (HRR) machinery, deleterious BRCA2 mutation enables synthetic lethality to a PARP inhibitor. Olaparib demonstrated clinical benefit in patients with deleterious mutations in HRR-related genes and most clearly in patients with BRCA2 mutations. Olaparib received the US FDA approval or mCRPC patients with a qualifying HRR gene mutation in May 2020. Rucaparib received an accelerated FDA approval for patients with BRCA1- or BRCA2-mutated mCRPC based on 43% objective response rate in a phase II study. To expand the application of a PARP inhibitor, several trials have evaluated various combination strategies with an androgen receptor signaling inhibitor, immunotherapy, radium-223, and others. While no PARP inhibitor combination regimen has been approved, promising data from a PARP inhibitor and an ASI combination have been reported. PARP inhibitor represents a standard treatment for patient with mCRPC with germline or somatic mutations in BRCA2 and other HRR pathway genes.

Histone lysine demethylase inhibition reprograms prostate cancer metabolism and mechanics

ObjectiveAberrant activity of androgen receptor (AR) is the primary cause underlying development and progression of prostate cancer (PCa) and castration-resistant PCa (CRPC). Androgen signaling regulates gene transcription and lipid metabolism, facilitating tumor growth and therapy resistance in early and advanced PCa. Although direct AR signaling inhibitors exist, AR expression and function can also be epigenetically regulated. Specifically, lysine (K)-specific demethylases (KDMs), which are often overexpressed in PCa and CRPC phenotypes, regulate the AR transcriptional program. MethodsWe investigated LSD1/UTX inhibition, two KDMs, in PCa and CRPC using a multi-omics approach. We first performed a mitochondrial stress test to evaluate respiratory capacity after treatment with MC3324, a dual KDM-inhibitor, and then carried out lipidomic, proteomic, and metabolic analyses. We also investigated mechanical cellular properties with acoustic force spectroscopy. ResultsMC3324 induced a global increase in H3K4me2 and H3K27me3 accompanied by significant growth arrest and apoptosis in androgen-responsive and -unresponsive PCa systems. LSD1/UTX inhibition downregulated AR at both transcriptional and non-transcriptional level, showing cancer selectivity, indicating its potential use in resistance to androgen deprivation therapy. Since MC3324 impaired metabolic activity, by modifying the protein and lipid content in PCa and CRPC cell lines. Epigenetic inhibition of LSD1/UTX disrupted mitochondrial ATP production and mediated lipid plasticity, which affected the phosphocholine class, an important structural element for the cell membrane in PCa and CRPC associated with changes in physical and mechanical properties of cancer cells. ConclusionsOur data suggest a network in which epigenetics, hormone signaling, metabolite availability, lipid content, and mechano-metabolic process are closely related. This network may be able to identify additional hotspots for pharmacological intervention and underscores the key role of KDM-mediated epigenetic modulation in PCa and CRPC.

A Systematic Review of Circulating Tumor Cells Clinical Application in Prostate Cancer Diagnosis.

Simple SummaryCell-dependent and cell-independent information drawn from the blood stream were merged into the attractive term “liquid biopsy” and tentatively applied to most segments of cancer management: detection, risk-stratification, personalization of care and follow-up. However, the robust science behind liquid biopsies has not been widely used, thereby remaining a latent and possibly undervalued instrument. Here, we conducted a systematic review of CTCs in prostate cancer management to summarize their use in clinical practice.The purpose of the review is to summarize the recent data on circulating tumor cells (CTC) use in clinical practice. We performed a systematic literature search using two databases (Medline and Scopus) over the past five years and the following terms: (CTC OR “circulating tumor cells” OR “liquid biopsy”) AND prostate. The primary outcome was CTC predictive value for prostate cancer (PC) progression and survival. The secondary outcomes were the CTC predictive value for therapy response and the results of CTC detection depending on the assessment method. In metastatic PC, the CTC count showed itself to be a prognostic marker in terms of clinically important features, namely survival rates and response to treatment. CTC concentration was significantly associated with the overall survival and progression-free survival rates. A strong association between the overall survival or progression-free survival rate and CTC concentration could be observed. Variant-7 androgen receptors-positive (AR-V7-positive) patients showed a poor response to androgen receptor signaling (ARS) inhibitors, but this did not compromise their response to taxanes. In localized PC, only positive Cluster of Differentiantion 82 protein (CD82+) correlated with a higher survival rate. CTC count and AR-V7 expression showed itself to be a valuable biomarker for survival in metastatic PC and response to ARS-inhibitors. CTC diagnostic performance for localized PC or for screening and early detection is not high enough to show additional value over the other biomarkers.

The Role of Epigenetic Change in Therapy-Induced Neuroendocrine Prostate Cancer Lineage Plasticity.

The androgen receptor (AR) signaling pathway is critical for growth and differentiation of prostate cancer cells. For that reason, androgen deprivation therapy with medical or surgical castration is the principal treatment for metastatic prostate cancer. More recently, new potent AR signaling inhibitors (ARSIs) have been developed. These drugs improve survival for men with metastatic castration-resistant prostate cancer (CRPC), the lethal form of the disease. However, ARSI resistance is nearly universal. One recently appreciated resistance mechanism is lineage plasticity or switch from an AR-driven, luminal differentiation program to an alternate differentiation program. Importantly, lineage plasticity appears to be increasing in incidence in the era of new ARSIs, strongly implicating AR suppression in this process. Lineage plasticity and shift from AR-driven tumors occur on a continuum, ranging from AR-expressing tumors with low AR activity to AR-null tumors that have activation of alternate differentiation programs versus the canonical luminal program found in AR-driven tumors. In many cases, AR loss coincides with the activation of a neuronal program, most commonly exemplified as therapy-induced neuroendocrine prostate cancer (t-NEPC). While genetic events clearly contribute to prostate cancer lineage plasticity, it is also clear that epigenetic events—including chromatin modifications and DNA methylation—play a major role. Many epigenetic factors are now targetable with drugs, establishing the importance of clarifying critical epigenetic factors that promote lineage plasticity. Furthermore, epigenetic marks are readily measurable, demonstrating the importance of clarifying which measurements will help to identify tumors that have undergone or are at risk of undergoing lineage plasticity. In this review, we discuss the role of AR pathway loss and activation of a neuronal differentiation program as key contributors to t-NEPC lineage plasticity. We also discuss new epigenetic therapeutic strategies to reverse lineage plasticity, including those that have recently entered clinical trials.

Abstract 2372: Targeting a metabolic compensatory mechanism for heat shock factor 1 inhibition in prostate cancer

Abstract While inhibition of androgen receptor signaling has greatly improved the outcome for many prostate cancer patients (PCa), there are limited therapeutic interventions for advanced and metastatic PCa. Heat shock factor 1 (HSF1) is activated by cellular stress and regulates transcription, proteome stability, and oxidative stress. HSF1 is elevated in aggressive cancers from many tissue types and has been directly implicated in driving cancer progression. We found that HSF1 predicts survival for metastatic castration resistant prostate cancer patients, who are unable to be treated by inhibition of the androgen receptor signaling. In comparison to benign prostate tissue, HSF1 had increased protein levels in primary PCa, castration resistant PCa, and small cell neuroendocrine carcinoma the most aggressive histologic type of PCa. Knockdown of HSF1 decreases cellular growth in PCa cell lines. In collaboration with the laboratory of Dr. Dennis Thiele, we identified a direct HSF inhibitor that targets nuclear HSF1 for degradation. Prostate cancer cells experience cellular senescence when treated with the HSF1 inhibitor. To better understand how HSF1 regulates PCa cellular growth, we investigated metabolic changes in response to the selective HSF1 inhibitor. We found that a rate limiting enzyme in the transsulfuration pathway was reproducibly altered after HSF1 knockdown and pharmacological inhibition. This is the first linkage of HSF1 and the transsulfuration pathway in cancer. The transsulfuration pathway is important for production of cellular energy in the form of pyruvate, so overactive HSF1 likely increases cellular growth through increased pyruvate production. The transsulfuration pathways also produces the antioxidant hydrogen sulfide. The increased production of antioxidants likely aid cancer cells in responding to the accumulation of reactive oxygen species that form in cancer cells from oncogene activation, decreased blood supply, and tumor hypoxia. We have identified novel HSF1 binding sites in the gene encoding this transsulfuration pathway enzyme, which regulate levels of gene expression. Small cell neuroendocrine carcinoma cells treated with both the selective HSF1 inhibitor and the transsulfuration pathway inhibitor have reduced growth and increased cellular death by caspase 3 cleavage. Targeting the transsulfuration pathway in combination with HSF1 inhibition may delay recurrence in PCa patients by killing tumor cells instead of solely inducing cellular senescence. Since HSF1 is increased in many forms of cancer, these data may provide novel therapeutic targets for aggressive cancer from many tissue types. Citation Format: J. Spencer Hauck, Xia Gao, William Butler, Lingfan Xu, Jiaoti Huang. Targeting a metabolic compensatory mechanism for heat shock factor 1 inhibition 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 2372.

Abstract 1527: Developing a multifaceted approach to target androgen receptor (AR) in castration resistant prostate cancer

Abstract Androgen receptor (AR) signaling is also regulated epigenetically by histone-modifying enzymes (histone deacetylases-HDACs) that result in the development and progression of prostate cancer (CaP). It has been established that the inhibition of HDACs suppresses CaP proliferation by inhibiting AR signaling via multiple mechanisms, including AR degradation. Hence, the goal of this study was to develop small molecules that employ multiple mechanisms to inhibit AR expression and promote its degradation, which would effectively control the emergence and treatment of castration-resistant prostate cancer (CRPC). Several rationally designed antiandrogen equipped HDAC inhibitors (HDACis) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo models. Results demonstrated that these compounds' target (HDAC and AR) binding properties translated into potent anticancer activity against AR+ CRPC cell lines and to a lesser extent in AR- CRPC cell lines. In addition, among these compounds, we found that SB-146, a nontoxic agent, not only inhibited the growth of AR+ CRPC cells at nanomolar IC50s without sparing toxicity to healthy prostate epithelial cells. Molecular analysis confirmed SB-146 downregulated the expressions of both AR and AR-spice variants (AR-SV) in CRPC cells. Further analyses revealed downregulation of AR downstream (PSA, HOXB13 and FOXA1) targets as well as AR-SV’s (NUP210 and SLC3A2) in CRPC cells. SB-146 was also able to overcome DHT-induced AR and AR-SV signaling in C4-2B and 22Rv1 cells. Assessment of SB-146’s therapeutic efficacy on in vivo models revealed that oral administration of this compound abrogated AR+ and AR-SV (C4-2B and 22Rv1) tumor growth in xenotransplanted nude mice models. Finally, immunohistochemical analysis confirmed SB-146’s target of both AR and AR-SVs on xenografted tumor tissues. Together these results confirm that SB-146 is a potent agent that inhibits AR signaling in CRPC cells. Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Subhasish Tapadar, Bocheng Wu, Oyelere Adegboyega, Chendil Damodaran. Developing a multifaceted approach to target androgen receptor (AR) in castration resistant 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 1527.

Androgen receptor blockade promotes response to BRAF/MEK-targeted therapy.

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed1,2. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.

Abstract 1264: CTC-based biomarker analysis and correlation with clinical response to PSMA-TRT in mCRPC

Abstract Prostate specific membrane antigen (PSMA)-targeted radionuclide therapies (PSMA-TRTs) is one of the most promising treatments for metastatic castration-resistant prostate cancer (mCRPC) upon progression on androgen receptor signaling inhibitors (ARPi). However, recent studies show that only about half of patients respond to PSMA-TRTs. Expression of PSMA on cancer cells via PET-imaging is of some use as a predictive biomarker of response to PSMA-TRTs, but there are patients with strong PSMA imaging who do not respond and some with poor PSMA imaging that do respond. PSMA imaging does not fully capture response predictability. Currently, the lack of predictive biomarkers of response limits optimal selection of the patient population most likely to benefit from to PSMA-TRTs. PSMA-TRTs radionuclides emit α or β radiations, which induce DNA damage in targeted cells, followed by immunogenic cell death (ICD), evidenced by calreticulin (CRT) plasma membrane accumulation. To investigate the role of PSMA expression and CRT membrane localization as a biomarker of response to PSMA-TRT, we isolated circulating tumor cells (CTCs) from patients with mCRPC, enrolled in a phase I dose escalation trial of 225Ac-J591 (NCT03276572). Thirty-two (32) patients were enrolled (median age 69. 5, range 52-89; PSA 149.1, range 4.8-7168); 78% and 68% of patients received ≥ 2 prior ARPi treatments or chemotherapy, respectively. 69% and 44% of patients experienced any or &amp;gt;50% PSA decline, respectively, with a median PFS of 5.1 months and median OS of 11.1 months. CTCs were isolated before treatment (baseline) from 30/32 of enrolled patients, and 12 weeks after treatment initiation (on-treatment) from 21/32 patients. Enriched CTCs via CD45 depletion were subsequently immunostained for cytokeratin (CK, standard epithelial marker), CD45 (standard marker to identify contaminating leukocytes), PSMA, calreticulin (CRT) and DAPI. CTCs were subjected to multiplex confocal microscopy and identified as CK+/CD45-/DAPI+ cells. For each patient, at each time point, the following parameters are scored: i) total number of CTCs, ii) number of PSMA+ CTCs, iii) number of CRT+ CTCs, iv) number of CTCs with plasma membrane CRT decoration. Percent of PSMA+ CTC at baseline and changes in percentage of CRT+ CTCs and CTCs with plasma membrane CRT decoration between baseline and on treatment timepoints are correlated with clinical response and time to progression upon 225Ac-J591 treatment. Our preliminary results indicate higher expression of plasma membranous CRT in on-treatment CTCs in responders (PFS&amp;gt;12 months; n= 39 CTCs from 3 patients) than in non-responders (PFS&amp;lt;3 months; n= 20 CTCs from 2 patients). Full analysis of CRT and PSMA expression is currently being completed. Citation Format: Giuseppe Galletti, Prerna Vatsa, Joseph R. Osborne, Neil H. Bander, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. CTC-based biomarker analysis and correlation with clinical response to PSMA-TRT in mCRPC [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 1264.

Abstract 2650: Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer

Abstract Background: The androgen receptor (AR), which plays a major role in prostate cancer (PCa), consists of a C-terminal ligand-binding domain (LBD), a hinge domain, a DNA-binding domain (DBD), and an N-terminal domain (NTD). Current AR antagonists and AR signaling inhibitors (ASIs), used in androgen deprivation therapy (ADT) and to prevent disease progression, often fail due to production of alternatively spliced AR variants (AR-Vs) that lack the LBD but retain the DBD, which is essential to AR transcriptional activity. This project examines a series of novel compounds designed to inhibit AR-DNA interaction and study how these compounds affect castration-resistant PCa (CRPC). Methods: Compounds potentially targeting the DBD were used to predict direction and position of compounds when docked to the AR. MTT assays, flow cytometry, immunoblots, qRT-PCR, luciferase assay, and immunofluorescence were used to test compounds on AR and PCa. Drug Affinity Responsive Target Stability (DARTS) tested potential binding of the compounds to the AR. Studies in intact mice were used to determine toxicity of the compounds and the maximum tolerated dose. Results: Of 48 compounds synthesized, C15 had the highest and most consistent efficacy overall. C15 decreased metabolic activity in 22Rv1 and CWR-R1 cells that express AR-Vs and in C4 and C4-2 that do not express AR-Vs. Further, C15 had a reduced effect in AR-null PC3 cells and minimal effect in normal human dermal fibroblast (NHDF) cells. In C4-2 and 22Rv1, C15 significantly reduced PSA mRNA and AR transcriptional activity, and in C4-2, C15 significantly reduced TMPRSS2 and Nrdp1 mRNA levels. C08 and C15 reduced pronase-induced AR proteolysis only when the AR DBD is present. Furthermore, C15 reduces AR binding at the PSA enhancer, but DHT stimulation may weaken this binding inhibition and reduction in AR transcriptional activity. However, combining C15 with an AR inhibitor targeting the LBD (enzalutamide, darolutamide, apalutamide) restores the full inhibitory potential and even reduces metabolic activity and AR transcriptional activity further than either drug alone in both C4-2 and 22Rv1s. No toxicity in intact mice was observed with C15 with the highest dose tested being 50 mg/kg. Conclusion: Our preliminary data demonstrate that C15 directly binds to the DBD of the AR and potently inhibits CRPC cells, including those that express AR-Vs missing the LBD. Although increased DHT levels may weaken C15’s inhibition of the AR DBD, addition of a LBD-targeting AR inhibitor improves the inhibitory potential of both drugs. Based on these results, we conclude that C15 may hold great clinical potential as it successfully inhibits CRPC by targeting the AR DBD with minimal toxicity, and it can improve the effects of standard-of-care LBD-targeting AR inhibitors by reducing AR-V-induced resistance. Citation Format: Thomas M. Steele, Elisabeth A. Messner, Maria Malvina Tsamouri, Salma Siddiqui, Brian Bennion, Ruiwu Liu, Paramita Ghosh. Targeting the DNA-binding domain of the androgen receptor in castration-resistant 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 2650.