Castration-resistant Prostate Cancer Models Research Articles

Voluntary Exercise Attenuates Tumor Growth in a Preclinical Model of Castration-Resistant Prostate Cancer.

To examine the effects of voluntary exercise training on tumor growth and explore the underlying intratumoral molecular pathways and processes responsible for the beneficial effects of VWR on tumor initiation and progression in a mouse model of Castration-Resistant Prostate Cancer (CRPC). Male immunodeficient mice (SCID) were castrated and subcutaneously inoculated with human CWR-22RV1 cancer cells to construct CRPC xenograft model before randomly assigned to either voluntary wheel running (VWR) or sedentary (SED) group (n=6/group). After three weeks, tumor tissues were collected. Tumor size was measured and calculated. mRNA expression of markers of DNA replication, Androgen Receptor (AR) signaling, and mitochondrial dynamics was determined by RT-PCR. Protein expression of mitochondrial content and dynamics was determined by western blotting. Finally, RNA-sequencing analysis was performed in the tumor tissues. Voluntary wheel running resulted in smaller tumor volume at the initial stage and attenuated tumor progression throughout the time course (P < 0.05). The reduction of tumor volume in VWR group was coincided with lower mRNA expression of DNA replication markers ( MCM2 , MCM6 , and MCM7 ), AR signaling ( ELOVL5 and FKBP5 ) and regulatory proteins of mitochondrial fission (Drp1 and Fis1) and fusion (MFN1 and OPA1) when compared to the SED group (P<0.05). More importantly, RNA sequencing data further revealed that pathways related to pathways related to angiogenesis, extracellular matrix formation and endothelial cell proliferation were downregulated. Three weeks of VWR was effective in delaying tumor initiation and progression, which coincided with reduced transcription of DNA replication, AR signaling targets and mitochondrial dynamics. We further identified reduced molecular pathways/processes related to angiogenesis that may be responsible for the delayed tumor initiation and progression by VWR.

B-255 Identification of the protein expression of Androgen Receptor variants using Targeted proteomics in clinical Castration Resistant Prostate Cancer models

Abstract Background Castration Resistant Prostate Cancer (CRPC) is a treatment resistant form of prostate cancer (PCa). Currently, there is not a way to identify which patients will develop this resistance before full blown CRPC develops. Therefore, all PCa treatment approaches are similar yet this results in tumor regression in some cases and progression in others. Targeting the androgen receptor (AR) is still the main focus of current therapies even in CRPC. Emergence of AR splice variants (AR-Vs) after initial treatment is thought to be one of the primary mechanisms of resistance. AR-Vs lack the ligand binding domain rendering Androgen Deprivation Therapy (ADT) ineffective in tumors expressing these variants. Recent work has identified the DNA and RNA species of ARVs in CRPC but investigation into whether the protein is translated are unknown. One exception is the approval of an antibody test that detects a specific AR-v, AR-v7, from the blood of PC patients and predicts ADT response. However, there are instances where a patient may not express ARv7 and are still resistant to ADT. This may suggest that other AR-Vs, not currently detected at the protein level, are important predictors for ADT response. Methods The primary goal of this work is to expand the protein identification of known and unknown AR-Vs that may predict response to ADT using targeted liquid chromatography tandem mass spectrometry (LC-MS/MS). We developed the AR-V targeted LC-MS/MS assay by first designing AR splice variants from unique splice regions via RNA-guided sequences translated to amino acid sequences. The AR-variants full amino acid sequences were then in-silico trypsin digested using Expasy software tool that generated tryptic peptides. Blast analysis of the AR-V peptides was performed where no overlap with other human proteins was observed. These AR-V peptide sequences were commercially synthesized along with heavy isotopically analogs to be used as internal standards in the LC-MS/MS method. The method examined 9 peptides and included 2 to 3 MRM transitions per peptide and calibration curves were constructed using unlabeled peptides. Specimens examined consisted of a panel of PCa cell lines (n= 6), CRPC patient derived xenografts tumors (PDX) (n=4) and a negative control cell line (no androgen receptor expression), Cos-1, which served with a dual function and was used as matrix for peptide MRM optimization experiments. Linearity and sensitivity for each peptide included in the method was examined using calibration curves. Results The targeted MRM approached was successfully achieved for the quantification of 9 peptides from 8 proteins in 6 PCa cell lines and 4 PDX tumor specimens. Out of the 8 proteins evaluated, we observed 3 known targets (AR-exon1, AR-V7 and AR-V12) and 4 novel AR-Vs in 22Rv1 PCa cell lines. Additionally, CRPC PDX were assessed where 3 novel AR-Vs were detected. Conclusions These preliminary results using a LC-MS/MS platform show promising identification and quantification of novel AR-Vs that have not been measured before at the protein level. Subsequent analysis of larger cohorts will further elucidate AR-Vs role in PCa treatment resistance mechanisms and possibly assist in future treatment approaches.

209 (PB197): Novel tumor microenvironment ameliorator E7130 shows substantial combination efficacy with enzalutamide in a preclinical model of castration-resistant prostate cancer.

209 (PB197): Novel tumor microenvironment ameliorator E7130 shows substantial combination efficacy with enzalutamide in a preclinical model of castration-resistant prostate cancer.

Defining splicing factor requirements for androgen receptor variant synthesis in advanced prostate cancer.

Resistance to androgen receptor (AR)-targeted therapies represent a major challenge in prostate cancer (PC). A key mechanism of treatment resistance in patients who progress to castrate-resistant PC (CRPC) is the generation of alternatively spliced androgen receptor variants (AR-Vs). Unlike full-length AR (FL-AR) isoforms, AR-Vs are constitutively active and refractory to current receptor-targeting agents hence drive tumour progression. Identifying regulators of AR-V synthesis may therefore provide new therapeutic opportunities in combination with conventional AR-targeting agents. Our understanding of AR transcript splicing, and the factors that control the synthesis of AR-Vs, remains limited. While candidate-based approaches have identified a small number of AR-V splicing regulators, an unbiased analysis of splicing factors important for AR-V generation is required to fill an important knowledge gap and furnish the field with novel and tractable targets for PC treatment. To that end, we conducted a bespoke CRISPR screen to profile splicing factor requirements for AR-V synthesis. MFAP1 and CWC22 were shown to be required for the generation of AR-V mRNA transcripts and their depletion resulted in reduced AR-V protein abundance and cell proliferation in several CRPC models. Global transcriptomic analysis of MFAP1-depleted cells revealed both AR-dependent and -independent transcriptional impact, including genes associated with DDR. As such, MFAP1 downregulation sensitised PC cells to ionising radiation suggesting therapeutically targeting AR-V splicing could provide novel cellular vulnerabilities which can be exploited in CRPC. Implications: We have utilised a CRISPR screening approach to identify key regulators of pathogenic AR splicing in prostate cancer.

Akkermansia muciniphila Metabolite Inosine Inhibits Castration Resistance in Prostate Cancer.

Prostate cancer (PCa) is initially sensitive to androgen deprivation therapy (ADT) but ultimately develops resistance and progresses to castration-resistant prostate cancer (CRPC) with a poor prognosis. This study indicated that some PCa patients and mice were more sensitive to ADT and entered CRPC later, which was related to the gut microbiota, especially the enrichment of Akkermansia muciniphila (AKK). Untargeted metabolomics analysis found that serum inosine level was upregulated in the treatment-sensitive group and significantly correlated with AKK. Furthermore, we revealed that intestinal permeability and serum lipopolysaccharide (LPS) levels increased in treatment-resistant mice. LPS stimulated the upregulation of p-NF-κB p65 and AR in tumors. Supplementing AKK metabolite inosine could alleviate intestinal barrier damage and reduce serum LPS level, ultimately inhibiting castration resistance via the LPS/NF-κB/AR axis. Finally, we constructed a predictive model for CRPC combining gut microbiota and clinical information (AUC = 0.729). This study revealed the potential mechanism of gut microbiota on CRPC and provided potential therapeutic targets and prognostic indicators.

Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

Castration-resistant prostate cancer (CRPC) is a frequently occurring disease with adverse clinical outcomes and limited therapeutic options. Here, we identify methionine adenosyltransferase 2a (MAT2A) as a critical driver of the androgen-indifferent state in ERG fusion-positive CRPC. MAT2A is upregulated in CRPC and cooperates with ERG in promoting cell plasticity, stemness and tumorigenesis. RNA, ATAC and ChIP-sequencing coupled with histone post-translational modification analysis by mass spectrometry show that MAT2A broadly impacts the transcriptional and epigenetic landscape. MAT2A enhances H3K4me2 at multiple genomic sites, promoting the expression of pro-tumorigenic non-canonical AR target genes. Genetic and pharmacological inhibition of MAT2A reverses the transcriptional and epigenetic remodeling in CRPC models and improves the response to AR and EZH2 inhibitors. These data reveal a role of MAT2A in epigenetic reprogramming and provide a proof of concept for testing MAT2A inhibitors in CRPC patients to improve clinical responses and prevent treatment resistance.

A phase 1, first-in-human, open-label study evaluating the safety, tolerability, pharmacokinetics, and efficacy of TT125-802 in patients with advanced solid tumors.

TPS3171 Background: TT125-802 is a selective, potent, orally bioavailable small molecule inhibitor of the bromodomain of CBP and p300, two highly homologues lysine acetyltransferases. CBP/p300 act as transcriptional co-activators with multiple domains and functions, including well-documented roles in cell cycle regulation and several critical tumorigenic pathways (1-3). TT125-802 has shown mono-therapy efficacy in preclinical models of castration-resistant prostate cancer, KRAS-mutated colorectal and non-small cell lung cancer (4,5). Furthermore, TT125-802 is capable to interfere with transcriptional resistance mechanisms limiting targeted therapies (6) which will be explored clinically upon determination of a safe, well-tolerated, and biologically active single-agent dose. Methods: This study aims to establish the safety, tolerability, and pharmacokinetic profile of TT125-802. The maximum tolerated dose/recommended dose for expansion (MTD/RDE) of repeat daily dosing for TT125-802 monotherapy will be determined in subjects with advanced solid tumors in a sequential escalating cohorts design, (1 cycle = 21 days) using the Bayesian Logistics Regression Model (BLRM) with sentinel patients (2-day window for first patient enrolled in each cohort). Cohorts will consist of 3 to 6 subjects who will be followed over a dose-limiting toxicity (DLT) period of at least 21 days. TT125-802 will be administered orally once daily; additional schedules may be evaluated. Dose escalation is planned to follow two-fold increments. It may be adapted upon dose level revision and guided by pharmacokinetic analysis. Additional subjects (up to 6) may be enrolled in at least two monotherapy cohorts deemed safe and tolerable to optimize the recommended phase 2 dose (RP2D). Up to a total of 20 additional subjects may be enrolled. Translational/pharmacodynamic endpoints: Exploratory endpoints will provide proof-of-principle and early biomarker data for CBP/p300 inhibition. These analyses will include: 1. Single-cell gene expression evaluation of peripheral blood mononuclear cells (PBMCs) at multiple time points, 2. anagen hair bulb bulk RNAseq analysis, and 3. paired tumor biopsies will be analyzed with single-cell RNA sequencing. Together, these investigations are expected to support optimal dose determination. Additionally, circulating tumor DNA might be evaluated to define molecular responses. Outlook: The recommended phase 2 dose will be used further to explore the efficacy of TT125-802 as monotherapy. TT125-802 will be evaluated with targeted therapies (for example, KRASG12C/D, BRAF, EGFR, etc., inhibitors) to exploit the blocking of transcriptional escape mechanisms driven by CBP/p300. 1. Iyer, 2007. 2. Dutta, 2016. 3. Attar, 2017. 4. AACR2023 posters #6268. 5. AACR poster #3907. 6. AACR2023 poster #3907. Clinical trial information: 2022-500849-24-00 .

P300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer.

Prostate cancer is an exemplar of an enhancer-binding transcription factor-driven disease. The androgen receptor (AR) enhanceosome complex comprised of chromatin and epigenetic coregulators assembles at enhancer elements to drive disease progression. The paralog lysine acetyltransferases p300 and CBP deposit histone marks that are associated with enhancer activation. Here, we demonstrate that p300/CBP are determinant cofactors of the active AR enhanceosome in prostate cancer. Histone H2B N-terminus multisite lysine acetylation (H2BNTac), which is exclusively reliant on p300/CBP catalytic function, marked active enhancers and was notably elevated in prostate cancer lesions relative to the adjacent benign epithelia. Degradation of p300/CBP rapidly depleted acetylation marks associated with the active AR enhanceosome, which was only partially phenocopied by inhibition of their reader bromodomains. Notably, H2BNTac was effectively abrogated only upon p300/CBP degradation, which led to a stronger suppression of p300/CBP-dependent oncogenic gene programs relative to bromodomain inhibition or the inhibition of its catalytic domain. In vivo experiments using an orally active p300/CBP proteolysis targeting chimera (PROTAC) degrader (CBPD-409) showed that p300/CBP degradation potently inhibited tumor growth in preclinical models of castration-resistant prostate cancer and synergized with AR antagonists. While mouse p300/CBP orthologs were effectively degraded in host tissues, prolonged treatment with the PROTAC degrader was well tolerated with no significant signs of toxicity. Taken together, our study highlights the pivotal role of p300/CBP in maintaining the active AR enhanceosome and demonstrates how target degradation may have functionally distinct effects relative to target inhibition, thus supporting the development of p300/CBP degraders for the treatment of advanced prostate cancer.

Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor

BackgroundProstate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation.MethodsIn this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database.ResultsGenome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC.ConclusionsOur findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Development and Characterisation of a New Patient-Derived Xenograft Model of AR-Negative Metastatic Castration-Resistant Prostate Cancer

As the treatment landscape for prostate cancer gradually evolves, the frequency of treatment-induced neuroendocrine prostate cancer (NEPC) and double-negative prostate cancer (DNPC) that is deficient for androgen receptor (AR) and neuroendocrine (NE) markers has increased. These prostate cancer subtypes are typically refractory to AR-directed therapies and exhibit poor clinical outcomes. Only a small range of NEPC/DNPC models exist, limiting our molecular understanding of this disease and hindering our ability to perform preclinical trials exploring novel therapies to treat NEPC/DNPC that are urgently needed in the clinic. Here, we report the development of the CU-PC01 PDX model that represents AR-negative mCRPC with PTEN/RB/PSMA loss and CTNN1B/TP53/BRCA2 genetic variants. The CU-PC01 model lacks classic NE markers, with only focal and/or weak expression of chromogranin A, INSM1 and CD56. Collectively, these findings are most consistent with a DNPC phenotype. Ex vivo and in vivo preclinical studies revealed that CU-PC01 PDX tumours are resistant to mCRPC standard-of-care treatments enzalutamide and docetaxel, mirroring the donor patient’s treatment response. Furthermore, short-term CU-PC01 tumour explant cultures indicate this model is initially sensitive to PARP inhibition with olaparib. Thus, the CU-PC01 PDX model provides a valuable opportunity to study AR-negative mCRPC biology and to discover new treatment avenues for this hard-to-treat disease.

Microwave hyperthermia enhances radiosensitization by decreasing DNA repair efficiency and inducing oxidative stress in PC3 prostatic adenocarcinoma cells

Purpose Radiotherapy (RT) is the primary treatment for prostate cancer (PCa); however, the emergence of castration-resistant prostate cancer (CRPC) often leads to treatment failure and cancer-related deaths. In this study, we aimed to explore the use of microwave hyperthermia (MW-HT) to sensitize PCa to RT and investigate the underlying molecular mechanisms. Methods We developed a dedicated MW-HT heating setup, created an in vitro and in vivo MW-HT + RT treatment model for CRPC. We evaluated PC3 cell proliferation using CCK-8, colony experiments, DAPI staining, comet assay and ROS detection method. We also monitored nude mouse models of PCa during treatment, measured tumor weight, and calculated the tumor inhibition rate. Western blotting was used to detect DNA damage repair protein expression in PC3 cells and transplanted tumors. Results Compared to control, PC3 cell survival and clone formation rates decreased in RT + MW-HT group, demonstrating significant increase in apoptosis, ROS levels, and DNA damage. Lower tumor volumes and weights were observed in treatment groups. Ki-67 expression level was reduced in all treatment groups, with significant decrease in RT + MW-HT groups. The most significant apoptosis induction was confirmed in RT + MW-HT group by TUNEL staining. Protein expression levels of DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways significantly decreased in RT + MW-HT groups. Conclusion MW-HT + RT treatment significantly inhibited DNA damage repair by downregulating DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways, leading to increased ROS levels, aggravate DNA damage, apoptosis, and necrosis in PC3 cells, a well-established model of CRPC.

Abstract LB217: Novel organoid models of lineage plasticity in castration resistant prostate cancer

Abstract The development of next-generation AR-targeted drugs, such as abiraterone and enzalutamide, has improved survival of patients with castration-resistant prostate cancer (CRPC). However, many patients eventually progress to therapy-resistant lethal prostate cancer. CRPC exhibits variant cellular states, including AR-pathway active prostate cancer (ARPC), AR activity-low prostate cancer (ARLPC), neuroendocrine prostate cancer (NEPC), amphicrine prostate cancer co-expressing AR and neuroendocrine genes (AMPC), and double-negative prostate cancer (DNPC) lacking both AR expression and neuroendocrine differentiation, indicating that lineage plasticity facilitates therapeutic resistance. While molecular characteristics of ARPC and NEPC are well described, other common forms of CRPC, such as DNPC which occurs in 20-25% of CRPC patients, remain poorly understood. Therefore, characterization of pre-clinical models of DNPC and identification of novel regulators of lineage plasticity in DNPC are urgent necessities for the development of effective treatment for lethal prostate cancer.In our studies, we have generated a new mouse model of CRPC in which Pten and Smad4 are deleted in distal luminal cells upon tamoxifen administration (Nkx3.1CreERT2/+; Ptenfl/fl; Smad4fl/fl; R26R-eYFP/+, NPS). We observed a molecular transition from ARPC to ARLPC and DNPC in the prostate tumors derived from NPS mice during tumor progression. Moreover, a combination of castration and enzalutamide treatment promoted a partial epithelial-mesenchymal transition (EMT) in DNPC. To identify the evolutionary trajectories of cancer progression and drivers of lineage plasticity in CRPC, we have established organoid lines from the prostate tumor cells of NPS mice. Single-cell RNA-sequencing (scRNA-seq) and marker validation analyses shown that these organoid lines display overt lineage plasticity, corresponding to ARPC, ARLPC, partial EMT-absent DNPC, and partial EMT-present DNPC. Orthotopic grafts derived from the established NPS organoid lines show consistent molecular phenotypes and confirm that AR-targeted therapies facilitate partial EMT in vivo. Furthermore, cross-species bioinformatic analyses using scRNA-seq datasets indicate that the established NPS organoid lines partially recapitulate the spectrum of lineage plasticity in human CRPC.In summary, we have established prostate cancer organoid lines from a novel mouse model of CRPC, which not only provide large and reproducible sources of two distinct forms of DNPC (partial EMT-absent DNPC and partial EMT-present DNPC) for molecular analyses, but also can capture the regulatory complexity of lineage plasticity in human CRPC. Therefore, NPS mice provide an excellent pre-clinical model of CRPC, especially DNPC, to identify novel regulators that drive lineage plasticity and to pursue assays for development of candidate drugs that can inhibit or even possibly convert therapy-resistant forms of CRPC to therapy-sensitive ones. Citation Format: Jinqiu Lu, Michael M. Shen. Novel organoid models of lineage plasticity in castration resistant prostate cancer [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 LB217.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Nuclear receptor NURR1 functions to promote stemness and epithelial-mesenchymal transition in prostate cancer via its targeting of Wnt/β-catenin signaling pathway

Dysregulated activation of Wnt/β-catenin signaling pathway is a frequent or common event during advanced progression of multiple cancers. With this signaling activation, it enhances their tumorigenic growth and facilitates metastasis and therapy resistance. Advances show that this signaling pathway can play dual regulatory roles in the control of cellular processes epithelial-mesenchymal transition (EMT) and cancer stemness in cancer progression. Aberrant activation of Wnt/β-catenin signaling pathway is shown to be common in prostate cancer and also castration-resistant prostate cancer (CRPC). However, the transcriptional regulators of this pathway in prostate cancer are still not well characterized. NURR1 (NR4A2) is an orphan nuclear receptor and plays an important role in the development of dopaminergic neurons. Previously, we have shown that NURR1 exhibits an upregulation in isolated prostate cancer stem-like cells (PCSCs) and a xenograft model of CRPC. In this study, we further confirmed that NURR1 exhibited an upregulation in prostate cancer and also enhanced expression in prostate cancer cell lines. Functional and molecular analyses showed that NURR1 could act to promote both in vitro (cancer stemness and EMT) and also in vivo oncogenic growth of prostate cancer cells (metastasis and castration resistance) via its direct transactivation of CTNNB1 (β-catenin) and activation of β-catenin to mediate the activation of Wnt/β-catenin signaling pathway. Moreover, we also demonstrated that NURR1 activity in prostate cancer cells could be modulated by small molecules, implicating that NURR1 could be a potential therapeutic target for advanced prostate cancer management.

Abstract 4750: The MNK inhibitor EB1 sensitizes AR-V7 positive castration-resistant prostate cancer to enzalutamide

Abstract Introduction: Resistance to androgen receptor signaling inhibitors (ARSI) like Enzalutamide is frequent in castration resistant prostate cancer (CRPC). The mechanisms driving drug resistance include the overexpression of androgen receptor (AR) and the presence of splice variants like androgen receptor splice variant 7 (AR-V7). Clinical studies (PROPHECY) have demonstrated that AR-V7 positive CRPC patients treated with Enzalutamide show poorer overall survival and thus lack an effective targeted line of treatment. Recently, AR signaling and the regulation of protein translation via the translation initiation factor 4E (eIF4E) and MAPK-interacting kinases (MNKs) have been shown to depend on each other and targeting eIF4E has been proposed to increase the efficacy of ARSI. Objectives: We have recently described EB1 as the first in class non-ATP competitive inhibitor of MNK1/2 (Bou-Petit et al. JMC, 2022) and have now tested its applicability in CRPC. In this work we describe the impact of EB1 on the efficacy of Enzalutamide treatment in CRPC. Methodology: Cell lines 22RV1 and LNCaP-95 were used as AR-V7 positive models of CRPC. Effects of EB1 and Enzalutamide alone and in combination on cell growth were studied through crystal violet assay. Drug combination Index was calculated following Chou-Talalay method using CompuSyn Software. Cell death was studied through propidium iodide (PI) staining. AR transcriptional activity was assessed through RT-qPCR of AR canonical target genes like PSA. A head-to-head study with eFT-508 (Tomivosertib, eFFECTOR Therapeutics, Inc.) as the most advanced Type I MNK inhibitor was performed. Lastly, spheroids obtained through centrifugation in ultra-low attachment plates were used for validating drug interaction in 3D growth and viability studies. Spheroid viability was assessed through PI and Calcein AM staining as well as MTT assay. Results: In vitro results demonstrate strong to moderate synergistic inhibition of cell growth through the induction of cell death by the combination of Enzalutamide and EB1 in AR-V7 positive CRPC cell lines, as well as reduction of AR transcriptional activity (PSA). Drug combination also reduced growth and viability of spheroids, indicating good tumor penetrating properties in 3D models and giving in vitro proof-of-concept for in vivo applications. Furthermore, drug synergy with Enzalutamide could only be observed with EB1 and not with Type-I MNK inhibitors like eFT-508, indicating that MNK1/2 functions beyond its kinase activity might be relevant for cancer progression. Conclusion: Here we demonstrate that the novel MNK inhibitor EB1 shows promising in vitro results, which cannot be obtained with Type-I MNK inhibitors. The combination of EB1 with the standard of care Enzalutamide provide a novel therapeutic opportunity for the treatment of CRPC, particularly in AR-V7 positive patients. Citation Format: Sara García-Ortega, Leticia Suárez-Cabrera, Joan Morote, Olga Méndez, Marta Cano-Galietero, Elena Muro-Blanc, Jose I. Borrell, Anna Santamaria, Santiago Ramón y Cajal, Stefan Hümmer. The MNK inhibitor EB1 sensitizes AR-V7 positive castration-resistant prostate cancer to enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4750.

Anticancer Activity of the Marine Triterpene Glycoside Cucumarioside A2-2 in Human Prostate Cancer Cells.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), treatment is inevitably hampered by the development of drug resistance. Thus, new drugs are urgently needed. We investigated the efficacy, toxicity, and mechanism of action of the marine triterpene glycoside cucumarioside A2-2 (CA2-2) using an in vitro CRPC model. CA2-2 induced a G2/M-phase cell cycle arrest in human prostate cancer PC-3 cells and caspase-dependent apoptosis executed via an intrinsic pathway. Additionally, the drug inhibited the formation and growth of CRPC cell colonies at low micromolar concentrations. A global proteome analysis performed using the 2D-PAGE technique, followed by MALDI-MS and bioinformatical evaluation, revealed alterations in the proteins involved in cellular processes such as metastatic potential, invasion, and apoptosis. Among others, the regulation of keratin 81, CrkII, IL-1β, and cathepsin B could be identified by our proteomics approach. The effects were validated on the protein level by a 2D Western blotting analysis. Our results demonstrate the promising anticancer activity of CA2-2 in a prostate cancer model and provide insights on the underlying mode of action.

Abstract A043: CDK8/19-regulated transcriptional reprogramming: a druggable driver of castration-resistant prostate cancer

Abstract Androgen deprivation therapy (ADT) is the mainstay of prostate cancer (PCa) treatment. However, most patients with aggressive PCa become resistant to ADT and develop castration-resistant prostate cancer (CRPC). Newer androgen receptor (AR)-targeting CRPC treatments extend survival by only a few months, and metastatic CRPC (mCRPC) remains an incurable disease. Multiple mechanisms of ADT resistance have been identified, among which non-genetic (transcriptional) mechanisms are prominent, due to the high transcriptomic plasticity of PCa and the role of tumor microenvironment (TME) as a determinant of ADT resistance. PCa progression involves changes in the expression of CDK8 and CDK19, enzymatic components of the cyclin-dependent kinase module (CKM), associated with the transcriptional Mediator complex and implicated in transcriptional reprogramming and immune surveillance. We have investigated the roles of CDK8/19 Mediator kinases in CRPC, through bioinformatic analysis of primary PCa and mCRPC and by analyzing the effects of genetic and pharmacological inhibition of CDK8 and CDK19 on tumor growth and gene expression. CDK8 and CDK19 were found to be differentially regulated by androgen, which downregulates CDK8 and upregulates CDK19. Accordingly, CDK8 is decreased and CDK19 increased in primary PCa, where CDK19 is expressed higher than in any other tissues or tumors. However, both CDK19 and CDK8, as well as the other components of the CKM, are upregulated in mCRPC, concordantly with the changes in AR-mediated transcriptional signaling. Selective Mediator kinase inhibitors did not suppress PCa models driven by canonical AR signaling but inhibited in vivo growth of cell line-based and patient-derived xenograft (PDX) models of CRPC. Genetic inactivation of CDK8 and CDK19 suppressed 22Rv1 CRPC tumors in surgically or chemically castrated (but not intact) male NSG mice, whereas the expression of CDK19 or CDK8 reversed this phenotype. Transcriptomic analysis revealed that Mediator kinase inhibition exerted the strongest effects on gene expression in tumors growing in castrated mice, increasing the transcriptomic effects of castration on most of the genes regulated by CDK8/19, while decreasing its effects on some of the genes implicated in PCa progression. A gene signature reflecting Mediator kinase activity in CRPC xenografts correlated with shorter overall survival among mCRPC patients. Systemic treatment with a CDK8/19 inhibitor also affected stromal gene expression; many of these effects were reproduced by Mediator kinase mutagenesis in tumor cells alone, suggesting that Mediator kinases of tumor cells are involved in shaping the TME. Prolonged (up to 300 days) CDK8/19 inhibition induced not only growth inhibition but also shrinkage of CRPC tumors in nude mice (which contain NK cells that are stimulated by CDK8/19 inhibition) and even produced cures in 18-27% of animals. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors for the treatment of the presently incurable CRPC. Citation Format: Jing Li, Thomas Hilimire, Yueying Liu, Lili Wang, Jiaxin Liang, Balazs Gyorffy, Vitali Sikitzhytski, Hao Ji, Li Zhang, Chen Cheng, Xiaokai Ding, Kendall Kerr, Charles Dowling, Gary Schools, Chang-Uk Lim, Eugenia Broude, George Wilding, Michael Lilly, Igor Roninson, Mengqian Chen. CDK8/19-regulated transcriptional reprogramming: a druggable driver of castration-resistant 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 A043.

Rational optimization of a transcription factor activation domain inhibitor

Transcription factors are among the most attractive therapeutic targets but are considered largely ‘undruggable’ in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.

Abstract A057: Small molecule microarray screening identifies novel androgen receptor ligands

Abstract The androgen receptor (AR) is a well-established oncogenic driver of prostate cancer, and AR-targeting hormone therapy has proven an efficacious treatment option for patients with metastatic prostate cancer. Due to the importance of AR in prostate cancer disease progression, we leveraged small molecule microarray (SMM) screening technology to identify compounds that bind to transcriptional complexes of a common AR splice-variant, AR-V7, present along with AR in castration-resistant prostate cancer cell lysates. A subset of SMM hits were identified to modulate the AR Transcription Regulatory Network (TRN) in hormone-sensitive, but not castration-resistant prostate cancer models using a functional cell-based transcription signature approach. Several AR SMM hits directly engaged the AR ligand binding domain in a biophysical assay. One of these AR SMM hits altered AR nuclear translocation and antagonized steroid receptor co-activator recruitment to AR but did not antagonize the glucocorticoid receptor. Leveraging structural data for known AR ligands, we propose a computational model for binding of this molecule to the known AR ligand binding domain. Together, the data demonstrates that the AR SMM lysate screen successfully identified structurally novel small molecules that modulate the AR TRN and bind the AR ligand binding domain. Citation Format: Marek J Kobylarz, Michelle G Shum, Wayne L Glore, Ava Saffren, Shannon Carpenter, Mulini Pingili, Andrew Clay, Tamara D Hopkins, Hua Gao, Anna C Schinzel, Marius S Pop, Nikolaus D Obholzer, David B Freeman, Christopher J Dinsmore, Benjamin W Trotter, Charles Y Lin, Peter B Rahl. Small molecule microarray screening identifies novel androgen receptor ligands [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 A057.