Enzalutamide-resistant Castration-resistant Prostate Cancer Research Articles

Moving Towards Precision Urologic Oncology: Targeting Enzalutamide-resistant Prostate Cancer and Mutated Forms of the Androgen Receptor Using the Novel Inhibitor Darolutamide (ODM-201)

Darolutamide (ODM-201) is a novel androgen receptor (AR) antagonist with a chemical structure distinctly different from currently approved AR antagonists that targets both wild-type and mutated ligand binding domain variants to inhibit AR nuclear translocation. Here, we evaluate the activity of darolutamide in enzalutamide-resistant castration resistant prostate cancer (CRPC) as well as in AR mutants detected in patients after treatment with enzalutamide, abiraterone, or bicalutamide. Darolutamide significantly inhibited cell growth and AR transcriptional activity in enzalutamide-resistant MR49F cells in vitro, and led to decreased tumor volume and serum prostate-specific antigen levels in vivo, prolonging survival in mice bearing enzalutamide-resistant MR49F xenografts. Moreover, darolutamide inhibited the transcriptional activity of AR mutants identified in the plasma of CRPC patients progressing on traditional therapies. In particular, darolutamide significantly inhibited the transcriptional activity of the F877L, H875Y/T878A, F877L/T878A, and the previously unreported T878G AR mutants, that transform enzalutamide into a partial agonist. In silico cheminformatics computer modeling provided atomic level insights confirming darolutamide antagonist effect in F877L and T878G AR mutants. In conclusion, our results provide a rationale for further clinical evaluation of darolutamide in enzalutamide-resistant CRPC, in particular in combination with circulating tumor DNA assays that detect AR mutants sensitive to darolutamide, in a precision oncology setting. Patient summaryIn this study we evaluated the novel drug darolutamide in preclinical models of prostate cancer. We found that darolutamide delays growth of enzalutamide-resistant prostate cancer, in particular in cells with mutated forms of the androgen receptor after previous treatment. Our data supports further evaluation of darolutamide in clinical trials.

BIRC6 Targeting as Potential Therapy for Advanced, Enzalutamide-Resistant Prostate Cancer.

Purpose: Enzalutamide resistance has emerged as a major problem in the management of castration-resistant prostate cancer (CRPC). Research on therapy resistance of CRPCs has primarily focused on the androgen receptor pathway. In contrast, there is limited information on antiapoptotic mechanisms that may facilitate the treatment resistance. The inhibitor of apoptosis proteins (IAP) family is well recognized for its role in promoting treatment resistance of cancers by inhibiting drug-induced apoptosis. Here, we examined whether BIRC6, an IAP family member, has a role in enzalutamide resistance of CRPCs and could provide a therapeutic target for enzalutamide-resistant CRPC.Experimental Design: Use of enzalutamide-resistant CRPC models: (i) the transplantable, first high-fidelity LTL-313BR patient-derived enzalutamide-resistant CRPC tissue xenograft line showing primary enzalutamide resistance, (ii) MR42D and MR49F CRPC cells/xenografts showing acquired enzalutamide resistance. Specific BIRC6 downregulation in these models was produced using a BIRC6-targeting antisense oligonucleotide (ASO-6w2). Gene expression was determined by qRT-PCR and gene expression profiling. Molecular pathways associated with growth inhibition were assessed via gene enrichment analysis.Results: Of eight IAPs examined, BIRC6 was the only one showing elevated expression in both enzalutamide-resistant CRPC models. Treatment with ASO-6w2 markedly suppressed growth of LTL-313BR xenografts and increased tumor apoptosis without inducing major host toxicity. Pathway enrichment analysis indicated that GPCR and matrisome signaling were the most significantly altered pathways. Furthermore, ASO-6w2 inhibited expression of prosurvival genes that were upregulated in the LTL-313BR line.Conclusions:BIRC6 targeting inhibited the growth of enzalutamide-resistant CRPC models and may represent a new option for clinical treatment of advanced, enzalutamide-resistant prostate cancer. Clin Cancer Res; 23(6); 1542-51. ©2016 AACR.

Abstract 4700: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer

Abstract Background: While treatment options for patients with castration-resistant prostate cancer (CRPC) are expanding, one American man is still predicted to die every 19 minutes from this disease this year. Moreover, more widespread use of novel and more potent AR-targeting agents has led to increased clinical frequency of virulent androgen and AR-independent CRPC subsets(Small, Huang et al. 2015). Currently, there are limited treatment options for men with CRPC who are resistant to these AR-targeting agents, clearly demonstrating an urgent need to develop more effective therapies for CRPC patients. Recent published reports demonstrate an important role for BET bromodomain chromatin reader proteins in prostate cancer models that are androgen or AR-dependent (Gao, Schwartzman et al. 2013; Wyce, Degenhardt et al. 2013; Asangani, Dommeti et al. 2014; Chan, Selth et al. 2015). However, there was limited information about the anti-tumor activity of this class of drugs in androgen-independent, enzalutamide-resistant, or AR-independent CRPC models. The studies reported herein were designed to address that deficit. Methods: We treated a panel of CRPC cell lines with dose escalation of the BET bromodomain inhibitor JQ1 and measured cell viability. To clarify molecular mechanisms that contribute to the anti-tumor effect, we treated CRPC cell lines with JQ1 and measured gene expression changes with RNA-sequencing. Finally, to determine the anti-tumor activity of JQ1 in vivo, we implanted enzalutamide resistant or AR-null CRPC xenografts in immunocompromised mice and treated them with JQ1. Results: All cell lines were sensitive to JQ1 with similar GI50 values (all < 1μM). Our analysis of RNA-seq data after JQ1 treatment identified a BET bromodomain response signature that was conserved across the population of cell lines. Further, we applied Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to identify novel Master Regulator transcription factors that mediate response to JQ1(Aytes, Mitrofanova et al. 2014). Finally, JQ1 treatment of enzalutamide resistant or AR-null CRPC xenografts implanted in vivo suppressed tumor growth without appreciable toxicity. Conclusions: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal CRPC. Our work using a broad panel of CRPC models sheds further light on pharmacodynamic markers of response and target proteins whose function is impacted by BET bromodomain inhibition. These findings may have implications for the design of BET bromodomain inhibitor clinical trials in men with CRPC and interpretation of on-target effects in those trials. Citation Format: Lina Gao, Daniel J. Coleman, Carly J. King, Jacob Schwartzman, Nicholas Wang, Amanda Esch, Joshua Urrutia, Archana Sehrawat, Laura M. Heiser, Joshi J. Alumkal. BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4700.

Abstract A150: Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling pathways

Abstract Background: Enzalutamide (ENZ) is the latest androgen receptor antagonist approved for treating castration-resistant prostate cancer (CRPC) and improves patient survival. However, increasing reports showing the development of ENZ-resistant tumor in patients and no known therapies were shown to be effective to-date. Our group has identified that BIRC6, an Inhibitor of Apoptosis (IAP) member, was elevated in CRPC. We developed antisense oligonucleotides (ASOs) that specifically target BIRC6 and demonstrated a significant inhibitory effect towards various CRPC models in vitro and in vivo. Most recently, we observed that BIRC6 expression was also elevated in an ENZ-resistant CRPC patient-derived xenograft (PDX) model. Thus, we hypothesize that anti-BIRC6 ASO may inhibit Enz-resistant CRPC. Materials and Methods: PDX model LTL313BR was a castration relapsed tumor line developed after androgen ablation in animal bearing LTL313B, a patient derived prostate cancer tumor line (androgen sensitive, AR+). LTL313BR was a typical CRPC (adenocarcinoma, AR+) and was resistant to Bicalutamide and Enzalutamide. The model was used to study the effect of anti-BIRC6 ASO on the growth of ENZ-resistant CRPC. Mice bearing LTL313BR tumors were randomized into scrambled control (Scrb) ASO or anti-BIRC6 ASO groups for 21-day treatment (n = 30 per group). Tumors were harvested 1 week after the end of treatment. Tumor volume and serum PSA was measured and the effect on tumor apoptosis was examined. Gene expression profiling was performed to investigate the mechanism of action of anti-BIRC6 ASO. Results: We demonstrated that anti-BIRC6 ASO significantly impeded the growth of Enz- resistant CRPC LTL313BR. Anti-BIRC6 ASO treated group showed median tumor volume of 385mm3 comparing to 521 mm3 in control ASO group, i.e. a 37% reduction. The marked tumor suppression was also coupled with significant reduction in serum PSA and induction of tumor apoptosis. Pathway enrichment analysis of gene expression profile indicates that anti-BIRC6 ASO altered gene expressions that inhibit pathways in mitogenic signalling, proliferation, cell migration, neutrophils chemotaxis and increased T-cell recruitment. Conclusion: Current study provides proof-of-principle data that anti-BIRC6 ASO may represent as novel therapeutic agent against ENZ-resistant CRPC. Citation Format: Iris Sze Ue Luk, Raunak Shrestha, Hui Xue, Yuwei Wang, Peter Gout, Colin Collins, Martin Gleave, Yuzhuo Wang. Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A150.

SiRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer.

Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model. Gene silencing of a luciferase reporter gene in the PC-3M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models. LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. LNP CLU-siRNA potently suppressed CLU and AR ASO-induced CLU and AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared with AR-ASO monotherapy. In subcutaneous ENZ-R LNCaP xenografts, combinatory treatment of LNP CLU-siRNA plus AR-ASO significantly suppressed tumor growth and serum PSA levels compared with LNP LUC-siRNA (control) and AR-ASO. LNP siRNA can silence target genes in vivo and enable inhibition of traditionally non-druggable genes like CLU and other promising cotargeting approaches in ENZ-R CRPC therapeutics.

Abstract LB-017: Minnelide reduces castration-resistant and enzalutamide-resistant prostate cancer via downregulation of androgen receptor-mediated signaling

Abstract Prostate cancer is the second leading cause of cancer death in men in western countries. Advanced prostate cancer is often resistant to hormonal treatment and systemic chemotherapy has limited efficacy. Androgen receptor (AR), a ligand dependent transcription factor plays pivotal role in the development and progression of prostate cancer. While majority of prostate cancers are initially androgen dependent and respond to androgen ablation therapy, most patients eventually recur with more aggressive castration-resistant prostate cancer (CRPC) where AR signaling is reactivated even in the absence of androgen stimulation. Therefore developing novel chemotherapeutic agents for castrate resistant prostate cancer (CRPC) treatment is critical to improve survival in men with CRPC. Triptolide, a diterpene triepoxide isolated from a chinese herb, is extremely effective against several cancers like pancreatic cancer, colorectal cancer and liver cancer both in vivo and in vitro. The water-soluble pro-drug of triptolide, Minnelide, downregulates HSP70 via inhibition of the activity of transcription factor Sp1. Since both Sp1 and HSP70 have been reported to be critical in functionality of AR, we assessed therapeutic potential of Minnelide on androgen dependent, CRPC in vitro and in vivo. Triptolide treatment resulted in dose- and time-dependent cell death in an androgen dependent cell line LNCaP, CRPC cell line C4-2 and enzalutamide resistant CRPC tumor cell line 22RV1. Triptolide treatment decreased expression of AR full length, AR splice variants and its downstream targets (PSA, NKX3.1) at the mRNA and protein levels. Further, reporter assay with AR responsive elements showed that triptolide decreased transcriptional activity of AR. Expression levels of Sp1 and HSP70 were also reduced following treatment with triptolide these cell lines. To test the efficacy of Minnelide in vivo, male athymic nude mice were castrated 7 days prior to implantation of enzalutamide resistant CRPC (22RV1) cells subcutaneously. The animals received daily intraperitoneal injection of Minnelide and tumor volume was measured weekly until tumor size reached 2cm3.Mice receiving daily injection of Minnelide had significantly smaller tumors than controls as early as two week of treatment (p = 0.008). Triptolide therapy inhibited enzalutamide resistant CRPC growth both in vitro and in vivo. Further, our studies for the first time showed that triptolide induces prostate tumor cell death by reducing expression of both full length AR and AR splice variants in a similar manner. Citation Format: Sumit Isharwal, Shrey Modi, Usman Barlass, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee, Badrinath Konety. Minnelide reduces castration-resistant and enzalutamide-resistant prostate cancer via downregulation of androgen receptor-mediated signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-017. doi:10.1158/1538-7445.AM2015-LB-017

MP46-15 LIPID NANOPARTICLE SIRNA POTENTLY SILENCES CLUSTERIN AND DELAYS PROGRESSION WHEN COMBINED WITH ANDROGEN RECEPTOR CO-TARGETING IN ENZALUTAMIDE RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2015MP46-15 LIPID NANOPARTICLE SIRNA POTENTLY SILENCES CLUSTERIN AND DELAYS PROGRESSION WHEN COMBINED WITH ANDROGEN RECEPTOR CO-TARGETING IN ENZALUTAMIDE RESISTANT PROSTATE CANCER Yoshiaki Yamamoto, Paulo Lin, Fan Zhang, Eliana Beraldi, Yoshihisa Kawai, Jeffrey Leong, Hideyasu Matsuyama, Pieter Cullis, and Martin Gleave Yoshiaki YamamotoYoshiaki Yamamoto More articles by this author , Paulo LinPaulo Lin More articles by this author , Fan ZhangFan Zhang More articles by this author , Eliana BeraldiEliana Beraldi More articles by this author , Yoshihisa KawaiYoshihisa Kawai More articles by this author , Jeffrey LeongJeffrey Leong More articles by this author , Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author , Pieter CullisPieter Cullis More articles by this author , and Martin GleaveMartin Gleave More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1578AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Suppression of androgen receptor (AR) signaling remains a therapeutic goal for castration resistant prostate cancer (CRPC) and despite newer potent AR pathway inhibitors, resistance frequently occurs. While co-targeting the AR with adaptive survival pathways is a rational goal, many biologically relevant genes including clusterin (CLU) are not druggable with small molecule inhibitors. While small interfering RNA (siRNA) offers the promise for very potent and specific gene silencing, in vivo delivery remains a major barrier. The most clinically advanced delivery of siRNA employs lipid nanoparticles (LNP). METHODS To improve siRNA delivery to tumors, we developed a LNP system and show that LNP-LUC siRNA silences its luciferase reporter gene in both subcutaneous and metatastic PC3-Luc xenograft models by the IVIS imaging system. Upon validation, the effect of LNP siRNA targeting CLU was used in combination with AR antisense oligonucleotides (ASO) in an enzalutamide-resistant (ENZ-R) CRPC LNCaP in vitro and in vivo models. RESULTS LNP CLU-siRNA inhibited AR-ASO induced up-regulation of CLU in vitro and in vivo, and significantly suppressed tumor growth and serum PSA levels in ENZ-R LNCaP xenografts compared to AR-ASO monotherapy. Interestingly, we revealed a stress-induced feed-forward loop involving AR-ASO-induced AKT and p90rsk, phosphoactivation of YB-1, and increased expression of CLU. CLU knockdown abrogates AR-ASO-induced phosphorylation of YB-1, AKT, and p90Rsk. CONCLUSIONS These data provide novel proof-of-principle that LNP siRNA can silence target genes in vivo and enable inhibiting traditionally non-druggable genes like CLU and other promising co-targeting approaches in ENZ-R CRPC therapeutics. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e549 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshiaki Yamamoto More articles by this author Paulo Lin More articles by this author Fan Zhang More articles by this author Eliana Beraldi More articles by this author Yoshihisa Kawai More articles by this author Jeffrey Leong More articles by this author Hideyasu Matsuyama More articles by this author Pieter Cullis More articles by this author Martin Gleave More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of lipid nanoparticle siRNA delivery systems on silencing clusterin and progression in enzalutamide resistant prostate cancer in vivo.

256 Background: Clusterin (CLU) is induced by androgen receptor (AR) pathway inhibition and its overexpression confers treatment resistance. Lipid nanoparticle (LNP) facilitates tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), currently with multiple products undergoing clinical evaluation. Gene silencing using small interfering RNA (siRNA) is a promising approach but in vivo delivery remains a major barrier. In our study, we investigated the efficacy siRNA tumor delivery using LNP systems in enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model. Methods: To validate the effect of LNP siRNA tumor delivery in vivo, gene silencing of a reporter gene, luciferase (LUC), in PC3-M-luc stable cell line was treated with LNP LUC-siRNA and examined for Luc activity by the IVIS imaging system. Next, we investigated the efficacy of LNP CLU-siRNA tumor delivery and LNP CLU-siRNA sensitized AR knockdown activity in ENZ-R CRPC LNCaP in vitro and in vivo models. Results: LNP LUC-siRNA exhibited LUC silencing effects in PC-3M-luc xenograft and metastatic models. LNP CLU-siRNA suppressed PSA and decreased AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro. LNP CLU-siRNA sensitized AR antisense oligonucleotides (ASO) activity, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared to AR-ASO monotherapy. In vivo,combinatory treatment of LNP CLU-siRNA and AR-ASO significantly suppressed tumor growth and serum PSA levels compared to LNP LUC -siRNA (control) and AR-ASO in ENZ-R LNCaP xenografts. Conclusions: The LNP CLU-siRNA systems sensitized AR knockdown resulting in inhibition of ENZ-R LNCaP cell growth, providing pre-clinical proof-of-principle as a promising AR co-targeting approach in ENZ-R CRPC.

Targeting the Androgen Receptor Confers In Vivo Cross-resistance Between Enzalutamide and Docetaxel, But Not Cabazitaxel, in Castration-resistant Prostate Cancer

Treatment options for metastatic castration-resistant prostate cancer (CRPC) have evolved with the established benefit of the novel androgen receptor (AR)-targeted agents abiraterone and enzalutamide in the prechemotherapy setting. However, concerns regarding cross-resistance between the taxanes docetaxel and cabazitaxel and these AR-targeted agents have arisen, and the optimal drug treatment sequence is unknown. We investigated the in vivo efficacy of docetaxel and cabazitaxel in enzalutamide-resistant CRPC, and mechanisms of cross-resistance between these agents. Castrated mice harboring enzalutamide-resistant tumors and enzalutamide-naïve tumors were treated with docetaxel and cabazitaxel. Tumor growth kinetics, AR nuclear localization, AR-regulated gene expression, Ki67 expression, and serum levels of prostate-specific antigen, docetaxel, and cabazitaxel were analyzed. Docetaxel inhibited tumor growth, AR nuclear localization, and AR-regulated gene expression in enzalutamide-naïve tumors, but did not in enzalutamide-resistant tumors, demonstrating in vivo cross-resistance. By contrast, cabazitaxel remained highly effective in enzalutamide-resistant tumors and demonstrated superior antitumor activity compared to docetaxel, independent of the AR pathway. These findings demonstrate that the AR pathway is able to confer in vivo cross-resistance between enzalutamide and docetaxel, but not cabazitaxel, in CRPC. Patient summaryWe found reduced efficacy of docetaxel, but not cabazitaxel, in enzalutamide-resistant prostate cancer.