Castration-resistant Prostate Cancer Xenograft Model Research Articles

DNA aptamer-conjugated lipid nanoparticle for targeted PTEN mRNA delivery to prostate cancer cells

The use of messenger RNA (mRNA) as a cancer vaccine and gene therapy requires targeted vehicle delivery to the site of disease. Here, we designed a mRNA-encapsulating lipid nanoparticle (LNP) conjugated with anti-programmed death-ligand 1 (PD-L1) DNA aptamer that delivers mRNA encoding a tumor suppressor gene, namely phosphatase and tensin homolog (PTEN), to castration-resistant prostate cancer (CRPC) cells expressing PD-L1 on the cell surface. The DNA aptamer-conjugated LNP-based mRNA delivery system (Apt-LNP[PTEN mRNA]) mediated efficient mRNA delivery and transfection in CRPC cells than LNPs without targeting ligands. Cancer-targeted PTEN mRNA delivery using Apt-LNPs achieved significantly higher PTEN expression via aptamer-mediated endocytosis in target cancer cells compared with non-targeted LNP delivery, resulting in significant downregulation of AKT phosphorylation. This enhanced PI3K/AKT pathway regulation, and in turn reduced cell migration after two days along with a 70 % decrease in cell viability, leading to effective apoptotic cell death. In a CRPC xenograft model, Apt-LNP[PTEN mRNA] led to an approximate 60 % reduction in tumor growth, which was attributable to the effective PTEN restoration and PI3K/AKT signaling pathway regulation. PTEN expression was significantly enhanced in CRPC tumor tissues, which abolished cancer cell tumorigenicity. These findings demonstrated the potential of Apt-LNPs for targeted mRNA delivery to cancer cells, thus providing a promising tool for targeted mRNA delivery to a range of cancers and tissues using a conventional LNP systems.

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.

Newly developed preclinical models reveal broad-spectrum CDK inhibitors as potent drugs for CRPC exhibiting primary resistance to enzalutamide.

Androgen-deprivation therapy is a standard treatment for advanced prostate cancer. However, most patients eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). In this study, we established new CRPC cell lines, AILNCaP14 and AILNCaP15, from LNCaP cells under androgen-deprived conditions. Unlike most pre-existing CRPC cell lines, both cell lines expressed higher levels of androgen receptor (AR) and prostate-specific antigen (PSA) than parental LNCaP cells. Moreover, these cells exhibited primary resistance to enzalutamide. Since AR signaling plays a significant role in the development of CRPC, PSA promoter sequences fused with GFP were introduced into AILNCaP14 cells to conduct GFP fluorescence-based chemical screening. We identified flavopiridol, a broad-spectrum CDK inhibitor, as a candidate drug that could repress AR transactivation of CRPC cells, presumably through the inhibition of phosphorylation of AR on the serine 81 residue (pARSer81 ). Importantly, this broad-spectrum CDK inhibitor inhibited the proliferation of AILNCaP14 cells both in vitro and in vivo. Moreover, a newly developed liver metastatic model using AILNCaP15 cells revealed that the compound attenuated tumor growth of CRPC harboring highly metastatic properties. Finally, we developed a patient-derived xenograft (PDX) model of CRPC and DCaP CR from a patient presenting therapeutic resistance to enzalutamide, abiraterone, and docetaxel. Flavopiridol successfully suppressed the tumor growth of CRPC in this PDX model. Since ARSer81 was found to be phosphorylated in clinical CRPC samples, our data suggested that broad-spectrum CDK inhibitors might be a potent candidate drug for the treatment of CRPC, including those exhibiting primary resistance to enzalutamide.

Targeting radioresistance and replication fork stability in prostate cancer.

The bromodomain and extraterminal (BET) family of chromatin reader proteins bind to acetylated histones and regulate gene expression. The development of BET inhibitors (BETi) has expanded our knowledge of BET protein function beyond transcriptional regulation and has ushered several prostate cancer (PCa) clinical trials. However, BETi as a single agent is not associated with antitumor activity in patients with castration-resistant prostate cancer (CRPC). We hypothesized novel combinatorial strategies are likely to enhance the efficacy of BETi. By using PCa patient-derived explants and xenograft models, we show that BETi treatment enhanced the efficacy of radiation therapy (RT) and overcame radioresistance. Mechanistically, BETi potentiated the activity of RT by blocking DNA repair. We also report a synergistic relationship between BETi and topoisomerase I (TOP1) inhibitors (TOP1i). We show that the BETi OTX015 synergized with the new class of synthetic noncamptothecin TOP1i, LMP400 (indotecan), to block tumor growth in aggressive CRPC xenograft models. Mechanistically, BETi potentiated the antitumor activity of TOP1i by disrupting replication fork stability. Longitudinal analysis of patient tumors indicated that TOP1 transcript abundance increased as patients progressed from hormone-sensitive prostate cancer to CRPC. TOP1 was highly expressed in metastatic CRPC, and its expression correlated with the expression of BET family genes. These studies open new avenues for the rational combinatorial treatment of aggressive PCa.

Redox-responsive nanoparticles enhance radiation therapy by altering multifaceted radio-resistance mechanisms in human castration-resistant prostate cancer cells and xenografts

IntroductionRadiation therapy (RT) is a major modality for the treatment of prostate cancer (PCa), especially castration-resistant PCa (CRPC). However, hypoxia, often seen in PCa tumors, leads to radiation-resistance. This work investigates the effect of a novel oxygen-generating polymer-lipid manganese dioxide nanoparticle (PLMDs) on improving RT outcomes in CRPC xenograft models by modulating the tumor microenvironment (TME) both before and after RT. Materials and methodsHuman PC3 and DU145 PCa cells were used to investigate clonogenic inhibition and DNA repair pathways in vitro. Tumor hypoxia and post-RT angiogenesis were evaluated in a PC3-bearing SCID mouse model. PC3 and DU145 xenografts were used to study the efficacy of PLMD in combination with single or fractionated RT. ResultsPLMD plus RT significantly inhibited clonogenic potential, increased DNA double-strand breaks, and reduced DNA damage repair in hypoxic PC3 and DU145 cells as compared to RT alone. PLMD significantly reduced hypoxia-positive areas, hypoxia induced factor 1α (HIF-1α) expression, and protein carbonyl levels (a measure of oxidative stress). Application of PLMD with RT decreased RT-induced angiogenic biomarkers by up to 3-fold. Treatment of the human CRPC xenografts with PLMD plus RT (single or fractionated doses) significantly prolonged median survival of the host compared to RT alone resulting in up to a 40% curative rate. ConclusionPLMD treatment modulated TME and sensitized hypoxic human CRPC cells to RT thus enhancing the efficacy of RT. These results confirmed the potential of PLMD as an adjuvant to RT for the treatment of hypoxic CRPC.

ISL1 promotes enzalutamide resistance in castration-resistant prostate cancer (CRPC) through epithelial to mesenchymal transition (EMT)

Abnormal expression of insulin gene enhancer-binding protein 1 (ISL1) has been demonstrated to be closely associated with cancer development and progression in several cancers. However, little is known about ISL1 expression in metastatic castration-resistant prostate cancer (CRPC). ISL1 has also been recognized as a positive modulator of epithelial–mesenchymal transition (EMT). In this study, we focused on ISL1 which showed maximum upregulation at the mRNA level in the enzalutamide-resistant cell line. Accordingly, we found that ISL1 was overexpressed in enzalutamide-resistant C4-2B cells and its expression was significantly related to EMT. Our findings reveal the important role of ISL1 in androgen receptor (AR)-dependent prostate cancer cell growth; ISL1 knockdown reduced the AR activity and cell growth. ISL1 knockdown using small-interfering RNA inhibited AR, PSA, and EMT-related protein expression in C4-2B ENZR cells. In addition, knock-down ISL1 reduced the levels of AKT and p65 phosphorylation in C4-2B ENZR cells and these suggest that knock-down ISL1 suppresses EMT in part by targeting the AKT/NF-κB pathway. Further, ISL1 downregulation could effectively inhibit tumor growth in a human CRPC xenograft model. Together, the present study shows that downregulation of ISL1 expression is necessary for overcoming enzalutamide resistance and improving the survival of CRPC patients.

Toppling the HAT to Treat Lethal Prostate Cancer

In this issue of Cancer Discovery, Welti and colleagues demonstrate a positive correlation between the expression of the histone acetyltransferase paralogs CBP and p300 with increased androgen receptor (AR) signaling and androgen deprivation therapy resistance in advanced prostate cancer. CCS1477, a selective inhibitor of p300/CBP bromodomain, disrupts AR- and MYC-regulated gene expression, suppresses tumor growth in vivo in multiple castration-resistant prostate cancer xenograft models, and modulates biomarker expression in early clinical evaluation, providing a novel therapeutic approach for AR-addicted advanced prostate cancer.See related article by Welti et al., p. 1118.

Discovery of a novel AR/HDAC6 dual inhibitor for prostate cancer treatment.

Androgen receptor (AR) and histone deacetylase 6 (HDAC6) are important targets for cancer therapy. Given that both AR antagonists and HDAC6 inhibitors modulate AR signaling, a novel AR/HDAC6 dual inhibitor is investigated for its anticancer effects in castration-resistant prostate cancer (CRPC). Zeta55 inhibits nuclear translocation of AR and suppresses androgen-induced PSA and TMPRSS2 expression. Meanwhile, Zeta55 selectively inhibits HDAC6 activity, leading to AR degradation. Zeta55 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a CRPC xenograft model. These results provide preclinical proof of principle for Zeta55 as a promising therapeutic in prostate cancer treatment.

Regulation of CEACAM5 and Therapeutic Efficacy of an Anti-CEACAM5-SN38 Antibody-drug Conjugate in Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive form of castration-resistant prostate cancer (CRPC) for which effective therapies are lacking. We previously identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a promising NEPC cell surface antigen. Here we investigated the scope of CEACAM5 expression in end-stage prostate cancer, the basis for CEACAM5 enrichment in NEPC, and the therapeutic potential of the CEACAM5 antibody-drug conjugate labetuzumab govitecan in prostate cancer. The expression of CEACAM5 and other clinically relevant antigens was characterized by multiplex immunofluorescence of a tissue microarray comprising metastatic tumors from 34 lethal metastatic CRPC (mCRPC) cases. A genetically defined neuroendocrine transdifferentiation assay of prostate cancer was developed to evaluate mechanisms of CEACAM5 regulation in NEPC. The specificity and efficacy of labetuzumab govitecan was determined in CEACAM5+ prostate cancer cell lines and patient-derived xenografts models. CEACAM5 expression was enriched in NEPC compared with other mCRPC subtypes and minimally overlapped with prostate-specific membrane antigen, prostate stem cell antigen, and trophoblast cell surface antigen 2 expression. We focused on a correlation between the expression of the pioneer transcription factor ASCL1 and CEACAM5 to determine that ASCL1 can drive neuroendocrine reprogramming of prostate cancer which is associated with increased chromatin accessibility of the CEACAM5 core promoter and CEACAM5 expression. Labetuzumab govitecan induced DNA damage in CEACAM5+ prostate cancer cell lines and marked antitumor responses in CEACAM5+ CRPC xenograft models including chemotherapy-resistant NEPC. Our findings provide insights into the scope and regulation of CEACAM5 expression in prostate cancer and strong support for clinical studies of labetuzumab govitecan for NEPC.

Abstract 6356: Discovery of seleno allyl hybrid compounds, the Se incorporated mimics of naturally occurring garlic based allylic compounds, as potential anticancer agents

Abstract Many synthetic derivatives of natural products have been found to exhibit biological activity towards cancer. One prominent example of such natural products are the garlic-based compounds. Given the well stablished preventive effects shown by garlic and some of its allylic components and a well-established chemopreventive and therapeutic effects shown by selenium (Se) compounds, we pursued to design Se-allyl hybrid compounds in order to increase efficacy as compared to naturally occurring allylic compounds. A total of 18 new selenoallyl (SeAl) compounds were designed following two different synthetic approaches. SeAl functionality was chosen to mimic the garlic bioactive allylic components. All the synthesized compounds were screened against colorectal (HT29 and HCT116) and mammary (MCF-7) cancer cell lines (48h treatment). Four most active compounds identified were submitted to the NCI Developmental Therapeutic Program (DTP). Two out of the four compounds were selected for the dose-response assay against a panel of 60 cancer cell lines. Both the compounds exhibited impressive cytotoxicity - lethal dose 50 (LD50) values below 9.2 µM in most of the melanoma and ovarian cancer cell lines tested (8 out of 9 for melanoma and 5 out of 7 for ovarian cancer cells for both compounds). Additionally, the Se-allyl derivative bearing an aspirin substituent presented a significant tumor size inhibition in a castration-resistant prostate cancer xenograft model at a dose of 4 mg/kg given intraperitoneally. Thus, this compound has the potential to be a promising candidate for prostate cancer treatment and potentially other cancers. To conclude, we firmly believe, based on the evidences obtained and presented herein, that structural modifications involving the hybridization approach with Se and naturally occurring allylic compounds could stand as a novel toolkit for anticancer drug development. Citation Format: Irene Talavera, Nora Astrain, Miguel Barajas, Ignacio Encío, Arun K. Sharma, Carmen Sanmartín, Daniel Plano. Discovery of seleno allyl hybrid compounds, the Se incorporated mimics of naturally occurring garlic based allylic compounds, as potential anticancer agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6356.

Imaging assisted evaluation of antitumor efficacy of a new histone deacetylase inhibitor in the castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is the most common cause of death in men. The effectiveness of HDAC inhibitors has been demonstrated by preclinical models, but not in clinical studies, probably due to the ineffectively accumulation of HDACI in prostate cancer cells. The purpose of this work was to evaluate effects of a novel HDACI (CN133) on CRPC xenograft model and 22Rv1 cells, and develops methods, PET/CT imaging, to detect the therapeutic effects of CN133 on this cancer. We designed and performed study to compare the effects of CN133 with SAHA on the 22Rv1 xenograft model and 22Rv1 cells. Using PET/CT imaging with [11C] Martinostat and [18F] FDG, we imaged mice bearing 22Rv1 xenografts before and after 21-day treatment with placebo and CN133 (1mg/kg), and uptake on pre-treatment and post-treatment imaging was measured. The anti-tumor mechanisms of CN133 were investigated by qPCR, western blot, and ChIP-qPCR. Our data showed that the CN133 treatment led to a 50% reduction of tumor volume compared to the placebo that was more efficacious than SAHA treatment in this preclinical model. [11C] Martinostat PET imaging could identify early lesions of prostate cancer and can also be used to monitor the therapeutic effect of CN133 in CRPC. Using pharmacological approaches, we demonstrated that effects of CN133 showed almost 100-fold efficacy than SAHA treatment in the experiment of cell proliferation, invasion, and migration. The anti-tumor mechanisms of CN133 were due to the inhibition of AR signaling pathway activity by decreased HDAC 2 and 3 protein expressions. Taken together, these studies provide not only a novel epigenetic approach for prostate cancer therapy but also offering a potential tool, [11C] Martinostat PET/CT imaging, to detect the early phase of prostate cancer and monitor therapeutic effect of CN133. These results will likely lead to human trials in the future.

Novel Dual BET and PLK1 Inhibitor WNY0824 Exerts Potent Antitumor Effects in CRPC by Inhibiting Transcription Factor Function and Inducing Mitotic Abnormality.

Castration-resistant prostate cancer (CRPC) is a lethal disease with few treatment alternatives once patients become resistant to second-generation antiandrogens. In CRPC, BET proteins are key regulators of AR- and MYC-mediated transcription, while the PLK1 inhibitor potentially downregulates AR and MYC besides influencing the cell cycle. Therefore, synchronous inhibition of BET and PLK1 would be a promising approach for CRPC therapy. This study developed a dual BET and PLK1 inhibitor WNY0824 with nanomolar and equipotent inhibition of BRD4 and PLK1. In vitro, WNY0824 exhibited excellent antiproliferation activity on AR-positive CRPC cells and induced apoptosis. These activities are attributable to its disruption of the AR-transcriptional program and the inhibition of the ETS pathway. Furthermore, WNY0824 downregulated MYC and induced mitotic abnormality. In vivo, oral WNY0824 administration suppressed tumor growth in the CRPC xenograft model of enzalutamide resistance. These findings suggest that WNY0824 is a selective dual BET and PLK1 inhibitor with potent anti-CRPC oncogenic activity and provides insights into the development of other novel dual BET- and PLK1-inhibiting drugs.

Hexane Insoluble Fraction from Purple Rice Extract Retards Carcinogenesis and Castration-Resistant Cancer Growth of Prostate Through Suppression of Androgen Receptor Mediated Cell Proliferation and Metabolism

Prostate cancer and castration-resistant prostate cancer (CRPC) remain major health challenges in men. In this study, the inhibitory effects of a hexane insoluble fraction from a purple rice ethanolic extract (PRE-HIF) on prostate carcinogenesis and CRPC were investigated both in vivo and in vitro. In the Transgenic Rat for Adenocarcinoma of Prostate (TRAP) model, 1% PRE-HIF mixed diet-fed rats showed a significantly higher percentage of low-grade prostatic intraepithelial neoplasia and obvious reduction in the incidence of adenocarcinoma in the lateral lobes of the prostate. Additionally, 1% PRE-HIF supplied diet significantly suppressed the tumor growth in a rat CRPC xenograft model of PCai1 cells. In LNCaP and PCai1 cells, PRE-HIF treatment suppressed cell proliferation and induced G0/G1 cell-cycle arrest. Furthermore, androgen receptor (AR), cyclin D1, cdk4, and fatty acid synthase expression were down-regulated while attenuation of p38 mitogen-activated protein kinase, and AMP-activated protein kinase α activation occurred in PRE-HIF treated prostate cancer cells, rat prostate tissues, and CRPC tumors. Due to consistent results with PRE-HIF in PCai1 cells, cyanidin-3-glucoside was characterized as the active compound. Altogether, we surmise that PRE-HIF blocks the development of prostate cancer and CRPC through the inhibition of cell proliferation and metabolic pathways.

A Comprehensive Analysis of FUT8 Overexpressing Prostate Cancer Cells Reveals the Role of EGFR in Castration Resistance.

The emergence of castration-resistance is one of the major challenges in the management of patients with advanced prostate cancer. Although the spectrum of systemic therapies that are available for use alongside androgen deprivation for treatment of castration-resistant prostate cancer (CRPC) is expanding, none of these regimens are curative. Therefore, it is imperative to apply systems approaches to identify and understand the mechanisms that contribute to the development of CRPC. Using comprehensive proteomic approaches, we show that a glycosylation-related enzyme, alpha (1,6) fucosyltransferase (FUT8), which is upregulated in CRPC, might be responsible for resistance to androgen deprivation. Mechanistically, we demonstrated that overexpression of FUT8 resulted in upregulation of the cell surface epidermal growth factor receptor (EGFR) and corresponding downstream signaling, leading to increased cell survival in androgen-depleted conditions. We studied the coregulatory mechanisms of EGFR and FUT8 expression in CRPC xenograft models and found that castration induced FUT8 overexpression associated with increased expression of EGFR. Taken together, our findings suggest a crucial role played by FUT8 as a mediator in switching prostate cancer cells from nuclear receptor signaling (androgen receptor) to the cell surface receptor (EGFR) mechanisms in escaping castration-induced cell death. These findings have clinical implication in understanding the role of FUT8 as a master regulator of cell surface receptors in cancer-resistant phenotypes.

Nuclear receptor ERRα contributes to castration-resistant growth of prostate cancer via its regulation of intratumoral androgen biosynthesis.

Enhanced intratumoral androgen biosynthesis and persistent androgen receptor (AR) signaling are key factors responsible for the relapse growth of castration-resistant prostate cancer (CRPC). Residual intraprostatic androgens can be produced by de novo synthesis of androgens from cholesterol or conversion from adrenal androgens by steroidogenic enzymes expressed in prostate cancer cells via different steroidogenic pathways. However, the dysregulation of androgen biosynthetic enzymes in CRPC still remains poorly understood. This study aims to elucidate the role of the nuclear receptor, estrogen-related receptor alpha (ERRα, ESRRA), in the promotion of androgen biosynthesis in CRPC growth.Methods: ERRα expression in CRPC patients was analyzed using Gene Expression Omnibus (GEO) datasets and validated in established CRPC xenograft model. The roles of ERRα in the promotion of castration-resistant growth were elucidated by overexpression and knockdown studies and the intratumoral androgen levels were measured by UPLC-MS/MS. The effect of suppression of ERRα activity in the potentiation of sensitivity to androgen-deprivation was determined using an ERRα inverse agonist.Results: ERRα exhibited an increased expression in metastatic CRPC and CRPC xenograft model, could act to promote castration-resistant growth via direct transactivation of two key androgen synthesis enzymes CYP11A1 and AKR1C3, and hence enhance intraprostatic production of dihydrotestosterone (DHT) and activation of AR signaling in prostate cancer cells. Notably, inhibition of ERRα activity by an inverse agonist XCT790 could reduce the DHT production and suppress AR signaling in prostate cancer cells.Conclusion: Our study reveals a new role of ERRα in the intratumoral androgen biosynthesis in CRPC via its transcriptional control of steroidogenic enzymes, and also provides a novel insight that targeting ERRα could be a potential androgen-deprivation strategy for the management of CRPC.

Abstract B016: CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation

Abstract Metastatic castration-resistant prostate cancer (CRPC) is an aggressive disease with high mortality rate, primarily resulting from the transcriptional addiction driven by Androgen Receptor (AR). First-line CRPC treatments typically target AR-signaling, but are rapidly bypassed, resulting in only a modest survival benefit with the FDA approved anti-androgen therapies such abiraterone or enzalutamide. Therefore, molecular approaches that more effectively block the AR-transcriptional program are urgently needed. Here, we present evidence demonstrating that AR transcriptional signaling is activated through a “phosphoswitch” catalyzed by cyclin-dependent-kinase 7 (CDK7). Specifically, CDK7 phosphorylates (in vitro and in vivo) the transcriptional co-activator MED1, a key subunit of Mediator complex, at Threonine 1457 to promote the formation of the AR-transcriptional complex at enhancers and super-enhancer (SE) sites. Furthermore, knockdown or inhibition of CDK7 with the recently developed covalent inhibitor THZ1 abolished T1457 phosphorylation that led to MED1 decruitment from the chromatin, attenuated AR-signaling and eliminated AR-addicted naïve or enzalutamide refractory prostate cancer cells. Interestingly, the reduced AR transcriptional output and cellular phenotypes associated with CDK7 knockdown or inhibition was reversed by T1457D phosphomimic suggesting MED1 as a major effector substrate of CDK7 transcription kinase. Finally, THZ1 demonstrated tumor regression in CRPC xenograft models in vivo. In summary, our identification of MED1 T1457 as a novel CDK7 substrate, which is essential for driving AR-mediated transcription, makes CDK7 a potential “non-oncogene dependency” in AR addicted advanced prostate cancer. Taken together, we will present data that strongly support the clinical evaluation of CDK7 specific inhibitors as a monotherapy or in combination with second generation anti-androgens in refractory castration-resistant prostate cancer. Conflict of Interest: None (P.S. This work is undergoing final revision for publication in Cancer Discovery) Citation Format: Reyaz Rasool, Ramakrishnan Natesan, qu deng, Shweta Aras, Priti Lal, Jessica Posimo, Nallasivam Palanisamy, Goutham Narla, Robert Den, Matthew Freedman, donita brady, irfan a asangani. CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B016. doi:10.1158/1535-7163.TARG-19-B016

Abstract LB-A13: LSD1 activates both PI3K/AKT and AR signaling in prostate cancer cells

Abstract Background: Lysine specific demethylase 1 (LSD1/KDM1A), a specific demethylase of mono- or di-methylated histone lysine 4 (H3K4me1,2, enhancer-associated signature), was first identified as a component of REST repressor complex composed of CoREST and histone deacetylases 1, 2 (HDAC1,2). In hematopoietic malignancies, targeting LSD1 has shown very promising clinical outcomes, potentially through promoting myeloid differentiation. In solid tumors, multiple mechanisms have been proposed for the antitumor effect of inhibiting LSD1. While LSD1 is well known for its transcription repressor activity, it also activates gene transcription through demethylating repressive histone marks, such as H3K9me1,2, and possibly other non-histone proteins. In particular, LSD1 functions as a major androgen receptor (AR) coactivator in prostate cancer (PCa) cells. Indeed, we reported that LSD1 interacts and colocalizes with FOXA1, a pioneer factor of AR, at AR-mediated enhancers, and this interaction may facilitate AR transcription activity. Nonetheless, since AR signaling is critical to PCa development and progression to the lethal stage of castration-resistant PCa (CRPC), studies from us and others highly suggest that targeting LSD1 may be a potential treatment strategy for CRPC, where AR signaling is commonly restored. However, whether LSD1 regulates other major tumor-promoting pathways in PCa cells remains to be determined. Identification of suchcritical LSD1 targets will provide important insights for developing more effective therapeutic strategy for treating PCa. Experiments and Results: To address this question, we conducted RNA-seq analyses of LSD1 inhibitor treated PCa cells. We first confirmed that prolonged LSD1 inhibitor treatment in the presence of androgens broadly impaired AR transcriptional output. Importantly, we found that the LSD1 inhibitor repressed genes were also enriched for PI3K/AKT pathway even in absence of androgen stimulation. We further confirmed that LSD1 inhibition and Knock-out significantly decreased AKT phosphorylation, which interestingly is independent of AR signaling, as targeting LSD1 also decreased AKT phosphorylation in AR negative PCa cells. Through functional annotation analyses and subsequent validations, we identified the regulatory subunit of PI3K, p85α and possibly its isoform p85β, as a critical transcriptional target of LSD1 that mediates its effect on PI3K/AKT pathway activation. Using a combined analysis with ChIP-seq against LSD1 and H3K4me2, we identified a regulatory region on the PIK3R1 gene loci that may be responsible for regulating its gene transcription. We further investigated the effect of LSD1 inhibitor treatment in LuCaP35CR, a patient-derived CRPC xenograft model which is TMPRSS2-ERG positive and PTEN-negative. Using this model, we found that targeting LSD1 impaired the tumor growth and suppressed PI3K/AKT signaling. Conclusion: our study suggests that LSD1 has dual functions in promoting PCa development: 1) it enhances AR signaling through its coactivator function, 2) it activates PI3K/AKT signaling through increasing p85 gene expression. Based on these findings, it is plausible that the effectiveness of LSD1 inhibitor treatment in CRPC may be due to inhibition of both AR signaling and PI3K/AKT signaling pathways. As LSD1 inhibitors are currently being tested in phase II clinical trials of leukemia, our studies can be rapidly translated into clinical trials of CRPC. Citation Format: zifeng wang, Shuai Gao, Changmeng cai. LSD1 activates both PI3K/AKT and AR signaling in prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A13. doi:10.1158/1535-7163.TARG-19-LB-A13

Androgen Receptor Interaction with Mediator Complex Is Enhanced in Castration-Resistant Prostate Cancer by CDK7 Phosphorylation of MED1.

In this issue of Cancer Discovery, Rasool and colleagues show that TF11H/CDK7 phosphorylates the MED1 component of the Mediator complex, which enhances its interaction with androgen receptor (AR), and that this phosphorylation is increased in prostate cancer that is resistant to castration and enzalutamide. A covalent CDK7-specific inhibitor (THZ1) impairs AR-mediated MED1 recruitment to chromatin, and can suppress enzalutamide resistance in vitro and induce tumor regression in a castration-resistant prostate cancer xenograft model, suggesting a novel therapeutic approach for advanced prostate cancer.See related article by Rasool et al., p. 1538.

Galeterone and The Next Generation Galeterone Analogs, VNPP414 and VNPP433-3β Exert Potent Therapeutic Effects in Castration-/Drug-Resistant Prostate Cancer Preclinical Models In Vitro and In Vivo.

These studies compared the efficacies of our clinical agent galeterone (Gal) and the FDA-approved prostate cancer drug, enzalutamide (ENZ) with two lead next generation galeterone analogs (NGGAs), VNPP414 and VNPP433-3β, using prostate cancer (PC) in vitro and in vivo models. Antitumor activities of orally administered agents were also assessed in CWR22Rv1 tumor-bearing mice. We demonstrated that Gal and NGGAs degraded AR/AR-V7 and Mnk1/2; blocked cell cycle progression and proliferation of human PC cells; induced apoptosis; inhibited cell migration, invasion, and putative stem cell markers; and reversed the expression of epithelial-to-mesenchymal transition (EMT). In addition, Gal/NGGAs (alone or in combination) also inhibited the growth of ENZ-, docetaxel-, and mitoxantrone-resistant human PC cell lines. The NGGAs exhibited improved pharmacokinetic profiles over Gal in mice. Importantly, in vivo testing showed that VNPP433-3β (at 7.53-fold lower equimolar dose than Gal) markedly suppressed (84% vs. Gal, 47%; p < 0.01) the growth of castration-resistant PC (CRPC) CWR22Rv1 xenograft tumors, with no apparent host toxicity. ENZ was ineffective in this CRPC xenograft model. In summary, our findings show that targeting AR/AR-V7 and Mnk1/2 for degradation represents an effective therapeutic strategy for PC/CRPC treatment and supports further development of VNPP433-3β towards clinical investigation.

SUN-LB002 CYP11A1 Inhibition as a Therapeutic Approach for the Treatment of Castration Resistant Prostate Cancer

Background: Prostate cancer is a major global challenge due to the increasing number of aging population and frequency of diagnosis. During the past decade new treatments have been targeted to the androgen signaling axis either by inhibiting the binding of androgens to androgen receptor (AR) and AR nuclear translocation, or by inhibiting androgen production via CYP17A1 enzyme. Despite the significant progress on the research and new therapies, CRPC is still incurable and there is urgent need for better, more effective treatments. ODM-208 is an oral, non-steroidal and selective inhibitor of CYP11A1 enzyme, suppressing the synthesis of all steroid hormones that could be potential agonistic AR ligands. Methods: The inhibition of CYP11A1 was measured in vitro by the formation of radiolabelled isocapronic acid in a human adrenal cortex cell line (H295R). The tumor growth inhibition of ODM-208 was studied in VCaP castration-resistant prostate cancer (CRPC) xenograft model. At the end of the xenograft study, plasma ACTH and LH, and key steroid hormone concentrations were analysed from plasma and target tissues. In addition, full length AR (AR-FL) and AR-V7 were analysed from the tumors by western blot and key enzymes of androgen biosynthesis, CYP17A1, AKR1C3, SRD5A1 were quantified by qPCR. In dogs an ACTH stimulation test was done. Results: ODM-208 potently inhibits CYP11A1 enzyme in vitro. In addition, in vivo in the VCaP CRPC xenograft model ODM-208 significantly inhibited tumor growth. Importantly, the amount of AR-FL and AR-V7 levels remained unchanged in the tumors after ODM-208 treatment. Slight increase of CYP17A1 and SRD5A1 enzyme levels was observed, indicating the activation of steroidogenesis in VCaP tumors in vivo. Treatment had no effect on plasma LH, whereas ACTH was significantly increased demonstrating reduction in glucocorticoid levels by negative feedback. In line with ACTH, all measured steroid hormones were significantly reduced both in plasma and target tissues. In dogs ACTH-stimulated cortisol production was significantly inhibited after single oral dose of ODM-208. Conclusions: ODM-208 shows promising antitumor activity in preclinical CRPC models and has favorable toxicological profile. Thus, ODM-208 might have potential for treating patients with CRPC. Based on the nonclinical results, a phase 1/2 clinical trial has been initiated. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.