CRPC Cell Research Articles

Targeting AR Variant-Coactivator Interactions to Exploit Prostate Cancer Vulnerabilities.

Castration-resistant prostate cancer (CRPC) progresses rapidly and is incurable. Constitutively active androgen receptor splice variants (AR-Vs) represent a well-established mechanism of therapeutic resistance and disease progression. These variants lack the AR ligand-binding domain and, as such, are not inhibited by androgen deprivation therapy (ADT), which is the standard systemic approach for advanced prostate cancer. Signaling by AR-Vs, including the clinically relevant AR-V7, is augmented by Vav3, an established AR coactivator in CRPC. Using mutational and biochemical studies, we demonstrated that the Vav3 Diffuse B-cell lymphoma homology (DH) domain interacted with the N-terminal region of AR-V7 (and full length AR). Expression of the Vav3 DH domain disrupted Vav3 interaction with and enhancement of AR-V7 activity. The Vav3 DH domain also disrupted AR-V7 interaction with other AR coactivators: Src1 and Vav2, which are overexpressed in PC. This Vav3 domain was used in proof-of-concept studies to evaluate the effects of disrupting the interaction between AR-V7 and its coactivators on CRPC cells. This disruption decreased CRPC cell proliferation and anchorage-independent growth, caused increased apoptosis, decreased migration, and resulted in the acquisition of morphological changes associated with a less aggressive phenotype. While disrupting the interaction between FL-AR and its coactivators decreased N-C terminal interaction, disrupting the interaction of AR-V7 with its coactivators decreased AR-V7 nuclear levels.Implications: This study demonstrates the potential therapeutic utility of inhibiting constitutively active AR-V signaling by disrupting coactivator binding. Such an approach is significant, as AR-Vs are emerging as important drivers of CRPC that are particularly recalcitrant to current therapies. Mol Cancer Res; 15(11); 1469-80. ©2017 AACR.

Abstract 1114: Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells

Abstract Androgen deprivation therapy is an efficient first line therapy for advanced prostate cancer. However, in most cases resistance inevitably develops resulting in metastatic castration-resistant prostate cancer (mCRPC). Abiraterone and enzalutamide are approved to treat mCRPC and act by inhibiting androgen biosynthesis and androgen receptor (AR) signaling, respectively. Poly (ADP-ribose) polymerase 1 (PARP1) is a key enzyme in the DNA damage repair pathway and has also been implicated in AR signaling cascade. Recent reports suggest significant efficacy combining anti-androgens and inhibitors of Poly (ADP-ribose) polymerase (PARP) particularly against cells harboring mutations in the DNA repair pathway. Here, we provide evidence of synergistic cytotoxicity combining PARP inhibition and androgen signaling ablation in the AR positive human CRPC cell line 22RV1 harboring mutation in the DNA repair gene BRCA2. The PARP inhibitor talazoparib (IC50 - 0.05 μM) was upto 20 times more potent than olaparib (IC50 – 1.0 μM) in inducing cell death in 22RV1 as seen by cell viability and clonogenic assays. When combined with AR antagonist enzalutamide (IC50 – 0.25 μM) or androgen synthesis inhibitor abiraterone (IC50 – 1.25 μM) both PARP inhibitors showed significant synergy, although talazoparib was more prominent owing to its superior potency. Higher concentrations of enzalutamide and abiraterone (>10μM) induced PARP cleavage and increased expression of γ-H2AX, a marker for DNA damage. Our results suggest that AR signaling inhibition involves PARP mediated apoptosis and highlight the crosstalk between PARP and AR pathways. In summary, combining anti-androgen therapy and PARP inhibitors carry significant therapeutic potential for CRPC patients harboring mutations in DNA repair pathway. Citation Format: Sahithi Pamarthy, Vinay Sagar, Rajita J. Vatapalli, Zachary Reinstein, Muhammad Zayd Ansari, Benedito A. Carneiro Filho, Francis J. Giles, Sarki A. Abdulkadir. Combining anti-androgen therapy and PARP inhibition results in synergistic cytotoxicity of metastatic castration-resistant prostate cancer (mCRPC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1114. doi:10.1158/1538-7445.AM2017-1114

Abstract 2969: Paracrine effect of the endothelium on prostate cancer cells

Abstract Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP. Citation Format: Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. Paracrine effect of the endothelium on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2017-2969

Abstract 1515: Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7)

Abstract Persistent androgen receptor (AR) signaling is key to the development and progression of metastatic castration resistant prostate cancer (mCRPC). This is in part due to expression of constitutively active AR splice variants like AR variant 7 (ARV7), confering resistance to current anti-androgens including enzalutamide (E) and abiraterone (A). To improve the outcome for patients with mCRPC, new therapeutic strategies to overcome AR and ARV7 oncogenic signaling are urgently required. The inhibition of co-factors modulating AR signaling are currently being investigated as novel strategies to treat mCRPC. One promising candidate is BRD4, a member of the BET protein family, that binds the AR on androgen response elements and facilitates the recruitment of the transcriptional machinery. BET inhibitors (BETi) have been shown to regulate AR and ARV7 signalling, however, the exact mechanism of ARV7 regulation remains unclear. As BETi are currently being explored in clinical trials of unstratified patients with mCRPC, we investigated their potential mechanism of action in CRPC cell lines, patient derived organoids (PDOs) and a patient derived mouse xenograft (PDX). Here we demonstrate that nuclear expression of BRD4 and ARV7 increases as patients develop resistance to E and/or A and inhibition of BRD4 by BETi is sufficient to block AR and ARV7 signalling in mCRPC. Both inhibition of BRD4 by BETi and genetic knockdown of BRD4 reduced the growth of CRPC cell lines and led to down-regulation of AR and ARV7 at the mRNA and protein level. To further investigate whether BETi is sufficient to inhibit ARV7 activity in patients with mCRPC, we treated patient derived organoids (PDOs) and a mouse xenograft (PDX) grown from metastatic biopsies of patients resistant to E and/or A with BETi. In this study 5 out of 10 PDOs were sensitive to BETi. Consistent with the cell culture experiments, BETi treatment of the PDX led to down-regulation of both ARV7 mRNA and protein expression. Mechanistically, BETi might inhibit pre-mRNA splicing of AR resulting in the observed decrease of ARV7 expression. However, RNAseq analysis of the ARV7 expressing CRPC cell line LNCaP95 demonstrated an increase in total splicing events including skipped exons, retained introns, mutually exclusive exons, alternative 5’ splice site and alternative 3’ splice site after BETi treatment. Despite this, focused analysis of splicing factors and spliceosome components identified a subset of eight splicing factors being down-regulated by BET inhibition including one yet uncharacterized factor that is crucial for ARV7 expression in LNCaP95 cells. Based on our results we propose a model that BETi mediated inhibition of this novel ARV7-mRNA splicing factor may lead to decreased splicing and subsequent expression of ARV7 at both mRNA and protein level; providing a novel approach to target ARV7 in mCRPC. Citation Format: Jonathan Welti, Adam Sharp, Ines Figueiredo, Wei Yuan, Daniel Nava Rodrigues, Veronica S. Gil, Eleanor Knight, Jian Ning, Jeff Francis, Antje Neeb, Gunther Boysen, Amanda Swain, Johann S. de Bono. Bromodomain and extra-terminal motif protein inhibitors (BETi) in metastatic castration resistant prostate cancer (mCRPC): A novel mechanism for regulating androgen receptor variant 7 (ARV7) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1515. doi:10.1158/1538-7445.AM2017-1515

MP83-10 CORRELATION BETWEEN CABAZITAXEL RESISTANCE AND EMT IN METASTATIC CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-10 CORRELATION BETWEEN CABAZITAXEL RESISTANCE AND EMT IN METASTATIC CASTRATION RESISTANT PROSTATE CANCER Hiroshi Hongo, Takeo Kosaka, Yota Yasumizu, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Hiroshi HongoHiroshi Hongo More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Yota YasumizuYota Yasumizu More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2578AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recently, cabazitaxel (CBZ) was identified as the most effective cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory CRPC. Accumulating evidence suggests EMT induced by androgen-deprivation therapy or docetaxel explains in part the aggressiveness of CRPC. The aim of this study was to screen for EMT inhibitory drugs by bioinformatic analysis and exploring the antitumor effect of CBZ for resistant CRPC. METHODS PC3 and DU145 were used in this study. We incubated the cell lines with gradually increasing concentrations of CBZ to establish CBZ-resistant cell lines. In addition, we performed screening tests for candidate drugs to inhibit EMT of metastatic CRPC. The combined effect of CBZ and a candidate EMT inhibitor for CBZ-resistant cell lines was determined using a cell viability assay. RESULTS We established a CBZ-resistant cell line, PC3-CBZR (Figure). PC3-CBZR cells underwent cell division with 3 nM CBZ. We compared the cytotoxic effect of CBZ on PC3 and PC3-CBZR cells using a cell viability assay. The IC50 of CBZ in PC3 and PC3-CBZR cells were 16.0 nM and 2.5 nM, respectively. Real-time PCR showed up-regulation of ZEB family and Vimentin and down-regulation of E-cadherin expression, suggesting PC3-CBZR cells had elevated metastatic potential. We screened for EMT inhibitors using The Broad institute′s connectivity map (CMAP) analysis, with human whole genome array data and identified a candidate drug, KOH513. The relative cell viabilities treated with 10 μM KOH513 in PC3 and PC3-CBZR cells were 60.0 ± 1.7%, 58.7 ± 0.7%, respectively. KOH513 was suggested to have an antitumor effect on CBZ-resistant prostate cancer. Real time PCR and immune fluorescence assay showed that KOH513 down-regulated ZEB family and Vimentin in PC3-CBZR cells at the protein and mRNA levels. We tested the efficacy of KOH513 in CBZ resistance using a cell viability assay. Single-agent administration of 6 nM CBZ or 3 μM KOH513 did not have an antitumor effect in PC3-CBZ cells. However, when treated with 6 nM CBZ and 3 µM KOH513 in combination, PC3-CBZ cell proliferation was significantly suppressed (relative cell viability 77.0 ± 1.7%). In the same manner, we established CBZ-resistant DU145 cells, DU145-CBZR. DU145-CBZR cells had also had an up-regulated EMT pathway, and cell proliferation was suppressed by EMT inhibitor KOH513. These results suggested that KOH513 had potential for reprogramming CBZ resistant CRPC cell lines into sensitive cell lines. CONCLUSIONS Targeting EMT signaling pathways with KOH513 can overcome CBZ resistance in CRPC. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1109-e1110 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Hiroshi Hongo More articles by this author Takeo Kosaka More articles by this author Yota Yasumizu More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B22: Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer

Abstract The anti-androgen bicalutamide is widely used in the treatment of advanced prostate cancer (CaP) in many countries, but its effect on castration resistant CaP (CRPC) is limited. We previously showed that resistance to bicalutamide results from increased activation of the mechanistic target of rapamycin (mTOR); indicating a role for mTOR inhibitors in CaP treatment. Interestingly, a clinical trial testing combinations of the mTOR inhibitor RAD001 with bicalutamide were initially effective (PSA decline>50%) in 62.5% CRPC patients, but many initial responders later developed resistance. Here we investigate causes for their difference in response. Evaluation of various CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E phosphorylation at S209 is associated with resistance to the combination. To recognize potential causes of resistance, we demonstrated using a human tumor xenograft mouse model, that bicalutamide use is associated with an increase in eIF4E(S209) phosphorylation. Investigations revealed that AR suppressed eIF4E phosphorylation, while the use of anti-androgens relieved this suppression, thereby triggering its increase. Additional investigation on the consequences of increased eIF4E phosphorylation in human prostatectomy samples revealed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67, indicating a role for eIF4E in tumor growth. Inhibition of eIF4E phosphorylation by siRNA suppression or treatment with CGP57380 (an inhibitor of MAPK interacting serine-threonine kinases Mnk1/2, the eIF4E upstream kinase,) sensitized CRPC cells to RAD001+bicalutamide, while eIF4E overexpression induced resistance. Our results demonstrate that bicalutamide treatment induced an increase in eIF4E phosphorylation by inhibition of AR which otherwise suppressed this phosphorylation. Increased eIF4E phosphorylation then induced resistance to the combination of anti-androgens with mTOR inhibitors by increasing proliferation. These results also indicate that resistance to RAD001+bicalutamide can be prevented by the concurrent use of a Mnk1/2 inhibitor. Citation Format: Leandro S. D'Abronzo, Michael Crapuchettes, Ryan Beggs, Ruth Vinall, Clifford Tepper, Salma Siddiqui, Maria Mudryj, Frank Melgoza, Blythe Durbin-Johnson, Ralph deVere White, Paramita Ghosh. Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors together with AR antagonists in advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B22.

Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells.

Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC.

Src promotes castration-recurrent prostate cancer through androgen receptor-dependent canonical and non-canonical transcriptional signatures.

Progression of prostate cancer (PC) to castration-recurrent growth (CRPC) remains dependent on sustained expression and transcriptional activity of the androgen receptor (AR). A major mechanism contributing to CRPC progression is through the direct phosphorylation and activation of AR by Src-family (SFK) and ACK1 tyrosine kinases. However, the AR-dependent transcriptional networks activated by Src during CRPC progression have not been elucidated. Here, we show that activated Src (Src527F) induces androgen-independent growth in human LNCaP cells, concomitant with its ability to induce proliferation/survival genes normally induced by dihydrotestosterone (DHT) in androgen-dependent LNCaP and VCaP cells. Src induces additional gene signatures unique to CRPC cell lines, LNCaP-C4-2 and CWR22Rv1, and to CRPC LuCaP35.1 xenografts. By comparing the Src-induced AR-cistrome and/or transcriptome in LNCaP to those in CRPC and LuCaP35.1 tumors, we identified an 11-gene Src-regulated CRPC signature consisting of AR-dependent, AR binding site (ARBS)-associated genes whose expression is altered by DHT in LNCaP[Src527F] but not in LNCaP cells. The differential expression of a subset (DPP4, BCAT1, CNTNAP4, CDH3) correlates with earlier PC metastasis onset and poorer survival, with the expression of BCAT1 required for Src-induced androgen-independent proliferation. Lastly, Src enhances AR binding to non-canonical ARBS enriched for FOXO1, TOP2B and ZNF217 binding motifs; cooperative AR/TOP2B binding to a non-canonical ARBS was both Src- and DHT-sensitive and correlated with increased levels of Src-induced phosphotyrosyl-TOP2B. These data suggest that CRPC progression is facilitated via Src-induced sensitization of AR to intracrine androgen levels, resulting in the engagement of canonical and non-canonical ARBS-dependent gene signatures.

Abstract 280: Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide

Abstract Metastatic castration-resistant prostate cancer (mCRPC) presents a daunting therapeutic challenge for patients who eventually progress after androgen ablation therapy. Although newer AR antagonists show a survival benefit, these patients ultimately relapse. Thus, it is critical to identify new therapies that will work in combination with AR antagonists or in AR-antagonist resistant settings. MOR209/ES414 is a bispecific ADAPTIR™ (modular protein technology) molecule currently under investigation in a phase 1 clinical trial in mCRPC, including patients that have progressed on enzalutamide. In pre-clinical studies, MOR209/ES414 has previously been shown to redirect T cell cytotoxicity against cells expressing Prostate Specific Membrane Antigen (PSMA). Here, we aimed to determine whether enzalutamide affects PSMA expression on CRPC cells in vitro, and could alter the effectiveness of MOR209/ES414 at inducing T-cell mediated tumor cell death. To address these questions, we utilized a CRPC cell line, 22Rv1, which has low expression of PSMA and is resistant to enzalutamide. Treatment of 22Rv1 cells with 10 μM enzalutamide for 3 weeks resulted in increased PSMA cell surface expression as measured by flow cytometry. 22Rv1 cells exposed to enzalutamide were also more sensitive to MOR209/ES414-mediated T-cell lysis, with a 25% increase in cytotoxicity over a 4 hour chromium release assay compared to 22Rv1 cells that were not exposed to enzalutamide. A concomitant decrease in EC50 for MOR209/ES414-mediated lysis from 0.8 pM to 0.5 pM was also observed in 22Rv1 cells exposed to enzalutamide. MOR209/ES414 and enzalutamide were next tested in combination using a PSMA-positive, enzalutamide-sensitive cell line, LNCaP cultured with primary T cells over several days. Suboptimal doses of MOR209/ES414 and enzalutamide were added to the cultures, and surviving LnCaP cells were quantitated 4 days later. Enzalutamide did not interfere with the ability of MOR209/ES414 to target T cells to LNCaP cells, and Combination Index analysis showed the activity of MOR209/ES414 and enzalutamide to be synergistic in vitro. These studies provide a rationale for further examination of combining MOR209/ES414 with enzalutamide, even in enzalutamide-resistant settings. Citation Format: Toddy Sewell, Michelle Blake, Jane A. Gross, Jan Endell, Johannes Weirather, John W. Blankenship. Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide. [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 280.

Abstract 288: Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel

Abstract Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Still mCRPC continues to be incurable. Furthermore, patients often experience severe side effects, prompting a re-evaluation of conventional regimen. Past studies have demonstrated promise for Low-Dose Metronomic (LDM) chemotherapy; schedule defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Using CRPC cell lines (PC3, Du145 and the LNCaP-derivative CL1), we have shown that cabazitaxel-treated cells have a significantly lower EC50 compared to docetaxel, with Du145 cells presenting the greatest differences. In cell cycle analysis, both low-dose taxanes increased the sub-G1 cells population. However, the sub-G1 increase was significantly greater in cabazitaxel- than in docetaxel-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cabazitaxel than docetaxel validating a higher efficacy due to increased apoptosis. As other possible cell death mechanisms, autophagy and necrosis were investigated. Beclin1 expression levels remain unchanged in all three cell lines. In contrast, necrosis was stimulated in all the taxanes-treated cell lines in a dose dependent manner. In vivo, LDM cabazitaxel was significantly more efficient in delaying tumor growth than docetaxel. This effect was markedly increased when cabazitaxel was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF). Other results showed that PEDF and LDM chemotherapy combination induces more phagocytosis of CRPC cells when compared to single treatments. In conclusion, our data demonstrate a higher efficacy of cabazitaxel on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC. Citation Format: Courtney L. Jarvis, Thomas Nelius, Dalia Martinez-Marin, Srirupa Cheerla, Stéphanie Filleur. Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel. [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 288.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features.

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features.

Resistance to docetaxel in prostate cancer is associated with androgen receptor activation and loss of KDM5D expression

The androgen receptor (AR) plays an essential role in prostate cancer, and suppression of its signaling with androgen deprivation therapy (ADT) has been the mainstay of treatment for metastatic hormone-sensitive prostate cancer for more than 70 y. Chemotherapy has been reserved for metastatic castration-resistant prostate cancer (mCRPC). The Eastern Cooperative Oncology Group-led trial E3805: ChemoHormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) showed that the addition of docetaxel to ADT prolonged overall survival compared with ADT alone in patients with metastatic hormone-sensitive prostate cancer. This finding suggests that there is an interaction between AR signaling activity and docetaxel sensitivity. Here we demonstrate that the prostate cancer cell lines LNCaP and LAPC4 display markedly different sensitivity to docetaxel with AR activation, and RNA-seq analysis of these cell lines identified KDM5D (lysine-specific demethylase 5D) encoded on the Y chromosome as a potential mediator of this sensitivity. Knocking down KDM5D expression in LNCaP leads to docetaxel resistance in the presence of dihydrotestosterone. KDM5D physically interacts with AR in the nucleus, and regulates its transcriptional activity by demethylating H3K4me3 active transcriptional marks. Attenuating KDM5D expression dysregulates AR signaling, resulting in docetaxel insensitivity. KDM5D deletion was also observed in the LNCaP-derived CRPC cell line 104R2, which displayed docetaxel insensitivity with AR activation, unlike parental LNCaP. Dataset analysis from the Oncomine database revealed significantly decreased KDM5D expression in CRPC and poorer prognosis with low KDM5D expression. Taking these data together, this work indicates that KDM5D modulates the AR axis and that this is associated with altered docetaxel sensitivity.

MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER Takeo Kosaka, Hiroshi Hongo, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Takeo KosakaTakeo Kosaka More articles by this author , Hiroshi HongoHiroshi Hongo More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2547AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cabazitaxel (CBZ) has become the most effective cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory CRPC. Accumulating evidence has suggested reactive oxygen species (ROS) production induced by taxanes in cancer cells may influence taxane-induced cell death or drug resistance. However, ROS production by CBZ or its mechanistic action remains to be characterized in CRPC. In this study, we investigated PCa cell sensitivity to docetaxel (DTX) or CBZ by measurement of ROS production. METHODS Three cell lines were used: LNCaP, a human PCa cell line that exhibits androgen-dependent proliferation; C4-2, a human PCa cell line that exhibits androgen-independent proliferation; and C4-2AT6, a CRPC cell line derived from C4-2. These cells were treated with increasing concentrations of docetaxel or CBZ in vitro. Gene expression profiles of CBZ- or DTX-treated cells were analyzed using a whole human genome microarray. FACS analysis was performed using a fluorogenic marker of ROS. Furthermore, we investigated the changes in expression of antioxidant enzymes induced by DTX and CBZ. RESULTS Cabazitaxel showed significantly higher cytotoxicity than docetaxel in human CRPC cells. FACS analysis revealed that treatment with cabazitaxel significantly increased ROS generation than docetaxel in C4-2AT6 cells, accompanied by higher expression of phospho-p38. Microarray analysis revealed significant changes in ROS regulation-related genes by CBZ. qPCR analysis revealed CBZ induced C4-2AT6 cell death, accompanied by significantly decreased expression of Sestrin-3 (SESN3). To investigate whether CBZ-mediated cell death was caused by induction of ROS generation, C4-2AT6 cells were incubated with CBZ in the presence of the antioxidant N-acetylcysteine (NAC), which was observed to reduce cabazitaxel-induced cytotoxicity. Microarray analysis revealed dynamic changes in ROS regulation-related genes by CBZ, and qPCR analysis demonstrated CBZ reduced mRNA expression of SESN3. We observed that ROS reduction by SESN3 was significantly inhibited by cabazitaxel in a dose-dependent manner, but not by docetaxel. When C4-2AT6 cells were treated with cabazitaxel in the presence of siRNAs against SESN3, significant cytotoxicity was observed, accompanied by enhanced ROS production. CONCLUSIONS Our results indicated that the higher sensitivity of human CRPC to CBZ, compared with DTX, involves ROS production via inhibition of SESN3 expression, an antioxidant enzyme. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1145 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takeo Kosaka More articles by this author Hiroshi Hongo More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract C1: Harnessing the pro-inflammatory tumor microenvironment of castrate-resistant prostate cancer to promote apoptosis

Abstract Introduction Castrate-Resistant Prostate Cancer (CRPC) represents a highly aggressive, currently incurable disease state. Overexpression of anti-apoptotic proteins including FLIP (FLICE-inhibitory protein) and inhibitors of apoptosis proteins (cIAP-1, cIAP-2 and XIAP) in prostate cancer have been associated with progression to castrate-resistant disease. These proteins are known to contribute to resistance to standard-of-care therapies and aid cancer progression through their anti-apoptotic activity. SMAC mimetics are small molecule compounds which mimic the activity of the endogenous inhibitor of IAPs known as SMAC (Second Mitochondrial-derived Activator of Caspase). SMAC mimetic activity is dependent on TNFα signalling and has also been shown to induce apoptosis in response to TRAIL therapy. Both TRAIL and TNFα are derived from immune cells that are frequently present in the tumour microenvironment, making SMAC mimetic therapy an attractive strategy for treating cancers associated with pro-inflammatory immune infiltrates. Methods A panel of prostate cancer cell lines were treated with SMAC mimetic alone, or in combination with exogenous TNFα or TRAIL. Cell viability was determined by MTT assay, and apoptosis was assessed using Western blotting and AnnexinV/PI flow cytometry. siRNA and Histone Deacetylase Inhbitor (HDACi) therapy were employed to reduce FLIP expression. The addition of exogenous TNFα, TRAIL or macrophage-conditioned media was used to model the pro-inflammatory microenvironment. Results Prostate cancer cells were found to be resistant to SMAC mimetic therapy alone and in the presence of TNFα or TRAIL. FLIP expression was found to be a resistance mechanism to SMAC-mimetic treatment. Depletion of FLIP expression by siRNA or HDACi therapy resulted in an increased sensitivity to TNFα, TRAIL and macrophage conditioned media alone and in combination with SMAC mimetics. Targeting FLIP alone may therefore be a more effective means of harnessing the pro-inflammatory microenvironment. Moreover, the apoptosis induced in the presence of macrophage conditioned media by SMAC mimetics in FLIP-depleted cells was demonstrated to be TNFα-dependent using TNFα neutralising antibodies. Conclusion Prostate cancer cell lines are inherently resistant to SMAC mimetic therapy even in the presence of TNFα or TRAIL. However, dual targeting of FLIP and IAPs sensitises CRPC cell lines to microenvironment-derived TNFα. Combined targeting of IAPs and FLIP, or single targeting of FLIP, may be an effective means of treating pro-inflammatory prostate cancer. Citation Format: Christopher McCann, Nyree Crawford, David Waugh, Daniel Longley. Harnessing the pro-inflammatory tumor microenvironment of castrate-resistant prostate cancer to promote apoptosis. [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 C1.

Abstract 16: Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necroptosis in C4-2B osseous metastatic castration-resistant prostate cancer cells

Abstract Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis in part due to enhanced aerobic glycolysis and biomass production, known as Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4μM SIM and 2mM MET (SIM + MET) led to significant G1-phase cell cycle arrest and decrease in percentage of DNA-replicating cells in S-phase by 24h; arrest was sustained throughout 96h treatment. SIM + MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagy as a mechanism of cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM + MET treatment led to Ripk1- and Ripk3-dependent necroptosis by 48-96h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necroptotic-induction and viability rescue with necrostatin-1 (Ripk1 activity inhibitor) and Ripk3-targeting siRNA. The necroptosis-inducing capacity of SIM + MET may reinvent these drugs as highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells. Citation Format: Melissa A. Babcook, R. Michael Sramkoski, Hisashi Fujioka, Firouz Daneshgari, Alexandru Almasan, Sanjeev Shukla, Sanjay Gupta. Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necroptosis in C4-2B osseous metastatic castration-resistant prostate cancer cells. [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 16. doi:10.1158/1538-7445.AM2015-16

Abstract 1865: TRIM24 acts as a transcriptional co-activator of the androgen receptor during prostate cancer progression

Abstract The steroid hormone androgen mediates a wide range of developmental, physiological and malignant responses by acting as an agonist for the androgen receptor (AR). AR in turn functions as a nuclear receptor transcription factor and executes specific gene expression programs in an androgen-dependent manner. Since the growth of prostate cancer cells initially depends on androgen, cancer therapy uses hormone-deprivation approaches to reduce the levels of androgen. While prostate cancer (PC) initially responds to androgen-ablation, most tumors progress to a castration resistant (CR) state insensitive to treatment. Both the androgen-dependent and the CRPC state depend on AR, which is probably activated by alternative molecular pathways in CRPC in response to low androgen levels. Here, we assessed the role of the transcriptional co-activator TRIM24 in mediating AR-dependent gene expression and growth in our CRPC cell model LNCaP-abl. We observed that knock-down of TRIM24 impairs CRPC cell growth, while TRIM24 and AR physically interact in these cells in the absence of androgen. When we identified TRIM24-dependent gene expression programs by combining genome-wide binding analyses with microarray studies, we found that TRIM24 and AR co-regulated many genes in CRPC cells. TRIM24- and AR-co-activated genes often function in mitosis and their expression is higher in patients with an increased chance of prostate cancer recurrence. These findings are corroborated by IHC-analyses of prostate cancer patients, where higher TRIM24 protein levels correlated with a greater chance of biochemical recurrence, higher Gleason scores and larger tumor sizes. Importantly, TRIM24 staining was positively correlated with AR staining. This supports a model where TRIM24 acts as an important activator of AR in the castration-resistant setting and therefore may harbor therapeutic potential. Citation Format: Anna C. Groner, Jonas de Tribolet-Hardy, Jean-Philippe Theurillat, Peter J. Wild, Myles Brown. TRIM24 acts as a transcriptional co-activator of the androgen receptor during prostate cancer progression. [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 1865. doi:10.1158/1538-7445.AM2015-1865

TNF signaling mediates an enzalutamide-induced metastatic phenotype of prostate cancer and microenvironment cell co-cultures.

The dramatic responses tumors display to targeted therapies are limited by acquired or pre-existing mechanisms of therapy resistance. We recently discovered that androgen receptor blockade by the anti-androgen enzalutamide paradoxically enhanced metastasis and that these pro-metastatic effects were mediated by the chemoattractant CCL2. CCL2 is regulated by TNF, which is negatively regulated by androgen signaling. Thus, we asked if TNF mediates the pro-metastatic effects of enzalutamide. We found that androgen withdrawal or enzalutamide induced TNF mRNA and protein secretion in castration resistant prostate cancer (C4-2) cells, but not in macrophage-like (THP1) or myofibroblast-like (WPMY1) cells. Androgen deprivation therapy (ADT) induced autocrine CCL2 expression in C4-2 (as well as a murine CRPC cell line), while exogenous TNF induced CCL2 in THP1 and WPMY1. TNF was most potent in myofibroblast cultures, suggesting ADT induces CCL2 via paracrine interactions within the tumor microenvironment. A soluble TNF receptor (etanercept) blocked enzalutamide-induced CCL2 protein secretion and mRNA, implying dependence on secreted TNF. A small molecule inhibitor of CCR2 (the CCL2 receptor) significantly reduced TNF induced migration, while etanercept inhibited enzalutamide-induced migration and invasion of C4-2. Analysis of human prostate cancers suggests that a TNF-CCL2 paracrine loop is induced in response to ADT and might account for some forms of prostate cancer therapy resistance.

Effect of cabazitaxel on the growth of human castration-refractory prostate cancer cells in vitro compared to docetaxel and on the anti-tumor properties of the angio-inhibitor PEDF in vivo.

205 Background: Docetaxel/DTX and cabazitaxel/CBZ have shown promise in the treatment of metastatic Castration-Refractory Prostate Cancer/mCPRC however, comparative studies are missing. Toxicities of these drugs are significant, urging the need to modify taxane regimens. Recently, low-dose metronomic/LDM treatments using conventional chemotherapeutic drugs have shown benefits in CPRC in improving the effect of anti-angiogenic agents. Previously, we have demonstrated that LDM-DTX in combination with PEDF curbs significantly CRPC growth, limits metastases formation and prolongs survival in vivo. In this study, we intended to compare the cytotoxic effect of CBZ and DTX on CRPC cells in vitro and CL1 tumors in vivo. Methods: PC3, DU145 cell lines were from ATCC.CL1 cells were obtained from androgen-deprived LNCaP cells. Cell proliferation was assessed by crystal violet staining and cell cycle analyses. In vitro cytotoxicity assays were performed on CL1 cells/RAW264.7 macrophages co-cultures treated with PEDF and increasing doses of taxanes. For the in vivo studies, CL1 cells were engineered to stably express the DsRed Express protein +/- PEDF. PEDF anti-tumor effects were assessed on s.c. xenografts treated with DTX (5mg/kg ip ev. 4 day) as reference, CBZ (5mg/kg ip ev. 4 days, 1mg/kg for 10 days, 0.5mg/kg q.a.d. and 0.1mg/kg daily) or placebo. Results: CBZ limits cell proliferation with a greater efficacy than DTX in all CRPC cell lines tested. DU145 presented the largest difference. High doses of taxane blocked tumor cells in mitosis, whereas LDM increased the SubG1 population. This effect was significantly higher in DU145 cells treated with CBZ. In vivo, 5mg/kg CBZ delayed tumor growth more efficiently than 5mg/kg DTX. PEDF/5mg/kg CBZ markedly delayed tumor growth compared to all treatments. Finally, engulfment of tumor cells by macrophages was higher in combined treatments suggesting an inflammation-related process. Conclusions: CBZ is more efficient than DTX both in vitro and in vivo.The data also reinforce PEDF as a promising anti-neoplasic agent in combination with LDM taxane chemotherapies.

Abstract 675: Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer

Abstract Inhibition of the androgen receptor (AR) results in activation of the PI-3K/mTOR pathway in PTEN deficient prostate cancer cells by reciprocal feedback inhibition (Carver BS, 2011). Enzalutamide or PI-3K/mTor inhibition result in feedback activation and combined inhibition abrogated this effect. We explored AR inhibition in CRPC cell lines with low, intermediate and high AR expression in the context of PI-3K/mTor inhibition to identify biomarkers of response and resistance. We utilized no/low (DU145, PC3), intermediate (LNCaP) and high (22Rv1,VCap) AR expressing prostate cancer (PC) cell lines to evaluate anti-tumor activity with 4 pan-PI3K/mTOR inhibitors (GDC-0980, GDC-0941, LY294002, PF-04691502), enzalutamide and abiraterone as single agents or various in combinations at clinically relevant doses. Cytotoxic activity of enzalutamide was more potent than abiraterone (IC50's was < 1µM and > 10µM) in LNCaP cells, however, the opposite was seen in DU145 and PC-3 cell lines. High AR expressing cells (22Rv1, VCap) were more sensitive to AR inhibition. PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 had IC50's in the range 50-500nM while LY294002 had an IC50 16-24 µM except for DU145. This cell line had increased IC50 values comparatively for PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 (1.5-3.75 µM) and LY29002 (48.5 µM). The combination index (CI) isobologram method indicated significant synergism for PI-3K/mTOR SMIs plus enzalutamide or abiraterone in all PC cell lines irrespective of AR status. Combination treatments were associated with increased apoptosis (Annexin-V staining and PARP-cleavage assay) in LNCaP and VCaP > 22Rv1 > DU145 and PC3. Cell cycle analysis by flow cytometry demonstrated that enzalutamide induces G1 arrest in LNCaP and DU145 cells and G2/M arrest in PC3 cells. PI-3K inhibition induces S arrest in LNCaP and DU145 and G2/M arrest in PC3 cells. The combination of enzalutamide plus PI-3K inhibition enhanced S arrest in LNCaP while in DU145 and PC3, G0/G1 arrest was prominent. PI-3K SMI plus enzalutamide completely inhibited pAkt308/473, pS6 and p4EBP1 in LNCaP cells, pAkt473 in PC3 cells and pS6 and p4EBP1 in 22Rv1 cells. Enzalutamide alone or in combination with PI-3K inhibition also suppressed AR expression in 22Rv1 cells. A 22Rv1 mouse xenograft model is evaluating safety and efficacy of the combination. This is a novel therapeutic strategy for CRPC patients to be evaluated in a clinical trial. Citation Format: Daruka Mahadevan, Carla Morales, Laurence S. Cooke, Bradley Somer, Wenqing Qi. Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2014-675

758O - Selinexor (Kpt-330), an Oral, Selective Inhibitor of Nuclear Export (Sine) Shows Anti-Prostate Cancer (Prca) Activity Preclinically & Disease Control in Patients (Pts) with Chemotherapy Refractory, Castrate-Resistant Prostate Cancer (Crpc)

ABSTRACT Aim: Exportin 1 (XPO1) is a nuclear export protein whose inhibition leads to the nuclear accumulation of tumor suppressor proteins (TSPs). XPO1 is overexpressed in many tumors including PrCa. Selinexor is an oral SINE that activates ≥10 TSPs with potent activity against various cancers including CRPC. Methods: Selinexor mediated nuclear localization of TSP & apoptosis induction was evaluated in CRPC cell lines & in pt derived tumors in vitro & xenografts (po 10 mg/kg QoDx3). Oral selinexor was given at 8 doses (twice weekly) 28-day cycle as part of an ongoing Phase 1 study in advanced solid tumors (KCP-330-002, NCT01607905). Response evaluation was every 2 cycles (CT & bone scans). Pts had progressing, chemotherapy refractory CRPC on study entry. Results: Selinexor induced nuclear accumulation of p53, p21, FOXO3a, & p27 in PrCa cells as well as nuclear retention of AR in androgen depleted PrCa cells. Oral selinexor showed strong (∼100%) inhibition of tumor growth in pt derived CRPC xenografts. 11 pts with CRPC (median 65 years; ECOG PS 0/1: 5/6; median prior regimens: 5.5 (range 2-10) received selinexor across 3 dose levels (35-65 mg/m2) & 8 were evaluable for response. All pts received taxanes & 5 pts either abiraterone and/or enzalutamide prior to selinexor. Grade 4 toxicities include leukopenia in 1 pt. The most common AEs (Grade 1/2/3) include: fatigue (18%/27%/18%), nausea (27%/18%/0%), anorexia (18%/27%/0%) & thrombocytopenia (9%/18%/9%). Supportive care with dexamethasone (9 pts, 8-24 mgs/weekly or equivalent), appetite stimulants & anti-emetics improved constitutional symptoms. Pharmacokinetics & pharmacodynamics showed dose-dependent increases in Cmax/AUC0-inf & in XPO1 mRNA. PSA & pain reductions were observed, 7 of 8 evaluable pts achieved prolonged SD (114-300+ days). 1 pt progressed during treatment. Conclusions: Selinexor shows potent activity in preclinical models of CRPC with activation of TSPs & retention of nuclear AR. Single agent oral selinexor induced long-term disease control in pts with chemotherapy-resistant CRPC. Future studies in pts with CRPC are planned in 2014. Disclosure: All authors have declared no conflicts of interest.