Splicing factor TRA2B enhances synthesis of androgen receptor variant AR-V7 in prostate cancer cells

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract In patients with castration-resistant prostate cancer (CRPC), clinical resistances such as androgen receptor (AR) mutation, AR overexpression, and AR splice variants (ARVs) limit the effectiveness of second-generation antiandrogens (SGAs). Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. Here, we found and identified a bifunctional small molecule Z15, which is both an effective AR antagonist and a selective AR degrader. Z15 could directly interact with the ligand-binding domain (LBD) and activation function-1 region of AR, and promote AR degradation through the proteasome pathway. In vitro and in vivo studies showed that Z15 efficiently suppressed AR, AR mutants and ARVs transcription activity, downregulated mRNA and protein levels of AR downstream target genes, thereby overcoming AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance in CRPC. In conclusion, our data illustrate the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment. Editor's evaluation The present study reports the discovery and preclinical evaluation of a novel therapeutic agent for the treatment of castration-resistance prostate cancer through inducing degradation of androgen receptor. The major strength of this study is the identification of a novel lead compound and its interesting in vitro and in vivo activities in prostate cancer models. https://doi.org/10.7554/eLife.70700.sa0 Decision letter eLife's review process Introduction Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death for men in western countries (Siegel et al., 2022; Sung et al., 2021). Advanced PCa initially responds to androgen deprivation therapy (ADT), but invariably fails and recurs as lethal castration-resistant prostate cancer (CRPC) (Harris et al., 2009; Desai et al., 2021). Androgen receptor (AR) signaling plays a crucial role in the progress and survival of CRPC (Dai et al., 2017). Second-generation antiandrogens (SGAs), such as enzalutamide (ENZa), abiraterone, apalutamide, and darolutamide, improve the overall survival time and decline prostate-specific antigen (PSA) levels in patients with CRPC (Sternberg et al., 2020; Armstrong et al., 2019; Smith et al., 2021; Smith et al., 2022; Ryan et al., 2015; de Bono et al., 2011). Despite the initial benefit of these agents, their success in treating CRPC has been eliminated by the emergence of drug resistance. Multiple possible mechanisms for the development of drug resistance have thus far been identified, including mutations in the AR LBD, amplification of AR, expression of AR splice variants (ARVs), and intra-tumoral de novo androgen synthesis (Buttigliero et al., 2015; Robinson et al., 2015; Karantanos et al., 2015). Therefore, more effective therapies are urgently required to conquer the SGAs drug resistance. Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. The first strategy is to develop new competitive antiandrogens targeting the AR hormone-binding pocket (HBP) site, such as darolutamide (Smith et al., 2022). Another strategy is to target the AR signaling axis beyond the HBP site, which includes activation function-1 (AF1), activation function-2, binding function 3, and the DNA binding site through active compounds, such as EPI-001, VPC-14449 (Caboni and Lloyd, 2013). Recently, down-regulating both AR protein and AR mRNA levels has attracted attention due to their potential in the discovery and development of new antiandrogens. The most exciting progress is AR degradation based on the proteolysis targeting chimeras (PROTACs) concept, with various of these AR PROTACs developed with a DC50 (drug concentration that results in 50% protein degradation) potency up to 1 nM. However, low cell permeability, poor pharmacokinetic properties, and complex chemical structures may restrict the clinical application of PROTAC drugs (He et al., 2020). What’s more, LBD-targeted AR PROTACs cannot degrade ARVs which were associated with unfavorable clinical outcomes in patients with CRPC (Fettke et al., 2020). The selective estrogen receptor degrader fulvestrant approved by the FDA in 2002 expanded treatment choices for advanced breast cancer (Bross et al., 2003), which gave rise to next-generation novel degraders with promising antitumor activity in recent years (Nardone et al., 2019). Bradbury et al., 2011 suggested that similar specific downregulation or degradation of AR might be proved beneficial in the treatment of CRPC. Therefore, selective AR degraders (SARDs) which could synthetically degrade and antagonize AR may be an efficient strategy to overcome the drug resistance in the antiandrogen therapy of CRPC. Based on structural modification of the AR antagonists and the tissue-selective AR agonist enobosarm, Miller et al. designed a series of SARDs, namely UT-155, UT-69, and UT-34, which could induce AR ubiquitin-proteasome degradation via binding to AF-1 of the AR to reduce its stability (Ponnusamy et al., 2017; Ponnusamy et al., 2019; Hwang et al., 2019). Notably, the degradation potency of these compounds for ARVs is quite limited. In the present study, we determined that Z15 screened by rational drug design as an AR antagonist and degrader via direct binding to the AR LBD and AR AF1, could overcome AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance of CRPC in vitro and in vivo. Results Identifying Z15 as an AR inhibitor To develop novel AR inhibitors and overcome antiandrogen resistance, we previously constructed a common molecular characteristic pharmacophore model, and screened ~7.5 million compounds from the ZINC lead-like database and ChemDiv database. About 47,202 compounds matched more than four features of the filtering model. Next, these compounds were docked into the HBP of the antagonistic AR. Then, compounds with the top 1000 docking scores were chosen for ADMET prediction by Discovery Studio v3.5. Finally, 80 hits with high drug-likeness were selected and purchased for further bioactivity evaluation (Figure 1—figure supplement 1 and Supplementary file 1a). To preliminarily evaluate the influences for AR transcriptional activity of these 80 candidates, human prostate cancer cells PC-3 co-transfected with wild-type AR (wt-AR) and PSA-luc were incubated with 5α-dihydrotestosterone (DHT) and 10 μM candidate compounds for 24 h. The cell lysates were collected and AR transcriptional activity was detected by dual-luciferase reporter assay. We identified 19 compounds that showed more than 25% AR transcription inhibition activity, among which compound Z15 (structure shown in Figure 1A) exhibited the most potent AR inhibition activity (Figure 1—figure supplement 2A). Nevertheless, the glucocorticoid receptor (GR) transcription inhibition activity of Z15 was quite feeble (Figure 1—figure supplement 2B–C). Figure 1 with 4 supplements see all Download asset Open asset Z15 specifically inhibits the transcription activity of AR and AR mutants. (A) Chemical structure of Z15. (B) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla, AR, and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 hr. (C) LNCaP, (D) VCaP, (E) and 22Rv1 cells co-transfected with Renilla and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 h. (F) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in LNCaP cells stimulated by 5 nM DHT, and treated with different concentrations of Z15 or ENZa for 24 hr. (G) Dual-luciferase reporter assays to measure MMTV-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla and MMTV promoter expression vector plasmids stimulated by 100 nM Dex, and treated with different concentrations of Z15 for 24 hr. (H) Dual-luciferase reporter assays to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla, AR_T877A mutation, and PSA promoter expression vector plasmids stimulated by 5 nM DHT treated with different concentrations of Z15 for 24 hr. (I) PC-3 cells co-transfected with Renilla, AR_F876L mutation, and PSA promoter expression vector plasmids, treated with different concentrations of Z15 for 24 hr. All experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DHT or Dex group. ENZa, enzalutamide; DHT, dihydrotestosterone; Dex, dexamethasone; Mif, mifepristone. Z15 selectively suppresses AR and AR mutant transcriptional activity To further investigate the AR inhibition potency of Z15, we optimized the synthesis route and prepared a sufficient amount of Z15 (Figure 1—figure supplement 3). Next, we performed a dual-luciferase reporter assay in several human PCa cell lines including wt-AR-transfected PC-3 and LNCaP cells. The results indicated that Z15 could inhibit DHT-induced transcriptional activities of both exogenous and endogenous AR in a dose-dependent manner (Figure 1B–C). Unexpectedly, Z15 showed potent AR transcription inhibition activity in AR overexpression and ENZa-insensitive VCaP cells (Figure 1D). In another ENZa resistance 22Rv1 cells which naturally express AR and ARV7, Z15 also inhibited DHT-activated AR transcriptional activity (Figure 1E). Moreover, the AR transcription inhibition IC50 (half-maximal inhibitory concentration) of Z15 in LNCaP cells was ~0.22 μM, which was comparable to ENZa (Figure 1F). We further detected the selectivity of Z15 in GR-positive PC-3 cells, the results indicated that Z15 hardly inhibited dexamethasone activated GR transcriptional activity compared to the GR antagonist mifepristone (Figure 1G). Then, we compared AR, GR, estrogen receptor (ER), and progesterone receptor (PR) transcription inhibition activities of Z15 by dual-luciferase reporter assay. The transcription inhibition IC50 of Z15 was 0.41 μM for AR (Figure 1—figure supplement 4A), over 20 μM for GR and ER (Figure 1—figure supplement 4B–C), and 9.29 μM for PR (Figure 1—figure supplement 4D), which suggests that Z15 is a highly selective AR inhibitor. AR LBD point mutations such as AR T877A (a flutamide-resistant mutation) and AR F876L (ENZa- and apalutamide-resistant mutation), are key causes leading to antiandrogen resistance. Dual-luciferase reporter assay results indicated Z15 could efficiently inhibit DHT-induced both AR T877A and AR F876L transcriptional activities (Figure 1H–I). Taken together, these data illustrate Z15 as a potent selective AR inhibitor both for wild-type and mutated ARs. Z15 inhibits the AR pathway Next, we assessed the influence of Z15 on LNCaP cells transcriptome by RNA-sequencing analysis. Obviously, Z15 dose-dependently inhibited a series of DHT-activated AR downstream genes (Figure 2A). Then, we detected three canonical AR downstream-regulated genes (PSA, PMEPA1, and TMPRSS2) by quantitative real-time PCR (qRT-PCR) assay. The results revealed that Z15 significantly inhibited the mRNA expression levels of these genes (Figure 2B), consistent with the findings of RNA-sequencing. Furthermore, Z15 also decreased DHT-induced PSA mRNA levels in the antiandrogen resistance 22Rv1 and VCaP cells (Figure 2C). Figure 2 with 3 supplements see all Download asset Open asset Z15 downregulates AR target genes and ARlevels. (A) LNCaP cells treated with vehicle, 0.5, or 5 μM Z15 in the presence of 5 nM DHT for 24 hr before performing RNA-sequencing. Heatmap shows the expression levels of AR target genes. (B) The mRNA levels of PSA, PMEPA1, and TMPRSS2 measured by quantitative-PCR and normalized to GAPDH in LNCaP cells treated with vehicle or different concentrations of Z15 in the presence of 5 nM DHT for 24 hr. (C) The mRNA levels of PSA measured by quantitative-PCR and normalized to GAPDH in 22Rv1 and VCaP cells treated with vehicle or different concentrations of Z15 in the presence of 5 nM DHT for 24 hr. (D) Western blot analysis of LNCaP cells treated with indicated concentrations of Z15 in the presence of 5 nM DHT for 24 hr, before cell lysing and determining PSA and AR protein levels. (E) Western blot analysis performed in 22Rv1 cells. (F) Western blot analysis performed in VCaP cells. (G) Western blot analysis of LNCaP cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr, before cell lysing, and determining AR protein levels. (H) Western blot analysis of 22Rv1 cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr, before cell lysing, and determining AR protein levels. Experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DHT group. We further detected the influence of Z15 on AR and PSA protein levels in LNCaP cells. As demonstrated in Figure 2D, Z15 reduced DHT-activated PSA protein levels significantly, which was in line with the qRT-PCR analysis. Surprisingly, AR protein levels were also downregulated by Z15, quite different from the effects of ENZa (Figure 2—figure supplement 1A–B). Notably, Z15 potently inhibited PSA and AR protein levels in ENZa resistance 22Rv1 and VCaP cells (Figure 2E–F and Figure 2—figure supplement 1C–G). Then, we evaluated the AR DC50 of Z15 in LNCaP and 22Rv1 cells. The AR DC50 of Z15 in LNCaP cells was 1.05 μM (Figure 2G and Figure 2—figure supplement 1H), while in 22Rv1 cells it was 1.16 μM and the ARV7 DC50 was 2.24 μM (Figure 2H and Figure 2—figure supplement 1I). In addition, we performed a 4D-label free proteomics study to analyze the effect of Z15 on global protein levels in LNCaP cells. Among 5334 quantifiable proteins, AR LBD-targeted PROTAC molecule ARV-110 significantly reduced 34 proteins and Z15 downregulated 69 proteins compared to the DHT group (Figure 2—figure supplement 2 file 1d-e). Both Z15 and ARV-110 reduced AR, KLK3, and TMPRSS2 protein levels significantly (Figure 2—figure supplement 2A–B). KEGG analysis also proved that these two compounds had a similar influence on the functional pathways (Figure 2—figure supplement 2C–D). Additionally, to verify the specificity of Z15 downregulated AR protein levels, we chose 3 AR pathway related but independent proteins GR, HSP90 (AR chaperonin), and cyclin-dependent kinases 7 (CDK7) as controls. Western blot analysis indicated that Z15 has no influence on GR, HSP90, and CDK7 protein levels in 22Rv1 cells (Figure 2—figure supplement 3). Collectively, these data suggest that Z15 is a novel specific AR pathway inhibitor, which may play a role as an AR antagonist as well as an AR and ARV7 degrader. Z15 inhibits DHT-induced AR nuclear translocation Androgen-binding initiates AR activation, induces its conformational change, and reveals the nuclear localization signal of AR. The hormone-bound AR dimerizes and translocates to the nucleus, where it binds to DNA and interacts with a series of transcriptional coregulators to regulate target gene expression. Accordingly, we investigated whether Z15 disturbed androgen-induced AR nuclear translocation. As shown in Figure 3A–B, the DHT treatment could promote the importing of AR into the nuclear compared to untreated group, while both ENZa and Z15 blocked DHT-induced AR nuclear translocation. This result proves that Z15 can inhibit DHT-induced AR nuclear translocation. Figure 3 Download asset Open asset Z15 inhibits AR nuclear localization. (A) Nuclear localization of AR in LNCaP cells treated with vehicle or 5 μM compounds in the presence of 5 nM DHT for 4 h. (B) Quantitative analysis of AR nuclear localization.Experiments were performed in triplicate. Z15 binds directly to AR LBD and AR AF1 Since the chemical structure of Z15 is remarkably different from that of previously reported AR antagonists, we next evaluated whether Z15 directly binds to AR in a similar manner as ENZa. The AR competitive binding assay was performed to demonstrate the direct interaction between Z15 and AR, whereby compounds in competition with the radioligand [3H] DHT in cytosolic lysates from LNCaP cells were measured. Synthetic androgen R1881 displayed strong binding potency to AR with an IC50 value of 0.45 nM, which indicated the feasibility of this assay system. The binding affinity between ENZa and AR was 121.2 nM. Interestingly, Z15 showed a comparable binding affinity to ENZa, with an IC50 value of 63.3 nM (Figure 4A). In addition, our fluorescence polarization assay demonstrated Z15 could compete with androgen binding to AR LBD (Figure 4—figure supplement 1). Besides, the biolayer interferometry (BLI) measurement also revealed that both ENZa and Z15 possess AR LBD binding ability (Figure 4B, Figure 4—figure supplement 2A). These data suggested that Z15 could antagonize AR by directly targeting the LBD region. AR LBD targeted compound ARV-110 has been shown as an efficient AR degrader in preclinical research, however, it could not induce ARV7 degradation in 22Rv1 cells (Figure 4—figure supplement 3A–C). Since Z15 could degrade both AR and ARV7, we wondered if Z15 could also bind to other regions of AR to induce ARV7 degradation. Hence, we investigated the binding affinity between Z15 and AR AF1, as AF1 is an important drug target region of AR. The surface plasmon resonance assay indicated that Z15 could directly bind to AR AF1 with a KD value of 0.93 μM (Figure 4C). Z15 was also detected to potently bind to AR AF1 with a comparable binding affinity to AR AF1 inhibitor UT-34 by BLI assay (Figure 4—figure supplement 2B–C). Unexpectedly, UT-34 could not induce ARV7 degradation in 22Rv1 cells from western blot analysis (Figure 4—figure supplement 3D–F). As a control, we did not find any binding potency between AR AF1 and ENZa even at 200 μM (Figure 4—figure supplement 2D). These data illustrate that Z15 potently inhibits ARV7 by directly binding to AR AF1. Figure 4 with 3 supplements see all Download asset Open asset Z15 directly binds to AR. (A) Competitive binding assay to detect binding affinity of R1881, ENZa, and Z15 to AR LBD, 1 nM radioligand [3H] DHT and LNCaP cytosol were used. (B) Biolayer interferometry measurements of Z15 binding to AR LBD. (C) Sensorgram and steady state fitted results of surface plasmon resonance assay to detect binding affinity between Z15 and AF1. Experiments were performed in triplicate. Z15 promotes AR degradation through the proteasome pathway We have shown that Z15 could reduce AR and ARV7 protein levels and conjectured that it is an AR degrader. To confirm this hypothesis, we detected the influence of Z15 on AR protein and mRNA levels in LNCaP cells without DHT treatment. Certainly, Z15 reduced AR protein levels in a dose-dependent manner without influencing the AR mRNA levels (Figure 5A and Figure 5—figure supplement 1A). Moreover, we observed similar effects of Z15 on AR protein and mRNA levels in ENZa resistance cell lines 22Rv1 (Figure 5B and Figure 5—figure supplement 1B–C) and VCaP cells (Figure 5C and Figure 5—figure supplement 1D). Western blot analysis for AR in LNCaP cells treated with protein synthesis inhibitor cycloheximide, showed that Z15 accelerated AR degradation (Figure 5D and Figure 5—figure supplement 1E). These data indicate that Z15 is indeed an AR degrader. Figure 5 with 1 supplement see all Download asset Open asset Z15 promotes AR degradation in proteasome pathway-dependent manner. A-C Western blot analysis of AR protein levels, and quantitative-PCR normalized to GAPDH of AR mRNA levels in LNCaP (A), 22Rv1 (B), and VCaP (C) cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr. (D) Western blot analysis of AR in LNCaP cells treated with 100 μg/mL CHX in the presence or absence of 5 μM Z15 for indicated time points. (E) Western blot analysis of AR protein levels in LNCaP and VCaP cells treated with 5 μM Z15 or/and 5 μM MG 132 for 8 hr. (F) Immunoprecipitation done using anti-AR and immunoblotting with anti-Myc antibody in 22Rv1 cells co-transfected with Myc-tag CW7-UB plasmids treated with or without 5 μM Z15 in the presence of 5 μM Mg132 for 12 hr. Input: immunoblot of lysates probed with AR antibody. Experiments were performed in triplicate. All results are shown as mean ± sd. CHX, cycloheximide. The ubiquitin-proteasome pathway (UPP) is the main participant that regulates intracellular protein degradation. To explore whether Z15 promoted AR degradation through UPP, LNCaP cells were treated with Z15 in the presence or absence of proteasome inhibitor MG132. Indeed, Z15 reduced the AR protein levels after 8 hr treatment, while AR protein levels reduction was counteracted by MG132. Similarly, Z15 induced AR protein decline was also counteracted by MG132 in VCaP cells (Figure 5E and Figure 5—figure supplement 1F–G). Furthermore, Z15 treatment strikingly induced ubiquitination of AR (Figure 5F). Together, these results indicate that Z15 degrades AR through the UPP. Z15 inhibits proliferation and induces in CRPC cell lines As Z15 exhibited AR and ARV7 inhibition and degradation we next investigated the effects of Z15 on cell proliferation activity in CRPC cell lines VCaP and 22Rv1 cells. In VCaP cells, Z15 showed comparable proliferation inhibition potency with ARV-110 However, in 22Rv1 cells, the proliferation inhibition activity of Z15 was than that of ARV-110 Both Z15 and ARV-110 displayed inhibition effects on the proliferation activity of PC-3 and cells (Figure To the effects of Z15 on CRPC cell activity, we 22Rv1 and PC-3 cells to 1 μM Z15 or ARV-110 for 2 As a Z15 significantly decreased the 22Rv1 cell compared to both and both Z15 and ARV-110 showed no influence on the PC-3 cell (Figure we proved that through and AR, Z15 could selectively inhibit the proliferation of AR and ARV7 CRPC cell Furthermore, based on PCa we PCa and treated the with 1 μM Z15 for 7 The results indicated that Z15 significantly inhibited PCa proliferation compared to the group (Figure What’s more, western blot analysis indicated that Z15 also promoted the of VCaP and 22Rv1 cells in a dose-dependent manner and while Z15 showed no influence on the of AR cells (Figure and Figure supplement Figure with 1 supplement see all Download asset Open asset Z15 selectively inhibits proliferation and induces of CRPC cells. (A) VCaP, and PC-3 cells treated with different concentrations of Z15 or ARV-110 for hr, cell proliferation detected by assay. (B) of PC-3 and 22Rv1 cells treated with or without 1 μM Z15 or ARV-110 for were (C) PCa treated with 1 μM Z15 or for 7 observed by (D) Western blot analysis of protein levels in VCaP cells treated with indicated concentrations of Z15 for 24 hr. (E) Western blot analysis of protein levels in 22Rv1 cells treated with indicated concentrations of Z15 for 24 hr. Experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs group. Z15 inhibits CRPC experiments proved that Z15 is a selective AR degrader and antagonist with activity in To evaluate the PCa inhibition activity of Z15 in we of 22Rv1 cells in the of an of were treated with vehicle control, 10 Z15, or 20 Z15 for were no effects observed in as assessed by the (Figure of with 10 and 20 Z15 both suppressed 22Rv1 and decreased the significantly (Figure In addition, western blot analysis indicated that AR, ARV7, and PSA protein levels in the were significantly in both 10 and 20 Z15 treatment (Figure Figure supplement analysis also revealed that Z15 reduced the and PSA protein levels in (Figure Taken together, our data indicate that Z15 could inhibit the of CRPC both in vitro and in vivo. Figure 7 with 1 supplement see all Download asset Open asset Z15 suppresses 22Rv1 progress in vivo. (A) from 22Rv1 cells treated with control, or 20 Z15 a for by (B) other (C) on the (D) Western blot analysis of AR, ARV7, and PSA protein levels in (E) analysis of proliferation and PSA levels in Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs group. Identifying Z15 as AR inhibitor Since Z15 showed CRPC inhibition we the and ZINC database to Z15 chemical structure compounds (Figure supplement Supplementary file with more than to Z15 were for further bioactivity Dual-luciferase reporter assay indicated that most of these compounds inhibited DHT-activated AR transcriptional activity at 1 μM for (Figure supplement that the group plays an role in the AR inhibitory activity of Z15 and its Western blot analysis revealed that these active also reduced AR and DHT-induced PSA protein levels (Figure supplement Furthermore, we detected the AR transcription inhibition IC50 of active Z15 Among these and exhibited the AR transcription inhibition while also showed comparable AR inhibition activity compared to Z15 (Figure Western blot analysis revealed that these active could reduce AR and PSA protein levels in a dose-dependent manner. Notably, of these compounds showed AR downregulation activity than Z15 (Figure and Figure supplement Together, these results indicate that through chemical structural modification to Z15, more and more selective AR degraders with AR inhibition activity might be found in the Figure 8 with 3 supplements see all Download asset Open asset Z15 comparable AR inhibition (A) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in LNCaP cells stimulated by 5 nM DHT, and treated with different concentrations of indicated compounds for 24 hr. (B) AR transcription inhibition (C) Western blot analysis of PSA and AR protein levels of LNCaP cells treated with indicated concentrations of Z15 and its in the presence of 5 nM DHT for 24 hr. Results are shown as mean ± sd. Experiments were performed in triplicate. Discussion SGAs are more in the clinical treatment of patients with CRPC. However, drug resistance by AR mutation, AR amplification, and has been reported to restrict the clinical of these therapies (Buttigliero et al., 2015; Robinson et al., 2015). AR a crucial target for CRPC therapeutic development of its key function in the progress of CRPC. In this study, we identified a compound Z15 that selectively inhibited AR transcriptional activity and significantly downregulated AR target genes at the mRNA and protein levels. studies proved that Z15 could bind directly to both AR LBD and AR AF1, as to androgen-induced AR nuclear which Z15 as an AR Moreover, Z15 could also degrade AR and ARV7 through the proteasome pathway (Figure several Z15 exhibited AR inhibition and downregulation potency than Z15, that Z15 is a promising lead compound for further chemical structure Figure Download asset Open asset The of Z15 inhibits the AR pathway and antiandrogen resistance. Z15 binds to both AR LBD and AR AF1, AR nuclear AR promotes AR and ARVs degradation through the proteasome as to overcome AR mutation, AR overexpression, and ARVs-induced antiandrogen resistance. AR amplification is a common in CRPC patients antiandrogens treatment. data showed that ENZa could hardly DHT-induced PSA levels in VCaP cells, which that AR is to overcome the drug resistance by AR rational to this

The rational drug design combining the bioassay identified a novel selective androgen receptor (AR) degrader for both AR and AR-VRs and illustrated the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment.

The rational drug design combining the bioassay identified a novel selective androgen receptor (AR) degrader for both AR and AR-VRs and illustrated the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment.

Prostate Cancer (PCa) is the third most common cause of death from cancer in men of all ages. Surgical or medical castration is one of the most common treatments for patients with advanced PCa; however a majority of patients develop castration resistant prostate cancer (CRPC), tumor relapse, which remains to be the second leading cause of cancer-related deaths of men in the US. Androgen receptor (AR) signaling is shown to play a critical role in the development and progression of PCa. Genetic aberrations within AR, including constitutively active AR splice variants and AR point mutations have been identified in CRPC. The most common AR splice variants lack the ligand-binding domain (LBD), which is often the target of CRPC therapies. Therefore, presence of these variants may act as a mechanism of resistance to AR-targeted therapies leading to the progression of prostate tumor growth. Additional PCa specific genetic aberrations include fusions between the androgen-related gene, TMPRSS2 and the ETS transcription factors, ERG (predominant) and ETV1. These fusion events are frequently associated with more aggressive prostate cancers leading to poorer prognosis. In this study, we developed TaqMan qRT-PCR assays to evaluate the presence of several previously identified AR splice variants, including ARV1, ARV3/V7, ARV567 and ARV8, AR somatic mutations, including L701H, V715M, H874Y and T877A, along with TMPRSS2 fusion genes, TMPRSS2:ERG and TMPRSS2:ETV1, in two independent PCa FFPET sample sets. The first sample set consisted of 42 Prostate adenocarcinomas ranging from stage II to stage IV. Results showed that ARV1 and ARV3/V7 were the most prevalent variants with 92% of all samples showing expression of either or both variant. TMPRSS2: ERG was present in 72% of all samples tested, with a high concordance to AR variant expression, prevalent in later stage (III/IV) PCa samples. The second sample set consisted of 8 prostate adenocarcinomas, including matched adjacent normal FFPET. Similar expression of the AR variants was observed in both the tumor and matched normal samples, however tumor prostate samples showed a higher and more prevalent expression (66.67%) of the TMPRSS2: ERG fusion gene than in the matched normal samples (33%). None of the four AR mutations evaluated were detected in either sample set. Overall, these findings demonstrate a strong presence of both AR splice variants and the TMPRSS2: ERG fusion gene in the prostate cancer patient population, supporting evidence for a functional role of these markers in PCa diagnosis and disease progression. Furthermore, presence of LBD negative AR splice variants indicates an attractive biomarker for stratification of the patient population resistant to AR targeted therapies. Citation Format: Dana S. Gaffney, Gabriela Martinez, Katherine Bell, Suso Platero, Deborah Ricci, Jayaprakash Karkera. Identification of androgen receptor (AR) splice variants, AR somatic mutations and TMPRSS2:ETS fusion genes in prostate cancer FFPET by qRT-PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2013-5

Androgen Receptor Modulation Optimized for Response—Splice Variant: A Phase 3, Randomized Trial of Galeterone Versus Enzalutamide in Androgen Receptor Splice Variant-7–expressing Metastatic Castration-resistant Prostate Cancer

To the Editor: There are no data regarding expressed and functional characterisation of cytoskeleton-related non-coding RNA has been reported in prostate cancer (PCa). Here, we report a cytoskeleton regulator RNA (CYTOR)-regulated process that mediates castration-resistant PCa (CRPC)-specific androgen receptor splice variant 7 (AR-V7) generation, and further explore the vulnerability of CRPC growth through CYTOR-targeted locked nucleic acid (LNA). We retrieved public castration-sensitive PCa (CSPC) datasets (n = 65), neuroendocrine PCa (NEPC) datasets (n = 49) and CRPC datasets (including two studies, n = 171 and n = 118).1 Across the above RNA-seq data, CYTOR was found to be upregulated in CRPC with low expression in CSPC and NEPC (Figure 1A). RNA in situ hybridisation (RISH) assays2 of our centre samples confirmed the public domain data (Figure 1B and Figure S1A). Consistent with tissue detection, androgen-influenced CYTOR revealed significant increase in our two continuous established castration-resistant cell lines: LNCaP-AI,3 C4-2 Enz-R (Figure 1C and Figure S1B–G). Additionally, progression was more common in CSPC with higher CYTOR expression (Figure 1D). Expression analysis of CYTOR in flash-frozen surgical specimens was conducted in 11 CRPC patients (Figure 1E). Patients with high expression of CYTOR received worse PSA response to subsequential enzalutamide than those with CYTOR low expression (Figure 1F). Then, gene functional assays suggested knockdown of CYTOR suppressed the cancer cells growth (Figure 1G–J). The above results hint the association of CYTOR with CRPC development and inferior clinical outcomes. As it is, the primary therapeutic intervention for advanced PCa is androgen-deprivation therapy (ADT) with the goal of castration to suppress androgen receptor (AR) signalling. Although most patients respond to ADT, some inevitably develop resistance and progress to CRPC because of AR-V7 expression.4 Extensively investigated AR-V7 is a typically truncated AR without the ligand-binding domain but retaining transcriptional-regulated activity to mediate ligand-independent AR signalling.5 RNA-seq analysis revealed many AR-V7 downstream genes were differentially regulated as CYTOR knockdown (Figure 2A, Table S2). Most of them were enriched in metabolic pathways (Figure 2B). We validated the downregulation of AR-V7 canonic-activated genes (Figure 2C) after silencing CYTOR. Interestingly, knockdown of CYTOR resulted in specific decrease of AR-V7 without concurrent decrease of full-length AR (AR-FL) (Figure 2D), suggesting the critical role of CYTOR in AR-V7 mRNA splicing process. Multiplexed RISH assays of CRPC specimens revealed colocalisation and positive correlation of CYTOR and AR-V7 (pre-mRNA accumulated in nuclei) (Figure 2E). Their positive correlation was also confirmed by RT-PCR in four flash-frozen specimens (Figure 2F). Because key RNA-binding protein families involved in alternative splicing may include serine/arginine-rich proteins (SR proteins) and heterogeneous nuclear ribonucleoproteins (hnRNPs), we conducted differential expression analysis of SR proteins and hnRNPs between LNCaP-AI and LNCaP cells by our published RNA-arrays (GSE124291), and screened six upregulated splicing factors in LNCaP-AI cells (>1.5-fold) (Figure 2G). We further confirmed the upregulation of three genes (Figure 2H and Figure S2A). By Human Splicing Finder,6 the similar consensus splice site value for splice junctions of intron 3/cryptic exon 3 (CE3) (80.38) (as in AR-V7) and intron 3/exon 4 (80.1) (as in AR-FL) (Figure S2B) suggested the existence of a mechanism for CRPC-specific CE3 splice site utilisation. Given the established role of SR proteins in binding to pre-mRNA that prevents exon skipping, and the classical role of hnRNPs as splicing repressors, we postulated that nuclear-localised SRSF4 and SRSF7 (Figure 2I) may repress CE3 skipping, thus ensuring the correct 5′ to 3′ linear order of exons (exon1-3/CE3) in AR-V7 mRNA. Indeed, knockdown of SRSF4 or SRSF7 resulted in decreased expression of AR-FL and AR-V7, while withoutimpact on CYTOR (Figure 2J). The catRAPID strength algorithm computed output suggested the high specificity of CYTOR–SRSF4 interaction and CYTOR–SRSF7 interaction, respectively (Figure 2K,L).7 RNA immunoprecipitation results revealed both SRSF4 and SRSF7 proteins interacted with CYTOR, AR-V7 pre-mRNA and AR-V7 mRNA (Figure 2K,L), indicating nuclear binding of SRSF4 and SRSF7 to AR-V7 pre-mRNA and CYTOR was responsible for AR-V7 generation, even though there was weak interaction of SRSF4 and SRSF7 (Figure 2M). According to the functional interaction of CYTOR and SRSF4/7 proteins, we hypothesised that CYTOR may recognise AR-V7 pre-mRNA to induce this splicing process. Toward this end, maximum entropy modeling was used to collect motifs in the intron3/CE3 flanking sequence and identified the 3′ motif (3′ site of intron 3) and the 5′ motif (first 20 bp of CE3) (Figure 3A–C).6 The complementary sequence of the 5′ motif in the sequence of CYTOR (5′-UUCCAACCGC-3′) suggested that CYTOR may recognise the 5′ motif of CE3 (5′-GGGUUGGCAA-3′) to initiate the splicing process (Figure 3C). Next, we designed an 18 bp antisense oligonucleotides (ASO) to the 5′ motif of CE3 (ASOCE3) to prevent the recognition. The ASOCE3 suppressed, in a concentration- and time-dependent manner, the expression of AR-V7 mRNA (Figure 3D). We then designed an 18 bp ASOCYTOR to the complementary sequence of CE3 5′ motif in CYTOR. ASOCYTOR inhibited expression of AR-V7 mRNA without interfering CYTOR expression (Figure 3E and Figure S2C). Also as shown in C4-2 Enz-R cells, the ASOCE3 and ASOCYTOR prevented the generation of AR-V7 mRNA (Figure 3F,G). To validate this splicing model, truncated mutant assays confirmed the pivotal role of 5′-UUCCAACCGC-3′ in CYTOR on AR-V7 expression (Figure S2D,E). Together, CYTOR/SRSF4/SRSF7 complex interacts with AR-V7 pre-mRNA to regulate its splicing by recognising a specific signal element in CE3 (Figure 3H). Then LNAs GapmeRCYTOR were designed to silence CYTOR (Figure 4A). In C4-2 Enz-R cells, AR-V7 expression was largely suppressed in parallel with the silenced pattern of CYTOR in a concentration- and time-dependent manner (Figure 4B–E). GapmeRCYTOR could attenuate the resistance of enzalutamide significantly in vitro (Figure 4F). We then established in vivo mouse models and found that enzalutamide significantly suppressed C4-2 tumours, shCYTOR- and GapmeRCYTOR-treated C4-2 Enz-R tumours (Figure 4G,H). The expressions of CYTOR and AR-V7 were validated in each group (Figure S2F,G). As such, our data suggested that on-target effect of CYTOR knockdown with GapmeRCYTOR can be used as an option in castration resistance to provide therapeutic efficacy. In conclusion, we propose the importance of a novel complex composed of CYTOR/SRSF4/SRSF7 that mediates AR-V7 generation, and a critical role in suppressing PCa progression by targeting CYTOR/AR-V7 axis with shRNA or preclinical LNA GapmerCYTOR. Some of the biospecimens used in the present study were provided by the Chungbuk National University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare, and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The authors wish to thank Ms. Eun-Ju Shim from the National Biobank of Korea at Chungbuk National University Hospital for the sample preparations and her excellent technical assistance. The authors declare they have no conflicts of interest. National Natural Science Foundation of China, Grant Numbers: 91959114, 81872106, 82072851, 81872100, 81972654, 82273262; National Natural Science Foundation of China, International (Regional) Cooperation and Exchange Program, Grant Number: 82061160493; Natural Science Foundation of Tianjin, Grant Numbers: 18PTLCSY00030, 21JCQNJC01700; Tianjin Key Medical Discipline (Specialty) Construction Project, Grant Numbers: TJYXZDXK-023A, TJYXZDXK-065B; The Second Hospital of Tianjin Medical University, Grant Number: 2020ydey01; Scientific Research Project of Tianjin Education Commission, Grant Number: 2021KJ225 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Understanding mechanisms by which advanced prostate cancer (PCa) progresses to a castration resistant PCa (CRPC) phenotype is crucial for tailoring targeted therapies for individual patients. Constitutively active androgen receptor (AR) splice variants that lack the ligand binding domain drive androgen independent growth in cell lines and xenograft models of PCa and their overexpression in CRPC metastases is associated with poor prognosis. However, the mechanism of AR splice variant overexpression in CRPC is not completely understood. We have shown that diverse genomic rearrangements within the AR gene are linked to AR splicing alterations in PCa cell lines, xenografts, and CRPC metastases. To test whether these AR gene rearrangements drive AR splice variant synthesis and a CRPC phenotype, we developed transcription activator-like effector nucleases (TALENs) for precision genome engineering in androgen-sensitive PCa cells. Engineering of specific AR gene rearrangements induced splicing alterations and efficient expression of AR splice variants. Functionally, these genome-engineered cells displayed constitutive, ligand-independent AR transcriptional activity and an androgen-independent growth profile. These fundamental properties of CRPC were blocked following siRNA-mediated knock-down of truncated AR variants. Overall, these data demonstrate that AR gene rearrangements induce fundamental aspects of the CRPC phenotype including constitutive, ligand-independent, AR splice variant activity. Overall, these data advance the concept of AR splice variants as key drivers of advanced disease, and provide new genome-engineered models that could be used to develop agents targeted to these AR-derived species. Citation Format: Michael D. Nyquist, Yingming Li, Scott M. Dehm. Creation of androgen independent prostate cancer cell lines using targeting nucleases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-136. doi:10.1158/1538-7445.AM2013-LB-136

BACKGROUND: The androgen-receptor splice variant 7 (AR-V7) has been implicated in the development of castration resistant prostate cancer (CRPC) and resistance to current therapies including enzalutamide and abiraterone. AR-V7 mRNA expression in circulating tumour cells of patients with CRPC correlated with treatment resistance. However, the importance of AR-V7 has been questioned in light of low AR-V7 mRNA levels relative to the full-length androgen receptor in CRPC and it is critically important to develop validated assays that confirm AR-V7 protein levels and its clinical importance in patients with CRPC. METHODS: Following validation of a monoclonal antibody, immunohistochemical analysis of nuclear AR-V7 (alongside a nuclear AR N-terminal domain antibody; AR-NTD) was performed in a patient cohort identified with matched therapy-naive hormone-sensitive primary prostate cancer (HSPC) and CRPC. We determined the levels of nuclear AR-V7 as patients progressed from HSPC to CRPC. We also determined if AR-V7 expression levels associated with overall survival from time of CRPC biopsy. RESULTS: In our patient cohort (n = 39), nuclear AR-V7 (p = &amp;lt;0.0001) and nuclear AR-NTD (p = 0.0006) increased significantly as patients progressed from HSPC to CRPC. Lower nuclear AR-V7 expression was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (18.7 vs 9.6 months; HR 0.36 [95% CI 0.17-0.62]; p = 0.002), 50th (13.0 vs 9.8 months; HR 0.60 [95% CI 0.29-1.07]; p = 0.09) or 75th (6.0 vs 11.5 months; HR 0.31 [95% CI 0.04-0.34]; p = 0.0004) percentile of AR-V7 expression. Similarly, a lower nuclear AR-V7 to nuclear AR-NTD ratio was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (17.8 vs 9.1 months; HR 0.42 [95% CI 0.22-0.78], p = 0.01), 50th (14.0 vs 8.8 months; HR 0.43 [95% CI 0.17-0.69], p = 0.005) and 75th (11.5 vs 7.3 months; HR 0.39 [95% CI 0.09-0.69], p = 0.009) percentile. Nuclear AR-NTD did not associate with overall survival from CRPC biopsy. CONCLUSION: We provide first evidence that expression of nuclear AR-V7 protein not only increases with emerging treatment resistance in CRPC but also is associated with overall survival from time of CRPC biopsy. These data support AR-V7 protein being key to CRPC progression and that agents targeting AR splice variants will be important to improve patient outcome in CRPC. Citation Format: Daniel Nava Rodrigues, Jon Welti, Adam Sharp, Shihua Sun, Ruth Riisnaes, Ines Figueiredo, Zafeiris Zafeiriou, Pasquale Rescigno, Johann S. de Bono, Stephen R. Plymate. Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic 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 4965.

In clinical samples, the expression of androgen receptor (AR) and of AR splice variant 7 (AR-V7) is higher in castration-resistant prostate cancer (CRPC) compared with that in hormone-sensitive prostate cancer (PCa). However, there are only a few reports on the ratio of the expression levels of AR-V7 to AR (AR-V7/AR) in prostate tissue. The present study evaluated AR-V7/AR expression in various types of human prostate tissues and CRPC cells. Pretreatment prostate tissue samples from patients with benign prostatic hyperplasia (BPH; n=18), Gleason score 7 (n=17), and Gleason score 8–10 (n=26) were collected at the time of prostate biopsy, and tissue samples from CRPC patients (n=10) were collected at the time of transurethral resection of the prostate. Furthermore, androgen-independent LNCaP cells were established. The mRNA expression levels of AR and AR-V7, cell proliferation and prostate-specific antigen (PSA) production were evaluated by reverse transcription quantitative PCR, MTS assay and chemiluminescent enzyme immunoassay, respectively. There was a significant difference in AR-V7/AR expression ratios between the CRPC group and the BPH and pre-treatment PCa groups (CRPC, 7%; BPH and pre-treatment PCa, 1%). Subsequently, we compared the AR and AR-V7 expression levels in CRPC samples with those in the pretreatment prostate tissues from the same patients. The results demonstrated that the AR-V7/AR ratio increased from 3 to 9% after CRPC onset. Furthermore, in vitro experiment demonstrated that AR-V7 expression in LNCaP cells was increased after transforming into CRPC cells. The AR-V7/AR ratio also increased from 0.05 to 0.3%. In addition, small interfering (si)-RNA-mediated knockdown of AR inhibited the proliferation of and PSA production from androgen-independent LNCaP cells; however, AR-V7 knockdown had no effect. Conversely, siRNA-mediated knockdown of both AR and AR-V7 inhibited the proliferation of VCAP cells. In summary, the findings from the present study demonstrated that AR-V7 expression and AR-V7/AR ratio were increased after the onset of CRPC, which had a limited role in CRPC cell proliferation. Further investigation is required to clarify the roles of AR other splice variants and AR-V7 in CRPC.

Identification of AR-V7 downstream genes commonly targeted by AR/AR-V7 and specifically targeted by AR-V7 in castration resistant prostate cancer

Background: Patients treated with Androgen Deprivation Therapy (ADT) have been shown to increase the expression of constitutively active Androgen Receptor (AR) splice variants (AR-Vs). These splice variants lack the ligand binding domain at the C-terminus of AR, required for binding to antiandrogen therapies. AR-V7 has also been correlated to the development and progression of castration resistant prostate cancer (CRPC). As not all circulating tumor cells (CTCs) contain AR-V7, we hypothesized that other AR-Vs are involved in the mechanism of resistance to ADT commonly seen in CRPC. To test this, we developed an mRNA quantitative assay targeting AR-Vs and then correlated our results with a targeted mass spectrometry (MS) based protein assay. This work will provide a feasible quantification of AR-V transcripts and that may provide a stratification for predicted responses to ADT treatment in patients that could be useful to clinicians. Methods: The absolute copy number of total AR, AR-V2, AR-V7, AR-V12, and AR-V23 were determined in eight different PCa cell lines and 48 patient derived xenografts (PDXs) using qRT-PCR assays. We performed siRNA targeting of these variants to evaluate primer specificity by evaluating knockdown efficiencies. These results will be correlated to targeted MS quantification of AR-Vs in each cell model. Results: We found that the most highly expressed variants in PCa cell lines were AR-V7 and AR-V12. In the LuCaP PDX model, AR-V12 was shown to have the highest expression of the AR-Vs tested. Specificity of AR-V knockdown was tested using AD-1 (which only expresses the full- length AR transcript) and R1-D567 (which only expresses AR-V567es, an AR-V12-like variant) cell lines. Conclusions: We were able to determine the landscape of AR-V mRNA expression in eight PCa cell lines and in 48 PDX tumor samples. This data will be correlated to protein expression currently being analyzed using a targeted MS method developed in our lab. Our work will be useful to help clinicians stratify PCa patients based on their AR-V expression profiles. This will establish a prognostic biomarker program that measures AR-V proteins in real time from clinical biopsy tissues, circulating tumor cells, or exosomes and informs the clinician on which course of treatment may be effective for each patient. Establishing a CRPC biomarker other than AR-V7 that can help explain the AR-V7 negative CRPC patients could eventually improve clinical application and predictive treatment outcomes. Citation Format: Gabrianne Larson, Zoi Sychev, Stephen Plymate, Eva Corey, Justin M. Drake. Androgen receptor variants mRNA absolute quantification in prostate cancer cell models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A005.

Continued androgen receptor (AR) signaling is an established mechanism underlying castration-resistant prostate cancer (CRPC), and suppression of AR signaling remains a therapeutic goal of CRPC therapy. Constitutively active androgen receptor splice variants (AR-Vs) lack the AR ligand-binding domain (AR-LBD), the intended target of androgen deprivation therapies (ADT) including new CRPC therapies such as abiraterone and MDV3100. Transcriptional programs directed by the canonical full-length AR (AR-FL) are well characterized, but those directed the AR-Vs are not well understood. In addition, AR-FL and AR-Vs are both increased in CRPC, underscoring the critical importance of dissecting the complex interplay between AR-FL and AR-Vs. In this study, we employed a set of detection and targeting tools that differentiate the AR-FL and AR-Vs to investigate the functional distinctions between AR-FL and AR-V. We show that suppression of endogenous AR-FL signaling by targeting AR-LBD leads to increased AR-V expression in two cell line models of CRPC. Importantly, treatment-induced AR-Vs activate a distinct expression signature enriched for cell cycle genes without requiring the presence of AR-FL. Conversely, activation of AR-FL signaling suppresses the AR-V-mediated transcriptional programs but activates genes mainly associated with macromolecular synthesis, metabolism, and differentiation. In prostate cancer cells and CRPC xenografts treated with MDV3100 and abiraterone, increased expression of two constitutively active AR-Vs, AR-V7 and ARV567ES, but not AR-FL, parallels increased expression of the AR-driven cell cycle gene UBE2C. In addition, protein expression of AR-V7, but not AR-FL, is positively correlated with UBE2C in clinical CRPC specimens. The cumulative in vitro and in vivo evidence support an adaptive shift toward AR-V-mediated signaling in at least a subset of CRPC tumors as the AR-LBD is rendered inactive, suggesting a functional dichotomy between AR-FL and AR-Vs in a therapeutic setting. The dynamic interplay between AR signaling mediated by AR-FL and AR-V should be further explored in rational development of novel CRPC therapies and multipurpose biomarkers for CRPC. Citation Format: Rong Hu, Changxue Lu, Elahe A. Mostaghel, Srinivasan Yegnasubramanian, Meltem Gurel, Clare Tannahill, Joanne Edwards, William Isaacs, Peter S. Nelson, Eric Bluemn, Stephen R. Plymate, Jun Luo. Transcriptional programs directed by the androgen receptor splice variants [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr IA21.

Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1335.

Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1335.

Androgen deprivation therapy is initially effective for treating patients with advanced prostate cancer; however, the prostate cancer gradually becomes resistant to androgen deprivation therapy, which is termed castration‐resistant prostate cancer (CRPC). Androgen receptor splice variant 7 (AR‐V7), one of the causes of CRPC, is correlated with resistance to a new‐generation AR antagonist (enzalutamide) and poor prognosis. Heat shock protein 70 (Hsp70) inhibitor is known to decrease the levels of full‐length AR (AR‐FL), but little is known about its effects against CRPC cells expressing AR‐V7. In this study, we investigated the effect of the Hsp70 inhibitors quercetin and VER155008 in the prostate cancer cell line LNCaP95 that expresses AR‐V7, and explored the mechanism by which Hsp70 regulates AR‐FL and AR‐V7 expression. Quercetin and VER155008 decreased cell proliferation, increased the proportion of apoptotic cells, and decreased the protein levels of AR‐FL and AR‐V7. Furthermore, VER155008 decreased AR‐FL and AR‐V7 mRNA levels. Immunoprecipitation with Hsp70 antibody and mass spectrometry identified Y‐box binding protein 1 (YB‐1) as one of the molecules regulating AR‐FL and AR‐V7 at the transcription level through interaction with Hsp70. VER155008 decreased the phosphorylation of YB‐1 and its localization in the nucleus, indicating that the involvement of Hsp70 in AR regulation might be mediated through the activation and nuclear translocation of YB‐1. Collectively, these results suggest that Hsp70 inhibitors have potential anti‐tumor activity against CRPC by decreasing AR‐FL and AR‐V7 expression through YB‐1 suppression.