Treatments Targeting the Androgen Receptor and Its Splice Variants in Breast Cancer.

Breast cancer is a major cause of death worldwide. The complexity of endocrine regulation in breast cancer may allow the cancer cells to escape from a particular treatment and result in resistant and aggressive disease. These breast cancers usually have fewer treatment options. Targeted therapies for cancer patients may offer fewer adverse side effects because of specificity compared to conventional chemotherapy. Signaling pathways of nuclear receptors, such as the estrogen receptor (ER), have been intensively studied and used as therapeutic targets. Recently, the role of the androgen receptor (AR) in breast cancer is gaining greater attention as a therapeutic target and as a prognostic biomarker. The expression of constitutively active truncated AR splice variants in breast cancer is a possible mechanism contributing to treatment resistance. Therefore, targeting both the full-length AR and AR variants, either through the activation or suppression of AR function, depending on the status of the ER, progesterone receptor, or human epidermal growth factor receptor 2, may provide additional treatment options. Studies targeting AR in combination with other treatment strategies are ongoing in clinical trials. The determination of the status of nuclear receptors to classify and identify patient subgroups will facilitate optimized and targeted combination therapies.

Drugging the Undruggable: Targeting the N-Terminal Domain of Nuclear Hormone Receptors.

This chapter focuses on the development of drugs targeting the N-terminal domain of nuclear hormone receptors, using progress with the androgen receptor as an example. Historically, development of therapies targeting nuclear hormone receptors has focused on the folded C-terminal ligand-binding domain. Therapies were traditionally not developed to target the intrinsically disordered N-terminal domain as it was considered "undruggable". Recent developments have now shown it is possible to direct therapies to the N-terminal domain. This chapter will provide an introduction of the structure and function of the domains of nuclear hormone receptors, followed by a discussion of the rationale supporting the development of N-terminal domain inhibitors. Chemistry and mechanisms of action of small molecule inhibitors will be described with emphasis on N-terminal domain inhibitors developed to the androgen receptor including those in clinical trials.

Structure-Activity Relationships for the Marine Natural Product Sintokamides: Androgen Receptor N-Terminus Antagonists of Interest for Treatment of Metastatic Castration-Resistant Prostate Cancer.

Synthetic analogues of the marine natural product sintokamides have been prepared in order to investigate the structure-activity relationships for the androgen receptor N-terminal domain (AR NTD) antagonist activity of the sintokamide scaffold. An in vitro LNCaP cell-based transcriptional activity assay with an androgen-driven luciferase (Luc) reporter was used to monitor the potency of analogues. The data have shown that the chlorine atoms on the leucine side chains are essential for potent activity. Analogues missing the nonchlorinated methyl groups of the leucine side chains (C-1 and C-17) are just as active and in some cases more active than the natural products. Analogues with the natural R configuration at C-10 and the unnatural R configuration at C-4 are most potent. Replacing the natural propionamide N-terminus cap with the more sterically hindered pivaloylamide N-terminus cap leads to enhanced potency. The tetramic acid fragment and the methyl ether on the tetramic acid fragment are essential for activity. The SAR optimized analogue 76 is more selective, easier to synthesize, more potent, and presumed to be more resistant to proteolysis than the natural sintokamides.

Keys to unlock androgen receptor translocation.

The androgen receptor (AR) is tightly linked to prostate cancer, but the mechanisms by which AR transactivation is dysregulated during cancer progression are not fully explored. Dagar et al. examined AR translocation to the nucleus to identify a link between heat shock protein 90 (HSP90) and protein kinase A (PKA). Their findings provide a potential mechanism of the initiation of AR transactivation and potential targets for developing and refining treatments for prostate cancer.

Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain.

Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis.

Targeting the N-Terminal Domain of the Androgen Receptor: A New Approach for the Treatment of Advanced Prostate Cancer.

: Despite the recent approval and widespread use of abiraterone acetate and enzalutamide for the treatment of castration-resistant prostate cancer (CRPC), this disease still poses significant management challenges because of various tumor escape mechanisms, including those that allow androgen receptor (AR) signaling to remain active. These AR-related resistance mechanisms include AR gene amplification or overexpression, constitutively active ligand-independent AR splice variants, and gain-of-function mutations involving the AR ligand-binding domain (LBD), among others. Therefore, the development of AR-targeted therapies that function independently of the LBD represents an unmet medical need and has the potential to overcome many of these resistance mechanisms. This article discusses N-terminal domain (NTD) inhibition as a novel concept in the field of AR-directed therapies for prostate cancer. AR NTD-targeting agents have the potential to overcome shortcomings of current hormonal therapies by inhibiting all forms of AR-mediated transcriptional activity, and as a result, may affect a broader AR population including mutational and splice variant ARs. Indeed, the first clinical trial of an AR NTD inhibitor is now underway. IMPLICATIONS FOR PRACTICE: Because of emerging resistance mechanisms that involve the ligand-binding domain of the androgen receptor (AR), there is currently no effective treatment addressing tumor escape mechanisms related to current AR-targeted therapies. Many patients still demonstrate limited clinical response to current hormonal agents, and castration-resistant prostate cancer remains a lethal disease. Intense research efforts are under way to develop therapies to target resistance mechanisms, including those directed at other parts of the AR molecule. A novel small-molecule agent, EPI-506, represents a new pharmaceutical class, AR N-terminal domain inhibitors, and shows preclinical promise to overcome many known resistance mechanisms related to novel hormonal therapies.

Androgen receptor targeted therapies in castration-resistant prostate cancer: Bench to clinic.

The androgen receptor is a transcription factor and validated therapeutic target for prostate cancer. Androgen deprivation therapy remains the gold standard treatment, but it is not curative, and eventually the disease will return as lethal castration-resistant prostate cancer. There have been improvements in the therapeutic landscape with new agents approved, such as abiraterone acetate, enzalutamide, sipuleucel-T, cabazitaxel and Ra-223, in the past 5 years. New insight into the mechanisms of resistance to treatments in advanced disease is being and has been elucidated. All current androgen receptor-targeting therapies inhibit the growth of prostate cancer by blocking the ligand-binding domain, where androgen binds to activate the receptor. Persuasive evidence supports the concept that constitutively active androgen receptor splice variants lacking the ligand-binding domain are one of the resistant mechanisms underlying advanced disease. Transcriptional activity of the androgen receptor requires a functional AF-1 region in its N-terminal domain. Preclinical evidence proved that this domain is a druggable target to forecast a potential paradigm shift in the management of advanced prostate cancer. This review presents an overview of androgen receptor-related mechanisms of resistance as well as novel therapeutic agents to overcome resistance that is linked to the expression of androgen receptor splice variants in castration-resistant prostate cancer.

Inhibitors of the transactivation domain of androgen receptor as a therapy for prostate cancer.

The androgen receptor (AR) is a modular transcription factor which functions as a master regulator of gene expression. AR protein is composed of three functional domains; the ligand-binding domain (LBD); DNA-binding domain (DBD); and the intrinsically disordered N-terminal transactivation domain (TAD). AR is transactivated upon binding to the male sex hormone testosterone and other androgens. While the AR may tolerate loss of its LBD, the TAD contains activation function-1 (AF-1) that is essential for all AR transcriptional activity. AR is frequently over-expressed in most prostate cancer. Currently, androgen deprivation therapy (ADT) in the form of surgical or chemical castration remains the standard of care for patients with high risk localized disease, advanced and metastatic disease, and those patients that experience biochemical relapse following definitive primary treatment. Patients with recurrent disease that receive ADT will ultimately progress to lethal metastatic castration-resistant prostate cancer. In addition to ADT not providing a cure, it is associated with numerous adverse effects including cardiovascular disease, osteoporosis and sexual dysfunction. Recently there has been a renewed interest in investigating the possibility of using antiandrogens which competitively bind the AR-LBD without ADT for patients with hormone sensitive, non-metastatic prostate cancer. Here we describe a class of compounds termed AR transactivation domain inhibitors (ARTADI) and their mechanism of action. These compounds bind to the AR-TAD to inhibit AR transcriptional activity in the absence and presence of androgens. Thus these inhibitors may have utility in preventing prostate cancer growth in the non-castrate setting.

Piperazate-Guided Isolation of Caveamides A and B, Cyclohexenylalanine-Containing Nonribosomal Peptides from a Cave Actinomycete.

Hybrid genome-mining/15N-NMR was used to target compounds containing piperazate (Piz) residues, leading to the discovery of caveamides A (1) and B (2) from Streptomyces sp. strain BE230, isolated from New Rankin Cave (Missouri). Caveamides are highly dynamic molecules containing an unprecedented ß-ketoamide polyketide fragment, two Piz residues, and a new N-methyl-cyclohexenylalanine residue. Caveamide B (2) exhibited nanomolar cytotoxicity against several cancer cell lines and nanomolar antimicrobial activity against MRSA and E. coli.

Rational optimization of a transcription factor activation domain inhibitor.

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

Advances in small molecule inhibitors of androgen receptor for the treatment of advanced prostate cancer.

OBJECTIVES: Current treatments for localized prostate cancer include brachytherapy, external beam radiation, surgery, and active surveillance. Unfortunately, 20-40% of prostate cancer patients will experience recurrence and require hormonal therapies. These therapies involve androgen ablation by chemical or surgical castration and application of antiandrogens. Hormonal therapy is initially effective, but will inevitably fail and the disease will progress to lethal castration-resistant prostate cancer (CRPC) from which patients succumb within 2 years. CRPC is considered to be dependent on transcriptionally active androgen receptors (AR). This article reviews recent advances in the discovery and development of small molecule inhibitors of AR. METHODS: A PubMed database search was performed for articles focused on small molecule inhibitors of AR for potential development for the treatment of prostate cancer. Compounds with broad effects on other pathways were not included. RESULTS: Currently, there are several novel antiandrogens being tested in the clinic that have improved affinity for the AR and work by different mechanisms to the current battery of approved antiandrogens that are discussed. Small molecule inhibitors that interact with regions other than the AR ligand-binding pocket have been also been discovered. These small molecules include allosteric inhibitors of the LBD, compounds that alter AR conformation, and antagonists to the AR NTD and are highlighted. CONCLUSIONS: CRPC is dependent upon transcriptionally active AR. Survival improvement may be achieved by complete blockade of all AR activity using novel small molecule inhibitors with unique mechanisms of action.

Androgen receptor and its splice variants in prostate cancer.

Androgen receptor (AR) is a transcription factor that becomes active upon binding to androgens via its ligand-binding domain (LBD) or in response to signaling cascades initiated by growth factors and cytokines. The activity of AR requires regions within the N-terminal domain (NTD) in a manner that is distinct from the activation of related steroid hormone receptors. Unequivocal evidence has been amassed to consider that the AR axis is the most critical pathway for the progression of prostate cancer. Qualitatively distinct insights into AR pathobiology have been garnered including that AR-regulated gene expression is stage-specifically modulated during disease progression and that the ligand requirement for AR activity could be rendered dispensable because of the expression of constitutively active AR splice variants that are devoid of LBD. The recent appreciation of the clinical challenge that stems from non-gonadal androgens that are not inhibited by traditional hormonal therapies has been tangibly translated into the development of more potent drugs that can potentially lead towards achieving an androgen-free environment. The pre-clinical evidence that proves that AR NTD is a druggable target also forecasts a further paradigm shift in the management of advanced prostate cancer. These advancements together with the identification of more robust AR antagonists and their promising clinical outcome have renewed the hope that targeting the AR pathway remains a sound strategy in the clinical management of prostate cancer. Here, we address these developments with a greater emphasis on the rapidly growing literature on AR splice variants.

Cyclin-dependent Kinase 4/6 Inhibitor Palbociclib in Combination with Ralaniten Analogs for the Treatment of Androgen Receptor-positive Prostate and Breast Cancers.

Androgen receptor (AR) has essential roles in the growth of prostate cancer and some breast cancers. Inhibition of AR transcriptional activity by targeting its N-terminal domain with ralaniten or an analog such as EPI-7170 causes accumulation of cells in the G1-phase of the cell cycle. Inhibition of cyclin-dependent kinases 4/6 with palbociclib also leads to accumulation of cells in the G1-phase. Here, a combination of EPI-7170 with palbociclib attenuated the in vivo growth of human castration-resistant prostate cancer xenografts that are resistant to antiandrogens. Cell-cycle tracing experiments in cultured cells revealed that EPI-7170 targeted cells in the S-phase, possibly through inducing DNA damage or impairing the DNA damage response, whereas palbociclib targeted the G1-S transition to delay the cell cycle. Combination treatment prevented cells in G1 and G2-M from progressing in the cell cycle and caused a portion of cells in the S-phase to arrest, which contributed to a twofold increase in doubling time to >63 hours compared with 25 hours in control cells. Importantly, sequential combination treatments with palbociclib administered first then followed by EPI-7170, resulted in more cells accumulating in G1 and less cells in the S-phase than concomitant combination which was presumably because each inhibitor has a unique mechanism in modulating the cell cycle in cancer cells. Together, these data support that the combination therapy was more effective than individual monotherapies to reduce tumor growth by targeting different phases of the cell cycle.

Differential Gene Expression Profiles between N-Terminal Domain and Ligand-Binding Domain Inhibitors of Androgen Receptor Reveal Ralaniten Induction of Metallothionein by a Mechanism Dependent on MTF1.

Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein-protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy.

Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

The androgen receptor (AR) is a validated therapeutic target for prostate cancer and has been a focus for drug development for more than six decades. Currently approved therapies that inhibit AR signaling, such as enzalutamide, rely solely on targeting the AR ligand-binding domain and, therefore, have limited efficacy on prostate cancer cells that express truncated, constitutively active AR splice variants (AR-Vs). The LNCaP95 cell line is a human prostate cancer cell line that expresses both functional full-length AR and AR-V7. LNCaP95 is a heterogeneous cell population that is resistant to enzalutamide, with its proliferation dependent on transcriptionally active AR-V7. The purpose of this study was to identify a LNCaP95 clone that would be useful for evaluating therapies for their effectiveness against enzalutamide-resistant prostate cancer cells. Seven clones from the LNCaP95 cell line were isolated and characterized using morphology, in vitro growth rate, and response to ralaniten (AR N-terminal domain inhibitor) and enzalutamide (antiandrogen). In vivo growth of the clones as subcutaneous xenografts was evaluated in castrated immunodeficient mice. All of the clones maintained the expression of full-length AR and AR-V7. Cell proliferation of the clones was insensitive to androgen and enzalutamide but importantly was inhibited by ralaniten, which is consistent with AR-Vs driving the proliferation of parental LNCaP95 cells. In castrated immunodeficient animals, the growth of subcutaneous xenografts of the D3 clone was the most reproducible compared to the parental cell line and other clones. These data support that the enzalutamide-resistant LNCaP95-D3 subline may be suitable as a xenograft tumor model for preclinical drug development with improved reproducibility.

Pin1 inhibition improves the efficacy of ralaniten compounds that bind to the N-terminal domain of androgen receptor.

Therapies for lethal castration-resistant prostate cancer (CRPC) are an unmet medical need. One mechanism underlying CRPC and resistance to hormonal therapies is the expression of constitutively active splice variant(s) of androgen receptor (AR-Vs) that lack its C-terminus ligand-binding domain. Transcriptional activities of AR-Vs and full-length AR reside in its N-terminal domain (NTD). Ralaniten is the only drug proven to bind AR NTD, and it showed promise of efficacy in Phase 1 trials. The peptidyl-prolyl isomerase Pin1 is frequently overexpressed in prostate cancer. Here we show that Pin1 interacted with AR NTD. The inhibition of Pin1 expression or its activity selectively reduced the transcriptional activities of full-length AR and AR-V7. Combination of Pin1 inhibitor with ralaniten promoted cell cycle arrest and had improved antitumor activity against CRPC xenografts in vivo compared to individual monotherapies. These findings support the rationale for therapy that combines a Pin1 inhibitor with ralaniten for treating CRPC.

Development of metastatic and non-metastatic tumor lines from a patient's prostate cancer specimen-identification of a small subpopulation with metastatic potential in the primary tumor.

BACKGROUND: Metastasis is the major cause of prostate cancer deaths. Tumor heterogeneity in both primary and metastatic prostate cancers is a major hurdle in elucidating the mechanisms of metastasis in this disease. To circumvent this obstacle and improve our understanding of prostate cancer metastasis, we developed multiple tumor tissue lines from one patient's primary prostate cancer specimen to examine differences in metastatic ability. METHODS: Pieces of tissue from different foci of a patient's primary prostate tumor were grafted into subrenal capsules of NOD-SCID mice and transplantable tumor sublines were established by serial passage. The metastatic ability of each subline was tested via orthotopic grafting into mice. Chromosomal alterations exclusively presented in a metastatic subline were examined by SKY and investigated for their presence in parental tissues by fluorescence in situ hybridization. RESULTS: Three transplantable sublines were developed, resembling the primary tumor histologically, exhibiting poor differentiation, and different growth rates. Importantly, the LTL-220N and LTL-221N sublines were non-metastatic, whereas the LTL-220M subline was spontaneously metastatic in vivo. SKY analysis showed limited but unique chromosomal alterations in each subline. Some chromosomal alterations, exclusively present in the metastatic LTL-220M subline, were also observed in a small portion of the parental cancer tissues. CONCLUSION: The results indicate that in primary prostate tumors metastatic potential can be confined to a minority of cancer cells. Subrenal capsule xenograft methodology can be used to dissect heterogeneous cancer cells in a patient's primary tumor and sublines derived from such cells provide valuable tools for investigating mechanisms underlying prostate cancer metastasis.

Novel biomarkers for prostate cancer including noncoding transcripts.

Levels of 27 transcripts were investigated as potential novel markers for prostate cancer, including genes encoding plasma membrane proteins (ADAM2, ELOVL5, MARCKSL1, RAMP1, TMEM30A, and TMEM66); secreted proteins (SPON2, TMEM30A, TMEM66, and truncated TMEFF2 (called POP4)); intracellular proteins (CAMK2N1, DHCR24, GLO1, NGFRAP1, PGK1, PSMA7, SBDS, and YWHAQ); and noncoding transcripts (POP1 (100 kb) from mRNA AK000023), POP2 (4 kb from mRNA AL832227), POP3 (50 kb from EST CFI40309), POP5 (intron of NCAM2, accession DO668384), POP6 (intron of FHIT), POP7 (intron of TNFAIP8), POP8 (intron of EFNA5), POP9 (intron of DSTN), POP10 (intron of ADAM2, accession DO668396), POP11 (87kb from EST BG194644), and POP12 (intron of EST BQ226050)). Expression of POP3 was prostate specific, whereas ADAM2, POP1, POP4, POP10, ELOVL5, RAMP1, and SPON2 had limited tissue expression. ELOVL5, MARCKSL1, NGFRAP1, PGK1, POP2, POP5, POP8, PSMA7, RAMP1, and SPON2 were significantly differentially expressed between laser microdissected malignant versus benign clinical samples of prostate tissue. PGK1, POP2, and POP12 correlated to clinical parameters. Levels of CAMK2N1, GLO1, SDBS, and TMEM30A transcripts tended to be increased in primary prostate cancer from patients who later had biochemical failure. Expression of GLO1, DHCR24, NGFRAP1, KLK3, and RAMP1 were significantly decreased in metastatic castration-recurrent disease compared with androgen-dependent primary prostate cancer. These novel potential biomarkers may therefore be useful in the diagnosis/prognosis of prostate cancer.

Discovery of drugs that directly target the intrinsically disordered region of the androgen receptor.

Introduction: Intrinsically disordered proteins (IDPs) and regions (IDRs) lack stable three-dimensional structure making drug discovery challenging. A validated therapeutic target for diseases such as prostate cancer is the androgen receptor (AR) which has a disordered amino-terminal domain (NTD) that contains all of its transcriptional activity. Drug discovery against the AR-NTD is of intense interest as a potential treatment for disease such as advanced prostate cancer that is driven by truncated constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD).Areas covered: This article presents an overview of the relevance of AR and its intrinsically disordered NTD as a drug target. AR structure and approaches to blocking AR transcriptional activity are discussed. The discovery of small molecules, including the libraries used, proven binders to the AR-NTD, and site of interaction of these small molecules in the AR-NTD are presented along with discussion of the Phase I clinical trial.Expert opinion: The lack of drugs in the clinic that directly bind IDPs/IDRs reflects the difficulty of targeting these proteins and obtaining specificity. However, it may also point to an inappropriateness of too closely borrowing concepts and resources from drug discovery to folded proteins.

Combination therapy with androgen receptor N-terminal domain antagonist EPI-7170 and enzalutamide yields synergistic activity in AR-V7-positive prostate cancer.

Resistance of castration-resistant prostate cancer (CRPC) to enzalutamide and abiraterone involves the expression of constitutively active, truncated androgen receptor (AR) splice variants (AR-Vs) that lack a C-terminal ligand-binding domain (LBD). Both full-length AR and truncated AR-Vs require a functional N-terminal domain (NTD) for transcriptional activity thereby providing rationale for the development of ralaniten (EPI-002) as a first-in-class antagonist of the AR-NTD. Here, we evaluated the antitumor effect of a next-generation analog of ralaniten (EPI-7170) as a monotherapy or in combination with enzalutamide in prostate cancer cells that express AR-V7 that were resistant to enzalutamide. EPI-7170 had 8-9 times improved potency compared to ralaniten. Enzalutamide increased levels of AR-V7 and expression of its target genes. Knockdown of AR-V7 restored sensitivity to enzalutamide, indicating a role for AR-V7 in the mechanism of resistance. EPI-7170 inhibited expression of genes transcriptionally regulated by full-length AR and AR-V7. A combination of EPI-7170 and enzalutamide resulted in synergistic inhibition of proliferation of enzalutamide-resistant cells that was consistent with results from cell cycle and clonogenic assays. In addition, this drug enhanced the antitumor effect of enzalutamide in enzalutamide-resistant CRPC preclinical models. Thus, a combination therapy targeting both the NTD and LBD of AR, and thereby blocking both full-length AR and AR-Vs, has potential for the treatment of enzalutamide-resistant CRPC.