Dr. Marianne Sadar, PhD

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.

Blocking androgen receptor (AR) transcriptional activity by androgen deprivation therapy (ADT) improves the response to radiotherapy for intermediate and high risk prostate cancer. Unfortunately, ADT, antiandrogens, and abiraterone increase expression of constitutively active splice variants of AR (AR-Vs) which regulate DNA damage repair leading to resistance to radiotherapy. Here we investigate whether blocking the transcriptional activities of full-length AR and AR-Vs with ralaniten leads to enhanced sensitivity to radiotherapy. Combination therapies using ralaniten with ionizing radiation were evaluated for effects on proliferation, colony formation, cell cycle, DNA damage, and Western blot analyses in human prostate cancer cells that express both full-length AR and AR-Vs. Ralaniten and a potent next-generation analog (EPI-7170) decreased expression of DNA repair genes whereas enzalutamide had no effect. FACS analysis revealed a dose-dependent decrease of BrdU incorporation with increased accumulation of γH2AX with a combination of ionizing radiation with ralaniten. An additive inhibitory effect on proliferation of enzalutamide-resistant cells was achieved with a combination of ralaniten compounds with ionizing radiation. Ralaniten and EPI-7170 sensitized prostate cancer cells that express full-length AR and AR-Vs to radiotherapy whereas enzalutamide had no added benefit.

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.

Inhibition of the androgen receptor (AR) is the mainstay treatment for advanced prostate cancer. Ralaniten (formally EPI-002) prevents AR transcriptional activity by binding to its N-terminal domain (NTD) which is essential for transcriptional activity. Ralaniten acetate (EPI-506) the triacetate pro-drug of ralaniten, remains the only AR-NTD inhibitor to have entered clinical trials (NCT02606123). While well tolerated, the trial was ultimately terminated due to poor pharmacokinetic properties and resulting pill burden. Here we discovered that ralaniten was glucuronidated which resulted in decreased potency. Long-term treatment of prostate cancer cells with ralaniten results in upregulation of UGT2B enzymes with concomitant loss of potency. This has proven to be a useful model with which to facilitate the development of more potent second-generation AR-NTD inhibitors. Glucuronidated metabolites of ralaniten were also detected in the serum of patients in Phase 1 clinical trials. Therefore, we tested an analogue of ralaniten (EPI-045) which was resistant to glucuronidation and demonstrated superiority to ralaniten in our resistant model. These data support that analogues of ralaniten designed to mitigate glucuronidation may optimize clinical responses to AR-NTD inhibitors.

Enzalutamide is a nonsteroidal antiandrogen for the treatment of metastatic castration-resistant prostate cancer (mCRPC) both before and after chemotherapy. Enzalutamide is more effective than its predecessor bicalutamide, which was analyzed in head-to-head studies of patients with CRPC. This family of nonsteroidal antiandrogens is now comprised of four drugs approved by the US Food and Drug Administration with two investigational drugs in clinical trials. Antiandrogens have been employed clinically for more than five decades to provide a rich resource of information. Steady-state concentration minimums (C{{sub}}min{{/sub}} or trough) in the range of ~1-13 μg/mL are measured in patients at therapeutic doses. Interestingly, enzalutamide which is considered to have strong affinity for the androgen receptor (AR) requires C{{sub}}min{{/sub}} levels >10 μg/mL. The sequence of antiandrogens and the clinical order of application in regard to other drugs that target the androgen axis remain of high interest. One novel first-in-class drug, called ralaniten, which binds to a unique region in the N-terminus domain of both the full-length and the truncated constitutively active splice variants of the AR, is currently in clinical trials for patients who previously received abiraterone, enzalutamide, or both. This highlights the trend to develop drugs with novel mechanisms of action and potentially differing mechanisms of resistance compared with antiandrogens. Better and more complete inhibition of the transcriptional activity of the AR appears to continue to provide improvements in the clinical management of mCRPC.

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.

Constitutively active splice variants of androgen receptor (AR-Vs) lacking ligand-binding domain (LBD) are a mechanism of resistance to androgen receptor LBD-targeted (AR LBD-targeted) therapies for metastatic castration-resistant prostate cancer (CRPC). There is a strong unmet clinical need to identify prostate cancer patients with AR-V-positive lesions to determine whether they will benefit from further AR LBD-targeting therapies or should receive taxanes or investigational drugs like EPI-506 or galeterone. Both EPI-506 (NCT02606123) and galeterone (NCT02438007) are in clinical trials and are proposed to have efficacy against lesions that are positive for AR-Vs. AR activation function-1 (AF-1) is common to the N-terminal domains of full-length AR and AR-Vs. Here, we provide proof of concept for developing imaging compounds that directly bind AR AF-1 to detect both AR-Vs and full-length AR. {{sup}}123{{/sup}}I-EPI-002 had specific binding to AR AF-1, which enabled direct visualization of CRPC xenografts that express full-length AR and AR-Vs. Our findings highlight the potential of {{sup}}123{{/sup}}I-EPI-002 as an imaging agent for the detection of full-length AR and AR-Vs in CRPC.

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.