BET bromodomain inhibitors—A novel epigenetic approach in castration-resistant prostate cancer

Abstract

The androgen receptor (AR) is central to the initiation and progression of prostate cancer, even after castration. There has been some success in therapies targeting AR signaling which have been shown to extend survival in men with castration-resistant prostate cancer (CRPC). However, durable responses to these therapies have been limited and there is a need to identify additional therapeutic targets within the AR-signaling network. Recently a group at University of Michigan Medical School outlined the potential for BET bromodomain protein inhibitors as a novel epigenetic approach to treatment of CRPC. In prostate cancer cell lines, BET bromodomain inhibitor, JQ1, was shown to induce apoptosis and down-regulate AR-regulated gene transcription. Bromodomain and the extra-terminal (BET) subfamily of human bromodomain proteins, with a focus on BRD4, were shown to play a major role in AR signaling and interact with AR via bromodomain (BD) 1/2. JQ1 inhibits this BRD4-AR bond, resulting in removal of RNA polymerase II from AR target genes, causing reduced AR gene transcription and subsequent diminished AR signaling. JQ1 lead to a significant reduction in tumor volume and weight in VCaP xenograft mice.

Keywords:

• androgen receptor
• BET bromodomain inhibitor
• castration-resistant prostate cancer
• gene transcription

Since Huggins and Hodges first description of prostate cancer's hormonal dependence in 1941, the cornerstone of prostate cancer treatment strategies have focused on blocking androgen-receptor signaling. Prostate cancer is initially sensitive to androgen deprivation therapy (ADT), which can induce marked tumor regression and normalize serum prostate specific antigen (PSA). However, resistance to treatment can emerge leading to castration-resistant prostate cancer (CRPC). CRPC is simply defined as progressive prostate cancer despite castrate serum testosterone levels (<50 ng/dL).¹ Unfortunately, most men who develop metastatic CRPC succumb to the disease.²

Progression to CRPC is now recognized as driven by deregulated androgen receptor (AR) signaling, partly due to over-expression of the receptor itself.³ AR amplification, mutation and alternative splicing have all been suggested as potential resistance mechanisms to anti-androgen treatments. Over half of CRPC patients have at least one of these aberrations in the AR pathway.² Although the AR remains critical in many cellular models of CRPC, it is also important to recognize that a variety of non–AR-dependent pathways may also contribute to prostate cancer growth in the absence of androgens.⁴

Recently, new therapies targeting AR signaling have been shown to extend survival in men with CRPC. The FDA approved abiraterone acetate, a potent inhibitor of CYP17 that reduces testosterone synthesis from cholesterol, in 2011 for metastatic CRPC following docetaxel, and in December 2012 approved an expanded indication for metastatic CRPC before chemotherapy.⁵ Phase III studies showed that abiraterone improved overall survival, progression free survival, delayed initiation of chemotherapy and doubled the time to first skeletal event.⁶

Enzalutamide (formerly MDV3100) is another novel endocrine treatment with reported significant anti-tumor activity. It is an AR-receptor-signaling inhibitor, blocking nuclear translocation, DNA binding, and co-activator recruitment. Enzalutamide significantly prolonged the survival of men with metastatic CRPC after chemotherapy in the Phase III AFFIRM trial.⁷ Recently, enzalutamide was assessed in metastatic CRPC prior to chemotherapy, in the Phase III PREVAIL trial Beer et al. showed decreased risk of radiographic progression and death, and delayed time to initiation of chemotherapy.⁸

Unfortunately, durable responses to these therapies have been limited and have raised new questions about how resistance develops, how to optimize combination therapy, and what other targets should be investigated. There is a need to identify additional therapeutic targets within the AR-signaling network.

In a paper published in Nature in June 2014, Asangani and colleagues investigated 2 selective small molecule inhibitors, JQ1 and I-BET762, and their potential activity in CRPC. These novel agents target the amino-terminal bromodomains of BRD4 and have been shown to exhibit anti-proliferative effects in a range of malignancies.² Members of the bromodomain and extra-terminal (BET) subfamily of human bromodomain proteins (BRD 2, 3, and 4) associate with acetylated chromatin and facilitate transcriptional activators.⁹ Recent research has established compelling evidence for targeting BRD 4 in cancer, elucidating a novel epigenetic approach for the blockade of oncogenic drivers in cancer.

JQ1 is a potent, selective small molecule inhibitor of BET bromodomains. It is a thieno-triazolo-1,4-diazepine that displaces BET bromodomains from chromatin by competitively binding to the acetyl-lysine recognition pocket.¹⁰

The authors have found that the bromodomain and extra-terminal (BET) family of chromatin readers might be a valuable target in the AR signaling network in CRPC. The treatment of 5 prostate cancer cell lines and one transformed normal epithelial cell line with JQ1 revealed that only the 3 cell lines that were positive for androgen signal (VCaP, LNCaP, 22RV) were sensitive to JQ1-induced apoptosis and cell cycle arrest. JQ1-induced apoptosis was shown to be dose-dependent in VCaP cells, by cPARP immunoblot analysis. These results were phenocopied by knockdown of BRD2, BRD3, and BRD4.

Quantitative PCR (qPCR) analysis of VCaP cells gene transcripts following treatment with varying concentrations of JQ1 showed that AR targets, PSA, ERG, and TMPRSS2, mRNA expression decreased in a dose-dependent manner, thus AR- regulated gene transcription was down-regulated by JQ1. This corresponds with immunoblot analyses of PSA, and ERG proteins in VCaP cells that demonstrated a dose-dependent decrease in protein expression in response to JQ1 treatment.

Gene set enrichment analysis (GSEA) of the AR target gene signature using microarray showed a significant repression of the wide range of genes involved in AR signaling upon JQ1 treatment, highlighting that JQ1 reduced the levels of transcription of the AR target genes in AR-positive cells. This suggests that BET family proteins are involved in AR-mediated transcriptional programs.¹¹

One of the proposed major targets for JQ1 is MYC, as MYC levels and MYC-associated gene transcription were reduced by JQ1. However loss of MYC did not completely phenocopy the effects of JQ1 treatment, nor did MYC expression rescue JQ1-mediated effects on cell growth. Thus, the authors showed that although MYC levels may be repressed by JQ1 in AR-positive cells, MYC does not seem to be a primary target for the anti-neoplastic effects of JQ1.¹¹

Asangani and colleagues went on to illustrate how BET family proteins affect AR-mediated transcription. They suggest that AR, BRD4 and RNA polymerase II likely form a large multi-protein complex, as these proteins predominantly co-eluted in a high-molecular weight complex on gel filtration chromography. The authors demonstrated that AR could interact with BRD2, BRD3, and BRD4. Focusing on BRD4, by immunoprecipitation and subsequent immunoblotting they elucidated that bromodomain 1 and 2 are necessary and sufficient for AR-BRD4 binding. The BRD4 construct containing bromodomain 1 and 2 maintained its ability to pull down AR at high salt concentrations, highlighting the strength of the AR-BRD4 bond. This BRD4 interaction with AR occurs in a concentration dependent manner, with an estimated dissociation constant (K_d) of 70nM.

Upon fine mapping of the interacting domains, NTD1b was found to be the precise site of bromodomain 1 and 2 binding on AR and treatment with JQ1 resulted in the disruption of NTD1b-BD1 binding in a concentration-dependent manner. The authors proved that in-vitro JQ1 inhibits this BRD4-AR bond at the BD1/2-NTD1b interaction, resulting in removal of RNA polymerase II from AR target genes, causing reduced AR gene transcription and subsequent diminished AR signaling.

Subsequently by chromatin immunoprecipitation followed by sequencing (ChIP–seq) Asangani et al. showed that JQ1 reduced the recruitment of AR to AR-responsive genes genome-wide almost as effectively as the direct AR antagonist enzalutamide. Furthermore, the authors observed a co-recruitment of AR and BRD4 at 2031 sites, with the strongest associations observed within promoters of AR-regulated genes.

When limiting their evaluation to AR and BRD4 coincident peaks, it was noted that JQ1 almost completely abrogated BRD4 recruitment to the AR-BRD4 shared loci. Gene expression analysis confirmed that this resulted in reduced transcription of target genes. In VCaP cells, which have both AR amplification and the transmembrane protease, serine 2 (TMPRSS2)–ETS-related gene (ERG) fusion gene, the expression of ERG and the ERG transcriptional program were blocked by JQ1 through the reduction of AR and BRD4 binding to the TMPRSS2 promoter/enhancer. ¹¹

The authors then compared the efficacy of JQ1 to enzalutamide in mice carrying VCaP. Though both drugs lead to decreased tumor volume and weight, JQ1 had the most pronounced effect, causing a significant reduction in both weight and volume. VCaP xenograft tumors are castration-sensitive but castration-resistant tumors can recur after treatment. Upon treatment of castration-resistant xenograft mice with JQ1 the tumor volume was significantly reduced in this subset as well.

This study highlights a need for future research into the mechanisms by which JQ1 functions in prostate cancer cells. It highlights the potential for BET bromodomain proteins as a novel target in CRPC and potentially in other cancers, thus warranting further investigation. By functioning downstream of AR, BET inhibition is less likely to be affected by acquired resistance associated with AR antagonists. This article acts as an indication for clinical evaluation of BET inhibitors in CRPC, either as a monotherapy or in combination with current FDA-approved anti-androgen therapies.

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