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

SIMPLE SUMMARY: Inhibition of the androgen receptor (AR) remains the mainstay treatment for prostate cancer. All current therapies involving AR inhibition either directly or indirectly target its ligand-binding domain (LBD). We have developed the first novel compounds which target the N-terminal domain, (NTD) a region which is essential for AR transcriptional activity. First-generation ralaniten (NCT02606123), and second-generation EPI-7386 (NCT04421222) remain the only AR-NTD inhibitors to progress to clinical trials. Here we aim to characterize differences between different classes of AR antagonists targeting the AR-LBD and the AR-NTD, as well as next generation AR-NTD inhibitors. An incidental finding was that ralaniten was uniquely associated with increased metallothionein expression which was independent of AR activity. Instead, expression of metallothionein genes was driven by MTF-1 indicating a potential off-target effect. Neither AR-LBD inhibitor enzalutamide nor second-generation AR-NTD inhibitor EPI-7170 had this effect. This work has important implications for the development of novel AR-NTD inhibitors. ABSTRACT: 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.