Research Approach: The overall hypothesis will be addressed by 3 Specific Aims. Specific Aim 1: To determine how NF90 cooperates with EZH2 to increase AR transcription. NF90 has never been studied in PCa. Our data showed that NF90 protein interacts with EZH2; NF90 knockdown reduces EZH2 binding at the AR promoter and decreases AR level. In this Aim, we strive to examine whether NF90 mediates EZH2-induced AR expression, map the minimal regions of NF90 and EZH2 protein that are responsible for their interaction, and determine whether dsRNA-binding mediates NF90/EZH2 interaction and thus AR gene induction using various NF90 mutants. Further, we will characterize the roles of NF90 in prostate tumorigenesis. Specific Aim 2: To investigate the efficacy of Enzalutamide (Enz) in combination with EZH2 inhibitor EPZ-6438 in CRPC using preclinical models Preliminary data showed that combinatorial approaches that target the dual roles of EZH2 simultaneously using its enzymatic inhibitor EPZ-6438 and AR antagonist enzalutamide are highly effective in suppressing CRPC cell growth. In this Aim, we will further confirm this in multiple cell lines and with various dose schemes in vitro and, most importantly, evaluate any synergistic effects in suppressing CRPC progression in vivo. Specific Aim 3: To develop next-generation small molecule EZH2 inhibitors capable of EZH2 protein degradation. Previous efforts have focused on developing active-site inhibitors of EZH2 which makes it very difficult to generate selectivity versus other methyl transferases, in addition to its limitation in targeting only the catalytic function of EZH2. A recent study demonstrated an attractive feature of the H1-receptor antagonist astemizole that targets the EZH2-EED protein-protein interaction leading to EZH2 degradation16. Unfortunately, astemizole has been shown to prolong the QT interval and cause arrhythmias, making it unsuitable as a cancer therapeutic. Here, we will first generate diverse new astemizole derivatives, which will be docked into the PRC2 crystal structure17 and selected for testing in fluorescence polarization (FP) and other in vitro screening assays. Our second strategy will be to use the PRC2 crystal structure along with recently reported mutational data18 which supports the binding site of astemizole to carry out a virtual high-throughput screen (vHTS) of 100 million compound Zinc database of compounds to identify new EZH2-EED inhibitor hit chemotypes, which will be purchased or synthesized and tested in our in vitro assays to determine which are valid hits. These hits will then be optimized using traditional hit-to-lead medicinal chemistry.
|Effective start/end date||8/1/17 → 7/31/22|
- U.S. Army Medical Research and Materiel Command (W81XWH-17-1-0406 P00003)
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