Alternative splicing of pre-mRNA is a fundamental mechanism to generate protein diversity that is often deregulated in cancer cells, producing aberrant proteins that promote growth and survival. Growth of prostate cancer (PCa) is driven by the androgen receptor (AR) activities. The standard care for metastatic PCa is androgen-deprivation therapy (ADT). However, ADT inevitably leads to castration-resistant PCa (CRPC) that, while still relies on the AR activities, is no longer hormone-sensitive. Among the many mechanisms underlying CRPC, is the generation of constitutively active AR variants (AR-Vs) through alternative splicing. Of note is AR- V7, which may play a causal role in PCa progression and treatment resistance. Until now, no FDA-approved agent can target these AR-Vs. Recently, we identified a pro-oncogenic role for histone demethylase KDM4B in PCa and several chemical inhibitors of KDM4B. In our preliminary studies, we found that KDM4B is necessary and sufficient to promote AR-V7 expression. KDM4B binds RNA and interacts with many trans-acting factors and may regulate alternative splicing of AR at both the pre-mRNA and chromatin levels. In addition, KDM4B may have other genome-wide alternatively spliced targets that are hallmarks of cancer. High KDM4B expression in human PCa patients predicts poor prognosis and correlates with elevated AR-V7 expression. Based on these scientific premises, we hypothesize that KDM4B may be a gene-specific alternative splicing regulator that dictates an oncogenic splicing pattern in CRPC and that targeting this enzyme could inhibit CRPC and re-sensitize CRPC to current ADT. We propose three specific aims to test this hypothesis.
Aim 1 : To determine the molecular mechanisms by which KDM4B regulates alternative splicing of AR-Vs. KDM4B may promote alternative splicing by recruiting the spliceosome to the 3'-splice site of alternative exons via binding to splicing regulatory elements (SREs) and by changing the chromatin structures around alternatively spliced exons. We will identify these SREs and determine the chromatin landscape around alternatively spliced exons.
Aim 2. To identify KDM4B-regulated genome-wide splice variants. Preliminary studies indicated that KDM4B may have additional alternatively spliced variants that are specific for PCa tumorigenesis. We will test this hypothesis by comparative profiling genome-wide KDM4B-targeted splice variants, their associated SREs and chromatin landscapes in both hormone-sensitive and refractory PCa cells.
Aim 3. To generate a clinical candidate(s) through optimization of KDM4B inhibitors. Our data indicated that the KDM4B inhibitor B3 may serve as a strong lead compound for further optimization to generate a clinical candidate agent. We will optimize B3 through iterative rounds of medicinal chemistry design, synthesis and testing. The notion that KDM4B is an oncogenic regulator of alternative splicing is novel. Understanding mechanism of action of KDM4B and identifying potent KDM4B inhibitors with in vivo efficacy will have significant clinical impact on the development of new therapies for CRPC with active oncogenic alternatively spliced variants.
The goals of this proposal are to understand how an epigenetic enzyme KDM4B generates alternatively spliced protein isoforms that are oncogenic and to develop KDM4B inhibitors that can block this process. Success of the proposal may develop effective treatments and address drug- resistance for men with high-risk of metastatic prostate cancer. !
