Targeting KDM4B, a novel alternative splicing regulator, in castration-resistant prostate cancer (CRPC)

前体mRNA的选择性剪接是产生蛋白质多样性的基本机制， 在癌细胞中失调，产生促进生长和存活的异常蛋白质。前列腺生长 前列腺癌（PCa）是由雄激素受体（AR）活性驱动的。转移性前列腺癌的标准治疗是 雄激素剥夺疗法（ADT）。然而，ADT不可避免地导致去势抵抗性PCa（CRPC）， 虽然仍然依赖于AR活动，但不再对广告敏感。在许多机制中， CRPC是通过选择性剪接产生组成型活性AR变体（AR-V）。值得注意的是， V7，其可能在PCa进展和治疗抗性中起因果作用。到目前为止，没有FDA批准的 最近，我们确定了组蛋白去甲基化酶KDM 4 B在AR-V中的促癌作用。 PCa和KDM 4 B的几种化学抑制剂。在我们的初步研究中，我们发现KDM 4 B是必要的， 并且足以促进AR-V7表达。KDM 4 B结合RNA并与许多反式作用因子相互作用 并且可以在前mRNA和染色质水平调节AR的选择性剪接。此外，KDM 4 B 可能有其他全基因组可变剪接靶点，这些靶点是癌症的标志。高KDM 4 B 在人PCa患者中的AR-V7表达预测不良预后并且与AR-V7表达升高相关。 基于这些科学前提，我们假设KDM 4 B可能是一种基因特异性选择性剪接， 一种决定CRPC致癌剪接模式的调节因子，靶向这种酶可以抑制 CRPC并使CRPC对当前ADT重新敏感。我们提出了三个具体目标来检验这一假设。目标1： 确定KDM 4 B调节AR-Vs选择性剪接的分子机制。 通过将剪接体募集到选择性外显子的3 '-剪接位点， 结合剪接调控元件（SRE），并通过改变周围的染色质结构， 剪接外显子我们将识别这些SRE并确定选择性剪接周围的染色质景观 外显子目标二。鉴定KDM 4 B调控的全基因组剪接变异体。初步研究表明， KDM 4 B可能具有对PCa肿瘤发生特异性的另外的可变剪接变体。我们将测试 通过比较分析全基因组KDM 4 B靶向剪接变体及其相关SRE， 和染色质的景观在这两个敏感性和难治性PCa细胞。目标3：为了生成临床 通过KDM 4 B抑制剂的优化来筛选候选物。我们的数据表明，KDM 4 B抑制剂B3可能 用作进一步优化以产生临床候选药剂的强先导化合物。我们将 通过反复的药物化学设计、合成和测试优化B3。这种理念 KDM 4 B是一种新的选择性剪接致癌调节因子。了解的作用机制 KDM 4 B和鉴定具有体内功效的有效KDM 4 B抑制剂将对KDM 4 B的治疗具有显著的临床影响。 开发针对具有活性致癌选择性剪接变体的CRPC的新疗法。

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.