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

Pre-mRNA的选择性剪接是产生蛋白质多样性的基本机制，通常 在癌细胞中解除调控，产生促进生长和存活的异常蛋白质。前列腺增生症 癌症(PCA)是由雄激素受体(AR)活性驱动的。转移性前列腺癌的标准护理是 雄激素剥夺疗法(ADT)。然而，ADT不可避免地会导致抗去势PCa(CRPC)， 虽然仍依赖AR的活动，但不再对激素敏感。在众多潜在的机制中 CRPC是通过选择性剪接产生的结构性活性AR变异体(AR-V)。值得注意的是AR- V7可能在PCa进展和治疗耐药中起因果作用。到目前为止，还没有FDA批准的 特工可以瞄准这些AR-V。最近，我们确定了组蛋白去甲基酶KDM4B的促癌作用。 PCA和几种KDM4B的化学抑制剂。在我们的初步研究中，我们发现KDM4B是必要的 并足以促进AR-V7的表达。KDM4B结合RNA并与多种反式作用因子相互作用 并可能在前mRNA和染色质水平上调节AR的选择性剪接。此外，KDM4B 可能有其他全基因组的选择性剪接靶点，这些都是癌症的标志。高KDM4B 人前列腺癌患者的表达预示着不良的预后，并与AR-V7的表达升高相关。 基于这些科学前提，我们假设KDM4B可能是一种基因特异性的选择性剪接 调控CRPC中致癌剪接模式的调节子，靶向该酶可以抑制 CRPC并使CRPC对当前的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： 目的：探讨KDM4B调控AR-VS选择性剪接的分子机制。KDM4B 可能通过将剪接体招募到选择性外显子的3‘-剪接点来促进选择性剪接 与剪接调控元件(SRE)结合并交替改变染色质结构 拼接的外显子。我们将鉴定这些SRE，并确定选择性剪接周围的染色质景观 外显子。目的2.鉴定KDM4B调控的全基因组剪接变异体。初步研究表明， KDM4B可能有额外的选择性剪接变异体，专用于PCa的肿瘤发生。我们将测试 这一假说是通过比较全基因组KDM4B靶向剪接变异体及其相关的SRE 以及激素敏感型和难治性前列腺癌细胞中的染色质景观。目标3.生成一个临床 候选者(S)通过对KDM4B抑制剂的优化。我们的数据表明KDM4B抑制剂B3可能 作为一种强有力的先导化合物进行进一步优化，以产生临床候选药物。我们会 通过药物化学设计、合成和测试的迭代循环来优化B3。有一种观点认为 KDM4B是一种新的选择性剪接的致癌调控因子。对其作用机制的认识 KDM4B和寻找具有体内疗效的有效KDM4B抑制剂将对 开发具有活性致癌或剪接变异体的CRPC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.