Targeting Drug-Resistant Castration Resistant Prostate Cancer via LIMK2

摘要 前列腺癌是美国男性癌症相关死亡的第二大常见原因，主要 由于耐去势前列腺癌(CRPC)的出现。我们的长期目标是找出有效的 针对CRPC的药物靶点和开发小分子抑制剂。我们的目标是确定潜在的 以LIMK2激酶为临床靶点，以高效、特异的LIMK2抑制剂Li-11为药物 苯扎鲁胺耐药CRPC的候选药物。核心假设是LIMK2是疾病特异性靶点 它在去势后上调，促进CRPC的出现、进展和对苯扎鲁胺的耐药性 通过上调AR、ARV7和PI3K信号通路。我们发现LIMK2在CRPC组织中高表达， 但在正常前列腺中低水平表达。LIMK2的诱导敲除或特异性抑制完全逆转 CRPC在体内致瘤，强烈支持LIMK2作为潜在的临床靶点。同样重要的是，Li-11 协同增强苯扎鲁胺在CRPC细胞中的疗效。在分子水平上，Li-11同时 耗尽AR和ARV7，抑制PI3K信号转导。因此，LIMK2针对驱动CRPC的三个关键节点 进展包括对苯扎鲁胺的耐药性。我们提出以下三个具体目标：(1) 剖析LIMK2激活CRPC中AR信号的分子机制。(2)调查 LIMK2激活CRPC中PI3K通路的分子机制(3)确定潜在的 在使用LI-11的苯扎鲁胺耐药CRPC和PDX模型中，LIMK2作为临床靶点。 创新：LIMK2是PCa的潜在临床靶点。这一假说是基于一项创新的 化学筛选，从而发现了LIMK2的几个直接底物。这些LIMK2衬底 解锁了CRPC中针对AR、ARV7和PI3K通路的治疗靶向的强大机制。 我们已经开发出一种高度有效和特异的LIMK2抑制剂(LI-11)，可以完全逆转体内的肿瘤形成 没有可检测到的毒性。LI-11将用于LIMK2作为细胞治疗靶点的临床前验证 在活体内。LIMK2水平可变的LuCaP PDX模型将用于确定LIMK2水平是否 与LI-11和苯扎鲁胺在体内的敏感性相关。将利用几个CRISPR工程细胞系 目的：探讨LIMK2基因S在慢性前列腺癌发病机制中的作用。 意义：成功完成拟议的研究将提供一种新的战略，以同时 通过LIMK2抑制导致CRPC进展和对苯扎鲁胺耐药的三个关键通路。活体内 研究将揭示LIMK2作为临床靶点和LI-11作为候选药物的潜力。PDX小鼠模型 不同的LIMK2水平将进一步揭示LIMK2水平是否可以作为一种新的预测标志物 确定CRPC治疗的疗效。数据进一步表明，靶向LIMK2可以防止 雄激素剥夺治疗后出现慢性前列腺癌。最后，LIMK2作为一种治疗药物的有效性 CRPC中的靶基因可能会增加治疗其他侵袭性癌症的几率，在这些癌症中，LIMK2基因上调。

Abstract Prostate cancer (PCa) is the second most common cause of cancer-related death in men in the US, primarily due to the emergence of castration resistant prostate cancer (CRPC). Our long-term goal is to identify effective drug targets and develop small molecule inhibitors for CRPC. Our objective here is to determine the potential of LIMK2 kinase as a clinical target, and LI-11 (a highly potent and specific LIMK2 inhibitor) as a drug candidate for enzalutamide-resistant CRPC. The central hypothesis is that LIMK2 is a disease-specific target that is upregulated upon castration and promotes CRPC emergence, progression and enzalutamide resistance via upregulation of AR, ARV7 and PI3K pathways. We show that LIMK2 is highly expressed in CRPC tissues, but minimally expressed in normal prostates. Inducible knockdown or specific inhibition of LIMK2 fully reverses CRPC tumorigenesis in vivo, strongly supporting LIMK2 as a potential clinical target. Equally importantly, LI-11 synergistically enhances the efficacy of enzalutamide in CRPC cells. At a molecular level, LI-11 simultaneously depletes AR and ARV7, and inhibits PI3K signaling. Thus, LIMK2 targets three critical nodes driving CRPC progression including enzalutamide-resistance in tandem. We propose the following three specific aims: (1) Dissect the molecular mechanisms by which LIMK2 activates AR signaling in CRPC. (2) Investigate the molecular mechanism of LIMK2-mediated activation of PI3K pathway in CRPC. (3) Determine the potential of LIMK2 as a clinical target in enzalutamide-resistant CRPC and PDX models using LI-11 in vivo. Innovation: LIMK2 is a potential clinical target for PCa. The hypothesis was formulated based on an innovative chemical screen, which led to the discovery of several direct substrates of LIMK2. These LIMK2 substrates unlocked a powerful mechanism for therapeutic targeting of AR, ARV7 and PI3K pathways in tandem in CRPC. We have developed a highly potent and specific LIMK2 inhibitor (LI-11) that fully reverses tumorigenesis in vivo with no detectable toxicity. LI-11 will be used for preclinical validation of LIMK2 as a therapeutic target in cells and in vivo. LuCaP PDX models with variable LIMK2 levels will be used to determine whether LIMK2 levels correlate with LI-11 and enzalutamide sensitivity in vivo. Several CRISPR engineered cell lines will be utilized for genetic interrogation of LIMK2’s role in CRPC pathogenesis. Significance: Successful completion of the proposed studies will provide a new strategy to simultaneously inhibit three critical pathways via LIMK2, which lead to CRPC progression and enzalutamide-resistance. In vivo studies will reveal the potential of LIMK2 as a clinical target and LI-11 as a candidate drug. PDX mouse models with different LIMK2 levels will further uncover whether LIMK2 levels can serve as a novel predictive marker for determining the efficacy of CRPC therapy. The data further indicate that targeting LIMK2 could prevent the emergence of CRPC following androgen deprivation therapy. Finally, the validation of LIMK2 as a therapeutic target in CRPC may improve the odds of treating other aggressive cancers, where LIMK2 is upregulated.