Targeting Drug-Resistant Castration Resistant Prostate Cancer via LIMK2

摘要 前列腺癌（PCa）是美国男性癌症相关死亡的第二大常见原因，主要是 由于去势抵抗性前列腺癌（CRPC）的出现。我们的长期目标是确定有效的 药物靶向和开发CRPC的小分子抑制剂。我们的目标是确定 LIMK 2激酶作为临床靶点，LI-11（一种高效特异性LIMK 2抑制剂）作为药物 Enzalutamide耐药CRPC的候选者。中心假设是LIMK 2是疾病特异性靶点 在去势后上调，并促进CRPC出现、进展和Enzalutamide耐药性 通过上调AR、ARV 7和PI 3 K途径。我们发现LIMK 2在CRPC组织中高度表达， 但在正常前列腺中表达最低。LIMK 2的诱导性敲低或特异性抑制完全逆转 CRPC体内肿瘤发生，强烈支持LIMK 2作为潜在的临床靶点。同样重要的是，LI-11 协同增强恩杂鲁胺在CRPC细胞中的功效。在分子水平上，LI-11同时 消耗AR和ARV 7，并抑制PI 3 K信号传导。因此，LIMK 2针对驱动CRPC的三个关键节点 包括enzalutamide耐药的进展。我们提出以下三个具体目标：（1） 剖析LIMK 2激活CRPC中AR信号传导的分子机制。(2)探讨 CRPC中LIMK 2介导的PI 3 K通路活化的分子机制。(3)确定的潜力 LIMK 2作为Enzalutamide耐药CRPC和PDX模型中的临床靶标，使用LI-11进行体内研究。 创新：LIMK 2是PCa的潜在临床靶点。该假设是基于一个创新的 化学筛选，导致发现了LIMK 2的几种直接底物。这些LIMK 2底物 揭示了CRPC中AR、ARV 7和PI 3 K通路串联治疗靶向的强大机制。 我们已经开发了一种高效和特异性的LIMK 2抑制剂（LI-11），可在体内完全逆转肿瘤发生 没有可检测到的毒性。LI-11将用于LIMK 2作为细胞中的治疗靶标的临床前验证 和体内。具有可变LIMK 2水平的LuCaP PDX模型将用于确定LIMK 2水平是否 与体内LI-11和Enzalutamide敏感性相关。将利用几种CRISPR工程化细胞系 用于LIMK 2在CRPC发病机制中的作用的遗传询问。 意义：成功完成拟议的研究将提供一个新的战略， 通过LIMK 2抑制导致CRPC进展和Enzalutamide耐药的三个关键途径。体内 研究将揭示LIMK 2作为临床靶点的潜力和LI-11作为候选药物的潜力。PDX小鼠模型 将进一步揭示LIMK 2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。这些数据进一步表明，靶向LIMK 2可以防止 雄激素剥夺治疗后出现CRPC。最后，LIMK 2作为治疗药物的验证 CRPC中的靶点可以提高治疗其他侵袭性癌症的几率，其中LIMK 2被上调。

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.