Elucidating Synergy of Enzalutamide and Src Kinase Inhibitors in Castration-Resistant Prostate Cancer

项目摘要 前列腺癌（PCa）是美国男性中最常见的非皮肤癌 人口治疗PCa的治疗剂，称为雄激素去甲肾上腺素治疗（ADT）， 已开发出靶向雄激素受体（AR）或雄激素合成途径。PCa进展为 由于酪氨酸激酶上调，存在ADT的去势抵抗性前列腺癌（CRPC） 活性和截短形式的AR称为AR剪接变体。虽然针对AR与更新的第二 第二代激素治疗处于CRPC治疗的最前沿，可能需要联合治疗 产生更深远的临床效益。我们实验室以前的工作表明，激酶 CRPC中磷酸化和活性升高，并已确立SRC激酶为关键激酶靶点 通过磷酸化蛋白质组引导的多组整合。该应用程序的目的是评估协同作用 Enzalutamide（第二代抗雄激素药物）与SRC激酶抑制剂在前列腺癌中的比较 模型中心假设是Enzalutamide与SRC激酶抑制剂配对， SRC上的AR全长活性和AR剪接变体活性下调，导致AR- 导致前列腺癌的存活，从而导致更大的前列腺癌细胞死亡。证据将提供给 支持以下目的的假设：1）阐明Enzalutamide与SRC之间的协同作用 激酶抑制剂在体外和体内。2)确定AR和SRC之间的结合或磷酸化， 有助于药物协同作用。3)确定由AR和SRC抑制改变的细胞信号传导的变化， 有助于药物协同作用。目标1的体外实验将涉及细胞活力测定以确定IC 50 每种治疗剂的剂量，并通过测定SRC激酶抑制剂和Enzalutamide之间的协同作用， 一组前列腺癌细胞系的组合指数方程。体内实验将评估 SRC激酶抑制剂和恩杂鲁胺在细胞系来源的异种移植物中的协同作用， 建立了表达活化SRC的小鼠前列腺组织重组癌模型， AR.目标2的实验涉及免疫测定和靶向磷酸蛋白质组学，以分析 在存在或不存在SRC激酶抑制剂的情况下，AR上感兴趣的残基上的结合或磷酸化。 这将涉及质谱法，以鉴定在存在下AR上磷酸化的关键残基。 恩杂鲁胺，然后对这些残基进行定点诱变，以确定是否存在磷酸化。 AR剪接变体有助于AR的配体非依赖性活性。目标3的实验涉及猎枪 磷酸蛋白质组学和RNA测序，以评估我们的药物给药后细胞信号传导的变化。 协同组合。拟议的研究是重要的，因为这将提供关键的临床前 数据可能会影响未来的临床试验如何设计，通过结合特定的激酶抑制剂与 Enzalutamide，最终实现增加致死性CRPC男性患者生存率的目标。

Project Summary Prostate Cancer (PCa) is the most commonly diagnosed non-skin cancer amongst the American male population. Therapeutics to treat PCa, which are known as Androgen Deprivation Therapies (ADTs), have been developed to target the androgen receptor (AR) or androgen synthesis pathways. PCa progresses into Castration-Resistant Prostate Cancer (CRPC) in the presence of ADTs due to upregulated tyrosine kinase activity and truncated versions of AR called AR splice variants. While targeting AR with newer second generation hormonal therapies is at the forefront of treatment of CRPC, combination therapy may be required to produce a more profound clinical benefit. Previous work from our laboratory has shown that kinase phosphorylation and activity are elevated in CRPC and have established SRC kinase as a key kinase target via phosphoproteome-guided multi-omic integration. The objective of the application is to evaluate the synergy between enzalutamide, a second generation anti-androgen, and SRC kinase inhibitors in prostate cancer models. The central hypothesis is that enzalutamide paired with SRC kinase inhibitors drives the downregulation of both AR full-length activity and AR splice variant activity on SRC, resulting in reduced AR- driven prostate cancer survival and hence greater prostate cancer cell death. Evidence will be provided to support the hypothesis in the following aims: 1) Elucidate the synergism between enzalutamide and SRC kinase inhibitors in vitro and in vivo. 2) Determine the binding or phosphorylation between AR and SRC that contribute to drug synergy. 3) Determine changes in cellular signaling altered by AR and SRC inhibition that contribute to drug synergy. In vitro experiments for Aim 1 will involve cell viability assays to determine IC50s of each therapeutic and to determine synergy between SRC kinase inhibitors and enzalutamide via the Combination Index Equation across a panel of prostate cancer cell lines. In vivo experiments will assess the synergy between SRC kinase inhibitors and enzalutamide in cell line derived xenografts and previously established mouse prostate tissue recombination cancer models expressing activated SRC with over-amplified AR. Experiments for Aim 2 involve immunoassays and targeted phosphoproteomics to analyze changes in binding or phosphorylation on residues of interest on AR in the presence or absence of SRC kinase inhibitors. This will involve mass spectrometry to identify key residues phosphorylated on AR in the presence of enzalutamide followed by site-directed mutagenesis of these residues to determine if the phosphorylation on AR splice variants contributes to ligand-independent activity of AR. Experiments for Aim 3 involve shotgun phosphoproteomics and RNA sequencing to assess changes in cellular signaling after administration of our synergistic combinations. The proposed research is significant because this will provide critical pre-clinical data that could influence how future clinical trials are designed via combining specific kinase inhibitors with enzalutamide, ultimately leading to the goal of increasing survival for men suffering from lethal CRPC.