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。治疗前列腺癌的治疗方法，即雄激素剥夺疗法(ADT)，有 已被开发用于靶向雄激素受体(AR)或雄激素合成途径。PCA进展为 耐去势前列腺癌(CRPC)伴ADTS的酪氨酸激酶上调 AR的活动和截断版本称为AR剪接变体。同时以更新的秒数瞄准AR 一代激素疗法是CRPC治疗的前沿，可能需要联合治疗 以产生更深刻的临床效益。我们实验室以前的研究表明，激酶 CRPC的磷酸化和活性升高，并将SRC激酶确立为关键的激酶靶点 通过磷酸蛋白质组引导的多组整合。应用程序的目标是评估协同效应 第二代抗雄激素药物苯扎鲁胺与SRC激酶抑制剂在前列腺癌治疗中的作用 模特们。中心假说是，苯扎鲁胺与SRC激酶抑制剂配对会推动 SRC上AR全长活性和AR剪接变异体活性下调，导致AR- 提高了前列腺癌的存活率，从而导致了更多的前列腺癌细胞死亡。证据将被提供给 支持以下假设：1)阐明苯扎鲁胺和SRC之间的协同作用 体外和体内的激酶抑制剂。2)确定AR与SRC之间的结合或磷酸化 促进药物协同效应。3)确定AR和SRC抑制改变的细胞信号变化 促进药物协同效应。AIM 1的体外实验将包括细胞存活率测定以确定IC50 以确定SRC激酶抑制剂和苯扎鲁胺的协同作用 一组前列腺癌细胞系的组合指数方程。活体实验将评估 SRC激酶抑制剂和苯扎鲁胺在细胞来源的异种移植和早期移植中的协同作用 高扩增表达活化SRC的小鼠前列腺组织重组癌模型的建立 阿。AIM 2的实验涉及免疫分析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，结合或磷酸化AR上感兴趣的残基。 这将涉及质谱学，以在存在的情况下识别AR上磷酸化的关键残基 然后对这些残基进行定点突变，以确定磷酸化是否在 AR剪接变异体有助于AR的配体非依赖性活性。AIM 3的实验涉及猎枪 磷酸蛋白质组学和RNA测序评估OUR给药后细胞信号的变化 协同组合。这项拟议的研究意义重大，因为这将为临床前提供关键的 可能影响未来临床试验设计的数据，方法是将特定的激酶抑制剂与 苯扎鲁胺，最终导致提高患有致命性CRPC的男性的生存率的目标。

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.