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

阐明恩杂鲁胺和 Src 激酶抑制剂在去势抵抗性前列腺癌中的协同作用

• 批准号: 10476997
• 负责人: Ralph E White
• 金额: $ 2.96万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10476997
• 关键词: Acetates; Aftercare; American; Androgen Antagonists; Androgen Receptor; Androgens; Animal Model; Automobile Driving; Binding; Biological Assay; Bypass; Cancer Etiology; Cancer Model; Cancer Patient; Cancer Survivorship; Cell Death; Cell Line; Cell Survival; Cessation of life; Clinical; Clinical Data; Clinical Trials Design; Combined Modality Therapy; Coupled; Data; Data Set; Diagnosis; Dose; Down-Regulation; Drug Synergism; Drug Targeting; Equation; Future; Generations; Genetic Transcription; Goals; Immunoassay; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Laboratories; Length; Ligands; Ligation; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measures; Medical; Mus; Nuclear Translocation; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Population; Post-Translational Protein Processing; Prostate; Prostate Cancer therapy; Protein Tyrosine Kinase; Publishing; RNA Splicing; Regulation; Research; Resistance; Shotguns; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Therapeutic; Tissue Recombination; Variant; Work; Xenograft procedure; abiraterone; androgen deprivation therapy; base; cancer survival; castration resistant prostate cancer; enzalutamide; experimental study; hormone therapy; in vivo; indexing; interest; kinase inhibitor; male; men; multiple omics; non-genomic; overexpression; phosphoproteomics; pre-clinical; prostate cancer cell; prostate cancer cell line; prostate cancer model; prostate cancer progression; src-Family Kinases; standard of care; synergism; transcriptome sequencing; tumor

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.

项目概要 前列腺癌 (PCa) 是美国男性中最常诊断出的非皮肤癌 人口。治疗 PCa 的疗法被称为雄激素剥夺疗法 (ADT)， 已开发用于靶向雄激素受体（AR）或雄激素合成途径。 PCa 进展为 由于酪氨酸激酶上调，ADT 存在下的去势抵抗性前列腺癌 (CRPC) AR 的活性和截短版本称为 AR 剪接变体。同时以更新的第二个瞄准 AR 新一代激素疗法是CRPC治疗的前沿，可能需要联合治疗 以产生更深远的临床效益。我们实验室之前的工作表明激酶 CRPC 中的磷酸化和活性升高，并已将 SRC 激酶确立为关键激酶靶标 通过磷酸化蛋白质组引导的多组学整合。该申请的目的是评估协同作用 恩杂鲁胺（第二代抗雄激素药物）和 SRC 激酶抑制剂在前列腺癌中的作用 模型。中心假设是恩杂鲁胺与 SRC 激酶抑制剂配对可驱动 下调SRC上的AR全长活性和AR剪接变体活性，导致AR- 驱动前列腺癌存活，从而导致更大的前列腺癌细胞死亡。将提供证据给 支持以下目标的假设：1）阐明恩杂鲁胺和 SRC 之间的协同作用 体外和体内激酶抑制剂。 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 AR 和 SRC 抑制所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 IC50 每种治疗方法的研究，并通过以下方法确定 SRC 激酶抑制剂和恩杂鲁胺之间的协同作用： 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和之前的协同作用 建立表达过度扩增的激活SRC的小鼠前列腺组织重组癌症模型 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 上感兴趣的残基的结合或磷酸化。 这将涉及质谱法来识别在存在 AR 的情况下磷酸化的关键残基 恩杂鲁胺，然后对这些残基进行定点诱变，以确定磷酸化是否 AR 剪接变体有助于 AR 的配体独立活性。 《目标 3》的实验涉及霰弹枪 磷酸蛋白质组学和 RNA 测序可评估施用我们的药物后细胞信号传导的变化 协同组合。拟议的研究意义重大，因为这将提供关键的临床前研究 可能影响未来临床试验如何设计的数据，通过将特定激酶抑制剂与 enzalutamide，最终实现提高患有致命性 CRPC 的男性生存率的目标。