AN ACTIVE ROLE OF ADAPTOR PROTEINS IN TYROSINE KINASE INHIBITOR RESISTANCE

描述(由申请人提供)：本项目的目标是确定接头蛋白在介导对受体酪氨酸激酶(RTK)抑制剂的耐药性中的积极作用。在田野里 在肺癌治疗方法中，针对突变形式的EGFR或EML4-ALK重排肺癌使用酪氨酸激酶抑制剂(TKI)已在晚期疾病患者的预后方面取得了重大改善。然而，内在或获得性耐药性仍然是治愈的持久障碍。肿瘤微环境中分泌的生长因子配体可以抵消由突变癌基因驱动的跨多种肿瘤细胞类型的TKI的影响。我们认为参与RTK信号转导的接头蛋白在这一过程中起着积极的作用。我们假设，适配器被TKI取代了驱动RTK，塑造了共表达的RTK信号，并影响了驱动耐药性的微环境配体的作用。我们的模型将在这个项目中进行测试，包括将目标驱动程序RTK重新定位到其他共表达的RTK的适配蛋白。这种将接头切换到共表达的RTK的过程可以通过接头的直接结合和两个RTK分子的二聚化来启动磷酸化。这允许在微量的生长因子配体存在的情况下完全激活RTK。在我们的实验室中，使用各种磷酸化蛋白质组学方法，包括用质谱仪直接测量磷酸化酪氨酸肽的丰度，我们一致地发现，在针对DRIVE癌基因的TKI处理细胞后，共表达的RTK中RTK酪氨酸磷酸化增加。我们的结果表明，在TKI处理共表达的RTK后，启动磷酸化可能与某些生长因子驱动TKI抵抗的能力有关。我们将使用质谱学和邻近连接分析来测试这一模型，以定量和定性地检测TKI治疗后细胞中的适配器和RTK复合体。具体目标1将表征TKI后共表达的RTK中接头蛋白的命运和蛋白复合体的变化。具体目标2将描述RTK：接头配对如何与生长因子配体拯救有关。具体目标3将表征接头蛋白在启动共表达的RTK的酪氨酸磷酸化中的作用。该项目的成功将确定接头蛋白在塑造TKI反应中的积极作用，并在未来产生预测性标记物，通过生长因子配体拯救来识别肿瘤对TKI的快速适应。这一假设是新颖的，因此需要检验。测量蛋白质复合体的复杂方法正在手中，进一步增加了成功的机会。

DESCRIPTION (provided by applicant): The goal of this project is to define an active role of adaptor proteins in mediating resistance to receptor tyrosine kinase (RTK) inhibitors. In the field of lung cancer therapeutics, use of tyrosine kinase inhibitors (TKI) directed against mutant forms of EGFR or EML4-ALK rearranged lung cancers has made major improvements in patient outcome with advanced disease. However, intrinsic or acquired drug resistance remains a persistent obstacle towards a cure. Growth factor ligands secreted in the tumor microenvironment can negate the effects of TKI across multiple tumor cell types driven by mutant oncogenes. We propose that adaptor proteins involved in RTK signaling are playing an active role in this process. We hypothesize that adaptors, displaced from driver RTK by TKI, shape co-expressed RTK signaling and influence the effects of microenvironmental ligands that drive resistance. Our model, to be tested in this project, consists of adaptor proteins being relocalized from the targeted driver RTK to other co-expressed RTK. This switching of adaptors to the co- expressed RTK enables priming phosphorylation mediated by direct binding of the adaptor and dimerization of two RTK molecules. This allows for full RTK activation in the presence of minute amounts of growth factor ligands. In our laboratory, using various phosphoproteomic approaches, including direct measurements of phosphotyrosine peptide abundance using mass spectrometry, we have consistently identified increased abundance of RTK tyrosine phosphorylation in co-expressed RTK following treatment of cells with TKI directed against the driver oncogene. Our results suggest that priming phosphorylation following TKI treatment of co- expressed RTK can be related to ability of some growth factors to drive TKI resistance. We will test this model using mass spectrometry and proximity ligation assays to quantitatively and qualitatively examine adaptor and RTK complexes in cells following TKI treatment. Specific aim 1 will characterize the fate of adaptor proteins and changes in protein complexes in co-expressed RTK following TKI. Specific aim 2 will characterize how RTK:adaptor pairing relates to growth factor ligand rescue. Specific aim 3 will characterize the role of adaptor proteins in priming tyrosine phosphorylation of co-expressed RTK. Success in this project will identify an active role in adaptor proteins in shaping TKI responses and can in the future yield predictive markers identifying tumors rapidly adapting to TKI through growth factor ligand rescue. The hypothesis is novel and therefore important to be tested. Complex approaches to measuring protein complexes are in hand further increasing the chances of success.