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衔接蛋白在介导对受体酪氨酸激酶（RTK）抑制剂的抗性中的积极作用。领域 在肺癌治疗中，针对EGFR或EML 4-ALK重排肺癌的突变形式的酪氨酸激酶抑制剂（TKI）的使用在晚期疾病患者的结果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续障碍。肿瘤微环境中分泌的生长因子配体可以抵消TKI在突变癌基因驱动的多种肿瘤细胞类型中的作用。我们建议，在RTK信号参与衔接蛋白在这一过程中发挥了积极的作用。我们假设，适配器，从驱动RTK由TKI取代，形状共表达RTK信号和影响的微环境配体驱动电阻的影响。我们的模型，将在这个项目中进行测试，由适配器蛋白被重新定位从目标驱动RTK到其他共表达的RTK。衔接子向共表达的RTK的这种转换使得能够引发由衔接子的直接结合和两个RTK分子的二聚化介导的磷酸化。这允许在存在微量生长因子配体的情况下完全RTK活化。在我们的实验室中，使用各种磷酸化蛋白质组学方法，包括使用质谱法直接测量磷酸酪氨酸肽丰度，我们一致地确定了在用针对驱动癌基因的TKI处理细胞后，共表达RTK中RTK酪氨酸磷酸化丰度增加。我们的研究结果表明，TKI处理后共表达的RTK的引发磷酸化可能与某些生长因子驱动TKI抗性的能力有关。我们将使用质谱法和邻位连接测定法来测试该模型，以定量和定性地检查TKI处理后细胞中的衔接子和RTK复合物。具体目标1将表征TKI后共表达RTK中衔接蛋白的命运和蛋白复合物的变化。具体目标2将描述RTK：衔接子配对与生长因子配体拯救的关系。具体目标3将表征衔接蛋白在引发共表达的RTK的酪氨酸磷酸化中的作用。该项目的成功将确定衔接蛋白在塑造TKI反应中的积极作用，并在未来产生预测标记物，识别通过生长因子配体拯救快速适应TKI的肿瘤。这一假设是新颖的，因此必须加以检验。测量蛋白质复合物的复杂方法正在进一步增加成功的机会。

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.