Protein phosphorylation downstream of mutant EGFR kinases

突变 EGFR 激酶下游的蛋白质磷酸化

• 批准号: 10014666
• 负责人: Udayan Guha
• 金额: $ 76.45万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014666
• 关键词: Adenocarcinoma Cell; Amino Acids; Biochemical; Bioinformatics; Biological Assay; Cations; Cell Culture Techniques; Cell Line; Cells; Cyclic AMP-Dependent Protein Kinases; Data; Data Analyses; Data Set; Digestion; EGF gene; ERBB2 gene; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Erlotinib; FRAP1 gene; Fractionation; Generations; Genetically Engineered Mouse; Goals; Human; Immunoprecipitation; In Vitro; Journals; KRAS2 gene; Label; Laboratories; Liquid Chromatography; Lung Adenocarcinoma; MAP Kinase Gene; MAPK14 gene; Malignant neoplasm of lung; Manuscripts; Mass Spectrum Analysis; Mediating; Membrane Proteins; Molecular; Mus; Mutation; Pathway interactions; Patients; Pattern; Phase; Phosphatidylinositols; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phosphotyrosine; Play; Proteins; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Publishing; Resistance; Resistance development; Role; Series; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Stable Isotope Labeling; Techniques; Testing; Trypsin; Tumor Suppressor Proteins; Tyrosine Kinase Inhibitor; Tyrosine Phosphorylation; Universities; Western Blotting; Xenograft procedure; bioinformatics tool; career; drug efficacy; exome sequencing; experimental study; in vivo; inhibitor/antagonist; knock-down; lung tumorigenesis; mTOR Inhibitor; mass spectrometer; member; mutant; novel; phosphoproteomics; proteogenomics; receptor recycling; resistance mechanism; side effect; tandem mass spectrometry; targeted treatment; titanium dioxide; trafficking; tumor

We asked the question: what are the changes in tyrosine phosphorylation of proteins upon EGF stimulation and tyrosine kinase inhibitor (TKI) inhibition in human lung adenocarcinoma cell lines expressing the mutant EGFRs? We used a 1st generation EGFR TKI, erlotinib, a 2nd generation EGFR TKI, afatinib, an irreversible EGFR and ERBB2 inhibitor, and two 3rd generation EGFR TKIs, osimertinib and rociletinib which are irreversible inhibitors. Various large-scale experiments were performed using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry. Lung adenocarcinoma cell lines used in these experiments for the 1st and 2nd generation EGFR TKIs were H3255 and 11-18 (L858R mutation), H1975 (L858R/T790M mutation), PC9 (E746-A750 Del EGFR). This part of the project is complete. We have validated the changes in phosphorylation of a subset of these proteins by immunoprecipitation, western blot experiments and tried to understand the significance of phosphorylation of these proteins. We also performed siRNA-mediated knockdown of proteins identified as phosphorylation targets of mutant EGFRs in lung adenocarcinoma cells harboring mutant EGFRs or mutant KRAS. It is interesting to note that several of the proteins whose tyrosine phosphorylation was inhibited by TKIs in mutant EGFR-expressing cells were also required for survival of EGFR mutant expressing cells but not KRAS mutant expressing cells. We followed two such targets: DAPP1 and SCAMP3. We have showed that both DAPP1 and SCAMP3 interact with mutant EGFRs. We have made several phosphorylation site specific mutants of these proteins and currently conducting biological assays to study the significance of altered phosphorylation of these proteins and the overall role played by these proteins in mutant EGFR-driven lung tumorigenesis. DAPPI1 (dual adapter for phosphotyrosine or 3-phosphoinositides) has not been implicated in EGFR signaling. However tyrosine phosphorylation at Y139 of DAPPI1 is stimulated upon EGF stimulation and inhibited 5-10 fold upon treatment with TKIs, erlotinib and afatinib, suggesting DAPPI1 may be an integral member of the EGFR signaling pathway. We have shown by biochemical experiments that DAPP1 interacts with EGFR. We have also shown that Y139 is a major phosphorylation site of DAPP1. We have planned experiments to assay whether EGFR is the kinase phosphorylating DAPP1, since there is some evidence that SRC may be the kinase involved. SCAMP3 (secretory career membrane protein) has been shown to interact with EGFR. We have demonstrated that mutant EGFRs phosphorylate specific sites in SCAMP3 more than WT EGFR. SCAMP3 is involved in receptor recycling. We are currently studying the role of SCAMP3 in mutant EGFR trafficking. We have also demonstrated that SCAMP3 is a tumor suppressor in EGFR mutant lung adenocarcinoma cell lines.A manuscript on the role of SCAMP3 and the phosphorylation targets downstream of mutant EGFRs is being submitted. Identification of Ser/Thr phosphorylation sites on downstream targets of mutant EGFRs and quantitation of phosphorylation changes upon TKI inhibition resistant to EGFR-directed TKIs This part of the project is complete. We have completed a series of experiments to identify Ser/Thr phosphorylation sites by SILAC labeling of adenocarcinoma cells and mass spectrometry. We used various fractionation techniques after in-solution trypsin digestion, such as strong cation exchange (SCX) or basic reverse phase. Around 30 fractions were then subjected to TiO2 enrichment for phosphopeptide isolation followed by reverse phase liquid chromatography and tandem mass spectrometry using an orbitrap elite mass spectrometer. H3255 lung adenocarcinoma cells harboring the L858R mutation and H1975 cells harboring L858R/T790M mutations were used in a triple-SILAC experiment. A total of around 8000 phosphosites were identified from H3255 cells and around 6000 sites identified in H1975 cells. We demonstrateddifferent patterns of dynamic phosphorylation changes upon EGF stimulation and TKI inhibition in these cells. We have analyzed this data set using several bioinformatic tools, including IPA and Ariadne pathway studio. Various canonical pathways such as p70S6, IRS, ERK/MAPK, mTOR, PKA, JAK/STAT were enriched in our dataset. We have collaborated with Dr. Andrea Califano in Columbia University to use their unique bioinformatics tools to interrogate the data to generate new hypothesis that can then be validated experimentally. Two manuscripts have been published in the journals Proteomics (2015) and Molecular and Cellular Proteomics (2017). We are currently performing quantitative mass spectrometry experiments to identify and quantify the proteome and phosphoproteome of lung adenocarcinoma cells sensitive and resistant to the 3rd generation EGFR TKIs, osimertinib and rociletinib. We have generated several human lung adenocarcinoma cell lines resistant to these TKIs by step-wise TKI treatment. We have finished the mass spectrometry analyses of the osimertinib and rociletinib-sensitive and resistant cells. We have performed SILAC mass spectrometry to quantitate the global proteome and phosphoproteome. Currently we are doing the same for osimertinib resistant cells. In parallel we have also generated in vivo resistance to osimertinib in mutant EGFR genetically engineered mouse (GEM) models. Osimertinib-resistant mouse tumors will be compared with sensitive tumors to identify mechanisms of osimertinib resistance. We have undertaken an integrated proteo-genomics approach to identifying the mechanisms of resistance to the 3rd generation EGFR TKIs, osimertinib and rociletinib. We have completed whole exome sequencing of cell lines and xenografts generated in our laboratory that are resistant to osimertinib. We are currently analyzing this data. In addition, we have completed global proteome quantitation and phosphoproteome quantitation of a subset of lung adenocarcinoma cell lines generated in our laboratory. We have identified specific kinases that are potentially more active in the osimertinib resistant cells compared to the sensitive cells- these include PI3K, AKT, mTOR, p38 MAPK, SGK1, among others. Further bioinformatic analysis of our mass spectrometry data identified dactolisib, a PI3K/mTOR inhibitor, and VX970, an ATR inhibitor. We validated the efficacy of these drugs in osimertinib and rociletinib resistant cells in vitro and in vivo in xenografts. One manuscript describing these findings with the 3rd generation EGFR TKis is being submitted.

我们提出这样的问题：在表达突变型 EGFR 的人肺腺癌细胞系中，EGF 刺激和酪氨酸激酶抑制剂 (TKI) 抑制后蛋白质的酪氨酸磷酸化发生了哪些变化？我们使用了第一代 EGFR TKI 厄洛替尼，第二代 EGFR TKI 阿法替尼，一种不可逆的 EGFR 和 ERBB2 抑制剂，以及两种第三代 EGFR TKI 奥西替尼和罗西替尼，这是不可逆的抑制剂。使用细胞培养物中氨基酸的稳定同位素标记 (SILAC) 和质谱法进行了各种大规模实验。这些实验中使用的第一代和第二代 EGFR TKI 的肺腺癌细胞系为 H3255 和 11-18（L858R 突变）、H1975（L858R/T790M 突变）、PC9（E746-A750 Del EGFR）。这部分项目已经完成。我们通过免疫沉淀、蛋白质印迹实验验证了这些蛋白质的一部分磷酸化的变化，并试图了解这些蛋白质磷酸化的重要性。我们还在携带突变 EGFR 或突变 KRAS 的肺腺癌细胞中对被确定为突变 EGFR 磷酸化靶标的蛋白质进行了 siRNA 介导的敲低。有趣的是，在突变型 EGFR 表达细胞中，酪氨酸磷酸化被 TKI 抑制的几种蛋白质也是 EGFR 突变型表达细胞生存所必需的，但 KRAS 突变型表达细胞则不需要。我们跟踪了两个这样的目标：DAPP1 和 SCAMP3。我们已经证明 DAPP1 和 SCAMP3 都与突变型 EGFR 相互作用。我们已经制作了这些蛋白质的几种磷酸化位点特异性突变体，目前正在进行生物测定，以研究这些蛋白质磷酸化改变的重要性以及这些蛋白质在突变型 EGFR 驱动的肺肿瘤发生中所起的总体作用。 DAPPI1（磷酸酪氨酸或 3-磷酸肌醇的双接头）尚未涉及 EGFR 信号转导。然而，DAPPI1 Y139 的酪氨酸磷酸化在 EGF 刺激下受到刺激，而在 TKI、厄洛替尼和阿法替尼治疗后被抑制 5-10 倍，这表明 DAPPI1 可能是 EGFR 信号通路的重要成员。我们通过生化实验证明DAPP1与EGFR相互作用。我们还表明 Y139 是 DAPP1 的主要磷酸化位点。我们计划进行实验来测定 EGFR 是否是磷酸化 DAPP1 的激酶，因为有一些证据表明 SRC 可能是所涉及的激酶。 SCAMP3（分泌生涯膜蛋白）已被证明与 EGFR 相互作用。我们已经证明突变型 EGFR 比 WT EGFR 更能磷酸化 SCAMP3 中的特定位点。 SCAMP3 参与受体回收。我们目前正在研究 SCAMP3 在突变型 EGFR 运输中的作用。我们还证明 SCAMP3 是 EGFR 突变型肺腺癌细胞系中的肿瘤抑制因子。一份关于 SCAMP3 的作用和突变型 EGFR 下游磷酸化靶点的手稿正在提交。突变型 EGFR 下游靶标上 Ser/Thr 磷酸化位点的鉴定以及对 EGFR 导向 TKI 耐药的 TKI 抑制时磷酸化变化的定量 该项目的这一部分已完成。我们已经完成了一系列实验，通过腺癌细胞的SILAC标记和质谱来鉴定Ser/Thr磷酸化位点。我们在溶液内胰蛋白酶消化后使用了各种分级技术，例如强阳离子交换 (SCX) 或碱性反相。然后对大约 30 个级分进行 TiO2 富集以分离磷酸肽，然后使用 Orbitrap Elite 质谱仪进行反相液相色谱和串联质谱分析。含有 L858R 突变的 H3255 肺腺癌细胞和含有 L858R/T790M 突变的 H1975 细胞用于三重 SILAC 实验。从 H3255 细胞中总共鉴定出约 8000 个磷酸位点，在 H1975 细胞中鉴定出约 6000 个位点。我们证明了这些细胞中 EGF 刺激和 TKI 抑制时动态磷酸化变化的不同模式。我们使用多种生物信息学工具（包括 IPA 和 Ariadne Pathway Studio）分析了该数据集。我们的数据集中丰富了各种经典途径，例如 p70S6、IRS、ERK/MAPK、mTOR、PKA、JAK/STAT。我们与哥伦比亚大学的 Andrea Califano 博士合作，使用他们独特的生物信息学工具来询问数据，以生成新的假设，然后可以通过实验验证。两篇手稿已发表在《蛋白质组学》（2015）和《分子与细胞蛋白质组学》（2017）杂志上。我们目前正在进行定量质谱实验，以鉴定和定量对第三代 EGFR TKI、奥希替尼和罗西替尼敏感和耐药的肺腺癌细胞的蛋白质组和磷酸蛋白质组。我们通过逐步 TKI 治疗产生了几种对这些 TKI 耐药的人肺腺癌细胞系。我们已经完成了奥希替尼和罗西替尼敏感和耐药细胞的质谱分析。我们进行了 SILAC 质谱分析来定量总体蛋白质组和磷酸化蛋白质组。目前我们正在对奥希替尼耐药细胞做同样的事情。与此同时，我们还在突变 EGFR 基因工程小鼠 (GEM) 模型中产生了对奥希替尼的体内耐药性。将对奥希替尼耐药的小鼠肿瘤与敏感肿瘤进行比较，以确定奥希替尼耐药的机制。我们采用了综合蛋白质基因组学方法来确定对第三代 EGFR TKI、奥希替尼和罗西替尼的耐药机制。我们已经完成了对我们实验室产生的对奥希替尼耐药的细胞系和异种移植物的全外显子组测序。我们目前正在分析这些数据。此外，我们还完成了我们实验室产生的肺腺癌细胞系子集的全局蛋白质组定量和磷酸蛋白质组定量。我们已经确定了与敏感细胞相比，在奥希替尼耐药细胞中可能更活跃的特定激酶，其中包括 PI3K、AKT、mTOR、p38 MAPK、SGK1 等。对我们的质谱数据进行进一步的生物信息分析，确定了 dactolisib（一种 PI3K/mTOR 抑制剂）和 VX970（一种 ATR 抑制剂）。我们在体外和体内异种移植物中验证了这些药物对奥西替尼和罗西替尼耐药细胞的疗效。正在提交一份描述第三代 EGFR TK 的这些发现的手稿。