Protein phosphorylation downstream of mutant EGFR kinases

突变 EGFR 激酶下游的蛋白质磷酸化

• 批准号: 10262315
• 负责人: Udayan Guha
• 金额: $ 41.57万
• 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/10262315
• 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; Deposition; 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; 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; p38 Mitogen Activated Protein Kinase; 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 demonstrated different 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 reviewed and deposited in bioRxiv.

我们提出的问题是：在表达突变型egfr的人肺腺癌细胞系中，在EGF刺激和酪氨酸激酶抑制剂（TKI）抑制下，酪氨酸磷酸化蛋白的变化是什么？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，它们是不可逆的抑制剂。利用细胞培养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。本实验中用于第一代和第二代EGFR TKIs的肺腺癌细胞系分别为H3255和11-18 （L858R突变）、H1975 （L858R/T790M突变）、PC9 （E746-A750 Del EGFR）。这部分工程已经完成。我们已经通过免疫沉淀、western blot实验验证了这些蛋白子集磷酸化的变化，并试图了解这些蛋白磷酸化的意义。我们还在含有突变egfr或突变KRAS的肺腺癌细胞中进行了sirna介导的蛋白敲除，这些蛋白被鉴定为突变egfr的磷酸化靶点。有趣的是，在表达EGFR突变体的细胞中，有几种酪氨酸磷酸化被TKIs抑制的蛋白也是表达EGFR突变体的细胞存活所必需的，而表达KRAS突变体的细胞则不需要这些蛋白。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个磷酸化位点特异性突变体，目前正在进行生物学分析，以研究这些蛋白质磷酸化改变的意义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-磷酸肌苷的双适配器）没有参与EGFR信号传导。然而，DAPPI1 Y139位点酪氨酸磷酸化在EGF刺激下被激活，在TKIs、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信号通路的一个组成部分。我们已经通过生化实验证明DAPP1与EGFR相互作用。我们还发现Y139是DAPP1的一个主要磷酸化位点。我们计划进行实验，以确定EGFR是否是磷酸化DAPP1的激酶，因为有一些证据表明SRC可能是参与的激酶。SCAMP3（分泌膜蛋白）已被证明与EGFR相互作用。我们已经证明突变型EGFR比WT型EGFR更能磷酸化SCAMP3中的特定位点。SCAMP3参与受体再循环。我们目前正在研究SCAMP3在突变体EGFR转运中的作用。我们也证明了SCAMP3在EGFR突变的肺腺癌细胞系中是一种肿瘤抑制因子。目前正在提交一份关于SCAMP3的作用和突变体egfr下游磷酸化靶点的手稿。鉴定突变体egfr下游靶点上的丝氨酸/苏氨酸磷酸化位点，定量测定抗egfr定向TKIs时TKI抑制的磷酸化变化。此部分项目已完成。我们已经完成了一系列的实验，通过腺癌细胞的SILAC标记和质谱鉴定丝氨酸/苏氨酸磷酸化位点。我们在溶液胰酶消化后使用了各种分离技术，如强阳离子交换（SCX）或碱性反相。然后对大约30个组分进行TiO2富集以分离磷酸肽，然后使用轨道rap精英质谱仪进行反相液相色谱和串联质谱分析。采用携带L858R突变的H3255肺腺癌细胞和携带L858R/T790M突变的H1975肺腺癌细胞进行三重silac实验。在H3255细胞中共鉴定出约8000个磷酸化位点，在H1975细胞中鉴定出约6000个磷酸化位点。我们在这些细胞中证明了EGF刺激和TKI抑制时动态磷酸化变化的不同模式。我们使用几种生物信息学工具，包括IPA和Ariadne pathway studio，分析了这些数据集。我们的数据集中丰富了p70S6、IRS、ERK/MAPK、mTOR、PKA、JAK/STAT等多种典型通路。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查询数据，以产生新的假设，然后可以通过实验验证。在《蛋白质组学》（2015）和《分子与细胞蛋白质组学》（2017）上发表论文2篇。我们目前正在进行定量质谱实验，以鉴定和量化对第三代EGFR TKIs、奥西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化蛋白质组。我们已经通过逐步TKI治疗产生了几种对这些TKI耐药的人肺腺癌细胞系。我们完成了对奥西替尼和罗西替尼敏感和耐药细胞的质谱分析。我们使用了SILAC质谱法来定量全局蛋白质组和磷蛋白质组。目前，我们正在对奥西替尼耐药细胞进行同样的治疗。同时，我们还在突变EGFR基因工程小鼠（GEM）模型中产生了对奥西替尼的体内耐药性。奥西替尼耐药小鼠肿瘤将与敏感肿瘤进行比较，以确定奥西替尼耐药机制。我们采用了一种综合的蛋白质基因组学方法来确定对第三代EGFR TKIs、奥西替尼和罗西替尼的耐药机制。我们已经完成了我们实验室产生的对奥西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，我们已经完成了我们实验室产生的肺腺癌细胞亚系的整体蛋白质组定量和磷酸化蛋白质组定量。我们已经确定了在奥西替尼耐药细胞中比敏感细胞更活跃的特定激酶，包括PI3K， AKT, mTOR, p38 MAPK， SGK1等。进一步的生物信息学分析我们的质谱数据确定了dactolisib， PI3K/mTOR抑制剂和VX970， 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。一篇描述这些发现的第三代EGFR TKis的手稿正在被审查并存放在bioRxiv上。