Protein phosphorylation downstream of mutant EGFR kinases

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？我们使用了两种TKIs，厄洛替尼，一种可逆的EGFR抑制剂和阿法替尼，一种不可逆的EGFR和ERBB2抑制剂。各种大规模的实验进行了SILAC和质谱。本实验使用的肺腺癌细胞系为H3255和11-18 （L858R突变）、H1975 （L858R/T790M突变）、PC9 （E746-A750 Del EGFR）。此外，等基因NR6 （3T3成纤维细胞的一种变体）和HBECs（人支气管上皮细胞）稳定表达WT EGFR、L858R EGFR、Del EGFR也被用于磷酸化研究。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。我们目前正在跟踪两个这样的目标：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。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。我们目前正在与哥伦比亚大学的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。2015年发表1篇-蛋白质组学，15(2-3):340-55（2015）。另一份已提交。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。我们完成了对罗西替尼敏感和耐药细胞的质谱分析。目前，我们正在对奥西替尼耐药细胞进行同样的治疗。与此同时，我们也在突变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。我们现在正在对耐药细胞中这些潜在活性激酶的tki进行迷你筛选，以验证激酶活性和奥希替尼耐药性。

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 two TKIs, erlotinib, a reversible EGFR inhibitor and afatinib, an irreversible EGFR and ERBB2 inhibitor. Various large-scale experiments were performed using SILAC and mass spectrometry. Lung adenocarcinoma cell lines used in these experiments were H3255 and 11-18 (L858R mutation), H1975 (L858R/T790M mutation), PC9 (E746-A750 Del EGFR). In addition isogenic NR6 (a variant of 3T3 fibroblasts) and HBECs (human bronchial epithelial cells) with stable expression of WT EGFR, L858R EGFR, Del EGFR were also used for phosphorylation studies. 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 are currently following 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. 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 are currently collaborating 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. One manuscript has been published in 2015 - Proteomics. 15(2-3):340-55 (2015). Another has been submitted. 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 rociletinib-sensitive and resistant cells. Currently we are doing the same for osimertinib resistant cells. In parallel we are also generating 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. We are now in the process of performing a mini-screen of TKIs of these potentially active kinases in the resistant cells to validate kinase activity and osimertinib resistance.