Comparison of substrate specificities of wild type and mutant EGFR kinases

1A：在蛋白质阵列上使用体外激酶测定法鉴定突变EGFR底物（预算的2%）我们正处于验证该筛选中鉴定的潜在靶点的最后阶段。我们有一个大约10种蛋白质的清单，我们已经用生物化学方法纯化了这些蛋白质，目前正在用野生型和突变型EGFR激酶进行体外激酶测定。我们已经进行了新的生物信息学分析，以选择这些蛋白质进行进一步验证。下文介绍了其中一些措施。我们试图通过蛋白质阵列的体外激酶试验来鉴定突变EGFR激酶的靶点。我们使用了由英杰公司开发的人类蛋白质阵列，最近与约翰霍普金斯医学研究所的朱恒博士的实验室合作，使用他们小组开发的定制蛋白质阵列。我们使用WTEGFR、L 858 R EGFR、L 858 R/T790 M EGFR在存在或不存在酪氨酸激酶抑制剂厄洛替尼的情况下对这些蛋白阵列进行体外激酶测定。我们确定了几种蛋白质，在体外磷酸化的突变激酶，但不是WTEGFR。例如，我们发现STK 3、PKC θ、PAK 3、MST 4（MASK）、TBK 1、LynA被L585 R EGFR磷酸化，但不被WT EGFR磷酸化。朱博士实验室的蛋白质阵列在载玻片上发现了大约20，000个全长人类蛋白质。我们已经完成了筛选，并分析了过去一年的激酶测定数据。我们目前正在验证一些突变EGFR的目标确定与此检测。我们已经采取了生物化学和遗传学方法来验证目标。一个这样的目标是蛋白质DLG 1（光盘大）。我们还从基于质谱的磷酸化蛋白质组学实验中鉴定了该蛋白质作为突变型EGFR的磷酸化靶点（见下文）。我们已经证明DLG 1确实与EGFR相互作用。这种相互作用和磷酸化的意义的进一步表征正在进行中。1B：比较与WT EGFR、L 858 R EGFR和Del E746-A750 EGFR相关的蛋白质的相互作用程度。该项目的这一部分已完成，未进一步完成。我们目前正在验证我们的激酶试验和基于质谱的相互作用试验筛选中获得的突变型EGFR的单个相互作用伴侣。进一步描述如下。两种最常见的TKI致敏肺癌特异性EGFR突变具有组成性活性。然而，两项针对厄洛替尼治疗患者的小型临床试验表明，携带Del EGFR的患者的预后优于携带L 858 R EGFR的患者。与携带L 858 R EGFR的患者相比，携带Del EGFR的患者反应更好，无进展生存期更长。我们进行了鉴定EGFR相互作用蛋白质的质谱法的方法。我们在NR 6细胞中表达WT EGFR、L 858 R EGFR和Del E746-A750 EGFR，NR 6细胞是小鼠3 T3成纤维细胞的一种变体，不表达内源性EGFR。我们使用稳定同位素标记与氨基酸在细胞培养物（SILAC）的差异标记细胞表达的每一个EGFR的变体。EGFR与EGFR胞外结构域特异性的单克隆抗体的免疫沉淀进行分离EGFR相互作用的蛋白质，进一步确定通过质谱。细胞的SILAC标记使得能够相对定量与WT EGFR和突变EGFR相互作用的蛋白质的相互作用程度。我们鉴定了几种与突变EGFR特异性相互作用的蛋白质。特别感兴趣的是与WT EGFR相比与L 858 R和Del EGFR相互作用更多的受体酪氨酸激酶（例如EPHA 2、DDR2、AXL），表明表达突变EGFR的细胞中受体酪氨酸激酶的串扰。目前，我们正在治疗一组没有EGFR和KRAS突变的肺癌细胞系，使用靶向活性激酶的药物，基于相互作用的伴侣和另一种基于Luminex的激酶磷酸化测定。我们已经证明了对由过度活跃的激酶预测的细胞系的敏感性。目前正在撰写一份手稿，以发表该项目的调查结果。

1A: Identify mutant EGFR substrates using in vitro kinase assays on protein arrays (2% of budget) We are in the final phases of validating potential targets identified in this screen. We have a list of around 10 proteins that we have purified biochemically and currently performing in vitro kinase assays with Wild type and mutant EGFR kinases. We have performed new bioinformatics analysis to select these proteins for further validation. Some of them are described below. We sought to identify targets of mutant EGFR kinases using in vitro kinase assays on protein arrays. We have used human protein arrays developed by Invitrogen and more recently collaborated with the laboratory of Dr. Heng Zhu at Johns Hopkins Medical Institute to use the custom protein arrays developed by their group. We used WTEGFR, L858R EGFR, L858R/T790M EGFR in presence or absence of tyrosine kinase inhibitor, erlotinib to perform in vitro kinase assays on these protein arrays. We identified several proteins that are phosphorylated in vitro by mutant kinases, but not WTEGFR. For example, we found STK3, PKCtheta, PAK3, MST4 (MASK), TBK1, LynA to be phosphorylated by L585R EGFR, but not WT EGFR. The protein arrays from Dr. Zhu laboratory have around 20,000 full-length human proteins spotted on glass slides. We have completed the screening and have analyzed the kinase assay data this past year. We are currently validating some of the targets of mutant EGFR identified with this assay. We have undertaken both biochemical and genetic approaches to validate the targets. One such target is the protein DLG1 (disc large). We also identified this protein as a phosphorylation target of mutant EGFR from our mass spectrometry-based phosphoproteomics experiments (see below). We have shown that DLG1 indeed interacts with EGFR. Further characterization of this interaction and the significance of the phosphorylation is underway. 1B: Compare the degree of interaction of proteins that associate with WT EGFR, L858R EGFR and Del E746-A750 EGFR This part of the project is complete and is not further being done. We are currently validating individual interaction partners of mutant EGFR obtained in our kinase assay and mass spectrometry based interaction assay screens. Further description is below. The two most common TKI-sensitizing lung cancer-specific EGFR mutations are constitutively active. However two small clinical trials of patients treated with erlotinib have shown that the prognosis of patients harboring the Del EGFR is better than those with L858R EGFR. Patients with Del EGFR respond better and have more prolonged progression free survival than those harboring the L858R EGFR. We undertook an approach of identifying EGFR-interacting proteins by mass spectrometry. We expressed WT EGFR, L858R EGFR and Del E746-A750 EGFR in NR6 cells, a variant of mouse 3T3 fibroblasts that do not express endogenous EGFR. We used stable isotope labeling with amino acids in cell culture (SILAC) to differentially label cells expressing each of the EGFR variants. Immunoprecipitation of EGFR with a monoclonal antibody specific to the extracellular domain of EGFR was performed to isolate EGFR-interacting proteins that were further identified by mass spectrometry. SILAC labeling of cells enabled relative quantitation of degree of interaction of proteins that interacted with both WT EGFR and mutant EGFRs. We identified several proteins that interacted specifically with mutant EGFRs. Of particular interest is receptor tyrosine kinases that interacted more with L858R and Del EGFR compared to WT EGFR (e.g. EPHA2, DDR2, AXL), suggesting cross talk of receptor tyrosine kinases in cells expressing mutant EGFRs. Currently we are treating a panel of lung cancer cell lines without the EGFR and KRAS mutations with drugs targeting the active kinases based on the interacting partners and another Luminex based phosphorylation assay of the kinases. We have demonstrated sensitivity on couple of cell lines predicted by the hyperactive kinases. A manuscript is being written to publish the findings of this project.