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分析鉴定突变的EGFR底物(预算的2%)我们正处于验证此屏幕中确定的潜在目标的最后阶段。我们有一个大约10种蛋白质的清单，我们已经通过生化纯化，目前正在进行野生型和突变型EGFR激酶的体外激酶分析。我们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。下面对其中一些进行了描述。我们试图使用蛋白质阵列上的体外激酶分析来确定突变的EGFR激酶的靶点。我们使用了由Invitgen开发的人类蛋白质阵列，最近又与约翰·霍普金斯医学院的朱恒博士的实验室合作，使用了他们团队开发的定制蛋白质阵列。我们使用WTEGFR、L858R EGFR、L858R/T790M EGFR在有或没有酪氨酸激酶抑制剂erlotinib的情况下对这些蛋白质阵列进行体外激酶分析。我们在体外鉴定了几种可被突变的激酶磷酸化的蛋白质，但没有发现WTEGFR。例如，我们发现STK3、PKCtheta、PAK3、MST4(MASK)、TBK1、Lyna可被L585R EGFR磷酸化，但WT EGFR不能。朱博士实验室的蛋白质阵列在玻片上发现了大约2万个全长的人类蛋白质。我们已经完成了筛查，并分析了过去一年的激酶检测数据。我们目前正在验证用这种方法鉴定的突变型EGFR的一些靶点。我们已经采取了生化和遗传方法来验证目标。其中一个靶点是蛋白质DLG1(大圆盘)。我们还通过基于质谱学的磷酸蛋白质组学实验(见下文)，将该蛋白确定为突变的EGFR的磷酸化靶点。我们已经证明DLG1确实与EGFR相互作用。这种相互作用的进一步表征和磷酸化的意义正在进行中。1B：比较与WT EGFR、L858R 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经完成，没有进一步完成。我们目前正在验证在我们的激酶分析和基于质谱学的相互作用分析筛选中获得的突变EGFR的单个相互作用伙伴。下面是进一步的描述。两个最常见的TKI致敏肺癌特异性EGFR突变在构成上是活跃的。然而，两项对接受厄洛替尼治疗的患者的小型临床试验表明，携带Del EGFR的患者的预后好于携带L858R EGFR的患者。与携带L858R EGFR的患者相比，Del EGFR患者的反应更好，无进展生存期更长。我们开展了一种利用质谱学鉴定EGFR相互作用蛋白的方法。我们在NR6细胞中表达了WT EGFR、L858R EGFR和Del E746-A750 EGFR，NR6是不表达内源性EGFR的小鼠3T3成纤维细胞的变种。我们使用细胞培养中氨基酸的稳定同位素标记(SILAC)来差异标记表达每一种EGFR变体的细胞。用一种针对EGFR胞外区的单抗对EGFR进行免疫沉淀，以分离与EGFR相互作用的蛋白，并用质谱仪进行进一步鉴定。细胞的SILAC标记使得能够相对定量与WT EGFR和突变的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的EGFR特异相互作用的蛋白质。与WT EGFR(如EphA2、DDR2、Ax1)相比，受体酪氨酸激酶与L858R和Del EGFR有更多的相互作用，这表明在表达突变的EGFR的细胞中存在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，使用基于相互作用伙伴的针对活性激酶的药物和另一种基于Luminex的激酶磷酸化分析。我们已经证明了对几个细胞系的敏感性，这些细胞系是由高活性的激酶预测的。正在撰写一份手稿，以公布这一项目的发现。

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.