Identification of potentially active tyrosine kinases in lung cancer

我们与布罗德研究所的托德戈卢布合作，进行了一项luminex测定，以定量酪氨酸激酶中的酪氨酸磷酸化。活性酪氨酸激酶通常也与总体酪氨酸磷酸化增加相关。我们的测定在多重测定中捕获超过150种酪氨酸激酶，并使用泛磷酸酪氨酸抗体（4G10）检测所有这些激酶中的酪氨酸磷酸化水平。我们用150例人肺腺癌肿瘤组织和相应的邻近正常肺组织来分析这些酪氨酸激酶的酪氨酸磷酸化。我们确定了大约30个特异性酪氨酸激酶在肿瘤组织中与邻近正常肺组织相比差异磷酸化。这种潜在活性酪氨酸激酶的实例是NTRK1、EPHA2、DDR。然后，我们与MSKCC的Marc Ladanyi博士合作完成了一项nanostring测定，以在多重测定中测量大多数酪氨酸激酶的转录表达。有趣的是，正如我们所预测的，几种酪氨酸激酶的表达至少在转录水平上没有变化，但是酪氨酸磷酸化有显著变化，酪氨酸磷酸化通常是激活的替代物。通过与Paul Meltzer博士在NCI内部计划中的合作，我们已经对大约1300个癌症相关基因进行了测序，其中包括我们的luminex检测中使用的酪氨酸激酶，以靶向方式识别特定突变。我们分析了测序数据，以确定所选基因的体细胞变化。体细胞突变数据已与luminex数据相关，以确定激酶中可能的体细胞变化，导致活性增加。正如预期的那样，我们已经通过Luminex检测在EGFR过度磷酸化患者中鉴定出EGFR突变。然而，对于大多数其他激酶，尚未鉴定出与过度磷酸化相关的特异性突变。这强调了使用酪氨酸磷酸化作为激酶活性的替代物来检查信号通路的重要性。我们已经确定了人肺腺癌细胞系中的酪氨酸磷酸化激酶是高的，但没有确定的突变，在这些激酶。这些细胞系用特异性激酶抑制剂处理，以确定细胞系是否依赖于特异性激酶信号传导途径存活和增殖。我们已经鉴定了被TRKA、MET和EPHB的激酶抑制剂抑制的细胞系。结合EGFR相互作用蛋白项目的数据，我们正在撰写描述这些结果的手稿。

In collaboration with Todd Golub at the Broad Institute, we have performed a luminex assay to quantitate tyrosine phosphorylation in tyrosine kinases. Active tyrosine kinases are also generally associated with increased overall tyrosine phosphorylation. Our assay captured more than 150 tyrosine kinases in a multiplex assay and detected the level of tyrosine phosphorylation in all these kinases using a pan-phosphotyrosine antibody (4G10). We used 150 human lung adenocarcinoma tumor tissue and corresponding adjacent normal lung tissue to profile tyrosine phosphorylation of these tyrosine kinases. We identified around 30 specific tyrosine kinases differentially phosphorylated in tumor tissue compared to adjacent normal lung. Examples of such potentially active tyrosine kinases are NTRK1, EPHA2, DDR. We then completed a nanostring assay in collaboration with Dr. Marc Ladanyi at MSKCC to measure transcript expression of majority of tyrosine kinases in a multiplex assay. Interestingly, as we had predicted, expression of several tyrosine kinases do not change at least at the level of trasnscription, however there is significant change in tyrosine phosphorylation, which is generally a surrogate of activation. In collaboration with Dr. Paul Meltzer in the NCI intramural program, we have sequenced around 1300 cancer-related genes, including the tyrosine kinases used in our luminex assay to identify specific mutations in a targeted manner. We have analyzed the sequencing data to identify somatic changes in the genes selected. The somatic mutation data has been correlated with the luminex data to identify possible somatic changes in kinases resulting in increased activity. We have identified, as expected EGFR mutations in patients with hyperphosphorylation of EGFR by luminex assay. However for most other kinases, no specific mutation has been identified that correlates with hyperphosphorylaiton. This underscores the importance of examining signaling pathways using tyrosine phosphorylation as a surrogate of kinase activity. We have identified human lung adenocarcinoma cell lines in which tyrosine phosphorylation of kinases is high, but there is no identified mutation in those kinases. These cell lines are being treated with the specific kinase inhibitors to determine if the cell line is dependant on the specific kinase signaling pathway for survival and proliferation. We have identified cell lines that are inhibited by kinase inhibitors of TRKA , MET and EPHB. In combination with the data from the EGFR interacting protein project we are writing a manuscript describing these results.