Identification of potentially active tyrosine kinases in lung cancer

与博德研究所的托德·戈卢布合作，我们进行了Luminex试验，以定量测定酪氨酸激酶中的酪氨酸磷酸化。活跃的酪氨酸激酶通常也与总的酪氨酸磷酸化增加有关。我们的方法在多重分析中捕获了150多个酪氨酸激酶，并使用泛磷酸酪氨酸抗体(4G10)检测了所有这些激酶中的酪氨酸磷酸化水平。我们使用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。我们鉴定了大约30个特定的酪氨酸激酶，与邻近的正常肺组织相比，肿瘤组织中存在差异磷酸化。这种潜在活性的酪氨酸激酶的例子有NTRK1、EphA2、DDR。然后，我们与MSKCC的Marc Ladanyi博士合作完成了纳米串测试，以在多重测试中测量大多数酪氨酸激酶的转录表达。有趣的是，正如我们预测的那样，几种酪氨酸激酶的表达至少在转录水平上没有变化，但酪氨酸磷酸化有显著的变化，它通常是激活的替代。通过与Paul Meltzer博士在NCI Intral计划中的合作，我们已经对大约1300个与癌症相关的基因进行了测序，包括Luminex测试中使用的酪氨酸激酶，以有针对性地识别特定的突变。我们已经分析了测序数据，以确定所选基因的体细胞变化。体细胞突变数据已经与Luminex的数据相关联，以确定导致活性增加的体细胞激酶变化的可能性。正如预期的那样，我们通过Luminex试验在EGFR过度磷酸化的患者中发现了EGFR突变。然而，对于大多数其他激酶，还没有发现与过度磷酸化相关的特定突变。这突显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。我们已经鉴定出人肺腺癌细胞系中有高酪氨酸磷酸化的激酶，但没有发现这些激酶的突变。这些细胞系正在接受特定的激酶抑制剂的治疗，以确定细胞系是否依赖特定的激酶信号通路来生存和增殖。我们已经确定了被TrkA、Met和EphB的激酶抑制剂抑制的细胞系。结合来自EGFR相互作用蛋白项目的数据，我们正在撰写一份手稿来描述这些结果。

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.