Mapping Tyrosine Kinases Based on a TK Activity-Representing Peptide Library Reveals a Role for SRC in H1975 Drug Resistance

Tyrosine kinases (TKs) are prominent
targets in cancer therapies,
and more than 30 TK inhibitors have been approved for treatments in
tumors with abnormal TK. Disappointingly, an incomplete response can
occur with the long-term use of TK inhibitors, known as cancer drug
resistance, which can be caused by kinome reprogramming. Hence, monitoring
the status of TKs is crucial for revealing the underlying drug resistance
mechanism. Here, we describe a TK activity-representing peptide library-based
multiple reaction monitoring (TARPL-MRM) strategy for directly inferring
TK activities. The strategy facilitated the assay of 87 human TKs
through target quantification of 301 phosphorylation sites. Using
this strategy, we demonstrated the heterogeneity of TK activity in
different non-small cell lung cancer (NSCLC) cell lines and assessed
the response of TK activities to the EGFR inhibitor AZD9291 in NSCLC
cells. We found that the acquired resistance of H1975 cells to AZD9291
requires SRC activity, and inhibition of SRC plays potential roles
in overcoming this resistance. In summary, our work reveals that this
strategy has the potential to become a powerful tool for TK studies,
clinical diagnostics, and the discovery of new therapeutic targets.

酪氨酸激酶（Tyrosine kinases，TKs）作为癌症治疗中的显著靶点，其重要性不言而喻。截至目前，已有超过30种酪氨酸激酶抑制剂被批准用于治疗存在异常酪氨酸激酶的肿瘤。然而，令人遗憾的是，长期使用酪氨酸激酶抑制剂可能导致不完全响应，即所谓的癌症药物耐药性，其产生可能与激酶组的重编程有关。因此，监测酪氨酸激酶的状态对于揭示潜在的药物耐药机制至关重要。本研究中，我们描述了一种基于酪氨酸激酶活性表征肽库的多种反应监测（TARPL-MRM）策略，该策略可直接推断酪氨酸激酶活性。该策略通过针对301个磷酸化位点的目标定量，实现了对87种人类酪氨酸激酶的检测。利用此策略，我们展示了不同非小细胞肺癌（NSCLC）细胞系中酪氨酸激酶活性的异质性，并评估了NSCLC细胞对EGFR抑制剂AZD9291的酪氨酸激酶活性响应。我们发现，H1975细胞对AZD9291的获得性耐药性需要SRC活性，抑制SRC可能在克服这种耐药性中发挥潜在作用。总之，我们的研究揭示了该策略有望成为酪氨酸激酶研究、临床诊断以及新治疗靶点发现的有力工具。