Kinome Reprogramming in Response to Targeted Kinase Inhibitors

描述(由申请人提供)：已经开发了一种新的方法来研究蛋白激酶的重新编程，允许在一次质谱分析中分析大约40%-60%的表达的亲和体。我们的方法利用复合抑制物珠(MIB)，由Sepharose珠和共价固定化的连接物适应的激酶抑制剂组成。这项技术允许对已知序列的激酶进行询问，但由于缺乏生物学或表型知识或试剂的可用性，对其研究不足。MIB/MS确定了对选定的MEK1/2抑制剂AZD6244的动态组反应特征，该抑制剂目前正在对三阴性乳腺癌进行临床测试。MEK1/2的唯一底物是ERK1和ERK2，但我们观察到在MEK抑制的情况下，各亚家族中的激酶活性发生了变化。Kinome评估显示，这是一种时间依赖性的重编程，涉及RAF和MEK的ERK反馈调节的早期丧失，从而允许MEK-ERK途径的上游重新激活。可以很容易地检测到PDGFR和DDR1等特异性受体酪氨酸激酶(RTK)MIB结合的时间依赖性变化，并提供了关键的实验观察，即MEK抑制是驱动多个RTK表达和激活的关键实验观察。C-Myc降解是介导动态组重编程的关键机制。多个RTK在MEK抑制反应中被激活的事实表明，使用单一的激酶抑制剂来阻止肿瘤的进展是困难的。本研究的目的包括：1.明确人类基础/低克隆蛋白样细胞系SUM159的激动组激活状态，以及基础/低克隆蛋白乳腺癌的临床前C3Tag GEMM。目前正在进行临床测试的MEK1/2和PI3K抑制剂单独或联合使用时，将定义激酶信号。2.明确MEK和PI3K抑制剂耐药肿瘤和细胞系中动态组重编程的机制。3.根据敏感和耐药肿瘤的动态组特征，开发合理的激酶抑制剂组合预测，用于在C3Tag GEMM中测试基础/Claudin-low乳腺癌。其目标是确定克服耐药性并导致细胞凋亡和肿瘤消退的联合疗法。

DESCRIPTION (provided by applicant): A novel approach has been developed to study the reprogramming of protein kinases "en masse" allowing ~40-60% of the expressed kinome assayed in a single mass spectroscopy run. Our methods utilize Multiplexed Inhibitor Beads (MIBs), consisting of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors. The technique allows interrogation of kinases known by sequence but which have been understudied due to lack of biologic or phenotypic knowledge or the availability of reagents. MIB/MS identified a kinome response signature to a select MEK1/2 inhibitor AZD6244 that is currently in clinical testing for triple negative breast cancer. The only defined substrate for MEK1/2 are ERK1 and 2, yet we observed changes in activity of kinases in every subfamily of the kinome in response to MEK inhibition. Kinome assessment showed a time-dependent reprogramming that involved an early loss of ERK feedback regulation of RAF and MEK that allowed upstream reactivation of the MEK-ERK pathway. The time dependent change in MIB binding of specific receptor tyrosine kinases (RTKs) such as PDGFR¿ and DDR1 was readily detected and provided the critical experimental observation that MEK inhibition was driving the expression and activation of multiple RTKs. c-Myc degradation was a key mechanism mediating kinome reprogramming. The fact that multiple RTKs are activated in response to MEK inhibition demonstrates the difficulty in using single kinase inhibitors to arrest tumor progression. The aims of this proposal include: 1. Define kinome activation state in the human basal/claudin-low like SUM159 cell line and the pre-clinical C3Tag GEMM for basal/claudin-low breast cancer. Kinase signatures will be defined in response to MEK1/2 and PI3K inhibitors alone and in combination, which are currently in clinical testing. 2. Define mechanisms of kinome reprogramming in MEK and PI3K inhibitor resistant tumors and cell lines. 3. Develop rational predictions of kinase inhibitor combinations based on kinome signatures of sensitive and resistant tumors for testing in the C3Tag GEMM for basal/claudin-low breast cancer. The goal is to define combination therapies that overcome resistance and cause apoptosis and tumor regression.