Kinome Reprogramming in Response to Targeted Kinase Inhibitors

描述（由申请人提供）：已开发了一种新方法来研究蛋白激酶"重组"的重编程，允许在单次质谱运行中测定约40 - 60%的表达激酶组。我们的方法利用多重抑制剂珠（MIBs），其由具有共价固定的、接头适配的激酶抑制剂的琼脂糖凝胶珠组成。该技术允许通过序列询问已知的激酶，但由于缺乏生物学或表型知识或试剂的可用性而未充分研究。MIB/MS鉴定了一种选择的MEK 1/2抑制剂AZD 6244的激酶组反应特征，该抑制剂目前正在三阴性乳腺癌的临床试验中。MEK 1/2的唯一确定的底物是ERK 1和2，然而我们观察到激酶组的每个亚家族中的激酶活性响应于MEK抑制的变化。激酶组评估显示时间依赖性重编程，其涉及RAF和MEK的ERK反馈调节的早期丧失，其允许MEK-ERK通路的上游再激活。特异性受体酪氨酸激酶（RTK）如PDGFR和DDR1的MIB结合的时间依赖性变化很容易检测到，并提供了关键的实验观察结果，即MEK抑制驱动多种RTK的表达和激活。c-Myc降解是介导激酶组重编程的关键机制。多个RTK响应于MEK抑制而被激活的事实证明了使用单一激酶抑制剂来阻止肿瘤进展的困难。本提案的目的包括：1.定义人基底/低密蛋白样SUM159细胞系和基底/低密蛋白乳腺癌临床前C3Tag GEMM中的激酶组激活状态。激酶特征将根据MEK 1/2和PI3K抑制剂单独使用和联合使用（目前正在进行临床试验）进行定义。2.定义MEK和PI3K抑制剂抗性肿瘤和细胞系中激酶组重编程的机制。3.基于敏感和耐药肿瘤的激酶组特征，开发激酶抑制剂组合的合理预测，用于在C3Tag GEMM中检测基底/低密蛋白乳腺癌。目标是确定克服耐药性并引起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.