Defining mechanisms of MEK1/2 inhibitor response in triple negative breast cancer

描述（由申请人提供）：三阴性乳腺癌（TNBC）是一种乳腺癌，其分子靶向治疗尚未获得临床批准，TNBC患者的治疗选择很少。在许多TNBC患者样品中观察到的高RAF/MEK/ERK活性促使研究使用MEK 1/2抑制剂作为潜在的治疗策略。用MEK 1/2抑制剂AZD 6244处理TNBC细胞系和小鼠模型引起激酶组重编程事件，其中诱导生长促进受体酪氨酸激酶（如PDGFRb）的活性和表达以抵消MEK 1/2抑制并重新激活ERK 1/2。用小分子抑制剂或siRNA靶向AZD 6244应答激酶不仅可以减弱ERK 1/2的再激活，还可以与AZD 6244协同作用，抑制TNBC细胞系的增殖并诱导其凋亡，并导致基因工程小鼠模型（C3-Tag）的肿瘤消退，这表明激酶组重编程有助于这些模型的耐药性。了解AZD 6244诱导的激酶组重编程的机制将为未来关于单独或联合使用MEK 1/2抑制剂治疗TNBC的研究提供信息。随着激酶和细胞因子表达的变化，AZD 6244处理引起的激酶组重编程是通过破坏控制激酶和细胞因子基因表达的转录因子的调节而启动的。转录因子c-Myc通过ERK 1/2介导的磷酸化而稳定，经AZD 6244处理后，在TNBC细胞中迅速降解。目的1将确定AZD 6244诱导的c-Myc（PDGFRb的已知转录调节因子）降解如何有助于AZD 6244处理细胞中PDGFRb表达和活性的增强。将应用c-Myc的诱导性敲低或蛋白酶体抑制来解决c-Myc稳定性在AZD 6244诱导的PDGFRb应答中的作用。目的2将定义在AZD 6244存在下ERK 1/2再激活的机制。将进行活性和非活性MEK 1/2的激酶试验、MEK亚型的选择性敲低和RAF/MEK/ERK通路磷酸化的定量蛋白质组学分析，以特别关注MEK 1/2如何逃脱AZD 6244的抑制以重新激活ERK 1/2。总之，这些目标将定义由ERK 1/2信号调节的转录因子如何介导对AZD 6244的激酶组重编程，以及重编程事件如何随后允许TNBC细胞克服MEK 1/2抑制。

DESCRIPTION (provided by applicant): Triple negative breast cancer (TNBC) is a form of breast cancer for which no molecularly targeted therapies have reached clinical approval, leaving few treatment options for TNBC patients. The observation of high RAF/MEK/ERK activity in many TNBC patient samples has prompted the investigation of using MEK1/2 inhibitors as a potential therapeutic strategy. Treating TNBC cell lines and mouse models with the MEK1/2 inhibitor AZD6244 provokes a kinome reprogramming event where the activity and expression of growth- promoting receptor tyrosine kinases, such as PDGFRb, are induced to offset MEK1/2 inhibition and reactivate ERK1/2. Targeting AZD6244-responsive kinases with small molecule inhibitors or siRNA not only attenuates the reactivation of ERK1/2, but also synergizes with AZD6244 to inhibit proliferation and induce apoptosis of TNBC cell lines and cause tumor regression of a genetically engineered mouse model (C3-Tag), suggesting that kinome reprogramming contributes to drug resistance in these models. Understanding the mechanisms of AZD6244-induced kinome reprogramming will inform future studies on the use of MEK1/2 inhibitors, alone or in combination, for treating TNBC. With changes in kinase and cytokine expression, kinome reprogramming caused by AZD6244 treatment is initiated by disrupted regulation of transcription factors governing kinase and cytokine gene expression. The transcription factor c-Myc, which is stabilized by ERK1/2-mediated phosphorylation, was shown to be rapidly degraded in TNBC cells following treatment with AZD6244. Aim 1 will determine how AZD6244-induced degradation of c-Myc, a known transcriptional regulator of PDGFRb, contributes to the enhancement of PDGFRb expression and activity in AZD6244-treated cells. Inducible knockdown of c-Myc or proteasomal inhibition will be applied to resolve the role of c-Myc stability in the AZD6244-induced PDGFRb response. Aim 2 will define the mechanism of ERK1/2 reactivation in the presence of AZD6244. Kinase assays with active and inactive MEK1/2, selective knockdown of MEK isoforms, and quantitative proteomics analysis of RAF/MEK/ERK pathway phosphorylation will be performed to focus specifically on how MEK1/2 can escape inhibition by AZD6244 to reactivate ERK1/2. Taken together, these aims will define how transcription factors regulated by ERK1/2 signaling mediate kinome reprogramming in response to AZD6244, and how the reprogramming events subsequently allow TNBC cells to overcome MEK1/2 inhibition.