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（Triple negative breast cancer， TNBC）是一种分子靶向治疗尚未获得临床批准的乳腺癌，留给TNBC患者的治疗选择很少。在许多TNBC患者样本中观察到高RAF/MEK/ERK活性，促使研究使用MEK1/2抑制剂作为潜在的治疗策略。用MEK1/2抑制剂AZD6244处理TNBC细胞系和小鼠模型会引发kinome重编程事件，其中诱导生长促进受体酪氨酸激酶（如PDGFRb）的活性和表达来抵消MEK1/2抑制并重新激活ERK1/2。用小分子抑制剂或siRNA靶向AZD6244反应性激酶，不仅可以减弱ERK1/2的再激活，还可以与AZD6244协同抑制TNBC细胞系的增殖和诱导凋亡，并引起基因工程小鼠模型（C3-Tag）的肿瘤消退，提示在这些模型中，kinome重编程有助于耐药。了解azd6244诱导的激酶重编程机制将为未来使用MEK1/2抑制剂单独或联合治疗TNBC的研究提供信息。随着激酶和细胞因子表达的改变，AZD6244处理引起的kinome重编程是通过破坏激酶和细胞因子基因表达的转录因子调控而启动的。通过erk1 /2介导的磷酸化稳定的转录因子c-Myc在AZD6244处理后在TNBC细胞中迅速降解。目的1将确定azd6244诱导的c-Myc（已知的PDGFRb转录调节因子）降解如何促进azd6244处理的细胞中PDGFRb表达和活性的增强。c-Myc的诱导下调或蛋白酶体抑制将被应用于解决c-Myc稳定性在azd6244诱导的PDGFRb反应中的作用。Aim 2将明确AZD6244存在时ERK1/2再激活的机制。将进行活性和非活性MEK1/2的激酶试验，选择性敲低MEK亚型，以及RAF/MEK/ERK途径磷酸化的定量蛋白质组学分析，以特别关注MEK1/2如何逃脱AZD6244的抑制，从而重新激活ERK1/2。综上所述，这些目标将定义ERK1/2信号调节的转录因子如何介导AZD6244对kinome的重编程，以及重编程事件随后如何使TNBC细胞克服MEK1/2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. PUBLIC HEALTH RELEVANCE: Triple negative breast cancer patients have a poor prognosis and limited treatment options. The RAF/MEK/ERK pathway is activated in many triple negative breast cancer patients, and use of MEK1/2 inhibitors is an attractive new strategy for treating these patients. This proposal aims to define how MEK1/2 inhibitors may provoke kinome reprogramming and drug resistance in triple negative breast cancer cells, and will contribute to the development of MEK1/2 inhibitors, alone or in combination with other therapeutics, for the treatment of triple negative breast cancer.