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

描述(申请人提供)：三重阴性乳腺癌(TNBC)是一种尚未获得临床批准的分子靶向治疗方法，为TNBC患者留下了很少的治疗选择。在许多TNBC患者样本中观察到高RAF/MEK/ERK活性，促使人们研究使用MEK1/2抑制剂作为一种潜在的治疗策略。用MEK1/2抑制剂AZD6244处理TNBC细胞株和小鼠模型会引发动态组重编程事件，其中促进生长的受体酪氨酸激酶(如PDGFRb)的活性和表达被诱导以抵消MEK1/2的抑制并重新激活ERK1/2。以小分子抑制剂或siRNA靶向AZD6244响应激酶不仅可以减弱ERK1/2的重新激活，而且还可以与AZD6244协同抑制TNBC细胞系的增殖和诱导凋亡，并导致基因工程小鼠模型(C3-Tag)的肿瘤退化。了解AZD6244诱导的动态组重编程的机制将为未来使用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治疗引起的动态组重编程是通过扰乱调控激酶和细胞因子基因表达的转录因子来启动的。经AZD6244处理后，转录因子c-Myc在TNBC细胞中迅速降解，该转录因子由ERK1/2介导的磷酸化稳定。目的1研究AZD6244对PDGFRb的转录调控因子c-Myc的降解如何促进AZD6244处理的细胞中PDGFRb的表达和活性。C-Myc的诱导敲除或蛋白酶体抑制将用于解决c-Myc稳定性在AZD6244诱导的PDGFRb反应中的作用。AIM 2将确定在AZD6244存在下ERK1/2重新激活的机制。对激活和失活的MEK1/2进行的激酶分析，MEK亚型的选择性敲除，以及RAF/MEK/ERK通路磷酸化的定量蛋白质组学分析将专门集中在MEK1/2如何逃脱AZD6244的抑制以重新激活ERK1/2。综上所述，这些目的将定义由ERK1/2信号调节的转录因子如何介导响应AZD6244的动态组重编程，以及重编程事件如何随后允许TNBC细胞克服MEK1/2抑制。 公共卫生相关性：三阴性乳腺癌患者预后较差，治疗选择有限。RAF/MEK/ERK通路在许多三阴性乳腺癌患者中被激活，使用MEK1/2抑制剂是治疗这些患者的一种有吸引力的新策略。这项建议旨在确定MEK1/2抑制剂如何在三阴性乳腺癌细胞中引起动态组重新编程和耐药性，并将有助于MEK1/2抑制剂的开发，单独或与其他疗法联合用于治疗三阴性乳腺癌。

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.