INTEGRATED PROTEOMICS AND GENOMICS TO IDENTIFY CRITICAL SIGNALING EVENTS IN RESI

在过去的两年里，转移性黑色素瘤的管理发生了范式转变。的 BRAF^（R）-特异性抑制剂如vemurafenib的开发导致了前所未有的反应率 50%以上。然而，由于获得性耐药，中位缓解持续时间较短（7个月）。到 克服这种阻力，仍然需要进一步了解潜在的分子信号传导 机制等 我们将描述已知耐药机制的分子细节和网络范围的特性 以及使用基于质谱（MS）的磷酸化蛋白质组学尚未表征的机制。使用 通过这种全球性的方法，我们将定量监测数千种蛋白质的信号变化。我们有 已建立的方法，用于解释所得到的系统范围的数据，以揭示信令网络如何 在出现耐药性时改变事件，并检测新的治疗信号靶标。 通过我们的磷酸化蛋白质组学方法，我们发现了一种由蛋白质介导的抗性机制。 BRAF的高度磷酸化和截短的剪接变体。我们将确定分子机制 通过突变和功能研究来巩固这种截短介导的抗性。在这些研究中，我们 将继续使用质谱作为一种无偏见的方法来监测我们的实验的影响， 扰动我们将通过磷酸缺陷突变体来确定磷酸化的作用， BF的？使用基于质谱的蛋白质共纯化测定的AF二聚化和蛋白质相互作用 我们已经应用我们的方法来研究与细胞凋亡相关的信号转导的系统范围的变化。 RTK介导的BRAF抑制剂抗性机制。综合基因组分析使我们能够 优先考虑几个候选信号事件，以促进抗性相关上皮细胞， 间充质样（EMT样）转变。通过额外的磷酸分析，我们将完善这个候选名单， 然后利用突变和功能研究来验证它们对抗性的信号贡献。 与PPG的研究人员一起，我们正在建立一个全面的黑色素瘤计划。我们的目标是 通过使用新技术学习的综合方法克服BRAF抑制剂耐药性 然后将这些知识转化回病人的护理中。这些研究具有更广泛的 这对其他恶性肿瘤也有影响，因为BRAF突变存在于所有癌症的7%中。 相关性（参见说明）： 专门针对驱动黑色素瘤的突变，已经允许开发治疗方法， 效率更高，副作用更少。不幸的是，细胞存活的复杂性几乎不可避免地 为失调的癌细胞提供了对初始治疗产生抗性的替代机会。通过 采取系统范围的方法，同时查看数千个事件，我们将描述这些 相互交织的机制，并提供了一个蓝图，使用鸡尾酒的靶向药物，以防止耐药性。

The past two years have seen a paradigm shift in the management of metastatic melanoma. The development of BRAF^(R)¿¿^-specific inhibitors like vemurafenib has resulted in unprecedented response rates above 50%. However, the median duration of response is short (7 months) due to acquired resistance. To overcome this resistance there remains a need to further understand the underlying molecular signaling mechanisms. We will characterize the molecular details and network-wide properties of known resistance mechanisms and of not-yet-characterized mechanisms using mass spectrometry (MS)-based phosphoproteomics. Using this global approach we will quantitatively monitor signaling changes in thousands of proteins. We have established methods for interpreting the resultant system-wide data to uncover how the network of signaling events is modified upon the emergence of drug resistance, and to detect new signaling targets for therapy. Through our phosphoproteomic approach we have uncovered a mechanism of resistance mediated by a highly phosphorylated and truncated splice variant of BRAF. We will determine the molecular mechanism undertying this truncation-mediated resistance through mutational and functional studies. In these studies we will continue to use mass spectrometry as an unbiased approach to monitor the effects of our experimental perturbations. We will determine the role of phosphorylation through phospho-deficient mutants, and the role of BF?AF dimerization and protein interactions using mass spectrometry-based protein co-purification assays We have applied our approach to investigate the system-wide changes in signaling associated with the RTK-mediated mechanism of BRAF inhibitor resistance. Integrated genomic analyses have allowed us to prioritize several candidate signaling events as promoting the resistance-associated epithelial to mesenchyme-like (EMT-like) transition. Through additional phosphoprofiling we will refine this candidate list, then use mutational and functional studies to validate their signaling contnbution to resistance. With the investigators of this PPG we are building a comprehensive melanoma program. Our goal is to overcome BRAF Inhibitor resistance through an integrated approach that uses new technologies to learn from patient tumors and then translates this knowledge back to patient care. These studies have broader implications on other malignancies, since the BRAF^ mutation is present In 7% of all cancers. RELEVANCE (See instructions): Specifically targeting the mutations that drive melanomas has allowed for the development of therapies with greater efficiency and fewer side effects. Unfortunately, the complexity of cell survival almost inevitably provides alternative opportunities for disregulated cancer cells to become resistant to the initial therapy. By taking a system-wide approach looking at thousands of events simultaneously, we will delineate these intertwined mechanisms and provide a blueprint for using a cocktail of targeted drugs to prevent resistance.