INTEGRATED PROTEOMICS AND GENOMICS TO IDENTIFY CRITICAL SIGNALING EVENTS IN RESI

在过去的两年里，转移性黑色素瘤的治疗模式发生了转变。这个 Vemurafenib等BRAF(R)特异性抑制剂的开发导致了前所未有的应答率 50%以上。然而，由于获得性耐药性，中位反应持续时间较短(7个月)。至 克服这种抗性仍然需要进一步了解潜在的分子信号转导 机械装置。 我们将描述已知抗性机制的分子细节和网络特性 以及使用基于质谱学(MS)的磷酸蛋白质组学的尚未表征的机制。vbl.使用 这种全球性的方法，我们将定量监测数千种蛋白质中的信号变化。我们有 已建立的方法用于解释得到的系统范围的数据，以揭示信令网络是如何 事件在出现耐药时被修改，并检测新的信号靶点用于治疗。 通过我们的磷酸蛋白质组学方法，我们已经揭示了一种由一种 高度磷酸化和截断的BRAF剪接变体。我们将确定分子机制 通过突变和功能研究来支持这种截断介导的抗性。在这些研究中，我们 将继续使用质谱学作为一种不偏不倚的方法来监测我们的实验效果 微扰。我们将通过缺磷突变体来确定磷酸化的作用，以及 基于质谱学的蛋白质共纯化方法研究BF、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 RTK介导的BRAF抑制剂耐药机制。综合基因组分析使我们能够 优先考虑几个候选信号事件，以促进耐药相关的上皮细胞 类间充质(类EMT)过渡。通过额外的磷光分析，我们将完善这份候选人名单， 然后利用突变和功能研究来验证它们对抗性的信号分布。 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集成方法克服BRAF抑制剂阻力 然后将这些知识转化为患者护理。这些研究具有更广泛的意义 对其他恶性肿瘤的影响，因为BRAF^突变存在于7%的癌症中。 相关性(请参阅说明)： 特别针对导致黑色素瘤的突变，使治疗方法的开发成为可能 更高的效率和更少的副作用。不幸的是，细胞生存的复杂性几乎不可避免 为不受调控的癌细胞提供替代机会，使其对最初的治疗产生抗药性。通过 采取系统范围的方法，同时查看数千个事件，我们将描述以下内容 相互交织的机制，并为使用靶向药物的鸡尾酒来防止耐药性提供了一张蓝图。

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.