MASSIVELY PARALLEL MEASUREMENT OF SRC KINASE ACTIVITY AND DRUG RESISTANCE IN VIV

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 能够促进生长并提供选择性优势的突变是癌症的根源。通过基于测序的癌症基因组学努力，癌症相关突变的识别大大加快了速度，但这些突变的功能后果往往尚不清楚。突变也经常是靶向癌症治疗失败的原因(例如，bcr-abl突变导致对伊马替尼产生耐药性)。因此，了解突变如何影响蛋白质功能和抑制物疗效，无论是在有效利用癌症基因组学数据还是在开发和部署靶向治疗方面都是至关重要的。目前，现有的蛋白质功能分析方法不能达到完全了解突变如何影响功能或抑制物耐药性所需的规模。为了满足这一需求，我们开发了一种基于蛋白质展示和高通量测序的方法，可以同时评估数十万个蛋白质突变的功能。我们正在努力使用这种方法来开发一种基于酵母的Src激酶的检测方法，这是一种关键的原癌基因产物，在结直肠癌和其他癌症中被不适当地激活。SRC激酶抑制是一种新兴的治疗策略，有20多个Src激酶抑制剂治疗结直肠癌、乳腺癌、肺癌和其他癌症的I期和II期临床试验正在进行中。我们建议在平行和体内量化几乎所有10,720个单一的Src激酶突变体的功能、调节和抵抗抑制的后果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Mutations that confer enhanced growth and provide a selective advantage are at the root of cancer. Identification of cancer-related mutations has been greatly accelerated by sequencing-based cancer genomics efforts, but the functional consequences of these mutations often remain unclear. Mutations also frequently underlie the failure of targeted cancer therapeutics (e.g. BCR-Abl mutations conferring resistance to imatinib). Therefore, understanding how mutations affect protein function and inhibitor efficacy is critical, both in making effective use of cancer genomics data and in the development and deployment of targeted therapies. Currently available methods for assaying protein function cannot achieve the scale necessary to produce a complete understanding of how mutations impact function or inhibitor resistance. To address this need, we have developed a method based on protein display and high-throughput sequencing that can assess the function of hundreds of thousands of mutants of a protein simultaneously. We are working to use this approach to develop a yeast-based assay for Src kinase, a critical proto-oncogene product that is inappropriately activated in colorectal and other cancers. Src kinase inhibition is an emerging therapeutic strategy, with over 20 phase I and II clinical trials of Src kinase inhibitors to treat colorectal, breast, lung and other cancers underway. We propose to quantify the consequences for function, regulation and resistance to inhibition of nearly all 10,720 single mutants of Src kinase in parallel and in vivo.