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.
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