Profiling Tumor Cells by SH2 Domain Binding Pattern

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

DESCRIPTION (provided by applicant): This proposal describes a novel proteomic method to profile cells on the basis of their binding; sites for Src Homology 2 (SH2) domains. This molecular diagnostic approach takes advantage of a basic signal transduction mechanism whereby cells receive information from the extracellular environment. Many signals, including those from growth factors, cytokines, and the extracellular matrix, are transmitted by changes in the tyrosine phosphorylation of intracellular proteins. These phosphorylated proteins in turn serve as binding sites for proteins containing a small protein-binding module termed the SH2 domain. All SH2 domains recognize tyrosine-phosphorylated sites, but each SH2 domain also has a distinctive binding specificity based on the residues surrounding the phosphotyrosine. Thus to a large extent the cytosol of the cell interprets its environment by the presence or absence of binding sites for specific SH2 domains. Recent innovations that increase the specificity of SH2 binding in vitro and decrease nonspecific background now make it feasible to use the pattern of binding sites for a panel of SH2 domain probes to build a profile or "fingerprint" for any cell, including tumor cells. Preliminary work using hematopoietic cell lines demonstrates that this is a robust and sensitive method which provides a unique molecular insight into the signaling machinery of the cell. The SH2 binding profiles will provide molecular diagnostic data that can be used to sub-classify histologically similar tumors, in much the same way that cDNA profiling is beginning to be used to classify tumors on the basis of their transcriptional activity. Moreover the SH2 profiling method has the added advantage that the information it provides will in many cases be directly relevant to the signaling derangements that contributed to the outgrowth of the tumor. Therefore SH2 binding profiles may not only have prognostic value, but also provide novel insights that will rationally guide therapy. SH2 binding proteins that are highly correlated with disease outcome can be identified and may serve as novel targets for drug discovery. In this proposal the SH2 profiling method will be applied samples from patients with hematopoietic and other malignancies, and any correlation between disease course and SH2 profile will be determined. Variants of the existing profiling method will also be pursued, with the aim of developing a high-throughput method to quantitate binding sites for tens to hundreds of different SH2 domains in a single binding reaction.