Thomas E. Smithgall, PhD

Our research focuses on non-receptor protein-tyrosine kinase structure, regulation and signal transduction in cancer, AIDS, and embryonic stem cell biology. 

Src family kinases, HIV Nef, and AIDS. Our group continues to explore the interaction of Src-family kinases with Nef, an HIV accessory protein essential for AIDS progression. Nef binds directly to the SH3 domains of several Src-family members found in HIV target cells (Hck, Lyn and c-Src), leading to constitutive kinase activation. In collaboration with the University of Pittsburgh Drug Discovery Institute, we are actively running in vitro and cell-based high-throughput screens of chemical libraries to identify selective inhibitors of the Nef:Hck complex. We have identified several new classes of Nef-dependent compounds with potent anti-HIV activity in vitro. These compounds represent exciting new tools for exploring the role of Nef-mediated Src-family kinase signaling in HIV pathogenesis as well as leads for anti-HIV drug development. In collaboration with the Department of Structural Biology, we are actively seeking to map the binding sites for these small molecule inhibitors within Nef and Nef:host cell kinase complexes. 

Src family kinases, Bcr-Abl and chronic myelogenous leukemia (CML). This project is focused on the interplay of Src-family kinases and Bcr-Abl in the pathogenesis and drug sensitivity of chronic myelogenous leukemia (CML). Selective targeting of Bcr-Abl, the tyrosine kinase oncoprotein responsible for CML, with the Abl kinase inhibitor imatinib is very effective in chronic phase CML. However relapse due to drug resistance is an emerging problem often due to Bcr-Abl kinase domain mutations that impact drug binding. Clinical imatinib resistance also results from hyperactivation of myeloid SFKs, particularly Hck and Lyn. We discovered that these Src-family kinases directly phosphorylate the Bcr-Abl SH3 domain, preventing the docking of this domain to its regulatory position within the SH2-kinase linker within the Abl kinase core. This finding suggests that Abl SH3:linker interactions persist in the context of Bcr-Abl, despite its constitutive kinase activity. Furthermore, this observation has led us to question whether enhanced SH3:linker interaction can allosterically inhibit both wild-type and drug-resistant forms of Bcr-Abl. To this end, we have developed Abl kinase variants in which SH3:linker interaction has been enhanced, and found that they show show dramatically reduced kinase activity and enhanced inhibitor sensitivity. We are currently developing screening assays to identify small molecules that induce this same allosteric effect. 

Chemical genetics of Src family kinases in ES cells. This project involves the role of Src-family kinases in the early fate determination of murine embryonic stem (ES) cells. A few years ago we discovered that selective pharmacological inhibitors of Src-family kinases induce either terminal differentiation or promote self-renewal of ES cells, suggesting the individual Src-family members control key signal pathways related to these biological outcomes. Because ES cells express multiple Src family members, we have taken a chemical genetics approach to the problem of individual kinase isoform functions in this cell type. This approach involves modified kinases with functionally silent kinase domain mutations that confer specific inhibitor sensitivity. Our plan is to introduce these inhibitor-sensitizing mutations into ES cells with standard gene-targeting approaches, thus allowing specific pharmacological assessment of the role of single kinase activities on developmental outcomes. A complementary approach involves Src kinases with engineered resistance to broad-spectrum inhibitors of the Src-family. This approach has enabled us to demonstrate a specific role for c-Src in the transition of ES cells to primartive ectoderm, one of the earliest steps in differentiation. Ultimately, we hope to identify inhibitors with the appropriate selectivity profiles to either promote self-renewal or guide desired developmental fates. 

The c-fes tyrosine kinase: Oncogene or tumor suppressor? While protein-tyrosine kinase activity is most commonly associated with tumor formation and progression, we discovered that expression of the c-Fes tyrosine kinase is commonly lost in colonic epithelial cells from colorectal cancer patients. Like many other tumor suppressor genes, the c-fes promoter region is epigenetically silenced by methylation in primary tumor specimens, but not in normal epithelial cells where the gene is expressed. We are now formally testing a tumor suppressor role for c-fes in APC/min mice, an animal model that is genetically predisposed to intestinal polyp formation. These experiments involve crossing APC/min animals into a fes-null background and looking for changes in tumor number, size and latency. In addition, we are actively pursuing discovery of small molecule inhibitors selective for c-Fes, which will be useful tools to explore the role of this pathway in colorectal cancer as well as other tumor sites.