Abstract The eight mammalian Src-family members are non-receptor protein-tyrosine kinases which are involved in nearly all cell signaling pathways. Hck and Fgr are members of this family expressed almost exclusively in myeloid hematopoietic cells and their progenitors. The overexpression of these kinases has been linked to the development of, and a poorer prognosis in, acute myeloid leukemia (AML). AML is a common form of blood cancer in adults, with nearly 20,000 new cases per year in the US. About one-third of AML cases have activating mutations in the Flt3 receptor tyrosine kinase, including point mutations and internal tandem duplications. Current treatments for this subset of AML patients include ATP-site kinase inhibitors, although acquired resistance mutations commonly develop within one year of the start of treatment. Inhibitors for Hck and Fgr are also emerging as a new approach to AML therapy. Our group recently identified small molecules that bind to the regulatory domains of Hck (unique-SH3-SH2-linker) as opposed to the ATP-binding site of the kinase domain. Binding data and docking models suggest that these compounds bind to a site that requires a specific 3D- conformation of the SH3 and SH2 domains. They also decrease the viability of an AML cell line that overexpresses active Hck and Fgr. Based on these results, we hypothesize that these compounds suppress AML cell growth by interfering with downstream signaling via the SH3 and SH2 domains. In addition, the compounds may allosterically influence the conformation of the active site to favor ATP-site inhibitor action. We aim to expand upon these findings using in vitro kinase and binding assays to explore selectivity within the Src- kinase family and possible synergy with ATP-site inhibitors. The anti-AML mechanism of action of the compounds will be tested in AML cell line models by correlating their effects on kinase activity and downstream substrate activation with growth suppression and apoptosis. Finally, we will determine the binding site for these putative allosteric inhibitors within Hck by X-ray crystallography. These experiments will provide crucial insight for the future development of these novel compounds as a treatment for AML, either as stand-alone therapy or in combination with existing ATP-site inhibitors. 1

Project Narrative Protein-tyrosine kinases are an important class of drug targets in cancer and other diseases. Virtually all clinically useful kinase inhibitors to date work by competing for ATP binding at the kinase domain active site. Because of the high degree of kinase sequence and structural homology, most inhibitors block multiple kinase targets, which clouds their mechanism of action and may contribute to side effects in a therapeutic setting. In this fellowship application, I propose investigation of a new type of inhibitor for protein-tyrosine kinases of the non-receptor class that takes advantage of a wealth of information regarding natural mechanisms of kinase regulation. Successful completion of this project has the potential to lead to new classes of drugs for the treatment of acute myeloid leukemia and other forms of cancer.