Insights into the inhibition of ETV6 PNT domain polymerization

ETV6 is a modular transcriptional repressor for which head-to-tail polymerization of its PNT (or SAM) domain facilitates cooperative binding to tandem DNA sites by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to the catalytic domain of one of several receptor protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, auto-phosphorylation, and aberrant stimulation of downstream signaling pathways, thereby leading to a diverse range of cancers. Inhibition of PNT domain polymerization through mutations renders the chimeric proteins non-oncogenic. This indicates that a small molecule inhibitor of polymerization could be a viable therapeutic against many ETV6-linked cancers. Protein-protein interactions are challenging to disrupt with small molecules, and thus I followed a multi-pronged approach for lead compound discovery. In Chapter 2, I describe work to obtain structural, dynamic, and thermodynamic insights into the PNT domain and its self-association properties. To this end, I characterized monomeric and heterodimeric forms of the PNT domain using nuclear magnetic resonance spectroscopy, X-ray crystallography, molecular dynamics simulations, amide hydrogen exchange, and alanine scanning mutagenesis in conjunction with surface plasmon resonance binding studies. Collectively, these studies defined “hot spot” regions critical to the PNT domain self-association interface. In Chapter 3, I discuss efforts undertaken to find inhibitors of ETV6 PNT domain polymerization using two high-throughput cellular approaches – a split luciferase reporter assay and a modified yeast two-hybrid assay – and a computational approach with the Bristol University Docking Engine (BUDE). Over 75 lead compounds from these assays were tested for binding to the PNT domain through NMR spectroscopy. None were found to bind to the PNT domain or inhibit its self-association. However, lessons learned from these screening assays may facilitate future high-throughput screening for or rational design of therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat