Defining Mechanisms Governing Myc Stability and its Modulation by Aurora Kinase A

Project Summary/Abstract. The Myc gene family encodes three highly conserved transcription factors (N-, c-, L-), known to be oncogenic drivers in many cancers. Dysregulation of Myc proteins is estimated to drive 30% of all cancers, attributed to inappropriate amplified Myc expression and dysregulated behaviors. c-Myc is prominent in a wide variety of cancers, due to its broad expression patterns, where N-Myc and L-Myc are tissue specific and heavily associated in neuroblastoma and small cell lung carcinoma, respectively. Due to its prevalent cancer involvement, Myc family transcription factors are considered an attractive anti-cancer target, but their disordered nature makes them poor drug targets. Protein-protein interactions of Myc with other regulatory partners creates an opportunity for therapeutic intervention via indirect targeting, yet structural details of many Myc interactions remain unclear. The N- and c-Myc binding partner Aurora Kinase A (AurA), a serine-threonine kinase, is hypothesized to stabilize Myc by preventing proper ubiquitin-mediated degradation by the SCFFbxw7 ubiquitin ligase complex. In this pro- posal, fluorescence anisotropy and time-resolved fluorescence will be utilized to fully assess binding of AurA at varied phosphorylation states and lengths of c-Myc. To structurally characterize a c-Myc/AurA complex I will pursue extensive training in X-ray crystallography alongside my experience using continuous-wave electron par- amagnetic resonance (CW-EPR) spectroscopy to study the interface of this interaction, proposed within. My preliminary work has supported the formation of an AurA/c-Myc/Fbxw7 complex, of which I will structurally char- acterize using continued training in cryogenic-electron microscopy (cryo-EM). Further characterization of c-Myc stabilization and ubiquitination patterns by AurA using my developed in vitro ubiquitination assays will result in a defined mechanism of AurA induced stabilization of c-Myc. The high conservation of regulatory protein binding domains across Myc family transcription factors suggests a role of AurA stabilization of L-Myc, already identified for N- and c-Myc. The work and training I receive studying the c-Myc/AurA interaction will build foundations for my future independent research on defining an impact of AurA on L-Myc stabilization, a widely understudied Myc family member. I will also use in cellular work to characterize the physiological protein interactions involved in L- Myc regulation and dysregulation, including kinases and SCF ubiquitin ligase components, currently unidentified. The extensive X-ray crystallography and cryo-EM training proposed within will add a strong structural biology foundation to the independent research program I plan to develop at an R1 institution. The focus of my lab will be rooted in characterizing the regulation mechanisms of disordered transcription factors in cancer and disease. I believe the extensive structural biology and biophysical background from proposed training and extensive ex- perience will make me a competitive candidate for a future tenure-track faculty role.