The Dynamic Structural Effects of Activating ERBB Kinase Somatic Mutations

The ERBB family of receptor tyrosine kinases, epidermal growth factor receptor (EGFR, ERBB1), ERBB2, ERBB3 and ERBB4, are transmembrane signaling proteins that regulate cellular processes such as cell survival, mobility, proliferation and differentiation. Normal activity of the ERBBs is essential for tissue growth and organ development. An array of somatic mutations of the ERBB genes have been linked to human malignancies. As a result, the ERBBs are important treatment targets, with multiple ERBB-based drugs currently in effective clinical use against e.g. lung, breast and colorectal cancers.

A comprehensive characterization of the ERBB somatic mutations identified in cancer samples is essential to unveil the molecular level impacts of the genetic alterations that could play a role in tumorigenesis. In this thesis, the structural consequences of four cancer-associated ERBB kinase mutations that aberrantly activate EGFR and ERBB2 proteins were explored. The mutations include three EGFR alterations, 746ΔELREA750 (ΔELREA), V769insASV and D770insNPG, and an ERBB2 missense mutation: E936K. The ERBB receptors and proteins in general are dynamic molecules. Hence, the possible structural changes exerted by the above activating mutations were examined by employing molecular dynamics simulations, which allow the assessment of time-dependent structural motions.

The simulations revealed that the EGFR ΔELREA deletion mutation stabilizes the active state EGFR conformation by conserving the states of key structural units, such as the αC helix and the Lys745 Glu762 salt bridge, which were disrupted in the wild-type EGFR. Furthermore, the deletion resulted in a structural change on the inactive EGFR state, an inward movement of the αC helix, which could drive a conformational change from the inactive towards the active EGFR state. The V769insASV and D770insNPG EGFR insertion mutations also led to the better stability of the active EGFR conformation relative to the wild-type EGFR. Moreover, the insertions obstructed the formation of an autoinhibitory interaction between Ala767 and Arg776 in the inactive EGFR conformation, which would predispose EGFR to transition to the catalytically active EGFR state. The ERBB2 E936K mutation affected the nature of interactions taking place at the ERBB2 homodimer and heterodimer interface, with a new inter-monomer ionic interaction being formed that strengthened the monomer-monomer binding. Consequently, the duration of the activated ERBB2 dimer would be extended, which fuels the phosphorylation of ERBB2.

Taken together, this thesis demonstrated that a series of structural changes are at play that collectively elicit the experimentally reported functional changes by these activating ERBB mutations. A thorough examination of the mutation-induced structural alterations furthers our knowledge on the role the mutations play in cancer progression and the results are essential knowledge when using structure-based, rational design of ligands that could inhibit ERBB kinase activity and subsequent receptor signaling. Such ligands have potential for further development towards a therapeutic agent in efforts to tackle cancers.