Kinase Dynamics Underlie Mechanisms of Sensitivity and Resistance of EGFR with LUAD Mutations to Tyrosine Kinase Inhibitors
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Pharmacology
First Advisor
Lemmon, Mark
Abstract
Tyrosine kinase inhibitors (TKIs) are used to treat non-small cell lung cancers (NSCLC) driven by epidermal growth factor receptor (EGFR) mutations in the tyrosine kinase domain (TKD). Although patients with common EGFR driver mutations in their tumour experience strong benefits in progression-free survival (PFS) and overall survival (OS) with these therapies, the best approach for patients with uncommon driver or TKI resistance mutations is less clear. In this dissertation, I studied the molecular mechanisms governing TKI sensitivity and resistance for a variety of common and uncommon EGFR variants seen in LUAD. Using structural modelling and hydrogen-deuterium exchange mass spectrometry (HDX-MS), I show that the osimertinib resistance mutation L718V likely functions to impair drug binding, and that kinase structural dynamics explain its differential impact on 3rd vs 1st and 2nd generation inhibitors. Next, I investigated the effect of LUAD mutations in the kinase N-lobe on dimerisation preferences. Using CHO cells to express full-length receptors, I found that these mutations lead to variant-specific changes in dimerisation selectivity. These studies suggest not only that altered dimerisation contributes to the mechanism of oncogenic activation of these variants, but also that precision medicine approaches are required for the use of therapeutic antibodies against EGFR or ErbB2/3 in LUAD to account for variant-specific differences in dimerisation. Next, we studied the heterogeneous group of exon 19 deletion mutations in EGFR that drive LUAD and found that these variants fall into two groups – one TKI sensitive (profile 2), and one TKI insensitive but with a preferential sensitivity to 2nd generation inhibitors (profile 1). We found that TKI sensitivity for the exon 19 variants is determined by KM, ATP, where elevated KM, ATP is defined by dynamic instability in the ATP-binding site that renders the profile 2 variants sensitive to inhibitors. KM, ATP in turn is defined by length of truncation of the β3-αC loop encoded by exon 19, which may be able to predict patients’ clinical benefit from 1st and 3rd generation inhibitors based on the specific mutation in the tumour. Finally, with the goal of identifying more effective inhibitors for the profile 1 exon 19 deletions that are less sensitive to standard-of-care osimertinib, I studied novel 3rd generation inhibitors. I found that these TKIs have different conformational preferences and potencies in cellular assays that are related to their different structural characteristics, despite their apparent chemical similarities to osimertinib. Upon finding that certain exon 20 insertion-directed TKIs effectively inhibit the ΔL747-A750InsP variant, I identified intriguing biochemical similarities between exon 20 insertion variants and profile 1 exon 19 deletion variants, potentially defined by their low KM, ATP. These observations may argue for the repurposing of exon 20 insertion-directed TKIs for the difficult-to-treat population of profile 1 exon 19 deletions. Overall, these analyses suggest that kinase dynamics of LUAD-associated variants of EGFR are the lynchpin underlying several different mechanisms of sensitivity and resistance to TKI inhibitors. Lastly, they demonstrate that study of individual mutations and mutational subsets can provide a path towards rational precision medicine approaches.
Recommended Citation
van Alderwerelt van Rosenburgh, Iris Katherine, "Kinase Dynamics Underlie Mechanisms of Sensitivity and Resistance of EGFR with LUAD Mutations to Tyrosine Kinase Inhibitors" (2023). Yale Graduate School of Arts and Sciences Dissertations. 997.
https://elischolar.library.yale.edu/gsas_dissertations/997
