UCSF

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. While ATP-competitive tyrosine kinase inhibitors (TKIs) offer growing potential for effectively targeting oncogenic MET, this too is limited by the emergence of resistance mutations with no treatment reference.

In chapter 1, I perform a parallel deep mutational scan (DMS) of MET intracellular kinase domain in two fusion protein backgrounds: wild type and METΔEx14. This comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C helix, pointing to differences in regulatory mechanisms between MET and other RTKs. Additionally, this study uncovered a β5 motif that acts as a structural pivot for kinase domain activation in MET and other TAM family of kinases. Lastly, a number of previously unknown activating mutations were identified, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

Building upon this foundation, in chapter 2, I extended this DMS to study TKI resistance in the kinase domain against 11 MET inhibitors to reveal unique and shared resistance mechanisms across inhibitor types. Leveraging this dataset as a reference, differential sensitivities to inhibitor pairs were explored to establish an experimental framework for assessing idiosyncratic resistance mutations, inhibitor combinations, and sequential treatments based on mutational sensitivity, location, and frequency within the screen, which hopefully can serve as a tool in future drug design.