Collections > Electronic Theses and Dissertations > Molecular Mechanisms of Patient-Derived KEAP1 Superbinder Mutants
pdf
Available after 31 December, 2019

In 30% of lung cancers, mutations in KEAP1 or NRF2 result in constitutive NRF2 activity. This promotes tumor progression, resistance to radio- and chemotherapy, and predicts poor patient outcome. Over 700 somatic mutations in the KEAP1 tumor suppressor gene have been identified in cancer, yet the mechanism and functional consequences of these mutations are unknown. This dissertation focuses on determining the phenotype and molecular profiles of patient-derived KEAP1 mutations. The objectives were to assign function for patient-derived KEAP1 mutations and to investigate the molecular mechanism(s) and phenotypes of functional classes of KEAP1 mutations. Through biochemical characterization of 18 KEAP1 mutations identified in lung squamous cell carcinoma, we defined a novel class of KEAP1 ‘superbinder’ mutants. These superbinder mutants had increased association with the transcription factor NRF2, yet could not fully suppress NRF2-dependent transcription of cytoprotective genes. Cell-based and in vitro studies determined that superbinder mutants ubiquitylated NRF2 but were impaired for NRF2 proteasomal degradation. Molecular biology techniques were employed to understand the mechanism and phenotypic consequences of the KEAP1 superbinder mutants. Through these studies, five core characteristics were attributed to the superbinder mutant class. First, superbinder residues are highly conserved and are among the most frequently mutated residues across a variety of cancer types. Second, KEAP1 superbinders increase NRF2 association but are not altered in their association with other KEAP1 substrates proteasomal chaperones, or ubiquitin receptors. Third, KEAP1 superbinders may impact KEAP1 tertiary structure thus stabilizing its interaction with the NRF2 degron. Fourth, KEAP1 superbinders sequester a pool of NRF2 in p62-dependent spherical clusters that are cleared by the cell. Furthermore, these clusters are comprised of a KEAP1 core surrounded by the autophagy adapter p62, phosphorylated p62 (pS351), polyubiqutin, and occasionally NRF2. Fifth, superbinders confer resistance to the DNA-damaging agent bleomycin in lung cancer cell lines stably overexpressing KEAP1 superbinder mutants. These studies expand our mechanistic understanding of KEAP1 superbinder mutants and provide insight into the dynamics and subcellular compartmentalization of the KEAP1-NRF2 complex.