Cancer-derived mutations in KEAP1 impair NRF2 degradation but not ubiquitination

Area of Science:

• Molecular Biology
• Cancer Biology
• Biochemistry

Background:

• Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor regulating cellular stress responses.
• Kelch-like ECH-associated protein 1 (KEAP1) acts as an E3 ubiquitin ligase, targeting NRF2 for degradation.
• Somatic mutations in KEAP1 are common in human cancers, suggesting a tumor suppressor role, but their functional impact remains unclear.

Purpose of the Study:

• To characterize the functional impact of 18 KEAP1 mutations found in lung squamous cell carcinoma.
• To investigate how these KEAP1 mutations affect NRF2 binding, degradation, and ubiquitination.
• To elucidate the mechanistic basis of KEAP1 mutations in cancer.

Main Methods:

• Characterization of 18 KEAP1 mutations from lung squamous cell carcinoma samples.
• Assessment of KEAP1-NRF2 binding affinity and NRF2 suppression activity.
• Proteomic analysis to identify KEAP1 mutants with altered NRF2 binding.
• Cell-based and in vitro biochemical assays to study NRF2 ubiquitination by mutant KEAP1.

Main Results:

• Four KEAP1 mutations behaved as wild-type, suggesting passenger events.
• Several mutations (R554Q, W544C, N469fs, P318fs, G333C) attenuated NRF2 binding and suppression.
• A subset of mutations (R320Q, R470C, G423V, D422N, G186R, S243C, V155F) enhanced KEAP1-NRF2 binding ('super-binders').
• 'Super-binder' mutants showed reduced NRF2 degradation, and R320Q retained NRF2 ubiquitination activity despite impaired suppression.

Conclusions:

• KEAP1 mutations in cancer exhibit diverse functional consequences on NRF2 regulation.
• Specific KEAP1 mutations can enhance NRF2 binding, leading to impaired NRF2 degradation.
• These findings reinforce the critical genetic interplay between KEAP1 and NRF2 in cancer pathogenesis and offer insights into KEAP1's molecular mechanisms.