Data from Cancer-Derived Mutations in KEAP1 Impair NRF2 Degradation but not Ubiquitination

Abstract

<div>Abstract<p>NRF2 is a transcription factor that mediates stress responses. Oncogenic mutations in <i>NRF2</i> localize to one of its two binding interfaces with KEAP1, an E3 ubiquitin ligase that promotes proteasome-dependent degradation of NRF2. Somatic mutations in <i>KEAP1</i> occur commonly in human cancer, where <i>KEAP1</i> may function as a tumor suppressor. These mutations distribute throughout the KEAP1 protein but little is known about their functional impact. In this study, we characterized 18 <i>KEAP1</i> mutations defined in a lung squamous cell carcinoma tumor set. Four mutations behaved as wild-type KEAP1, thus are likely passenger events. R554Q, W544C, N469fs, P318fs, and G333C mutations attenuated binding and suppression of NRF2 activity. The remaining mutations exhibited hypomorphic suppression of NRF2, binding both NRF2 and CUL3. Proteomic analysis revealed that the R320Q, R470C, G423V, D422N, G186R, S243C, and V155F mutations augmented the binding of KEAP1 and NRF2. Intriguingly, these “super-binder” mutants exhibited reduced degradation of NRF2. Cell-based and <i>in vitro</i> biochemical analyses demonstrated that despite its inability to suppress NRF2 activity, the R320Q “superbinder” mutant maintained the ability to ubiquitinate NRF2. These data strengthen the genetic interactions between <i>KEAP1</i> and <i>NRF2</i> in cancer and provide new insight into KEAP1 mechanics. <i>Cancer Res; 74(3); 808–17. ©2013 AACR</i>.</p></div>