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Summary: Clinical evidence has revealed that high-level activation of NRF2 caused by somatic mutations in NRF2 (NFE2L2) is frequently detected in esophageal squamous cell carcinoma (ESCC), whereas that caused by somatic mutations in KEAP1, a negative regulator of NRF2, is not. Here, we aspire to generate a mouse model of NRF2-activated ESCC using the cancer-derived NRF2L30F mutation and cancer driver mutant TRP53R172H. Concomitant expression of NRF2L30F and TRP53R172H results in formation of NRF2-activated ESCC-like lesions. In contrast, while squamous-cell-specific deletion of KEAP1 induces similar NRF2 hyperactivation, the loss of KEAP1 combined with expression of TRP53R172H does not elicit the formation of ESCC-like lesions. Instead, KEAP1-deleted cells disappear from the esophageal epithelium over time. These findings demonstrate that, while cellular NRF2 levels are similarly induced, NRF2 gain of function and KEAP1 loss of function elicits distinct fates of squamous cells. The NRF2L30F mutant mouse model developed here will be instrumental in elucidating the mechanistic basis leading to NRF2-activated ESCC.

Summary: The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses. : The Keap1-Nrf2 system plays a central role in the oxidative stress response. Suzuki et al. show that the H2O2 sensor of Keap1 is distinct from those used for electrophilic inducers. Keap1 exploits Cys226, Cys613, and Cys622/624 residues for sensing H2O2, and these residues set up an elaborate fail-safe mechanism. Keywords: Keap1, Nrf2, reactive cysteine residues, oxidative stress response