Deciphering the Role of Reductive Stress in Non Small Cell Lung Cancer

Project Summary
Control of the redox homeostasis is essential to cancer cell proliferation and requires the delicate maintenance
of oxidative and reductive metabolic pathways. This equilibrium is controlled by signal transduction pathways
and imbalances lead to redox stress that potently blocks cancer growth. Much work has focused on the role of
oxidative stress in cancer proliferation, however, the converse– reductive stress and its impact on malignant
cells is poorly understood. We have studied the role of redox control in cancer in the context of modification of
proteinaceous cysteines by reactive oxygen species and the NRF2 transcription factor pathway. NRF2 functions
as the master regulator of the cellular antioxidant response and promotes the expression of key metabolic and
detoxification genes to generate a reductive environment and negate oxidative stress. NRF2 is activated in many
cancers including ~30% of non small cell lung cancers (NSCLC) through mutation of its negative regulator
KEAP1. While NRF2 has been extensively studied in KEAP1-mutant NSCLCs, we wondered what role this
pathway plays in the proliferation of NSCLC cell lines which are wildtype (WT) for KEAP1. To this end, we
pharmacologically activated NRF2 in 50+ NSCLC cell lines (WT for KEAP1) and measured their proliferation.
Unexpectedly, we find that in ~16% of NSCLC cell lines, NRF2 activation results in a severe block in proliferation.
A genome wide CRISPR screen identifies that genes involved in mitochondrial metabolism, mitochondrial fusion
and the electron transport chain (ETC) are major sensitizers to NRF2 activation when lost and can function as
companion biomarkers for NRF2-sensitivity. In line with the generation of reductive stress following NRF2
activation, key cysteines on enzymes involved in mitochondrial metabolism and mitochondrial fusion are reduced
as determined by chemical proteomic platforms. To explain these surprising biological characteristics we
propose the following hypothesis: NRF2 activation in a subset of NSCLC cell lines promotes an overly reductive
environment that decreases the activity of key enzymes in mitochondrial metabolism and mitochondrial
respiration and fusion. The inactivation of these pathways synergize to block cell growth. In this grant application,
we build on our research surrounding NRF2 sensitization and mechanistically characterize the role of reductive
stress in NSCLC proliferation. In this grant application, we will comprehensively define KEAP1-dependence by
identifying NRF2 regulation of mitochondrial metabolism/fusion at the protein, cellular and organismal levels.
The research proposed herein, takes full advantage of a series of recently conceived methods: chemical
proteomics, genome-wide CRISPR screens and untargeted metabolomics, which have previously been
deployed in isolation, to be used in an integrated manner to effectively dissect how protein reduction underlies
protein malfunction and KEAP1-dependence. These studies will not only provide a comprehensive
understanding of NRF2/KEAP1 biology but may also lay the foundation for developing translational therapeutics
to benefit lung cancer patients with deregulated NRF2 signaling.

Grant Number: 5R37CA260062-04
NIH Institute/Center: NIH
Principal Investigator: Liron Bar-Peled