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A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer
Chang Jiang1, Nathan P Ward1, Nicolas Prieto-Farigua1
1Department of Cancer Physiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Abstract:
The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2•-) -generating drug β-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, β-Lapachone did not generate mitochondrial O2•- but rather SOD2 loss enhanced the efficacy of β-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized cells to β-Lapachone, demonstrating that these effects may be translated to increase ROS sensitivity therapeutically.
Insights
Researchers identified vulnerabilities in non-small cell lung cancer (NSCLC) with mutated KEAP1/NRF2. Loss of mitochondrial superoxide dismutase 2 (SOD2) surprisingly enhanced chemotherapy efficacy by disrupting iron-sulfur proteins and ATP production.
Area of Science:
- Biochemistry
- Oncology
- Molecular Biology
Background:
- Nuclear factor erythroid 2-related factor 2 (NRF2) is a key redox regulator often hyperactivated in non-small cell lung cancer (NSCLC).
- NRF2 hyperactivation in NSCLC correlates with resistance to chemotherapy and radiation therapy.
- Identifying specific vulnerabilities in KEAP1/NRF2-mutant NSCLC is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To identify antioxidant enzyme genes that, upon loss, sensitize KEAP1/NRF2-mutant NSCLC cells to the superoxide-generating drug β-Lapachone.
- To elucidate the mechanisms by which the loss of specific antioxidant enzymes impacts cellular response to oxidative stress and drug treatment.
Main Methods:
- A CRISPR-Cas9-based negative selection screen was employed to identify genes whose deletion sensitized NSCLC cells to β-Lapachone.
- Cells with KEAP1/NRF2 mutations were treated with sub-lethal concentrations of β-Lapachone.
- Analysis focused on identifying antioxidant enzyme genes that were essential for cell survival under these conditions.
Main Results:
- The screen identified known antioxidant pathways, including the pentose phosphate pathway, thioredoxin system, and glutathione reductase, as expected.
- Mitochondrial superoxide dismutase 2 (SOD2) was identified as a top hit, indicating its importance in NSCLC resistance.
- Contrary to initial expectations, loss of SOD2 enhanced β-Lapachone efficacy not through increased mitochondrial reactive oxygen species (ROS), but via disruption of iron-sulfur protein function, impaired mitochondrial ATP production, and reduced NADPH generation.
Conclusions:
- Loss of SOD2 confers sensitivity to β-Lapachone in NSCLC by affecting mitochondrial function and metabolic pathways, rather than direct ROS generation.
- Inhibition of mitochondrial electron transport activity can sensitize NSCLC cells to β-Lapachone, suggesting a potential therapeutic approach.
- Targeting mitochondrial function represents a promising strategy to overcome chemoresistance in KEAP1/NRF2-mutant NSCLC.
