PHF10 is a Novel Substrate of Keap1 to Protect Non-Small-Cell Lung Cancer (NSCLC) Cells Against Oxidative Stress and Confer Ferroptosis Resistance
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Xuan Lu1, Ningning Kang2, Yajun Chen1
1School of Life Science, Hefei Normal University, Hefei, China.
Purpose:
Keap1 mutations mainly caused NRF2-dependent anti-oxidative stress responses, yet whether there are other downstream substrates and pathways remains unknown. This study aimed to uncover the role of Keap1 mutations in regulating PHF10-NRF2 axis in NSCLC and ferroptosis evasion.
Materials And Methods:
Tandem affinity purification with mass spectrometry was used to screen peptides. Co-IP and ubiquitination assays were used to confirm the Keap1-PHF10 axis. A series of analyses in cell lines, patient samples, and xenograft models were conducted to uncover the functional dependency between Keap1 and NRF2. Transmission electron microscope was used to detect mitochondrion swelling under ferroptosis.
Results:
Here, we reported that Keap1 binds and promotes polyubiquitination and degradation of PHF10, a subunit of the PBAF complex. NSCLC-associated Keap1 mutations are incapable of degrading PHF10, and thus induces PHF10 proteins stability. PHF10 ablation shows synthetic lethality in Keap1-deficient NSCLC cells. Mechanistically, PHF10 interacts with NRF2 to activate its downstream targets and enhance the NRF2-dependent anti-oxidative stress capacity in NSCLC. PHF10 recruits SMARCA2, one core cBAF subunit, to increase chromatin accessibility in NRF2-binding transcriptional regions. Cancer-associated Keap1 mutants confer resistance to ROS-induced cell death via accumulating PHF10-SMARCA2 complex. Increased PHF10 further induced ferroptosis resistance in Keap1-deficient NSCLC. Lastly, we utilized one small molecule inhibitor, SMARCA2-IN-8, to inhibit progression of Keap1-deficient NSCLC murine models.
Conclusion:
Together, our study highlight the synthetic lethal relationship between Keap1 and PHF10, and provide targeting PHF10-SMARCA2 complex as an effective option to hit Keap1-deficient NSCLC.
Insights
Keap1 mutations stabilize PHF10, promoting NRF2 activity and ferroptosis evasion in NSCLC. Targeting the PHF10-SMARCA2 complex offers a synthetic lethal strategy for Keap1-deficient NSCLC.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Keap1 mutations primarily activate NRF2-dependent antioxidant responses.
- The full spectrum of Keap1 mutation downstream effects and pathways remains largely unexplored.
- Understanding these pathways is crucial for developing targeted therapies in Non-Small Cell Lung Cancer (NSCLC).
Purpose of the Study:
- To investigate the role of Keap1 mutations in regulating the PHF10-NRF2 axis.
- To elucidate the mechanisms by which Keap1 mutations contribute to ferroptosis evasion in NSCLC.
- To identify potential therapeutic targets in Keap1-mutated NSCLC.
Main Methods:
- Tandem affinity purification coupled with mass spectrometry for peptide screening.
- Co-immunoprecipitation and ubiquitination assays to validate the Keap1-PHF10 interaction.
- In vitro and in vivo studies using NSCLC cell lines, patient samples, and xenograft models.
- Transmission electron microscopy to assess mitochondrial changes during ferroptosis.
Main Results:
- Keap1 binds and promotes PHF10 polyubiquitination and degradation; NSCLC-associated Keap1 mutations impair this process, stabilizing PHF10.
- PHF10 ablation exhibits synthetic lethality in Keap1-deficient NSCLC cells.
- PHF10 interacts with NRF2, enhancing its downstream targets and antioxidant capacity, and recruits SMARCA2 to increase chromatin accessibility at NRF2-binding sites.
- Accumulation of the PHF10-SMARCA2 complex confers resistance to reactive oxygen species-induced cell death and promotes ferroptosis resistance in Keap1-deficient NSCLC.
- Inhibition of SMARCA2 using a small molecule inhibitor (SMARCA2-IN-8) suppressed tumor progression in NSCLC murine models.
Conclusions:
- Keap1 and PHF10 share a synthetic lethal relationship in NSCLC.
- The PHF10-SMARCA2 complex is a key mediator of ferroptosis evasion in Keap1-deficient NSCLC.
- Targeting the PHF10-SMARCA2 complex represents a promising therapeutic strategy for Keap1-deficient NSCLC.
