Related Experiment Video
Updated: Jan 10, 2026
Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
Published on: May 23, 2025
Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression
Fidan Seker-Polat1, Magdalena Rogozinska1, Youngho Ban2
1Department of ObGyn, Northwestern University Feinberg School of Medicine, Robert Lurie Comprehensive Cancer Center, Chicago, IL, USA.
Abstract:
Cancer cells rapidly induce PD-L1 expression in response to inflammatory cytokines such as IFNγ from cytotoxic T cells. Increased surface PD-L1 is a primary mechanism of cancer cells evading cytotoxic T-cell-mediated immune clearance. Identifying how cancer cells increase PD-L1 expression may yield clinically relevant immune checkpoint regulators. However, the key regulators and molecular mechanisms mediating rapid PD-L1 induction are yet to be understood entirely. To identify targetable mechanisms controlling cytokine-induced PD-L1 expression, we performed functional CRISPR gene KO screening with a custom-designed sgRNA library that targets "druggable" genes. We performed the screening in 6 different cancer lines: 3 ovarian (OVCAR4, CaOV3, and SKOV3) and three pancreatic cancer (MiaPaca2, ASPC1 and KP4) cell lines. The screening recovered the known regulators of PD-L1 expression and uncovered several novel regulators of PD-L1 that control its expression in all cell lines or in a cancer-type-specific fashion. For example, while genetic or pharmacological depletion of CSNK1A1 results in reduced PD-L1 expression in ovarian cancer cells, CDK1 depletion modulates PD-L1 in pancreatic cancer cell lines. Significantly, we discovered that KEAP1 depletion or pharmacological inhibition diminishes PD-L1 in all cell lines tested (n = 6). Mechanistically, KEAP1 depletion-mediated reduced PD-L1 is due to transcriptional repression of the PD-L1 gene by NRF2 activation. As such, depletion of NRF2 restores PD-L1 expression, while its overexpression leads to diminished PD-L1 expression. Supporting this, pharmacological NRF2 activation resulted in significant antitumor immunity with increased cytotoxic effector T cell infiltration and reduced exhausted T cells, resulting in smaller xenografted tumors. These findings establish the KEAP1/NRF2 axis as a novel and potentially druggable mechanism of IFNγ-meditated PD-L1 expression in cancer cells.
Insights
Researchers identified the KEAP1/NRF2 pathway as a key regulator of PD-L1 expression in cancer cells. Targeting this axis may offer new strategies for cancer immunotherapy by enhancing anti-tumor immunity.
Area of Science:
- Cancer Biology
- Immunology
- Genetics
Background:
- Cancer cells express PD-L1 to evade immune attack by cytotoxic T cells.
- Understanding the regulation of PD-L1 is crucial for developing effective cancer immunotherapies.
Purpose of the Study:
- To identify novel, targetable regulators of cytokine-induced PD-L1 expression in cancer cells.
- To elucidate the molecular mechanisms controlling PD-L1 upregulation.
Main Methods:
- Functional CRISPR gene knockout screening in ovarian and pancreatic cancer cell lines.
- Targeting "druggable" genes using a custom sgRNA library.
- Assessing PD-L1 expression changes and mechanistic pathways.
Main Results:
- CRISPR screening identified known and novel regulators of PD-L1, including CSNK1A1 (ovarian) and CDK1 (pancreatic).
- KEAP1 depletion or inhibition significantly reduced PD-L1 expression across all tested cancer cell lines.
- KEAP1 depletion leads to NRF2 activation, which transcriptionally represses PD-L1; NRF2 modulation inversely affects PD-L1 levels.
- Pharmacological NRF2 activation enhanced anti-tumor immunity and reduced tumor growth in vivo.
Conclusions:
- The KEAP1/NRF2 axis is a critical regulator of IFNγ-mediated PD-L1 expression in cancer.
- This pathway represents a novel and potentially druggable target for cancer immunotherapy.
