Abstract LB286: Keap1 loss promotes cancer stemness and anti-PD-1 resistance in head and neck cancer

Abstract Background: KEAP1 is a central negative regulator of NRF2-dependent antioxidant and metabolic programs, and alterations in KEAP1/NRF2 signaling occur in approximately 20% of head and neck squamous cell carcinoma (HNSCC). However, the role of KEAP1/NRF2 signaling in HNSCC and responsiveness to immunotherapy remains incompletely defined. Methods: KEAP1 was knocked out in the murine oral cancer cell line MOC22 using CRISPR/Cas9. Cell proliferation and motility were assessed using an MTT-based growth assay and an in vitro migration assay. Tumor growth was evaluated in an immunocompetent syngeneic model using wild-type (WT) and KEAP1-knockout (KO) MOC22 cells. Stem-like features were assessed by flow cytometric quantification of the EpCAM+CD44+ tumor-cell population. To evaluate immunotherapy response, tumor-bearing mice were treated with anti-PD-1 or isotype control IgG and intratumoral CD8+ T-cell responses were profiled. Ferroptotic susceptibility in vivo was assessed using readouts of oxidative stress and iron/glutathione homeostasis, including cellular reactive oxygen species (ROS), cytoplasmic labile iron, and glutathione (GSH) regulation in tumor cells. Results: KEAP1 KO in MOC22 cells resulted in constitutive activation of antioxidant signaling. KEAP1 loss increased migratory capacity and modestly reduced proliferation in vitro. In contrast, KEAP1-deficient tumors grew more rapidly in vivo and exhibited an increased EpCAM+CD44+ tumor-cell fraction, consistent with enhanced stem-like properties. KEAP1-deficient tumors were significantly resistant to anti–PD-1 therapy. Mechanistically, anti–PD-1 treatment increased tumor-infiltrating CD8+ T cells in both genotypes; however, in WT tumors this was associated with tumor-cell lipid peroxidation–mediated ferroptotic death, reflected by increased cellular ROS, increased cytoplasmic iron accumulation, and dysregulated GSH. In KEAP1 KO tumors, tumor cells exhibited significantly reduced ROS and cytoplasmic labile iron in vivo, consistent with attenuated ferroptotic execution despite CD8+ T-cell infiltration. Conclusions: Collectively, these data support a model in which KEAP1 loss promotes stem-like tumor phenotypes in vivo and contributes to anti-PD-1 resistance by limiting CD8+ T cell-mediated ferroptotic tumor cell death through reinforced redox and iron-buffering programs. Citation Format: Khandoker Ifaz Ahmed, Qu Deng, Xue Xiong, Savallya Boyini, Yash Shah, Zhibin Cui. Keap1 loss promotes cancer stemness and anti-PD-1 resistance in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB286.