KEAP1 mutation in lung adenocarcinoma promotes immune evasion and immunotherapy resistance

Abstract

Lung cancer treatment has benefited greatly through advancements in immunotherapies. However, immunotherapy often fails in patients with specific mutations like KEAP1, which are frequently found in lung adenocarcinoma. We established an antigenic lung cancer model and used it to explore how Keap1 mutations remodel the tumor immune microenvironment. Using single-cell technology and depletion studies, we demonstrate that Keap1-mutant tumors diminish dendritic cell and T cell responses driving immunotherapy resistance. This observation was corroborated in patient samples. CRISPR-Cas9-mediated gene targeting revealed that hyperactivation of the NRF2 antioxidant pathway is responsible for diminished immune responses in Keap1-mutant tumors. Importantly, we demonstrate that combining glutaminase inhibition with immune checkpoint blockade can reverse immunosuppression, making Keap1-mutant tumors susceptible to immunotherapy. Our study provides new insight into the role of KEAP1 mutations in immune evasion, paving the way for novel immune-based therapeutic strategies for KEAP1-mutant cancers.

Keywords: CD103 DC; CP: Cancer; CP: Immunology; KEAP1; LUAD; NRF2; NSCLC; T cell; adenocarcinoma; immune surveillance; immunotherapy; lung cancer.

Figure 1.. Loss of Keap1 promotes immune evasion and accelerated tumor growth in a novel antigenic model of LUAD

(A) Schematic of our antigenic H-Y-driven orthotopic mouse model. (B) Growth kinetics (left) of male KP tumors established in female or male hosts with endpoint lung weights (right). (C) Growth kinetics (left) of male KP cells grown in female hosts following antibody-mediated depletion of CD4 or CD8 T lymphocytes with endpoint lung weights (right). (D) Growth kinetics (left) and endpoint lung weights (right) of male KP cells injected into male hosts following antibody-mediated depletion of CD4 or CD8 T lymphocytes. (E) Growth kinetics of Keap1 wild-type and mutant KP cells in females. (F) Representative images of H&E and NQO1 immunohistochemical staining of Keap1 wild-type and mutant KP tumors in female hosts. (G) Growth kinetics of Keap1 wild-type and mutant KP cells injected into male hosts. (H) Lung weight measured on day 26 in female and male mice bearing Keap1 wild-type and mutant tumors. (I) Growth kinetics (left) and endpoint luminescence (right) of Keap1 wild-type and mutant male cells injected into immunodeficient (NSG) female mice. (J) Schematic of KP Keap1^+/+, Keap1^fl/+, or Keap1^fl/fl GEMM mice infected with 20K TU Cre-expressing lentivirus. (K) Representative H&E and NQO1 immunohistochemical staining of Keap1^+/+, Keap1^fl/+, and Keap1^fl/fl KP tumors and tumor area quantification of mice infected with Cre-expressing lentivirus and sacrificed 3.5 months post infection. Scale bars, 2 mm. Each experimental subgroup had n ≥ 6 mice. Each symbol represents an individual mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001.

Figure 2.. Absence of CD103 DC-mediated anti-tumor immune responses in Keap1-mutant tumors

(A) UMAP of DC subclusters identified by scRNA-seq colored by cell type. (B) UMAP of the distribution of DC subclusters in Keap1 wild-type (WT, blue) and mutant (MUT, red) lung tumors. (C) Bar plot showing distribution of the DC clusters in Keap1 wild-type and mutant mouse lung tumors. (D) Representative flow cytometry plots for CD103 versus CD11b within a CD11c⁺MHCII⁺ DC gate. (E) Percentage of CD103 and CD11b DCs out of total tissue-infiltrating immune cells (CD45⁺CD45circ⁻) in normal (non-tumor) lung and lungs with orthotopic Keap1 wild-type and mutant tumors. (F) Percentage of CD103 and CD11b DCs out of total tissue-infiltrating immune cells (CD45⁺CD45circ⁻) in normal (non-tumor) lung and lungs with autochthonous genetically engineered mouse model (GEMM) Keap1 wild-type and mutant tumors. (G) Percentage of CD86, CD44, and CD80 among CD103 DCs in wild-type and mutant Keap1 tumors. FMO (fluorescence-minus-one) control shown for all markers. (H) Confocal images of lung tumor sections from Keap1 wild-type and mutant KP tumors. GFP (tumors) is shown in green, circulating CD45⁺ cells are shown in blue, CD11c is shown in red, and CD103 is shown in yellow. Scale bars, 100 μm. Images are representative of individual tumors from ≥5 mice per genotype. (I) Quantification of tissue-infiltrating CD103 DCs (CD11c⁺CD103⁺CD45circ⁻) in tumor (GFP⁺) areas. Each symbol represents an individual tumor. (J) Growth kinetics of Keap1 wild-type and mutant lung orthotopic tumors in C57BL/6J and Batf3^−/− female mice measured by bioluminescence. (K) Quantification of tumor burden (tumor area/total lung area) of Keap1 wild-type and mutant tumors in C57BL/6J and Batf3^−/− based on H&E staining. Scale bars, 2 mm. Each experimental subgroup had at least n = 6 mice. (L) Representative image of the LUAD tissue microarray. Scale bars, 1 mm. Three regions of interest per tumor sample were quantified. Pancytokeratin-positive staining is shown in green, negative is shown in magenta, and DAPI is shown in blue. (M) Boxplots showing expression of CD11c/HLA-DR and CD11C/HLA-DR/CD14/CD68/CD163 protein modules in KEAP1 wild-type (n = 19) and mutant (n = 19) patient tumor samples from tumor microarray containing LUAD patient samples. The microarray was stained and analyzed using the Nanostring GeoMx platform. Expression is shown for pancytokeratin-positive areas. (N) Boxplots showing expression for individual proteins: β-2-microglobulin and HLA-DR. Each symbol represents an individual mouse or human tumor core. *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 3.. Keap1-mutant tumors suppress CD8 T cell responses and promote T cell exhaustion

(A) UMAP of T cell subclusters identified by scRNA-seq, colored by cell type. (B) UMAP of the distribution of T cell subclusters in Keap1 wild-type (WT, blue) and mutant (MUT, red) lung tumors. (C) Bar plot showing distribution of T cell clusters in Keap1 wild-type and mutant mouse lung tumors. (D) Percentage of CD3, CD4, and CD8 T cells among tissue-infiltrating immune cells (for CD3) or of tissue-infiltrating TCRβ⁺ T cells in non-tumor lung (normal) and lung with Keap1 wild-type or mutant orthotopic tumors. Each symbol represents an individual mouse. Each experimental subgroup had n ≥ 4 mice. (E) Percentage of CD3, CD4, and CD8 T cells among tissue-infiltrating immune cells (for CD3) or of tissue-infiltrating TCRβ⁺ T cells in non-tumor lung (normal) and lung with Keap1 wild-type or mutant GEMM tumors. Each symbol represents an individual mouse. Each experimental subgroup had n ≥ 4 mice. (F) Percentage of CD69, PD1, CD44⁺CD62L⁻, or Ki67 among CD8⁺ T cells in wild-type and mutant Keap1 tumors. Each symbol represents an individual mouse. Each experimental subgroup had n ≥ 5 mice. (G) Percentage of intracellular IFN-γ- or TNF-α-positive cells among the CD8⁺ T lymphocytes in wild-type and mutant Keap1 tumors. Each symbol represents an individual mouse. Each experimental subgroup had n ≥ 5 mice. (H) Growth kinetics of Keap1 wild-type and mutant tumors measured by bioluminescence in female hosts upon CD8 T cell depletion. (I) Representative images of lung tumor burden and quantification by H&E staining. Scale bars, 2 mm. (J) Representative H&E (top) and immunofluorescence (bottom) images of KEAP1 wild-type and mutant human tumors. DAPI is shown in blue, CD3 in green, CD8 in red, Foxp3 in magenta, and PD1 in orange. Scale bars, 20 μm. Quantification of T cells (CD3⁺), CD8 T cells (CD8⁺), and PD1⁺ CD8 T cells (CD8⁺PD1⁺) in KEAP1 wild-type and mutant human tumors is shown at the bottom. Tumor area was identified based on H&E staining. *p < 0.05; **p < 0.01.

Figure 4.. Keap1 mutation drives immunotherapy resistance in antigenic Kras-driven lung adenocarcinoma mouse model

(A) Growth kinetics of Keap1 wild-type (WT, left) and mutant (MUT, right) KP cells injected intravenously in female C57BL/6J hosts upon treatment with anti-PD1 monoclonal antibody or isotype control. Each experimental subgroup had n ≥ 6 mice. (B) Representative images of lung tumor burden and quantification (tumor area/total lung area) by H&E staining of Keap1 wild-type and mutant orthotopic tumors treated with either isotype or α-PD1. Scale bars, 2 mm. Each experimental subgroup had n ≥ 6 mice. (C) Waterfall plots showing the percentage of tumor growth of Keap1 wild-type and mutant lung tumors treated with isotype control or anti-PD1. (D) Percentage of CD103 and CD11b DCs among tissue-infiltrating immune cells in the lungs of animals with Keap1 wild-type or mutant tumors treated with anti-PD1 or isotype control. (E) Percentage of CD3⁺ lymphocytes among tissue-infiltrating immune cells. Each experimental subgroup had n ≥ 5 mice. (F and G) Percentages of Ki67⁺ cells (F) or intracellular IFN-γ- and TNF-α-positive cells within the CD8 T cell gate in Keap1 wild-type and mutant tumor-bearing mice treated with anti-PD1 or isotype control. Each experimental subgroup had at least n = 5 mice. Each symbol represents an individual mouse. *p < 0.05; **p < 0.01.

Figure 5.. NRF2 pathway drives immune evasion in Keap1-mutant tumors, and glutamine modulation synergizes with ICB to reverse immunosuppression and immunotherapy resistance

(A) Growth kinetics of Nrf2-wild-type and Nrf2-deficient Keap1-mutant KP cells injected intravenously into female C57BL/6J hosts measured by bioluminescence. (B) Percentage of CD103 and CD11b DCs among tissue-infiltrating immune cells in the lungs of animals with wild-type or Nrf2-deficient Keap1-mutant tumors. (C) Percentage of CD3 lymphocytes among tissue-infiltrating immune cells and percentage of intracellular IFN-γ- and TNF-α-positive cells among the CD8 T lymphocytes. Each experimental subgroup had n ≥ 5 mice. (D) Activated OTI CD8 T cells were co-cultured with LucOS-expressing Keap1-mutant and wild-type KP cell lines in the presence of CB-839 (n = 3). After 24 h, the bioluminescent signal was measured and plotted normalized to the zero T cell condition. (E) Representative images of lung tumor burden and quantification by H&E staining of Keap1-mutant tumors treated with vehicle/isotype control, α-PD1, CB-839, or combination of α-PD1 and CB-839. Histological samples were collected from a sample other than those used for survival studies, and all tissues came from the same time point. Scale bars, 2 mm. (F) Survival of C57BL/6J female mice injected with Keap1-mutant KP cells and treated with vehicle/isotype control, α-PD1, CB-839, or a combination of α-PD1 and CB-839, with n ≥ 11. (G) Percentage of CD103 and CD11b DCs among tissue-infiltrating immune cells in the lungs of animals with Keap1-mutant tumors treated with vehicle/isotype control, α-PD1, CB-839, or a combination of α-PD1 and CB-839. (H) Percentage of CD3⁺ lymphocytes among tissue-infiltrating immune cells. (I) Percentage of intracellular IFN-γ- and TNF-α-positive cells among the CD8 T lymphocytes infiltrating the lungs of animals with Keap1-mutant tumors treated with vehicle/isotype control, α-PD1, CB-839, or a combination of α-PD1 and CB-839. Each symbol in the bar plots represents an individual mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.