KRAS-G12D mutation drives immune suppression and the primary resistance of anti-PD-1/PD-L1 immunotherapy in non-small cell lung cancer

Abstract

Background: Although immune checkpoint inhibitors (ICIs) against programmed cell death protein 1 (PD-1) and its ligand PD-L1 have demonstrated potency towards treating patients with non-small cell lung carcinoma (NSCLC), the potential association between Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene substitutions and the efficacy of ICIs remains unclear. In this study, we aimed to find point mutations in the KRAS gene resistant to ICIs and elucidate resistance mechanism.

Methods: The association between KRAS variant status and the efficacy of ICIs was explored with a clinical cohort (n = 74), and confirmed with a mouse model. In addition, the tumor immune microenvironment (TIME) of KRAS-mutant NSCLC, such as CD8⁺ tumor-infiltrating lymphocytes (TILs) and PD-L1 level, was investigated. Cell lines expressing classic KRAS substitutions were used to explore signaling pathway activation involved in the formation of TIME. Furthermore, interventions that improved TIME were developed to increase responsiveness to ICIs.

Results: We observed the inferior efficacy of ICIs in KRAS-G12D-mutant NSCLC. Based upon transcriptome data and immunostaining results from KRAS-mutant NSCLC, KRAS-G12D point mutation negatively correlated with PD-L1 level and secretion of chemokines CXCL10/CXCL11 that led to a decrease in CD8⁺ TILs, which in turn yielded an immunosuppressive TIME. The analysis of cell lines overexpressing classic KRAS substitutions further revealed that KRAS-G12D mutation suppressed PD-L1 level via the P70S6K/PI3K/AKT axis and reduced CXCL10/CXCL11 levels by down-regulating high mobility group protein A2 (HMGA2) level. Notably, paclitaxel, a chemotherapeutic agent, upregulated HMGA2 level, and in turn, stimulated the secretion of CXCL10/CXCL11. Moreover, PD-L1 blockade combined with paclitaxel significantly suppressed tumor growth compared with PD-L1 inhibitor monotherapy in a mouse model with KRAS-G12D-mutant lung adenocarcinoma. Further analyses revealed that the combined treatment significantly enhanced the recruitment of CD8⁺ TILs via the up-regulation of CXCL10/CXCL11 levels. Results of clinical study also revealed the superior efficacy of chemo-immunotherapy in patients with KRAS-G12D-mutant NSCLC compared with ICI monotherapy.

Conclusions: Our study elucidated the molecular mechanism by which KRAS-G12D mutation drives immunosuppression and enhances resistance of ICIs in NSCLC. Importantly, our findings demonstrate that ICIs in combination with chemotherapy may be more effective in patients with KRAS-G12D-mutant NSCLC.

Keywords: KRAS-G12D; PD-L1; chemo-immunotherapy; immunotherapy; non-small cell lung carcinoma; tumor-infiltrating lymphocyte.

FIGURE 1

Relationships of KRAS oncogene substitutions with the efficacy of immunotherapy in NSCLC. (A) Kaplan‐Meier survival curves of patients with NSCLC receiving anti‐PD‐1 monotherapy based on different subtypes of KRAS mutations concerning PFS. (B) Kaplan‐Meier survival curves of patients with NSCLC receiving anti‐PD‐1 monotherapy between the two subgroups (G12D and non‐G12D) concerning PFS. (C) Boxplots evaluating DCB of patients with NSCLC harboring different substitutions of KRAS mutations after anti‐PD‐1 monotherapy. (D) Boxplots evaluating DCB of patients with NSCLC between the two subgroups (G12D mutation and non‐G12D mutations). (E‐F) Western blotting (E) and flow cytometry (F) analysis of PD‐L1 protein level in normal lung cell line and NSCLC cell lines. (G) The tumoricidal activity of CD8⁺ T cells cocultured with five NSCLC cell lines harboring KRAS mutations after treatment with isotype control and nivolumab (200 μg/mL) for 48 h (The blue marker indicates the cells with KRAS‐G12D mutation, and the red indicates the cells without KRAS‐G12D mutation). Abbreviations: G12D, samples with KRAS‐G12D mutation; non‐G12D, samples without KRAS‐G12D mutation; DCB, patients with durable clinical benefit; non‐DCB, patients without durable clinical benefit; PFS, progression‐free survival.

FIGURE 2

Mouse models to study the association between PD‐L1 blockade efficacy and KRAS oncogene substitutions. (A) Scheme for constructing the mouse model with different subtypes of KRAS mutations and the dosing schedule. (B) The mice bearing LA795 cells with different subtypes of KRAS mutations received anti‐PD‐L1 monoclonal antibody (mAb) treatment. Anti‐PD‐L1 mAb did not reduce tumor growth significantly in the mice with KRAS‐G12D mutation compared to other subtypes of KRAS mutations (left lane). Representative images of tumor nodules in each treated group (right lane). (C–D) The distributions of IHC score of PD‐L1 level (C) and CD8⁺ T cells (D) in each treated group from B before and after PD‐L1 blockade, as well as the representative immunohistochemistry images. **, P < 0.01; ***, P < 0.001.Abbreviations: WT, mice without KRAS mutation; G12A, mice with KRAS‐G12A mutation; G12C, mice with KRAS‐G12C mutation; G12D, mice with KRAS‐G12D mutation; G12V, mice with KRAS‐G12V mutation; Pre‐ICIs, before using anti‐PD‐L1 monoclonal antibody; On‐ICIs, after using anti‐PD‐L1 monoclonal antibody; IHC, immunohistochemistry.

FIGURE 3

Tumor microenvironment and transcriptome data of KRAS‐mutant NSCLC patients with or without KRAS‐G12D point mutation. (A‐C) IHC analysis of PD‐L1 level (A), CD8⁺ TILs (B), and tumor microenvironment based on PD‐L1 and CD8⁺ TILs (C) according to KRAS oncogene substitutions in a cohort of 112 NSCLC specimens with KRAS mutations. (D) Volcano plot of significantly down‐ and up‐regulated genes in LUAD samples with KRAS‐G12D mutation. (E) The distributions of remarkably dysregulated immune‐related genes among the five classes based on immune response. (F‐G) GO analysis (F) and KEGG analysis (G) of the biological pathways enriched for significantly dysregulated immune‐related genes. (H) Heatmap plot of significantly dysregulated immune‐related genes. (I) IHC analysis of protein levels of CXCL10 and CXCL11 in LUAD samples with or without KRAS‐G12D mutation. Abbreviations: G12D, samples with KRAS‐G12D mutation; non‐G12D, samples without KRAS‐G12D mutation; TIL, tumor infiltrating lymphocyte; GO, Gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; IHC, immunohistochemistry.

FIGURE 4

Down‐regulation of PD‐L1, CXCL10, CXCL11, and HMGA2 levels in LUAD samples with KRAS‐G12D mutation. (A) The sequence of Beas‐2B cells confirmed the presence of wild‐type KRAS gene. (B) Western blotting analyzed the KRAS protein level in Beas‐2B cells with heterozygous KRAS gene knockout. (C) Western blotting analysis of flag, KRAS, and KRAS‐G12D protein levels in Beas‐2B cells stably transfected with empty vector (VEC), vector coding for wild‐type KRAS (WT), mutant KRAS‐Gly12Ala (G12A), mutant KRAS‐Gly12Cys (G12C), mutant KRAS‐Gly12Asp (G12D), or mutant KRAS‐Gly12Val (G12V). (D) KEGG analysis of the biological pathways enriched for significantly dysregulated genes in cell lines with the KRAS‐G12D mutation compared to other substitutions of the KRAS gene. (E‐F) The conjoint analysis (E) and heatmap plot (F) of significantly dysregulated genes in LUAD samples and cell lines with KRAS‐G12D mutation compared with other KRAS mutations. (G) Cross‐correlogram based on Pearson's r values among PD‐L1, CXCL10, CXCL11 and the significantly dysregulated genes in LUAD samples with KRAS mutations. (H) Cross‐correlogram based on Pearson's r values among HMGA2 level and infiltration of six tumor‐infiltrated immune cells in LUAD samples with KRAS mutations via TIMER. (I) IHC analysis of correlations among levels of PD‐L1, CXCL10, CXCL11, and HMGA2 and infiltration of CD8⁺ T cells in LUAD samples with KRAS mutations. (J) IHC analysis of HMGA2 protein level in LUAD samples with KRAS mutation. (K) Western blotting analysis of protein levels of PD‐L1, CXCL10, CXCL11, and HMGA2 in Beas‐2B cells with KRAS mutation. (L) Western blotting analysis of protein levels of PD‐L1, CXCL10 and CXCL11 in control cells or HMGA2‐overexpressing Beas‐2B‐G12D, SK‐LU‐1, and A427 cells. Abbreviations: KO, knockout; NC, normal control; G12D, samples with KRAS‐G12D mutation; non‐G12D, samples without KRAS‐G12D mutation; IHC, immunohistochemistry; H‐VEC, cells transfected with empty vector; H‐OE, cells transfected with vector coding for HMGA2.

FIGURE 5

KRAS‐G12D mutation suppressed PD‐L1 protein level via the P70S6K/PI3K/AKT axis. (A) Western blotting analysis of PD‐L1 and kinase signaling pathways in Beas‐2B cells with KRAS mutation. (B) Western blotting analysis of protein levels of PD‐L1, CXCL10, CXCL11, and HMGA2 and kinase signaling pathway in cell lines with KRAS mutation after treatment with the mTOR inhibitor rapamycin (0.5 μmol/L) for 24 h. Abbreviations: G12A, Beas‐2B cells stably transfected with vector coding for mutant KRAS‐Gly12Ala (G12A); G12C, Beas‐2B cells stably transfected with vector coding for mutant KRAS‐Gly12Cys (G12C); G12D, Beas‐2B cells stably transfected with vector coding for mutant KRAS‐Gly12Asp (G12D); G12V, Beas‐2B cells stably transfected with vector coding for mutant KRAS‐Gly12Val (G12V).

FIGURE 6

Paclitaxel upregulated protein levels of chemokines CXCL10/CXCL11 via HMGA2. Western blotting analysis of protein levels of CXCL10, CXCL11 and HMGA2 in Beas‐2B‐G12D (A), SK‐LU‐1 (B), A427 (C) cells, and KRAS‐G12D‐mutant organoids (D) treated with paclitaxel. Abbreviations: Beas‐2B‐G12D, Beas‐2B cells stably transfected with vector coding for mutant KRAS‐Gly12Asp (G12D); PTX: paclitaxel; PDO‐G12D, a patient‐derived organoid model with KRAS‐G12D mutation.

FIGURE 7

PD‐L1 blockade combined with paclitaxel elicited synergistic anti‐tumor immune responses in the KRAS^G12D‐mutant mouse model. (A) Scheme for constructing a mouse model with the KRAS‐G12D mutation and dosing schedule. (B) The mice bearing LA795 cells with KRAS‐G12D mutation (n = 5) received treatment with paclitaxel and/or anti‐PD‐L1 monoclonal antibody (mAb). Control mice were treated with vehicle control. The combination of anti‐PD‐L1 mAb and paclitaxel significantly weakened tumor growth compared to the other treated group in KRAS^G12D‐mutant mice. (C) Representative images of tumor nodules in each treated group from B.(D‐G) The distributions of IHC score of PD‐L1 (D), CD8⁺ T cell (E), IHC scores of CXCL10 (F) and CXCL11 (G) in each treated group, as wells as the representative IHC images. *, P > 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Abbreviations: IHC, immunohistochemistry.

FIGURE 8

Patients with KRAS‐G12D mutations showed favorable clinical benefits from chemo‐immunotherapy. (A‐B) Boxplots evaluating tumor response (A) and durable clinical benefit (B) of patients with NSCLC harboring KRAS‐G12D mutations after PD‐1/PD‐L1 inhibitor monotherapy or chemo‐immunotherapy. (C‐D) Kaplan‐Meier survival curves of patients with NSCLC harboring KRAS‐G12D mutations after initiation of PD‐1/PD‐L1 blockade monotherapy treatment or chemo‐immunotherapy concerning progression‐free survival (C) and overall survival (D). Abbreviations: ICIs, immune checkpoint inhibitors; ICIs+Chemo, chemo‐immunotherapy; PR, patients with partial response; SD, patients with stable disease; PD, patients with progressive disease; DCB, patients with durable clinical benefit; non‐DCB, patients with no durable clinical benefit; PFS, progression‐free survival; OS, overall survival.