The LDH-H3K18La-Nur77 Axis Potentiates Immune Escape in Small Cell Lung Cancer

Abstract

Small cell lung cancer (SCLC) remains a therapeutic challenge due to its aggressive nature and limited response to immunotherapy. This study identifies lactate-induced histone lactylation as a novel epigenetic mechanism in SCLC, contributing to immune escape and poor therapeutic outcomes. By identifying the LDH-H3K18La-Nur77 axis, new insights into the metabolic regulation of immune responses in SCLC are offered. Multi-omics analysis, including metabolomics and TCR sequencing, is used to compare serum samples and immune cell profiles from SCLC patients. Data from Shanzhong cohort (n = 222), along with a validation cohort from the IMpower133 study (n = 264), demonstrates that LDH levels are associated with poorer outcomes following immunotherapy. ChIP-qPCR and luciferase reporter assays reveal that lactate-induced histone lactylation at H3K18La induces transcriptional activation of Nur77 in naïve CD8⁺ T cells, leading to tonic TCR signaling, which impairs antigen recognition and prevents effective anti-tumor activity. In preclinical SCLC models, lactate inhibition reduces Nur77 expression, restores T cell function, and enhances the efficacy of PD-1 blockade, leading to a decreased tumor burden and improved survival. This study uncovers a novel mechanism of immune escape in SCLC through lactate-driven histone lactylation and provides the first evidence that targeting lactate metabolism can enhance immunotherapy effectiveness.

Keywords: H3K18La; Nur77; SCLC; lactate; naïve CD8+T cells.

Figure 1

Metabolomic and prognostic significance of lactate and LDH in SCLC patients. a) A principal component analysis (PCA) of serum‐targeted metabolomics data comparing small cell lung cancer (SCLC) patients and healthy donors (HD). b) Volcano plot of the detected metabolites in serum metabolomics (SCLC patients versus HD). Significantly differential metabolites are colored in red (upregulated) and green (downregulated); the others are colored in gray. c) The VIP score of identified differential metabolites. d) Violin plots comparing metabolite levels between SCLC patients and HD. e) Kaplan‐Meier survival curves showing the overall survival (OS) of extensive‐stage small cell lung cancer (ES‐SCLC) patients from Shandong Cancer Hospital and Institute (Shanzhong) cohort treated with immune checkpoint inhibitors (ICIs) stratified by serum LDH levels. f) Kaplan‐Meier survival curves showing the OS of ES‐SCLC patients from IMpower133 cohort treated with atezolizumab (Atezo) or with non‐Atezo stratified by serum LDH levels.

Figure 2

Extensive tonic TCR signaling and naïve CD8⁺ T cell responsiveness negatively correlate with elevated LDH levels. a) CIBERSORT analysis of RNA‐seq data from the IMpower133 cohort. b) Schematic workflow of TCR sequencing (TCRseq) performed on peripheral blood mononuclear cells (PBMCs) from 24 SCLC patients. c) The correlation between LDH expression and the TCR singleton index. d) Flow cytometry analysis of Nur77 and TCR expression in naïve CD8⁺ T cells after exposure to conditioned medium (CM) from H446 with or without lactate. e,f) Western blotting and RT‐qPCR analysis of the Nur77 levels in naïve CD8⁺ T cells after exposure to CM from H446 with or without lactate. g) Histograms show expression of the indicated activation markers of cells stimulated for 24 h with anti‐CD3 (0.25 µg mL⁻¹) and APCs in CD8⁺ T cells after exposure to CM from H446 with or without lactate. Cells were gated on viable CD8⁺ T cells. h,i) Representative Contour plots and violin diagrams show the percentage of proliferated CFSE‐labeled T cells after exposure to CM from H446 with or without lactate. j,k) Contour plots and violin diagrams depict the secretion of IFN‐γ and TNF‐α by CD8⁺ T cells after exposure to CM from H446 with or without lactate. Data, mean ± S.E.M. of three independent experiments. n.s., not significant.

Figure 3

Effects of lactate inhibition on Nur77 expression and functional reprogramming of naïve CD8⁺ T cells in SCLC. a) Flow cytometry analysis of Nur77 and TCR expression in naïve CD8⁺ T cells after exposure to CM from H446 with or without lactate inhibitor (LAi). b) Violin diagrams display the mean fluorescence intensity (MFI) for Nur77 and TCR. c,d) Western blotting and RT‐qPCR analysis of the Nur77 protein levels in naïve CD8⁺ T cells after exposure to CM from H446 with or without LAi. e,f) Representative histograms and scatter graphs show the percentage of proliferated CFSE‐labeled T cells after exposure to CM from H446 with or without LAi. g) Histograms show expression of the indicated activation markers of cells stimulated for 24 h with anti‐CD3 (0.25 µg mL⁻¹) and APCs in CD8⁺ T cells after exposure to CM from H446 with or without LAi. Cells were gated on viable CD8⁺ T cells. h) Violin diagrams display the MFI for CD69 and CD25. i,j) Contour plots and violin diagrams depict the secretion of IFN‐γ and TNF‐α by CD8⁺ T cells after exposure to CM from H446 with or without LAi. Data, mean ± SEM of three independent experiments. n.s., not significant.

Figure 4

Therapeutic effects of combining lactate inhibition with αPD‐1 therapy in an orthotopic SCLC mouse model. a) Schematic representation of the four experimental groups: control, αPD‐1, LAi, and combination of αPD‐1 and LAi. b) H&E staining of the orthotopic lung tumor model in mice. c) In vivo bioluminescent images of four groups at the indicated time points. d,e) Bioluminescence intensity was measured at 5 days post‐injection of luciferase‐expressing KP3 cells into mouse lungs, comparing four different treatment groups. f) Comparison of tumor weight across treatment groups. g) Comparison of body weight in mice across four groups. h) Kaplan‐Meier survival analysis of the mice in different treatment groups. i) The mediastinal lymph nodes were analyzed by flow cytometry for naïve CD8⁺ T cells (CD44^lowCD62L^high). Cells were gated on viable CD8⁺ T cells. j) The mediastinal lymph nodes were analyzed by flow cytometry for Nur77 expression on naïve CD8⁺ T cells. k,l) Orthotopic tumors were analyzed by flow cytometry for IFN‐γ and TNF‐α expression on CD8⁺ T cells. Scatter graphs represent the means ± S.E.M. for each group. c–k) n = 8 for each group. One‐way ANOVA statistical tests were adopted for more than two groups. Log‐rank tests were used in h. n.s., not significant.

Figure 5

Role of histone lactylation in regulating Nur77 expression and its clinical implications in SCLC. a) IGV tracks for Nur77 from ChIP‐seq were shown. b) ChIP‐qPCR was conducted on naïve CD8⁺ T cells treated with LA (0 mm, 10 mm) for 24 h. c) Deletion analysis identified H3K18La‐enrichment region in the Nur77 promoter (left panel), along with the relative luciferase activity measured (right panel). d) Western blotting analysis of PanKla, H3K18La and Nur77 levels in naïve CD8⁺ T cells under H446, H69 CM or LA conditions. e) Western blotting analysis of PanKla, H3K18La and Nur77 levels in naïve CD8⁺ T cells under H446, H69 CM or LAi conditions. f) Representative images of Immunofluorescence staining for Nur77 expression in lymph nodes of ES‐SCLC patients (n = 10) stratified by LDH. Scale bars: 40 µm. g) Representative images of IHC staining for H3K18La expression in lymph nodes of ES‐SCLC patients (n = 10) stratified by LDH. Scale bars: 50 µm. h,i) The OS curves of ES‐SCLC patients (n = 10) from Shandong Cancer Hospital with low and high Nur77 g) or H3K18La h) levels were generated using Kaplan‐Meier method and the log‐rank test.

Figure 6

NR4A1 ablation remodels the immunosuppressive microenvironment to enhance anti‐PD‐1 efficacy in SCLC. a) Schematic representation of the experimental design: Nr4a1 ^−/− and Nr4a1 ^+/+ mice treated with αPD‐1 or IgG2a isotype control. b) In vivo bioluminescent images of mice from different treatment groups. c) Quantitative analysis of bioluminescence intensity after injection of luciferase‐expressing KP3 cells into the lungs. d) Representative images of lung orthotopic tumors in four groups. e) Kaplan‐Meier survival analysis of the mice in different treatment groups. f) Flow cytometry analysis of naïve CD8⁺ T cells and Nur77⁺naïve CD8⁺ T cells in mediastinal lymph nodes of mice. g) Scatter graphs show the frequencies of naïve CD8⁺ T cells and Nur77⁺naïve CD8⁺ T cells in mediastinal lymph nodes of mice from different treatment groups. h) Flow cytometry analysis of IFN‐γ and TNF‐α expression on CD8⁺ T cells in tumor tissues of mice. i) Scatter graphs show the frequencies of IFN‐γ, TNF‐α‐secreting cells in orthotopic tumors. j) Representative images of Immunofluorescence staining for CD8⁺CD25⁺ cells in tumor tissues of mice from different treatment groups. CD8: white, CD25: green, Scale bars, 50 µm. k) Representative images of IHC staining of Nur77 and H3K18La in tumor tissues of mice from different treatment groups. Scale bars, 50 µm. l) Quantitative analysis of Nur77 and H3K18La expression. b–j) n = 8 for each group. One‐way ANOVA statistical tests were adopted for more than two groups. Log‐rank tests were used in e. n.s., not significant.

Figure 7

The LDH‐Nur77 axis modulates immune checkpoint therapy efficacy in ES‐SCLC. a) Flow cytometry analysis shows the number of naïve CD8⁺ T cells (CD45RA⁺ CCR7⁺) in the peripheral blood (PB) of ES‐SCLC patients stratified by LDH levels. b,c) Histograms and graph bars show expression of TCR and Nur77 on naïve CD8⁺ T cells in the PB of ES‐SCLC patients. d) Contour plots and graph bars depict the secretion of IFN‐γ and TNF‐α by CD8⁺ T cells in the PB of ES‐SCLC patients. e) Representative images of multiplex immunohistochemistry (mIF) staining of CD8, CD45RA, CCR7, TCR and Nur77 expression in lymph nodes between ES‐SCLC patients with low‐ and high‐LDH level. Scale bars, 70 µm, left panel; 50 µm, right panel. f) Two ES‐SCLC patients treated with anti‐PD‐1 therapy representing a responder (Pt.8) and non‐responder (Pt.12) case were analyzed. Tumor diameter based on the CT imaging was annotated by the radiologist with a red line. g) The difference in tumor diameter for 18 patients from Qilu Hospital of Shandong University, where those with increased tumor diameter are colored by red. h) Quantitative correlation between the change in tumor diameter and LDH levels. The correlation coefficient and the p values were computed based on Pearson method. n.s., not significant.

Figure 8

Model of the proposed mechanism of LDH‐H3K18La‐Nur77 axis. SCLC tumor‐derived lactate initiates a signaling cascade in naïve CD8⁺ T cells resulting in Histone H3K18 lactylation dependent up‐regulation of Nur77 expression, inducing increased levels of tonic TCR signaling. These naïve CD8⁺ T cells were hyporesponsive to agonist TCR stimulation, inhibiting antitumor T cell activity, and thus limits the efficacy of immunotherapy. Inhibition of lactate with lactate inhibitor will overcome lactate‐mediated immunosuppression and sensitize tumors to immunotherapy.