Dysregulated gene expression of SUMO machinery components induces the resistance to anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes

Abstract

Background: The majority of patients with lung cancer exhibit drug resistance after anti-PD-1 immunotherapy, leading to shortened patient survival time. Previous studies have suggested an association between epigenetic abnormalities such as methylation and clinical response to anti-PD-1 immunotherapy, while the role of SUMOylation in resistance to anti-PD-1 antibody immunotherapy is still unclear.

Methods: Here, the mRNA expression of 15 SUMO machinery components in PBMC from lung cancer patients receiving anti-PD-1 immunotherapy were analyzed using real-time PCR. Base on the percentage change in mRNA levels, the relationship between the expression of SUMO machinery components and outcomes of anti-PD-1 immunotherapy, and the influencing factors of SUMOylation were evaluated. PBMC was treated with different concentrations of 2-D08 (a specific inhibitor of SUMOylation) in vitro, and analyzed the activation and the death rates of lymphocyte subsets by flow cytometry analysis.

Results: A predictive method, base on the gene expression of three SUMO machinery components (SUMO1, SUMO3 and UBE2I), were developed to distinguish non-responders to PD-1 inhibitors. Furthermore, the number of lymphocytes in peripheral blood significantly reduced in the dysregulated SUMOylation groups (the percentage change >100 or -50 ~ -100 groups). In vitro studies confirmed that lightly low SUMOylation level improved the activation status of T and NK lymphocytes, but extremely low SUMOylation level lead to the increased death rates of lymphocytes.

Conclusion: Our findings implied that dysregulated gene expression of SUMO machinery components could induce the resistance of anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes. These data might provide effective circulating biomarkers for predicting the efficacy of anti-PD-1 immunotherapy, and uncovered a novel regulatory mechanism of resistance to anti-PD-1 immunotherapy.

Keywords: PD-1; Sumoylation; lymphocyte; peripheral blood; resistance.

Figure 1

Dysregulated gene expression of SUMO machinery components in PBMC was associated with the prevalence rates of NR. (A–C) The relative mRNA expression of SUMO1 (A), SUMO3 (B) and UBE2I (C) in PBMC from responders (n=53) and non-responders (n=52). Data were expressed as mean ± 95%CI. (D–F) The percentage change in the relative mRNA expression of three SUMO machinery components (SUMO1, SUMO3 and UBE2I) was associated with the prevalence rates of NR.

Figure 2

The mRNA levels of three SUMO machinery components predicted clinical response to anti-PD-1 immunotherapy. ROC analysis based on SUMO1 (blue), SUMO3 (green), UBE2I (purple) and combined three genes (red) for the diagnosis of non-responders was shown.

Figure 3

Association between the percentage change in UBE2I mRNA level and different white blood cell populations in peripheral blood of lung cancer patients. (A–D) The absolute counts of white blood cell (WBC), neutrophils (NEU), monocytes (MON) and lymphocytes (LYM) were compared among different groups base on the percentage change of UBE2I. (E–H) The percentages of lymphocyte subsets were compared among different groups base on the percentage change of UBE2I. Student’s paired t-test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4

The relationship between the SUMOylation inhibition and the phenotype of lymphocyte. (A) Flow-cytometry dot plots showed the strategy for gating peripheral blood lymphocyte cells with CD69⁺ and Annexin V⁺ phenotype. (B-E) The expression of CD69 and Annexin V were analyzed on CD45⁺CD3⁺CD4⁺ T cells (B), CD45⁺CD3⁺CD8⁺ T cells (C), CD45⁺CD3^-CD56⁺ NK cells (D) and CD45⁺CD3^-CD19⁺ B cells (E). Student’s paired t-test, * P < 0.05, ** P < 0.01, *** P < 0.001.