Fig 2: eEF2K inhibition synergistically enhanced the therapeutic efficacy of PD-1 blockade in vivo. (A–E) C57BL/6 mice were implanted with B16F10 cells and co-treated with NH125 and PD-1 mAb. (A) A schematic view of the treatment protocol. (B) Photopraphs of B16F10 tumors harvested after euthanizing the mice. n=6 for each group. (C) The tumor growth of B16F10 cells in NH125 and/or anti-PD-1 antibody-treated C57BL/6 mice. (D) Plots for tumor weight. (E) CD8α and granzyme B in mouse tumor tissues of each group were determined by immunofluorescence. (F–I) C57BL/6 mice were implanted with Ctrl or sheEF2K-transfected B16F10 cells, and received PD-1, CD8α mAb treatment or IgG isotype control. (F) A schematic view of the treatment plan. (G) Photopraphs of mice tumors of each group at the end of the experiment. (H) Curves of tumor growth. (I) Plots for tumor weight. ∗∗P<0.01; ∗∗∗p<0.001. (J) A proposed model for eEF2K-induced GSK3β-phosphorylation-dependent PD-L1 stabilization and immunoregulation in melanoma. eEF2K overexpression in cancer cells phosphorylates GSK3β at serine 9 for GSK3β inactivation, leading to PD-L1 stabilization, enhanced PD-1 interaction and subsequent immunosuppressive microenvironment as a consequent. Therapeutic depletion or inhibition of eEF2K maintains GSK3β activity for phosphorylation-dependent proteasome degradation of PD-L1, thereby decreasing the cancer cells PD-L1 expression level and synergistically enhancing the therapeutic efficacy of PD-1 mAb therapy. eEF2K, eukaryotic elongation factor 2 kinase; GSK3β, glycogen synthase kinase 3 beta; mAb, monoclonal antibody; PD-1, programmed cell death protein-1; PD-L1, programmed death ligand-1.

Fig 3: Inhibitory effects of C1 on TNBC cells are dependent on eEF2K expression. A) The relative eEF2K mRNA expression in different TNBC cell lines was determined by qRT‐PCR. B) The expressions of eEF2K protein in different TNBC cell lines was determined by western blotting. GAPDH was used as a loading control. C) Anti‐proliferation effects of C1 in different TNBC cell lines. D) Western blotting was employed to examine eEF2K expression in non‐target (shNT) or stable eEF2K knockdown (sheEF2K 1#, 2#) MDA‐MB‐231, and HCC1806 cells. E) Effects of C1 and sheEF2K on proliferation of MDA‐MB‐231 and HCC1806 cells. F) CCK‐8 assay for the cell viability of control or eEF2K‐KD cells treated with C1. The results are displayed as means ± SDs (n = 3). *** p < 0.001, t‐test. G) Colony‐formation assay for proliferation of control and eEF2K‐KD cells treated with C1. The results are displayed as means ± SDs (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. n.s., no significant, t‐test.

Fig 4: Depletion of eEF2K suppresses B16F10 xenograft tumor growth and promotes T cell activity. Ctrl and two sheEF2K (#1 and #2)-transfected B16F10 cells were injected subcutaneously into 6-week-old male Balb/c nude mice and C57BL/6 mice, the tumor sizes were measured on the days as indicated. (A, B) Subcutaneous tumors from B16F10 xenograft Balb/c nude mice were excised and photographs were taken at the termination of the experiment (A) and tumor weights were measured (B). Tumor inhibition rate (%, TIR), 67.5%. (C, D) Subcutaneous tumors from B16F10 xenograft C57BL/6 mice were excised and photopraphs were taken at the termination of the experiment (C) and xenograft tumor weights were measured (D). TIR, 80.7%. Data represents the mean±SD of tumor weights of each group (n=6). (E) FACS of CD8+ in CD3+ and GZMB+CD8+ in CD8+ TILs from B16F10 xenografts. (F) Representative images of IF staining of eEF2K, p-GSK3β/S9 and PD-L1 of sheEF2K and Ctrl B16F10 xenografts. (G–K) ShNT and sheEF2K-transfected B16F10 cells were injected subcutaneously into 6-week-old male C57BL/6 mice, and received CD8α mAb treatment or IgG isotype control. (G) A schematic view of the treatment plan. (H) Photopraphs of mice tumors of each group at the end of the experiment. (I) Curves of tumor growth. (J) Plots for tumor weight. (K) FACS of CD8+ in CD3+ TILs from B16F10 xenografts. ∗P<0.05; ∗∗p<0.01; ∗∗∗p<0.001. eEF2K, eukaryotic elongation factor 2 kinase; GSK3β, glycogen synthase kinase 3 beta; IF, immunofluorescence; PD-L1, programmed death ligand-1; mAb, monoclonal antibody; TIL, tumor-infiltrating lymphocyte.

Fig 5: C1 promotes the degradation of eEF2K. A) Western blotting was used to detect the expression of eEF2K in MDA‐MB‐231, HCC1806, and BT549 cells treated with C1 at 0, 0.05, 0.1, 0.2, 0.4, and 0.8 µm for 48 h. GAPDH was used as a loading control. Relative eEF2K protein levels were shown as the means ± SDs, * p < 0.05, ** p < 0.01, *** p < 0.001, t‐test. B) MDA‐MB‐231 and HCC1806 cells were stimulated with 0.2 µm C1 for 48 h with or without 20 µm MG132, and eEF2K levels were assessed by western blotting. GAPDH was used as a loading control. C) MDA‐MB‐231 and HCC1806 cells were stimulated with 0.2 µm C1 for 48 h in the presence or absence of 20 µg mL−1 CHX. eEF2K levels were assessed by western blotting. GAPDH was used as a loading control.