Targeting oxidative phosphorylation to increase the efficacy of immune-combination therapy in renal cell carcinoma

Abstract

Background: Immune checkpoint inhibitors (ICIs) are the standard of care for metastatic renal cell carcinoma (RCC); however, most patients develop de novo or acquired resistance to ICIs. Oxidative phosphorylation (OXPHOS) has been rarely explored as a potential target for correcting ICI resistance.

Methods: We systematically analyzed RNA sequencing and clinical data from CheckMate, JAVELIN Renal 101, and NCT01358721 clinical trials, and clinicopathological data of 25 patients from Tongji Hospital to investigate the relationship between OXPHOS and ICI resistance. The Ndufb8-knockdown Renca cell line was derived to determine the effect of OXPHOS on RCC immunotherapy in vivo.

Results: An analysis of the CheckMate series data revealed that high OXPHOS levels are risk factors for ICI in patients with RCC, but are affected by thevon Hippel-Lindau protein (VHL) and hypoxia-inducible factor-1α status. This result is consistent with correlation between clinicopathological characteristics and prognostic observations at our institute. Knockdown of the mitochondrial complex I subunit Ndufb8 of the Renca cell line had no effect on cell growth and migration in vitro, but slowed down cell growth in vivo. Among anti-programmed death ligand 1 (PD-L1)-treated BALB/c mice, shNdufb8 Renca tumors grew slower than shControl Renca tumors and the corresponding mice survived longer. Flow cytometry revealed that CD8⁺ T cells in shNdufb8 Renca tumors, which were exposed to a lower degree of hypoxia and expressed less programmed death-1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3), secreted more interferon-γ after stimulation. Immunofluorescence demonstrated that the shNdufb8 Renca tumors had a higher proportion of CD8⁺ T cells and the proportion of these cells was lower in the hypoxic area.

Conclusions: OXPHOS is a reliable predictor of immunotherapy response in RCC and is more pronounced in metastatic lesions. RCC cells generate a hypoxic tumor microenvironment and inhibit T-cell function through oxidative metabolism, thereby leading to immunotherapy resistance.

Keywords: Immunotherapy; Metabolic Networks and Pathways; Renal Cell Carcinoma.

Figure 1

OXPHOS predicts response and PFS in ICB-treated renal cell carcinoma cases in CheckMate studies. (A) GSEA of differentially expressed genes in PD and SD cases when compared with PR and CR cases indicated that the OXPHOS pathway was significantly upregulated. (B) The OXPHOS pathway enrichment score display (p<0.001). (C) High OXPHOS levels were associated with poorer PFS in the nivolumab arm (p=0.039). (D) OXPHOS were not associated with PFS in the everolimus arm (p=0.088). (E) High OXPHOS were associated with poorer OS in the nivolumab arm (p=0.038). (F) OXPHOS were not associated with OS in the everolimus arm (p=0.59). CR, complete response; GSEA, gene set enrichment analysis; ICB, immune checkpoint blockade; OS, overall survival; OXPHOS, oxidative phosphorylation; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease.

Figure 2

The OXPHOS predicts PFS in each group of NCT01358721. (A) Higher OXPHOS in the TKI-resistant arms (arms A, B, and C) were associated with poorer PFS (p=0.045). (B) OXPHOS were not associated with PFS in the TKI-naïve arm (arm D) (p=0.077). (C) Higher OXPHOS in the TKI-resistant arms (arms B and C) were associated with poorer PFS (p=0.0088). OXPHOS, oxidative phosphorylation; PFS, progression-free survival; TKI, tyrosine kinase inhibitors.

Figure 3

Expression of OXPHOS-related protein in different objective-responses groups of Tongji cases. A significant difference was noted in the objective response rate between the high and low expression cases of OXPHOS-related proteins, and the expression was negatively correlated with CD8 and PD-L1 (magnification: 10×20). OXPHOS, oxidative phosphorylation; PD, progressive disease; PD-L1, programmed death ligand 1; PR, partial response; SD, stable disease.

Figure 4

Prediction of objective response rate and PFS by immunohistochemistry grouping of Tongji cases. (A) PFS was significantly worse in the group with a high expression of OXPHOS-related proteins, for example, NDUFB8 (p=0.0033). (B) The proportion of PR patients in the NDUFB8 high-expression group was significantly reduced (p=0.005, Fisher’s exact test). (C) The PFS of the PD-L1 high-expression group was better than that of the low-expression group, although the difference was insignificant (p=0.12). (D) No significant difference was noted in the objective response rate between the PD-L1 high and low expression groups (p=0.593). (E) The PFS of the CD8 high-expression group was significantly better than that of the low-expression group (p=0.0023). (F) The proportion of PR patients in the CD8 high-expression group was significantly higher than that in the low-expression group (p=0.005). OXPHOS, oxidative phosphorylation; PD, progressive disease; PD-L1, programmed death ligand 1; PFS, progression-free survival; PR, partial response; SD, stable disease.

Figure 5

Knockdown of NDUFB8 does not affect the proliferation and migration of RENCA. (A) RNA-knockdown efficiency was tested by qPCR. The knockdown efficiency of shNdufb8 1 was 70%, while that of shNdufb8 2 was 56%. (B) Western blotting was performed to test the knockdown efficiency at the protein level. The knockdown efficiency of shNdufb8 1 was found to be 61%, while that of shNdufb8 2 was 22%. After Ndufb8 was knocked down, the expression of B2m, a constituent molecule of antigen major histocompatibility complex I, was significantly downregulated. (C) The shNdufb8 Renca cell line showed less HIF-1α accumulation than shControl Renca after 8 hours of culture in a sealed environment. (D) The OD 450 after CCK-8 addition was measured at 24 hours and 48 hours, respectively, and there was no statistically significant difference in the proliferation of shNdufb8 and shControl Renca cells at both times. (E) The colony-formation experiment performed to compare the proliferation ability of shNdufb8 and shControl Renca cells revealed that the number of colonies formed by shNdufb8 1 Renca was slightly more than that of shControl Renca cells, although there was no statistically significant difference in the size of the colonies between the two cells. (F) Edu detects the proliferative ability. Edu and Hoechst 33342 mark the replicating DNA molecules and double-stranded DNA (nuclei), respectively, that is, mark proliferating cells and all cells. No significant difference was noted in the proportion of proliferating cells between shNdufb8 1 Renca and shControl Renca (magnification: ×200). (G) The cell scratch assay compared the migration ability of shNdufb81 and shControl Renca cells, with no statistically significant difference in the 72 hours migration rate between the two cells. B2m, beta-2 microglobulin; HIF-1α, hypoxia-inducible factor-1α; qPCR, quantitative PCR.

Figure 6

Ndufb8 knocking down increases the efficacy of immune checkpoint inhibitor in renal cell carcinoma in vivo. Tumor growth curves of the four groups of mice (A–D). (E) A comprehensive graph of tumor growth curve revealed that the growth rate of shNdufb8+IgG group was significantly slower than that of shControl+IgG (p<0.001), that of the shControl+anti-PD-L1 group was significantly slower than that of the shControl+IgG group (p<0.001), while that of the shNdufb8+anti-PD-L1 group was significantly slower than that of the shControl+anti-PD-L1 group (p<0.05). The growth velocity of the shNdufb8+anti-PD-L1 group was significantly slower than that of the shNdufb8+IgG group (p<0.001). Two-way analysis of variance tests were performed using GraphPad Prism V.9.0.0. (F) The survival of the mice in the shNdufb8+IgG group was significantly better than that in the shControl+IgG group (p=0.0027). (G) The survival of the mice in the shControl+anti-PD-L1 group was significantly better than that in the shControl+IgG group (p=0.0027). (H) The survival of mice in the shNdufb8+anti-PD-L1 group was significantly better than that in the shControl+anti-PD-L1 group (p=0.0048). (I) The survival of mice in the shNdufb8+anti-PD-L1 group was significantly better than that in the shNdufb8+IgG group (p<0.001). (J) The proportion of positive hypoxyprobe in the shNdufb8 group was significantly lower than that in the shControl group (p<0.05), while the proportion of tumor-infiltrating CD8⁺ T cells positive for IFN-γ was significantly higher (p<0.05). (K) The proportion of PD-1⁺ and TIM-3⁺ positive tumor-infiltrating CD8+T cells in the shNdufb8 group was significantly lower than that in the shControl group (p<0.05). (L) Immunofluorescence revealed that tumor-infiltrating CD8⁺T lymphocytes (red fluorescence) in the shNdufb8 group were significantly more than those in the shControl group. The infiltrated CD8⁺ T cells (red fluorescence) in the hypoxic area (green fluorescence) in the shNdufb8 group were significantly lower than those in the non-hypoxic area. *p<0.05, **p<0.01. DAPI, 4′,6-diamidino-2-phenylindole; IFN, interferon; PD-L1, PD-1, rogrammed death protein 1; pPD-L1, programmed death ligand 1; T-cell immunoglobulin domain and mucin domain 3.