AMPK-dependent Parkin activation suppresses macrophage antigen presentation to promote tumor progression

Abstract

The constrained cross-talk between myeloid cells and T cells in the tumor immune microenvironment (TIME) restricts cancer immunotherapy efficacy, whereas the underlying mechanism remains elusive. Parkin, an E3 ubiquitin ligase renowned for mitochondrial quality control, has emerged as a regulator of immune response. Here, we show that both systemic and macrophage-specific ablations of Parkin in mice lead to attenuated tumor progression and prolonged mouse survival. By single-cell RNA-seq and flow cytometry, we demonstrate that Parkin deficiency reshapes the TIME through activating both innate and adaptive immunities to control tumor progression and recurrence. Mechanistically, Parkin activation by AMP-activated protein kinase rather than PTEN-induced kinase 1 mediated major histocompatibility complex I down-regulation on macrophages via Autophagy related 5-dependent autophagy. Furthermore, Parkin deletion synergizes with immune checkpoint blockade treatment and Park2^-/- signature aids in predicting the prognosis of patients with solid tumor. Our findings uncover Parkin's involvement in suppressing macrophage antigen presentation for coordinating the cross-talk between macrophages and T cells.

Fig. 1.. Loss of Parkin in mice leads to reduced tumor burden in multiple cancers.

(A and B) Tumor volume (A) and survival (B) of WT (n = 9) and Park2^−/− (n = 9) mice bearing subcutaneous MC38 tumors. (C and D) Tumor volume (C) and survival (D) of WT (n = 16) and Park2^−/− (n = 17) mice bearing subcutaneous Hepa1-6 tumors. (E) Survival analysis of WT (n = 8) and Park2^−/− (n = 10) mice in an in situ model of hepatocellular carcinoma. (F) Representative images of hepatocellular carcinoma tumors. (G) Plot showing the overall survival (OS) of WT (n = 12) and Park2^−/− (n = 10) mice intracranially inoculated with CT2A glioma cells. GBM, glioblastoma multiforme. (H) Representative histology of murine brains with intracranial tumors (tumor indicated by arrows). Images are representative of at least three images for one indicated animal. Scale bars, 2 cm. (I) t-distributed stochastic neighbor embedding (t-SNE) plot of 40,091 cells from WT or Park2^−/− mice bearing MC38 tumors (pooled cells of two mice per group) analyzed by single-cell RNA sequencing (scRNA-seq). DCs, dendritic cells. (J) Proportions of major cell types in (I). (K) Percentages of T cells, CD8⁺ T cells, and CD4⁺ T cells in subcutaneous MC38 tumors in WT (n = 5) or Park2^−/− (n = 4) mice. (L and M) IL-2 (L) and IFN-γ (M) in tumor interstitial fluids of WT (n = 7) and Park2^−/− (n = 8) mice. (N to P) Tumor volumes of WT and Park2^−/− MC38–bearing mice treated with (N) phosphate-buffered saline (PBS) and (O) anti-CD4 and (P) anti-CD8 antibodies. The numbers of mice are indicated. The results are depicted as the means ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Fig. 2.. Parkin deficiency leads to greater antitumor activity of TILs.

(A) Annotated Uniform Manifold Approximation and Projection (UMAP) plot of T cells from tumors in WT and Park2^−/− mice. T_H1, T helper cell 1; T_H17, T helper cell 17; IST, Interferon-response CD4⁺ T cell. (B) Percentages of early activated_CD8, effector_CD8, Ifng_CD8, and T_regs in WT and Park2^−/− mice. (C) Volcano plot of differentially expressed genes (edgeR) of T cells from Park2^−/− and WT mice (thresholds: at least 1.2 fold change, adjusted P < 0.05). (D) Trajectory analysis of T cells in WT and Park2^−/− mice. (E) Expression of genes and a signature associated with T cell exhaustion differ in the three trajectory branches. (F) Relative proportions of T cells from WT and Park2^−/− mice in different trajectory branches. (G) Enriched signatures of cytotoxic, cytolytic, and cytokine effector CD8⁺ T cells from WT and Park2^−/− mice. (H and I) Expression levels of IFN-γ (H) and CD107a (I) in TILs by fluorescence-activated cell sorting (FACS) analysis. T cells were isolated from MC38 tumors in WT (n = 4) or Park2^−/− (n = 6) mice and stimulated with phorbol 12-myristate 13-acetate and ionomycin. (J) Experiment design. sc, subcutaneously; iv, intravenously. (K and L) Subcutaneous tumor volume (K) and OS (L) of Rag1^−/− mice inoculated with MC38 cells and transplanted with WT or Park2^−/− splenic CD8⁺ T cells from tumor-bearing mice. The numbers of mice are indicated. (M and N) Subcutaneous tumor volume (M) and OS (N) of Rag1^−/− mice inoculated with MC38 cells and transplanted with WT or Park2^−/− splenic CD8⁺ T cells from naïve mice. The numbers of mice are indicated. (O and P) Expression levels of the IFN-γ in splenic CD8⁺ T cells from tumor-bearing (O) or naïve mice (P) (n = 6 per group). The results are depicted as the means ± SEM, *P < 0.05 and ***P < 0.001.

Fig. 3.. Parkin deficiency enhances macrophage function and T cell activation.

(A) Annotated UMAP plot showing scRNA-seq analysis of myeloid cells from tumors in WT and Park2^−/− mice. (B and C) Differential strength of interactions between CD8⁺ T cells and myeloid cells in WT and Park2^−/− mice. (D) Heatmap of the expression levels of selected genes in TAMs of WT and Park2^−/− mice bearing MC38 tumors in scRNA-seq dataset. (E) Tumor volume of WT and Park2^−/− mice treated with anti–colony-stimulating factor 1 receptor (anti-CSF1R) antibodies or left untreated after inoculation of MC38 cells. The numbers of mice are indicated. (F) Tumor volume of control (Ctrl; n = 12) and Park2^fl/fl-Lyz2-Cre^+/− (n = 13) mice bearing subcutaneous MC38 tumors. CKO, conditional KO. (G) Percentages of T cells, CD8⁺ T cells in subcutaneous MC38 tumors in control (n = 5), or Park2^fl/fl-Lyz2-Cre^+/− (n = 5) mice. (H) Experiment design. (I) Tumor volume of WT mice inoculated with Hepa1-6 cells and transplanted with WT or Park2^−/− bone marrow–derived macrophages (BMDMs). The numbers of mice are indicated. (J) Transcripts from macrophages in tumors from WT and Park2^−/− mice in scRNA-seq dataset were assessed for enrichment of signatures of costimulation, antigen presentation, inflammation, and M1-type macrophages. (K) Bubble heatmap showing the interactions of selected ligand-receptor pairs between TAMs from WT or Park2^−/− mice and T cell subpopulations based on scRNA-seq analysis of the MC38 model. The results are depicted as the means ± SEM, **P < 0.01 and ***P < 0.001.

Fig. 4.. Parkin suppresses antigen presentation of macrophages.

(A) Enrichment analysis of up-regulated genes in Park2^−/− macrophages from macro_CD80 and macro_C1qc macrophage subgroups. GO_BP, Gene Ontology: Biological process; GO_CC, Gene Ontology: Cellular component; GP_MF, Gene Ontology: Molecular function. TAP, TAP protein is a heterodimeric peptide transporter consisting of the subunits TAP1 and TAP2. (B) ECDF plots of enrichment score for proinflammation and MHC-I antigen presentation signatures in WT (black) or Park2^−/− (red) macro_CD80 and macro_C1qc macrophages. (C) Dot plot of selected genes from M1 macrophage signatures. (D) Dot plot of selected genes from antigen presentation signature. (E) Representative images and proportions of OT-I T cells stimulated with BMDMs from (n = 3 per group). (F) MHC-I expression of TAMs in subcutaneous MC38 tumors (n = 4 per group). (G and H) Representative images and proportions of OT-I T cells stimulated with TAMs for 72 hours (n = 3 per group) (G) or control and Park2^fl/fl-Lyz2-Cre^+/− mice (n = 5 per group) (H). (I) Representative images and proportions of IFN-γ⁺ OT-I T cells after cocultured with BMDMs (n = 6 per group). (J) Secreted IFN-γ of OT-I T cells after cocultured with TAMs for 72 hours (n = 5 per group). (K) Heatmap of the expression levels of genes related to IFN-γ production of in MC38 tumors from WT and Park2^−/− mice in bulk RNA-seq dataset. (L) Tumor volume of WT and Park2^−/− MC38–bearing mice with IFN-γ blockade. The numbers of mice are indicated. (M) Productive clonality in WT or Park2^−/− TILs (n = 3 per group). (N) ECDF plot of enrichment score for memory T cell signature in WT (black) or Park2^−/− (red) T cells. (O) Dot plot of selected genes in memory T cell signature. (P) Tumor growth of WT (n = 9) or tumor-free Park2^−/− (n = 7) mice rechallenged with fivefold numbers of Hepa1-6 cells. The results are depicted as the means ± SEM, *P < 0.05 and ***P < 0.001.

Fig. 5.. AMPK-dependent Parkin activation suppresses MHC-I presentation via autophagy.

(A and B) Tumor growth (A) and OS (B) of WT and Pink1^−/− mice bearing subcutaneous MC38 tumors (n = 5 per group). (C and D) Representative image (C) and surface levels of MHC-I (D) on control and Parkin full-length Raw264.7 cells (n = 6 per group). Comp-FL2-A, Compensated Signal Fluorescence Channel 2 Area; PE-A, Phycoerythrin Area Parameter. (E) Total cellular levels of Parkin and MHC-I on control and Parkin full-length Raw264.7 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Surface levels of MHC-I on control, Parkin full-length, Parkin S65A, S108A, and C430S mutant Raw264.7 cells (n = 9 per group). (G) Expression and activity of the AMPK signaling in immune subpopulations. (H) Surface levels of MHC-I on Parkin full-length, Parkin S65A, S108A, and C430S mutant Raw264.7 cells treated without (NC) or with inhibitors of AMPK (AMPKi) (n = 3 per group). (I and J) mRNA expression levels of H2-K1 and H2-Q1 on control and Parkin full-length Raw264.7 cells (n = 3 per group). (K) Surface levels of MHC-I on control and Parkin full-length Raw264.7 cells treated without or with inhibitors of autophagy [CQ and bafilomycin A1 (BafA1)] (n = 6 per group). (L and M) Western blot of MHC-I expression in control and Parkin full-length Raw264.7 cells treated with BafA1. (N) Tumor growth of WT and Atg5^fl/fl-Lyz2-Cre^+/− mice bearing subcutaneous MC38 tumors (n = 5 per group). (O) Representative image and percentages of T cells in MC38 tumors (n = 5 per group). (P) MHC-I expression of TAMs in MC38 tumors from control and Atg5^fl/fl-Lyz2-Cre^+/− mice (n = 5 per group). (Q) Representative images and proportions of proliferating OT-I T cells stimulated with TAMs for 72 hours (h) (n = 4 per group). The results are depicted as the means ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.. The absence of Parkin enhances the efficacy of ICB therapy, and Parkin is a potential therapeutic target for cancer immunotherapy.

(A) Correlation analysis between the expression of Park2 and the infiltration of activated CD8⁺ T cells in various tumors. UCS, Uterine carcinosarcoma; THCA, Thyroid carcinoma; SARC, Sarcoma; PRAD, Prostate Adenocarcinoma; LGG, Low-grade gliomas; KIRP, Kidney renal papillary cell carcinoma; KIRC, Kidney Renal Clear Cell Carcinoma; HNSC, Head and neck squamous cell carcinoma; GBM, Glioblastoma multiforme; COAD, Colon adenocarcinoma; BRCA, Breast Invasive Carcinoma; BLCA, Bladder Urothelial Carcinoma. (B) Expression of Park2 in patients with mUC who received neoadjuvant anti–PD-1 therapies (GSE26359337). (C) Tumor volume of WT and Park2^−/− MC38–bearing mice administered intraperitoneally with anti-mouse PD-L1 or left untreated on days 9, 12, 15, and 18. The numbers of mice are indicated. (D to K) Correlation of the Park2^−/− signature score with overall survival (OS), dextran sulfate sodium (DSS), and progression free interval (PFI) in adenoid cystic carcinoma (ACC) (D), breast cancer (BRCA) (E), colon adenocarcinoma (COAD) (F), liver hepatocellular carcinoma (LIHC) (G), prostate adenocarcinoma (PRAD) (H), rectum adenocarcinoma (READ) (I), skin cutaneous melanoma (SKCM) (J), and stomach adenocarcinoma (STAD) (K). Low Park2^−/− signature, blue curve; high Park2^−/− signature, red curve. The results are depicted as the means ± SEM, *P < 0.05 and **P < 0.01.