Demethylzeylasteral induces PD-L1 ubiquitin-proteasome degradation and promotes antitumor immunity via targeting USP22

Abstract

Programmed cell death ligand-1 (PD-L1) is a T cell inhibitory immune checkpoint molecule that interacts with programmed cell death-1 (PD-1) to promote immune escape of tumor cells. Compared with antibody therapies, small molecule drugs show better prospects due to their advantages such as higher bioavailability, better tissue penetration, and reduced risk of immunogenicity. Here, we found that the small molecule demethylzeylasteral (Dem) can significantly downregulate the expression of PD-L1 in colorectal cancer cells and enhance the killing effect of T cells on tumor cells. Mechanistically, Dem binds to the deubiquitinating enzyme USP22 and promotes its degradation, resulting in increased ubiquitination and degradation of PD-L1 through the proteasome pathway. In addition, Dem increased the activity of cytotoxic T cells and reduced the number of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in tumor-infiltrating lymphocytes (TILs), thereby activating the tumor immune microenvironment and inhibiting the growth of subcutaneous MC38 tumors in C57BL/6 mice. Moreover, we also found that the combination of Dem and CTLA4 antibodies can further improve the efficacy of antitumor therapy. Our study reveals the mechanism by which Dem promotes PD-L1 degradation and suggests that the combination of Dem and CTLA4 antibodies may improve the efficacy of immunotherapy.

Keywords: Antitumor immunity; Colorectal cancer; Demethylzeylasteral; Deubiquitination; Immune checkpoint blockade; PD-L1; Tumor-infiltrating lymphocytes; USP22.

Graphical abstract

Figure 1

Dem can downregulate PD-L1 expression in CRC cells. (A) Process diagram of the screening of small molecules that can downregulate the expression of PD-L1 in RKO cells from the compound library. (B) A total of 225 molecules were screened in the compound library. RKO cells were treated with the drugs at 10 μmol/L for 24 h. The hit compounds that induced a decrease in PD-L1 levels are shown in blue. A depth of blue represents a decreased level of PD-L1. The reduction in PD-L1 levels in RKO cells treated with 225 drugs was measured via Western blotting. The corresponding small molecule names can be found in Supporting Information Table S1. (C, D) RKO and HT29 cells were treated with different concentrations of Dem for 24 h, after which total PD-L1 was analyzed by immunoblotting (IB). The results of the quantitative IB analysis are shown below. (E, F) RKO and HT29 cells were treated with Dem (10 or 20 μmol/L) for the indicated times and subjected to IB analysis of total PD-L1. The results of the quantitative IB analysis are shown below. (G) RKO and HT29 cells were treated with different concentrations of Dem for 24 h in the absence or presence of IFN-γ (50 ng/mL), and the plasma membrane PD-L1 concentration was detected via flow cytometry. (I) RKO and HT29 cells were treated with Dem for the indicated times in the absence or presence of IFN-γ (50 ng/mL), and the plasma membrane PD-L1 concentration was detected via flow cytometry. (H, J) Quantitative analysis of plasma membrane PD-L1 expression. ^##P < 0.01 compared with DMSO group; ∗∗∗P < 0.001 compared with the IFN-γ DMSO group. (K) Immunofluorescence staining images of RKO cells. PD-L1 is labeled in red, and nuclei are labeled in blue with DAPI. Scale bar = 100 μm. (L) The quantitative results of immunofluorescence staining. (M) Images of EdU-stained cells showing the effect of Dem on RKO cell proliferation (represented by green fluorescence) and nuclei are labeled in blue with Hoechst. Scale bar = 200 μm. (N) The quantitative results of EdU staining. The data shown are the mean ± standard error of the mean (SEMs); n = 3; n.s P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the DMSO group.

Figure 2

Dem can enhance the killing ability of T cells and inhibit tumor growth in mice with normal immune function in vivo. (A, B) Jurkat cells were activated with 1 mg/mL phytohemagglutinin (PHA) and 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and cocultured with RKO and HT29 cells for 24 h in the presence of Dem. The surviving tumor cells were visualized by crystal violet staining. The quantitative results for the surviving cells are shown on the right (n = 3). ^##P < 0.01 compared with the RKO or HT29 DMSO group; ∗∗P < 0.01 and ∗∗∗P < 0.001 compared with the RKO + Jurkat cell or HT29+Jurkat cell group. Scale bar = 200 μm. A subcutaneous tumor model was constructed in C57BL/6 mice (C) or nude mice (D), and the process of drug administration was performed. (E–H) Effect of Dem (1, 2, or 4 mg/kg) treatment on tumor growth in C57BL/6 mice (n = 5). Tumors were harvested at the 12-day follow-up (E), tumor volume (F), tumor weight (G), and body weight (H). (I–L) Effect of Dem (4 mg/kg) treatment on tumor growth in nude mice (n = 5). Tumors were harvested at the 10-day follow-up (I), tumor volume (J), tumor weight (K), and body weight (L). (M) IHC staining results for PD-L1, c-caspase-3, CD8, and Foxp3. The quantitative results of IHC staining are shown on the right. Scale bar = 100 μm. The data shown are the mean ± standard error of the mean (SEM); n.s P > 0.05, ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared with the saline group.

Figure 3

Dem promotes PD-L1 degradation via the ubiquitin/proteasome pathway. (A, B) Quantitative RT-PCR analysis of the mRNA level of PD-L1 in RKO cells treated with different concentrations of Dem for 24 h and treated with 10 μmol/L Dem for the indicated times. (C) IB analysis of PD-L1 levels in RKO cells treated with Dem or CHX (25 μg/mL) alone or in combination for the indicated durations. The quantitative results are shown below. (D) KEGG enrichment of the top 20 signaling pathways identified via proteomics. (E–H) IB analysis of PD-L1 levels in RKO cells treated with Dem in the absence or presence of the inhibitors MG132 (E), CQ (F), Baf (G), and 3-MA (H). The results of the quantitative IB analysis are shown below. (I) Immunofluorescence detection of PD-L1 expression on the cell membrane in RKO cells treated with Dem in the absence or presence of the inhibitor MG132 (1 μmol/L) or CQ (20 μmol/L). PD-L1 is labeled in red, and nuclei are labeled in blue with DAPI. The quantitative results are shown on the right side. Scale bar = 200 μm. (J) Ubiquitinated PD-L1 in RKO cells were immunoprecipitated (IP) and subjected to IB analysis with a ubiquitin antibody. The cells were pretreated with MG132 (10 μmol/L) for 6 h. (K) Expression of PD-L1 and USP22 after transfection of the USP22 plasmid into RKO cells. (L) Expression of PD-L1 and USP22 after transfection of USP22 interfering RNA into RKO cells. The quantitative results are shown on the right. The data shown are the mean ± standard error of the mean (SEM); n = 3; n.s P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the DMSO group.

Figure 4

Dem binds to USP22 and promotes its degradation. (A) CETSA was used to determine the thermal instability of the interaction of USP22 with Dem at a series of temperatures ranging from 37 to 75 °C under short drug exposure (5 min). (B, C) CETSA was used to determine the thermal instability of the interaction of USP22 with Dem after different incubation times at room temperature. The quantitative results are shown below. (D, E) CETSA was used to determine the thermal instability of the interaction of USP22 with different concentrations of Dem at room temperature. The quantitative results are shown below. (F, G) The DARTS assay determined the instability of USP22 at different concentrations of Dem when the ratio of pronase to protein was 1:600. The quantitative results are shown below. (H) Molecular docking of Dem to USP22. (I–K) Cellular MST assay of GFP-tagged USP22 upon overexpression of wild-type USP22 (I) or disrupted USP22 mutants (Arg371 and His374) (J, K). (L–O) Immunoblot analysis of USP22 levels in RKO cells treated with different concentrations of Dem for 24 h and treated with 10 μmol/L Dem for the indicated times. The quantitative results are shown below. (P, Q) Quantitative RT-PCR analysis of the mRNA level of USP22 in RKO cells treated with different concentrations of Dem for 24 h (P) and treated with 10 μmol/L for the indicated times (Q). The data shown are the mean ± standard error of the mean (SEM); n = 3; n.s P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the DMSO group.

Figure 5

The combination of Dem and CTLA4 antibodies inhibits tumor growth in C57BL/6 mice. (A) Construction of a subcutaneous tumor model in C57BL/6 mice and the process of drug administration. (B–E) Effects of treatment with saline, anti-PD-L1, anti-CTLA4, Dem, Dem + anti-PD-L1, Dem + anti-CTLA4 or anti-PD-L1+anti-CTLA4 on tumor growth in C57BL/6 mice (n = 5). Tumor volume (B). Tumors were harvested at the 12-day follow-up (C). Tumor weight (D). Body weight (E). (F) Flow cytometry was used to detect GzmB⁺, Gr-1⁺ CD11b⁺, and Foxp3⁺ CD25⁺ cells in these groups. The quantitative results of flow cytometry are shown on the right (n = 3). The data shown are the mean ± standard error of the mean (SEM); n.s P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the saline group.

Figure 6

The association between PD-L1 and USP22 expression in CRC tissues. (A) The survival of CRC patients stratified by the expression of PD-L1 or USP22 was compared by two-side log-rank analysis. (B) Scatter plots showing the correlation between PD-L1 or USP22 expression and infiltrating CD8⁺ T cells or Tregs in colon adenocarcinoma patients as determined by TIMER 2.0. (C) Representative IHC images of PD-L1 and USP22 staining in paracancerous tissues (PT) and cancer tissues (CT) from CRC patients. Scale bar = 100 μm.