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. 2024 Feb 20;5(2):101357.
doi: 10.1016/j.xcrm.2023.101357. Epub 2024 Jan 17.

Benzosceptrin C induces lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting DHHC3

Qun Wang  1 , Jinxin Wang  2 , Dianping Yu  1 , Qing Zhang  1 , Hongmei Hu  1 , Mengting Xu  1 , Hongwei Zhang  1 , Saisai Tian  2 , Guangyong Zheng  1 , Dong Lu  1 , Jiajia Hu  3 , Mengmeng Guo  1 , Minchen Cai  1 , Xiangxin Geng  1 , Yanyan Zhang  1 , Jianhua Xia  1 , Xing Zhang  1 , Ang Li  4 , Sanhong Liu  5 , Weidong Zhang  6
Affiliations

Benzosceptrin C induces lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting DHHC3

Qun Wang et al. Cell Rep Med. .

Abstract

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blockade has become a mainstay of cancer immunotherapy. Targeting the PD-1/PD-L1 axis with small molecules is an attractive approach to enhance antitumor immunity. Here, we identified a natural marine product, benzosceptrin C (BC), that enhances the cytotoxicity of T cells to cancer cells by reducing the abundance of PD-L1. Furthermore, BC exerts its antitumor effect in mice bearing MC38 tumors by activating tumor-infiltrating T cell immunity. Mechanistic studies suggest that BC can prevent palmitoylation of PD-L1 by inhibiting DHHC3 enzymatic activity. Subsequently, PD-L1 is transferred from the membrane to the cytoplasm and cannot return to the membrane via recycling endosomes, triggering lysosome-mediated degradation of PD-L1. Moreover, the combination of BC and anti-CTLA4 effectively enhances antitumor T cell immunity. Our findings reveal a previously unrecognized antitumor mechanism of BC and represent an alternative immune checkpoint blockade (ICB) therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Keywords: DHHC3; PD-L1; benzosceptrin C; cancer immunotherapy; colorectal cancer.

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Conflict of interest statement

Declaration of interests All authors declare that they have no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1
BC can promote the degradation of PD-L1 in RKO cells (A) Brief description of our drug screening and validation workflow. (B) Screening of colon cancer cell lines with high expression of PD-L1. (C) Screening of 300 molecules in the natural compound library. RKO cells were treated with the drugs at 10 μM for 24 h. Berberine (Ber) was used as a positive control. The hit compounds that induced a decrease in PD-L1 levels are shown in blue. The depth of blue represents a decreased level of PD-L1. The reduction in PD-L1 levels in RKO cells treated with 300 drugs was measured by western blot. (D) Chemical structure of BC. (E and F) RKO and HCT116 cells were treated with different concentrations of BC for 24 h or treated with 10 μM BC for the indicated times. (G) Quantitative results of BC on PD-L1 in RKO and HCT116 cells are shown. (H) RKO and HCT116 cells were treated with BC (1, 5, and 10 μM) for 24 h, and plasma membrane PD-L1 was detected by flow cytometry. The IFN-γ (50 ng/mL) function is to increase PD-L1 expression in tumor cells. (I) RKO and HCT116 cells were treated with BC (10 μM) for 24 h. Immunofluorescence staining showed PD-L1 labeling in red and nuclei in blue with Hoechst. Scale bar, 200 μm. (J) RKO and HCT116 cells were treated with BC (1, 5, and 10 μM) for 24 h, and the effect of the drug on cell viability was determined with a CCK-8 kit. (K) RKO and HCT116 cells were treated with BC (1, 5, and 10 μM) for 24 h, and the effect of the drug on cells was detected with an EdU kit. The data shown are the mean value ± standard error of the mean (SEM; t test). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the PBS group.
Figure 2
Figure 2
BC can attenuate the ability of tumor cells to bind PD-1, enhance the cytotoxicity of T cells, and mediate a T cell-dependent antitumor effect (A) PD-L1/PD-1 binding assay in RKO cells treated with BC (10 μM, 24 h). The nuclei were stained with Hoechst. Scale bar, 100 μm. (B) Bound PD-1 was calculated according to the intensity of green fluorescence (n = 3, t test). ∗∗∗p < 0.001 compared with the DMSO group. (C) PD-L1/PD-1 blockade assay performed with RKO cells treated with 1, 5, and 10 μM BC for 12 h. Jurkat NFAT-luciferase reporter cells (10,000 cells/well) were added, and the cells were cocultured for 4 h. Data are presented as the fold induction over the untreated control (n = 3, t test). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the DMSO group. (D‒G) T cells were cocultured with RKO and HCT116 cells in 12-well plates for 24 days in the presence of BC, and the surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensity are shown (n = 3, one-way ANOVA). ##p < 0.01 compared with the RKO and HCT116 DMSO groups; ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the RKO + T cell or HCT116 + T cell groups. (H) MC38 cells were injected into C57BL/6 mice on day −5, and BC was administered as indicated. (I) Ex vivo observation of the tumors from the treated mice (0, 5, 25, and 50 mg/kg). (J) C57BL/6 mice with MC38 tumors were treated intraperitoneally (i.p.) with PBS or BC, and tumor growth was monitored. (K) Comparison of the weight of the tumors from the mice treated with PBS or BC. (L) The body weight curves of the mice measured every 2 days. (M) Expression of PD-L1 in the tumors of mice treated with PBS or BC. (N) Nude mice bearing MC38 tumors received daily i.p. injections of PBS or BC (50 mg/kg) for 12 days (O–Q) (O) Ex vivo observation of the tumors from the treated mice (0 and 50 mg/kg), and (P) tumor growth and (Q) tumor weight were measured. (R) Representative immunohistochemistry (IHC) staining results for PD-L1, CD3, cleaved caspase 3, and FOXP3 in PBS- or BC (50 mg/kg)-treated C57BL/6 mice.Representative positive expressions are indicated by arrows. Scale bar, 10 μm. (S) Quantification of IHC staining. The data shown are the mean value ± SEM (one-way ANOVA). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the PBS group.
Figure 3
Figure 3
BC induces lysosome-dependent degradation of PD-L1 (A‒C) Quantitative RT-PCR was used to analyze the mRNA level of PD-L1 in RKO cells treated with BC at different concentrations or at different times or in RKO cells treated with BC (10 μM) and 50 ng/mL IFN-γ for 12 h. (D) Immunoblotting detecting the PD-L1 abundance in RKO cells treated with DMSO or BC (10 μM) for the indicated time periods in the presence of CHX (50 mg/mL). (E) Quantification of the PD-L1 intensity from (D). (F‒M) The degradation of PD-L1 in RKO cells was evaluated by lysosome (CQ, Baf), proteasome (MG132), and autophagy (VM) inhibitors, and quantification of the intensity was determined by the relative level of PD-L1. (N) LysoTracker red staining in RKO cells treated with BC (10 μM) or Torin1 (1 μM) for 12 h (scale bar, 200 μm). (O) Quantification of the LysoTracker intensity from (N). (P) The degradation of PD-L1 in RKO cells was evaluated by lysosome (CQ) and proteasome (MG132) inhibitors. (Q) Quantification of PD-L1 intensity from (P). (R‒W) Flow cytometry measuring PD-L1 expression in RKO cells pretreated with the indicated concentrations of MG132, Baf, or CQ, followed by BC treatment for 12 h. Quantification of the MFI of PD-L1 is shown in (S), (U), and (W). The data shown are the mean value ± SEM (t test). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the PBS group.
Figure 4
Figure 4
BC affects the palmitoylation of PD-L1 by DHHC3 acetyltransferase (A) Heatmap showing the expression of mRNAs in BC-treated RKO cells. (B) Volcano plot showing the significantly upregulated (red dots) and downregulated (blue dots) mRNAs in BC-treated RKO cells. (C) KEGG analysis of differentially expressed mRNAs in RKO cells. (D) Heatmap showing proteomics in RKO cells after BC treatment. (E) Flowchart of the DARTS experiment. (F) The expression of different DHHCs in colorectal cancer (CRC) was determined according to the immunohistochemistry results of the human protein profile. (G) Venn diagram showing the expression and distribution of different DHHCs in CRC, including the expression level and subcellular distribution. (H) Expression of PD-L1 in RKO cells transfected with siRNA from different DHHCs. (I) Expression of PD-L1 after transfection of DHHC3 interfering RNA into RKO, HCT116, and MC38 cells. (J) Immunofluorescence staining showed PD-L1 expression after transfection of DHHC3 interfering RNA into RKO cells. (K) Expression of PD-L1 after transfection of the DHHC3 plasmid into RKO, HCT116, and MC38 cells. (L) The reciprocal coimmunoprecipitation of PD-L1 and DHHC3 revealed a physical interaction between endogenously expressed PD-L1 and DHHC3 in RKO cells. (M) Immunofluorescence staining showed PD-L1 expression after transfection of the DHHC3 plasmid into HCT116 cells. (N) RKO cells were immunostained for PD-L1 and DHHC3. The white arrows indicates the interaction between PD-L1 and DHHC3. Scale bars, 10 μm.
Figure 5
Figure 5
BC directly binds to and inhibits DHHC3 activity (A) The CETSA determined the thermal stabilization of the DHHC3 interaction with BC at a series of temperatures from 30°C to 50°C. (B) Quantification of DHHC3 in CETSA in Figure 5A. (C) Stability of different concentrations of BC on DHHC3 at 45°C. (D and F) Quantification of the DHHC3 intensity of (C) and (E). ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the control group (one-way ANOVA). (E) Stability of different concentrations of BC on DHHC3 when the ratio of Pronase to protein was 1:300. (G) Molecular docking of BC to DHHC3. (H) The cellular MST assay of GFP-tagged DHHC3 upon overexpression of wild-type DHHC3 and the disruptive mutants. (I‒L) The degradation of PD-L1 or the C272A mutant in HCT116 cells was evaluated by CHX assay in the presence of inhibitors for CQ, MG132, and 3-MA. Quantification of the PD-L1 intensity is shown on the right. (M‒O) Representative immunofluorescence images show the colocalization between heterotopic PD-L1 and Rab11b, Rab7b, and Lamp1. (P) Schematic diagram of PD-L1 transport from the plasma membrane to vesicles, early endosomes, circulating endosomes (labeled with Rab11), late endosomes (Rab7b), and lysosomes (Lamp1). (Q) Statistical results of the colocalization between PD-L1 and Rab11/Rab7b/Lamp1 in RKO cells treated with BC or DMSO. The data shown are the mean value ± SEM (one-way ANOVA). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the control group.
Figure 6
Figure 6
Combined BC and CTLA4 inhibited tumor growth C57BL/6 mice bearing MC38 cells were treated with PBS, anti-PD-L1, anti-CTLA4, BC, BC + anti-PD-L1, anti-PD-L1 + anti-CTLA4, and BC + anti-CTLA4. (A) MC38 cells were injected into C57BL/6 mice on day −5, and BC, anti-PD-L1, and anti-CTLA4 were administered as indicated. (B–E) The MC38 tumor volume (B), tumor weight (C), mouse weight (D), and tumor images (E) were measured for 16 days. (F–H) Flow cytometry detecting GzmB+ (F), Gr-1+CD11b+ (G), and FOXP3+CD25+ (H) in the PBS, anti-PD-L1, anti-CTLA4, BC, BC + anti-PD-L1, anti-PD-L1 + anti-CTLA4, and BC + anti-CTLA4 groups. Quantification of GzmB+, Gr-1+CD11b+, and FOXP3+CD25+ populations in the PBS, anti-PD-L1, anti-CTLA4, BC, BC + anti-PD-L1, anti-PD-L1 + anti-CTLA4, and BC + anti-CTLA4 groups is shown on the right. (I) Representative IHC staining results for CD3, FOXP3, PD-L1, Ki-67, and cleaved caspase-3 in PBS-, anti-PD-L1-, anti-CTLA4-, BC-, BC + anti-PD-L1-, anti-PD-L1 + anti-CTLA4-, and BC + anti-CTLA4-treated C57BL/6 mice. Scale bar, 10 μm. Quantification of IHC staining is shown on the right. The data shown are the mean value ± SEM (one-way ANOVA). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the control group.
Figure 7
Figure 7
The association between DHHC3 and PD-L1 expression in CRC tissues (A) Expression levels of DHHC3 and PD-L1 proteins in paracancerous and tumor tissues from six colon cancer patients. (B and C) The expression of CD274 (B) and DHHC3 (C) in cancer tissues and paracancerous tissues. (D) Representative IHC images of PD-L1 and DHHC3 staining in paracancerous and CRC tumor samples. (E) Correlation between colorectal cancer and DHHC3. (F) Correlation between total cancer and DHHC3. (G) The survival of CRC patients stratified by the expression of DHHC3 or PD-L1 was compared by two-sided log-rank analysis.
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