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. 2020 Nov 3;2(1):192-205.
doi: 10.1039/d0cb00157k. eCollection 2021 Feb 1.

A peptidic inhibitor for PD-1 palmitoylation targets its expression and functions

Han Yao  1 , Chushu Li  1 , Fang He  2 , Teng Song  2 , Jean-Philippe Brosseau  3 , Huanbin Wang  1 , Haojie Lu  4 , Caiyun Fang  4 , Hubing Shi  5 , Jiang Lan  5 , Jing-Yuan Fang  1 , Jie Xu  2
Affiliations

A peptidic inhibitor for PD-1 palmitoylation targets its expression and functions

Han Yao et al. RSC Chem Biol. .

Abstract

Programmed cell death protein 1 (PD-1) is a crucial anticancer target, but the relatively low response rate and acquired resistance to existing antibody drugs highlight an urgent need to develop alternative targeting strategies. Here, we report the palmitoylation of PD-1, discover the main DHHC enzyme for this modification, reveal the mechanism of its effect on PD-1 protein stability, and rationally develop a peptide for targeting PD-1 expression. Palmitoylation promoted the trafficking of PD-1 to the recycling endosome, thus preventing its lysosome-dependent degradation. Palmitoylation of PD-1, but not of PD-L1, promoted mTOR signaling and tumor cell proliferation, and targeting palmitoylation displayed significant anti-tumor effects in a three-dimensional culture system. A peptide was designed to competitively inhibit PD-1 palmitoylation and expression, opening a new route for developing PD-1 inhibitors and combinatorial cancer immunotherapy.

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Conflict of interest statement

The authors declare no conflicts of interest related to this work. Data and material availability: all data needed to evaluate the conclusions in the paper are present in the paper or the ESI.† The plasmids require a material transfer agreement from Fudan University, China.

Figures

Fig. 1
Fig. 1. PD-1 is palmitoylated at C192. (A) Prediction of the PD-1 palmitoylation site at Cys192 using the MDD-Palm algorithm, with the matched motif in the inset and topology model on the right. (B) Schematic representation of the Click-IT procedure used to detect PD-1 palmitoylated proteins. (C and D) Click-IT assay in RKO, NB4 and Molt-4 cells showing the palmitoylation level of endogenous PD-1 in the three tumor cells. *** P < 0.001, ANOVA test. n = 3 independent experiments. (E) Click-IT was performed on cells transiently expressing wildtype PD-1 or Cys192Ser (C192S) mutated PD-1 to demonstrate that no palmitoylation was detected in the C192S PD-1 mutant. (F) Click-IT was performed on cells transiently expressing wildtype PD-1 or Cys284Ala (C284A) mutated PD-1 to demonstrate that no palmitoylation change was detected in the C284A PD-1 mutant.
Fig. 2
Fig. 2. Palmitoylation of PD-1 stabilizes its protein level. (A) Expression of PD-1 in a panel of cancer cell lines by Western blot using the anti-PD-1 specific antibody. (B) The expression and cell membrane localization of PD-1 are reduced in cells treated with 2-BP (palmitoylation inhibitor) while they increased in cells treated with PalmB (palmitoylation agonist), as shown by immunofluorescence. Images represent one of three independent experiments. Scale bars, 10 μm. (C and D) The treatment with 2-BP and PalmB, respectively, decreased and increased PD-1 expression in RKO, A375, HCT116, HepG2, LoVo, and SW1116 cells. *** P < 0.001, ** P < 0.01, * P < 0.05, ANOVA test. n = 3 independent experiments.
Fig. 3
Fig. 3. PD-1 is palmitoylated by DHHC9. (A) Western blot showing the effect of two independent siRNAs targeting the indicated DHHC enzymes on DHHC protein levels in A375 cells with the specific antibody for each DHHC enzyme. (B) Western blot showing the expression of PD-1 in A375 cells treated by two independent siRNAs targeting the indicated DHHC enzymes. (C) Western blot showing the expression of PD-1 in RKO and HCT116 cells treated by two independent siRNAs targeting DHHC9. Statistics shown on the right, *** P < 0.001, ** P < 0.01, ANOVA test. n = 3 independent experiments. (D) Immunoblot showing the expression of PD-1 in different cells overexpressing DHHC9. Statistics shown on the right, *** P < 0.001, ** P < 0.01, ANOVA test. n = 3 independent experiments.
Fig. 4
Fig. 4. Palmitoylation promotes the binding of PD-1 to RAB11. (A and B) The interactions of RAB11:WT PD-1 and RAB11:mutant PD-1 (C192S) are shown by co-IP in HCT116 cells. The antibody for (A) PD-1 and (B) RAB11 was used for pulldown. Statistics shown on the right, *** P < 0.001, ** P < 0.01, NS, P > 0.05, ANOVA test. n = 3 independent experiments. (C and D) The interactions of RAB11:WT PD-1 and RAB11:mutant PD-1 (C192S) are shown by co-IP in RKO (C) and LoVo cells (D). Disruption of palmitoylation by the overexpression of PD-1 C192S or by 2-BP treatment significantly decreased the binding between PD-1 and RAB11, as shown by co-IP. Statistics shown on the right, *** P < 0.001, ** P < 0.01, NS, P > 0.05, ANOVA test. n = 3 independent experiments. (E and F) Colocalization between PD-1 and RAB11 in RKO (E) and A375 cells (F) by immunofluorescence (right panels). White arrows indicate the regions of the recycling endosome marked by RAB11, and the intensity profiles of PD-1 and RAB11 in the inset are quantified and plotted on the right. Upon 2-BP treatment, the enrichment of PD-1 on the recycling endosome was prevented. Images represent one of three independent experiments. Scale bars, 10 μm. (G) The degradation of PD-1 was evaluated by cycloheximide (CHX)-chase assay. Treatment with 2-BP reduces the PD-1 level and this decrease is rescued by the lysosomal inhibitor chloroquine (CQ). (H) Degradation rates of wild-type PD-1 and the C192S mutant, as determined by CHX-chase assay.
Fig. 5
Fig. 5. Palmitoylation promotes tumor-intrinsic PD-1 signaling. (A) Interaction between endogenous PD-1 and S6 in tumor cells by co-IP. (B–D) The interactions between PD-1 and S6/eIF4E were significantly decreased by depleting DHHC9 (B), 2-BP treatment (C) and mutating Cys192 (D), as shown by co-IP. (E) Modulating the expression level of PD-1 by 2-BP and PalmB impacts the phosphorylation level of the mTOR effectors eIF4E (left panels) and S6 (right panels). (F) Modulating the expression level of PD-1 by 2-BP and PB in a dose-dependent manner correlates with the phosphorylation of the mTOR effectors eIF4E (left panel) and S6 (right panel). (G) Overexpression of PD-1 other than its mutant C192S increased the phosphorylation level of eIF4E and S6 in cancer cells. (H) Phosphorylation of eIF4E and expression of PD-1 in RKO cells treated with siRNAs specific for DHHC9. Statistics shown on the right, *** P < 0.001, ANOVA test. n = 3 independent experiments. (I) The phosphorylation of eIF4E and S6 is increased in HepG2 cells overexpressing DHHC9. Statistics shown on the right, *** P < 0.001, ** P < 0.01, ANOVA test. n = 3 independent experiments. (J) The phosphorylation of eIF4E and S6 is not affected by PD-L1 silencing or PD-L1 overexpression.
Fig. 6
Fig. 6. Targeting palmitoylation suppressed tumor-intrinsic PD-1 functions. (A) CCK8 proliferation assay showing that the proliferation of cancer cells treated with siRNAs for DHHC9 is decreased. *** P < 0.001, ** P < 0.01, ANOVA test. n = 3 independent experiments. (B) Treatment of HCT116 cells with 2-BP decreases the proliferation of cells as detected by CCK8 assay. (C) Overexpressing the C192S mutant (C192S OE) decreases cell proliferation compared to WT PD-1 (PD-1 OE) in LoVo cells. (D) Overexpressing WT PD-1 (PD-1 OE) increases anchorage-free colony formation assay (statistics shown on the right, PD-1 C192S compared to WT, and WT compared to Vector. ** P < 0.01, ANOVA test. n = 3 independent experiments) and targeting DHHC9 decreases it compared to control conditions (statistics shown on the left, *** P < 0.001, ANOVA test. n = 3 independent experiments). (E) CCK8 proliferation assay showing that the proliferation of cancer cells was not affected by the transfection of siRNAs for PD-L1 or PD-1 plasmid. NS, P > 0.05, ANOVA test. (F) CCK8 proliferation assay showing that PD-1 and its C284A mutant but not its C192S mutant or PD-L1 could rescue the effect of 2-BP or si-DHHC9 on tumor cell proliferation. *** P < 0.001, NS, P > 0.05, ANOVA test. n = 3 independent experiments.
Fig. 7
Fig. 7. Competitive inhibitor of PD-1 palmitoylation. (A) Sequence of the designed PD1-PALM peptide. (B) Click-IT assay showing the decreased palmitoylation of PD-1 in RKO, HCT116 and HepG2 cells treated by the PD1-PALM peptide. (C) Expression of PD-1 in SW480 (left panel) and RKO (right panel) cells treated with different concentrations of the PD1-PALM peptide. (D) Reciprocal co-immunoprecipitation assay of exogenous DHHC9 and Flag-AVICSRAARG-GFP (P2) in HCT116 cells. (E) Co-immunoprecipitation assay of endogenous DHHC9 and Flag-AVICSRAARG-GFP (P2) in A375 and RKO cells. (F) Inhibitory effect of PD1-PALM in the 3D tumor culture model of HCT116 cells. Statistics shown on the right, *** P < 0.001, ** P < 0.01, NS P > 0.05, ANOVA test. n = 3 independent experiments.
Fig. 8
Fig. 8. The intrinsic function and regulation of palmitoylated PD-1 in cancer cells. Schematic model showing the roles of palmitoylation in promoting PD-1 expression and intrinsic mTOR signaling to facilitate tumor growth.
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References

    1. Fourcade J. et al., Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J. Exp. Med. 2010;207:2175–2186. doi: 10.1084/jem.20100637. doi: 10.1084/jem.20100637. - DOI - DOI - PMC - PubMed
    1. Wang Y. et al., Regulation of PD-L1: Emerging Routes for Targeting Tumor Immune Evasion. Front. Pharmacol. 2018;9:536. doi: 10.3389/fphar.2018.00536. doi: 10.3389/fphar.2018.00536. - DOI - DOI - PMC - PubMed
    1. Postow M. A. Callahan M. K. Wolchok J. D. Immune Checkpoint Blockade in Cancer Therapy. J. Clin. Oncol. 2015;33:1974–1982. doi: 10.1200/JCO.2014.59.4358. doi: 10.1200/JCO.2014.59.4358. - DOI - DOI - PMC - PubMed
    1. Meng X. et al., FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells. Nature. 2018;564:130–135. doi: 10.1038/s41586-018-0756-0. doi: 10.1038/s41586-018-0756-0. - DOI - DOI - PubMed
    1. Kleffel S. et al., Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth. Cell. 2015;162:1242–1256. doi: 10.1016/j.cell.2015.08.052. doi: 10.1016/j.cell.2015.08.052. - DOI - DOI - PMC - PubMed