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Repurposing screen identifies Amlodipine as an inducer of PD-L1 degradation and antitumor immunity


Cancer cell expression of PD-L1 leads to T cells exhaustion by transducing co-inhibitory signal, and further understanding the regulation of PD-L1 in cancer cells may provide additional therapeutic strategies. Here by drug repurposing screen, we identified amlodipine as a potent inhibitor of PD-L1 expression in cancer cells. Further survey of calcium-associated pathways revealed calpain-dependent stabilization of the PD-L1 protein. Intracellular calcium delivered an operational signal to calpain-dependent Beclin-1 cleavage, blocking autophagic degradation of PD-L1 accumulated on recycling endosome (RE). Blocking calcium flux by amlodipine depleted PD-L1 expression and increased CD8+ T-cell infiltration in tumor tissues but not in myocardium, causing dose-dependent tumor suppression in vivo. Rescuing PD-L1 expression eliminated the effects of amlodipine, suggesting the PD-L1-dependent effect of amlodipine. These results reveal a calcium-dependent mechanism controlling PD-L1 degradation, and highlight calcium flux blockade as a potential strategy for combinatorial immunotherapy.

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Fig. 1: Amlodipine decreased PD-L1 expression post-translationally by blocking intracellular calcium flux.
Fig. 2: Amlodipine facilitated the autophagic degradation of PD-L1.
Fig. 3: Calpain modulated Beclin-1-dependent stabilization of PD-L1.
Fig. 4: Inactivated calpain facilitated the autophagic degradation of PD-L1 from recycling endosomes.
Fig. 5: Amlodipine suppressed PD-1 binding and growth of tumor cells.
Fig. 6: Amlodipine promoted antitumor immunity by decreasing PD-L1 expression in vivo.
Fig. 7: The calcium flux inhibitor Amlodipine induces PD-L1 degradation by promoting selective autophagy of recycling endosome.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary information files.


  1. 1.

    Sun C, Mezzadra R, Schumacher TN. Regulation and function of the PD-L1 checkpoint. Immunity. 2018;48:434–52.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Kaplon H, Reichert JM. Antibodies to watch in 2019. mAbs. 2019;11:219–38.

    CAS  PubMed  Google Scholar 

  3. 3.

    Syn NL, Teng MWL, Mok TSK, Soo RA. De-novo and acquired resistance to immune checkpoint targeting. Lancet Oncol. 2017;18:e731–41.

    PubMed  Google Scholar 

  4. 4.

    Kon E, Benhar I. Immune checkpoint inhibitor combinations: current efforts and important aspects for success. Drug Resist Updat. 2019;45:13–29.

    PubMed  Google Scholar 

  5. 5.

    Burr ML, Sparbier CE, Chan YC, Williamson JC, Woods K, Beavis PA, et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature. 2017;549:101–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Wang H, Yao H, Li C, Shi H, Lan J, Li Z, et al. HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity. Nat Chem Biol. 2019;15:42–50.

    CAS  PubMed  Google Scholar 

  7. 7.

    Cha JH, Yang WH, Xia W, Wei Y, Chan LC, Lim SO, et al. Metformin promotes antitumor immunity via endoplasmic-reticulum-associated degradation of PD-L1. Mol Cell. 2018;71:606–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Maher CM, Thomas JD, Haas DA, Longen CG, Oyer HM, Tong JY, et al. Small-molecule Sigma1 modulator induces autophagic degradation of PD-L1. Mol Cancer Res. 2018;16:243–55.

    CAS  PubMed  Google Scholar 

  9. 9.

    Wang X, Wu WKK, Gao J, Li Z, Dong B, Lin X, et al. Autophagy inhibition enhances PD-L1 expression in gastric cancer. J Exp Clin Cancer Res. 2019;38:140.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Zhang Z, Zhou L, Xie N, Nice EC, Zhang T, Cui Y, et al. Overcoming cancer therapeutic bottleneck by drug repurposing. Signal Transduct Target Ther. 2020;5:113.

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol. 2008;182:685–701.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Ylä-Anttila P, Vihinen H, Jokitalo E, Eskelinen E-L. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy. 2009;5:1180–5.

    PubMed  Google Scholar 

  13. 13.

    Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol. 2009;11:1433–7.

    CAS  PubMed  Google Scholar 

  14. 14.

    Hamasaki M, Furuta N, Matsuda A, Nezu A, Yamamoto A, Fujita N, et al. Autophagosomes form at ER–mitochondria contact sites. Nature. 2013;495:389–93.

    CAS  PubMed  Google Scholar 

  15. 15.

    Yamashita SI, Kanki T. How autophagy eats large mitochondria: autophagosome formation coupled with mitochondrial fragmentation. Autophagy. 2017;13:980–1.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Yen W-L, Shintani T, Nair U, Cao Y, Richardson BC, Li Z, et al. The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy. J Cell Biol. 2010;188:101–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Guo Y, Chang C, Huang R, Liu B, Bao L, Liu W. AP1 is essential for generation of autophagosomes from the trans-Golgi network. J cell Sci. 2012;125:1706–15.

    CAS  PubMed  Google Scholar 

  18. 18.

    Longatti A, Lamb CA, Razi M, Yoshimura S-i, Barr FA, Tooze SA. TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J Cell Biol. 2012;197:659–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Puri C, Renna M, Bento Carla F, Moreau K, Rubinsztein David C. Diverse autophagosome membrane sources coalesce in recycling endosomes. Cell. 2013;154:1285–99.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Knævelsrud H, Carlsson SR, Simonsen A. SNX18 tubulates recycling endosomes for autophagosome biogenesis. Autophagy. 2013;9:1639–41.

    PubMed  Google Scholar 

  21. 21.

    Pavel M, Rubinsztein DC. Mammalian autophagy and the plasma membrane. FEBS J. 2017;284:672–9.

    CAS  PubMed  Google Scholar 

  22. 22.

    Johansen T, Lamark T. Selective autophagy mediated by autophagic adapter proteins. Autophagy. 2011;7:279–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Pimentel-Muiños FX, Boada-Romero E. Selective autophagy against membranous compartments: canonical and unconventional purposes and mechanisms. Autophagy. 2014;10:397–407.

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Anding AL, Baehrecke EH. Cleaning house: selective autophagy of organelles. Dev Cell. 2017;41:10–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Beese CJ, Brynjólfsdóttir SH, Frankel LB. Selective autophagy of the protein homeostasis machinery: ribophagy, proteaphagy and ER-phagy. Front Cell Dev Biol. 2020;7:373.

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Kim I, Lemasters JJ. Mitophagy selectively degrades individual damaged mitochondria after photoirradiation. Antioxid Redox Signal. 2010;14:1919–28.

    Google Scholar 

  27. 27.

    Puri C, Vicinanza M, Ashkenazi A, Gratian MJ, Zhang Q, Bento CF, et al. The RAB11A-positive compartment is a primary platform for autophagosome assembly mediated by WIPI2 recognition of PI3P-RAB11A. Dev Cell. 2018;45:114–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Loi S, Dushyanthen S, Beavis PA, Salgado R, Denkert C, Savas P, et al. RAS/MAPK activation is associated with reduced tumor-infiltrating lymphocytes in triple-negative breast cancer: therapeutic cooperation between MEK and PD-1/PD-L1 immune checkpoint inhibitors. Clin Cancer Res. 2016;22:1499–509.

    CAS  PubMed  Google Scholar 

  29. 29.

    Kang S-H, Keam B, Ahn Y-O, Park H-R, Kim M, Kim TM. et al. Inhibition of MEK with trametinib enhances the efficacy of anti-PD-L1 inhibitor by regulating anti-tumor immunity in head and neck squamous cell carcinoma. Oncoimmunology. 2018;8:e1515057.

    PubMed  PubMed Central  Google Scholar 

  30. 30.

    Liu L, Mayes PA, Eastman S, Shi H, Yadavilli S, Zhang T, et al. The BRAF and MEK inhibitors dabrafenib and trametinib: effects on immune function and in combination with immunomodulatory antibodies targeting PD-1, PD-L1, and CTLA-4. Clin Cancer Res. 2015;21:1639–51.

    CAS  PubMed  Google Scholar 

  31. 31.

    Mohan N, Hosain S, Zhao J, Shen Y, Luo X, Jiang J. et al. Atezolizumab potentiates Tcell-mediated cytotoxicity and coordinates with FAK to suppress cell invasion and motility in PD-L1(+) triple negative breast cancer cells. Oncoimmunology. 2019;8:e1624128.

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Cao D, Qi Z, Pang Y, Li H, Xie H, Wu J, et al. Retinoic acid-related orphan receptor C regulates proliferation, glycolysis, and chemoresistance via the PD-L1/ITGB6/STAT3 signaling axis in bladder cancer. Cancer Res. 2019;79:2604–18.

    CAS  PubMed  Google Scholar 

  33. 33.

    Geng Y, Liu X, Liang J, Habiel DM, Kulur V, Coelho AL, et al. PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis. JCI Insight. 2019;4:e125326.

    PubMed Central  Google Scholar 

  34. 34.

    Li J, Yu T, Yan M, Zhang X, Liao L, Zhu M, et al. DCUN1D1 facilitates tumor metastasis by activating FAK signaling and up-regulates PD-L1 in non-small-cell lung cancer. Exp Cell Res. 2019;374:304–14.

    CAS  PubMed  Google Scholar 

  35. 35.

    Pan M-R, Wu C-C, Kan J-Y, Li Q-L, Chang S-J, Wu C-C, et al. Impact of FAK expression on the cytotoxic effects of CIK therapy in triple-negative breast cancer. Cancers. 2019;12:E94.

    PubMed  Google Scholar 

  36. 36.

    Ota K, Azuma K, Kawahara A, Hattori S, Iwama E, Tanizaki J, et al. Induction of PD-L1 expression by the EML4-ALK oncoprotein and downstream signaling pathways in non-small cell lung cancer. Clin Cancer Res. 2015;21:4014–21.

    CAS  PubMed  Google Scholar 

  37. 37.

    Hong S, Chen N, Fang W, Zhan J, Liu Q, Kang S. et al. Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients. Oncoimmunology. 2015;5:e1094598.

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Ma L, Lv J, Dong Y, Zhang X, Li X, Zhang H, et al. PD-L1 Expression and Its Regulation in Lung Adenocarcinoma with ALK Translocation. Interdiscip Sci. 2019;11:266–72.

    CAS  PubMed  Google Scholar 

  39. 39.

    Bulsara KG, Cassagnol M. Amlodipine. StatPearls. StatPearls Publishing: Treasure Island (FL); 2020.

  40. 40.

    Kayamori H, Shimizu I, Yoshida Y, Hayashi Y, Suda M, Ikegami R, et al. Amlodipine inhibits vascular cell senescence and protects against atherogenesis through the mechanism independent of calcium channel blockade. Int Heart J. 2018;59:607–13.

    CAS  PubMed  Google Scholar 

  41. 41.

    Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA, et al. Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Cell Rep. 2017;19:1189–201.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wang L, Tassiulas I, Park-Min KH, Reid AC, Gil-Henn H, Schlessinger J, et al. ‘Tuning’ of type I interferon-induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages. Nat Immunol. 2008;9:186–93.

    CAS  PubMed  Google Scholar 

  43. 43.

    Nair JS, DaFonseca CJ, Tjernberg A, Sun W, Darnell JE Jr, Chait BT, et al. Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-gamma. Proc Natl Acad Sci USA. 2002;99:5971–6.

    CAS  PubMed  Google Scholar 

  44. 44.

    Koide Y, Ina Y, Nezu N, Yoshida TO. Calcium influx and the Ca2+-calmodulin complex are involved in interferon-gamma-induced expression of HLA class II molecules on HL-60 cells. Proc Natl Acad Sci USA. 1988;85:3120–4.

    CAS  PubMed  Google Scholar 

  45. 45.

    Bootman MD, Chehab T, Bultynck G, Parys JB, Rietdorf K. The regulation of autophagy by calcium signals: Do we have a consensus? Cell Calcium. 2018;70:32–46.

    CAS  PubMed  Google Scholar 

  46. 46.

    Sun F, Xu X, Wang X, Zhang B. Regulation of autophagy by Ca(2). Tumour Biol. 2016;37:15467–76.

    CAS  PubMed Central  Google Scholar 

  47. 47.

    Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH, et al. Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 2008;4:295–305.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 2007;3:542–5.

    CAS  PubMed  Google Scholar 

  49. 49.

    Zhou C, Zhong W, Zhou J, Sheng F, Fang Z, Wei Y, et al. Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells. Autophagy. 2012;8:1215–26.

    CAS  PubMed  Google Scholar 

  50. 50.

    Yu T, Guo F, Yu Y, Sun T, Ma D, Han J, et al. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell. 2017;170:548–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Goll DarrelE, Thompson ValeryF, Li Hongqi, Wei Wei, Cong Jinyang. The calpain system. Physiol Rev. 2003;83:731–801.

    CAS  PubMed  Google Scholar 

  52. 52.

    Xia HG, Zhang L, Chen G, Zhang T, Liu J, Jin M, et al. Control of basal autophagy by calpain1 mediated cleavage of ATG5. Autophagy. 2010;6:61–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol. 2006;8:1124–32.

    CAS  PubMed  Google Scholar 

  54. 54.

    Zhao Q, Guo Z, Deng W, Fu S, Zhang C, Chen M, et al. Calpain 2-mediated autophagy defect increases susceptibility of fatty livers to ischemia-reperfusion injury. Cell Death Dis. 2016;7:e2186.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Russo R, Berliocchi L, Adornetto A, Varano GP, Cavaliere F, Nucci C, et al. Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death Dis. 2011;2:e144.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Marie N, Lindsay AJ, McCaffrey MW. Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner. Biochem J. 2005;389:223–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Blander JM. Regulation of the cell biology of antigen cross-presentation. Annu Rev Immunol. 2018;36:717–53.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Yao H, Lan J, Li C, Shi H, Brosseau J-P, Wang H, et al. Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours. Nat Biomed Eng. 2019;3:306–17.

    CAS  PubMed  Google Scholar 

  59. 59.

    Li CW, Lim SO, Chung EM, Kim YS, Park AH, Yao J, et al. Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1. Cancer Cell. 2018;33:187–.e110.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Li CW, Lim SO, Xia W, Lee HH, Chan LC, Kuo CW, et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat Commun. 2016;7:12632.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Shaughnessy M, Lamuraglia G, Klebanov N, Ji Z, Rajadurai A, Kumar R, et al. Selective uveal melanoma inhibition with calcium channel blockade. Int J Oncol. 2019;55:1090–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Roderick HL, Cook SJ. Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat Rev Cancer. 2008;8:361–75.

    CAS  PubMed  Google Scholar 

  63. 63.

    Bong AHL, Monteith GR. Calcium signaling and the therapeutic targeting of cancer cells. Biochim Biophys Acta Mol Cell Res. 2018;1865:1786–94.

    CAS  PubMed  Google Scholar 

  64. 64.

    Barcelo C, Siso P, Maiques O, de la Rosa I, Marti RM, Macia A.T-type calcium channels: a potential novel target in melanoma. Cancers. 2020;12:391.

    PubMed Central  Google Scholar 

  65. 65.

    Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA. 2003;100:15077–82.

    CAS  PubMed  Google Scholar 

  66. 66.

    Cao Y, Klionsky DJ. Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res. 2007;17:839–49.

    CAS  PubMed  PubMed Central  Google Scholar 

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This work was supported by National Natural Science Foundation of China (No: 82030104, 81874050, 81572326), Basic Research Projects of Shanghai Science and Technology Innovation Action Plan (20JC1410700); National Key R & D Program of China (2016YFC0906002, 2016YFC0906002), Tang Scholar (JX), and Startup Research Funding of Fudan University.

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CL, HY, and HW performed experiments and analyzed data. JYF provided supports on study resources. CL and JX wrote the paper. JX conceived the study.

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Correspondence to Jie Xu.

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Li, C., Yao, H., Wang, H. et al. Repurposing screen identifies Amlodipine as an inducer of PD-L1 degradation and antitumor immunity. Oncogene 40, 1128–1146 (2021).

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