Potentiating CD8⁺ T cell antitumor activity by inhibiting PCSK9 to promote LDLRmediated TCR recycling and signaling

Abstract

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8⁺ T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8⁺ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8⁺ T cell LDLR level and TCR signaling via tumor cellderived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8⁺ T cells by alleviating the suppressive effect on CD8⁺ T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.

Key words： LDLR; PCSK9; TCR; CD8⁺ T cells; tumor microenvironment; cancer immunotherapy

References

1 Abifadel M, Varret M, Rabès JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D (2003) Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 34:154–156

2 Alcover A, Alarcón B, Di Bartolo V (2018) Cell biology of T cell receptor expression and regulation. Annu Rev Immunol 36:103–125

3 Almeida L, Lochner M, Berod L, Sparwasser T (2016) Metabolic pathways in T cell activation and lineage differentiation. Semin Immunol 28:514–524

4 Baumann T, Dunkel A, Schmid C, Schmitt S, Hiltensperger M, Lohr K, Laketa V, Donakonda S, Ahting U, Lorenz-Depiereux B (2020) Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal. Nat Immunol 21:555–566

5 Bensinger SJ, Bradley MN, Joseph SB, Zelcer N, Janssen EM, Hausner MA, Shih R, Parks JS, Edwards PA, Jamieson BD, Tontonoz P (2008) LXR signaling couples sterol metabolism to proliferation in the acquired immune response. Cell 134:97–111

6 Bian Y, Li W, Kremer DM, Sajjakulnukit P, Li S, Crespo J, Nwosu ZC, Zhang L, Czerwonka A, Pawlowska A (2020) Cancer SLC43A2 alters T cell methionine metabolism and histone methylation. Nature 585:277–282

7 Blom DJ, Hala T, Bolognese M, Lillestol MJ, Toth PD, Burgess L, Ceska R, Roth E, Koren MJ, Ballantyne CM (2014) A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. N Engl J Med 370:1809–1819

8 Brody T, Brody T (2018) FDA’s drug review process and the package label: strategies for writing successful FDA submissions. Academic Press, London

9 Bunse L, Pusch S, Bunse T, Sahm F, Sanghvi K, Friedrich M, Alansary D, Sonner JK, Green E, Deumelandt K (2018) Suppression of antitumor T cell immunity by the oncometabolite (R)-2-hydroxyglutarate. Nat Med 24:1192–1203

10 Cascone T, McKenzie JA, Mbofung RM, Punt S, Wang Z, Xu C, Williams LJ, Wang Z, Bristow CA, Carugo A (2018) Increased tumor glycolysis characterizes immune resistance to adoptive T cell therapy. Cell Metab 27:977–987

11 Chang CH, Qiu J, O’Sullivan D, Buck MD, Noguchi T, Curtis JD, Chen Q, Gindin M, Gubin MM, van der Windt GJ (2015) Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell 162:1229–1241

12 Chapman NM, Boothby MR, Chi H (2020) Metabolic coordination of T cell quiescence and activation. Nat Rev Immunol 20:55–70

13 Cunningham D, Danley DE, Geoghegan KF, Griffor MC, Hawkins JL, Subashi TA, Varghese AH, Ammirati MJ, Culp JS, Hoth LR (2007) Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat Struct Mol Biol 14:413–419

14 Draghiciu O, Lubbers J, Nijman HW, Daemen T (2015) Myeloid derived suppressor cells-an overview of combat strategies to increase immunotherapy efficacy. Oncoimmunology 4:e954829

15 Dugnani E, Pasquale V, Bordignon C, Canu A, Piemonti L, Monti P (2017) Integrating T cell metabolism in cancer immunotherapy. Cancer Lett 411:12–18

16 Ecker C, Guo L, Voicu S, Gil-de-Gomez L, Medvec A, Cortina L, Pajda J, Andolina M, Torres-Castillo M, Donato JL (2018) Differential reliance on lipid metabolism as a salvage pathway underlies functional differences of T cell subsets in poor nutrient environments. Cell Rep 23:741–755

17 Fu CM, Jiang AM (2018) Dendritic cells and CD8 T cell immunity in tumor microenvironment. Front Immunol 9:3059

18 Gaus K, Chklovskaia E, Fazekas de St Groth B, Jessup W, Harder T (2005) Condensation of the plasma membrane at the site of T lymphocyte activation. J Cell Biol 171:121–131

19 Geltink RIK, Kyle RL, Pearce EL (2018) Unraveling the complex interplay between T cell metabolism and function. Annu Rev Immunol 36:461–488

20 Go GW, Mani A (2012) Low-density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis. Yale J Biol Med 85:19–28

21 He BB, Peng WB, Huang J, Zhang H, Zhou YS, Yang XL, Liu J, Li ZJ, Xu CL, Xue MX (2020) Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein Cell 11:518–524

22 Ho PC, Bihuniak JD, Macintyre AN, Staron M, Liu X, Amezquita R, Tsui YC, Cui G, Micevic G, Perales JC (2015) Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses. Cell 162:1217–1228

23 Hobbs HH, Russell DW, Brown MS, Goldstein JL (1990) The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 24:133–170

24 Hu Z, Ott PA, Wu CJ (2018) Towards personalized, tumour-specific, therapeutic vaccines for cancer. Nat Rev Immunol 18:168–182

25 Ishida Y, Agata Y, Shibahara K, Honjo T (1992) Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J 11:3887–3895

26 Jeon H, Blacklow SC (2005) Structure and physiologic function of the low-density lipoprotein receptor. Annu Rev Biochem 74:535–562

27 Kalluri R (2016) The biology and function of fibroblasts in cancer. Nat Rev Cancer 16:582–598

28 Kidani Y, Elsaesser H, Hock MB, Vergnes L, Williams KJ, Argus JP, Marbois BN, Komisopoulou E, Wilson EB, Osborne TF (2013) Sterol regulatory element-binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity. Nat Immunol 14:489–499

29 Kishton RJ, Sukumar M, Restifo NP (2017) Metabolic regulation of T cell longevity and function in tumor immunotherapy. Cell Metab 26:94–109

30 Kuhnast S, van der Hoorn JW, Pieterman EJ, van den Hoek AM, Sasiela WJ, Gusarova V, Peyman A, Schafer HL, Schwahn U, Jukema JW, Princen HM (2014) Alirocumab inhibits atherosclerosis, improves the plaque morphology, and enhances the effects of a statin. J Lipid Res 55:2103–2112

31 Kumar V, Donthireddy L, Marvel D, Condamine T, Wang F, Lavilla-Alonso S, Hashimoto A, Vonteddu P, Behera R, Goins MA (2017) Cancer-associated fibroblasts neutralize the anti-tumor effect of CSF1 receptor blockade by inducing PMN-MDSC infiltration of tumors. Cancer Cell 32:654–668.e655

32 Kwon HJ, Lagace TA, McNutt MC, Horton JD, Deisenhofer J (2008) Molecular basis for LDL receptor recognition by PCSK9. Proc Natl Acad Sci USA 105:1820–1825

33 Leach DR, Krummel MF, Allison JP (1996) Enhancement of antitumor immunity by CTLA-4 blockade. Science 271:1734–1736

34 Leone RD, Zhao L, Englert JM, Sun IM, Oh MH, Sun IH, Arwood ML, Bettencourt IA, Patel CH, Wen J (2019) Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science 366:1013–1021

35 Lintner NG, McClure KF, Petersen D, Londregan AT, Piotrowski DW, Wei L, Xiao J, Bolt M, Loria PM, Maguire B (2017) Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain. PLoS Biol 15:e2001882

36 Liu X, Bao X, Hu M, Chang H, Jiao M, Cheng J, Xie L, Huang Q, Li F, Li CY (2020) Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer. Nature 588:693–698

37 Ma L, Wang L, Nelson AT, Han C, He S, Henn MA, Menon K, Chen JJ, Baek AE, Vardanyan A (2020) 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression. Cancer Lett 493:266–283

38 Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P (2017) Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol 14:399–416

39 Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507–1517

40 Maxwell KN, Fisher EA, Breslow JL (2005) Overexpression of PCSK9 accelerates the degradation of the LDLR in a postendoplasmic reticulum compartment. Proc Natl Acad Sci USA 102:2069–2074

41 Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314:126–129

42 Neelapu SS, Tummala S, Kebriaei P, Wierda W, Gutierrez C, Locke FL, Komanduri KV, Lin Y, Jain N, Daver N (2018) Chimeric antigen receptor T-cell therapy—assessment and management of toxicities. Nat Rev Clin Oncol 15:47–62

43 Newton RH, Shrestha S, Sullivan JM, Yates KB, Compeer EB, Ron-Harel N, Blazar BR, Bensinger SJ, Haining WN, Dustin ML (2018) Maintenance of CD4 T cell fitness through regulation of Foxo1. Nat Immunol 19:838–848

44 Patel CH, Powell JD (2017) Targeting Tcell metabolism to regulate T cell activation, differentiation and function in disease. Curr Opin Immunol 46:82–88

45 Poirier S, Mayer G, Benjannet S, Bergeron E, Marcinkiewicz J, Nassoury N, Mayer H, Nimpf J, Prat A, Seidah NG (2008) The proprotein convertase PCSK9 induces the degradation of low density lipoprotein receptor (LDLR) and its closest family members VLDLR and ApoER2. J Biol Chem 283:2363–2372

46 Proto JD, Doran AC, Subramanian M, Wang H, Zhang M, Sozen E, Rymond CC, Kuriakose G, D’Agati V, Winchester R (2018) Hypercholesterolemia induces T cell expansion in humanized immune mice. J Clin Investig 128:2370–2375

47 Raal FJ, Honarpour N, Blom DJ, Hovingh GK, Xu F, Scott R, Wasserman SM, Stein EA (2015) Inhibition of PCSK9 with evolocumab in homozygous familial hypercholesterolaemia (TESLA Part B): a randomised, double-blind, placebo-controlled trial. Lancet 385:341–350

48 Raal FJ, Hovingh GK, Blom D, Santos RD, Harada-Shiba M, Bruckert E, Couture P, Soran H, Watts GF, Kurtz C (2017) Long-term treatment with evolocumab added to conventional drug therapy, with or without apheresis, in patients with homozygous familial hypercholesterolaemia: an interim subset analysis of the open-label TAUSSIG study. Lancet Diabetes Endocrinol 5:280–290

49 Rafiq S, Hackett CS, Brentjens RJ (2020) Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17:147–167

50 Riddell SR, Greenberg PD (1990) The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J Immunol Methods 128:189–201

51 Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee W, Yuan J, Wong P, Ho TS (2015) Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348:124–128

52 Shi X, Bi Y, Yang W, Guo X, Jiang Y, Wan C, Li L, Bai Y, Guo J,Wang Y (2013) Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 493:111–115

53 Stanford SM, Rapini N, Bottini N (2012) Regulation of TCR signalling by tyrosine phosphatases: from immune homeostasis to autoimmunity. Immunology 137:1–19

54 Stein EA, Honarpour N, Wasserman SM, Xu F, Scott R, Raal FJ (2013) Effect of the proprotein convertase subtilisin/kexin 9 monoclonal antibody, AMG 145, in homozygous familial hypercholesterolemia. Circulation 128:2113–2120

55 Sukumar M, Liu J, Ji Y, Subramanian M, Crompton JG, Yu Z, Roychoudhuri R, Palmer DC, Muranski P, Karoly ED (2013) Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function. J Clin Investig 123:4479–4488

56 Sun D, Wang J, Han Y, Dong X, Ge J, Zheng R, Shi X, Wang B, Li Z, Ren P (2020) TISCH: a comprehensive web resource enabling interactive single-cell transcriptome visualization of tumor microenvironment. Nucleic Acids Res. https://doi.org/10.1093/nar/gkaa1020

57 Togashi Y, Shitara K, Nishikawa H (2019) Regulatory T cells in cancer immunosuppression- implications for anticancer therapy. Nat Rev Clin Oncol 16:356–371

58 van der Merwe PA, Dushek O (2011) Mechanisms for T cell receptor triggering. Nat Rev Immunol 11:47–55

59 Wang F, Beck-García K, Zorzin C, Schamel WW, Davis MM (2016) Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol. Nat Immunol 17:844–850

60 Wang W, Zou W (2020) Amino acids and their transporters in T cell immunity and cancer therapy. Mol Cell 80:384–395

61 Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH, Ariyan CE, Gordon RA, Reed K (2013) Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med 369:122–133

62 Wu W, Shi X, Xu C (2016) Regulation of T cell signalling by membrane lipids. Nat Rev Immunol 16:690–701

63 Xu CQ, Gagnon E, Call ME, Schnell JR, Schwieters CD, Carman CV, Chou JJ, Wucherpfennig KW (2008) Regulation of T cell receptor activation by dynamic membrane binding of the CD3 epsilon cytoplasmic tyrosine-based motif. Cell 135:702–713

64 Yang W, Bai Y, Xiong Y, Zhang J, Chen S, Zheng X, Meng X, Li L, Wang J, Xu C (2016) Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism. Nature 531:651–655

65 Zech T, Ejsing CS, Gaus K, de Wet B, Shevchenko A, Simons K, Harder T (2009) Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling. EMBO J 28:466–476

66 Zelcer N, Hong C, Boyadjian R, Tontonoz P (2009) LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor. Science 325:100–104

67 Zhang DW, Lagace TA, Garuti R, Zhao Z, McDonald M, Horton JD, Cohen JC, Hobbs HH (2007) Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation. J Biol Chem 282:18602–18612

68 Zhang Y, Ertl HC (2016) Starved and asphyxiated: how can CD8(+) T cells within a tumor microenvironment prevent tumor progression. Front Immunol 7:32

69 Zhang Y, Kurupati R, Liu L, Zhou XY, Zhang G, Hudaihed A, Filisio F, Giles-Davis W, Xu X, Karakousis GC (2017) Enhancing CD8 (+) T cell fatty acid catabolism within a metabolically challenging tumor microenvironment increases the efficacy of melanoma immunotherapy. Cancer Cell 32:377–391