Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

English version České info

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10⁻⁹). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10⁻⁹), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Vyšlo v časopise: Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway. PLoS Genet 9(5): e32767. doi:10.1371/journal.pgen.1003487
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003487

Souhrn

Zdroje

1. McInnesIB, SchettG (2011) The pathogenesis of rheumatoid arthritis. N Engl J Med 365: 2205–2219.

2. PlengeR (2010) GWASs and the age of human as the model organism for autoimmune genetic research. Genome Biol 11: 212.

3. SanseauP, AgarwalP, BarnesMR, PastinenT, RichardsJB, et al. (2012) Use of genome-wide association studies for drug repositioning. Nat Biotechnol 30: 317–320.

4. CollinsFS (2011) Reengineering translational science: the time is right. Sci Transl Med 3: 90cm17.

5. PamukcuB, LipGY, SnezhitskiyV, ShantsilaE (2011) The CD40-CD40L system in cardiovascular disease. Ann Med 43: 331–340.

6. GiuntaB, Rezai-ZadehK, TanJ (2010) Impact of the CD40-CD40L dyad in Alzheimer's disease. CNS Neurol Disord Drug Targets 9: 149–155.

7. ChatzigeorgiouA, LyberiM, ChatzilymperisG, NezosA, KamperE (2009) CD40/CD40L signaling and its implication in health and disease. Biofactors 35: 474–483.

8. MacDonaldKP, NishiokaY, LipskyPE, ThomasR (1997) Functional CD40 ligand is expressed by T cells in rheumatoid arthritis. J Clin Invest 100: 2404–2414.

9. BernerB, WolfG, HummelKM, MullerGA, Reuss-BorstMA (2000) Increased expression of CD40 ligand (CD154) on CD4+ T cells as a marker of disease activity in rheumatoid arthritis. Ann Rheum Dis 59: 190–195.

10. KyburzD, CarsonDA, CorrM (2000) The role of CD40 ligand and tumor necrosis factor alpha signaling in the transgenic K/BxN mouse model of rheumatoid arthritis. Arthritis Rheum 43: 2571–2577.

11. MauriC, MarsLT, LondeiM (2000) Therapeutic activity of agonistic monoclonal antibodies against CD40 in a chronic autoimmune inflammatory process. Nat Med 6: 673–679.

12. SeniorK (2000) Anti-CD40 allays rheumatoid arthritis in mice. Lancet 355: 2054.

13. DurieFH, FavaRA, FoyTM, AruffoA, LedbetterJA, et al. (1993) Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261: 1328–1330.

14. ZhengX, SuzukiM, ZhangX, IchimTE, ZhuF, et al. (2010) RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis. Arthritis Res Ther 12: R13.

15. TellanderAC, MichaelssonE, BrunmarkC, AnderssonM (2000) Potent adjuvant effect by anti-CD40 in collagen-induced arthritis. Enhanced disease is accompanied by increased production of collagen type-II reactive IgG2a and IFN-gamma. J Autoimmun 14: 295–302.

16. RaychaudhuriS, RemmersEF, LeeAT, HackettR, GuiducciC, et al. (2008) Common variants at CD40 and other loci confer risk of rheumatoid arthritis. Nat Genet 40: 1216–1223.

17. ConleyME, DobbsAK, FarmerDM, KilicS, ParisK, et al. (2009) Primary B cell immunodeficiencies: comparisons and contrasts. Annu Rev Immunol 27: 199–227.

18. Margolles-ClarkE, KenyonNS, RicordiC, BuchwaldP (2010) Effective and specific inhibition of the CD40-CD154 costimulatory interaction by a naphthalenesulphonic acid derivative. Chem Biol Drug Des 76: 305–313.

19. GrammerAC, SlotaR, FischerR, GurH, GirschickH, et al. (2003) Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J Clin Invest 112: 1506–1520.

20. KalunianKC, DavisJCJr, MerrillJT, TotoritisMC, WofsyD (2002) Treatment of systemic lupus erythematosus by inhibition of T cell costimulation with anti-CD154: a randomized, double-blind, placebo-controlled trial. Arthritis Rheum 46: 3251–3258.

21. DavisJCJr, TotoritisMC, RosenbergJ, SklenarTA, WofsyD (2001) Phase I clinical trial of a monoclonal antibody against CD40-ligand (IDEC-131) in patients with systemic lupus erythematosus. J Rheumatol 28: 95–101.

22. HuangW, SinhaJ, NewmanJ, ReddyB, BudhaiL, et al. (2002) The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum 46: 1554–1562.

23. GerritseK, LamanJD, NoelleRJ, AruffoA, LedbetterJA, et al. (1996) CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci U S A 93: 2499–2504.

24. EarlyGS, ZhaoW, BurnsCM (1996) Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response. J Immunol 157: 3159–3164.

25. KalledSL, CutlerAH, FerrantJL (2001) Long-term anti-CD154 dosing in nephritic mice is required to maintain survival and inhibit mediators of renal fibrosis. Lupus 10: 9–22.

26. KalledSL, CutlerAH, DattaSK, ThomasDW (1998) Anti-CD40 ligand antibody treatment of SNF1 mice with established nephritis: preservation of kidney function. J Immunol 160: 2158–2165.

27. WangX, HuangW, SchifferLE, MiharaM, AkkermanA, et al. (2003) Effects of anti-CD154 treatment on B cells in murine systemic lupus erythematosus. Arthritis Rheum 48: 495–506.

28. MohanC, ShiY, LamanJD, DattaSK (1995) Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J Immunol 154: 1470–1480.

29. JacobsonEM, ConcepcionE, OashiT, TomerY (2005) A Graves' disease-associated Kozak sequence single-nucleotide polymorphism enhances the efficiency of CD40 gene translation: a case for translational pathophysiology. Endocrinology 146: 2684–2691.

30. FehrmannRS, JansenRC, VeldinkJH, WestraHJ, ArendsD, et al. (2011) Trans-eQTLs Reveal That Independent Genetic Variants Associated with a Complex Phenotype Converge on Intermediate Genes, with a Major Role for the HLA. PLoS Genet 7: e1002197 doi:10.1371/journal.pgen.1002197.

31. ZellerT, WildP, SzymczakS, RotivalM, SchillertA, et al. (2010) Genetics and beyond–the transcriptome of human monocytes and disease susceptibility. PLoS ONE 5: e10693 doi:10.1371/journal.pone.0010693.

32. FairfaxBP, MakinoS, RadhakrishnanJ, PlantK, LeslieS, et al. (2012) Genetics of gene expression in primary immune cells identifies cell type-specific master regulators and roles of HLA alleles. Nat Genet 44: 502–510.

33. PluvinetR, PetrizJ, TorrasJ, Herrero-FresnedaI, CruzadoJM, et al. (2004) RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells. Blood 104: 3642–3646.

34. ZhangJH, ChungTD, OldenburgKR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4: 67–73.

35. AnWF, TollidayN (2010) Cell-based assays for high-throughput screening. Mol Biotechnol 45: 180–186.

36. ShawSY, BlodgettDM, MaMS, WestlyEC, ClemonsPA, et al. (2011) Disease allele-dependent small-molecule sensitivities in blood cells from monogenic diabetes. Proc Natl Acad Sci U S A 108: 492–497.

37. SubramanianA, TamayoP, MoothaVK, MukherjeeS, EbertBL, et al. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102: 15545–15550.

38. AlmawiWY, MelemedjianOK (2002) Negative regulation of nuclear factor-kappaB activation and function by glucocorticoids. J Mol Endocrinol 28: 69–78.

39. RubegniM, SacchettiG, BruniG, De MauroG, ProvvediD (1981) A double-blind evaluation of oral indoprofen versus ASA in osteoarthritic patients: influence on haemostatic parameters and clinical effects. Eur J Rheumatol Inflamm 4: 41–48.

40. KartnerN, YaoY, LiK, CrastoGJ, DattiA, et al. (2010) Inhibition of osteoclast bone resorption by disrupting vacuolar H+-ATPase a3-B2 subunit interaction. J Biol Chem 285: 37476–37490.

41. ShiotaN, KovanenPT, EklundKK, ShibataN, ShimouraK, et al. (2010) The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice. Br J Pharmacol 159: 626–635.

42. InglisJJ, CriadoG, AndrewsM, FeldmannM, WilliamsRO, et al. (2007) The anti-allergic drug, N-(3′,4′-dimethoxycinnamonyl) anthranilic acid, exhibits potent anti-inflammatory and analgesic properties in arthritis. Rheumatology (Oxford) 46: 1428–1432.

43. DadgostarH, ZarnegarB, HoffmannA, QinXF, TruongU, et al. (2002) Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes. Proc Natl Acad Sci U S A 99: 1497–1502.

44. KlareskogL, CatrinaAI, PagetS (2009) Rheumatoid arthritis. Lancet 373: 659–672.

45. ScottDL, WolfeF, HuizingaTW (2010) Rheumatoid arthritis. Lancet 376: 1094–1108.

46. KathiresanS, VoightBF, PurcellS, MusunuruK, ArdissinoD, et al. (2009) Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nat Genet 41: 334–341.

47. CohenJ, PertsemlidisA, KotowskiIK, GrahamR, GarciaCK, et al. (2005) Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat Genet 37: 161–165.

48. KotowskiIK, PertsemlidisA, LukeA, CooperRS, VegaGL, et al. (2006) A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am J Hum Genet 78: 410–422.

49. CohenJC, BoerwinkleE, MosleyTHJr, HobbsHH (2006) Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 354: 1264–1272.

50. AbifadelM, VarretM, RabesJP, AllardD, OuguerramK, et al. (2003) Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 34: 154–156.

51. SteinEA, GipeD, BergeronJ, GaudetD, WeissR, et al. (2012) Effect of a monoclonal antibody to PCSK9, REGN727/SAR236553, to reduce low-density lipoprotein cholesterol in patients with heterozygous familial hypercholesterolaemia on stable statin dose with or without ezetimibe therapy: a phase 2 randomised controlled trial. Lancet 380: 29–36.

52. SteinEA, MellisS, YancopoulosGD, StahlN, LoganD, et al. (2012) Effect of a monoclonal antibody to PCSK9 on LDL cholesterol. N Engl J Med 366: 1108–1118.

53. UdaM, GalanelloR, SannaS, LettreG, SankaranVG, et al. (2008) Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of beta-thalassemia. Proc Natl Acad Sci U S A 105: 1620–1625.

54. SankaranVG, MenneTF, XuJ, AkieTE, LettreG, et al. (2008) Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322: 1839–1842.

55. XuJ, PengC, SankaranVG, ShaoZ, EsrickEB, et al. (2011) Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing. Science 334: 993–996.

56. BauerDE, KamranSC, OrkinSH (2012) Reawakening fetal hemoglobin: prospects for new therapies for the beta-globin disorders. Blood

57. HuangR, SouthallN, WangY, YasgarA, ShinnP, et al. (2011) The NCGC pharmaceutical collection: a comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci Transl Med 3: 80ps16.

58. MacarronR, BanksMN, BojanicD, BurnsDJ, CirovicDA, et al. (2011) Impact of high-throughput screening in biomedical research. Nat Rev Drug Discov 10: 188–195.

59. EyreS, BowesJ, DiogoD, LeeA, BartonA, et al. (2012) High-density genetic mapping identifies new susceptibility loci for rheumatoid arthritis. Nat Genet 44: 1336–1340.

60. StahlEA, RaychaudhuriS, RemmersEF, XieG, EyreS, et al. (2010) Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet 42: 508–514.

61. PurcellS, NealeB, Todd-BrownK, ThomasL, FerreiraMA, et al. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81: 559–575.

62. PriceAL, PattersonNJ, PlengeRM, WeinblattME, ShadickNA, et al. (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38: 904–909.

63. RivasMA, BeaudoinM, GardetA, StevensC, SharmaY, et al. (2011) Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease. Nat Genet 43: 1066–1073.