title: Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.
abstract: 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.
authors.affiliation: Division of Rheumatology, Immunology, and Allergy and Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.forename: Dorothée
authors.initials: D
authors.lastname: Diogo
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.forename: Jim
authors.initials: J
authors.lastname: Spoonamore
authors.forename: Vlado
authors.initials: V
authors.lastname: Dancik
authors.forename: Lude
authors.initials: L
authors.lastname: Franke
authors.forename: Fina
authors.initials: F
authors.lastname: Kurreeman
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Rossin
authors.forename: Grant
authors.initials: G
authors.lastname: Duclos
authors.forename: Cathy
authors.initials: C
authors.lastname: Hartland
authors.forename: Xuezhong
authors.initials: X
authors.lastname: Zhou
authors.forename: Kejie
authors.initials: K
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Philip L
authors.initials: PL
authors.lastname: De Jager
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Siminovitch
authors.forename: Alexandra
authors.initials: A
authors.lastname: Zhernakova
authors.forename: Soumya
authors.initials: S
authors.lastname: Raychaudhuri
authors.forename: John
authors.initials: J
authors.lastname: Bowes
authors.forename: Steve
authors.initials: S
authors.lastname: Eyre
authors.forename: Leonid
authors.initials: L
authors.lastname: Padyukov
authors.forename: Peter K
authors.initials: PK
authors.lastname: Gregersen
authors.forename: Jane
authors.initials: J
authors.lastname: Worthington
authors.forename: Namrata
authors.initials: N
authors.lastname: Gupta
authors.forename: Paul A
authors.initials: PA
authors.lastname: Clemons
authors.forename: Eli
authors.initials: E
authors.lastname: Stahl
authors.forename: Nicola
authors.initials: N
authors.lastname: Tolliday
authors.forename: Robert M
authors.initials: RM
authors.lastname: Plenge
mesh_terms: D000483:Alleles; D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics; D001172:Arthritis, Rheumatoid / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D019013:CD40 Antigens / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D040641:Quantitative Trait Loci / Q000235:genetics; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology
chemical_list: D018941:Antigens, CD19; D019013:CD40 Antigens; D016328:NF-kappa B; D054852:Small Molecule Libraries