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.
Highlights
Rheumatoid arthritis (RA) is a common autoimmune disease for which there is no known cure
A current challenge in human genetics is to follow-up ‘‘hits’’ from genome-wide association studies (GWAS) to guide drug discovery for complex traits
We demonstrate that the rheumatoid arthritis (RA) risk allele is a gain-of-function allele that increases the amount of CD40 on the surface of primary human B lymphocyte cells from healthy control individuals
Summary
Rheumatoid arthritis (RA) is a common autoimmune disease for which there is no known cure. Risk alleles help calibrate the amount of target modulation that is tolerable in humans, as gain-of-function and loss-of-function mutations in the same gene can be assessed for clinical phenotypes in carriers of these mutations. Consistent with these concepts, known drug targets that are safe and effective in humans appear on the list of genes identified by genome-wide association studies (GWAS) of common diseases [3], which suggests that other GWAS hits represent targets worthy of further investigation [4]
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