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.

Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases

“…53,54). The CD40 ligand is a particularly compelling candidate, as the locus encoding the CD40 receptor is an established GWAS locus in RA and MS, has been functionally studied in cell culture and animal models, and was the focus of a recent large-scale RA drug-screening effort 55 .…”

Revealing pathway cross-talk related to diabetes mellitus by Monte Carlo Cross-Validation analysis

“…CD40 signaling in professional antigenpresenting cells, including B cells, macrophages, and dendritic cells, is critical for the efficient activation of humoral and cell-mediated immune responses [25][26][27]. It was verified that CD40 signaling pathway represents a good example of a pathway for which human genetics helped to guide drug development in rheumatoid arthritis [28]. Additionally, another study has shown that CD40 pathway activation status may predict the antitumor activity of CD40-stimulating therapeutic drugs [29].…”

Immunogenetics of rheumatoid arthritis: Understanding functional implications

“…Other examples of functional studies to elucidate the mechanism of action of the putative causal variant include PADI4 [49], TNFAIP3 [53], IL6R [54], NFKBIE [55], CD5 [56] (Table 1) as well as TYK2, CCR6, IL2RA and CD40. The CD40 variant leads to increased cell surface expression of CD40 protein on B cells, leading to enhanced NF-kB pathway activation [57]. Chemokine receptor 6 (CCR6) is considered as a surface marker for Th17 cells [58].…”