Molecular and cellular pathophysiology of NF-κB signaling defects due to disease-causing NFKB2 sequence variants in humans

• Applicant: Professor Dr. Bodo Grimbacher
• Subject Area: Clinical Immunology and Allergology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555820492

NFKB1 and NFKB2 mutations account for the largest subgroup of monogenic-defined antibody deficiencies. Heterozygous NFKB2 germline mutations were first described in DAVID syndrome, a clinical condition with an early-onset variable immunodeficiency and autoimmune ACTH insufficiency and may cause profound clinical problems due to B- and T cell dysfunction, including hypogammaglobulinemia, chronic viral or opportunistic infections, and additional autoimmune phenomena. NFKB2 encodes p100, one of the 4 core modules of the non-canonical NF-κB pathway, which integrates numerous signaling cues during immune cell maturation. p100 dimerizes with REL proteins and inhibits the canonical NF-κB pathway. Pathway activation triggers conversion of the inhibitory cytoplasmic precursor p100 into the DNA-binding subunit p52 and its translocation into the nucleus. Most known pathogenic NFKB2 mutations disable the C-terminal phosphorylation and/or ubiquitination of p100. The disease results from an excessive inhibitory activity - mediated by the unprocessed p100 on both the canonical and the non-canonical pathway - and from insufficient p52. In contrast, central truncating mutations in NFKB2 bypass the p100 precursor stage, and predict the direct expression of p52-like proteins, while haploinsufficiency causes a milder phenotype. We recently discovered additional protein damages caused by missense mutations, including intensified protein decay, loss-of-DNA binding, and loss-of-dimerization, with an interesting genotype-phenotype correlation. In this project, we aim to decipher the molecular and cellular injuries originating from the diverse ‘types’ of pathogenic NFKB2 mutations. Using cell culture overexpression systems, we will first characterize the protein defects per se by analyzing interaction and pathway cross-inhibition activities. The underlying NF-κB signaling flaws will be next dissected in cell line models with endogenous NFKB2 expression: aberrant endogenous NF-κB activities will be tracked in B-, T- and monocytic cells, genetically engineered using the CRISP/Cas9-technique to introduce harmful NFKB2 mutations. To understand the pathogenic mechanisms emanating from dysregulated gene expression patterns, we will determine transcriptional signatures specific for distinct ‘types’ of pathogenic NFKB2 mutations and for distinct severities of the disease phenotypes. B cell subsets will be analyzed by SUMseq, enabling simultaneous chromatin accessibility and gene expression profiling. Data will be integrated to uncover transcription factor-enhancer-gene networks characteristic for each protein defect cluster. With our research, we expect to accelerate diagnosis and precise prognosis in affected patients. Moreover, understanding the distinct roles of each of the NF-B subunits, will aid the development of targeted and specific therapeutic interventions, not only for patients with immunodeficiencies, but also for patients with autoimmunity.

• DFG Programme: Research Grants