A BAFF-R Mutation Associated with Non-Hodgkin Lymphoma Exhibits Altered TRAF Binding and Reveals New Insights Into Proximal BAFF-R Signaling

Abstract

468 The requirement for BAFF and BAFF-R in normal human and murine B cells is well studied, but there is also significant evidence to suggest that BAFF plays an important role in malignant B cell proliferation and survival. Serum BAFF levels are elevated in patients with non-Hodgkin lymphoma (NHL) and high BAFF levels correlate with aggressive disease and a poor response to therapy. There is also increasing genetic evidence suggesting an association between the development of human disease and genetic variation in genes encoding BAFF and its receptors. Mutations in TNFRSF13B (TACI) were identified in patients with familial common variable immunodeficiency (CVID) and IgA deficiency and we have found that single nucleotide polymorphisms (SNP) in TNFSF13B (BAFF) are associated with elevated BAFF levels and risk for developing NHL. To build upon these findings we sequenced BAFF and its receptors; TNFSF13B, TNFRSF13B, TNFRSF17(BCMA), and TNFRSF13C (BAFF-R) in NHL patients to identify novel genetic variants that may be associated with NHL risk. Among 40 individual samples (20 controls and 20 follicular lymphoma (FL) cases) that were bi-directionally sequenced we identified a heterozygous cytosine to thymidine transition in 1 patient specimen at position 475 (C475T) of TNFRSF13C. The C475T transition encodes a missense substitution of tyrosine for histidine in codon 159 (H159Y) in the highly conserved cytoplasmic tail of BAFF-R, adjacent to the TRAF3 binding motif PVPAT. We next expanded our analysis of BAFF-R H159Y and analyzed NHL tumor biopsies for the presence of the mutation. 4/41 (10%) follicular lymphomas (FL), 2/42 (5%) diffuse large B cell lymphomas, 1/22 (5%) lymphoplasmacytic lymphomas (LPL), and 1/24 (4%) mucosal associate lymphoid tissue lymphomas carried the heterozygous mutation. The BAFF-R H159Y mutation was not detected in any of the normal control DNA from healthy donors (n=100). Given its close proximity to the TRAF3 binding site in the cytoplasmic domain of BAFF-R we first wanted to determine if the H159Y mutation altered BAFF induced signaling. We generated cell lines that express HA-tagged wildtype BAFF-R, BAFF-R with the H159Y mutation, or BAFF-R with an ablated TRAF3 binding site as a negative control. Analysis of cells expressing H159Y BAFF-R demonstrates that this mutation results in increased BAFF-R-mediated NFκB1 and NF-κB2 activation. The enhanced signal activated by BAFF-R H159Y is coupled with a several fold increase in TRAF3, TRAF2, and TRAF6 recruitment to BAFF-R and increased IgM production. We further demonstrate that recruitment of TRAF6 to BAFF-R is not unique to the mutant H159Y BAFF-R, but is also an important and necessary feature of BAFF-R signaling in normal B cells. Collectively, our data identify a novel lymphoma-associated mutation in BAFF-R and describe exciting new aspects of BAFF-R signaling that are important for understanding normal B cell homeostasis and function, as well as pathogenic BAFF-R contributions to human disease. Disclosures: No relevant conflicts of interest to declare.

CVID-Associated B Cell Activating Factor Receptor Variants Change Receptor Oligomerization, Ligand Binding and Signaling Responses

“…It changes the conserved His159 against Tyr affecting a region, which is conserved between birds and mammals (Supplementary Table S2), as it is directly adjacent to the TNF receptor-associated factor (TRAF3) recognition motif PVPAT. The H159Y variant results in a hyperactive receptor with enhanced recruitment of TRAF3 and TRAF6 (22,25). H159Y has an increased prevalence in patients suffering from Sjögren's syndrome-related lymphoma, non-Hodgkin lymphoma and multiple sclerosis (22,25,26).…”

“…The H159Y variant results in a hyperactive receptor with enhanced recruitment of TRAF3 and TRAF6 (22,25). H159Y has an increased prevalence in patients suffering from Sjögren's syndrome-related lymphoma, non-Hodgkin lymphoma and multiple sclerosis (22,25,26). H159Y and P21R are frequently found in linkage disequilibrium (23,26) suggesting that this allelic combination had a selective advantage.…”

TRAF3, ubiquitination, and B‐lymphocyte regulation

“…BAFF-R activates the PI3K-Akt and NF-jB2 survival pathways (58)(59)(60). Upon activation, BAFF-R recruits TRAF molecules including TRAF2, TRAF3, and TRAF6 to activate NF-jB1 and NF-jB2 pathways (5,18,61). TRAF2, cIAP1/2, and TRAF3 appear to negatively regulate BAFF-R mediated NF-jB2 activation (4, 5, 18).…”

“…In the context of BAFF‐R signaling to B cells, TRAF6 is important for activation of the classical NF‐κB1 pathway; in the absence of TRAF6 binding, activation of this pathway is abrogated . TRAF6 associates with Act1 in response to IL‐17 in epithelial cells .…”

“…Thus, prolonged or abnormal signaling through CD40 or BAFF‐R can result in the enhanced survival seen in genetically TRAF3‐deficient B cells. In this regard, it is interesting to note that a human CD40 polymorphism resulting in enhanced TRAF3 binding is associated with amplified signaling to B cells , and a mutant BAFF‐R isolated from a human B‐cell lymphoma exhibits both enhanced TRAF3 association and increased B‐cell signaling .…”