NIK and the Alternative NF-κB Pathway in Human Lymphomas.

Abstract 3943

Poster Board III-879

The nuclear factor κB (NF-κB) family of transcription factors is required for the development of T and B lymphocytes and the regulation of the innate and adaptive immune response. Deregulated NF-κB activity has been associated with a number of malignancies and several types of lymphomas depend on NF-κB activity for cell proliferation and survival. A number of studies have been published in the last few years demonstrating the importance of the alternative NF-κB pathway in lymphomas. The NF-κB-Inducing Kinase (NIK or MAP3K14) is a serine/threonine kinase that is essential for the activation of the alternative NF-κB pathway. NIK induces the phosphorylation of the NF-κB member p100, which is followed by the processing of p100 to p52 and its subsequent nuclear translocation. Gene expression data from 106 lymphoma samples with different diagnosis (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), mucosa-associated lymphoid tissue (MALT), nodal marginal zone lymphoma (NMZL) and chronic lymphocytic leukemia (CLL)) were previously generated using Agilent oligonucleotide microarrays. Data analysis indicated variability in NIK expression among the different lymphoma samples, showing a higher expression in CLL and FL samples. NIK expression was closely associated with the expression of other members of the alternative NF-κB pathway, such as NFKB2, RELB and CD40. To investigate the correlation between NIK gene expression and functional pathways, we performed a gene set enrichment analysis (GSEA). We found that the expression of NIK was positively and significantly correlated with several biologically important pathways in both lymphomagenesis and normal leukocyte development and function, such as B- and T-cell receptor pathways, CD40 signaling pathway, and the classical and alternative NF-κB pathways. Twenty seven lymphoma-derived cell lines were examined by Western blot for expression of NIK and p100/p52. The pathway was found to be frequently activated in these cell lines since high levels of p52 were detected in the majority of MCL (5/9 cell lines), Hodgkin lymphoma (3/3), CLL (1/1), DLBCL (7/9) and T-cell lymphoma (5/5) cell lines. Two-thirds of these p52-positive cell lines also expressed NIK and three of them expressed a truncated form of p100. The expression of NIK and p52 was also associated with Epstein-Barr virus (EBV) infection, given that four out of five EBV-positive cell lines showed elevated levels of these proteins. p100/p52 expression was immunohistochemically analyzed using tissue microarrays including paraffin-embedded tissues from totally 356 DLBCL patients. Examination of these samples indicated that the alternative pathway is activated in a subset of these tumors, with nuclear p52 expressed in 17% of the cases. A significant positive correlation between EBV-infection and p52 expression was established and the majority of the p52-positive cases also expressed nuclear p50, suggesting that both the alternative and classical NF-κB pathways are frequently activated in the same tumors. Alternative and classical NF-κB activation was more frequently present in the non-GC DLBCL type, but also observed in a significant proportion of the GC-DLBCL cases. Furthermore, array based comparative genomic hybridization (aCGH) revealed mono-allelic loss of TRAF3, a negative regulator of NIK, in 5 out of 22 DLBCL cases. Taken together, our results show that the alternative NF-κB pathway is activated in a subset of human lymphoma samples and cell lines. This highlights the relevance of NIK as a potential therapeutic target in lymphoid malignancies.