Abstract 2400

Poster Board II-377

Tumor necrosis factor superfamily member BAFF-R has been shown to transduce a powerful survival signal in B lineage cells upon ligation by BAFF, its soluble high affinity ligand. The availability of BAFF in lymphoid organs and its detectable levels in the blood suggest that it is the control of receptor expression by the B cell rather than ligand production by supportive cells that limits the activity of this survival signal. The presence of BAFF-R on the surface of B cells from the transitional stages of development through maturity mirrors the expression of BAFF-R on the malignancies thought to arise from these various developmental stages and may enable B lineage malignancies to receive an anti-apoptotic signal from BAFF that reinforces and supports the expansion of the malignant cells. This additional survival signal may be crucial in indolent malignancies like chronic lymphocytic leukemia (CLL), a cancer characterized more by the prolonged survival of the cancerous cells than by their abundant proliferation. While there are ongoing efforts to thwart this survival pathway with antibody-based therapeutics directed toward BAFF and BAFF-R, understanding the regulation of BAFF-R expression may open a more proximal, potent, and novel therapeutic avenue. Rel/NF-kB family member c-Rel is an appealing candidate BAFF-R regulator, since its nuclear localization and expression within B lineage cells roughly correlate with BAFF-R expression, predominating at nuclear kB sites as a dimer with p50 in mature B cells and declining in plasma cells. Recently published evidence has shown that B cell receptor (BCR)-induced c-Rel expression can modulate BAFF-R in murine transitional B cells, providing a critical increase in BAFF-R expression and thus, BAFF-responsiveness, during B cell development (Castro, I. J Immunol. 2009; 182(12): 7729). A role for the BCR is especially provocative in the context of CLL, which is commonly characterized by lower levels of surface BAFF-R despite a generally activated phenotype and, in the unmutated subtype, typically increased expression of genes downstream of the BCR. In related studies, we have analyzed BAFF-R promoter activity in BAFF-R expressing B cells vs. myeloma cell lines, which generally lack expression of this receptor, and have indeed demonstrated differential promoter activity of the region directly upstream of the BAFF-R gene. The goal of our current study was to focus on the role of the transcription factor, c-Rel, in regulating BAFF-R expression. While in silico tools identifying transcription factor binding sites in the three kilobases upstream of the BAFF-R gene found numerous possible c-Rel binding sites, only three c-Rel sites remained under the most stringent search conditions. An initial chromatin immunoprecipitation (ChIP) experiment with an anti-c-Rel antibody suggested that the binding site within the first five hundred bases upstream of the transcriptional start site may be bound by c-Rel: ChIP with RAMOS B cells showed a 6-fold increase in the precipitated DNA of the region over the isotype-matched antibody control. Electrophoretic mobility shift assays (EMSAs) using peripheral blood (PB) B cell nuclear extracts both from normal donors and CLL patients as well as malignant cell lines, demonstrate the different levels of binding to the putative c-Rel sites identified in silico and support the hypothesis that c-Rel directly regulates BAFF-R expression in these cells. These assays, which demonstrated the affinity of c-Rel for the putative promoter sites upstream of the BAFF-R gene, led to the transfection of new luciferase reporter constructs into malignant B and plasma cell lines, which demonstrate the effects both of amplifying and of mutating the c-Rel binding sites in our reporter assays. We supplemented these assays with experiments measuring the production of c-Rel in these cells both by quantitative RT-PCR, which demonstrated a twenty-fold decrease in plasma cell line ALMC-1 and a two-fold decrease in CLL cell c-Rel mRNA as compared to normal PB B cells, and by Western blot. Finally, we used a c-Rel expression vector to directly increase BAFF-R expression in the malignant cell lines. By identifying c-Rel expression and activation as a possible mechanism of direct control of BAFF-R expression in human B cells, we reveal a critical new rationale for the use of NF-kB inhibitors as chemotherapeutic adjuvants in B cell cancers.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.