BAFF and APRIL Protect CLL B Cells from Apoptosis through Activation of NF-κB Canonical Pathway.

B cell activating factor belonging to the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) are members of the TNF super family that are potent regulators of normal B cell development and function. BAFF binds to three distinct TNF receptors: BCMA (B cell maturation antigen), TACI (transmembrane activator and CAML interactor) and BAFF-R (also called BR3). Two of these receptors, TACI and BCMA, also bind APRIL. Some TNF super family proteins are known to induce activation of nuclear factor kB (NF-κB). Recent studies revealed two major pathways for NF-κB activation. One is the canonical pathway, which is regulated by the inhibitor of κB (IκB), and the alternative pathway, which is dependent upon the processing of NF-κB2/p100 to p52. The relative role of each pathway in enhancing CLL cell survival have not been well described. The aim of this study was to clarify the mechanism of CLL cell survival supported by BAFF and APRIL. CLL cells express BCMA, TACI, and BR3 by flow cytometry. CLL cells were cultured alone, with recombinant BAFF (rBAFF), or recombinant APRIL (rAPRIL). After 24 hours in culture, cytoplasmic and nuclear extracts were evaluated for p100/p52, p65, and phospho-IκBα by immunoblot analysis. When CLL cells were cultured with rBAFF or rAPRIL, the viability was higher compared to CLL cells in control cultures without such factors, indicating that BAFF or APRIL could protect CLL cells from spontaneous apoptosis. rBAFF could induce translocation of both p52 and p65 to the nucleus. On the other hand, rAPRIL could induce only p65 translocation to the nucleus, meaning that rBAFF could activate both the canonical and the alternative pathway of NF-κB, but rAPRIL could activate only the canonical pathway. These data suggest that signaling from BR3, but not from BCMA or TACI, can activate the alternative pathway. To verify this, CLL cells were cultured with rBAFF and anti-BR3, which can block BAFF binding to BR3. Anti-BR3 at 10μ M could completely inhibit p52 translocation to the nucleus induced by rBAFF. This data indicate that signaling from BR3 is necessary and sufficient to activate the alternative pathway in CLL cells. We examined the role of each pathway in the survival of CLL cells. 10μ M of anti-BR3, which can block activation of the alternative pathway, did not impair survival of CLL cells cultured with rBAFF. On the other hand, an IKKβ inhibitor, 5-(4-fluorophenyl)-2-ureido-thiophene-3 carboxylic acid amide (UTC), which can block the canonical pathway but not the alternative pathway, blocked the capacity of these factors to enhance CLL survival. UTC had less effect on survival induced by rBAFF on isolated normal B cells of healthy donors. We then transfected super repressor IkB (SR- IκB) to CLL B cells. SR- IκB transfected CLL cells underwent apoptosis more readily than control cells transfected with empty vector 24 hours after transfection. The survival of SR-IκB transfected cells could not be enhanced by rBAFF or APRIL. Collectively, BAFF and APRIL protect CLL B cells from apoptosis. Anti-apoptotic effects of these factors apparently are mediated via activation of the NF-κB canonical pathway. We speculate that inhibitors of IKKβ that inhibit the canonical pathway might have therapeutic activity in this disease.