Networking Modules of Canonical and Alternative NFkB Signaling in Growth and Survival Regulations of Large B Cell Lymphomas

Abstract

Constitutive NF-kB activation is considered a “hallmark” in B-cell malignancies, especially in large B-cell lymphoma (LBCL), a common but heterogenous Non-Hodgkin's lymphoma type. However, the mechanism(s) of activation and interactions of NF-kB components in the nuclear compartment of B-cell lymphomas are still poorly defined. Our findings demonstrate that both the canonical and the alternative NF-kB pathways are constitutively activated in both LBCL cell lines and primary lymphoma cells. NF-kB DNA binding ELISA analysis showed that the canonical NF-kB components p65 and c-rel as well as the non-canonical NF-kB components p52 and relB are constitutively activated in 14 LBCL cell lines and in 14 primary lymphoma patients' cells (both ABC-and GCB-like subtypes). Based on the ELISA data, we found that the p65/c-rel ratio is relatively higher in ABC-like LBCL subtype when compared to GCB-like LBCL subtype, but the alternative NF-kB members' p52 and rel-B are differentially activated in both GCB- and ABC-like LBCL subtypes. Micro-tissue array (MTA) studies confirmed that both NF-kB pathways are constitutively active in LBCL biopsy-cores. The components of both NF-kB pathways were also found to merge and form functional “hybrid” NF-kB complexes in the nuclear compartment. Besides the common hetero-dimeric complexes occur between p50, p65, and c-rel of the canonical NF-kB pathway and between p52 and relB of the alternative NF-kB pathway, components of both pathways also merged to form “hybrid” dimeric complexes, such as p52-relB, p52-c-rel, relB-p65, and relB-c-rel in LBCL cells. Interestingly, p65 and c-rel binds to relB and p52, respectively, with higher affinity in GCB- than in ABC-like LBCL subtype. Our data also showed that activation of both NF-kB pathways and their cross-interaction are not restricted to only neoplastic B cells but that they are also activated and utilized in highly proliferative activated normal B-lymphocyte. Specific siRNA that target individual NF-kB members p65, c-rel, and rel-B have inhibitory activity in lymphoma cell growth, but similar p52 siRNAs do not have inhibitory activity. These studies indicated that in both LBCL cell lines and patient samples (GCB-and ABC-like subtypes), both the canonical and the alternative NF-kB pathways are not only constitutively activated but also interact to form multimeric NF-kB complexes in the nuclear compartment, giving rise to an NF-kB signaling module that not only controls lymphoma cell growth and survival but likely contributes to the heterogeneity of the disease processes. These findings reveal additional aspects of molecular heterogeneity and complexity in NF-kB signaling mechanisms and interacting transcriptional modules, contributing to the pathophysiology of LBCL. These results also highlight the critical importance of multiple constitutively active NF-kB signaling pathways in LBCL and may also contribute to therapeutic refractoriness, particularly in relapsed/refractory LBCLs.