A Constitutivly Active NFkB Non Canonical Pathway Is Maintained by RelB Repression of TRAF2 to Prevent Autophagy-Mediated Cell Death in Hodgkin Lymphomas

Abstract 428

NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell processes such as proliferation, apoptosis and inflammation. The REL or NFkB family is comprised of hetero or homo dimeric combinations of five members: RELA (p65), RELB, c-REL, NFKB1 (p50) and NFkB2 (p52).

Despite considerable evidence supporting the role of the REL members in the immune system and lymphomagenesis, it has never been accurately established whether specific NFkB dimers control a particular set of target genes that account for the biological functions known to be mediated by these TFs. Furthermore, it is not clear whether NFkB controls different targets in distinct subsets of germinal center (GC) B cell derived lymphomas.

We performed loss of function experiments delivering specific shRNAs to knock down each REL member in lymphoma cell lines modeling sequential stages of the GC B cell maturation: Mantle Cell Lymphoma (MCL), Diffuse Large B Cell Lymphoma [DLBCL including ABC-Like (Activated B Cell), GCB-Like (Germinal Center B Cell) and PMBL (Primary Mediastinal B cell Lymphoma)], Burkitt Lymphoma (BL) and Hodgkin Lymphoma (HL). All the NHL subsets showed a dependency on RelA and c-Rel to survive but were largely unaffected by RelB depletion. Only HL cells were dramatically sensitive to RelB knock-down.

ChIP-seq analysis uncovered an extensive NFkB network in BJAB (GCB), HBL-1 (ABC), U-H01 (HL) and human primary centroblasts. This data shaped two network architectures: a RelB hierarchical network in HL and a c-rel hierarchy in NHL. Out of 4,581 genes that had relB binding (+/− 2kB TSS) in HL cells, more than 70% were only relB targets in HL as compared to NHL cells.

Microarray analysis of these cell lines following RelA, RelB or c-Rel knockdown was merged with our ChIP-seq data. The overlap of RelB peaks and RelB shRNA downregulated targets showed that RelB directly controlled cell cycle regulators and apoptosis inhibitors including CCND3, CDK6, BCL2 and BCLxL. Interestingly, RelB maintained HL cell viability through BCL2 induction since expression of a BCL2 cDNA rescued HL cell lines from the shRNA RelB lethality.

Strikingly, the RelB induced BCL2 and BCLxL protein down regulation in HL cells does not kill these cells by predicted apoptotic mechanisms. Morphological and molecular evidence indicated that RelB inhibition lead to cell death by autophagy. Electron microscopy revealed intact nucleus with lack of condensed chromatin and citosolic vacuoles. After RelB inhibition, the well known autophagy marker L3C, is completely degraded meanwhile ATG7 protein expression, a key player in the autophagosomes development, is enhanced. RelB suppressed BCL2/BCL-xL dependent autophagic cell death by maintaining high levels of these two pro-survival molecules interacting with the autophagy executing protein Beclin-1. The interaction is disrupted by the RelB shRNA.

We found that RelB is engaged in a positive regulatory loop that keeps the non canonical arm of the NFkB pathway on.

Activation of the alternative pathway centers on the modulation of NIK (NFkB inducing kinase). In physiological conditions, NIK undergoes constitutive ubiquitin-dependent degradation through the TRAF-2/TRAF-3/c-IAP-1/c-IAP-2 complex, which keeps its abundance below the threshold required for its function. We found that HL cell lines lack TRAF-2 or TRAF-3 protein expression which leads to stabilization and accumulation of NIK. RelB knock down is followed by up-regulation and/or re-expression of TRAF members and depletion of NIK protein. Remarkably, lymph nodes of HL patient biopsies showed NIK protein citosolic expression by IHC in the Reed-Sternberg Cells, indicating stabilization and accumulation of NIK that accounts for constitutive NFkB-2/p100b processing to release active p52-RelB dimers.

In summary, we found that HL is the only GC B cell derived malignancy that relies on RelB for viability, and thus differentiates Hodgkin Lymphomas from non-Hodgkin lymphomas. These data argue that each REL member has specific and unique functions and targets, as we predicted from the lack of similarities in their transactivation domains. Of clinical relevance, HL patient samples reproduced the alternative NFkB pathway molecular alterations that we initially observed in HL cell lines.

Furthermore, the unique Hodgkin Lymphoma RelB dependency makes the non canonical NF-kB signaling pathway an attractive therapeutic target.