Dichotomy in NF-κb Signaling and Chemoresistance in IgV_H Mutated Versus Unmutated CLL Cells Revealed by CD40Ligand/CpG Stimulation

Introduction and aim IgV_H mutated (M) and unmutated (UM) Chronic Lymphocytic Leukemia (CLL) cells differ in their response to the TLR-9 ligand CpG. Whereas CpG induces proliferation in UM CLL it induces apoptosis in M CLL cells obtained from peripheral blood (PB). Yet, PB CLL cells are generally not representative of the cycling fraction which resides in lymph node (LN) proliferation centers. In vitro CD40 stimulation of CLL cells enhances NF-κB activity, upregulates anti-apoptotic proteins Bcl-XL and Bfl-1 and results in chemoresistance. These characteristics are highly similar to, and provide a model for, the situation in LN. In the present study we analyzed the effect of CD40 ligation on CpG responses of both IgVH M and UM CLL cells.

Methods An in vitro co-culture system of PB CLL cells with 3T3 fibroblasts expressing human CD40L was employed to which soluble CpG was added. Cells, RNA and protein fractions were analyzed with respect to:

1. apoptosis, proliferation and drug sensitivity and

2. NF-κB signaling and downstream targets.

Results In both prognostic subgroups 72 hours of CD40 stimulation results in chemoresistance to different classes of drugs (fludarabine, velcade and roscovitine), as we reported before. However, an important difference in chemoresistance was seen when soluble CpG was added to the culture; UM CLL cells remained drug resistant, but M CLL cells now became sensitive. A corresponding dichotomy in NF-κB signaling occurred upon stimulation with CD40L/CpG compared to CD40L alone. M CLL cells showed gradually declining classical and alternative NF-κB activation and the downstream target Bcl-XL, whereas in UM CLL cells NF-κB and Bcl-XL levels remained equal. Interestingly, after treatment with ABT-737, a BH3 mimetic which is very effective against Bcl-XL, also UM CLL cells became sensitive for cytostatic drugs underscoring the importance of anti-apoptotic Bcl-XL. Induction of TNF-α secretion, also a downstream effect of NF-κB activation, took place similarly in both subgroups upon stimulation with CD40L and CpG. We hypothesize that the source of the dichotomy NF-κB activation lays in upstream signaling determinants such as cIAP1/2, NIK and TRAF family members.

Conclusions Our data uncover a novel distinction in NF-κB activation and drug susceptibility in mutated versus unmutated CLL cells in a relevant model for LN proliferation centers. Together, they provide a rationale for the development of novel drug regimens targeting the NF-κB pathway to treat UM CLL patients.