Dichotomy in NF-κB signaling and chemoresistance in immunoglobulin variable heavy-chain-mutated versus unmutated CLL cells upon CD40/TLR9 triggering

Abstract

Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavychain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-jB (NF-jB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-X L levels, respectively. A dichotomy in NF-jB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-X L levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-X L and remained chemoresistant. The pivotal contribution of Bcl-X L to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-X L levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-jB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-X L levels.

The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

“…Such heterogeneity of response has not been observed when CD40L was used to increase immunogenicity of CLL cells, by stably transducing CD40L either in stromal cells 42 or directly in CLL cells 30,43 to crosslink CD40 on transduced and nontransduced bystander CLL cells. Conceivably, the strength of the signal delivered to the leukemic cells was higher than that provided by the soluble form of CD40L that we have used in this work.…”

“…Recently, correlations between functional features of CD40 ligation and IGHV gene mutational status have been reported in some instances. 42,45 This discrepancy could be simply explained by the different number of cases tested and/or by differences in the experimental models used, as CD40 stimulation was studied together with additional stimuli like cytokines 45 or Toll-like receptors ligands. 42 A final answer will be only obtained with a prospective study involving hundreds of cases to eliminate any selection bias.…”

“…42,45 This discrepancy could be simply explained by the different number of cases tested and/or by differences in the experimental models used, as CD40 stimulation was studied together with additional stimuli like cytokines 45 or Toll-like receptors ligands. 42 A final answer will be only obtained with a prospective study involving hundreds of cases to eliminate any selection bias. Such a study will also help elucidating whether the capacity to respond to CD40 stimulation remains stable over time and can predict the outcome at any given moment during the disease course or whether it modifies indicating a functional switch that may herald a clinical turning point.…”

The miR-17∼92 family regulates the response to Toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes

“…4,8,43 In the case of TLR9 signaling, these distinct cellular responses have been associated with differences in intracellular signal transduction. 8,9,20,43 In particular, CpG-stimulated UM CLL cells exhibit greater and prolonged activation of several important downstream signaling molecules, including the transcription factor NF-kB and the kinases Akt, ERK and JNK. Prolonged activation of these downstream signaling molecules has been associated with a greater capacity to traverse the G1/S checkpoint of the cell cycle, as evidenced by [3H]thymidine or BrdU incorporation.…”

“…According to bioinformatics tools for global analysis of gene function, among the 50 top-ranked GO categories that were selected as containing differentially expressed genes, 20 (40%) were GO categories related to proliferation and cell cycle control (for example, cell division, DNA replication, mitosis and G1/S transition of cell cycle), apoptosis (for example, regulation of apoptosis, induction of apoptosis and anti-apoptosis) and activation of the NF-kB cascade (Supplementary Table S4). 8,20 When performed on M CLL cells, GEP analysis found a set of 379 differentially expressed genes between CpG-stimulated and unstimulated M CLL cells (Supplementary Figure S2 and Supplementary Table S5). Among these genes, only a minority (13 genes) was in common with the genes differentially expressed between CpG-stimulated and unstimulated UM CLL cells (Supplementary Tables S3 --S5).…”