Overexpression of TOSO in CLL Is Triggered by B-Cell Receptor Signaling and Associated with Progressive Disease.

Abstract

Resistance towards apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals like B-cell receptor stimulation (BCR) are considered to be responsible for accumulation of malignant B cells in CLL . TOSO, also known as Fas-inhibitory molecule 3 (FAIM3), was identified as overexpressed candidate gene in CLL based on re-evaluation of publicly available microarray data sets. Based on primary CLL samples from 106 patients, TOSO expression was compared to healthy donor B cells using quantitative real-time PCR, western-blot, flow cytometry and immunohistochemistry. To reveal underlying mechanisms of TOSO overexpression, B-cell receptor (BCR) and CD40Ligand stimulation as well as bone marrow stroma cell co-incubation was performed. Apoptotic resistance was assessed by annexin V/7-AAD flow cytometry in context of CH11-Fas-agonistic antibody. TOSO was identified to exhibit elevated relative expression of 6.8 compared to healthy donor B cells using quantitative real-time PCR (p=0.004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy and unmutated IgVH status. CD38+ CLL subsets harboring proliferative activity showed significantly enhanced TOSO expression. Immunohistochemistry revealed upregulation of TOSO in lymph nodes of CLL patients. In lymph nodes derived from healthy donors TOSO was detected in single plasmocytoid cells within the germinal center and in the marginal zone. No specific staining was seen in follicular lymphomas. CLL-specific upregulation of TOSO was confirmed by RT-PCR, samples of follicular lymphomas, DLBCL, marginal zone lymphoma and Hodgkin cell lines did not reveal TOSO up-regulation. We evaluated functional mechanisms of aberrant TOSO expression in CLL cells and identified TOSO expression significantly being induced by BCR-stimulation compared to control cells (relative expression (RE) 8.25 vs. 4.86, p=0.013). In contrast, CD40L signaling significantly reduced TOSO expression (RE 2.60; p=0.007). Spontaneous apoptosis of CLL cells was significantly reduced by BCR-stimulus, in CD40Ligandstimulated CLL samples a slight sensitization towards Fas-mediated apoptosis was seen. In summary, we show that the anti-apoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38+ cell subset. Both association with unmutated IgVH and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL. Down-regulation of TOSO by CD40Ligand in the context of CD40Ligand-mediated Fas-sensitization of CLL reveal TOSO as a new anti-apoptotic factor in CLL. CLL-specific over-expression of the transmembrane protein might further offer new therapeutic strategies in CLL treatment.

Novel X‐linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL‐mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis

“…Only patients without prior therapy or patients who had a period of at least 6 months since their last chemotherapy were included in this study. Fresh CLL samples were enriched by applying B‐RosetteSep (StemCell Technologies, Vancouver, BC, USA) and Ficoll‐ Hypaque (Seromed, Berlin, Germany) density gradient purification, resulting in purity of more than 98% of CD19/CD5 CLL cells (Pallasch et al , 2008a). Control cells were isolated from healthy blood donors using untouched depletion (naive B‐cell isolation kit II; Miltenyi, Bergisch‐Gladbach, Germany) resulting in at least 90% purity of B cells.…”

“…Control cells were isolated from healthy blood donors using untouched depletion (naive B‐cell isolation kit II; Miltenyi, Bergisch‐Gladbach, Germany) resulting in at least 90% purity of B cells. Isolated cells were cultured as previously described (Pallasch et al , 2008a, 2009). This study was approved by the ethics committee of the University of Cologne and blood samples were given with informed consent according to the Helsinki protocol.…”

Sustained NF‐kappaB activity in chronic lymphocytic leukemia is independent of genetic and epigenetic alterations in the TNFAIP3 (A20) locus

“…Control cells were isolated from healthy blood donors using untouched depletion (naive B‐cell isolation Kit II; Miltenyi, Bergisch‐Gladbach, Germany) resulting in at least 90% purity of B cells. Isolated cells were cultured as previously described 18, 19. This study was approved by the ethics committee of the University of Cologne; blood samples were given with informed consent according to the Helsinki protocol.…”

Fas apoptosis inhibitory molecules: more than death‐receptor antagonists in the nervous system

“…In the context of cancer, FAIM3 was shown to be overexpressed in chronic lymphocytic leukemia (Proto-Siqueira et al 2008) and associated with disease progression (Pallasch et al 2008;Pallasch and Wendtner 2009). Protein and mRNA expression analyses demonstrated that high levels of FAIM3 expression were a unique feature of chronic lymphocytic leukemia cells and not present in other B-cell lymphomas (Pallasch and Wendtner 2009).…”