Annelieke Jaspers

c-Abl Kinase Inhibitors Overcome CD40 Mediated Drug Resistance in CLL; Implications for Therapeutic Targeting of Chemoresistant Niches

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the increased drug resistance and searched for novel strategies to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro CD40 stimulation, which resulted in up-regulation of antiapoptotic Bcl-xL, A1/Bfl-1, and Mcl-1 proteins, and afforded resistance to various classes of drugs (fludarabine, bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of Bim-EL protein, but ERK inhibition did not prevent drug resistance. Drugs combined with sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects per individual CD40-stimulated CLL. The antiapoptotic profile of CD40-triggered CLL resembled BCR-Abl-dependent changes seen in chronic myeloid leukemia (CML), which prompted application of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib, prevented the entire antiapoptotic CD40 program in CLL cells, and restored drug sensitivity. These effects also occurred in CLL samples with dysfunctional p53. Importantly, ex vivo CLL LN samples also displayed strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels. These data indicate that CLL cells in chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl inhibitors.

Enhanced formation and survival of CD4+ CD25hi Foxp3+ T-cells in chronic lymphocytic leukemia

Recently, it has been described that patients with chronic lymphocytic leukemia (CLL) have increased numbers of regulatory T (Treg) cells. In the present study, we analysed the mechanism behind Treg cells expansion in CLL. Neither analysis of the T-cell receptor repertoire nor CD45 isoform expression of Treg cells from patients with CLL provided evidence for chronic (tumor) antigenic stimulation as a possible cause for Treg cells expansion in CLL. We found evidence however for increased formation of Treg cells via CD70 costimulation, because we observed that CD40 ligand activated CLL cells (which might be considered a model of lymph node CLL cells) strongly induced CD70-dependent formation of Treg cells. Reverse transcription-multiplex ligation-dependent probe amplification assay expression analysis of 34 apoptosis-regulating genes showed that in comparison with other CD4+ T-cells, Treg cells from both healthy individuals (HD) and patients with CLL had a high expression of pro-apoptotic Noxa and a low expression of anti-apoptotic Bcl-2. Strikingly, Bcl-2 levels of Treg cells in patients with CLL were significantly higher than in HD. Finally, the different apoptotic profile resulted in differences at the functional level, because Treg cells from patients with CLL were more resistant to drug-induced apoptosis than Treg cells from HD. In conclusion, Treg cells in CLL may accumulate both by increased formation, facilitated by CD27-CD70 interaction in the lymph node proliferation centres, and decreased sensitivity to apoptosis because of a shifted Noxa-Bcl-2 balance.

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

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 heavy-chain-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-κB (NF-κB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-XL levels, respectively. A dichotomy in NF-κB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-XL levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-XL and remained chemoresistant. The pivotal contribution of Bcl-XL to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-XL levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-κB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-XL levels.

In vivo Dynamics of Stable Chronic Lymphocytic Leukemia Inversely Correlate with Somatic Hypermutation Levels and Suggest No Major Leukemic Turnover in Bone Marrow

Although accumulating evidence indicates that chronic lymphocytic leukemia (CLL) is a disease with appreciable cell dynamics, it remains uncertain whether this also applies to patients with stable disease. In this study, (2)H(2)O was administered to a clinically homogeneous cohort of nine stable, untreated CLL patients. CLL dynamics in blood and bone marrow were determined and compared with normal B-cell dynamics in blood from five healthy individuals who underwent a similar (2)H(2)O labeling protocol. Average CLL turnover rates (0.08-0.35% of the clone per day) were approximately 2-fold lower than average B-cell turnover rates from healthy individuals (0.34-0.89%), whereas the rate at which labeled CLL cells in blood disappeared (0.00-0.39% of B cells per day) was approximately 10-fold lower compared with labeled B cells from healthy individuals (1.57-4.24% per day). Leukemic cell turnover variables inversely correlated with the level of somatic hypermutation of the CLL clone (IgVH mutations). Although CLL cells in bone marrow had a higher level of label enrichment than CLL cells in blood, no difference between proliferation rates and proapoptotic and antiapoptotic profiles of CLL cells from these compartments was observed. These data suggest that, in stable disease, there is a biological relationship between the degree of somatic hypermutation of the CLL clone and its dynamics in vivo. Furthermore, in contrast to lymph nodes, the bone marrow does not seem to be a major CLL proliferation site.

Detection of p53 dysfunction in chronic lymphocytic leukaemia cells through multiplex quantification of p53 target gene induction.

Detection of p53 dysfunction in chronic lymphocytic leukaemia cells through multiplex quantification of p53 target gene induction

Influence of pressure on the curiepoint of a 70–30% Ni−Cu alloy

Summary The effect of pressure on the resistance phenomena near the Curiepoint of a Cu−Ni alloy has been determined. The Curie temperature increases by increasing pressure 6.10−5 degree per Atm. The result is discussed in connection with recent theories on ferromagnetism.