No abstract

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 in-

No abstract

In chronic lymphocytic leukemia (CLL), proliferation centers reside in lymph node (LN) and possibly also bone marrow, where the environment protects CLL cells from apoptotic and cytotoxic triggers. This protective milieu may well contribute to the lack of curative chemotherapy in CLL, and our recent analysis of the distinct profiles of apoptosis regulators in CLL LN versus peripheral blood supports this notion (Smit et al., Blood 2007, 109: 1660). The aim of the present study was to define the molecular basis for the increased drug resistance and to search for novel strategies to circumvent it. To mimic the situation in CLL LN, we applied prolonged in vitro CD40 stimulation of CLL cells, which results in strong upregulation of anti-apoptotic Bcl-xL and Mcl-1. Moreover, we now also report a gradual reduction of Bim at the protein level, further contributing to the anti-apoptotic profile. Using specific inhibitors (PD98059 and MG132), we found that the decrease in Bim is due to ERK-mediated phosporylation and subsequent proteasomal degradation. ERK inhibition during CD40 triggering abrogated the decrease in Bim levels, but did however not re-establish sensitivity to various drugs (fludarabine, Velcade, Roscovitine). In chronic myeloid leukemia (CML), changes in Bcl-xL, Mcl-1 and Bim levels similar to those observed in our CLL/CD40 system are known to depend on BCR-Abl signaling. Therefore, we next applied c-Abl inhibitors Gleevec or Dasatinib in conjunction with CD40. Both drugs caused a profound reversal of most protective CD40 effects; ERK activity, Bim, Bcl-xL and Mcl-1 levels as well as sensitivity to subsequent drug treatment were restored to pre-CD40 values. These effects also occurred in CLL samples with dysfunctional p53 (n=3). Importantly, in CLL LN samples we also found strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels, suggesting that there might be a c-Abl dependent survival pathway in proliferation centers. These data provide a molecular basis for combination strategies that could target refractory niches in CLL, using therapeutics that function independently of p53.

Mainly based on the observation that overexpression of CD23 in B-CLL cells is regulated by Notch2, deregulation of the Notch pathway has been suggested to contribute to the pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). The aim of the present study was to assess a possible functional role of the Notch pathway in CLL. To this end we performed two kind of experiments. First we co-cultured primary CLL cells with either L cells or OP9 cells expressing the Notch ligands DeltaLike1 (DL1) and Jagged1 (Jag1). This did not affect cell survival in CLL. Next we evaluated the effect of the g-secretase inhibitors GSI-1 and GSI-9 (DAPT) which have been shown to block intracellular processing of all four Notch receptors. Here we encountered a surprising dichotomy: whereas DAPT was unable to induce apoptosis in CLL, it did inhibit lineage commitment of early thymic precursors by means of DL-1 triggering of the Notch1 receptor (Dontje et al, Blood 15 March 2006, Vol. 107, No. 6). In contrast we found that GSI-1 was a potent inducer of apoptosis in CLL, but did not affect Notch1 dependent lineage commitment, indicating that GSI-1-induced apoptosis in B-CLL is not due to inhibition of Notch signaling. Instead, we observed efficient GSI-1 mediated blocking of the proteasome, measured using a 20S proteasomal activity assay (Fig. 1). The blocking activity was equivalent to that observed with two well known proteasome inhibitors, Bortezomib and MG-132. In contrast DAPT had no effect on proteasome activity. Furthermore, GSI-1-induced apoptosis was associated with a transcription-independent accumulation of the BH3-only protein Noxa. The pivotal role of Noxa in GSI-1 mediated apoptosis was demonstrated via RNAi in a model system. Importantly, p53 functionality proved not to be required for GSI-1-induced apoptosis. In summary, we have shown that GSI-1 efficiently blocks the proteasome and that this induces p53-independent apoptosis in B-CLL. Therefore, GSI-1, or similar compounds that inhibit g-secretase activity, may be an effective treatment option in B-CLL. Figure 1: Proteasome activity upon GSI-1 treatment The enzymatic activity of the 20S proteasome was measured after adding Bortezomib (20 nM), MG-132 (0.5 μM), GSI-1 (5 μM), DAPT (5 μM), Roscovitine (25 μM) and Fludarabine (100 μM) to cytoplasmic extracts from freshly isolated B-CLL patients. After 15 minutes incubation, the fluorogenic proteasome substrate LVVY-AMC was added. Monitoring the increase in fluorescence over time allowed quantification of the enzymatic activity. Figure 1:. Proteasome activity upon GSI-1 treatment The enzymatic activity of the 20S proteasome was measured after adding Bortezomib (20 nM), MG-132 (0.5 μM), GSI-1 (5 μM), DAPT (5 μM), Roscovitine (25 μM) and Fludarabine (100 μM) to cytoplasmic extracts from freshly isolated B-CLL patients. After 15 minutes incubation, the fluorogenic proteasome substrate LVVY-AMC was added. Monitoring the increase in fluorescence over time allowed quantification of the enzymatic activity.