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.

Author notes

Disclosure: No relevant conflicts of interest to declare.