Regulation of NOTCH2 Signaling in B-CLL: Involvement of PKC-δ.

We have previously shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002 May 15;99(10):3742–7). NOTCH2 plays a determining role in the development/homeostasis of CD5+ B1 B-cells and of the related marginal zone (MZ) B2 B-cells, suggesting a potential role for NOTCH2 in B-CLL leukemogenesis. Using electrophoretic mobility shift assays (EMSA) we demonstrate that freshly isolated B-CLL patient samples (n=30) express an activated form of nuclear NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 surface expression, cytogenetics).

Although the majority of cultured B-CLL samples lose their N2IC activity within one day, DNA-bound N2IC complexes could be maintained by low concentrations of the PKC-stimulating phorbol esther PMA (1ng/ml). This was accompanied by the upregulation of CD23. The effect of PMA on N2IC activation and CD23 expression was abrogated by the PKC-δ inhibitor Rottlerin. Since wild type NOTCH2 signaling is regulated through binding to its ligand followed by γ-secretase mediated cleavage and release of the intracellular domain (N2IC), we induced NOTCH2 signaling by PMA in the presence or absence of the γ-secretase inhibitors (GSI) DAPT and compound E. Results demonstrated that the PMA-induced NOTCH2 activity is resistant to GSI treatment in 24 out of 30 B-CLL cases (80%). This suggests that the leukemic cells from the majority of B-CLL patients express an activated form of N2IC which is independent from γ-secretase cleavage and, thus, do not reqire NOTCH2 ligands for signaling.

In conclusion, the results suggest that PKC-δ is involved in the activation/nuclear translocation of N2IC in B-CLL cells. The data may also suggest that deregulated NOTCH2 signaling is an early step in the development of B-CLL and might be critically involved in the aberrant response of the malignant clone to cell fate modulating stimuli acting through PKC.