Regulation and function of Notch2 in B-CLL

Members of the Notch gene family encode transmembrane receptors that control differentiation, proliferation and apoptotic programs in response to ligands expressed on neighbouring cells. Activation of Notch causes cleavage and translocation of the intracellular domain (NotchIC) to the nucleus, where it activates the transcription of CBF1 responsive genes. In our previous study we elucidated the mechanisms leading to the overexpression of the transmembrane glycoprotein CD23, a striking feature of B-cell chronic lymphocytic leukemia (B-CLL) cells. Bandshift assays visualized a prominent transcription factor complex (C1) which binds sequence specific to five different CBF1 sites in the CD23a core promoter region. Using Epstein Barr virus (EBV) infected BL41 cells as a model for CBF1 mediated CD23 expression, C1 was found to be EBV inducible. Since CBF1 is the nuclear target of Notch signaling, we investigated the expression pattern of different members of the Notch gene family and found that Notch2 is a component of C1 and appears to be overexpressed in B-CLL cells. The expression of constitutive active Notch forms is known to lock cells into an immature state of differentiation and to inhibit apoptosis in leukemic cell lines, two attributes which would be consistent with the nature of B-CLL cells. Therefore, Notch2 might not only account for B-CLL specific CD23 expression but also for other phenotypic changes, characteristic for this neoplastic B-cell type.To elucidate the role of Notch2 and its possible implications in the pathogenesis of B-CLL, we plan to perform gain of function experiments by transducing primary B-cells and several CD23 negative B-cell lines, representing different stages of differentiation, with a retroviral expression vector containing the coding region for Notch2IC. Subsequently, putative Notch2 target genes will be identified by standard cDNA microarrays.These experiments are destined to correlate the gene expression signature of B-CLL lymphocytes with that of Notch2IC transduced B-cells. They should also help to clarify the role of Notch2 in relation to B-cell transformation and should lead to new insights into the molecular pathways and phenotypic changes regulated by Notch2 in B-lymphocytes. Since little is known about the role of Notch2 signaling in B-cell development, it is also planned to analyze purified sub-populations of normal B-cells and of B-CLL related B-cell neoplasias for differences in their Notch2 expression. Furthermore, B-CLL samples will be analyzed for the expression of mutated constitutive active Notch2 proteins.

B-cell chronic lymphocytic leukemia is characterized by the relentless accumulation of CD5/CD19/CD23 positive monoclonal B-cells which are unable to undergo apoptosis in vivo. Using the approach of reverse genetics, we have recently shown that Notch2 signaling is involved in the overexpression of CD23 in B-CLL cells. The Notch2 oncogene has a determining function in the development and maintainance of CD5 positive B1a B-cells suggesting a potential role for Notch2 in B-cell leukemogenesis. Here, we present evidence that B-CLL cells express a ligand independent neoplastic form of nuclear Notch2 (Notch2IC). The transcriptional activity of Notch2IC was found to be dependent on PKC-d, thereby explaining the aberrant regulation of CD23 through phorbolesthers (TPA) and IFN- in this leukemia. Moreover, our data indicates that promoter bound Notch2IC correlates with B-CLL cell survival suggesting that Notch2 has an anti-apoptotic function in B-CLL. Since B-CLL cells are locked in an anergic state, we next asked whether Notch2 modulates B-cell receptor signaling and found that retrovirally transduced Notch2IC rescues B-cells from surface immunoglobulin M (sIgM) mediated apoptosis (activation induced cell death or AICD). The relevance of this finding was supported by the fact, that B-CLL cells express the AICD mediator TR3/nur77 which is known to be inhibited from its apoptotic function by the expression of constitutive active NotchIC proteins. In summary, our data provide evidence that B- CLL cells express a neoplastic form of Notch2IC which might protect the malignant clone from peripheral negative selection.