Chronic Lymphocytic Leukemia
Dr. Martina Seiffert and Dr. rer. nat. Claudia Kalla
Fig. 1: FACS Analysis of primary B-CLL cells transfected with GFP-specific mRNA. Transfected cells were stained with 7-AAD 24 hours after transfection with Amaxa B cell Nucleofection Kit.
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Understanding the pathogenesis of B-CLL to develop novel targeted therapies
B cell chronic lymphocytic leukemia (B-CLL), which is the most common leukemia in adults in the Western World, is an incurable disease, and its pathogenesis is only partly understood. In about 80% of all B-CLL patients specific genomic aberrations can be detected, but only in a few of the more frequently affected chromosomal regions, tumor-associated genes have been identified so far. One of the major efforts in our group is therefore the identification and functional characterization of genes in chromosomal regions which are commonly affected in B-CLL patients by deletion, amplification or translocation. For this purpose matrix CGH data, microarray-based expression and epigenetic profiles, and mutational analysis of primary B-CLL cells, B-CLL cell lines and normal B cells have been acquired and extensively evaluated. The results lead us to a list of candidate genes with potential impact in B-CLL pathogenesis, which are currently analyzed in a variety of functional assays.
The leukemic cells in B-CLL show prolonged survival in vivo, but when removed from their natural microenvironment, these cells rapidly die by spontaneous apoptosis, unless cocultured with stromal cells. To analyze the role of microenvironmental factors in the pathogenesis of B-CLL, we have established a coculture model, in which B-CLL cell survival is maintained for up to several months. Via microarray-based expression profiling and Cytokine Antibody Arrays we have analyzed differences in gene expression of B-CLL and stromal cells before and after coculture. Presently, we perform extensive functional analysis of identified candidate genes, testing a variety of biological performances, like survival, apoptosis, migration, and proliferation. For this purpose we use siRNA-mediated Knock-down, as well as transient transgenic expression of selected genes in B-CLL cell lines and primary cells. Of importance for these studies is our previously established transfection protocol for primary B-CLL cells, which are notoriously resistant to most gene transfer techniques.
The B-CLL/stroma cocultures will be further used for compound screens in search for apoptosis-inducing reagents. The screens will include selected inhibitor molecules for identified signalling molecules or pathways, known and novel chemotherapeutics, as well as a library of biological active substances produced by various fungi species. A broader understanding of genomic and cellular abnormalities, as well as microenvironmental factors influencing B-CLL pathogenesis, will further help to test for useful combinations of therapeutics to achieve synergistic effects in the treatment of the patients.
Collaborations
Prof. Dr. Hartmut Döhner, University Clinic Ulm
Prof. Dr. Stephan Stilgenbauer, University Clinic Ulm
CLL
- Translocation t(X;11)(q13;q23) in B-cell chronic lymphocytic leukemia disrupts two novel genes.
- Analysis of 11q22-q23 deletion target genes in B-cell chronic lymphocytic leukaemia: evidence for a pathogenic role of NPAT, CUL5, and PPP2R1B.
- Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids.