A9 (new in 2nd funding period): Role of BCR signaling in chronic lymphocytic leukemia
Expression of the B cell receptor (BCR) is a key feature of most B cell neoplasms including chronic lym-phocytic leukemia (CLL) suggesting that BCR signaling is required for CLL pathogenesis. However, the ex-act role of the BCR, the underlying mechanisms of its activation and the nature of the downstream pathways that induce survival remain unclear. In the proposed project, we aim at the dissection of signaling pathways that are indispensable for the maintenance of CLL cells. For this purpose, we will use a transgenic mouse strain that expresses a tamoxifen-inducible mb1-CreERT2 (or CD79a-CreERT2) thereby enabling efficient B-cell specific activation/inactivation of individual genes. By combining the mb1-CreERT2 allele with loxP-flanked alleles for BCR components such as µHC or Igβ, we will inactivate BCR expression in B cells of the Tcl1 transgenic mice, which develop a CLL-like disease. By monitoring cell survival and characterizing the changes in downstream signaling pathways we aim to identify the factors and signaling modules critical for maintenance of transformed B cells.
An essential module for the regulation of proliferation, differentiation and survival of B cells is induced by the phosphoinositide 3-kinase (PI3K) downstream of BCR signaling. In CLL, PI3K signaling is constitutively active and its inhibition appears to be an efficient therapeutic approach, although the exact role in the sur-vival of CLL B cells is unclear. Available data indicate that the transcription factors (TF) NFAT and FOXP1 belong to the PI3K downstream elements that are involved in the pathogenesis of CLL. For instance, NFATc1 (also known as NFAT2) is constitutively active in CLL B cells, while recurrent mutations in the splice factor SF3B1 lead to enhanced expression of FOXP1 in CLL. Most importantly, FOXP1 was recently shown to prevent the induction of apoptosis in diffuse large B-cell lymphoma and to directly suppress genes such as IRF4 and PRDM1 (encoding Blimp1) in human memory B cells, thereby preventing the terminal differenti-ation of these cells. In this proposal, we will investigate FOXP1 expression in primary CLL B cells and whether it is induced by PI3K signaling. Furthermore, we will test whether FOXP1 represses PRDM1 in CLL B cells and whether NFATc1 is involved in this repression. LoxP-flanked alleles for inactivation of the genes encoding NFATc1 and FOXP1 are available and will be used together with the mb1-CreERT2 allele for induci-ble deletion in Tcl1-transgenic mice. Importantly, our preliminary data show that inducible PRDM1 deletion is sufficient for the induction of a CLL-like disease in mice suggesting that FOXP1-mediated repression of PRDM1 may well be a crucial step in the CLL pathogenesis.
In addition to investigating the importance of BCR expression for CLL B cells and characterizing the CLL-specific downstream signaling pathways, we will analyse the molecular mechanisms that induce BCR signal-ing in CLL B cells. Using a unique reconstitution system, we have recently shown that CLL-derived BCRs possess the exceptional capacity for cell-autonomous signaling independent of external antigen. Crystallo-graphic analyses confirmed our model that CLL-BCRs bind to intrinsic motifs in nearby BCRs on the very same cell. By combining crystallography with the measurement of autonomous signaling of wild type and mutated receptors in our unique reconstitution system, we will generate a structure-function relationship for representative CLL-BCRs. Together, we will generate new animal models and employ classical as well as cutting-edge approaches of biochemistry and molecular/cellular immunology and hope to achieve a com-prehensive characterization of the signaling pathways that are activated by BCR signaling and are important for CLL pathogenesis.
For a current list of project-related publications, please go to this page