Apoptosis resistance concomitant with aberrant upregulation of pro-survival pathways is a main pathogenic mechanism in development and maintenance of chronic lymphocytic leukemia (CLL). Our group recently identified TOSO to be significantly overexpressed in CLL compared to other B cell lymphomas or healthy B cells. Interestingly, TOSO is thought to exert pro-survival signaling, although it remains still enigmatic, how TOSO is regulated and why TOSO is expressed extremely heterogeneous on different B cells entities. Moreover, we previously detected elevated TOSO expression to be associated with progressive disease, including unmutated IgVH status of the B cell receptor (BCR). Since the BCR is a driving force in CLL, TOSO expression was investigated after BCR crosslinking and resulted in an increase of TOSO. To date, the TOSO promoter has not been described yet. Here, we firstly identified the TOSO proximal region to exert promoter activity. Moreover, in silico analysis and phylogenetic footprinting exhibited existence of transcription factor binding sites for NF-κB and BCL6. In luciferase reporter assays, including targeted mutagenesis, NF-κB was confirmed as novel inducer of TOSO expression. Whereas BCL6 binding, confirmed by ChIP and luciferase assays, was shown to exert repressing activity on the TOSO promoter. Although it can explained now how TOSO is regulated by the BCR, the reason for its distinct basal expression levels in normal B cells and other B cell malignancies still remained unclear. Our data illustrate for the first time that DNA hypomethylation of the TOSO promoter is a conspicuous characteristic in CLL patients compared to healthy donors. Indeed, the methylation status seems to play a major role, since the methylation level correlates with TOSO expression also in other B cell lymphomas.
Moreover, it is indispensible to clarify the biologic significance of TOSO, particularly in the CLL relevant B cells. Therefore, we generated a B cell-specific knockout mouse model and identified impaired B cell development characterized by diminished B cell count. Gene expression analysis and flow cytometry revealed a decrease of the B-cell activating factor receptor (BAFF-R). BAFF-R ligation is known to promote B cell survival in particular via degradation of IκBα and translocation of NF-κB to the nucleus, thus activating the NF-κB pathway. Thus, BAFF-R decrease caused by TOSO depletion might lead to the detected reduction of B lymphocytes, which corresponds to the previous observations.
Taken together, this work reveals a counteractive TOSO regulation by transcription activator NF-κB and transcription repressor BCL6 in a BCR-dependent manner in B cells. Moreover, we detected CLL-specific hypomethylation of the TOSO promoter, which is supposed to be causative for elevated TOSO level in CLL. Moreover, our results might reveal a new function of TOSO in pro-survival signaling and B cell homeostasis, supporting the anti-apoptotic feature of TOSO in B cells.
Identifying the regulating mechanisms and biological function of the anti-apoptotic TOSO, is an essential step towards elucidation of the underlying molecular causes for the development of CLL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.