ALBERT

Beschreibung: MicroRNAs (miRNA) play a key role in cellular regulation and, if deregulated, in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). RNAs from primary cells of 50 treatment-naive CLL patients and peripheral B cells of 14 healthy donors were applied to miRNA expression profiling using bead chip technology. In CLL cells, a set of 7 up- and 19 down-regulated miRNAs was identified. Among the miRNAs down-regulated in CLL cells, 6 of 10 miRNA promoters examined showed gain of methylation compared with normal B-cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3′ untranslated region of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site-directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107, and miR-424. Although expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells compared with the levels in healthy donor B cells. In summary, we could demonstrate disruption of miRNA-mediated translational control, partly due to epigenetic transcriptional silencing of miRNAs, with subsequent overexpression of the oncogenic transcription factor PLAG1 as a putative novel mechanism of CLL pathogenesis.

Beschreibung: Abstract 3898 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. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3463 Poster Board III-351 MicroRNAs play a key role in cellular regulation and if deregulated in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). Both deregulations of miRNAs as well as the identification of their functional relevant targets and regulatory circuits in CLL pathogenesis are only partly understood and remain to be elucidated. RNAs from primary cells of 50 treatment-naïve CLL patients and peripheral B-cells of 14 healthy donors were applied to miRNA-expression profiling using bead chip technology. The majority of patients presented with Binet stage A disease and showed a favorable risk profile as assessed by clinical and molecular features. Comparing the total number of miRNA being expressed a significantly lower number of miRNA was detected in CLL compared to normal B cells. The predominance of down-regulated miRNAs in CLL cells was accompanied by highly significantly lower total number of miRNAs expressed above the detection threshold in CLL patients (19.8% vs 23.5%; p

Beschreibung: Abstract 2433 We have previously shown that silencing of the tumor suppressor gene, DAPK1, by genetic and epigenetic mechanisms is associated with the pathogenesis of CLL. To elucidate genes and pathways involved in DAPK1 transcriptional regulation, genome-wide siRNA screens were performed using a novel transgene reporter system involving a stably-integrated BAC construct expressing luciferase under the control of the DAPK1 locus. c-FOS and other members of the FOS gene family were identified as positive regulators, implicating the AP-1 pathway in DAPK1 transcription. A subsequent comprehensive examination of the FOS, JUN and ATF gene families in CLL and healthy B cells was performed. As the expression of the AP-1 gene family is highly subject to fluctuations in stress and mitogenic stimuli, freshly isolated cells were cultured using autologous serum conditions, allowing for a uniform establishment of baseline gene expression. This work reveals an alteration of the relative composition of AP-1 transcription factors in CLL, demarked by a substantial reduction in c-FOS gene expression. We find that the inducibility of c-FOS in CLL following MAPK activation by TPA (ERK-MAPK) is impaired 7.0-fold relative to healthy B cells and is completely abrogated following anisomycin (p38-MAPK) stimulation. The level of c-FOS induction following TPA activation can be used to clearly segregate CLL patients into two non-overlapping groups, those with low (mean=4.0-fold reduced expression; range=3.0–7.2; n=9) and very low (mean=21.7-fold reduced expression; range=17.6–36.7; n=8) following one hour induction versus healthy B cells. The very low c-FOS induction cases are characterized by poor prognostic indicators (IGHV unmutated (0/9 in low vs. 5/8 in very low); 17p, 6q deletion (0/9 in low vs. 3/8 in very low); and a substantially shorter time to progression (102.7±40.4 months in low vs. 10.8±8.6 in very low). Patients with very low c-FOS induction also demonstrate elevated c-MYC induction following activation. TPA-dependent ERK phosphorylation and activation of other immediate early genes, such as EGR1 and c-JUN, is intact in both CLL groups, absolving MAPK pathway dysfunction as a relevant c-FOS silencing mechanism. Reduced c-FOS expression can be partially explained, in the majority of very low c-FOS inducible cases, by elevated levels of miR-155 and miR-221 targeting the c-FOS transcript. Reduced expression of c-FOS and the resulting reconfiguration of AP-1 transcription factor composition may be involved in the pathogenesis of CLL and the subsequent silencing of DAPK1. Disclosures: No relevant conflicts of interest to declare.