The death-associated protein kinase 1 (DAPK1) : prognostic relevance in pediatric acute lymphoblastic leukemia (ALL) and evaluation as a therapeutic target
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Date
2018-03-26
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Dissertation
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Abstract
In the present study, we referred to previous published data that identified different engraftment phenotypes of patient-derived ALL-samples (Acute Lymphoblastic Leukemia) transplanted on NOD/SCID mice (Non Obese Diabetes/ Severe Combined Immunodeficiency), which demonstrated a connection between patient outcome and engraftment properties. A gene expression analysis of patient-derived xenograft samples identified altered expression levels of apoptosis-related genes, including the pro-apoptotic molecule DAPK1 (Death-Associated Protein Kinase 1) which was significantly downregulated in the TTLshort/ early relapse group (TTL = time to leukemia) compared to TTLlong on transcript level. As a first result of the present study we could confirm these findings and found reduced expression levels of DAPK1 protein in TTLshort compared with TTLlong in the same cohort as well. Out of a group of ten ALL-cell lines we identified two BCP-ALL (B-Cell Precursor-Acute Lymphoblastic Leukemia) (REH, NALM-6) and one mature B-ALL (TANOUE) as low expressing DAPK1 ALL cell lines and one T-ALL (JURKAT) that shows moderate expression levels of DAPK1. Treatment experiments with the DNMT-Inhibitor Decitabine were performed, showing a time- and dose-dependency of Decitabine-induced cell death in all four cell lines. Furthermore, we analyzed expression levels of DAPK1 upon Decitabine-treatment in all four cell lines. Upregulation of DAPK1 transcript expression was observed in all four cell lines whereas DAPK1 protein levels were only increased in REH, NALM-6 and JURKAT cells. Assessing Annexin/PI staining, mitochondrial cytochrome c release and subsequent caspase activation we showed that Decitabine induced apoptosis in BCP-cell lines REH and NALM-6. In line with the cell line data in patient-derived xenograft samples also a dose- and time dependent cell death induction upon Decitabine treatment was found. Moreover, DAPK1 gene expression was markedly enhanced in patient-derived xenograft samples, upon exposure to 0.5 μM Decitabine. However, analysis of DAPK1 promoter methylation performed in cooperation with the research group of C. Plass in Heidelberg revealed no significant difference of methylation status comparing TTLshort and TTLlong. Accordingly to this contradictory result, promoter hypermethylation seems to be not the reason for reduced DAPK1 gene expression in the TTLshort group. Therefore, it is also not clear whether increased DAPK1 expression and apoptosis induction observed upon Decitabine treatment is due to demethylation of the DAPK1 specific promoter. We additionally analyzed SMAD1 (SMA + MAD; SMA = Small Body Size; MAD = Mothers Against Decapentaplegic) expression levels, since SMAD-molecules were previously described to possibly interact with DAPK1 and SMAD1 was also identified to be downregulated in TTLshort phenotype. Expression of both SMAD1-transcript and protein levels revealed to be significantly downregulated in the TTLshort/ early relapse group and were found to be significantly associated with engraftment times (TTL) and a significant correlation was found for DAPK1 and SMAD1 protein levels. Our results demonstrate that DAPK1 may have relevance for risk stratification and prognosis estimation in pediatric high-risk ALL. Furthermore, the integration of Decitabine in treatment regimen of relapsed acute lymphoblastic leukemia might be a promising approach.
Taken together, low DAPK1 expression might contribute to lower apoptosis signaling in TTLshort ALL and increased expression upon Decitabine exposure might point to methylation as regulating mechanism, thus indicating a future marker for prognostication and target for therapy. However, additioinal investigations, particularly with respect to Decitabine and the specific role of DAPK1 in inducing apoptosis (inhibitor studies) need to be addressed before.
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Faculties
Medizinische Fakultät
Institutions
UKU. Klinik für Kinder- und Jugendmedizin
UKU. Klinik für Innere Medizin III
UKU. Klinik für Innere Medizin III
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DFG Project uulm
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Keywords
DAPK1, Leukämie, Epigenetik, Apoptosis, Methylierung, Leukemia; Genetics, Death-associated protein kinases, Methylation, Apoptosis, Epigenomics, Precursor cell lymphoblastic leukemia-lymphoma, DDC 610 / Medicine & health