Analysis of functional properties of the Acute Promylocytic Leukemia (APL) oncogene PML-RARalpha using a novel ex-vivo assay
Zusammenfassung der Projektergebnisse
Acute Promyelocytic Leukemia (APL) is the most differentiated form of the Acute Myeloid Leukemia (AML) and is characterized by chromosomal translocations. In 98 %of APL cases, the t(15;17) translocation is found. It leads to the expression of the PML-RARα (hPR) fusion protein. Treatment of APL with arsenic trioxide (ATO) or all-trans retinoic acid (ATRA) leads to a complete remission in 90 % of the patients. We are trying to identify common underlying mechanisms of leukemogenesis, which could be targeted in other cancerous diseases. For this, we use APL as a “model disease” to study the mechanisms of leukemogenesis. The functions of PML are very species specific. To study these in murine cells, we consequently used a mouse-adapted fusion protein of murine PML and human RARα (mPR). The functional domains of mPR were inactivated by site-directed mutagenesis. To study their impact on leukemogenesis, we performed a series of tests to analyze three main gain of function properties of PML-RARα: disruption of PML Nuclear Bodies (PML NBs), transcriptional repression, and immortalization of primary murine bone marrow. Thereby we found the RING-Finger and dimerization domain to be essential for the transcriptional repression. Both mutations led to a loss of a prospective SDS-resistant mPR-Dimer band at 230 kDa in Western Blot along with loss of SUMOylation. The loss of the prospective mPR- Dimer band in the RING-Finger mutant implicates a role of this domain in the self-association of mPR. The disruption of the PML Nuclear Bodies (PML NBs) is historically an important property in APL and plays a role in preventing the onset of cellular senescence. We were able to show, that the RING-Finger, SUMOylation, dimerization und DNA binding of mPR are essential for its PML NB-disrupting phenotype. This correlated perfectly with the observation, that these mutants are no longer able to immortalize primary murine bone marrow, as it was seen with mPR. To analyze the immortalizing properties of the different mutants, we established an in vitro immortalization assay (LCI-Assay). With this assay we could show for the first time, that the oncogenic potential of hPR differs in varying mouse strains used. For the validation of the in vitro results we performed at the end an exemplary mouse experiment. With the combination of the used in vitro methods we are able to analyze the oncogenic potential of different mutants. Hence, with the combination of these methods it is possible to reduce the number of money- and time-consuming animal experiments.
Projektbezogene Publikationen (Auswahl)
-
(2014). The PML domain of PML-RARalpha blocks senescence to promote leukemia. Proceedings of the National Academy of Sciences of the United States of America 111, 12133-12138
Korf, K., Wodrich, H., Haschke, A., Ocampo, C., Harder, L., Gieseke, F., Pollmann, A., Dierck, K., Prall, S., Staege, H., et al.
-
(2015). Analyse der onkogenen Eigenschaften von PML-RARa in der Akuten Promyelozyten-Leukämie. Dissertation, Universität Hamburg
Korf, K.
