Abrogation of the DNA-Damage Response in De Novo AML Versus MDS Cell Lines.

Background: The increasing evidence that activation of the DNA-damage-response (DDR) pathway in preneoplastic lesions of solid neoplasms hinders tumorigenesis prompted us to evaluate the same hypothesis in cell line models representing different subtypes of MDS or AML.

Methods: myeloid cell lines corresponding to different subgroups of MDS, having or not progressed to AML (p39, MOLM-13) and de novo AML (MV4-11, and KG-1) were assessed for their capacity to induce a DDR. Using the quintessential inducer of a DDR, the cell lines were gamma-irradiated (2, 5 and 10Gy) and their ability to arrest the cell cycle in G2/M and to undergo apoptosis were assessed.

Results: in contrast to the de novo AML-derived KG-1 cells - the irradiated MDS/AML-derived cell lines P39 and MOLM-13 exhibited an earlier and more pronounced arrest of the cell cycle in G2/M, as well as a higher percentage of apoptotic cells. Whereas 10 Gy induced a G2/M-arrest in about 30% of KG-1 cells, this percentage increased to about 60% in P39 and MOLM-13 cells. Comparably, whereas KG-1 cells exhibited about 15% of apoptotic cells following irradiation with 10Gy, the percentage reached 30% in P39 and MOLM-13 cells. Assessing the underlying molecular determinants we demonstrated that irradiated P39 and MOLM-13 cells retained the ability to activate crucial mediators of the DDR, whereas this ability was decreased in the de novo AML cell line KG-1. In particular, in comparison to KG-1 cells, irradiated P39 and MOLM-13 cells exhibited a more pronounced upregulation of the crucial cell cycle/checkpoint regulators Chk1-P-Ser317 and Cdk1-P-Tyr15. Noteworthy, MDS cell lines with an increased G2/M-arrest exhibited a higher expression of nucleophosmin - which serves as a shuttling protein between the nucleus and the cytoplasm - and were able to retain this increased expression upon irradiation. Moreover, elevated expression of NPM in MDS cell lines corresponded to higher PCNA-expression, which serves as an essential component of the DNA-repair machinery. To further investigate the functionality of the DDR-pathway, the different cell lines were gamma-irradiated in the presence of the ATM-inhibitor KU-55933 and the Chk1 inhibitor UCN-01. Whereas inhibition of Chk1 (and to a lesser extent inhibition of ATM) was able to hinder G2/M-progression in irradiated MDS cells, it remained without effect in the AML cell lines KG-1 and MV4-11. Noteworthy, whereas irradiated P39/MDS cells hindered to activate Chk1 decrease expression of Polo-like-kinase-1 (Plk-1) - which not only governs mitotic entry and exit, but also functions as an oncogene - KG-1 cells maintained a high expression of Plk-1, comparable to that observed in non-irradiated cells.

Conclusion: we demonstrated the different capacities of MDS (or AML post MDS) and de novo AML cell lines to activate the DDR and thus provide evidence that the DDR-pathway is abrogated in de novo AML as compared to MDS cells.