Further Evidence That MDS and AML Cells Exhibit a Differential Capacity To Activate the DNA-Damage Response (DDR) Pathways.

Background: Findings, that activation of the DDR-pathway in preneoplastic lesions of solid neoplasms slows tumorigenesis, could also underly progression of MDS to AML. We previously showed (ASH 2006, abstr n° 2649), that - in contrast to the de novo AML-derived KG-1 cells - irradiated MDS/AML-derived cell lines P39 and MOLM-13

• exhibit earlier and greater cell cycle arrest in G2/M and a higher percentage of apoptotic cells;

• upregulate the crucial checkpoint kinase Chk1-P-Ser317;

• decrease G2/M-arrest upon inhibition of Chk1 (and to a lesser extent upon inhibition of ATM).

Methods: We further analyzed this differential checkpoint regulation in MDS versus AML cell lines by assessing irradiation-induced apoptosis in the presence of Chk1- and ATM-inhibitors and the functional role of H2AX, situated below the crucial checkpoint kinase Chk1. Myeloid cell lines P39, MOLM-13, MV4-11, and KG-1 were g-irradiated and their ability to undergo apoptosis (DIOC/PI staining) quantified in the absence and presence of the Chk1 inhibitor UCN-01 and the ATM inhibitor KU-55933. Protein expression of activated H2AX was quantified by immunoblot analysis with an anti-H2AX-P-Ser139 antibody. Histological staining of bone marrow biopsies from patients with low and high risk MDS, and AML was made with an anti-H2AX-P-Ser139 antibody. Staining was considered positive if at least 20% of the biopsy surface exhibited 50% or more stained cells.

Results: In the presence of the ATM inhibitor irradiated KG-1 cells retained their apoptosis-resistance, and irradiated P39, MOLM-13 and MV4-11 cells exhibited the same degree of apoptosis as seen in the absence of the inhibitor. In contrast, inhibition of Chk1 (newly) established apoptosis-sensitivity in KG-1 cells (which showed about 30% of DIOC-low cells following 24 hours of irradiation with 10 Gy), while having only minor effects on the other cell lines. Determining the concomitant activation status of H2AX, which is a downstream target of Chk1, showed that irradiation induced a comparable expression of H2AX-P-Ser139 in MDS and AML cell lines. Co-incubation with the Chk1 inhibitor was associated with lower expression of activated H2AX in the irradiated MDS cell lines P39 and MOLM-13 as compared to irradiated KG-1 cells. Histological staining of marrow biopsies with an anti-H2AX-P-Ser139 antibody demonstrated that activation of H2AX is less frequent in low risk MDS (1/10 samples) than in high risk MDS (9/18 samples) and AML (4/6 samples).

Conclusion: Further analyzing the different capacities of MDS and AML cells to employ DDR-pathways, we provide additional insights into the functional relevance of ATM and Chk1 inhibition in the apoptotic DDR, and give preliminary evidence for a differential activation of H2AX in irradiated cell lines and patient samples of MDS and AML.