Selective Suppression of the DNA Damage Response in Acute Myeloid Leukemia Versus Myelodysplastic Syndrome.

Background: Activation of the DNA-damage-response (DDR)-pathway in preneoplastic lesions of solid neoplasms hinders tumorigenesis and needs to be abolished for transformation, and could also underlie progression of MDS to AML. We previously showed (ASH 2007, abstr n° 2438) in a panel of myeloid cell lines, that

• γ-irradiation induces apoptosis and G2/M-arrest in the MDS-derived cell lines P39 and MOLM-13, but to a considerably lesser extent in AML cell lines MV4-11 and KG-1;

• this DDR is mediated by a concomitant increase in the phosphorylation of ATM, Chk-1 and H2AX in MDS-derived cell lines;

• inhibition of ATM by KU-55933 establishes radiosensitivity in MV4-11, but not KG-1 cells, whereas inhibition of Chk-1 by UCN-01 confers radiosensitivity in both AML cell lines Having thus demonstrated the differential capacity of myeloid cell lines representing different subtypes of MDS and AML to activate the DDR pathways, we here tested the hypothesis that this response is determined by the endogenous activation pattern of DDR existing already before activation by an exogenous stimulus.

Methods: The endogenous pattern of DDR in the cell lines P39 and KG-1 was evaluated by immunoflourescence staining of P-ATM-Ser1981, P-Chk-1-Ser317, P-Chk-2-Ser68, and γ-H2AX (P-Ser139). The expression pattern in ex vivo material was determined by staining bone marrow biospies of normal controls (n=2), MDS (n=27; IPSS : Int1: n=7, Int2: n=14, High: n=6) and AML (n=5) patients (pts). Bone marrow (BM) biopsies were stained by immunohistochemistry for expression of P-ATM-Ser1981, P-Chk-1-Ser317, P-Chk-2-Ser68, and γ-H2AX and results were correlated with clinical parameters.

Results: We show that the AML (KG-1)- but not MDS (P39)-derived cells exhibit signs of an endogenous activation of the DDR as demonstrated by the presence of about 10% P-ATM+ and γ-H2AX+ cells. Immunohistochemical assessment of BM biopsies provides evidence that

• normal controls exhibit <5% P-ATM+, γ-H2AX+, P-Chk-1+ and P-Chk-2+ cells;

• the percentage of P-ATM+ cells increases in samples from AML pts (median: 75% P-ATM+) as compared to high-risk MDS samples (IPSS high: median 42% P-ATM+, Int-2: median: 26% P-ATM+) and significantly correlates with the percentage of BM blasts (p<0,01);

• the frequency of γ-H2AX+ cells is heterogeneous in all subgroups of AML and MDS and is not correlated with the percentage of BM blasts;

• whereas intermediate-1 MDS samples contain as little P-Chk-1+ and P-Chk-2+ as healthy controls, staining for checkpoint kinases increased in intermediate-2 and high-risk MDS (median P-Chk-1+: 32%, median P-Chk-2+: 51%) yet declined to near-to-background levels in AML samples (median P-Chk-1+: 2%, median P-Chk-2+: 8%).

P-Chk-1+ and P-Chk-2+ expression are not correlated with the percentage of BM blasts. Nevertheless P-Chk-1+ expression significantly correlates with the P-Chk-2+ expression (p<0,001)

Conclusions: We here provide novel evidence that the heterogeneity of the DDR response in cell lines is determined by the endogenous activation pattern of the DDR. We corroborate this observation by assessement of the DDR pattern in BM biopsies of MDS and AML demonstrating the selective suppression of the DDR in AML (as compared to MDS) due to the uncoupling between activated ATM and inactive checkpoint kinases.