p53 signaling in response to increased DNA damage sensitizes AML1-ETO cells to stress-induced death

Abstract

Chromosomal translocation (8;21) is present in 10% to 15% of patients with acute myeloid leukemia. Expression of the AML1-ETO (AE) fusion protein alone is not sufficient to induce leukemia, but the nature of the additional genetic alterations is unknown. It is unclear whether AE facilitates acquisition of these cooperating events. We show that AE down-regulates genes involved in multiple DNA repair pathways, potentially through a mechanism involving direct binding at promoter elements, and increases the mutation frequency in vivo. AE cells display increased DNA damage in vitro and have an activated p53 pathway. This results in increased basal apoptosis and enhanced sensitivity to DNA damaging agents. Intriguingly, microarray data indicate that t(8;21) patient samples exhibit decreased expression of DNA repair genes and increased expression of p53 response genes compared with other acute myeloid leukemia (AML) patient samples. Inhibition of the p53 pathway by RNAi increases the resistance of AE cells to DNA damage. We thus speculate that AML1-ETO may facilitate accumulation of genetic alterations by suppressing endogenous DNA repair. It is possible that the superior outcome of t(8;21) patients is partly due to an activated p53 pathway, and that loss of the p53 response pathway is associated with disease progression.

Figure 1

AE associates with the POLE and OGG1 promoters and promotes DNA damage accumulation. (A) Graphic of the 5′ regulatory regions of the OGG1 and POLE genes. Black ovals represent RUNX1-binding sites; arrows indicate orientation. Numbers represent distance between the indicated regions, in base pairs. (B) RUNX1-binding sites were amplified by PCR after chromatin immunoprecipitation. The RUNX1 site present in the p14^ARF promoter was used as a positive control. Water was used as a negative control. One percent of lysate input used for immunoprecipitation (IP) is amplified as an additional control. Control IP has no Ab and specific IP was with anti-HA Ab (AE contains the HA epitope). (C) MIT and AE cells were stained for the DNA damage marker γH2A.X; nuclei are stained with DAPI. (D) Cells positive for γH2A.X were scored in 4 control, 4 AE, and 3 MLL-AF9 cultures. (E) Percentage of cells with DNA damage in 3 pairs of AE and control MIT cultures during the first 5 weeks after transduction. (F) Cells from samples in panel E were cultured under 2.6% oxygen starting at week 2 after transduction. Percentage of cells positive for γH2A.X was scored at weeks 3 and 5 after transduction. Error bars represent SD. (G) Amount of reactive oxygen species was measured by flow cytometry in AE and MIT cultures stained with H₂DCFDA probe. MIT cells treated with 300 μM H₂O₂ for 20 minutes were used as a positive control. The staining was repeated multiple times with no consistent difference between the 2 cultures.

Figure 2

AE increases the mutation frequency in vivo. Mutation frequency of AE and control MIGR1 murine bone marrow cells after 3 months in vivo. Data represent averages of 8 mice (MIGR1) and 7 mice (AE) from 3 separate transductions. The hatched area represents mutations with no change in plasmid size (primarily point mutations); the remainder are translocations or deletions. Error bars represent SEM.

Figure 3

AE cells have an activated p53 pathway, increased apoptosis, and increased sensitivity to DNA damage. (A) Western blot analysis showing higher levels of p53 and p21 proteins in 3 pairs of AE and MIT cultures at week 5 after transduction. (B) RQ-PCR analysis shows increased expression levels of p53 target genes DAPK1 and TP53I3. Three pairs of AE and MIT cultures were analyzed at weeks 1 and 5 after transduction. (C) BrdU staining of AE and MIT cultures shows a decreased percentage of cells in early S phase after irradiation, demonstrating a functional G₁ checkpoint. (D) Staining of AE and MIT cells for an early mitotic antigen MPM-2 shows a decreased number of cells in M phase after irradiation, demonstrating a functional G₂ checkpoint. Cultures used for experiments shown in panels C,D were 4 weeks old, and flow panels are representative of at least 3 separate stainings. (E) AnnexinV staining reveals increased levels of apoptosis in AE cultures compared with MIT cultures. Six AE and 5 MIT cultures were analyzed at week 5 after transduction. (F) AE and MIT cultures 5 weeks after transduction do not show differences in cell cycle. BrdU was incorporated and cells were stained with Anti-BrdU Ab and 7-AAD. An average and standard deviation from 3 separate experiments are shown. (G-I) AE cells show increased sensitivity to ionizing irradiation, mitomycin C (MMC), and AraC compared with MIT cells. Proliferation assays were performed on paired cultures at 5 weeks after transduction, 72 hours after irradiation, or after 72 hours of incubation with MMC or AraC.

Figure 4

Knock-down of p53 in AE cells increases resistance to DNA damage and interferes with the G1 checkpoint. (A) RQ-PCR analysis shows decreased TP53 gene expression in AE cells expressing shRNA targeting p53 (AE-p53sh) compared with AE cells expressing scrambled sequence (AE-scrambled). (B) Western blot analysis shows reduction of total p53 and p21 proteins and decreased level of induction after irradiation in AE-p53sh compared with AE-scrambled cells. Cells were irradiated (5 Gy) and samples were analyzed after 5 hours. (C) The AE-p53sh cells have reduced induction of p53 target genes after irradiation. Bars represent a ratio of the target gene expression in irradiated to nonirradiated samples. (D) BrdU incorporation demonstrates a faulty G1 checkpoint in AE-p53sh compared with AE-scrambled and as well as in Kasumi-1 cells after irradiation. (E) Staining for early mitotic antigens using MPM-2 antibodies demonstrates intact initiation of the G2 checkpoint in all cultures. (F,G) The p53 knockdown increases resistance of AE cells to ionizing radiation and to AraC. Proliferation assays were performed at 72 hours after irradiation or after 72 hours of incubation with AraC. Experiments were repeated at least twice with similar results. Error bars represent SD, and numbers on plots are percentages of total cells.

Figure 5

Expression of p53 target genes segregates t(8;21) (and inv16) AML samples from the remaining AML patient samples. (A) Seven p53 target genes were chosen, based on published literature, and used in a principal component analysis on the published dataset of 130 AML patient samples (Table 2). CBF leukemias segregate from the other patient samples in a principle component analysis based on the expression of these p53 target genes. (B) Analysis of variance (7-way ANOVA) shows that t(8;21) patient samples have a significantly increased group mean expression level for the p53 target genes compared with other patient groups, excluding inv(16). Each dot represents differences in group mean value based on the 7 p53 target genes (y-axes) between t(8;21) AML and the other AML cytogenetic subtypes (noted on the x-axis). The bars represent 95% confidence intervals.