Abstract
Genomic instability is the driving force of disease progression to frank leukemia. Evidence suggests that aberrant repair of double strand breaks (DSB) by non homologous end-joining (NHEJ), a major repair pathway in mammalian cells, can lead to chromosomal instability and cancer. We previously reported significantly increased error-prone NHEJ in preleukemic syndromes, and a variety of myeloid malignancies, and demonstrated that these cells harbor constitutive DNA damage. We postulated that increased NHEJ misrepair may be a response to the increased DNA damage. Here, we have studied a mouse model for myeloproliferative/myelodysplastic syndrome (MPD/MDS) to determine whether the frequency of DNA damage and aberrant NHEJ repair may be an indicator for genomic instability as the disease progresses. Transgenic mice bearing mutant NRAS and BCL-2 driven by the MRP8 promoter, which directs expression of the transgene to committed myeloid progenitors and neutrophils, have a relatively mild phenotype with an increase of immature neutrophils. The BCL2 mice have an increase in marrow blasts, but have normal blood counts. Transgenic mice harboring both mutant NRAS and BCL2 genes results in a disease phenotype morphologically resembling human late MDS (FAB subtypes refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt) or chronic myelomonocytic leukaemia (CMML)) with increased marrow blasts. We show that the bone marrow and spleen from the NRAS and BCL2 mice demonstrate an increase in the frequency of NHEJ misrepair activity, compared with normal (FVBN) mice (NRAS: 7.6% vs 3.7%, BCL2: 6.5% vs 3.7%, n=3). Strikingly, the NRAS +BCL2 double transgenic mice show a large and significant increase in NHEJ misrepair activity (19.02%, n=3, p<0.001), compared with controls and single transgenics. Using an immunofluorescence-based assay for DNA damage, dependent on BrdU incorporation, we find that the magnitude of DNA damage mirrors NHEJ activity. Chromatin fibers from both NRAS and BCL2 mice demonstrate an increase in the frequency of DNA damage, compared to normal mice (NRAS: 35% vs 8%, mean [n=3]), (BCL2 22% vs 8%, [n=3]). However, this damage increases even further in RAS +BCL2 mice (62% vs NRAS/BCL2 28%, [FVBN] 8%, n=3, p<0.001). This DNA damage co-localizes with the variant histone γH2AX, a key protein in the repair of DSB. DNA damage and γH2AX also co-localize with the NHEJ protein Ku86 emphasizing that DNA damage is linked to repair by NHEJ in situ. Given that activated RAS produces increased reactive oxygen species (ROS), an established source for DSB, we considered whether ROS accounted for some of this DNA damage. We find that cells from transgenic mice show an increase (up to 2-fold) in ROS, compared with controls. The same is true for FDCP1 murine cells transduced with NRAS and BCL2, and treatment with the antioxidant, N-acetyl cysteine results in an up to 50% decrease in ROS, DNA damage and concomitant NHEJ misrepair activity. Our data suggest that increased DNA damage and error-prone repair may be a platform for the creation of increased genomic instability with disease progression in MPD/MDS in mice. Decreased DNA damage and error-prone repair with antioxidant treatment suggests a mechanism for the amelioration of the activities that drive disease progression.
Genomic instability is an early event driving chromatin reorganization and escape from oncogene-induced senescence
