CML Progenitor Cells Have Chromsomal Instability and Display Increased DNA Damage at DNA Fragile Sites.

Chronic myelogeneous leukemia (CML) is a two stage disease which progresses to blast crisis over a period of 3–5 years in untreated patients. The BCR/ABL oncogene induces the hyperproliferation associated with chronic phase CML but whether BCR/ABL induces chromosomal instability leading to blast crisis has been controversial. We have previously demonstrated that BCR/ABL delays the repair of DNA double strand breaks and increases chromosomal instability in a murine cell line. Furthermore, we have demonstrated in cell lines that BCR/ABL disrupts the function of the DNA damage sensing protein, ataxia telangiectasia and rad 3 related (ATR). One of the functions of ATR is to maintain the stability of DNA fragile sites, late replicating sites in the chromosome that are frequently involved in translocations. To determine if BCR/ABL affects the stability of DNA fragile sites in Ba/F3 cells that do or do not express BCR/ABL, cells were incubated in low dose aphidicolin for 24 hours to induce fragile site breakage. BCR/ABL expressing cells, but not control cells, demonstrated fragile site damage consistent with a disruption of ATR function in BCR/ABL expressing cells. In order to determine if primary patient cells display a genomic instability phenotype, we have analyzed the response to DNA damage in CD34+ cells from normal volunteers and from CML patients seen at the University of Pennsylvania Cancer Center. We first examined the DNA repair response by treating cells for two hours with etoposide. Both normal cells and CML progenitor cells demonstrate DNA double strand breaks as measured by the comet assay, a quantitative assay for DNA double strand breaks. However, in Ph+ cells from the patient sample there was a delay in the repair of DNA double strand breaks as indicated by a significant increase in the olive tail moment at 2 hours and 24 hours after treatment with etoposide. In addition, we analyzed the effect of a two hour exposure to etoposide on chromosome stability as measured by spectral karyotyping (SKY). Normal CD34+ cells and CD34+ cells from patients were treated with etoposide and then allowed to recover for 48 hours before analysis of metaphase spreads. Normal cells demonstrated no spontaneous DNA damage and, after etoposide treatment and repair, demonstrated only modest levels of DNA damage (2 translocations and 5 numerical alterations per 14 metaphases analyzed). In contrast, Ph+ cells demonstrated spontaneous DNA damage in these cell conditions. Furthermore, after etoposide treatment Ph+ cells demonstrated high levels of DNA damage with 9 translocations and 12 numerical alterations in 13 metaphases. These results suggest that Ph+ progenitor cells from patients with CML demonstrate chromosomal instability and suggest a mechanism for progression from CML chronic phase to blast crisis. Full analysis of additional patient samples will be presented. Taken together, we propose that BCR/ABL disrupts ATR function in cell lines and primary cells leading to an increase in chromosomal instability that leads to CML blast crisis.