c-Abl inhibition affects DNA repair kinetics and genetic stability

4871 Imatinib mesylate (Gleevec) is the most effective medical therapy for the treatment of chronic myeloid leukemia (CML), a disorder characterized by the expression of Bcr-Abl. However, complete eradication of malignant cells by Imatinib is rare. Long-term, maybe even lifelong therapy is therefore probably required to avoid or at least delay advanced CML. Imatinib inhibits both Bcr-Abl and the physiological non-receptor tyrosine kinase c-Abl. c-Abl is conserved in all eukaryotes and is activated by a variety of signals including DNA damage. We therefore investigated possible effects of long term Imatinib treatment on gamma irradiation induced DNA damaging in Bcr-Abl negative cells. For this, different hematopoietic cell lines but also primary lymphocytes from normal donors were pre-treated with or without Imatinib (1-3µM) and exposed to gamma-irradiation. We evaluated these cells for proliferation, induction of apoptosis, DNA repair kinetics, and the frequency of loss of function mutations in the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. An increased frequency of HPRT mutations was observed in Bcr-Abl negative cell lines pretreated with Imatinib after low-dose gamma-irradiation. An enhanced number of 6-Thioguanine-resistant colonies was also detected in c-Abl-/- MEFs and in MEFs expressing a kinase deficient variant of c-Abl confirming that the c-Abl kinase activity is critical for genetic stability. Neither growth rate nor induction of apoptosis was altered by Imatinib in gamma-irradiated cells. However, we found that pre-incubation with Imatinib caused a significant slow-down of DNA strand break repair as measured by the COMET assay both in cell lines and in PBMCs from healthy volunteers. A comparable delay of DNA repair was also observed with Dasatinib. To verify that the c-Abl kinase activity is necessary for proper DNA strand break repair we performed experiments with MEFs expressing either a kinase-active or defective c-Abl variant and with cells expressing a c-AblT315I Imatinib resistant mutant. MEFs expressing kinase defect c-Abl showed a delay in DNA strand break repair comparable to that observed in wt-c-Abl cells treated with Imatinib. In addition, Imatinib had no effect on the kinetics of DNA repair in Imatinib insensitive T315I-c-Abl cells. In summary, our study demonstrates that the catalytic function of c-Abl is essential for efficient strand break repair and for maintaining genetic stability in non-malignant cells. These in-vitro data indicate that long-term treatment with Imatinib may affect cellular DNA damage response.