Mutations in the BCR/ABL Kinase Encoding the Resistance to Imatinib Mesylate Do Not Modify the Sensitivity to Genotoxic Treatment.

Imatinib mesylate (IM), a selective inhibitor of ABL kinase activity, revolutionized the treatment of BCR/ABL-positive leukemias. Unfortunately, clinical and experimental observations reveal that resistance to IM is a rising problem, which obscures an otherwise very successful oncogene-targeted therapy. IM resistance can be achieved by point mutations in the kinase domain of BCR/ABL and have been detected in 50–90% of patients with acquired resistance to IM, including ~23% of the IM-naive patients. Strategies to enhance the effect of IM and eventually overcome the resistance are dose escalation, addition of a growth factor, and combinations with novel tyrosine kinase inhibitors like AMN107 and dasatinib, or with inhibitors targeting downstream BCR/ABL effectors, e.g. PI-3k. Unfortunately, resistance to other small molecule inhibitors is likely to appear, as well. Therefore, we tested the sensitivity of leukemia cells expressing IM-resistant BCR/ABL mutants to genotoxic agents. Baf3 cells expressing similar levels of p210BCR/ABL wild-type (WT) and Y253F, Y253H, E255K, E255V, T315I, M351T, and H396P mutants were established. These mutants were selected since they represent several functionally distinct ABL kinase domain regions, including P-loop (Y253F/H and E255K/V), the site of a hydrogen bond with IM (T315I), the activation loop hinge (M351T), and the activation loop (H396P). In addition, they exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to IM. Western analysis demonstrated similarity, but also differences in the patterns of tyrosine phosphorylated proteins in total cell lysates. As expected, these clones displayed different level of sensitivity to IM: T315I>Y253H>E255V>E255K=Y253F>M251T=H396P>WT. However, leukemia cells expressing the WT and IM-resistant BCR/ABL kinase mutants demonstrated similar sensitivity to cytotoxic drugs such as hydroxyurea (HU), mitomycin C (MMC), and N-Methyl N’-nitro-N-nitrosoguanidine (MNNG). Surprisingly, clones expressing IM-resistant BCR/ABL kinase mutants were more sensitive to γ-irradiation than their WT counterparts. γ-H2AX (histone H2AX phosphorylated on S139) nuclear foci, which serve as marker of DNA double-strand breaks (DSBs), the most lethal DNA lesions, were detected by immunofluorescence. Leukemia cells expressing WT and IM-resistant p210BCR/ABL proteins accumulated similar numbers of DSBs after MMC treatment and γ-irradiation. This observation suggests that leukemia cells expressing IM-resistant p210BCR/ABL mutants may be eliminated by genotoxic treatment, especially γ-irradiation. In addition, IM-resistant BCR/ABL kinase mutants, in contrast to the WT kinase may not be able to modulate the mechanisms protecting from apoptosis induced by γ-irradiation.