BCR/ABL Oncogenic Kinase Stimulates DNA Oxidative Damage To Induce Self-Mutagenesis Causing Resistance to Imatinib Mesylate.
Authors: Koptyra Mateusz , Falinski Rafal , Nowicki Michal O. , Blasiak Janusz , Skorski Tomasz 
Published: October 2019

Abstract
Imatinib mesylate (IM) [Gleevec, STI571], a selective inhibitor of ABL kinase activity, revolutionized the treatment of BCR/ABL-positive leukemias. Unfortunately, resistance to IM has become a rising clinical problem. Although the resistance could be achieved by various mechanisms, mutations in the BCR/ABL kinase domain seem to play a major role. Identification of the mechanisms causing these mutations may have a significant impact on the effectiveness of IM therapy. Mutations usually result from enhanced DNA damage and/or deregulated mechanisms responsible for DNA repair. Much endogenous DNA damage arises from intermediates of oxygen reduction generating potentially damaging molecules called reactive oxygen species (ROS). We report here that BCR/ABL-mediated transformation is associated with the kinase-dependent stimulation of ROS production and elevation of DNA oxidative damage. Immunofluorescence visualized more 8-oxoG nuclear lesions and enzymatic assays detected more oxidized bases in DNA in BCR/ABL-transformed cells (CML patient cells and leukemic cell lines) in comparison to normal cells. To examine the potential effect of ROS on mutations in BCR/ABL kinase freshly transformed 32D-BCR/ABL cells (no mutations in BCR/ABL) were cultured in the absence (control) or presence of IM (to inhibit BCR/ABL kinase and ROS production), PDTC (ROS scavenger) or IM+BSO (to inhibit BCR/ABL kinase and elevate ROS because BSO prevents its decay) for 8 weeks in medium supplemented with IL-3; then IM-resistance was examined. About 0.1% of control cells displayed IM-resistance, which was dramatically reduced in the presence of IM or PDTC, implicating BCR/ABL kinase-dependent ROS-mediated mechanism. IM+BSO treatment resulted in partial inhibition of IM-resistance, suggesting that another BCR/ABL-induced mechanism may collaborate with ROS in mutagenesis. IM-resistance was associated with mutations in the sequence encoding bcr/abl kinase. The majority of detected mutations were GC→AT transitions causing amino acid substitutions; most of them are already proven to be clinically relevant. Similar conditions were applied to study the mutagenesis in Na+K+ ATPase (mutations may cause the resistance to ouabain) in 32D parental and freshly transformed 32D-BCR/ABL cells. BCR/ABL increased the ouabain resistance rate ~12-times, which was associated with mutations in the alpha1 subunit of Na+K+ ATPase, essential for ouabain resistance. Again the majority of mutations were GC→AT transitions. To confirm that similar mechanisms of BCR/ABL-dependent mutagenesis operate in living organisms, IM-resistance was examined in SCID mice injected with freshly transformed 32D-BCR/ABL cells and fed with normal or vitamin E-rich (VE) diet (to decrease ROS) for 8 weeks. Leukemia cells harvested from bone marrow and spleen of the mice fed with VE diet displayed ~2 -fold reduction in the frequency of IM-resistance in comparison to the animals obtaining regular food. Mutations in the BCR/ABL kinase were detected in IM-resistant clones. Altogether, these studies suggest that BCR/ABL kinase facilitates ROS-dependent mutagenesis of in different genes, including bcr/abl gene, and that this process is essential for IM-resistance. Early treatment with IM can prevent mutagenesis, but if mutations occur it will select the outgrowth of mutant IM-resistant cells. In addition, reduction of ROS should greatly diminish the mutagenesis in BCR/ABL kinase associated with IM-resistance.