BCR/ABL Kinase Inhibits Mismatch Repair To Reduce Apoptosis and Induce Point Mutations.

Clinical and experimental findings indicate that BCR/ABL-positive leukemias display genomic instability leading to point mutations in ABL kinase domain and other genes (e.g. p53), eventually causing resistance to imatinib and progression of the disease. Mismatch repair system (MMR) maintains genomic stability by removing replication errors and oxidative lesions from DNA, thus preventing point mutations. In addition, MMR induces apoptosis, thereby eliminating cells with too many errors. Single-base mismatches are recognized by the MSH2-MSH6 heterodimer. Upon mismatch binding, the MSH2-MSH6 heterodimer associates with the MLH1-PMS2 heterodimer, which is essential for repair. MLH1-PMS2 complex couples the mismatch recognition step to downstream processes that include the removal of the mismatch from the nascent DNA strand, re-synthesis of the degraded region, and ligation of the remaining nick. Ouabain-resistance test was employed to investigate mutagenesis caused by BCR/ABL kinase. Cells become resistant to ouabain when mutations arise in the gene encoding Na+/K+ ATPase. Mutagenesis was induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) - a known mutagen, which induces O6-methylation of guanine and thymine; resulting DNA lesions are recognized by the MMR pathway. We report that BCR/ABL-positive cells are less sensitive to MNNG than normal counterparts. In addition, BCR/ABL-positive cells surviving the treatment with MNNG displayed 15-fold higher mutation frequency than parental counterparts suggesting a negative effect of BCR/ABL on MMR. To assess MMR activity quantitatively, we employed an in vivo assay using the plasmid substrate containing cDNA for enhanced green fluorescent protein (EGFP) corrupted by a T-G mismatch in the start codon. MMR restores T-A match and EGFP expression. The efficacy of MMR was reduced 2-fold in BCR/ABL-positive cell lines and CD34+ CML cells in comparison to normal counterparts. MMR activity was enhanced after inhibition of BCR/ABL kinase with imatinib. Western blotting and immunofluorescence studies indicated that the total and nuclear expression of MMR proteins (MSH2, MSH6, MLH1, PMS2) did not differ in BCR/ABL-positive and normal cells, but their nuclear co-localization in response to MNNG was severely impaired in the former cells. Co-localization of MSH2 and MSH6 proteins, forming a heterodimer homologous to bacterial MutS and responsible for mismatch recognition, remained similar in parental and leukemia cells upon MNNG treatment. However, co-localization of MLH1 and PMS2 proteins, forming a heterodimer homologous to bacterial MutL, which is essential for mismatch repair, and of MSH2 (MutS member) and MLH1 (MutL member) was detected in non-transformed cells, but not in BCR/ABL-positive leukemia cells including CD34+ CML primary cells. The defects in interaction of MMR proteins in leukemia cells were reversed by inhibition of BCR/ABL kinase by imatinib. These results suggest that BCR/ABL kinase inhibits MMR activity to prevent cell death and/or to induce mutator phenotype responsible for point mutations, including these encoding for resistance to imatinib, nilotinib and dasatinib and others contributing to more malignant phenotype of the disease.