Enhanced Phosphorylation of Nbs1, a Member of DNA Repair/Checkpoint Complex RAD50-Mre11-Nbs1, Can Be Targeted Simultaneously with BCR/ABL Kinase To Eliminate Leukemia Cells.

Growing evidence indicate that ABL kinase inhibitors may need partner drugs to cure BCR/ABL-positive leukemias. Genotoxic drugs have been successfully combined with imatinib mesylate to increase its anti-leukemia activity in vitro. Although BCR/ABL-positive cells may accumulate even higher levels of DNA damage in comparison to their normal counterparts the former cells repair the lesions more proficiently and eventually survive. Therefore, targeting the mechanisms responsible for survival of leukemia cells after genotoxic treatment may increase the chances to eradicate BCR/ABL-positive leukemias. Nbs1, a member of the Rad50/Mre11/Nbs1 complex, is phosphorylated by ATM on Serine 343 (S343) in response to DNA double strand breaks (DSBs) to regulate intra-S and G2/M cell cycle checkpoints and DNA repair. Here we show that BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC, BCR/FGFR, and NPM/ALK, stimulate Nbs1 expression by protection from caspase-dependent degradation and induction of c-Myc-dependent transactivation. Downregulation of Nbs1 in BCR/ABL positive cells using siRNA increased their sensitivity to mitomycin C (MMC). Enhanced phosphorylation of Nbs1 on S343 (pNbs1) was detected by Western analysis in BCR/ABL-positive leukemia cells (CD34+ CML patient cells and leukemic cell lines) treated with various cytotoxic drugs (MMC, hydroxyurea = HU, cisplatin - CPL) in comparison to normal counterparts. This effect is associated with increased ATM kinase activity in BCR/ABL cells treated with MMC. In addition, immunofluoresence studies demonstrated an increase of the pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. DNA damage-dependent enhancement of pNbs1 appears to be a broad phenomenon because it was also detected in MMC-treated tumor cells expressing other FTKs. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells and BCR/ABL-transformed cell lines displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase, indicating an intact intra-S phase checkpoint. Expression of the Nbs1-S343A phosphorylation-less mutant downregulated pNbs1 and disrupted intra-S phase checkpoint resulting in reduced accumulation of BCR/ABL leukemia cells in S phase after MMC treatment. This effect was associated with an increase of the sensitivity of leukemia cells to genotoxic treatment (MMC, HU, CPL). A combinatorial strategy was employed targeting enhanced Nbs1 phosphorylation and the deregulated BCR/ABL tyrosine kinase activity, using the Nbs1-S343A phosphorylation-less mutant and a sub-optimal concentration of STI571 eliminating ~50% of leukemia cells, respectively. Targeting both BCR/ABL kinase activity and Nbs1 phosphorylation in combination significantly sensitizes B/A-positive cells to MMC treatment, nearly eradicating all leukemia cells.