Chronic myeloid leukemia chronic phase (CML-CP) CD34 ؉ cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylationless mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression. (Blood. 2011;118(4):1062-1068) IntroductionChronic myeloid leukemia in chronic phase (CML-CP) is a myeloproliferative disorder characterized by the presence of the Philadelphia (Ph) chromosome that results from a (9;22)(q34;q11) reciprocal translocation that juxtaposes the c-abl oncogene 1 (ABL1) gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22, generating the BCR-ABL1 fusion oncogene. Most CML-CP patients are currently treated with tyrosine kinase inhibitor (TKI) imatinib designed to block the enzymatic action of the ABL1 tyrosine kinase. Approximately 60%-70% of patients achieve and maintain a complete cytogenetic response (CCyR) 5 years after initiating imatinib treatment. Two "second-generation" TKIs, dasatinib and nilotinib, are effective at inducing or restoring CCyR in 40%-50% of patients who appear to have failed primary treatment with imatinib. However approximately 20% of patients presenting with CML-CP fail to respond to both imatinib and a subsequent second-generation TKI; their prognosis is poor because of a higher risk of disease progression. In addition, because TKIs do not eradicate the disease, the patients in CCyR may carry 10 6 to 10 9 leukemia cells, and even BCR-ABL1-negative patients in complete molecular response may have up to 10 6 leukemia cells. 1 These cells may be the "time-ticking bombs" eventually exploding into TKI-resistant clone and/or CML-blast phase (CML-BP) clone on accumulation of additional genetic aberrations. 2 Clinical observations and experimental findings clearly demonstrated that genomic instability in CML is driven, at least in part, by BCR-ABL1 kinase. 2,3 However, TKI-treated CML pat...

Bloom protein (BLM) is a 30 -5 0 helicase, mutated in Bloom syndrome, which plays an important role in response to DNA double-strand breaks and stalled replication forks. Here, we show that BCR/ABL tyrosine kinase, which also modulates DNA repair capacity, is associated with elevated expression of BLM. Downregulation of BLM by antisense cDNA or dominantnegative mutant inhibits homologous recombination repair (HRR) and increases sensitivity to cisplatin in BCR/ABLpositive cells. Bone marrow cells from mice heterozygous for BLM mutation, BLM Cin/ þ , transfected with BCR/ ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. BCR/ABL promotes interactions of BLM with RAD51, while simultaneous overexpression of BLM and RAD51 in normal cells increases drug resistance. These data suggest that BLM collaborates with RAD51 to facilitate HRR and promotes the resistance of BCR/ABL-positive leukemia cells to DNA-damaging agents.

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A genome-wide screen suggested that BCR/ABL kinase might stimulate WRN, a member of the RecQ-like DNA helicases family. The Werner syndrome protein (WRN) exerts DNA helicase and 3′-5′ exonuclease activities. Inactivating mutations in the WRN gene causes Werner syndrome, characterized by premature aging, genomic instability and cancer predisposition. The WRN helicase unwinds unusual DNA structures, which can occur physiologically, or can be accidentally generated during DNA repair (double-stranded DNA with mismatched tails, bimolecular G4 quartets and Holliday junctions). In addition, WRN physically interacts with components of two major systems for DNA double-strand breaks (DSBs) repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here we demonstrated that BCR/ABL regulates the expression of WRN mRNA and protein in CML primary cells and BCR/ABL-transformed cell lines. BCR/ABL kinase-induced WRN expression is mediated by c-MYC, but not STAT5 - dependent transcription as well as by inhibition of caspases-dependent cleavage. In addition, immunoprecipitation and pull-down studies indicated that BCR/ABL interacts directly with WRN resulting in its tyrosine phosphorylation. Mutation analysis revealed that multiple domains/amino acid residues of BCR/ABL and WRN are involved in the interaction. BCR/ABL-positive leukemia cells exerted an enhanced WRN-dependent helicase activity. In addition, immunoprecipitation and double-immunofluorescence co-localization studies demonstrated an elevated interaction between WRN and RAD51 in BCR/ABL cells undergoing genotoxic stress in comparison to parental counterparts. Altogether, it is likely that WRN is involved in DSBs repair by HR in leukemia cells. More detailed studies are underway to pinpoint the role of WRN in DNA damage response in BCR/ABL-transformed cells.