The transition of CML from chronic phase to blast crisis is characterized by the accumulation of molecular and chromosomal abnormalities (64), but the molecular mechanisms underlying this genetic instability are poorly understood (65). In the past two to three years, few studies have directly addressed the relationship between BCR-ABL expression and levels/activity of proteins involved in DNA repair, particularly the repair of DNA double-strand breaks (21).
In the first study investigating such a relationship, Deutsch et al. (66) looked at the effects of BCR-ABL on the catalytic subunit, DNA-PKCS, of the DNA-PK complex formed with the heterodimeric Ku protein. Repair of DSBs by the DNA-PK-dependent pathway [nonhomologous end-joining (NHEJ) recombination] is the preferred pathway utilized by human cells (67), and in mice, NHEJ deficiency accelerates lymphoma formation and promotes the development of soft tissue sarcomas that possess clonal amplifications, deletions, and translocations (68,69). In BCR-ABL-expressing cells (including primary CML cells), levels of DNA-PKCS were markedly downregulated (66) and were reversed by proteasome inhibitors, suggesting the activation of a BCR-ABL-dependent pathway leading to enhanced proteasome-dependent protein degradation (66). Downregulation of DNA-PKCS levels was associated with a higher frequency of chromosomal abnormalities after exposure of BCR-ABL-expressing cells to ionizing radiation (IR) and increased radiosensitivity (66). Such an increase was, however, modest probably due to enhanced survival of BCR-ABL-expressing cells caused by the activation of multiple antiapoptotic pathways. The same group also reported an association of BCR-ABL expression in primary CML samples and in established cell lines with downregulation of BRCA-1 (70), a protein involved in the surveillance of genome integrity (71,72). Downregulation of BRCA-1 was more evident in cell lines in which levels of BCR-ABL were more abundant and correlated with increased chromosome aberrations after DNA damage. However, these findings have not been yet confirmed in a large cohort of primary samples obtained from CML patients, and a causal link between decreased repair of DSBs and disease progression has not been demonstrated. Another group identified BCR-ABL-dependent pathways leading to enhanced expression/ activity of RAD51 (73), a protein that participates in homologous recombination repair (HRR) (74). Expression of BCR-ABL increased the efficiency of HRR in a RAD51-dependent manner as well as resistance to apoptosis induced by drugs like mitomycin C and cisplatin, which promote DSBs (73). In light of enhanced high-fidelity HRR promoted by RAD51 (74), it seems counterintuitive that the increased expression/ activity of RAD51 (and other paralogues) in BCR-ABL-expressing cells might be associated with genomic instability. Together, the apparently opposite effects of BCR-ABL on RAD51, DNA-PKCS, and BRCA-1 may not be mutually exclusive and may all be involved in promoting genomic instability associated with defective repair of DSBs. Deregulation of the DNA repair mechanisms and the acquisition of mutations in genes critically important for the regulation of proliferation are expected to activate control checkpoints (i.e., p53 expression), which may lead to the elimination of cells with damaged DNA. However, the ability of BCR-ABL to regulate multiple antiapoptotic pathways, perhaps in a dose-dependent manner and in specific subsets of progenitor cells, is likely to allow survival of cell populations carrying mutations that promote their proliferation and maintenance. In this regard, appears to the unrestrained BCR-ABL kinase activity induce an increase in reactive oxygen species (ROS), which can cause chronic oxidative
DNA damage resulting in the accumulation of DSBs (75). These lesions are repaired by BCR-ABL-stimulated HRR and NHEJ mechanisms; however, a high mutation rate is detected in HRR, and large deletions are found in NHEJ products in BCR-ABL-expressing cells, but not in the normal counterparts (75). Interestingly, the BCR-ABL-dependent increase in the levels of oxidative DNA damage is also responsible for the emergence of clinically relevant mutations in the kinase domain of BCR-ABL itself (76). Conversely, Dierov et al. (77) reported that BCR-ABL interacts with the ATR protein (atoxic teleoegectasia related) in the nucleus and suppresses its activity, implicating this mechanism in the increased number of DSBs after etoposide treatment, and suggesting a delay in DNA double strand break repair after genotoxic stress. Furthermore, the same group has examined the occurrence of chromosomal abnormalities in BCR-ABL expressing cells after recovery from DNA damage (41) and found that BCR-ABL-transduced BaF3 cells compared to parental cells exhibit an increase in chromosomal abnormalities, including chromatid damage, after DNA repair (41). Similar studies in primary human cells show increased frequency of translocations in CML samples versus normal hematopoietic progenitors (M. Carroll, personal communication).
From the data discussed earlier, it seems that the mechanism(s) leading to increased genomic instability is(are) still not yet well defined. However, it seems likely that BCR-ABL expression alters the cellular response to genotoxic stress and predisposes cells to genetic (e.g., point mutations) and gross chromosomal alterations (e.g., translocations), which, undoubtedly, contribute to the aggressiveness of CML blast crisis cells.
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