Fanconi Anemia D2 (FANCD2) Is Required To Overcome Reactive Oxygen Species (ROS) - Induced DNA Double-Strand Breaks in BCR/ABL-Positive Leukemias.

BCR/ABL-mediated transformation is associated with elevation of ROS which, in addition to enhancing the cytoplasmic signaling pathways, may increase the number of oxidative DNA lesions including DSBs. Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts implicating the role in repair of ROS-dependent DSBs. In addition, higher levels of monoubiquitinated FANCD2 protein was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. This speculation is supported by impaired transformation potential of the BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied with delayed leukemogenesis in SCID mice. Growth ability of BCR/ABL-positive FANCD2−/− cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, however, the former cells accumulate more DSBs evaluated by neutral comet assay and detected by γ-H2AX foci immunostaining. This effect could be reversed by the expression of FANCD2 S222A, but not K561R mutant, again implicating HR in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by vitamin E and N-acetylcysteine reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in BCR/ABL-dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.