Abstract

We showed before that BCR/ABL-mediated leukemogenesis is associated with the elevation of reactive oxygen species (ROS), which in addition to enhancing signaling pathways may increase the number of oxidative DNA lesions including DNA double-strand breaks (DSBs) [Blood, 2005, 2006]. CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain higher number of DSBs induced by ROS in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cell-enriched populations display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Thus, BCR/ABL-positive leukemia cells appear to accumulate an excess of ROS-induced DSBs, which may cause apoptosis if not repaired. We reported before that homologous recombination repair (HRR), involving RAD51 protein, plays a pivotal role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS [Blood, 2005]. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 by FANCL ubiquitinase and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 (FANCD2-Ub) interacts with RAD51 to facilitate HRR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint. We detected an increased amount of FANCD2-Ub in BCR/ABL-positive leukemia cell line and CD34+ CML-CP and CML-BC cells in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of ROS with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). Therefore we postulate that BCR/ABL kinase-dependent ROS-induced FANCD2- Ub may play a role in leukemic transformation. This hypothesis is supported by impaired transformation potential of BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. Restoration of the expression of FANCD2 protein in FANCD2−/− cells rescued the transforming potential of BCR/ABL kinase. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. Again BCR/ABL kinase did not exert a negative effect on the proliferation of FANCD2-reconstituted cells. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied by delayed leukemogenesis in SCID mice. Growth potential of BCR/ABL-positive FANCD2−/− could be rescued by co-expression of FANCD2 wild-type and S222A mutant, but not the K561R mutant. This observation supports our hypothesis that K561 monoubiquitination, but not S222 phosphorylation of FANCD2 might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, elevated expression of FANCD2-Ub may facilitate this process. This speculation is supported by the observation that although BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, the former cells accumulate more DSBs evaluated by neutral comet assay and immunostained gamma-H2AX nuclear foci. This effect could be reversed by the expression of FANCD2 S222A mutant, but not K561R mutant, again implicating FANCD2-Ub 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 ROS by VE and NAC 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 FANCD2-Ub, but not FANCD2-phosphoS222 may play an important role in BCR/ABL-dependent leukemogenesis probably due to its ability to interact with RAD51 and facilitate HRR of the numerous ROS-induced DSBs.

Disclosures: No relevant conflicts of interest to declare.

Author notes

Corresponding author