BCR/ABL Requires Fanconi Anemia D2 (FANCD2) Protein to Transform Hematopoetic Stem Cells.
Authors: Stoklosa Tomasz , Koptyra Mateusz , Hoser Grazyna , Seferynska Ilona , Glodkowska Eliza , Skorski Tomasz 
Published: October 2019

Abstract
Abstract 3249
Poster Board III-1
Fanconi D2 protein (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) and phosphorylated on S222 (FANCD2-phosphoS222) in response to DNA double-strand breaks (DSBs). FANCD2-Ub interacts with RAD51 to facilitate homologous recombination repair (HRR), and FANCD2-phosphoS222 activates the S phase checkpoint. We detected an increased amount of FANCD2-Ub in CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) patients and in BCR/ABL-positive cell lines 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 reactive oxygen species (ROS) with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). In addition mitomycin C routinely used for diagnostic testing in Fanconi anemia, strongly elevated FANCD2-Ub in CD34+ CML cells. Therefore we postulate that FANCD2-Ub may play a role in BCR/ABL transformation. In support for this hypothesis, we observed that clonogenic potential of BCR/ABL-positive murine leukemia stem cells (LSCs)-enriched FANCD2-/- Sca1+Kit+lin- bone marrow cells was reduced by approximately 10-fold in comparison to BCR/ABL-positive FANCD2+/+ counterparts; non-transformed -/- and +/+ cells displayed similar clonogenic potential stimulated by SCF and GM-CSF. Restoration of FANCD2 expression “rescued” the impaired clonogenic activity of BCR/ABL-positive FANCD2-/- cells. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Negative effect of BCR/ABL kinase on FANCD2-/- cell growth was reversed by expression of exogenous FANCD2. The in vitro growth defect of BCR/ABL-positive FANCD2-/- cells was accompanied by delayed leukemogenesis in SCID mice. These results suggest that FANCD2, a key regulator of DNA damage response, may play an important role in the initiation and/or maintenance of BCR/ABL-positive leukemias. We showed before that CD34+ CML-CP and CML-BC cells contain higher number of ROS-induced DSBs in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that LSC-enriched (CD34+CD38-) CML-CP and CML-BC cells display more DSBs than normal counterparts. Thus, BCR/ABL-mediated leukemogenesis is associated with accumulation of an excess of ROS-induced DSBs, which if not repaired, may induce apoptosis. We hypothesize that FANCD2 is necessary to “protect” leukemia cells from potentially lethal effect of BCR/ABL-induced oxidative DNA damage (including DSBs) at early stages of transformation and possibly also during the progression to CML-BC. This suggestion is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DSBs in comparison to +/+ counterparts. This effect did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect of BCR/ABL-positive FANCD2-/- cells. Accumulation of excessive DNA damage (DSBs) and impairment of growth potential of BCR/ABL-positive FANCD2-/- cells could be prevented by expression of FANCD2 wild-type (proficient in DNA repair and S phase checkpoint) and S222A phosphorylation-deficient mutant (proficient in DNA repair, but deficient in S phase checkpoint), but not the K561R monoubiquitination-deficient mutant (deficient in DNA damage, but proficient in S phase checkpoint). Since FANCD2-Ub interacts with RAD51 to promote HRR and BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, it is plausible that elevated expression of FANCD2-Ub may facilitate DSB repair to protect leukemia cells from lethal effects of DSBs. In concordance, co-localization of FANCD2-Ub and RAD51 was readily detected in the nuclei of BCR/ABL-positive leukemia cells. In conclusion our results indicate that FANCD2 plays an important role during the induction and perhaps also progression of Philadelphia-chromosome positive leukemias due to its ability to facilitate the repair of numerous, potentially lethal DSBs.

Disclosures:
No relevant conflicts of interest to declare.