Letters to the Editor
1447
Unraveling the role of FANCD2 in chronic myeloid leukemia tein.5,6 Our results evidenced for the first time that a disruption of the FA/BRCA pathway in BCR-ABL1 cells---in particular the impaired formation of nuclear FANCD2 foci---should have an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies. In their study, Koptyra et al.4 showed that in response to reactive oxygen species or MMC exposure, FANCD2-Ub was upregulated both in CD34 þ CML cells and in BCR-ABL1 cell lines. These authors showed that either the inhibited expression or monoubiquitination of FANCD2 reduced the clonogenic potential of CD34 þ CML cells and delayed the leukemogenesis of a lymphoblastic cell line after transplantation in recipient mice. Additionally, Koptyra et al.4 showed that FANCD2-Ub protects BCR-ABL1 cells from the potential lethal effect of an excess of ROSinduced double-strand breaks (DSBs), indicating that FANCD2-Ub should have an important role in BCR-ABL1 leukemogenesis. In contrast to our results, Koptyra et al.4 associated the upregulated expression of FANCD2-Ub in BCR-ABL1 cells with an enhanced nuclear foci formation by FANCD2-Ub in the Mo7e cell line.4 The authors discussed that their data were seemingly in contrast with our results, arguing that most of our measurements were performed in BCR-ABL1-positive CB-CD34 þ cells 16 h after MMC treatment. According to these authors, such time would correspond to late stages of DNA-damage response, where most MMC-induced DSBs would be already repaired in BCR-ABL1-positive cells. Aiming to investigate whether the inhibited formation of FANCD2 foci observed in our study with BCR-ABL1 cells could be related to the hypothesis proposed by Koptyra et al.,4 the kinetics of FANCD2 and also of gH2AX (surrogate marker of DSBs) foci formation after MMC exposure was determined both in BCR-ABL1-transduced CB CD34 þ cells and also in primary CD34 þ cells from CML patients.
Recent studies have offered new clues to understand the role of the Fanconi anemia (FA) pathway in the etiology and the progression of sporadic cancer (reviewed in Lyakhovich and Surralles1 and Valeri et al. 2) This is particularly true in the case of chronic myeloid leukemia (CML), where two different studies have recently shown the role of FANCD2, a critical protein in the FA pathway, in the genetic instability3 and the leukemogenesis4 induced by the BCR-ABL1 oncogene. Using human hematopoietic cells of different origin, our data showed that BCR-ABL1 inhibits the formation of FANCD2 nuclear foci, but not the expression nor the monoubiquitination of this protein, either spontaneously or after exposure to DNA crosslinking drugs, such as mitomycin C (MMC).3 Although the inhibited formation of FANCD2 foci in cells from CML patients or from the BCR-ABL1-transfected megakaryoblastic leukemia Mo7e cell line could be a consequence of secondary events related to the genetic instability of these cells, our data in fresh cord blood CD34 þ (CB-CD34 þ ) cells transduced with the MIN210 retroviral vector are of particular relevance, as they directly implicate the BCR-ABL1 oncogene in the inhibited formation of FANCD2 foci in primary human hematopoietic stem/progenitor cells. In that study, we also showed that both the impaired formation of FANCD2 nuclear foci and also the centrosomal and chromosomal aberrations---measured by chromatid-type breakages---induced by BCR-ABL1, were significantly reverted by the ectopic expression of BRCA1, in good consistency with previous data showing a downregulated expression of BRCA1 by the BCR-ABL1 oncopro-
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Leukemia (2012) 26, 1447--1448; doi:10.1038/leu.2012.32; published online 2 March 2012
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Figure 1. Kinetic analysis of gH2AX and FANCD2 nuclear foci in BCR-ABL1-transduced cord blood CD34 þ cells and in bone marrow CD34 þ cells from a CML patient. In (a, b) cord blood CD34 þ cells transduced with the control MINR1 retroviral vector (only expressing DNGFR as a marker) or with the MIN210 retroviral vector (carrying the BCR-ABL1 and the DNGFR) were immunoselected to assure 495% transduced cells. In (c) fresh peripheral blood CD34 þ cells from a CML patient at diagnosis were used. The figure shows the proportion of cells with gH2AX and FANCD2 foci at different time points after exposure to 40 nM of MMC. In each condition, samples not exposed to MMC were also analyzed as a control. In each Figure, representative microphotographies showing nuclear gH2AX, FANCD2, and co-localized gH2AX and FANCD2 foci are included. Accepted article preview online 6 February 2012
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Leukemia (2012) 1402 -- 1448
Letters to the Editor
1448
added to CD34 þ cells. Again, a marked inhibition in the formation of FANCD2 foci in BCR-ABL1 cells was observed (data not shown). All these studies confirm our previous observations3 and reinforce our main conclusion that BCR-ABL1 inhibits the formation of FANCD2 foci, in a process that is particularly significant after DNA damage. Taken together the results of our previous study3 (reinforced by data from Figures 1 and Supplementary Figure 1 from this correspondence) and the results presented by Koptyra et al.,4 it can be proposed that FANCD2 would have a dual role in the progression of CML (see Figure 2). Although the upregulated expression of FANCD2, followed by their monoubiquitination in K561, would facilitate the survival and/or growth of CML cells, as proposed by Koptyra et al,4 the inhibited formation of FANCD2 nuclear foci by the BCR-ABL1 oncoprotein would contribute to the genetic instability of CML cells.3 Elucidating the pathways by which FANCD2-Ub could somehow participate in the survival/ growth of CML cells independently of its chromosomal stability function---directly related to the generation of nuclear foci at sites of DNA damage---would further clarify the role of FANCD2 in the leukemia progression of CML. Figure 2. Proposed model of the role of FANCD2 in the leukemia progression of CML cells.
CONFLICT OF INTEREST The authors declare no conflict of interest.
As shown in Figure 1a, a progressive increase in the percentage of control cells (CB-CD34 þ cells transduced with the MINR1 RV carrying the DNGFR marker) with gH2AX foci was observed after MMC exposure. A parallel kinetics was observed regarding the formation of FANCD2 foci in these cells. In CB-CD34 þ cells transduced with the MIN210 RV (carrying the BCR-ABL1 plus the DNGFR marker), also a gradual increase in the proportion of cells with gH2AX foci was observed (up to 16 h post MMC treatment; Figure 1b). Remarkably, most of these cells did not show FANCD2 foci (Figure 1b). Moreover, identical observations were obtained when CD34 þ cells from a CML patient at diagnosis were analyzed (Figure 1c). Our data in Figure 1 demonstrate that the inhibited formation of FANCD2 foci in BCR-ABL1 cells is occurring in cells with a high number of DSBs. This contrasts the suggestion of Koptyra et al.4 who proposed that the inhibited formation of FANCD2 foci reported in our study was a consequence of conducting our analyses at late stages of DNA damage response, where most MMC-induced DSBs could be already repaired. Because the discrepancy in FANCD2 foci formation in our study and in Koptyra’s study might depend on the antibodies used for FANCD2 analyses, we investigated in healthy and also in BCR-ABL1 CD34 þ cells (both BCR-ABL1-transduced CB CD34 þ cells and CD34 þ cells from CML patients at diagnosis) the generation of FANCD2 foci with these two antibodies. As shown in Supplementary Figure 1, almost identical results were obtained when either of these antibodies was used. In additional studies, a much higher dose of MMC (1.50 mM) as used in Koptyra’s experiments was
A Valeri1, P Rı´o1, X Agirre2, F Prosper2 and JA Bueren1 1 Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energe´ticas, Medioambientales y Tecnolo´gicas (CIEMAT) and Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Madrid, Spain and 2 Fundacio´n para la Investigacio´n Me´dica Aplicada (CIMA), Clı´nica Universidad de Navarra, Pamplona, Spain E-mail:
[email protected]
REFERENCES 1 Lyakhovich A, Surralles J. Disruption of the Fanconi anemia/BRCA pathway in sporadic cancer. Cancer Lett 2006; 232: 99 -- 106. 2 Valeri A, Martinez S, Casado JA, Bueren JA. Fanconi anaemia: from a monogenic disease to sporadic cancer. Clin Transl Oncol 2011; 13: 215 -- 221. 3 Valeri A, Alonso-Ferrero ME, Rio P, Pujol MR, Casado JA, Perez L et al. Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells. PloS One 2010; 5: e15525. 4 Koptyra M, Stoklosa T, Hoser G, Glodkowska-Mrowka E, Seferynska I, Klejman A et al. Monoubiquitinated Fanconi anemia D2 (FANCD2-Ub) is required for BCR-ABL1 kinase-induced leukemogenesis. Leukemia 2011; 25: 1259 -- 1267. 5 Deutsch E, Jarrousse S, Buet D, Dugray A, Bonnet ML, Vozenin-Brotons MC et al. Down-regulation of BRCA1 in BCR-ABL-expressing hematopoietic cells. Blood 2003; 101: 4583 -- 4588. 6 Wolanin K, Magalska A, Kusio-Kobialka M, Podszywalow-Bartnicka P, Vejda S, McKenna SL et al. Expression of oncogenic kinase Bcr-Abl impairs mitotic checkpoint and promotes aberrant divisions and resistance to microtubuletargeting agents. Mol Cancer Ther 2010; 9: 1328 -- 1338.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Leukemia (2012) 1402 -- 1448
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