Published in Volume
121, Issue 1
(January 4, 2011)J Clin Invest.
Copyright © 2011, American Society for Clinical Investigation
Cytokinesis failure and attenuation: new findings in Fanconi anemia
Division of Hematology, The Children’s Hospital of Philadelphia, and University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Philip J. Mason, Room 302E, Leonard and Madlyn Abramson, Pediatric Research Center, The Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104-4318, USA. Phone: 267.426.9327; Fax: 215.590.4834; E-mail:
First published December 22, 2010
The hallmarks of the rare inherited disorder Fanconi anemia (FA) are progressive bone marrow failure and susceptibility to cancer. The former is the major cause of death for patients with FA, as it usually occurs earlier in life than cancer development. Despite spectacular advances in unraveling the molecular details of FA, the origin of the bone marrow failure that is central to this condition for most patients has long been puzzling and controversial. Two studies recently published in the JCI, including one in this issue, will add to the debate. They also highlight the fact that studying rare disorders can elucidate important new clinical and biological principles.
Fanconi anemia (FA) is a recessive, inherited disorder that occurs with an incidence of approximately 1 per 350,000 births, that is, relatively rarely. One of the major hallmarks of FA is bone marrow failure (BMF), which is the major caused of death in patients with FA, but it is also associated with an increased susceptibility to leukemia and certain cancers (1). FA is caused by mutations in one of fourteen or more genes (the so-called FA genes), whose products cooperate to ensure the repair of interstrand crosslinks that form during DNA replication (i.e., during the S phase of the cell cycle) (2). Failure to repair these lesions efficiently leads to the activation of S and G2 cell cycle checkpoints that trigger cell cycle arrest (3, 4). Failure of cells, particularly HSCs and blood cell precursors, to progress through the cell cycle has been thought to contribute to the BMF experienced by patients with FA (5). Despite intensive investigation of the nature and function of the FA genes, the precise mechanism(s) underlying BMF and cancer susceptibility in FA has not been fully elucidated and remains controversial. The same can be said of other inherited BMF syndromes, such as dyskeratosis congenita, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome (6). In these diseases, BMF develops slowly over the course of many years, making the process difficult to investigate in a cell line or even in a short-lived mouse model.
Two remarkable studies recently published in the JCI, including one in this issue, report findings that will change the way we think about BMF in FA and may eventually change the way patients with the disease are cared for and treated (7, 8). In a recent issue of the JCI, Vinciguerra et al. (7) reported that some FA cells proceed past the G2 checkpoint with DNA that is incompletely replicated and complete mitosis with sister chromatids in daughter nuclei still connected by ultrafine DNA bridges. It is at the cytokinesis stage, the point of division of the cytoplasm into two daughter cells, that problems arise. As reported in the second paper (8), which is published in this issue of the JCI, Ceccaldi et al. found that a proportion of FA patients, who live longer than others and do not have severe BMF, have developed a clone of blood cells that is still deficient in repairing interstrand crosslinks but no longer activates the G2 checkpoint to trigger cell cycle arrest. This is due to a somatically acquired dampening of the signaling pathway that regulates the G2/M transition, although the precise molecular change was not defined.
Failure of cytokinesis as a possible mechanism of BMF
The impetus for the study by Vinciguerra et al. (7) was the recent finding that chromatin foci containing monoubiquitinated FA, complementation group D2 (FANCD2) and FANCI, which assemble during S phase and disassemble late in S phase when DNA repair is complete, sometimes persist during and after mitosis at fragile sites in which DNA replication is slow (9, 10). In these cases, the sites in sister chromatids in which these foci are located are connected by ultrafine bridges (UFBs). These bridges are coated by two DNA helicases: Bloom syndrome, RecQ helicase-like (BLM), the protein mutated in Bloom syndrome (a rare autosomal recessive disorder characterized by a high frequency of chromosomal breaks and rearrangements), and Plk1-interacting checkpoint helicase (PICH), a checkpoint helicase (11). Crosslinking agents, such as mitomycin C (MMC), to which FA cells are exquisitely sensitive, increase the number of UFBs.
These findings led Vinciguerra et al. to study the effect of FA mutant phenotypes on the behavior of UFBs (7). They found that the number of UFBs was increased in fibroblasts and cell lines from patients with FA and in HeLa cells depleted of specific FA proteins. They went on to show that late in mitosis the UFBs were coated with the FA protein FANCM, which is important in recruiting the FA core complex to the sites of crosslinks and in facilitating the monoubiquitination of FANCD2 and FANCI. FANCM mutant cell lines do not ubiquitinate FANCD2 and are MMC sensitive (12). Interestingly, a FANCM mutant that does not have ATPase activity corrected the ubiquitination defect but not the MMC sensitivity or the increased number of UFBs, suggesting that ATPase activity is needed for preventing or resolving the bridges. The consequence of the increased number of UFBs is an increased number of binucleate or multinucleate cells arising from cytokinesis failure. Multinucleate cells were also generated by knock down of several FA proteins. BLM knock down and the FANCM ATPase mutation also result in an increase in the number of multinucleate cells (13). Can cytokinesis failure contribute to BMF in FA? Mice null for Fancg or Fancd2 show a HSC defect but not overt BMF. HSCs purified from these mice showed a higher percentage of multinucleate cells than did controls. Moreover, bridges joining the nuclei stained with lamin-associated polypeptide 2, a nuclear membrane protein that accumulates around chromatin at the end of mitosis. The fate of cells failing cytokinesis is either apoptosis or cell cycle arrest and senescence. In the mice, an increase in apoptosis correlated with the increase in multinucleate cells (Figure 1).
Cytokinesis failure in FA. PICH-BLM UFBs are ultrafine DNA bridges that have FANCD2-FANCI foci at their extremities and are most likely derived from unresolved replication intermediates. The paper by Vinciguerra et al. (7) shows that hematopoietic cells from patients with FA have an increased number of UFBs between daughter nuclei that may inhibit cytokinesis, leading to binucleated or multinucleated cells. These cells may undergo apoptosis, contributing to BMF, or may lead to genomic instability and malignancy.
This body of work reported by Vinciguerra and colleagues (7) highlights the importance of the FA proteins in cell division, in which they are shown to be important in the resolution of UFBs that arise from unresolved replication intermediates. The question now is how important is the observed failure of cytokinesis in BMF or in progression to cancer? FANC mutant cells show increased chromosomal breaks at fragile sites (14), providing a connection between UFBs and genomic instability. If genomic instability or multinucleated cells result in apoptosis in HSCs, as Vinciguerra and colleagues observed (7), UFBs could contribute to BMF. Furthermore, if the cells are allowed to progress by somatic mutations compromising checkpoint regulation, this may lead to the development of cancer. However, BLM-deficient cells behave like the FA cells, in terms of the formation of UFBs and multinucleate cells, and patients with Bloom syndrome do not develop BMF. This implies that there may be a qualitative difference in the resolution of UFBs in Bloom syndrome versus FA, due to the presence or absence of FANC proteins at the UFB termini; alternatively, there might be an intrinsic difference in the downstream response, as discussed by Vinciguerra and colleagues (7).
Attenuation of BMF for the price of leukemia later in life
The study by Ceccaldi et al. began with the observation that a small number of patients have only two of the usual three criteria for FA (8). Like those of other FA patients, the peripheral blood cells (stimulated lymphocytes) of these patients lacked the crucial monoubiquitination of FANCD2 and showed increased chromosomal breaks after MMC treatment. But, unlike the cells from most FA patients, they did not show the characteristic G2 arrest after MMC treatment. A cohort of 97 patients was examined in this way, and this phenotype was seen in 17 patients. Most of these patients were among the oldest in the cohort, their blood counts were almost normal, and five of them developed myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The fact that this “attenuation” of the G2 checkpoint in response to DNA damage was an acquired characteristic was apparent, because the MMC-induced G2 arrest was still operational in fibroblasts from the same patients and because two patients were observed to convert to the attenuated phenotype from typical FA over the course of the analysis.
Attenuation was accompanied by clonal hematopoiesis, as demonstrated by a biased X-inactivation (8), implying that all peripheral blood cells derived from a single progenitor cell in which a molecular event took place, giving rise to attenuated G2 arrest (Figure 2). However, three out of six classical FA patients also showed clonal hematopoiesis, as is commonly seen in individuals with inherited BMF syndromes (15). Arrest at the G2 checkpoint is modulated by a pathway involving ataxia telangiectasia and Rad3 related (ATR) phosphorylation, p53 stabilization, and CHK1 checkpoint homolog (CHK1) expression (16). p53 and CHK1 levels were low in attenuated cells from six out of the nine patients with this phenotype, and CHK1 mRNA levels were low in attenuated cells from most of them. The search for the molecular explanation did not come up with any mutation or deletion in the P53 or CHK1 genes or any alterations in their methylation pattern. However, of two micro RNAs previously shown to target CHK1 expression, one of them, miR-15a, was present at a higher level in attenuated patients. That this might be important in the attenuation mechanism was supported by the authors’ demonstration that CHK1 had a higher turnover, as assessed by proteosome inhibition studies, in attenuated cells (8). Verification that FA-induced arrest at the G2 checkpoint can be regulated by the ATR and CHK1 pathway was provided by experiments with a HeLa cell line with a silenced FANCD2 gene. Knocking down ATR or CHK1 in these cells removed the arrest at the G2 checkpoint. A CHK1 inhibitor was also shown to attenuate EBV lines from patients with the classical FA phenotype, while a CHK2 inhibitor had no effect. These cells were also protected from MMC-induced death by the CHK1 inhibitor. The cost of the attenuated FA phenotype in these patients was a higher risk of malignancy. Of the 97 patients in the cohort, five had excess blasts or overt myeloid leukemia. All of these had the attenuated phenotype. In four of these patients, blasts could be purified, and they all had very low CHK1 levels. Interestingly, low CHK1 levels are characteristic of AML generally, not only when arising from FA (17).
Clonal attenuation of a G2 checkpoint in FA.
Hematopoietic cells from patients with FA exhibit slow growth and accumulate in G2, leading to BMF. Ceccaldi et al. (8) show that, in some patients, blood counts are normalized due to the appearance of a clone of cells, in which the arrest at the G2 checkpoint does not take place. Hematopoiesis in these patients is greatly improved, but the trade off is an increased susceptibility to later development of MDS and AML. This scenario, identified in FA, is likely to be a theme, applicable not only to many forms of inherited BMF syndromes but possibly to other regenerative disorders associated with an increased risk of malignant transformation.
There are several important aspects of this work by Ceccaldi et al. (8). The attenuation phenotype may explain the very different clinical outcomes sometimes seen in two members of the same family who carry the same causative mutation, particularly in the absence of any evidence of genetic reversion (18). In addition, attenuation, identified by the dissociation of chromosomal breakage and G2 arrest after exposure to DNA crosslinking agents, is associated with a higher chance of transformation to malignancy, thus identifying patients who, although milder in their presentation of BMF, might need continuous, intensified tumor surveillance. Finally, the study by Ceccaldi et al. (8) identifies a druggable target, the G2 checkpoint, that, when suppressed, improves blood cell production and may prolong the survival of FA patients if the increased incidence of cancer can be controlled. If the checkpoint could be activated or restored, it could, in principle, be used to limit the growth of cancer cells by inducing cell cycle arrest and cell death.
Conclusions and prospects
Most previous studies have focused on the role of the FANC proteins in S phase. The papers by Vinciguerra et al. (7) and Ceccaldi et al. (8) provide important insights into different aspects of FA pathogenesis that converge on events in the G2 phase of the cell cycle and during mitosis. Vinciguerra et al. showed that in FA there is an increase in the number of cells that enter mitosis with incompletely replicated DNA and that this can lead to the formation of multinucleated cells. These, in turn, can lead to BMF by apoptosis or to genomic instability and cancer. Ceccaldi et al. showed that, in FA patients, clones of blood cells can arise in which the G2 checkpoint has been attenuated and no longer slows cell cycle progression. This leads to an improvement in blood counts and lifespan but carries with it an increased risk of malignancy. Both papers illustrate that studying a rare disease can lead to the discovery of important clinical and biological principles, with implications far beyond the original investigation. In this case, DNA repair proteins are shown to be involved in the resolution of a new kind of ultrafine DNA bridge that, if unresolved, is associated with cytokinesis failure and cell death, providing a possible additional mechanism for BMF. The Ceccaldi paper, on the contrary, demonstrates that overcoming a cell cycle checkpoint is associated with an improvement in BMF, highlighting the importance of the activated G2 checkpoint in the pathogenesis of BMF.
Philip J. Mason’s work is supported in part by NIH/NCI grant R01 CA106995, and Monica Bessler’s work is supported in part by NIH/NCI grant R01 CA105312.
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