Review Article
Why does the bone marrow fail in Fanconi anemia?
Juan I. Garaycoechea and K. J. Patel
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom
The inherited bone marrow failure (BMF)
syndromes are a rare and diverse group
of genetic disorders that ultimately result
in the loss of blood production. The molecular defects underlying many of these
conditions have been elucidated, and great
progress has been made toward understanding the normal function of these
gene products. This review will focus on
perhaps the most well-known and genetically heterogeneous BMF syndrome:
Fanconi anemia. More specifically, this
account will review the current state of
our knowledge on why the bone marrow
fails in this illness and what this might
tell us about the maintenance of bone
marrow function and hematopoiesis.
(Blood. 2014;123(1):26-34)
Historical timeline
It is now 85 years since the Swiss pediatrician Guido Fanconi ﬁrst
described the case of 3 brothers with aplastic anemia associated with
developmental defects.1 It was later found that the condition was
inherited as a Mendelian recessive trait, thus setting the ground for
the eventual determination of the molecular defect driving “Fanconi
anemia” (FA). Over time, a single unifying feature of the disease
emerged: cells from afﬂicted individuals were extremely sensitive to
agents that cross-link the 2 strands of DNA together.2,3 Cross-linking
chemicals, such as mitomycin C (MMC) and diepoxybutane, are a
diverse class of molecules that are potently cytotoxic. Thus, the
pivotal observation that cells derived from FA children accumulate
broken chromosomes and die rapidly when exposed to crosslinking agents deﬁned a cell autonomous molecular defect underpinning this condition. This linked FA to a failure to resolve a speciﬁc
class of genomic damage. Armed with cross-linker–induced chromosome breakage as a simple cellular test for FA, clinicians rapidly
identiﬁed many families throughout the world affected with this
illness.4 In 1992, the ﬁrst FA gene (FANCC) was identiﬁed by
expression cloning and complementation of a patient-derived cell
line.5 However, the coding sequence of the FANCC gene revealed
nothing of how it may function to prevent FA; the FANCC
polypeptide is completely devoid of any domain signatures. In the
decades that followed, we came to realize that FA was genetically
heterogeneous, and a worldwide race ensued to identify the many
genes that constitute the 16 FA complementation groups.
At the same time as the complex molecular genetics was unraveling, studies were underway to understand the molecular function
of the FA gene products. The key questions to resolve were how the
known components prevented the cellular chromosome breakage
phenotype and, more challengingly, the clinical phenotype. Early
studies indicated that the FANCC protein localized to the cytoplasm.6,7
This unexpected result immediately suggested that this FA protein
might not exert a direct inﬂuence on DNA repair. It also brought into
question whether the chromosomal breakage defect in FA cells was
sufﬁcient or indeed the primary reason for bone marrow failure
(BMF). Credence for this view came from a body of work indicating
that FA patient–derived cells were sensitive to certain cytokines.8,9
This unusual observation could not be easily reconciled with
defective DNA repair as the primary reason why the bone marrow
fails in FA. More recent studies indicated a more direct role
for certain FA proteins in modulating signaling responses10,11
(Figure 112-16).
On the other hand, the majority of the FA gene products were
shown to form a large nuclear complex, which also includes FANCC,
because it was later found that FANCE clearly promotes its import
into the nucleus.17 This multiprotein complex modiﬁed the key
downstream FA gene product FANCD2, by conjugation of a single
ubiquitin molecule (monoubiquitination). This critical modiﬁcation
could be stimulated by DNA damage, resulting in the localization of
the FANCD2 protein to sites of nuclear DNA damage.18 Further
strong support for the view that the primary function of the FA gene
products was in the repair of DNA damage came from the eventual
identiﬁcation of mutations in bona ﬁde DNA repair genes in rare FA
patient complementation groups.19-21 Thus, 2 hypotheses (ie, defective DNA repair or abnormal cytokine response) emerged as rival
and contrasting explanations of why the bone marrow fails in FA.
However, a key limitation was that they both lacked evidence that
linked either of them directly to BMF. Subsequently, we set out the
merits of these 2 hypotheses in the origins of marrow failure in FA.
We review and assess the current state of knowledge and evidence to
support each of them in this context. Finally, we discuss recent work,
which has identiﬁed naturally derived aldehydes as drivers of BMF
in FA.
Submitted September 17, 2013; accepted October 28, 2013. Prepublished
online as Blood First Edition paper, November 7, 2013; DOI 10.1182/blood2013-09-427740.
© 2014 by The American Society of Hematology
26
FA gene products have a clear function in
DNA repair
All 16 FA gene products clearly function in a common process to
maintain genomic stability and to repair DNA interstrand cross-links
(ICLs) (Figure 1). The majority of the FA proteins (FANCA, B, C,
E, F, G, L, and M) assemble to form a large nuclear complex termed
the “FA core complex.”22 The core complex functions as a multisubunit E3 ubiquitin ligase with FANCL catalyzing the monoubiquitination of FANCD2 and FANCI after DNA damage18,23
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WHY DOES THE BONE MARROW FAIL IN FANCONI ANEMIA?
27
Figure 1. Multifunctionality of the FA proteins. This figure summarizes the known FA proteins and their functions. All FA proteins cooperate in the FA pathway to repair
cross-linked DNA. Most of them have additional functions in other DNA repair transactions.12-14 Finally, some of them have been found to modulate cytokine responses or
interact with REDOX sensing proteins, independently of their role in DNA repair.15,16,82-84
(Figure 2). Loss of any one of the components of the core complex
leads to its destabilization, abrogation of E3 ligase activity, and
therefore, loss of robust FANCD2/FANCI ubiquitination in response
to DNA damage. Ubiquitinated FANCD2 and FANCI form a
heterodimer that is recruited to chromatin where it participates in
the DNA repair process. However, an ICL is a complex lesion,
requiring that multiple steps be removed. How then do the FA
proteins participate in this removal?
A comprehensive genetic analysis of DNA cross-link repair was
carried out in a tractable genetic system offered by chicken DT40
cells. These studies revealed that the FA proteins must work in
cooperation with 3 other key repair processes: excision repair,
translesion synthesis, and double-strand break repair through
homologous recombination (HR).24,25 The discovery that mutations
in bona ﬁde HR genes (like BRCA2) and in components of the
excision nuclease machinery (SLX4 and, most recently, XPF) can
all result in FA conﬁrmed the observations made in genetic
systems.19,20,26,27 However, the order and the regulation of these
various processes could not be further deﬁned using such genetic
approaches, and the precise mechanism of how the FA proteins
participated in the repair of a DNA cross-link remained elusive.
The solution of the structure of FANCD2/FANCI provides some
insight into this question. This showed that both proteins form
a binding interface for both single- and double-stranded DNA; the
DNA structures that form when replicating DNA are stalled at
a cross-link.28
A key insight into how replication-coupled DNA cross-link
repair is achieved came from work using Xenopus egg extracts. In
this cell-free system, the replication of a plasmid bearing a single
cross-link can be followed with single nucleotide resolution in
vitro.29 First, replication stops before reaching the cross-linked
DNA. One fork advances up to the cross-link, and at this point, the
cross-link is excised. A translesion polymerase synthesizes across
the adducted DNA creating an intact duplex strand of DNA.29,30
This duplex is then used to repair the double-strand break using
HR.31 When FANCD2 is depleted from the nuclear extract or its
monoubiquitination is abrogated, then cross-link repair is impeded
because of a failure to initiate the nuclease incision step. This work
unequivocally showed that the Fanconi pathway is critical for
promoting endonucleolytic cleavage of cross-linked DNA, and
that its loss leads to a speciﬁc molecular defect in DNA repair.
However, our DNA is not normally exposed to nitrogen mustard,
the chemical used to create this cross-link, so the question remained as
to what the FA DNA repair pathway is repairing in a physiological
context.
BMF in FA is attributable to a stem cell defect
Ultimately, most FA patients develop BMF culminating in pancytopenia. Blood counts at birth are typically normal, but at the
median age of 7, hematologic complications arise. This initially
manifests as low platelet counts, thrombocytopenia, followed by
leukopenia before eventually developing into aplastic anemia.32,33 The fact that all blood lineages eventually become deﬁcient
strongly implies hematopoietic stem cell (HSC) dysfunction. Indeed,
FA patients have very low numbers of CD341 cells, a bone marrow
fraction that is enriched for HSCs and is capable of producing all
blood components upon transplantation.34 More recent work has
shown signiﬁcant depletion of the CD341 fraction in very young FA
patients, well before the onset of pancytopenia.35 The very low
number of CD341 cells in young FA patients points to a prenatal
origin of the stem cell defect. In fact, knockdown of FA genes in
human embryonic stem cells resulted in defective hematopoiesis.36
However, because of the limitations of studying the dynamics of
hematopoiesis in humans, numerous labs set out to establish murine
models of FA. Although BMF can be induced in these mice with the
use of the cross-linking agent MMC,37 these mouse knockouts do not
recapitulate the full severity of FA because they do not spontaneously
develop the hematologic phenotype.38 In a few instances, mice deﬁcient
in the Fanconi pathway show hematologic dysfunction.39,40 However,
importantly, FA knockout mice do show quantiﬁable lower numbers
of HSCs coupled with a reduced ability to reconstitute blood
production in irradiated recipients after transplantation.41,42 Furthermore, Fancc2/2 embryos show reduced numbers of stem and progenitor pools with compromised repopulation activity.43 Collectively,
28
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Is unresolved DNA damage the initiator
of BMF?
Despite the overwhelming genetic and biochemical evidence for the
role of FA gene products in repairing DNA cross-links, direct
evidence to show that this function is essential to preserve bone
marrow function has been lacking. Some circumstantial support
comes from the fact that FA patients are cancer prone and blood
malignancies from these patients often harbor gross chromosomal
changes. Both myelodysplastic syndrome (MDS) and acute myeloid
leukemia (AML) are thought to originate at the stem or progenitor cell level, as the longevity and proliferative capacity of
these cells renders them susceptible to the accumulation and
transmission of mutations. Such cytogenetic changes can only
occur as a consequence of misrepair of DNA damage, suggesting
that FA patients are prone to genetic instability within their HSC
and progenitor compartments.32,44-49
There are other aspects of the FA phenotype that might give us
a further clue as to which function of the FA gene products results in
the main feature of the human illness. FA is phenotypically heterogeneous; some patients present with a complex spectrum of congenital
abnormalities, whereas others only have a mild phenotype. The
hematologic features characteristic of the disease also vary; some
patients succumb to marrow failure as early as age 3, whereas others
might never develop it. With a few exceptions, most attempts at
correlating genotype-phenotype have proved unsuccessful so far.
However, what is clear is that children with severe developmental
abnormalities also present with early onset of BMF.50 This observation, together with the fact that the HSC defect starts in utero,
suggests that both developmental abnormalities and the contraction
of the stem cell pool could be triggered by the same cause.
One of the main cellular responses to DNA damage is the induction
of p53, which then initiates a response that results in cell cycle arrest
and, in certain instances, apoptosis. Genetic ablation or deﬁciency of
Figure 2. Current model of ICL repair by the FA pathway. (A) When DNA containing an ICL undergoes replication, the leading strands of 2 converging replication
forks stop at the lesion. The FA core complex is recruited to chromatin, where it
subsequently monoubiquitinates its 2 substrates, FANCD2 and FANCI. (B) Next, the 2
sister chromatids are uncoupled via dual incisions on either side of the ICL, possibly by
SLX4-XPF-ERCC1. (C) Subsequently, a translesion DNA polymerase (TLS pol)
extends the nascent strand beyond the ICL. (D) Finally, 2 fully repaired DNA duplexes
are generated through the action of nucleotide excision repair (NER) on the top duplex
and homologous recombination (HR) on the bottom duplex. The remaining FA
proteins (FANCD1/BRCA2, FANCO/RAD51C, FANCN/PALB2, and FANCJ/BRIP1)
are thought to be involved in these recombination transactions.
these data suggest that FA patients have an impaired HSC pool. This
defect begins in utero, worsens during childhood, and results in
spontaneous BMF early in life (Figure 3).
Figure 3. Progressive attrition of HSCs underlies BMF in FA. (A) The progressive
decline of HSC numbers in FA patients leads to BMF early in life. The HSC defect is
likely to start in utero. (B) Fanconi-deficient mice do not recapitulate the main feature of
FA. For this reason, their usefulness as disease models has been questioned.
However, FA-deficient mice do display a quantifiable defect in their HSC pool as well
as reduced ability to reconstitute blood production in irradiated recipients, which is also
present before birth. Even in the absence of BMF, it would be interesting to see if
Fanconi-deficient mice display age-dependent attrition in the quality of their HSC pool,
as has been observed for other DNA repair–deficient mice.
BLOOD, 2 JANUARY 2014 x VOLUME 123, NUMBER 1
p53 enables cells to tolerate DNA damage, so it is no surprise that p53
deﬁciency can partially rescue DNA cross-linker sensitivity in FAdeﬁcient cells. The lack of p53 also accelerates tumor formation in
Fancd22/2 and Fancc2/2 mice, suggesting that the FA DNA repair
pathway and p53 cooperate to suppress tumorigenesis.51,52 Most
recently, primary bone marrow cells from FA patients and Fancd22/2
and Fancg2/2 mice were shown to have elevated levels of p53, and
this was attributable to unresolved DNA damage. Finally, deletion or
knockdown of p53 also rescued the defects of FA human and mouse
hematopoietic progenitors.35 Although p53 inactivation allows
HSCs to survive DNA damage, a note of caution must be sounded:
this comes at the enormous cost of enhanced genomic instability and
tumor formation. Collectively, these data strongly indicate that
unresolved DNA damage in FA cells induces p53, which might then
lead to HSC depletion and hence progressive BMF in FA. Knockdown experiments and gene expression analysis initially suggested
that the p53-driven HSC elimination was mediated by p21 and cell
cycle arrest.35 However, genetic ablation of p21 does not rescue the
hematologic phenotype of Fancd22/2 mice, arguing against this
approach.53 Although it is possible that the p53 response in HSCs differs
between mice and humans, future genetic studies should address the
contribution of p53-dependent apoptosis to BMF in FA patients. In
support of this, a recent study has demonstrated an apoptotic response to
cytokinesis failure in FA-deﬁcient HSCs. It remains to be established
whether Fanconi proteins have a distinct role during mitosis or the DNA
bridges in FA-deﬁcient cells are a consequence of unresolved DNA
damage. However, this is one mechanism of HSC depletion that may
contribute to BMF in FA patients.54
The bone marrow is one of the most radiosensitive tissues in the
body, being the ﬁrst organ system to fail following total body
irradiation. It is DNA damage to the HSCs that ultimately limits the
regeneration of hematopoiesis.55 Genetic evidence for the consequences of DNA damage on HSC function and bone marrow
homeostasis is provided by the severe consequences of disrupting
many different DNA repair pathways in mice.56-58 Mice bearing null
or hypomorphic alleles for genes involved in DNA damage response
(ATM2/2, conditional ATR2/2), nonhomologous end joining (Ku702/2 ,
Ku802/2 , DNA-PKc2/2 , DNA-PKcs3A/3A, LigIV Y288C/Y288C),
HR (Rad50S/S), nucleotide excision repair (XPDTTD), and mismatch repair (Msh22/2) show severe defects both in the quantity
and functional quality of their HSC pools. In some cases, this
resulted in spontaneous BMF (DNA-PKcs3A/3A, LigIV Y288C/Y288C,
Rad50S/S).
It is likely that the main driver of BMF in FA is DNA damage to
the HSC pool. Genetic instability within FA-deﬁcient HSCs results
in the progressive attrition of this vital cell population or the genesis
of neoplastic clones. This notion is supported by the observation that
a strong p53 response mediates the FA hematologic phenotype and
by the fact that different DNA repair deﬁciencies, unrelated to FA,
also have profound consequences on HSC function.
What is the cause of DNA damage in HSCs?
Should unresolved DNA damage lead to HSC attrition in FA, then
a key question is how such damage arises in the physiological setting.
Without resolution of this key question, a DNA damage hypothesis
for the cause of BMF would be based solely on the circumstantial
evidence presented previously. Genomic DNA is intrinsically unstable, being subject to both spontaneous and enzymatic degradation.
Moreover, metabolism can generate a battery of reactive molecules
WHY DOES THE BONE MARROW FAIL IN FANCONI ANEMIA?
29
that are capable of attacking DNA, the most ubiquitous being reactive
oxygen species (ROS).
The ﬁrst clue that such molecules might contribute to the endogenous burden of DNA damage driving the FA phenotype was the
hypersensitivity of patient-derived cell lines. ROS arise in cells as
a consequence of normal cellular metabolism, namely, the electron
transport chain and lipid peroxidation, but they also have important
roles in cell signaling. ROS can damage DNA, RNA, and proteins,
and they may also contribute to the aging process. It was demonstrated
long ago that the frequency of chromosomal aberrations in FA
lymphocyte cultures depends on oxygen tension.59 Since then, many
groups have shown that FA-deﬁcient cells grow better under hypoxic
conditions than in ambient oxygen concentration.60 More recently,
the use of low oxygen tension allowed the generation of FA-deﬁcient
iPS lines.61 Superoxide dismutases are a set of enzymes that
metabolize superoxide radicals (·O2-) to molecular oxygen and
hydrogen peroxide (H2O2), thus providing a defense against
oxygen toxicity. Genetic evidence linking oxidative stress to
bone marrow dysfunction in FA comes from Sod12/2 Fancc2/2
double mutant mice.62 These double knockout mice showed bone
marrow hypocellularity and decreased numbers of colony-forming
units. However, these mice showed normal numbers of HSCs as seen
by ﬂow cytometry and did not display developmental defects or
chromosomal aberrations typical of FA.
In search of a source of endogenous DNA damage, our laboratory
has focused on simple aldehydes. Small aldehydes such as formaldehyde and acetaldehyde are ubiquitously found in the environment and are also by-products of cellular metabolism. They are also
highly reactive, being able to form DNA adducts in vitro and
in vivo.63,64 Moreover, FA-deﬁcient cells have been shown to be
hypersensitive to both of these compounds and to accumulate
double-strand breaks and chromosomal aberrations.65-68 Finally,
acetaldehyde treatment induces the activation of the FA pathway
in wild-type cells.69
To genetically test if endogenous aldehydes are a source of DNA
damage, our laboratory generated mice harboring disruptions of the
key FA gene Fancd2 in combination with aldehyde dehydrogenase 2
(Aldh2). Aldh2 oxidizes acetaldehyde to acetate, thereby preventing
the accumulation of this genotoxic agent. Therefore, if metabolically
produced aldehydes are indeed DNA-damaging agents normally
counteracted by the FA pathway, then the simultaneous disruption of
both Aldh2 and Fancd2 should have a synergistic effect on the
phenotype of these mice.65 Most strikingly, the presence of aldehyde
catabolism in the mother was found to be essential for the development of Aldh22/2Fancd22/2 embryos. Surprisingly, if the mother
expressed Aldh2 (Aldh21/2), then double mutant mice could be born,
suggesting that the mother was capable of breaking down
acetaldehyde and compensating for the Aldh2 deﬁciency in the fetus.
When born, the Aldh22/2Fancd22/2 mice had increased prevalence
of developmental abnormalities such as kinked tails or eye defects.
Most Aldh22/2Fancd22/2 mice succumbed to acute lymphoblastic
leukemia within the ﬁrst 6 months of life. In an attempt to link
acetaldehyde to the phenotype of double knockout mice, these
animals were challenged with ethanol, a precursor of acetaldehyde. The treatment not only potentiated the developmental
abnormalities but also precipitated myelotoxicity in adult double
mutant mice.
Analysis of the hematopoietic compartment of double knockout
mice revealed a profound defect in the stem and progenitor cell pool,
with a more than 600-fold reduction in their HSC pool.70 It has been
known for a long time that HSCs possess high levels of aldehyde
dehydrogenase activity, and this was shown, in the case of mouse
30
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BLOOD, 2 JANUARY 2014 x VOLUME 123, NUMBER 1
Figure 4. A 2-tier protection mechanism preserves
HSC function. (A) HSCs display high Aldh2 activity
that protects their genome against the toxic effects of
reactive aldehydes. If aldehydes should evade this
protection mechanism, then DNA damage is dealt with
by the FA DNA repair pathway. (B) In Aldh22/2Fancd22/2
mice, the HSC pool is exposed to a greater burden of
reactive aldehydes, and in the absence of DNA repair,
DNA damage persists and leads to neoplastic transformation or HSC loss and spontaneous BMF.
HSCs, to be attributable to Aldh2. When this protection was taken
away, the HSCs were particularly compromised, as Aldh22/2
Fancd22/2 HSCs seemed to be far more sensitive to acetaldehyde
than more mature progenitors. This increased sensitivity also
correlated with the accumulation of DNA damage, as measured by
the induction of g-H2AX. Most strikingly, a small group of Aldh22/2
Fancd22/2 mice that did not get leukemia developed spontaneous
BMF. All of this data put together led us to the conclusion that BMF in
FA patients could result from endogenous aldehyde-induced toxicity,
which then leads to the depletion of HSCs (Figure 4). Despite this
recent discovery, the question remains as to why single FA knockout
mice so poorly replicate the human hematologic phenotype. It is only
upon exposure to the exogenous cross-linking agent MMC or by
taking away aldehyde catabolism in Fancd22/2 mice that the key
features of this human illness can be recapitulated. We provide some
speculations on this paradox.
First, FA patients show an age-dependent decrease in the frequency
of CD341 cells, which correlates with the worsening of clinical signs
and results in the onset of hematologic abnormalities at the median age
of 7.32 It is possible that the 2-year life span of mice does not allow for
the complete exhaustion of the stem cell pool. It would be interesting to
analyze Fanc-deﬁcient mice to ask if there are age-related changes in
the quantity/quality of the HSC pool (Figure 3). In support of this, it was
shown that mice deﬁcient in different genomic maintenance pathways
(NER, nonhomologous end joining [NHEJ], and telomere maintenance) show a severe decrease in the functional capacity of their HSCs
with age.71 Additionally, Fancc2/2 HSCs were found to perform
poorly at serial transplantation.43
Second, laboratory mice spend their lives in a highly controlled
environment with the same food source throughout their lives. It is
possible that this limits their exposure to exogenous DNA-damaging
agents. Finally, the murine metabolism could produce less endogenous genotoxins, or, alternatively, the protection against them
could be higher in mice than in humans. The metabolic rate of mice
is much greater than that of larger mammals.72 This could result in
the more rapid and efﬁcient detoxiﬁcation of toxic metabolites. It is
possible that it is necessary to take this protection away in order to
reveal the FA phenotype in mice.
An important prediction is that mutations in ALDH2 will lead to
more severe clinical features in FA patients. In humans, ALDH2 is
mutated in ;1 billion people. This dominant-negative polymorphism
(E487K) causes a dramatic decrease in ALDH2 activity, and, because it is most common in Southeast Asia, it is known as the
Asian ﬂushing syndrome.73 Hira and colleagues have recently determined the ALDH2 genotype of a group of Japanese FA patients.
They found that ALDH2 deﬁciency dramatically accelerates BMF
and increases the frequency of malformation in some tissues. Most
strikingly, those patients entirely deﬁcient for ALDH2 developed
BMF within the ﬁrst 7 months of life. These results unequivocally
conﬁrm that reactive aldehydes play an important role in the pathogenesis of FA.74
Although Aldh2 commonly oxidizes acetaldehyde, it has broad
substrate speciﬁcity, being able to oxidize other aldehydes including
4-hydroxynonenal, acrolein, propionaldehyde, and butyraldehyde.75
It remains to be clariﬁed if endogenous acetaldehyde is solely
responsible for the phenotype observed in Aldh22/2Fancd22/2
mice. Conversely, Aldh2 is 1 member of a family of 18 enzymes,
any of which could also be offering protection against various
aldehydes.76 Finally, there is a synthetic lethal interaction between
FANCD2 and alcohol dehydrogenase 5 (ADH5) in the chicken B-cell
line DT40.68 Adh5 is responsible for the detoxiﬁcation of formaldehyde, the simplest and most toxic aldehyde, which can be produced
close to DNA by means of DNA and histone demethylation. It will be
interesting to see what the consequences of Adh5 disruption in
Fanconi-deﬁcient mice are.
Do the FA gene products modulate
cytokine responses?
So far, we have discussed the very strong biochemical and genetic
evidence for a function of the FA genes in DNA repair and how
this function relates to BMF. However, we have already alluded to
the unusual sensitivity that FA cells display to certain inﬂammatory
cytokines.8,9 Three crucial observations underpin this discovery:
First, FA cells are prone to apoptosis when exposed in vitro to
tumor necrosis factor a (TNF-a) and interferon g (IFN-g).9,77,78
Second, exposing FA-deﬁcient mice to these cytokines results in
bone marrow dysfunction.79,80 Third, elevated levels of these
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WHY DOES THE BONE MARROW FAIL IN FANCONI ANEMIA?
31
Figure 5. Models for BMF in FA. (A) The accumulation of DNA damage in the HSC pool leads to p53dependent HSC depletion by diminishing the ability
of HSCs to proliferate and self-renew. Endogenously
generated aldehydes are an important source of such
damage. (B) The molecular basis for cytokine overproduction and hypersensitivity by FA cells remains
unclear. However, some FA proteins were proposed to
have a direct role in hematopoiesis and to regulate the
response to inflammatory signals, independent of DNA
repair. (C) Reconciliation of the roles of FA proteins in
hematopoiesis. A defect in DNA repair could be the
initiating event that would lead to cell death, tissue
injury, and the production of inflammatory cytokines.
These could then contribute to bone marrow dysfunction in a number of ways. TNF-a and IFN-g can cause
apoptosis in stem cells and progenitors, mediated, for
example, by the Fas receptor.78 Alternatively, TNF-a
could produce additional DNA damage by the induction
of reactive molecules, like ROS.85 Finally, inflammatory
cytokines like TNF-a and IFN-g have classically been
considered to inhibit HSC and limit stem cell function.
However, recent studies are challenging this view and
propose a role for inflammatory signals in the direct
regulation of HSCs.86 It is conceivable that these
signals could lead to the differentiation of HSCs, either
directly or indirectly, causing them to encounter new
DNA damage in the context of replication.
and other cytokines have been reported in FA patients and are
overproduced by FA-deﬁcient cells in vitro.13,81 These observations
underpin a general hypothesis that the hematopoietic phenotype of
FA is attributable to the overproduction of precisely the cytokines to
which FA cells are hypersensitive.
Most work on understanding the hypersensitivity of FA cells to
cytokine overproduction has been conducted with the FANCC gene
product, as well as cells derived from this complementation group
and Fancc2/2 knockout mice. This is probably because of the fact
that FANCC was the ﬁrst FA gene to be cloned and its product was
initially shown to localize to the cytoplasm. FANCC has been
proposed to have a completely separate role in hematopoiesis and to
modulate cytokine responses by suppressing protein kinase R activation and its interactions with the chaperone HSP70 and STAT1,
a key effector of IFN-g signaling82-84 (Figure 5B85,86). It has been
suggested that understanding all potential functions of FA proteins
outside of DNA repair is a prerequisite for deﬁning the pathogenesis
of marrow failure in FA. On the other hand, BMF is a universal
feature of FA, and this will be explained not by the independent
functions of some FA proteins, but by understanding the function
common to all 16 FA proteins (Figure 1).
Alanine scanning mutation of FANCC identiﬁed a putative conserved motif that mediates these interactions. Mutation of this motif
renders cells resistant to cross-linkers but sensitive to inﬂammatory
cytokines, suggesting that the cytokine sensitivity and DNA repair
function of FANCC can be decoupled. Cells bearing a naturally
occurring mutation of FANCC (c.67delG, previously c.322delG)
carry a hypomorphic 50-kDa FANCC polypeptide reinitiated at
methionine 55 (FP-50 or M55).87 The M55 polypeptide preserves
the conserved motif required for normal STAT1 activation and
HSP70 interaction but does not rescue the hypersensitivity to
MMC. However, patients with the c.67delG (c.322delG) mutation
have milder congenital malformations but share the hematologic
phenotype of other FA patients.88 This critical genetic observation
32
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GARAYCOECHEA and PATEL
does not support a role for the conserved STAT1/HSP70 binding
motif in BMF in FA. Additionally, it remains to be explained how
the aberrant signaling by FANCC might contribute to cytokine
hypersensitivity and BMF in patients from other complementation
groups.8,80 Although it is clear that FA cells are sensitive to
inﬂammatory cytokines, it has not been emphatically established
that this is independent of defective DNA repair or, indeed, that
this points to a distinct role for the FA proteins in limiting the
production and/or in resistance to these molecules. Hopefully, in the
future, genetic dissection of the various components (cytokine, cytokine receptors, and downstream effector molecules), in combination
with FA knockout mice, should clarify these potentially important
questions.
The molecular basis for the overproduction of inﬂammatory
cytokines in FA is unclear. A key question in this respect is whether
this is a primary defect attributable to the function of the FA proteins
or if this is a secondary phenomenon. In fact, high levels of cytokines can be induced by cytotoxic agents.89 Recent work from
Matsui and colleagues has, for the ﬁrst time, looked at cytokine
production by bone marrow cells from patients with different inherited BMF syndromes. This work contradicts previous studies
because no evidence for the overproduction of TNF-a or IFN-g by
unstimulated Fanconi-deﬁcient T cells could be found. Cells from
FA patients tended toward higher cytokine levels following lipopolysaccharide treatment, but this was not speciﬁc to FA cells and
was seen in bone marrow samples from all syndromes.90 It is
therefore plausible that raised cytokines levels are a generic
response to bone marrow dysfunction, chronic inﬂammation, and
tissue injury (Figure 5C).
Future directions
Over the past decade, much evidence for the role of FA gene
products in DNA repair has been established. However, until
recently, we have had little insight into what damages the DNA in
the ﬁrst place. The identiﬁcation of reactive aldehydes as potent
genotoxins in FA-deﬁcient HSCs strengthens the link between
DNA repair and the onset of BMF in FA. It will be crucial to deﬁne
the sources of these reactive molecules and how they can be
neutralized. However, it is not yet clear if these chemicals are the
only or indeed main drivers of endogenous DNA damage in HSCs.
Similar genetic approaches may reveal a role for other reactive
molecules as endogenous genotoxins.
At present, we do not completely understand the precise nature
of the DNA damage caused by aldehydes that is repaired by the FA
pathway, and whether it is direct or indirect. For instance, is the
lesion a DNA-DNA cross-link, DNA-protein cross-link, or simply
an adducted base? At a more mechanistic and fundamental level,
much work needs to be done to determine the biochemical and
structural basis of how endogenous DNA damage is repaired by the
FA proteins. All these potentially fertile avenues of research look
set to keep this fascinating rare bone failure syndrome at the center
of basic and translational biomedical research.
Acknowledgments
The authors would like to thank the members of the Patel laboratory
for critically reading the manuscript.
Authorship
Contribution: J.I.G. and K.J.P. wrote the paper and had ﬁnal
approval of the submitted manuscript.
Conﬂict-of-interest disclosure: The authors declare no competing ﬁnancial interests.
Correspondence: Juan I. Garaycoechea, MRC Laboratory of
Molecular Biology, Francis Crick Ave, Cambridge Biomedical
Campus, Cambridge CB2 0QH, United Kingdom; e-mail: juang@
mrc-lmb.cam.ac.uk; and K. J. Patel, MRC Laboratory of Molecular Biology, Francis Crick Ave, Cambridge Biomedical
Campus, Cambridge CB2 0QH, United Kingdom; e-mail: kjp@
mrc-lmb.cam.ac.uk.
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