Ribosomopathies and the paradox of cellular hypo- to hyperproliferation
Kim De Keersmaecker 1,2, Sergey O. Sulima 1,2 and Jonathan D. Dinman 3
1 Department of Oncology, KU Leuven, Leuven, Belgium
2 Center for the Biology of Disease, VIB, Leuven, Belgium
3 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park
MD, USA
CORRESPONDENCE
Kim De Keersmaecker, Campus Gasthuisberg O&N4, box 602, Herestraat 49, 3000 Leuven.
Kim.DeKeersmaecker@cme.vib-kuleuven.be
Jonathan D. Dinman, Microbiology Building #231, Department of Cell Biology and Molecular
Genetics, University of Maryland, College Park MD 20742 USA. dinman@umd.edu
WORD COUNTS:
Abstract (max 200): 137
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1
ABSTRACT
Ribosomopathies are largely congenital diseases linked to defects in ribosomal proteins or
biogenesis factors. Some of these disorders are characterized by hypoproliferative phenotypes
such as bone marrow failure and anemia early in life, followed by elevated cancer risks later in
life. This transition from hypo- to hyperproliferation presents an intriguing paradox in the field
of hematology known as ‘Dameshek’s riddle.’ Recent cancer sequencing studies also revealed
somatically acquired mutations and deletions in ribosomal proteins in T-cell acute lymphoblastic
leukemia (T-ALL) and solid tumors, further extending the list of ribosomopathies and
strengthening the association between ribosomal defects and oncogenesis. In this perspective,
we summarize and comment on recent findings in the field of ribosomopathies. We explain how
ribosomopathies may provide clues to help explain Dameshek’s paradox and highlight some of
the open questions and challenges in the field.
2
INTRODUCTION
Ribosomopathies are a collection of disorders of ribosome dysfunction characterized by a
defect in ribosome biogenesis, typically due to a mutation in a ribosomal protein or biogenesis
factor. They are rare diseases, with the most frequent having incidences in the range of 1 in 50
000 to 1 in 200 000 live births. These include Diamond-Blackfan Anemia (DBA) and 5qsyndrome that are associated with mutations in ribosomal proteins, as well as diseases linked
to defects in ribosome biogenesis factors such as Schwachman-Diamond syndrome (SDS) and
Treacher Collins syndrome (TCS). An example of a rarer ribosomopathy is X-linked
dyskeratosis congenita. All of these diseases are characterized by distinct clinical phenotypes
and patients present with one or several of a wide variety of developmental abnormalities
including
hematopoietic
defects
(e.g.
anemia
and
other
cytopenias),
craniofacial
malformations, short stature, mental and motor retardation. Although diverse, these
phenotypes can all be categorized as cellular hypoproliferative defects. Intriguingly, some
ribosomopathy patients are also at increased risk of developing leukemias and solid tumors,
i.e.
hyperproliferative cellular
defects.1,2,3
For
example: DBA
is
characterized
by
hypoproliferation phenotypes including macrocytic anemia, short stature, craniofacial defects
and thumb abnormalities beginning early in life. In addition, these patients have been reported
to have a 5-fold higher incidence of cancers, with the highest predisposition to colon cancer
(36-fold higher incidence than in the general population), osteosarcoma (33-fold higher) and
acute myeloid leukemia (28-fold higher).1 Although these associations with elevated cancer
risks have been described in DBA, it is important to remark that this is a rare disease and that
the number of cancer cases studied remains limited. Further studies demonstrating a causal
link between ribosomal defects and elevated cancer risks in DBA are needed. Additionally, it is
important to note that elevated cancer risks have not been demonstrated in all ribosomopathies,
e.g. TCS has not been linked to any higher cancer predisposition.
It is not our aim here to give an extensive overview of ribosomopathies or an in depth
description of the exact clinical manifestations of the different diseases. Therefore, we refer to
excellent reviews written by others.4,5,6 It is important to point out that ribosomopathies
represent only a subset of congenital bone marrow failure syndromes: marrow failure and
myelodysplasia with associated risk for transformation can also be caused by other classes of
congenital and non-congenital defects such as mutations in genes involved in DNA repair
(mutations in Fanconi anemia pathway),7 telomere regulation,8 epigenetics and splicing9. These
are however not the subjects of this perspective.
In this perspective, we summarize and comment on recent findings in the field of
ribosomopathies. To begin, we highlight the rapid expansion of the ribosomopathy field due to
the discovery of somatic mutations in ribosomal proteins in T-cell acute lymphoblastic leukemia
(T-ALL) and other cancer samples. Next, we summarize the state-of-the-art of the molecular
biology underlying the hypo- and hyperproliferative phenotypes in ribosomopathies and provide
3
a model that may partially explain the transition from one to the other (Dameshek’s riddle).
Finally, we discuss some of the open questions and future perspectives in the field.
'TO BE' OR 'NOT TO BE' A RIBOSOMOPATHY: TOWARDS A DEFINITION
The birth of the field of ribosomopathies dates back to 1999, when Draptchinskaia and
colleagues described mutations in the ribosomal protein coding gene RPS19/eS19 (please note
that the second nomenclature refers to the universal ribosomal protein nomenclature which
was recently introduced to conform to atomic resolution ribosome structures from all three
domains of life)10 in DBA.11 Since then, the list of ribosomopathies has continued to grow. One
example is the recent discovery of congenital mutations in ribosomal protein SA (RPSA/uS2)
in patients with isolated congenital asplenia.12 Another important expansion of the list of
ribosomopathies comes from the recent application of unbiased next generation sequencing
based mutation screening to cancer samples. Whereas previously all ribosomopathies (with
the exception of the 5q- syndrome, linked to somatic loss of the RPS14/uS14 gene) have been
associated with congenital genetic defects, genome wide sequencing efforts of cancer samples
revealed that some of the genes associated with such congenital defects in ribosomopathies
can also be targets of somatic mutation in cancers. For example, we recently identified
inactivating mutations in the gene encoding ribosomal protein L5 (RPL5/uL18) in patients with
T-ALL, some of which are identical to mutations described in DBA patients.13 Rare somatic
mutations in T-ALL patients were also identified in RPL11/uL5, another gene that is affected in
DBA.14 Interestingly, somatic defects in ribosomal proteins that have not been implicated in
congenital ribosomopathies have now also been identified in T-ALL, gastric and ovarian cancer.
These include somatic mutations in the RPL10/uL16 and RPL22/eL22 genes.13,15,16,17
Consistent with the observation that patients with congenital ribosomopathies are predisposed
to develop various tumor types, the presence of somatic ribosome defects is emerging as a
common occurrence in cancers.18 In the near future, completion of ongoing cancer sequencing
projects will deepen our understanding of the spectrum of ribosomal mutations in cancer and
may also identify ribosome biogenesis factors implicated in carcinogenesis.
The discovery of somatic ribosome defects in cancer samples calls into question the current
definition of ‘ribosomopathy’. Should the definition be limited to diseases and syndromes
involving congenital mutations in ribosomal protein or biogenesis factor genes only, or should
it be broadened to include somatically acquired mutations as well? Precedent for the latter
definition may be found in 5q- syndrome. The observations that not all diseases typically
accepted as ribosomopathies are associated with elevated cancer risks and that clinical
hypoproliferative phenotypes have not been noted in cancers with somatic ribosome mutations
suggests that the ‘ribosomopathies’ should be broadly defined. However, to err on the side of
caution, we do think that a causal link between the ribosome defect and associated disease
4
should have been demonstrated before calling a disease a ribosomopathy. Therefore, we
propose a conservative definition of ribosomopathies as ‘any disease associated with a
mutation in a ribosomal protein or biogenesis factor impairing ribosome biogenesis in which a
defect in ribosome biogenesis or function can be clearly linked to disease causality’. It is not
clear at this point if a disease like cartilage hair hypoplasia falls under this definition. So far
there has been no clear demonstration of a role for RNase MRP, the gene affected in cartilage
hair hypoplasia, in ribosome biogenesis in humans. Moreover, it is currently unclear whether
ribosome dysfunction is the cellular basis for the disorder. This definition also leaves ribosome
mutant cancers in a gray zone: although loss of an RPL22/eL22 allele is able to accelerate
tumor formation in a mouse model of T-cell leukemia,15 the causal role of other somatic
ribosome mutations described in cancer remains to be shown.
It is worth noting that somatic mutations in ribosomal proteins and biogenesis factors have only
been described in a limited set of cancer samples to date. However, several cancer types are
characterized by recurrent large deletions in which no tumor suppressor gene has yet been
identified and which contain a ribosomal protein gene that is considered intrinsically
‘uninteresting.’ Our recent findings suggest that defects in ribosomal proteins and other
synthesis factors should not be overlooked in cancer genotypes. Additionally, it has been
clearly established by many research groups that protein synthesis appears deregulated in the
majority, if not all, cancer samples. This is not surprising: this process plays such a central role
in the cell that the signaling cascades and transcriptional regulators that are prominently altered
in cancer also (de)regulate translation. Indeed, aberrations affecting the PI3K/AKT/mTOR
pathway, the tumor suppressor protein p53 (TP53) pathway and the MYC transcription factor
in cancer also impact cellular protein translation by deregulating expression of components of
the translation machinery and by influencing the set of mRNAs which are most efficiently
translated in the cell.19,20 However, whether altered translation in these instances is the cause
of or consequence of cellular transformation is not clear. Therefore, we think it is not appropriate
at this point to call such tumors ribosomopathies.
HOW RIBOSOMOPATHIES CAN OFFER NEW INSIGHTS INTO DAMESHEK’S RIDDLE
In a 1967 editorial, the founding editor-in-chief of Blood, William Dameshek, posed the following
riddle: 'What do aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and 'hypoplastic'
leukemia have in common?'.21 Here, Dameshek described the intriguing paradox in which
patients who initially develop a hypoproliferative disease such as aplastic anemia tend to be at
higher risk of hyperproliferative diseases such as PNH (clonal expansion of red cells) or acute
leukemia (clonal expansion of white cells) later in life. Although not explicitly described by
Dameshek, this observation of a class of diseases characterized in the early stages by a
hypoproliferative disorder followed by the propensity to develop into a hyperproliferative
disease fits the clinical presentation of certain ribosomopathies. As such, Dameshek’s riddle
5
may be more broadly applicable than initially postulated.
The challenge is to explain this paradox. Dameshek proposed that a sufficiently severe insult
to the marrow - whether chemical, ionizing radiation, viral or by a congenital defect - may result
in a variable degree of injury with a variable degree of hypoplasia. In some cases, abnormal
clones of abnormal leukocytes (hypoplastic leukemia) or red cells (PNH) could conceivably
arise during the process of repair.21 In other words, Dameshek proposed that an initial insult
first results in hypoproliferative disease, and that something involved in the process of its repair
provides a subset of cells with the capacity to proliferate excessively. This idea was conceived
in 1967, before the molecular genetics era. Remarkably, his model remains consistent with the
models that are currently being proposed to explain Dameshek’s riddle. The differences lie in
the depth of our understanding of the molecular nature of the 'initial insult', the identities of the
signaling cascades that lead to impaired proliferation, and the nature of the molecular 'repair
processes' that may underlie the transition to hyperproliferation.
As discussed above, the initial insult occurring in ribosomopathies are defects in ribosomal
protein genes and biogenesis factors. Ribosome biogenesis is a complex multistage process.22
Given the central role of the ribosome in the genetic program, there is strong pressure in cells
to identify and remove malfunctioning ribosomes before they are released into the cytoplasmic
pool of actively translating ribosomes. It is becoming clear that this process involves quality
control mechanisms that operate at each step of the process.23,24 This has two consequences.
The first is that cells may not produce enough functional ribosomes to support their metabolic
demands. This is particularly germane to hematopoietic cells, whose normally high proliferative
rates require large amounts of ribosomes. This alone may account for cellular hypoproliferation.
In addition, too few functional ribosomes due to insufficient quantities of one or more ribosomal
proteins may also result in the release of excess free ribosomal proteins into the cytoplasm.
This is a molecular stress signal. The most well-documented research in this area focuses on
cytoplasmic release of RPL5/uL18 and RPL11/uL5. These have been shown to bind to and
inhibit the murine double minute 2 protein (MDM2, or HDM2 in humans), a ubiquitin ligase that
directs degradation of the TP53 protein. HDM2 inhibition results in TP53 stabilization, initiating
a series of cellular signaling cascades leading to cell cycle arrest and apoptosis, adding to
cellular hypoproliferation.25,26,27,28,29,30 This research only represents the tip of the iceberg: TP53
independent cell cycle arrest and apoptosis mechanisms have been described that may also
contribute to the ribosomopathic-stress induced hypoproliferation.31,32,33,34
Although the human ribosome consists of 81 ribosomal proteins, only a fraction of these have
been described as mutated or deleted in ribosomopathies. Similarly, only a restricted set of
biogenesis factors have been implicated in ribosomopathies to date.4,5 This is most likely
explained by the fact that mutation or deletion of many ribosomal proteins and biogenesis
factors would be incompatible with cell survival and affected cells would thus not live long
6
enough to produce the ribosomopathic phenotype. Indeed, the ribosomal proteins currently
implicated in ribosomopathies encode proteins located on the cytoplasmic/solvent-accessible
surfaces of the ribosome.35 None of these proteins stabilize interdomain rRNA interactions, nor
are they located in regions of the ribosome that directly mediate mRNA decoding or peptidyl
transfer, leaving ribosomopathic ribosomes functional enough to support viability. Two typical
trans-acting ribosomopathogenic factors, Shwachman–Bodian–Diamond syndrome protein
(SBDS) and dyskerin (DKC1), are also consistent with this idea: SBDS is involved in the late,
cytoplasmic quality control of pre-60S subunits,24 and DKC1 catalyzes pseudouridylation of
rRNA. Although rRNA modifications occur early during rRNA transcription, these are thought
to merely “fine-tune” ribosome structure rather than playing central roles in domain
folding.36,37,38
The next issue concerns the nature of the 'repair process' in Dameshek’s model. Not
surprisingly, this is less well understood as it is more complex. To approach this, we have turned
to molecular evolutionary theory. As described above, the original mutation results in too few
cells available to perform their physiological function, e.g. production of red blood cells. From
an evolutionary standpoint, this places pressure on the dwindling population of hematopoietic
stem cells, selecting for mutations that enable cells to produce enough ribosomes to meet their
protein synthetic requirements. One such class of mutants are those that repair the original
defect (revertants). A second class, referred to as second site suppressors, are mutations that
either inactivate or bypass the ribosome biogenesis quality control apparatus. Cells harboring
this class of mutations would produce enough ribosomes to satisfy their translational
requirements, enabling them to out-compete those having only the original mutation. While this
solves the hypoproliferation problem, the tradeoff is that these cells must utilize defective
ribosomes. Emerging research indicates that these ribosomes have specific defects in
translational fidelity, i.e. their ability to faithfully translate the genetic code. Indeed, our
laboratories have begun to identify a causal chain of events detailing how ribosomopathy
associated defects such as RPL10/uL16 R98S or Cbf5p-D95A (a catalytically impaired mutant
of Cbf5p, the yeast homologue of DKC1) cause subtle changes in ribosome structure that alter
the basic biochemistry of mutant ribosomes, and how this in turn affects specific aspects of
translational fidelity, altering the expression of specific sets of genes. 35,38 For RPL10/uL16
R98S, we also showed that these changes in gene expression confer new stresses on this
population of cells, hastening the selection for additional compensatory mutations that
eventually result in cellular immortalization, i.e. hyperproliferation. 35 This is a classic ‘deal with
the devil’: the process that initially enables cells to fulfill their physiological function eventually
leads to their uncontrolled growth and cancer.
The current challenge is to identify the critical second site suppressors. Once identified, rational
strategies can be designed to reverse their effects. The ideal samples would be tumors
collected from patients that developed a ribosome defect associated malignancy. Such patient
7
samples are however scarce because, fortunately, not all patients develop malignancies.
Moreover, cancer samples are often characterized by genetic instability and accumulation of
defects, making it difficult to pinpoint the exact nature of the original hypoproliferation
suppressor mutation. This is where the molecular genetics of model organisms becomes a
powerful tool. Recent work from our laboratories provides insight into the molecular nature of
the 'repair process'. This began with identification of a recurrent arginine to serine mutation
(R98S) in ribosomal protein RPL10/uL16 in T-ALL patient cells. Intriguingly, while T-ALL
represents a hyperproliferative disease, introduction of this mutation into yeast and mammalian
cells impairs ribosome biogenesis and cellular proliferation causing the hypoproliferative
phenotype.13 In depth analysis of the RPL10/uL16 R98S mutant using the yeast genetic model
revealed that this mutation does not completely inhibit ribosome biogenesis: this is why they
are alive, albeit barely. Importantly, allowing cells to grow over many generations selected for
a population of cells with normal growth rates. Genetic analysis revealed that this was due to
acquisition of a compensatory mutation in a ribosome biogenesis factor that bypassed a critical
quality control checkpoint. Importantly, while this second site mutation rescued the
hypoproliferative defect, it did not correct the underlying structural, biochemical and
translational fidelity defects and altered gene expression profiles conferred by the original
RPL10/uL16 R98S mutation (Figure 1A, B).35 While we have not been able to identify
analogous mutations in biogenesis factors in RPL10/uL16 R98S positive T-ALL samples, this
may be due to the paucity of knowledge pertaining to human biogenesis factors, i.e. mammalian
ribosome biogenesis factors are still poorly defined. Additionally, yeast and human cells are
sufficiently different that mutations in alternative classes of genes may be required to rescue
human cell proliferation defects. For example, human cells may mutate components of the
TP53 pathway or of the other pathways that initiate hypoproliferation upon ribosome
biogenesis-induced stress (Figure 1C). It would be highly interesting to perform polysome
profiling on non-cancerous versus cancerous tissue from patients with a congenital ribosome
defect to determine whether a compensatory mutation was acquired that corrects the
biogenesis defect (Figure 1B) or whether the biogenesis defect is still present, as predicted by
the model shown in Figure 1C.
Zebrafish are also becoming an attractive model to study ribosome mutation associated
cancers. Seventeen zebrafish lines have been described harboring heterozygous inactivation
of different ribosomal protein genes that are predisposed to develop malignancies. These
tumors have much in common with those observed in Tp53 null zebrafish, and tumors induced
by ribosomal protein haploinsufficiency showed a specific translation defect impairing Tp53
protein production.39 Unfortunately, a hypoproliferative phenotype has not been described in
these fish, and thus it remains unclear whether or not defective Tp53 translation can actually
cause a switch from a hypo- to a hyperproliferative phenotype in this model system. A mouse
model on RPL22/eL22 haploinsufficiency supports the idea that a ribosomal protein defect may
contribute to a hyperproliferative phenotype provided that additional mutations are present.
8
RPL22/eL22 has been described as a target for heterozygous somatic mutations and deletions
in T-ALL and in microsatellite instability-positive gastric and endometrial cancer.15,40,17 Whereas
Rpl22/eL22
+/-
mice do not develop any malignancies within 25 weeks, crossing of these mice
to a strain with T-cell specific expression of myristoylated Akt2 (MyrAkt2) accelerates the
latency of T-cell lymphoma development from 19 weeks (MyrAkt2- Rpl22/eL22+/+) to 11 weeks
(MyrAkt2- Rpl22/eL22+/-).15 Although very interesting, these data still do not identify the nature
of the extra mutations that human cancer cells acquire to be transformed in the context of a
ribosomal defect. It is also worth remarking that although loss of 1 copy of Rpl22/eL22 is
sufficient to accelerate T-cell lymphoma development in a MyrAkt2 background, loss of both
Rpl22/eL22 copies is needed to observe a hyperproliferative phenotype of arrested
development of alphabeta-lineage T cells at the beta-selection checkpoint.15,41
FUTURE PERSPECTIVES AND CONCLUSIONS
A long list of genes associated with ribosomopathies has been generated over the past three
decades. However, it is clear that we are still far from the goal of a complete understanding of
the pathogenesis of ribosomopathies. Essential information regarding the cellular changes
associated with ribosomal mutations remains to be discovered. While mRNA expression
studies may provide certain clues,42,43,44,45 given that protein translation is the central function
of ribosomes, it may be essential to complement these studies by a detailed analysis of the
effects on protein production. This necessity is illustrated by the recent finding that in a
substantial fraction of DBA cases, one consequence of the ribosomal protein mutations is
failure of translation of the transcription factor GATA-1, an essential factor for development of
erythropoiesis beyond the CFU-E stage. This may be because the long form of GATA-1 is
difficult to translate and is impaired in DBA cells.46 These translational defects of GATA-1 were
found because of the rarity of GATA-1 mutations in DBA. However, additional translation
defects in DBA and other ribosomopathies have probably not been discovered because of a
lack of thorough comparative proteomic studies of normal versus ribosomopathic cells.
Unfortunately, this is a classic example of a problem waiting for a technological advance;
proteomics technologies are still evolving and analyzing an entire proteome of human cells is
not yet within reach of research labs. At this juncture, sequencing of polysome associated RNA
undergoing translation is an option.47,48,49 The best approximation at this point is ribosome
profiling technology, which allows not only identification of ribosome protected (= translated)
mRNA fragments but also analysis of alternative start codon usage, read-through beyond stop
codons and of translational kinetics.50,51 Additionally, although the need for cooperating
mutations appears to be clear, questions remain regarding the nature of these mutations, the
moment when they are acquired and the amount required to stimulate hyperproliferation. One
attractive possibility accounting for the varying penetrance of cancers in different
ribosomopathy patients may lie in their genetic backgrounds, i.e. cooperating mutations may
be inherited as allelic variants that are normally maintained in the population.
9
Yet another intriguing paradox regarding ribosomopathies is the issue of phenotypic tissue
specificity: why are such a wide spectrum of clinical presentations with typical tissue-specific
pathologies observed for each disease, despite the fact that they all share a common defect in
the same biochemical process? While patients with congenital ribosomopathies are born with
the same ribosome defect in each cell of their body, not all tissues are affected and only
particular disease symptoms are developed.5 Interestingly, although mutations in several
ribosomal proteins and biogenesis factors give rise to anemia in different ribosomopathies,
each disease also exhibits additional unique features. Even within the same ribosomopathy,
mutations in different ribosomal protein genes promote different types of birth defects. For
instance, most patients with RPL5/uL18 mutations have a cleft palate, whereas most individuals
with RPL11/uL5 mutations do not have any craniofacial defects.52 As such, patients with genetic
disorders that are linked to mutations in ribosomal proteins show remarkably specific
phenotypes, suggesting that ribosomal proteins have unique functions in different tissues.
Dissecting the molecular and biochemical defects of different types of mutant ribosomes can
contribute to the exciting emerging field of ‘specialized ribosomes’ – ribosomes whose
specificity may be controlled and fine-tuned by tissue-specific and developmentally regulated
signals. Alternatively, essential tissue-specific roles of ribosomal proteins outside of the
ribosome (extra-ribosomal functions) may explain the tissue-specific phenotypes. Indeed, there
is a growing body of evidence that ribosomal proteins do not only function in translation and
that they can also regulate transcription and enzyme activity in the cell.53
In conclusion, many potential explanations for cellular transformation exist and are important
subjects for investigation. We speculate that the ribosomal protein and biogenesis mutants in
ribosomopathies allow the ribosome to become mature enough to support some degree of
translation, yet still cause sufficient perturbations of ribosomal function to effect the changes in
gene expression associated with ribosomopathic phenotypes. Indeed, we showed that yeast
cells expressing a T-ALL associated RPL10/uL16 R98S mutant or a Cbf5p-D95A mutant
promote specific defects in translation fidelity.35,38 Based on our work on RPL10/uL16 R98S, 35
we also hypothesize that compensatory mutations might be likely drivers of transformation, and
are hence particularly important because they allow bypass of the cellular proliferation defects
due to defective ribosome biogenesis. One class of compensatory mutations comprises mutant
biogenesis factors which suppress the assembly but not the functional defects of mutant
ribosomes. This leads to increased cellular utilization of defective ribosomes with altered
fidelity. Additional experimental work is needed to validate this model in other systems and for
other ribosomal defects. Moreover, while bypassing the ribosomal assembly quality control
through mutated biogenesis factors may provide one window to oncogenesis, it is likely that
other classes of oncogenic secondary mutations exist. Indeed it is becoming clear that
cooperating
secondary
development.54
mutations/suppressors
are
frequently
required
for
disease
Transformation merely represents an endpoint that is likely made more
10
accessible by evolutionary selection for secondary mutations in a variety of cellular pathways.
Determining the nature of the pathways and the mutations is an exciting and promising field for
investigation.
11
ACKOWLEDGEMENTS
KDK receives a postdoctoral fellowship of FWO Vlaanderen. SOS is recipient of an EMBO longterm postdoctoral fellowship. This work was partially supported by a grant from the National
Institutes of Health to JDD (R01 HL119438) and by an FWO grant (G.0840.13), a Stichting
Tegen Kanker (F25) and an ERC starting grant (GA 334946) to KDK.
AUTHORSHIP CONTRIBUTIONS
KDK, SOS and JDD wrote and edited the manuscript.
DISCLOSURE OF CONFLICTS OF INTEREST
The authors declare no competing financial interests
REFERENCES
1.Vlachos A, Rosenberg PS, Atsidaftos E, Alter BP, Lipton JM. Incidence of neoplasia in
Diamond Blackfan anemia: a report from the Diamond Blackfan Anemia Registry. Blood.
2012;119(16):3815-9.
2.Rosenberg PS, Alter BP, Bolyard AA, et al. The incidence of leukemia and mortality from
sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy.
Blood. 2006;107(12):4628-35.
3.Alter BP, Giri N, Savage SA, et al. Malignancies and survival patterns in the National Cancer
Institute inherited bone marrow failure syndromes cohort study. Br. J. Haematol.
2010;150(2):179-88.
4.Narla A, Ebert BL. Ribosomopathies: human disorders of ribosome dysfunction. Blood.
2010;115(16):3196-205.
5.Armistead J, Triggs-Raine B. Diverse diseases from a ubiquitous process: the ribosomopathy
paradox. FEBS letters. 2014;588(9):1491-500.
6.Ruggero D, Shimamura A. Marrow failure: a window into ribosome biology. Blood.
2014;124(18):2784-2792.
7.Longerich S, Li J, Xiong Y, Sung P, Kupfer GM. Stress and DNA repair biology of the Fanconi
anemia pathway. Blood. 2014;124(18):2812-2819.
8.Townsley DM, Dumitriu B, Young NS. Bone marrow failure and the telomeropathies. Blood.
2014;124(18):2775-2783.
9.Bejar R, Steensma DP. Recent developments in myelodysplastic syndromes. Blood.
2014;124(18):2793-2803.
10.Ban N, Beckmann R, Cate JHD, et al. A new system for naming ribosomal proteins. Curr.
Opin. Struct. Biol. 2014;24:165-9.
11.Draptchinskaia N, Gustavsson P, Andersson B, et al. The gene encoding ribosomal protein
S19 is mutated in Diamond-Blackfan anaemia. Nat. Genet. 1999;21(2):169-75.
12.Bolze A, Mahlaoui N, Byun M, et al. Ribosomal protein SA haploinsufficiency in humans with
isolated congenital asplenia. Science. 2013;340(6135):976-8.
12
13.De Keersmaecker K, Atak ZK, Li N, et al. Exome sequencing identifies mutation in CNOT3
and ribosomal genes RPL5 and RPL10 in T-cell acute lymphoblastic leukemia. Nat. Genet.
2013;45(2):186-190.
14.Tzoneva G, Perez-Garcia A, Carpenter Z, et al. Activating mutations in the NT5C2
nucleotidase gene drive chemotherapy resistance in relapsed ALL. Nature medicine.
2013;19(3):368-71.
15.Rao S, Lee S, Gutierrez A, et al. Inactivation of ribosomal protein L22 promotes
transformation by induction of the stemness factor, Lin28B. Blood. 2012;120(18):3764-73.
16.Wang K, Kan J, Yuen ST, et al. Exome sequencing identifies frequent mutation of ARID1A
in molecular subtypes of gastric cancer. Nat. Genet. 2011;43(12):1219-23.
17.Novetsky AP, Zighelboim I, Thompson DM, et al. Frequent mutations in the RPL22 gene
and its clinical and functional implications. Gynecol. Oncol. 2012;128(3):470-4.
18.Kandoth C, McLellan MD, Vandin F, et al. Mutational landscape and significance across 12
major cancer types. Nature 2013;502(7471):333-9.
19.Ruggero D, Pandolfi PP. Does the ribosome translate cancer? Nature Reviews Cancer.
2003;3(3):179-192.
20.Stumpf CR, Ruggero D. The cancerous translation apparatus. Current opinion in genetics
& development. 2011;21(4):474-83.
21.Dameshek W. Riddle: what do aplastic anemia, paroxysmal nocturnal hemoglobinuria
(PNH) and “hypoplastic” leukemia have in common? Blood. 1967;30(2):251-254.
22.Strunk BS, Karbstein K. Powering through ribosome assembly. RNA. 2009;15(12):2083104.
23.Matsuo Y, Granneman S, Thoms M, et al. Coupled GTPase and remodelling ATPase
activities form a checkpoint for ribosome export. Nature 2013;505(7481):112-6.
24.Lo K, Li Z, Bussiere C, et al. Defining the pathway of cytoplasmic maturation of the 60S
ribosomal subunit. Molecular cell. 2010;39(2):196-208.
25.Dai M, Lu H. Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal
protein L5. J. Biol. Chem. 2004;279(43):44475-82.
26.Marechal V, Elenbaas B, Piette J, Nicolas JC, Levine AJ. The ribosomal L5 protein is
associated with mdm-2 and mdm-2-p53 complexes. Mol. Cell. Biol. 1994;14(11):7414-20.
27.Zhang Y, Wolf GW, Bhat K, et al. Ribosomal protein L11 negatively regulates oncoprotein
MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway. Mol. Cell. Biol.
2003;23(23):8902-12.
28.Lohrum MAE, Ludwig RL, Kubbutat MHG, Hanlon M, Vousden KH. Regulation of HDM2
activity by the ribosomal protein L11. Cancer Cell. 2003;3(6):577-87.
29.Bursać S, Brdovčak MC, Pfannkuchen M, et al. Mutual protection of ribosomal proteins L5
and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress.
Proc. Natl. Acad. Sci. U.S.A. 2012;109(50):20467-72.
30.Bursac S, Brdovcak MC, Donati G, Volarevic S. Activation of the tumor suppressor p53 upon
impairment of ribosome biogenesis. Biochim. Biophys. Acta. 2014;1842(6):817-830.
13
31.Donati G, Montanaro L, Derenzini M. Ribosome biogenesis and control of cell proliferation:
p53 is not alone. Cancer research. 2012;72(7):1602-7.
32.Narla A, Payne EM, Abayasekara N, et al. L-Leucine improves the anaemia in models of
Diamond Blackfan anaemia and the 5q- syndrome in a TP53-independent way. Br. J.
Haematol. 2014;167(4):524-528.
33.Singh SA, Goldberg TA, Henson AL, et al. p53-Independent cell cycle and erythroid
differentiation defects in murine embryonic stem cells haploinsufficient for Diamond Blackfan
anemia-proteins: RPS19 versus RPL5. PloS one. 2014;9(2):e89098.
34.Heijnen HF, van Wijk R, Pereboom TC, et al. Ribosomal Protein Mutations Induce
Autophagy through S6 Kinase Inhibition of the Insulin Pathway. PLoS genetics.
2014;10(5):e1004371.
35.Sulima SO, Patchett S, Advani VM, et al. Bypass of the pre-60S ribosomal quality control
as a pathway to oncogenesis. Proc. Natl. Acad. Sci. U.S.A. 2014;111(15):5640-5645.
36.Decatur WA, Fournier MJ. rRNA modifications and ribosome function. Trends in biochemical
sciences. 2002;27(7):344-51.
37.Baxter-Roshek JL, Petrov AN, Dinman JD. Optimization of ribosome structure and function
by rRNA base modification. PloS one. 2007;2(1):e174.
38.Jack K, Bellodi C, Landry DM, et al. rRNA pseudouridylation defects affect ribosomal ligand
binding and translational fidelity from yeast to human cells. Molecular cell. 2011;44(4):660-6.
39.MacInnes AW, Amsterdam A, Whittaker CA, Hopkins N, Lees JA. Loss of p53 synthesis in
zebrafish tumors with ribosomal protein gene mutations. Proc. Natl. Acad. Sci. U.S.A.
2008;105(30):10408-13.
40.Nagarajan N, Bertrand D, Hillmer AM, et al. Whole-genome reconstruction and mutational
signatures in gastric cancer. Genome Biol. 2012;13(12):R115.
41.Anderson SJ, Lauritsen JPH, Hartman MG, et al. Ablation of ribosomal protein L22
selectively impairs alphabeta T cell development by activation of a p53-dependent checkpoint.
Immunity. 2007;26(6):759-72.
42.Ebert BL, Pretz J, Bosco J, et al. Identification of RPS14 as a 5q- syndrome gene by RNA
interference screen. Nature. 2008;451(7176):335-9.
43.Aspesi A, Pavesi E, Robotti E, et al. Dissecting the transcriptional phenotype of ribosomal
protein deficiency: implications for Diamond-Blackfan Anemia. Gene. 2014;545(2):282-9.
44.Devlin EE, Dacosta L, Mohandas N, Elliott G, Bodine DM. A transgenic mouse model
demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated
with Diamond-Blackfan anemia. Blood. 2010;116(15):2826-35.
45.Rujkijyanont P, Adams S, Beyene J, Dror Y. Bone marrow cells from patients with
Shwachman-Diamond syndrome abnormally express genes involved in ribosome biogenesis
and RNA processing. Br. J. Haematol. 2009;145(6):806-15.
46.Ludwig LS, Gazda HT, Eng JC, et al. Altered translation of GATA1 in Diamond-Blackfan
anemia. Nature medicine. 2014;20(7):748-53.
14
47.Horos R, Ijspeert H, Pospisilova D, et al. Ribosomal deficiencies in Diamond-Blackfan
anemia impair translation of transcripts essential for differentiation of murine and human
erythroblasts. Blood. 2011;119(1):262-72.
48.Pereboom TC, Bondt A, Pallaki P, et al. Translation of branched-chain aminotransferase-1
transcripts is impaired in cells haploinsufficient for ribosomal protein genes. Exp. Hematol.
2014;42(5):394-403.e4.
49.Hesling C, Oliveira CC, Castilho BA, Zanchin NIT. The Shwachman-Bodian-Diamond
syndrome associated protein interacts with HsNip7 and its down-regulation affects gene
expression at the transcriptional and translational levels. Experimental cell research.
2007;313(20):4180-95.
50.Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling
strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA
fragments. Nature protocols. 2012;7(8):1534-50.
51.Ingolia NT, Lareau LF, Weissman JS. Ribosome profiling of mouse embryonic stem cells
reveals the complexity and dynamics of mammalian proteomes. Cell. 2011;147(4):789-802.
52.Gazda HT, Sheen MR, Vlachos A, et al. Ribosomal protein L5 and L11 mutations are
associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients. Am.
J. Hum. Genet. 2008;83(6):769-80.
53.Lindström MS. Emerging functions of ribosomal proteins in gene-specific transcription and
translation. Biochemical and biophysical research communications. 2008;379(2):167-70.
54.Ishimura R, Nagy G, Dotu I, et al. RNA function. Ribosome stalling induced by mutation of
a CNS-specific tRNA causes neurodegeneration. Science. 2014;345(6195):455-459.
FIGURE LEGENDS
Figure 1. Model explaining transition from hypo- to hyperproliferation phenotypes in
ribosomopathies. (A) In the initial phase of the disease, a mutation in a ribosomal protein or
ribosome biogenesis factor (symbolized by the red star on the 60S subunit) impairs proper
ribosome biogenesis, resulting in lower concentrations of assembled ribosomes. This ribosome
biogenesis defect impairs proper proliferation of the cells by activating the TP53 pathway and/or
by TP53 independent mechanisms. At this stage, functional ribosomes are still assembled to a
certain extent. These ribosomes are however intrinsically defective and may induce
translational changes/shifts in the cells. (B + C) The impaired proliferation imposes strong
pressure on cell populations, selecting for cells that acquire a compensatory mutation rescuing
the impaired proliferation. The nature of this compensatory mutation is currently unclear. Cells
may acquire a mutation in a biogenesis factor rescuing the biogenesis defect (B). Alternatively,
the signaling pathways inducing the proliferation impairment upon a biogenesis defect (TP53
or other) may be crippled by a compensatory mutation (C). In both scenarios, after acquiring
the compensatory mutation, defective ribosomes would still be formed that alter the
translational capacity/fidelity leading to the cell obtaining a clonal advantage over other cells.
15
However, these ribosomes are intrinsically defective, leading to altered gene expression
programs. More speculative parts in the figure are indicated by dashed arrows.
16
17