Molecular

Cancer
Research

Review

The Changing Mutational Landscape of Acute Myeloid

Leukemia and Myelodysplastic Syndrome
s-Cardama3
Connie A. Larsson1,4, Gilbert Cote2, and Alfonso Quinta

Abstract
Over the past few years, large-scale genomic studies of patients with myelodysplastic syndrome (MDS) and
acute myelogenous leukemia (AML) have unveiled recurrent somatic mutations in genes involved in epigenetic
regulation (DNMT3A, IDH1/2, TET2, ASXL1, EZH2 and MLL) and the spliceosomal machinery (SF3B1,
U2AF1, SRSF2, ZRSR2, SF3A1, PRPF40B, U2AF2, and SF1). The identication of these mutations and their
impact on prognostication has led to improvements in risk-stratication strategies and has also provided new
potential targets for the treatment of these myeloid malignancies. In this review, we discuss the most recently
identied genetic abnormalities described in MDS and AML and appraise the current status quo of the dynamics
of acquisition of mutant alleles in the pathogenesis of AML, during the transformation from MDS to AML, and in
the context of relapse after conventional chemotherapy.
Implications: Identication of somatic mutations in AML and MDS suggests new targets for therapeutic
development. Mol Cancer Res; 11(8); 81527.
2013 AACR.

Introduction
Acute myelogenous leukemia (AML) and myelodysplastic
syndrome (MDS) are hematologic disorders caused by
defects in programs that regulate the differentiation potential
and self-renewing capacity of myeloid cells originating from
multipotent hematopoietic stem cells (HSC). AML is characterized by an abnormal expansion of hematopoietic precursor cells with limited or abnormal differentiation. MDS
represents a heterogeneous group of clonal disorders characterized by an expansion of poorly differentiated hematopoietic precursors with limited self-renewing capacity and
ineffective hematopoiesis, dysplastic changes, enhanced apoptosis, and a propensity to transform into AML. Recurrent
chromosomal structural aberrations have been linked with
distinct outcomes and continue to represent one of the most
important risk factors when patients are stratied by level
of risk before therapy. However, approximately 50% of
patients with AML or MDS are cytogenetically normal and
lack recurrent structural abnormalities, which suggests other
molecular events in the pathogenesis of these diseases. With
the application of global DNA sequencing, somatic gene
mutations have been found to be more common than
previously expected. For example, in cytogenetically normal
Authors' Afliations: Departments of 1Genetics, 2Endocrine Neoplasia and
Hormonal Disorders, and 3Leukemia, and 4The University of Texas Graduate
School of Biomedical Sciences Program in Genes and Development, The
University of Texas MD Anderson Cancer Center, Houston, Texas
s-Cardama, Department of LeuCorresponding Author: Alfonso Quinta
kemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 428, Houston, TX 77030. Phone: 713-745-4009;
Fax: 713-792-5640; E-mail: aquintas@mdanderson.org
doi: 10.1158/1541-7786.MCR-12-0695

2013 American Association for Cancer Research.

www.aacrjournals.org

AML (CN-AML) the discovery of recurrent mutations in

three different genes, NPM1, FLT3, and CEBPA, has led to
improvements in prognostication, minimal residual disease
monitoring, and molecular characterization within these
subsets (13). As a result, these mutations are thought to
be primary genetic events contributing to the pathogenesis of
AML (4). Among these, mutations to NPM1 and CEBPA
were included as provisional entities in the 2008 World
Health Organization (WHO) classication of "AML with
recurrent genetic abnormalities" and account for more than
50% of AML patients with normal karyotype (5). The
literature covering mutations to these genes and their implication in leukemogenesis is extensive. Therefore, this review
does not focus on NPM1, CEBPA, and FLT3 mutations
except in the context of mutations to genes in this review. We
focus more on recently identied novel genetic alterations
and provide an updated report on mutated genes that are well
described in MDS and AML.
Recently, the use of high-throughput massive parallel
sequencing (i.e., next-generation sequencing) platforms has
led to the identication of novel somatic mutations that also
have important prognostic value and/or potential as therapeutic targets. Several reports have been published in the past
4 years describing the sequence of 26 AML genomes (12
M1-AML, 13 M3-AML, and 1 therapy-related AML with
complex karyotype) as well as exome sequencing of 14 cases
of M5-AML (612). These reports have unveiled mutations
in epigenetic regulator genes such as TET2, IDH1, IDH2,
DNMT3a, and EZH2. Importantly, sequence analysis of
patients with myeloid malignancies revealed a considerable
overlap of mutated genes between patients with cytogenetically normal MDS (CN-MDS) and CN-AML. These mutations may have important therapeutic consequences because
drugs that inuence the epigenetic regulation of genes [i.e.,

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Larsson et al.

DNA hypomethylating agents and histone deacetylase

(HDAC) inhibitors] are widely used for the treatment of
patients with AML or MDS. In addition, recent studies have
reported the presence of mutations in genes that encode
components of the splicing machinery in patients with MDS
and AML (Table 1). Here, we discuss the most exciting
recent developments about recurrent mutations in AML and
MDS, the prognostic impact of such mutations (Table 2),
and their potential as therapeutic targets as well as the
dynamics of mutant alleles during the progression from
MDS to AML and between genomes at diagnosis and after
failure to standard therapy.
Epigenetic Deregulation in the Pathogenesis of
MDS and AML
Regulation of gene expression through epigenetic reprogramming is fundamental to development and cellular
differentiation of higher eukaryotes. DNA methylation and
histone modications that change the conformation of
chromatin stably alter the gene expression potential through
mechanisms that do not change the sequence of the genome.
Notably, many malignancies, including AML and MDS,
exhibit aberrant DNA methylation and altered histone
modications that result in the alteration of gene expression,
such as silencing of tumor suppressors and/or activation of
oncogenes. The main epigenetic modication in humans is

the methylation of DNA, in which a methyl group is

covalently attached to the 50 carbon of cytosine [50 -methylcytosine (5mC)] within a CpG dinucleotide. There are
regions of the genome that are rich in CpG dinucleotides
(i.e., CpG islands), which generally span the 50 regulatory
region of genes and are present in approximately half of all
human genes. The methylation status of promoter CpG
islands is modulated by DNA methyltransferases (DNMT)
and dictates the outcome of gene expression (Fig. 1). Under
normal conditions, CpG islands are unmethylated, which is
characteristic of active gene expression. Conversely, promoter CpG methylation is a hallmark of gene repression and,
under normal conditions, is generally restricted to X-chromosome inactivation in women and imprinted germlinespecic and tissue-specic gene (13). Using mouse models,
the specic roles of different DNMTs have been elucidated
and well characterized. Dnmt3a/ mice displayed normal
levels of DNA methylation and were born normal but
eventually succumbed to developmental defects and died
by 4 weeks of age. Dnmt3b/ mice exhibited slightly
lower levels of DNA methylation and were not viable.
However, de novo DNA methylation was severely impaired
in Dnmt3a/; Dnmt3b/ double mutants, showing that
DNMT3a and DNMT3b have overlapping functions in de
novo DNA methylation and embryonic development (14).
Dnmt1/ mice are embryonic lethal and displayed a global
reduction in DNA methylation levels, which is consistent

Table 1. Frequency of gene mutations in patients with MDS or AML

Gene

Gene function

Mutation description

DNMT3A

De novo DNA methylation

IDH1/2

Catalyzes interconversion of
isocitrate and a-KG

TET2

Oxidizes 5-mC to 5-hmC, a

demethylation intermediate
Progressive trimethylation of
H3K27

Recurrent heterozygous missense

mutation at R882
IHD1-heterozygous R132
missense IDH2-heterozygous
R172 or R140 missense mutation
Primarily biallelic mutations
resulting in loss of function
Inactivating mutations and
overexpression observed in
myeloid malignancies
Primarily heterozygote, monoallelic
truncating mutations that deletes
the PHD domain
Translocations involving MLL that
result in a fusion protein or partial
tandem duplication mutations of
MLL
Predominantly heterozygous
missense mutation at R625,
H662, and K700
Heterozygous missense mutation
at P95
Heterozygous missense mutation
at S34 and Q157

EZH2a

ASXL1

Transcription factor that maintains

HOX expression

MLL

H3K4 methyltransferase

SF3B1

Spliceosomal gene important for

HOX gene repression

SRSF2

Spliceosomal gene implicated in

genomic stability
Spliceosomal gene implicated in
proper splicing of genes

U2AF35

Mutation frequency
in MDS (%)

Mutation frequency in
CN-AML (%)

1825

411

619

1926

2427

6.4

Rare

14

5.2

Rare

10 (de novo),
30 (secondary)

80% in MDS-RARS and 6% in MDS

without RS

35

2% in MDS-RARS and 12% in

MDS w/o RS
12% (only in MDS without RS)

78
11

Abbreviations: PHD, plant homeodomain; RS, ringed sideroblasts.

a
EZH2 mutations are generally activating mutations in other malignancies.

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Mutations in MDS and AML

Table 2. Prognostic implications of gene mutations in patients with MDS or AML

Gene

Disease

Patients, n

% with mutation

Prognosis of patients carrying mutation vs. wild type

Study

DNMT3Aa

AML
AML

415
605

23
12

(21)
(22)

AML

398

23

MDS

150

AML
AML

783
318

7
27

MDS
MDS

439
96

20
23

86

13

Worse OS [HR, 1.38 (95% CI, 1.132.05); P 0.022]

Worse OS (median, 15.0  1.9 mo; P < 0.001); worse EFS
(median, 8.0  1.2 mo; P 0.01)
Improved survival to high-dose daunorubicin in patients
enrolled in ECOG E1900 carrying mutant DNMT3a
compared with patients with wild-type DNMT3a
Worse OS (log-rank P 0.005), worse EFS (P 0.009),
higher rate of transformation to AML (P 0.007)
No impact on response or OS
Inferior OS (median, 20.4 vs. 62.2 mo; P 0.173), shorter
EFS (median, 6.7 vs. 18.7 mo; P 0.009), shorter time to
relapse (median, 10.3 vs. 41.3 mo; P 0.005)
No impact on survival
Independent prognostic factor for OS [HR, 5.2 (95% CI, 1.6
16.3); P 0.005]
No impact on OS, but favorable response to azaciditine
(82% mutant vs. 45% wild type; P 0.007)
Worse DFS in patients with NPM1 mutations without FLT3
mutations
Worse OS in patients carrying wild-type FLT3 and mutant
NPM1 alleles
Lower complete remission rate and shorter OS in patients
with CN-AML carrying NPM1 mutations
Worse OS [HR, 1.38 (95% CI, 1.001.89); P 0.006]
Independent prognostic factor for shorter OS [HR, 1.52
(95% CI, 1.122.4); P 0.010]
Worse OS [HR, 2.13 (95% CI, 1.363.33); P 0.001]
Translocations involving the MLL gene portend very poor
OS (median, 15.0  1.9 mo; P < 0.001) and EFS (median,
8.0  1.2 mo; P < 0.01)
Favorable prognosis, longer EFS [HR, 0.1 (95% CI, 0.00.7);
P 0.02]
Shorter OS [median, 17 vs. 39% at 5 y (HR, 1.76; 95% CI,
1.03.1); P 0.049]; shorter 5-y DFS [median, 39 vs. 69%
(HR, 2.5; 95% CI, 1.225.1); P 0.012]
More rapid transformation to AML [HR, 2.53 (95% CI, 0.97.13); P 0.079]; no impact on OS [HR, 1.49 (95% CI,
0.544.1); P 0.44]

TET2a

MDS or AML
IDH1/2a

AML

358

14 in IDH1, 19 in IDH2

AML

893

6 in IDH1, 11 in IDH2

AML

520 and 805

9-16 in IDH1 3-7 in IDH2

ASXL1

MDS
AML

439
882

14
5

EZH2a
MLL

MDS
AML

439
605

6
14

SF3B1

MDS

354

20

SRSF2a

MDS

193

12

U2AF1a

MDS

193

(20)

(23)
(30)
(24)

(26)
(31)
(32)
(35)
(78)
(37, 38)
(26)
(45)
(26)
(22)

(59)
(65)

(65)

Abbreviations: CI, condence interval; DFS, disease-free survival; EFS, event-free survival; OS, overall survival.
a
Prognostic value remains controversial and is not currently used in clinical practice.

with the notion that DNMT1 is essential for maintaining

hemi-methylated DNA during replication (13). 5-mC, a
mark of DNA methylation, can be further modied by the
a-ketoglutarate (a-KG)dependent oxygenases TET1,
TET2, and TET3, which catalyze the conversion of 5-mC
to 50 -hydroxymethylcytosine (5-hmC; ref. 15). a-KG is
obtained from isocitrate in a process catalyzed by isocitrate
dehydrogenase 1 (IDH1) in the cytoplasm and by IDH2 in
the mitochondria. Sequencing analysis of MDS and AML
patient samples revealed mutations in genes important for
DNA methylation such as TET2, IDH1/2, and DNMT3A.
Not surprisingly, these mutations are consistent with aberrant
DNA methylation patterns being a common feature of MDS
and AML (7, 16).

www.aacrjournals.org

Mutations in genes involved in DNA methylation

DNMT3A. DNMT3A, which encodes a DNMT and is
related to the DNMT1 and DNMT3b genes, has been found
mutated in 18% to 25% of patients with AML and approximately 8% of those with MDS. The most common mutation observed is a missense mutation that results in a
substitution of arginine-882 to histidine, cysteine, proline,
or serine (7, 17). Patients with DNMT3A R882 missense
mutations have reduced DNA methylation compared with
matched AML patients wild-type for DNMT3A (18). This
reduction in DNA methylation suggests that the R882
mutation reduces the methyltransferase activity of the
enzyme in a dominant-negative manner, which is further
supported by the fact that all R882 mutations are

Mol Cancer Res; 11(8) August 2013

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Larsson et al.

Methylated cytosine
Hydroxymethylated cytosine
Unmethylated cytosine

Gene repression
CpG island

Gene
Gene activation

CpG island

CpG island

heterozygous (7). Surprisingly, however, differences in gene

expression, methylation patterns, or altered total 5-mC
content could not be robustly linked to DNMT3A mutational status (7, 19). Serial transplantations of Dnmt3a-null
HSCs, which were derived and puried from a conditional
Dnmt3a knockout mouse, into wild-type recipient mice
resulted in a competitive advantage of mutant cells over the
wild-type ones, suggesting that loss of Dnmt3a may contribute to clonal dominance. Reduction in DNA methylation at sites that correlated with increased expression of
multipotency genes and downregulation of differentiation
factors was also observed in the differentiated progeny of
Dnmt3a null HSCs (19). This suggests that loss of de novo
DNA methylation impairs the differentiation potential of
HSCs, providing a possible explanation of how DNMT3A
mutations contribute to the pathogenesis of MDS and AML.
DNMT3A mutations also tend to cluster in patients carrying
NPM1, FLT3, and/or IDH1 mutations (20). In a study
including AML patients under the age of 60 years,
DNMT3A mutations were observed in 23% of patients and
were associated with worse overall survival (OS) and relapsefree survival than that observed in patients with wild-type
DNMT3A alleles (21). Similar results were observed in a
cohort of 1185 Chinese patients with AML (22). DNMT3A
mutations, typically R822H, were also noted in patients
with all subtypes of MDS. Similar to AML, DNMT3A
mutations correlated with shorter survival and increased risk
of transformation to AML (23).
TET2. Uniparental disomy and deletions spanning
chromosome 4q24 were rst described in patients with
myeloid malignancies (16). Sequencing of the commonly
deleted region unveiled acquired somatic mutations to TET2
(the ten-eleven-translocation gene 2) in patients with myeloid cancers, including patients with MDS (19%26%) or
AML (24%27%; refs. 16, 24, 25). When they occur, TET2
mutations are present in the vast majority of bone marrow
precursor hematopoietic cells (including CD34 progeni-

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Mol Cancer Res; 11(8) August 2013

Figure 1. Role of proteins encoded

by genes mutated in MDS or AML
in DNA methylation. DNMTs
catalyze the addition of a methyl
group to cytosine. IDH enzymes
are responsible for catalyzing the
reversible conversion of isocitrate
to a-KG and CO2 in a two-step
reaction. In turn, a-KG is required
by the TET proteins to oxidize 5mC to 5-hmC, an intermediate in
DNA demethylation. Mutant IDH
proteins metabolize a-KG to 2-HG,
an oncometabolite that
competitively inhibits the
enzymatic activity of TET proteins.

Gene

tors; ref. 25). TET2 mutations can be observed concomitantly with NPM1, FLT3, JAK2, RUNX1, CEBPA, CBL,
and KRAS mutations but are mutually exclusive with
IDH1/2 mutations (24, 26). Frequently, patients will carry
more than one TET2 mutation (16). Missense mutations
frequently occur in regions that are evolutionary conserved
and are typically C-terminus truncation mutations that
result in loss of TET2 protein function (25). TET2 catalyzes
the oxidation of 5-mC to 5-hmC, which has been shown to
be a demethylation intermediary (27, 28). The role of TET2
mutations in the pathogenesis of MDS and AML, however,
is not well understood, and published reports are often
conicting. In general, loss of TET2 function leads to a
global decrease in 5-hmC levels and accumulation of 5-mC,
which is a feature of DNA hypermethylation that results in
gene repression (28). Surprisingly, an analysis of 88 patients
with different myeloid malignancies harboring TET2 mutations revealed that a reduction in 5-hmC levels was associated with CpG hypomethylation, contradicting the paradigm that loss of TET2 function results in hypermethylation
of differentially methylated CpG sites (27). Furthermore,
the dysplastic phenotype of MDS cannot be attributed to
TET2 mutations alone. Experiments using mouse models
have shown that loss of Tet2 in the hematopoietic compartment results in an increase in HSC self-renewal with
enhanced HSC function and expression of myeloid-specic
and self-renewal gene programs, leading to progressive
myeloproliferation and extramedullary hematopoiesis
(Table 3; ref. 29). In addition, TET2 mutations are relatively
frequent in patients with myeloproliferative neoplasms, in
whom dysplastic features are very infrequently present. The
impact of TET2 mutations on the outcomes of patients with
AML or MDS remains controversial. In one study, among
patients with AML who had achieved complete remission
after induction chemotherapy, the incidence of TET2 mutations was 17% but had no impact on survival. This nding is
further supported by another study in 783 younger adult

Molecular Cancer Research

Mutations in MDS and AML

Table 3. Mouse models genetically engineered to express gene mutations encountered in patients with
MDS or AML
Gene (reference)

Mouse model

Phenotype

Strategy

DNMT3A & DNMT3B (14)

Dnmt3a/, Dnmt3b/, and

[Dnmt3a/, Dnmt3b/] straight KO

Intercrossed Dnmt3a and Dnmt3b

heterozygotes, which are grossly
normal and fertile, to generate nulls

DNMT3A (19)

Dnmt3a/ conditional KO

Dnmt3a/ died by 4 wk of age.

Dnmt3b/ are embryonic lethal.
Dnmt3 double mutants severely
impaired in de novo DNA methylation
and died by E11.5
Dnmt3a loss in HSCs leads to
expansion of morphologically normal
HSC with improper differentiation

IDH1 (34)

Idh1LSL/WT R132H conditional KI

Increase in hematopoietic progenitors,

splenomegaly, and anemia with
extramedullary hematopoiesis

TET2 (29)

Tet2f/f conditional KO

EZH2 (50)

Ezh2/ conditional KO

EZH2 (54)

Ezh2/ conditional KO in a myeloid

leukemia mouse model

ASXL1 (42)

Asxl1tm1Bc/tm1Bc KO

ASXL1 (43)

NRasG12D/Asxl1 shRNA

Biallelic deletion of Tet2 results in

progressive myeloproliferation,
increase HSC self-renewal, CMML
phenotype
Loss of Ezh2 results in development of
T-cell leukemia with almost complete
penetrance in less than 1 year;
implicates Ezh2 as a tumor
suppressor
Increased numbers of differentiated
leukemic cells leukemic and
perturbed leukemic progression after
Ezh2deletion suggesting Ezh2 is
oncogenic in AML
Asxl1 loss displays mild phenotype;
differentiation defects of
hematopoietic precursors
Asxl1 loss causes myeloproliferation
with myeloid inltration in spleen and
liver resulting in splenomegaly and
hepatomegaly and impaired survival

SF3B1 (64)

Sf3b1/ mutant

Sf3b1 haploinsufciency leads to

various skeletal defects;
homozygous null are embryonic
lethal

Transplantation of puried Mx1-Cre;

Dnmt3a/ HSCs and deletion of
Dnmt3a induced 4 weeks after
transplanting
Expression of R132H mutation in all
hematopoietic cells or specically
myeloid cells by crossing Idh1lsl/WT
with VavCre or LysM-Cre,
respectively
Deletion of Tet2 in the hematopoietic
compartments by crossing Tet2/
mice with different tissue-specic
Cre mice
Ezh2/ mice crossed with Mx-Cre Tg.
Deletion of the set domain induced by
poly(I)poly(c) injections

Transplanted MLL-AF9transformed
GMPs from Cre-ERT;Ezh2/ mice
into lethally irradiated mice; deletion
of Ezh2 by tamoxifen injections
Insertion of a PGKneo cassette
upstream from the PHD domain to
disrupt Asxl1 expression globally.
Transplantation of bone marrow cells
expressing NRasG12D in
combination with Asxl1 short hairpin
RNA construct or empty vector with
GFP into lethally irradiated mice
Mutant allele generated by replacing 4
exons with neo gene to disrupt gene
expression globally

Abbreviations: KO, knockout; KI, knockin.

patients with AML, in which TET2 mutations were identied in 7% of cases but had no impact on response or OS
(30). Another study found that 20% of patients with MDS
had TET2 mutations, clustering in cases of CN-MDS, and
not affecting survival (26). However, other groups have
reported a favorable impact of TET2 mutations on survival
in MDS (31). Yet, other groups have reported that TET2
mutations may have a detrimental effect on survival in AML,
particularly among patients with favorable molecular risk
(i.e., carriers of CEPBA and NPM1 mutations; ref. 24).
Intriguingly, the presence of TET2 mutations seems to
improve the response rates of patients with high-risk MDS
or AML to azacitidine, although no impact on OS has been

www.aacrjournals.org

yet observed (32). Because of the inconsistent reports on the

prognosis of patients with MDS or AML harboring TET2
mutations, the prognostic value of TET2 mutations remains
controversial and therefore it is not currently used as a
prognostic indicator in clinical practice.
IDH1 and IDH2. IDH1 and IDH2 are metabolic
enzymes that catalyze the interconversion of a-KG and
isocitrate. Mutations to either IDH1 or IDH2 share a common feature of acquiring a neomorphic enzymatic phenotype where a-KG is reduced to 2-hydroxyglutarate (2-HG).
2-HG is presumed to be an oncometabolite that contributes
to the tumorigenic process by competitively inhibiting the
enzymatic activity of dioxygenases that require a-KG as a

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Larsson et al.

cofactor (refs. 15; Fig. 1). Notably, the TET proteins are
dioxygenases that metabolize a-KG, and high levels of 2-HG
inhibit the enzymatic activity of the TET proteins. Mutations mainly map to codons R132 of IDH1 and R140 or
R172 of IDH2. All IDH mutations reported thus far occur
in the active catalytic site and participate in isocitrate
binding (8, 15). An analysis of 385 patients with de novo
AML revealed that those harboring an IDH1 or IDH2
mutation displayed global DNA hypermethylation and
impaired hematopoietic differentiation, correlating with an
increase in stem and progenitor cell markers (15). Mutations
to IDH1 are always heterozygous and exclusively occur at
residue 132, with an arginine to histidine being the most
common amino acid substitution. It is important to note
that IDH mutations are mutually exclusive with TET2
mutations, suggesting a common pathogenetic nexus leading to TET2 inactivation through alterations in either
enzyme (24). Although the mechanism for the hypermethylation and leukemogenesis in IDH-mutant AML is not entirely known, it has been linked to downregulation of a-KG
levels, which are needed by a variety of enzymes for their
function, such as TET proteins, thus affecting differentiation of myeloid cells, or the Jumonji family of histone lysine
demethylases (which demethylate H3K9 and H3K36),
thus resulting in increased methylation of lysine residues
(33). Recently, a mouse model that specically expresses the
Idh1R132H missense mutation in the hematopoietic or in
the myeloid compartment was generated to gain a better
understanding of how IDH1/2 mutations promote leukemogenesis. These mice showed an increase in hematopoietic
progenitors and developed splenomegaly and anemia with
extramedullary hematopoiesis, which occurs in 10% to 15%
of patients with MDS bearing an IDH1/2 mutation who
progress to AML. Furthermore, expression of the mutant in
the myeloid compartment led to hypermethylated histones
and patterns of aberrant DNA methylation that are consistent with those observed in patients with AML with IDH1/2
mutations (34).
The impact of IDH mutations in the prognosis of AML
was investigated in patients with CN-AML, with frequencies
of 14% and 19% for IDH1 and IDH2, respectively (35).
IDH mutations tend to cluster in cases with mutant NPM1
but wild-type FLT3. In that setting, IDH1 mutations were
associated with worse disease-free survival (DFS; ref. 35).
Large studies from the United Kingdom, France, and Germany reported IDH mutations in 8% to 16% of patients,
which were associated with worse complete remission rates
and shorter survival among patients with CN-AML carrying
NPM1 mutations (3638). An analysis of 398 patients younger than 60 years randomly assigned to receive standard-dose
or high-dose daunorubicin in the Eastern Cooperative Oncology Group (ECOG) E1900 trial revealed that the favorable
effect of NPM1 mutations was limited to patients with
simultaneous expression of NPM1 and IDH1 or IDH2
mutations (20). IDH1/IDH2 mutations are less frequent in
MDS (4%11%), compared with AML, but have been linked
to shorter OS compared with unmutated patients (39).
However, as with TET2 mutations, the prognostic value of

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Mol Cancer Res; 11(8) August 2013

IDH mutations remains controversial and needs to be further

evaluated both experimentally and in additional cohorts.
Importantly, given that mutant IDH proteins acquire neomorphic enzymatic activity, they might lend themselves to
inhibition by molecules designed to inhibit the synthesis of
2-HG.
Mutations in chromatin remodeling genes
Although DNA methylation is the best characterized
epigenetic modication, a combination of different highthroughput sequencing applications has recently revealed
recurrent mutations to chromatin remodelers in MDS and
AML, suggesting a role in neoplastic transformation (Table
1). This led to the identication of recurrent mutations to
genes that encode histone modiers, such as ASXL1 (additional sex-comb like-1), and EZH2. ASXL1, and EZH2
encode proteins that are members of the polycomb-group
(PcG) family. PcG family members form protein complexes
that function to maintain the transcriptionally repressive
state of genes, such as the clustered (class I) homeobox genes
that encode HOX proteins, which are frequently overexpressed in AML and confer a poor prognosis. For instance,
the H2.0-like homeobox (HLX) gene is overexpressed by
2- to 6.8-fold in 87% of patients with AML compared with
CD34 cells of healthy donors, and increased HLX expression correlated with inferior survival (40).
ASXL1. ASXL1 has opposing functions in maintaining
both the activation and suppression of HOX genes, depending on the histone-modifying protein that interacts with
ASXL1 (41). Mutations in ASXL1 that map to the Cterminus of the protein have been identied in 3% to
22% of patients with MDS or AML, and correlate with
poor OS (Table 2; ref. 20, 26). In vivo studies using Asxl1
knockout mice showed that loss of ASXL1 resulted in a mild
phenotype characterized by differentiation defects in the
myeloid and lymphoid progenitors without ever progressing
to a more severe disease state that is typically observed in
patients with MDS and AML harboring an ASXL1 mutation
(42). It is possible that loss of ASXL1 is compensated for by
homologous proteins with redundant functions, such as
ASXL2 and ASXL3, thus resulting in a milder phenotype.
However, mutations conferring loss of both ASXL1 loci are
rare events. In fact, 90% of ASXL1 mutations are truncating
mutations that result in the loss of the plant homeodomain
(PHD) of the protein, yet the function of this mutant protein
remains unclear (26). It is possible that a truncating mutation in ASXL1 results in gain-of-function or dominantnegative activity of the mutated protein. Recently, using a
panel of human myeloid leukemia cell lines, it was shown
that leukemia cells with homozygous ASXL1 frameshift/
nonsense mutations had undetectable ASXL1 protein levels,
whereas leukemia cells with heterozygous ASXL1 mutations
showed a reduction in ASXL1 protein levels. Furthermore,
knockdown of ASXL1 in these cell lines is associated with
global loss of the trimethylation mark of H3K27 and
upregulation of HOXA gene expression (43). Interestingly,
ASXL1 is a cofactor essential for the enzymatic activity of
BAP1, and coimmunoprecipitation assays showed that

Molecular Cancer Research

Mutations in MDS and AML

ASXL1 forms a complex with BAP1 in human myeloid

leukemia cells that are wild-type for ASXL1. Selective BAP1
haploinsufciency in hematopoietic cells in mice induces a
myelodysplastic phenotype reminiscent of chronic myelomonocytic leukemia (CMML), suggesting than the ASXL1
BAP1 axis may play an important role in MDS (44).
Contrary to this, loss of BAP1 in human leukemia cell lines
with wild-type ASXL1 did not lead to an increase in HOXA
gene expression, suggesting that leukemic transformation
as a result of ASXL1 loss occurs through a mechanism that is
independent of BAP1 (43). In a study involving 439 patients
with MDS, ASXL1 mutations were found in 14% of patients
and were independent prognostic factors for survival [HR
for death from any cause, 1.38; 95% condence interval
(CI), 1.001.89; ref. 26). ASXL1 mutations were found in
5% of patients with AML and were inversely associated
with FLT3-ITD and mutually exclusive with NPM1 mutations, and represented an unfavorable prognostic factor for
survival (45).
EZH2. EZH2 makes up the catalytic component of
the polycomb repressive complex 2 (PRC2), where it interacts with EED and SUZ12. Mutations to any of these genes
have been described in early T-cell precursor acute lymphoblastic leukemia (T-ALL; ref. 46). The SET domain of
EZH2 catalyzes the progressive trimethylation of H3K27
(H3K27me3) to induce the repression of target genes
(47). EZH2 is overexpressed in many different epithelial
cancers and lymphomas, whereas inactivating mutations
are observed in myeloid malignancies, suggesting that
H3K27me3 alterations have biologic consequences that are
tissue specic (48). In patients with MDS, mutations to
EZH2 are generally missense or truncating mutations that
disrupt or delete the SET domain, leading to a reduction of
H3K27me3 levels (48, 49). Another study reported that
biallelic deletion of Ezh2 in the HSCs of mice was sufcient to cause T-ALL, which occurred with almost complete penetrance in less than 1 year (50). PRC proteins
play a signicant role in regulating HOX gene expression,
providing a possible causal link between inactivating
mutations to EZH2 and its implication in myeloid malignancies. Certain HOX proteins, such as HOXA7-11, are
frequently overexpressed in AML, and in vivo studies
showed that overexpression of HOXA10 in HSCs was
sufcient to cause AML in mice (51, 52). One study
showed that repression of certain HOX genes involves both
promoter CpG methylation and NSPc1-mediated H2A
ubiquitination in a process that requires the cooperation
of EZH2, DNMT3A, and NSPc1, a component of PRC1.
Trimethylation of H3K27 is required for the recognition
and binding of DNMT3A and NSPc1, and loss of H3K27
trimethylation, as a result of EZH2 depletion, led to
impairments in NSPc1-mediated H2A ubiquitination and
CpG methylation in HOXA gene clusters (53). EZH2
mutations are observed in 6% of patients with MDS and
are associated with worse OS compared with patients with
wild-type EZH2 (26, 48, 49). This is not surprising, as
EZH2 maps to chromosome 7, which is a genomic area
frequently lost in MDS and associated with very poor

www.aacrjournals.org

prognosis. Interestingly, EZH2 mutations are infrequently found in patients with AML (20). In fact, loss of Ezh2
perturbs the progression of AML by promoting the differentiation of leukemic cells in mice transplanted with
MLL-AF9transformed granulocyte-macrophage progenitors (GMP), suggesting that EZH2 is oncogenic in AML
(54).
MLL. MLL (mixed lineage leukemia) is another SET
domain-containing protein that represents one of the rst
identied epigenetic modiers that is frequently altered in
leukemia. MLL is translocated in 10% of AML cases and in
30% of secondary AML. MLL is recruited to many promoters where it mediates H3K4 methyltransferase activity,
resulting in activated gene expression (55). It has been
speculated that mislocalized activity of the H3K79 histone
methyltransferase, DOT1L, is the main leukemogenic driver
in cases of AML carrying MLL rearrangements. Importantly,
the novel DOT1L inhibitor EPZ004777 has been shown to
selectively inhibit H3K79 methylation, resulting in blocked
expression of leukemogenic genes with negligible impact on
non-MLLtranslocated cells (56). These data suggest that
pharmacologic inhibition of DOT1L may represent a potential therapeutic option for patients with MLL rearrangements.
BCOR. The genome of a female patient with AML and
devoid of mutations to FLT3, NPM1, and CEBPA was
sequenced as a means to gain a better understanding of the
molecular characterization of the subgroup of patients with
CN-AML who lack NPM1 and CEBPA mutations. Further
mutational screenings and additional sequencing of CNAML cases identied recurrent mutations to BCOR, which
encode a corepressor of BCL6. Mutations to BCOR occur at
a frequency of 3.8% in unselected CN-AML cases. However, in the subgroup of CN-AML that is least characterized,
BCOR mutations are observed in 17% of cases. This subgroup includes patients with no MLL-PTD and lacking
mutations to CEBPA, NMP1, FLT3, or IHD1/2 (4). In
addition, a single BCOR mutation has been observed in a
small MDS examination that included 26 patient samples
(57). BCOR has been shown to interact with HDACs, and
mutations to BCOR are associated with DNMT3A mutations, implicating a cooperative epigenetic mechanism for
AML pathogenesis. Furthermore, this study showed that
BCOR mutations portend inferior survival (4).
Mutations in spliceosomal genes
The spliceosome plays a fundamental role in the processing of nascent RNA transcripts. Spliceosomes are complexes
of small nuclear RNAs and proteins that remove (splice)
introns and unconventional exons from primary transcripts
(pre-mRNA) to assemble recognized exons (splicing) into
the mature mRNA (Fig. 2). Disease-associated aberrant
RNA splicing is a well-described paradigm that is primarily
gene specic and due to mutations in cis-regulatory
sequences that guide spliceosomal recognitions of exons
(58). However, a growing number of mutations are known
to target components of the spliceosome, and thus potentially to impart global rather than gene-specic RNA splicing
changes. In patients with myeloid malignancies, somatic

Mol Cancer Res; 11(8) August 2013

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Larsson et al.

Strong constitutive

Alternatively recognized

Commitment
complex E

Branch
point

8-20 nt
Py-rich tract

Splicing U2 snRNP
ATP
complex A

Branch
point

U12 dependent

Commitment
complex E

Exon

Splicing U2 snRNP
ATP
complex A

SF1
ADP

8-20 nt
Py-rich tract

Branch Shorter of Disrupted

Py-rich tract
point

Exon

Exon

Splicing
complex A

SF1
ADP

Branch Shorter of Disrupted

point
Py-rich tract

Exon

Branch Sequence varies

point based on G vs. C

Exon

Figure 2. MDS and AML mutations target spliceosomal genes associated with initial 30 splice site recognition. Somatic mutations have been found in all
major genes involved in 30 splice site recognition and spliceosome commitment. This includes SF1, U2AF2, and U2AF1, which are critical for commitment
complex E formation in U2-dependent splicing, as well as SF3A1, SF3B1, and ZRSR2, proteins that are essential for stabilization of splicing complex
A. It is important to note that SRSF2 serves as a generalized enhancer of 30 splice recognition facilitating splice site recognition through stabilization of U2AF1
interaction with the AG dinucleotide. Because SRSF2 functions through binding of exonic splicing enhancer (ESE) sequences it acts on only a subset of
exons. ZRSR2, which is the only spliceosomal gene predominately targeted by inactivating nonsense mutations, is essential for U2AF-independent
recognition of U12-mediated RNA splicing.

mutations have been described in eight core spliceosomal

genesSF3B1, SRSF2, U2AF35 (U2AF1), ZRSR2, SF3A1,
PRPF40B, U2AF65 (U2AF2), and SF1. Mutations were
highly recurrent in SF3B1, SRSF2, ZRSR2, and U2AF1,
whereas mutations in the other spliceosomal genes (U2AF2,
SF1, PRPF40B, and SF3A1) occurred with a frequency of
1% or less (57, 5961). Mutations to spliceosomal genes are
highly specic for patients with refractory anemia with ring
sideroblasts (RARS; 84.9%), or MDS without ringed sideroblasts (43.9%), and CMML (54.5%) compared with
patients with de novo AML (6.6%) or myeloproliferative
neoplasms (9.6%). The spectrum of spliceosomal mutations
also seems to be specic to disease type (57). As mutations to
U2AF2, SF1, PRPF40B, and SF3A1 are rare events in MDS
and AML, there will be no further discussion in regards to
these genes within this review. This review focuses on the
four most frequently mutated splicing genes, SF3B1, SRSF2,
ZRSR2, and U2AF1, occurring in all subtypes of MDS. It is
important to note that with the exception of ZRSR2, the
majority of the mutations described to date are targeted
missense rather than nonsense or frameshift mutations,
suggesting a gain-of-function rather than a reduction in
splicing factor levels. Because these genes play a fundamental
role in the recognition of the 30 splice site during early
spliceosome formation, functional changes in these proteins,
such as in their ability to stabilize U2 interactions, have the
potential to differentially affect both alternative and constitutive splicing decisions.
SF3B1. The SF3B1 gene encodes the 155-kDa subunit
of the SF3B complex, which serves to target the U2snRNP
complex to the branch point during complex A formation
(Fig. 2). Mutations are found in more than 80% of patients
with RARS, whereas it is mutated in only 6% of patients with
MDS without ringed sideroblasts, specically implicating
SF3B1 mutation in the pathogenesis of this subtype of

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Mol Cancer Res; 11(8) August 2013

MDS (57, 62). Mutations to SF3B1 confer a more favorable

prognosis in MDS, MDS/MPN, and RARS cases and are
associated with lower risk of progression to AML compared
with patients with wild-type SF3B1 (59, 62). SF3B1 mutations are predominantly heterozygous missense mutations at
residues K700, K666, R625, and H662, but the specic
effect of these mutations on protein function remains
unknown. It is hypothesized that mutations in SF3B1 alter
branch point and 30 splice site recognition, which contributes to changes in the mature mRNA pool. Indeed, a recent
examination of splicing of 81,564 exons in 9,069 genes
revealed that 423 exons in 350 genes were differentially used
in mutants compared with a representative healthy donor.
Notable genes with aberrant RNA splicing included ASXL1,
CBL, EZH, and RUNX. Some consequences of the aberrant
splicing pathways were structural differences in iron distribution contributing to the pathogenesis of RARS (63).
Importantly, SF3B1 is involved in repressing HOX genes
by physically interacting with PcG proteins, suggesting a
potential role for SF3B1 in epigenetic regulation. Loss of one
Sf3b1 allele leads to developmental defects characterized by
various skeletal alterations as a result of impaired Hox gene
silencing in murine models. Furthermore, Mll mutations
rescue the Sf3b1/ phenotype (64). It is important to
emphasize that translocations or insertions involving MLL,
resulting in an MLL gene fusion, occur in about 10% of
adult AML and confer a poor prognosis.
SRSF2. SRSF2 is a member of the SR family of RNAbinding proteins that predominantly function to enhance
exon recognition by binding regulatory splicing sequences.
At the 30 splice site region, SRSF2 facilitates the recruitment
of both U2AF1 and SF3A2, which is consistent with the
model whereby gene mutation alters regulation of exon
recognition (Fig. 2). For the myeloid diseases, SRSF2 is
currently the second most mutated splicing gene. Mutations

Molecular Cancer Research

Mutations in MDS and AML

to SRSF2 are almost always heterozygous missense mutations that specically occur at P95 and are more prevalent in
CMML cases (28%) when compared with patients with
RARS (1.5%), MDS without ringed sideroblasts (11.6%),
and AML (7%; ref. 57). MDS harboring an SRSF2
mutation is associated with a higher rate of transformation
to AML with shorter progression time and lower OS
compared with MDS with wild-type SRSF2 (65). Depletion
of SRSF2 leads to an accumulation of DNA damage and
genomic instability and to induction of G2M cell-cycle
arrest, uncovering pathways that may be targeted as a result
of aberrant RNA splicing (66). Thus, gain-of-function
mutations could provide a compelling reason as to why
mutations to SRSF2 confer a poor prognosis. Furthermore,
SRSF2 mutations are associated with concomitant mutations in IDH1 and RUNX1, a transcription factor that is
frequently mutated and/or misspliced in MDS (61). Both
mutations and missplicing of RUNX1 are associated with
inferior event-free survival (EFS) and OS. The fact that
SRSF2 mutations frequently co-occur with mutations in
RUNX1, which also happens to be abnormally spliced when
SRSF2 is mutated, may explain why deregulations in these
two genes lead to a similar phenotype and prognosis.
ZRSR2. ZRSR2 is a member of the U2AF1-related
protein family, which functions in early spliceosome assembly through direct interactions with the U2AF 65-kDa
subunit (U2AF2). Unlike the other members of this group,
ZRSR2 does not seem capable of functionally replacing
U2AF1, and while required its precise role in U2-dependent
splicing is unclear (67). An important consideration, however, is that ZRSR2 plays a different role in assembly of the
minor U12-dependent spliceosome, where it is capable of
completely replacing the U2AF complex (68). Mutations of
the ZRSR2 gene occur at similar frequency in MDS (8%)
and CMML (8), and unlike other spliceosomal gene
mutations they do not occur in select "hot spots" (60).
Instead, nearly all reported mutations are nonsense or
frameshift mutations, clearly establishing a loss-of-function
role. In one study, patients with ZRSR2 mutations
seemed to cluster with RAEB-1 and RAEB-2, MDS
subtypes with further associated pronounced thrombocytopenia (60). Furthermore, these mutations were associated
with a higher AML transformation rate and poor OS. A
specic link to U12-dependent gene splicing remains to
be established.
U2AF1. The U2AF1 gene encodes the 35-kDa subunit
of U2AF and functions to recognize the AG dinucleotide
marking the end of the intron. For introns with a weak
U2AF2-binding sequence, U2AF1 is critical for splice site
recognition (69). U2AF1-related proteins, ZRSR1, ZRSR2,
and U2AF1L4, substitute for U2AF1 in some spliceosomal
reactions, clearly dening the potential to affect a subset of
genes (68). Mutations to U2AF1 were not observed in
patients with RARS but occurred with relatively equal
frequencies in MDS without ringed sideroblasts (11.6%),
CMML (23%), and AML (11.0%) cases (57, 59, 62, 70).
Mutations to U2AF1 occur exclusively at codons S34 or
Q157 and correlate with a more rapid transformation from

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MDS to AML, although their impact on OS remains unclear (65). Expression of mutant U2AF1 in HeLa cells leads
to a reduction in cell proliferation and enhanced apoptosis,
which are common features of MDS (57). Much like SRSF2
mutations, mutations to U2AF1 are implicated in abnormal
splicing of certain genes, namely RUNX1 and TET2, and are
also associated with mutations in ASXL1 and TET2. Furthermore, expression of mutant U2AF1 resulted in upregulation of nonsense-mediated RNA decay (NMD) pathway
genes, indicating that U2AF1 mutations lead to increases
in RNA splicing defects that require targeting for degradation (61).
Overall, mutations to SF3B1, SRSF2, ZRSR2, and U2AF1
are the most frequent gene aberrations occurring in all
subtypes of MDS and are mutually exclusive (57, 5962).
TP53 mutations in the pathogenesis of MDS/AML
TP53 is the tumor suppressor more frequently mutated in
human cancer. Mutations to TP53 occur in approximately
10% of de novo MDS and AML cases, whereas it is mutated
in more than 25% of therapy-related MDS (t-MDS) and
AML (t-AML) cases. Although the presence of TP53 mutations has long been recognized as a poor risk factor for
survival, novel aspects of TP53 biology have been linked to
the pathogenesis of myeloid malignancies. It is now widely
recognized that patients with TP53 mutations typically have
a complex karyotype (CK; ref. 71). It has been recently
shown that the events leading to complex genomic rearrangements in CK-AML are orchestrated by a one-step catastrophic event termed chromothripsis (Fig. 3; ref. 72).
Genomic rearrangements caused by chromothripsis are
characterized by complex somatic rearrangements with
alternating copy-number states affecting one or a few chromosomes. Two scenarios describing the events leading to
p53-mediated chromothripsis have been discussed. One
scenario describes critical telomere shortening, followed by
chromosomal end-to-end fusion, and subsequent breakage
as the events leading to complex interchromosomal rearrangements. The fact that progressive telomere shortening
occurs as a function of patient age may also explain why
chromothripsis is more frequent in advanced aged TP53mutated AML and why the frequency of CK-AML increases
with age. The other scenario postulates that chromothripsis
occurs when double-stranded DNA breaks, caused by ionizing radiation or DNA-damaging agents, are subjected to
repair by the error-prone nonhomologous end joining
(NHEJ) pathway, the dominant repair pathway in p53decient (e.g., mutated) cells (72).
TP53 function is also implicated in the pathogenesis of
5q syndrome. In vitro studies showed that downregulation
of RPS14, a 40S ribosomal subunit, in human bone marrow
cells phenocopied the erythroid failure observed in patients
with 5q syndrome (73). Deletion of the Cd74-Nid67
interval (syntenic to the 5q syndrome commonly deleted
region that contains RPS14) in the bone marrow of mice
resulted in elevated levels of p53 and increased bone marrow
apoptosis. Crossing Cd74-Nid67/ mice with p53-null
mice (74) or pharmacologic inhibition of p53 rescues the

Mol Cancer Res; 11(8) August 2013

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Larsson et al.

Normal HSCs

sAML

MDS

Chemotherapy
TET2 mutation

FLT3 mutation

HSC pool expansion

dysplasia

Myeloproliferation

Founding clone

Subclone
on
tati
mu
1
NX
Resistant founding
RU
clone

Relapse

Chromothripsis
TP53 mutation

AS
XL
1m
uta
tio
n

Resistant subclone
Normal HSC

Catastrophic chromosome
breakage

Rearrangement of chromosome
as a result of repair attempt

Relapse
Cure

Figure 3. Schema representing a potential path of acquisition of mutant disease alleles leading to MDS, transformation to AML, and relapse after failure
to respond to standard chemotherapy. Normal HSCs randomly acquire hundreds of nonpathogenetic mutations in a time-dependent fashion. At some point,
a "driver" mutation is acquired (e.g., TET2) that confers a growth advantage to a "founding clone." Previously acquired background mutations (i.e.,
"passenger") migrate with the driver mutation and are present in all its progeny. Driver mutations disturb normal myeloid maturation and induce dysplastic
features clinically compatible with MDS. Subsequent acquisition of mutant alleles in the founding clone (e.g., FLT3) may result in leukemic transformation
(i.e., secondary AML). Patients with AML receive induction to remission cytotoxic chemotherapy followed by consolidation chemotherapy. A fraction
of patients are cured upon eradication of the founding clone as well as "subclones" with a more complex mutational make-up than the former. However, a
signicant fraction of patients relapse after chemotherapy due to resistance and subsequent reexpansion of resistant clones. This sequence of events is
driven by the acquisition of new mutations (e.g., RUNX1, ASXL1, and TP53) either in the founding clone or in a subclone of the founding clone as a
consequence of therapy-induced selection pressure or as a result of DNA damage directly induced by cytotoxic chemotherapy. TP53 mutations have
been associated with a phenomenon termed chromothripsis, which may occur at any point during the pathogenesis of AML. The latter induces local
chromosome fragmentation resulting from DNA double-strand breaks, likely repaired by NHEJ. The net result is a conglomerate of complex chromosomal
rearrangements that adds a new layer of genomic complexity to that provided by the numerous point mutations present in AML genomes and provides
the genetic basis for tumor growth and resistance to therapy.

erythroid defect (75), thereby denitely implicating p53

in the pathogenesis of this syndrome.
Dynamics of mutated alleles in MDS and AML
Although the use of next-generation sequencing platforms has shed invaluable new light into the mutational
landscape in MDS and AML, several questions remain to
be answered. First, available studies only provide a snapshot of the mutational make-up of MDS or AML but do
not address the mutational clonal hierarchy within the
bulk of leukemic cells or aid in establishing which mutations bear real weight in the pathogenesis of the disease
(i.e., "driver" vs. "passenger" mutations). Second, serial
mutational screenings will be necessary to establish the

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Mol Cancer Res; 11(8) August 2013

genetic lesions involved in the progression from MDS

to AML. Third, extensive work is warranted to exploit
the available wealth of information for diagnostic, prognostic, and therapeutic purposes.
A recent study has shown that hematopoietic stem/
progenitor cells (HSPC) of normal individuals contain
hundreds of random mutations, that the number of
mutations is positively correlated with age, and that the
mutational spectrum between normal healthy HSPCs and
that of AML genomes is very similar. This suggests that
normal HSPCs accumulate random mutations over time
that are irrelevant for AML pathogenesis but will be
transmitted (i.e., passengers) to all AML clones if and
when a driver mutation is acquired and provides a growth

Molecular Cancer Research

Mutations in MDS and AML

advantage for an HSPC. Furthermore, a comparative

analysis of 12 FAB-M1 CN-AML and 12 FAB-M3 AML
genomes identied 13 recurring mutations in M1-AML,
including NPM1, DNMT3a, and IDH1, which only
coexisted in M1-AML, suggesting that the latter are likely
M1-AML-initiating mutations rather than cooperating
mutations (12). Not surprisingly, an analysis of the
dynamics of mutated alleles during the progression from
MDS to AML in 7 patients by high-throughput sequencing, DNA copy-number analysis, and microarray-based
gene-expression studies found that the MDS clones also
contained hundreds of acquired mutations. More importantly, the AML arising from them were derived from at
least one subclone acquiring new driver mutations or
genomic rearrangements and that 85% to 90% of MDS
and secondary AML unfractionated bone marrow cells
were clonal, even with a myeloblast count of less than 5%
(76). In this group of patients, 11 genes were found
mutated recurrently: CDH23, NPM1, PTPN11, RUNX1,
SMC3, STAG2, TP53, U2AF1, UMODL1, WT1, and
ZSWIM4 (76). It is also important to consider that
mutation of spliceosomal genes may serve as complete
genetic drivers of tumorigenesis, a concept originating
from the observation that SRSF1 is capable of inducing
tumor formation (77). It is clear that mutations involving
spliceosomal genes specically affect genes known to be
sequentially targeted by individual mutations.
Although most patients with AML respond to initial
induction chemotherapy, a large number of them will relapse
and exhibit refractoriness to subsequent therapy that almost
invariably leads to the patient's death. It is therefore important
to understand the genetics underlying primary tumorigenesis
as well as disease recurrence. The genome sequences of 8
patients with AML at diagnosis have been compared from the
same patients at the time of relapse by deep sequencing (44).
Such analysis unveiled novel mutations in AML such as those
in WAC, SMC3, DIS3, DDX41, and DAXX. More importantly, two patterns of clonal evolution at relapse were
identied. These include one in which the founding clone
at diagnosis acquired new mutations that were later identied
in clones at relapse, and another one in which a subclone of the
founding clone withstood initial therapy and acquired additional mutations that led to clonal expansion and relapse (Fig.
3). In both instances, initial therapy failed to eradicate all or
specic subclones from the founding clone. Interestingly,
some of the mutations observed at relapse were found to be
due to DNA damage secondary to cytotoxic chemotherapy,
highlighting potential deleterious effects of current AML
therapeutic regimens (44).
Perspective and Conclusions
Available mutational data challenge the conceptual "twohit" dictum in AML and MDS, whereby the pathogenesis of
these diseases is driven by a mutation in a gene-regulating cell
growth or proliferation that is complemented by a second
mutation in a transcription factor regulation myeloid maturation and depicts a much more complex picture about the

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pathogenesis of these malignancies. It is now becoming more

recognized that mutations driving neoplastic transformation
and progression are not restricted to tumor suppressors,
oncogenes, or pathway-specic genes involved in growth
signaling, proliferation, migration, and survival. Regulatory
proteins that are not dened to a specic pathway but have
functional roles in the regulation of global processes (i.e.,
transcription and translation) are frequently targeted to
functionally deregulating specic mutations in many different blood disorders, including AML and MDS. Crossexaminations of the epigenetic prole in patients with
myeloid cancers harboring mutations to epigenetic genes
suggest that aberrations to the epigenome play a signicant
role in the pathogenesis of these malignancies. This is not
surprising considering that many of these epigenetic regulators are important for maintaining the repressive state of
genes involved in growth, stem cell renewal, and survival.
Importantly, epigenetic-modifying agents such as HDAC
inhibitors and hypomethylating agents have already shown
clinical activity in patients with AML and MDS. More
importantly, the number of different genetic lesions in
epigenetic regulators provides researchers with more potential therapeutic targets and currently remains an area of focus
in cancer drug discovery. Furthermore, a high proportion of
patients with MDS carry somatic mutations in several
different spliceosome genes, namely SF3B1, SRSF2, and
U2AF1. Although the roles of these genes in RNA splicing
are well described, little is known about their roles in the
context of hematopoietic function or how splicing mutations
contribute to the pathogenesis of MDS and in some cases,
the transformation to AML. With the accumulating data
correlating spliceosome mutations with mutations in epigenetic regulators, it is arguable that abnormal RNA splicing
parallels aberrant epigenetic reprogramming in the pathogenesis of MDS or that a direct link may exist between RNA
splicing and epigenetic regulation. The overlap and frequency of mutations to these genes and the data correlating
splicing mutations with the missplicing of epigenetic regulators recurrently mutated in myeloid malignancies further
supports this notion. Studying the interplay between concomitantly mutated genes will undoubtedly shed new light
into the mechanisms whereby normal myeloid cells become
malignant and will aid in developing more biologically
informed therapeutic approaches.
Disclosure of Potential Conicts of Interest
No potential conicts of interest were disclosed.

Authors' Contributions
Conception and design: C.A. Larsson, A. Quintas-Cardama
Development of methodology: A. Quintas-Cardama
Acquisition of data (provided animals, acquired and managed patients, provided
facilities, etc.): A. Quintas-Cardama
Writing, review, and/or revision of the manuscript: C.A. Larsson, G. Cote,
A. Quintas-Cardama
Administrative, technical, or material support (i.e., reporting or organizing data,
constructing databases): A. Quintas-Cardama
Study supervision: A. Quintas-Cardama

Acknowledgments
The authors thank Amanda Wasylishen for critical review of the article.

Mol Cancer Res; 11(8) August 2013

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Larsson et al.

Grant Support
Work in the authors' laboratory is supported by the NIH (grant 5T32CA00929932) and the American Legion Auxiliary Fellowship.

Received December 18, 2012; revised March 12, 2013; accepted April 11, 2013;
published OnlineFirst May 3, 2013.

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