See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/370059144

Epigenetic regulation by ASXL1 in myeloid malignancies

Article  in  International Journal of Hematology · April 2023

DOI: 10.1007/s12185-023-03586-y

CITATION READS

1 50

2 authors, including:

Feng-Chun Yang
University of Texas Health Science Center at San Antonio
231 PUBLICATIONS   7,931 CITATIONS   

SEE PROFILE

All content following this page was uploaded by Feng-Chun Yang on 23 May 2023.

The user has requested enhancement of the downloaded file.

International Journal of Hematology
https://doi.org/10.1007/s12185-023-03586-y

PROGRESS IN HEMATOLOGY

Stem cell regulation and dynamics in myeloid malignancies

Epigenetic regulation by ASXL1 in myeloid malignancies

Feng‑Chun Yang1,2   · Joel Agosto‑Peña1,2

Received: 2 February 2023 / Revised: 2 March 2023 / Accepted: 22 March 2023

© Japanese Society of Hematology 2023

Abstract
Myeloid malignancies are clonal hematopoietic disorders that are comprised of a spectrum of genetically heterogeneous
disorders, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic
leukemia (CMML), and acute myeloid leukemia (AML). Myeloid malignancies are characterized by excessive prolifera-
tion, abnormal self-renewal, and/or differentiation defects of hematopoietic stem cells (HSCs) and myeloid progenitor cells
hematopoietic stem/progenitor cells (HSPCs). Myeloid malignancies can be caused by genetic and epigenetic alterations
that provoke key cellular functions, such as self-renewal, proliferation, biased lineage commitment, and differentiation.
Advances in next-generation sequencing led to the identification of multiple mutations in myeloid neoplasms, and many new
gene mutations were identified as key factors in driving the pathogenesis of myeloid malignancies. The polycomb protein
ASXL1 was identified to be frequently mutated in all forms of myeloid malignancies, with mutational frequencies of 20%,
43%, 10%, and 20% in MDS, CMML, MPN, and AML, respectively. Significantly, ASXL1 mutations are associated with a
poor prognosis in all forms of myeloid malignancies. The fact that ASXL1 mutations are associated with poor prognosis in
patients with CMML, MDS, and AML, points to the possibility that ASXL1 mutation is a key factor in the development of
myeloid malignancies. This review summarizes the recent advances in understanding myeloid malignancies with a specific
focus on ASXL1 mutations.

Keywords  ASXL1 · Myeloid malignancies · Epigenetic regulation · Mutation

Identification of ASXL1 mutation in human myeloproliferative neoplasms (MPN), chronic myelomono-

diseases
ASXL1 mutations in myeloid malignancies
Myeloid malignancies are clonal hematopoietic disorders
characterized by excessive proliferation, abnormal self-
renewal, and/or differentiation defects of hematopoietic stem
cells (HSCs) and myeloid progenitor cells hematopoietic
stem/progenitor cells (HSPCs) [1–3]. Myeloid malignancies
are comprised of a spectrum of genetically heterogeneous
disorders, including myelodysplastic syndromes (MDS),
* Feng‑Chun Yang
yangf1@uthscsa.edu
1
Department of Cell Systems and Anatomy, University
of Texas Health Science Center at San Antonio,
San Antonio, TX 78229, USA
2
Mays Cancer Center, University of Texas Health Science
Center at San Antonio, San Antonio, TX 78229, USA
cytic leukemia (CMML), and acute myeloid leukemia
(AML). MDS occurs as a result of disordered insufficient
development of blood cells, to a greater or lesser extent,
within the bone marrow, which can evolve to bone marrow
failure and variable rates of transformation to AML. Mye-
loid malignancies can be fatal in the majority of patients.
Advances in next-generation sequencing led to the identi-
fication of multiple mutations in myeloid neoplasms, and
many new gene mutations were identified as key factors in
driving the development of myeloid malignancies. Targeted
therapies are desperately needed.
Additional sex comb-like 1 (ASXL1) is one of the most
frequently mutated genes in a variety of myeloid malignan-
cies, including ~ 15–21% of MDS [4, 5], ~ 8–10% of MPN
[6, 7], ~ 43–49% of CMML [8, 9], ~ 7–8% of juvenile myelo-
monocytic leukemia (JMML) [10, 11], and ~ 3–10% of AML
[12, 13]. ASXL1 is a human homolog of the Drosophila asx
gene, which maps to chromosome 20q11.21. ASXL1 encodes
the additional sex combs–like protein 1 (ASXL1). It belongs

13
Vol.:(0123456789)

to the polycomb group (PcG) and trithorax complexes family
[14, 15]. Significantly, ASXL1 mutations are associated with
aggressiveness and poor prognosis in all myeloid malignan-
cies, including high-risk MDS, CMML, MPN, and second-
ary AML (sAML) [5, 7, 9, 12, 16]. ASXL1 mutations are
associated with a reduced time to progression in AML and
constitute an independent prognostic marker in patients with
MDS [5]. ASXL1 mutations also remained an independent
adverse prognostic marker for time to AML progression in
CMML patients. [9]
ASXL1 mutations have also been described in some rare
myeloid malignancies. Mastocytosis is a heterogeneous
group of disorders which are characterized by abnormal
growth and accumulation of mast cells in one or more organ
systems. Traina et al. reported that ASXL1 mutations are
found in two of eight systemic mastocytosis patients [17].
With a sixty-two SM-AHNMD patient cohort, Damaj et al.
reported that 14% patients harbored ASXL1 mutations and
ASXL1 mutation remained an independent prognostic factor
that negatively affected overall survival. [18]
Interestingly, ASXL1 mutations have only been sporadi-
cally observed in lymphoid leukemia [20]. Compared with
ASXL1 mutations found in all spectrum of myeloid malig-
nancies, ASXL2 mutations are restricted in a very specific
subset of AML, ~ 23% of AML with t(8;21) translocation
[21, 22]. Interestingly, ASXL1 and ASXL2 mutations are
mutually exclusive in t(8;21) AML, suggesting that the
mutations may have convergent downstream and/or have
synthetic lethal effects with each other.
Most ASXL1 mutations in myeloid malignancies are
frameshift or nonsense mutations in exon 12 (last exon)
before the PHD finger. Mutant ASXL1 transcripts are pre-
dicted to produce C-terminally truncated ASXL1 protein by
escaping from nonsense-mediated-decay [16, 23, 24]. These
mutations are always heterozygous, suggesting that they
either have dominant-negative or gain-of-function effects.
The most commonly detected ASXL1 mutation is ASXL1
NM_015338.5:c.1934dup;p.Gly646Trpfs*12 (ASXL1
c.1934dupG), a frameshift mutation in ASXL1 exon 12 due
to a one nucleotide expansion of a contiguous repeat of
eight guanine nucleotides (GGG​GGG​GG) that results in a
truncated ASXL1 protein lacking the PHD finger [25, 26].
ASXL2 mutations in myeloid malignancies, at least in AML
with t(8;21)/RUNX1-RUNX1T1, are out-of-frame frameshift
mutations, exclusively heterozygous, and occur exclusively
in exons 11 and 12. [21]
Inoue et al. reported that the truncating ASXL1 mutant
can be detected in MDS cells, which may play a role in
MDS pathogenesis [23]. Thol et al. reported that, in an
MDS cohort, ASXL1 frameshift mutations were inde-
pendently associated with an unfavorable prognosis, and
patients harboring ASXL1 frameshift mutations not only
have a shorter overall survival, but also have reduced time
13
F.-C. Yang, J. Agosto‑Peña
to AML progression in univariate and multivariate analyses.
[5] Despite this being widely acknowledged, it continues to
remain controversial as to whether truncating mutations in
ASXL1 result in gain- or loss-of-function, or if they confer
dominant-negative activity in vivo. Hence, several groups
including ours have investigated whether the presence of
the truncated forms of ASXL1 protein induces myeloid
malignancies.
ASXL1 mutations in clonal hematopoiesis
Clonal hematopoiesis with somatic mutations is observed in
at least 10% of people over 65 years of age, without apparent
hematologic disorders. Age-related clonal hematopoiesis is
a common premalignant condition and is associated with
increased overall mortality and increased risk of hemato-
logic cancer, cardiovascular diseases, and atherosclerosis.
Genes mutated in clonal hematopoiesis are similar to those
in myeloid malignancies. ASXL1 is one of the most fre-
quently involved somatic mutations detected in individuals
with age-related clonal hematopoiesis [27–29]. A clinical
study by Yoshizato et al. showed that clonal hematopoie-
sis was prevalent in aplastic anemia, and ASXL1 mutations
are common in this population [19]. While these patients
respond to immunosuppressive therapy, some still develop
MDS and AML.
Several clinical studies demonstrate that ASXL1 muta-
tions have been repeatedly identified in individuals with
clonal hematopoiesis of indeterminate potential (CHIP),
a condition whose frequency increases with advanced age
[27–29], indicating that ASXL1 is one of the earliest genetic
events during the process of myeloid transformation. Close
monitoring of clonal hematopoiesis by both deep sequencing
and SNP array will need to be combined with clinical evalu-
ation for prognosis and to guide the management of patients
with aplastic anemia.
In myeloid malignancies, clonal heterogeneity can evolve
upon disease progression and/or relapse AML. The shared
somatic mutations in all leukemic cells reflect its clonal ori-
gin, eg. the founding clone, and additional mutations pre-
sented in subpopulations of cells define subclones in leu-
kemia. Sub-clonal mutations have been shown to be highly
enriched for epigenetic regulators. Therefore, understand-
ing the mechanism by which ASXL1 mutations contribute to
myeloid transformation is clinically important given the fact
that ASXL1 is a key epigenetic regulator in hematopoiesis.
Acquired aplastic anemia is a nonneoplastic bone marrow
failure. Acquired aplastic anemia is an immune-mediated
bone marrow aplasia, which has been known to associate
with clonal hematopoiesis upon marrow recovery. More
than 70% of patients with aplastic anemia develop somatic
mutations in their hematopoietic cells [30].Patient age at the
diagnosis of aplastic anemia is known as one of the strongest
Epigenetic regulation by ASXL1 in myeloid malignancies
predictors of clonal hematopoiesis with MDS-associated
somatic mutations. Advances in cytogenetic and next-gen-
eration sequencing demonstrated that clonal hematopoiesis
develops at a striking prevalence of 80–100% in adults with
aplastic anemia [19, 31–33]. Aplastic anemia patients have
a disproportionate number of mutations in epigenetic regula-
tors ASXL1, BCOR, and BCORL1. [19, 32, 34]
ASXL1 mutations in Bohring‑Opitz syndrome (BOS)
In addition to somatic mutations in hematological malig-
nancies, ASXL1 mutations have been discovered in in
approximately 75% Bohring-Opitz syndrome (BOS) patients
[35–37]. BOS is a heterogeneous genetic condition charac-
terized by severe developmental delay, characteristic crani-
ofacial appearance, fixed contractures of the upper limbs,
abnormal posture, feeding difficulties, severe intellectual
disability, fetal microsomia, and failure to thrive [38–40].
Most patients die in early childhood due to developmen-
tal deficits, unexplained bradycardia, obstructive apnea, or
pulmonary infections [38]. In 2011, Hoischen et al. identi-
fied de novo nonsense mutations of ASXL1 in patients with
BOS [35]. Following this report, several groups reported that
ASXL1 mutations account for around 50% of BOS patients.
Bohring-Opitz syndrome occurs sporadically, suggesting
that de novo mutations may be the underlying cause of this
disorder. The majority of ASXL1 mutations in BOS are de
novo frameshift and nonsense mutations. Regardless of the
clinical significance, it remains unknown whether ASXL1
mutations in BOS results in a loss-of-function, gain-of-func-
tion, or exert a dominant role. Deletion of Asxl1 (Asxl1−/−) in
mice leads to abnormalities of axial skeletal patterning (pos-
terior and anterior), which suggests disruption of both PcG
and TrxG activities. [41–43]. Thus, as regulators of PcG and
TrxG activities, the ASXL proteins are crucial in maintain-
ing the lineage-specific gene expression patterns during cell
division and replication.
Mouse models of ASXL1
Mouse models have been pivotal in understanding the
mechanisms by which they contribute to biological func-
tions and pathogenesis of diseases. Several mouse mod-
els have been developed by different research groups
(Table  1), which allow for investigating the impact of
ASXL1 alteration in hematopoiesis and myeloid malig-
nancies. [15, 24, 44–47] Fisher et al. reported that Asxl1
is required for normal hematopoiesis in an Asxl1-mutant
mouse model. [43, 48] However, these Asxl1-mutant mice
did not reveal a profound HSC/HPC cellular phenotype
or MDS/leukemia, likely due to an incomplete loss-of-
function of Asxl1. [48] We and others have reported that
homozygous or hematopoietic-specific loss of Asxl1
results in the abnormal self-renewal capacity of HSCs and
the development of MDS or MDS/MPN-like disease, with
HSC/HPCs from the latter exhibiting decreased global lev-
els of H3K27me3 and the increased expression of poste-
rior Hoxa genes. [44–46]
In human AML, c.1934dupG; p.G646WfsX12 is the most
common ASXL1 mutation in myeloid malignancy patients
[12]. Hsu and colleagues generated a mouse model (ASX-
L1tm/+) which bears human-like Asxl1 mutation [49]. They
found that ASXL1tm/+ hematopoietic cells had higher short-
term in vitro proliferation capacities, and bone marrow cells
of Asxl1 G643WfsX12 heterozygotes showed compromised
long-term in vivo repopulation and self-renewal capabilities.
ASXL1 G643WfsX12 mutation facilitated engraftment of
bone marrow cell overexpressing MN1, a proto-oncogene
[49]. However, these mice do not show development of
blood malignancies, indicating that ASXL1 G643WfsX12
alone was not sufficient for development of blood malignan-
cies in mice.
Nagase et al. generated a conditional Asxl1-mutant knock-
in mouse model (mimicking ASXL1 E635RfsX15) [50].
They found that expression of the C-terminal truncated
ASXL1 mutant in vivo led to myeloid skewing, thrombocy-
tosis, age-dependent anemia, and dysplasia [50]. They also
found that bone marrow cells from these mice had substan-
tial reductions in the levels of H3K4me3 and H2AK119Ub,
while the level of H3K27me3 was similar to that of control
cells. Mechanistically, they found that ASXL1 E635RfsX15-
KI HSCs displayed substantial reductions in H3K4me3 and
H2AK119Ub without significant reductions in H3K27me3,
distinct from the effects of Asxl1 loss [50]. Chromatin
immunoprecipitation followed by next-generation sequenc-
ing analysis demonstrated opposing effects of wild-type and
mutant Asxl1 on H3K4me3.
We established a Vav1 promoter driven Asxl1Y588X trans-
genic mouse model (Asxl1Y588XTg) expressing the analogous
protein product of the mutant ASXL1Y591X, frequently seen in
human patients. Asxl1Y588XTg mice had shortened survival
rates, and predisposition to a spectrum of myeloid malig-
nancies, closely recapitulating the characteristics of myeloid
malignancy patients with ASXL1-truncation mutations [47].
We found that ASXL1 truncation exerts an oncogenic role
in HSCs, at least in part through the gained interaction with
BET bromodomain-containing protein 4 along with altered
gene expression. More importantly, Asxl1Y588XTg BM cells
are sensitive to BET bromodomain inhibitors, suggesting
potential novel therapies targeting the cells bearing the trun-
cation mutation of ASXL1 through the use of BET inhibitors.
Despite the significant impact of ASXL1 mutations in leuke-
mia, the underlying mechanisms by which they contribute to
myeloid malignancies remain unknown, which hinders the
development of targeted therapeutics.
13

13
Table 1  Summary of mouse models used to study the functions of Asxl1 in hematopoietic systems
Mouse model
Asxl1fl/fl;Mx1Cre or VavCre
Asxl1-MT (derived from the
most common ASXL1 mutation,
1934dupG;G646WfsX12)
Asxl1−/− and Asxl1±
Asxl1tm/+  + MN1
(mimics the most common
mutation c.1934dupG;
p.G646WfsX12)
Asxl1±;Nf1±
Asxl1Y588XTg
Asxl1-MT KI (mimics the well-
described human ASXL1 muta-
tion, p.E635RfsX15)
VavCre;Asxl1-MTfl/fl
Asxl1G643fs/+
HSC phenotype
Increased LT-HSC and LSK
Not evaluated
Decreased LSK fractions in
Asxl1−/−
Promoted stem cell activities
(increased long-term colony
cells
Increased LT-HSC
Increased ST-HSC and LSK
fractions; and enhanced HSC in spleen and liver; > 20% blast
self-renewal
Decreased HSPSC-LSK, MPP and Mild leukopenia, and anemia,
increased LT-HSC
Decreased LT-HSC and LSK
fractions
Decreased LT-HSC and LSK
Disease phenotype Disease type Histone modification References
Leukopenia, anemia, erythroid MDS-like disease Decreased H3K27me3 [45]
dysplasia, multiple cytopenia
Multilineage myelodysplasia, MDS-like disease, occasional Decreased H3K27me3 [24]
pancytopenia progression to AML
Multiple cytopenias; dysplastic MDS-like and MDS/MPN-like Reduced H3K4me3 and [46]
features; myeloid cell infiltration disease H3K27me3 in Asxl1−/−
in spleen and liver
facilitates engraftment of cells None observed Increased H3K27me3 [49]
overexpressing MN1
Hepatosplenomegaly, anemia, MDS, MDS/MPN, myeloid Increased H3K4me3 [110]
thrombocytopenia, myeloid leukemia
infiltration in spleen and liver
Anemia; myeloid cell infiltration AML, MPN, MDS, and MDS/ Increased H3K27ac and [47]
MPN H3K122ac
cells in BM of leukemia mice
CHIP Decreased H3K4me3, [50], [158]
thrombocytosis, hypocellular H2AK119Ub, H3K27me3
bone marrow, myeloid skewing
Decreased RBC and increased No disease alone Reduced H3K4me3 and [50]
platelets H2AK119ub
Leukocytosis, anemia, thrombocy- MDS, MDS/MPN like disease Decreased H2AK119Ub [159]
tosis, disrupted splenic architec-
ture, myeloid and perivascular
infiltration in liver
F.-C. Yang, J. Agosto‑Peña
Epigenetic regulation by ASXL1 in myeloid malignancies
ASXL1 structural characterization
The human  ASXL  gene family consists of three mem-
bers, ASXL1, ASXL2, and ASXL3, which encodes ASXL1,
ASXL2, and ASXL3 proteins, respectively. [15, 51, 52]
Mutations in Asx enhance the phenotypes of both Poly-
comb group (PcG) and Thirotorax group (TrxG) gene
mutations [43]. PcG and TrxG proteins are regulated by
Enhancers of Trithorax and Polycomb (ETP) proteins,
which recruit PcG and TrxG complexes to target chroma-
tin [53, 54]. PcG and TrxG proteins modulate the expres-
sion of numerous genes by regulating histone methylation
to maintain repressive and active chromatin states at tar-
get loci, respectively. They preserve epigenetic memory
and maintain the expression patterns of cell lineage-
defining genes during cell division and replication. PcG
proteins maintain the inactive state of Hox genes, while
TrxG proteins keep Hox genes activated [55].Hox genes
are known to be critical for stem cell functions and, as
a result, dysregulated Hox protein expression results in
leukemogenesis [55]. ASXL1 contains 1541 amino acids
[15], ASXL2 contains 1435 amino acids [51], whereas,
ASXL3 has 2248 amino acids [52]. The three ASXL fam-
ily member proteins share conserved domains, including
a N-terminal ASXN domain, an ASXH domain, ASXM1
and ASXM2 domains in the middle region and a PHD
domain in the C-terminal region [15, 51, 52]. While there
is ∼40% amino acid homology between ASXL1, ASXL2,
and ASXL3, this homology increases to ∼70% in the con-
served domains of ASXL proteins including the ASXN,
ASX homology (ASXH), ASXM1, and ASXM2 domains
and the carboxy-terminal cysteine cluster plant homedo-
main (PHD) [15, 51, 52]. The ASXN domain is similar to
the Forkhead-box (FOX) domain, which is also known as
the winged helix-turn-helix domain, of FOXA3, FOXK1,
FOXO1 and FOXO4. [15, 51, 52, 56] The ASXH domain
has been shown as a deubiquitinase adaptor domain (DEU-
BAD), spanning from 238 amino acid to 390 amino acid of
ASXL1. The DEUBAD domain is critical for the binding
between ASXL1 and the deubiquitinating enzyme (DUB)
BRCA1-associated protein 1 (BAP1) [57–60]. The C-ter-
minal plant homeodomain (PHD) finger is located at the
very end of the ASXL proteins and is a putative histone-
interacting PHD domain. [15, 51, 52]
ASXL1 structural characterization
The human  ASXL  gene family consists of three mem-
bers, ASXL1, ASXL2, and ASXL3, which encodes ASXL1,
ASXL2, and ASXL3 proteins, respectively. [15, 51, 52]
Mutations in Asx enhance the phenotypes of both Poly-
comb group (PcG) and Thirotorax group (TrxG) gene
mutations [43]. PcG and TrxG proteins are regulated by
Enhancers of Trithorax and Polycomb (ETP) proteins,
which recruit PcG and TrxG complexes to target chromatin
[53, 54]. PcG and TrxG proteins modulate the expression
of numerous genes by regulating histone methylation to
maintain repressive and active chromatin states at target
loci, respectively. They preserve epigenetic memory and
maintain the expression patterns of cell lineage-defining
genes during cell division and replication. PcG proteins
maintain the inactive state of Hox genes, while TrxG pro-
teins keep Hox genes activated [55].Hox genes are known
to be critical for stem cell functions and dysregulated Hox
protein expression results in leukemogenesis [55]. ASXL1
contains 1541 amino acids [15], ASXL2 contains 1435
amino acids [51], whereas, ASXL3 has 2248 amino acids
[52]. The three ASXL family member proteins share con-
served domains, including a N-terminal ASXN domain,
an ASXH domain, ASXM1 and ASXM2 domains in the
middle region and a PHD domain in the C-terminal region
[15, 51, 52]. While there is ∼40% amino acid homology
between ASXL1, ASXL2, and ASXL3, this homology
increases to ∼70% in the conserved domains of ASXL
proteins including the ASXN, ASX homology (ASXH),
ASXM1, and ASXM2 domains and the carboxy-terminal
cysteine cluster plant homedomain (PHD) [15, 51, 52].
The ASXN domain is similar to the Forkhead-box (FOX)
domain, which is also known as the winged helix-turn-
helix domain, of FOXA3, FOXK1, FOXO1 and FOXO4.
[15, 51, 52, 56] The ASXH domain has been shown as a
deubiquitinase adaptor domain (DEUBAD), spanning from
238 amino acid to 390 amino acid of ASXL1. The DEU-
BAD domain is critical for the binding between ASXL1
and the deubiquitinating enzyme (DUB) BRCA1-asso-
ciated protein 1 (BAP1) [57–60]. The C-terminal plant
homeodomain (PHD) finger is located at the very end of
the ASXL proteins and is a putative histone-interacting
PHD domain. [15, 51, 52]
ASXL1 interacts with other nuclear proteins
to regulate hematopoiesis
ASXL1 interacts with polycomb repressive complex
2 (PRC2)
The polycomb repressive complex-2 (PRC2) complex
consists of four core components, including enhancer of
zeste homolog 1 (EZH1) or its paralog-EZH2, suppres-
sor of zeste 12 homolog (SUZ12), embryonic ectoderm
development (EED), and retinoblastoma-binding protein 4
and 7 (RBBP4/7) [61, 62]. As a component of the PRC2
13

complex, EZH2 is a histone methyltransferase that initi-
ates trimethylation of lysine 27 in histone 3 (H3K27me3), a
repressive chromatin mark [44, 57, 63]. The C-terminal SET
(Su(var)3–9, Enhancer-of-zeste and Trithorax) domain of
EZH2 catalyzes the methylation of H3K27. SUZ12 and EED
of the PRC2 complex are required for the catalytic activity
of EZH2 [64–66]. RBBP4 and AEBP2 are two additional
subunits of the PRC2 complex, which maintain the integ-
rity of PRC2 by stimulating EZH2 methytransferate activ-
ity on H3K27 [67]. The pathological role of deregulated
EZH2 has been well documented in both human and mice.
EZH2 overexpression are found in human cancers, such as
breast, prostate, and bladder tumors [68]. Coincidentally,
somatic loss-of-function mutations of EZH2 occur in MDS
[63, 69]. EZH2 mutations in lower-risk MDS are correlated
with a significantly worse prognosis [70]. Deletion of Ezh2
in mice results in the development of multi-forms of hemato-
logical malignancies, such as acute T-cell lymphoblastic leu-
kemia (T-ALL), lymphoma, and myeloid malignancies [71,
72, 73]. Loss of Ezh2 promotes JAK2V617F mutant-associated
myelofibrosis [74–76]. These findings indicate that EZH2
plays a tumor suppressive role in MDS and MPN.
ASXL1 has been reported to exert its epigenetic regula-
tory function by activating or repressing the transcription of
genes involved in proliferation and differentiation through its
cooperative effect with other chromatin modifiers to regu-
late histone modification [44, 57, 77]. ASXL1 is required to
maintain homeotic gene activation and silencing. [15, 43]
Being a member of the enhancer of trithorax (trxG) and
polycomb (PcG) group genes in Drosophila, Asx is associ-
ated with histone modification [14, 78]. PcG proteins sustain
the ‘off state’ while trxG proteins sustain the ‘on state’ of
the clustered Hox (Homeotic box) genes [79]. It has been
reported that the PcG and trxG genes are required for normal
hematopoiesis and mutations of these genes disturb self-
renewal of hematopoietic stem cells (HSCs) in mammals
[80, 81]. Using myeloid leukemic cell lines, Abdel-Wahab
and colleagues reported that that ASXL1 physically interacts
with PRC2, which is likely important for the recruitment
of the PRC2 complex [44]. Therefore, ASXL1 exerts the
repressive regulation of its target genes through H3K27me3
by physically interacting with other PRC2 complex proteins
and regulates epigenetic marks and expression of genes criti-
cal for HSCs/hematopoietic progenitor cells (HPCs) through
interaction with polycomb complex proteins and various
transcription activators and repressors.
ASXL1 interacts with cohesin protein complex
Cohesin is an evolutionarily conserved macromolecu-
lar complex. Cohesin protein complex is composed of
multiple-subunit proteins, including three core subunits
(SMC1A, SMC3, and RAD21), and multiple regulatory
13
F.-C. Yang, J. Agosto‑Peña
proteins (i.e., STAG1, STAG2, PDS5A, PDS5B, NIPBL,
WAPL, and Sororin) [82–84]. Cohesin protein complex
forms a ring-shaped structure to embrace chromatin,
cohere sister chromatids, and prevent their premature sepa-
ration. [85, 86] It has been reported that cohesin complex
proteins are also involved in DNA replication, DNA repair
and dynamic restructuring of chromosomes during cell
division, and gene regulation. [87–92]
Using mouse model system, we reported that ASXL1
interacts with the three core proteins (SMC1A, SMC3,
and RAD21) of cohesin. [93] Deletion of Asxl1 increases
frequency of cells with nuclear bridging and prominent
disrupted sister chromatid separation in myeloid cells. We
also showed that expressing ASXL1 amino acids 401 to
587 (i.e., the cohesin binding region of ASXL1) disrupts
cohesin and endogenous ASXL1 interaction and increases
the frequency of cells with nuclear bridging and prema-
ture sister chromatid separation. Cohesin works together
with CCCTC-binding factor (CTCF) to facilitate long-
range interactions in the genome and gene regulation [94].
The interaction between cohesin complex and CTCF is
required for CTCF-anchored loops and contributes to
the positioning of cohesin at CTCF binding sites [95]. It
has also been reported that cohesin and NIPBL-MAU2
form an active holoenzyme that interacts with DNA either
pseudo-topologically or non-topologically to extrude
genomic interphase DNA into loops [96]. Our ChIP-seq
analysis on HSC/HPCs revealed a significant overlap of
ASXL1/SMC1A/RAD21 binding sites, and Asxl1 loss
resulted in the decreased genomic occupancy of cohesin
and the dysregulation of several ASXL1/SMC1A/RAD21
common target genes, including those that have a role in
apoptosis, proliferation, and myelopoiesis and/or leukemo-
genesis (e.g., Stat3, Cbfb, and Fus) [93]. Our study dem-
onstrates that ASXL1-cohesin interaction is required to
maintain normal cell morphology, chromatid separation,
and gene expression in hematopoietic cells [93]. Recent
studies indicate that cohesin mutations confer a clonal
advantage by altering the chromatin and transcriptional
state of HSC/HPCs, perturbing the balance between self-
renewal and differentiation and that cohesin mutations
could confer resistance to inflammatory signals, increased
self-renewal and clonal expansion [97, 98]. While clinical
studies demonstate significant impact of somatic mutations
of ASXL1 in clonal hematopoiesis and myeloid malig-
nancies [28, 29], the impact of cohesin complex in the
ASXL1 mutation-related clonal hematopoiesis remains to
be determined.
Chromatin looping is known to bring promoters and
enhancers in proximity to activate gene transcription [99].
Whether ASXL1 is also involved in chromatin looping via
cohesin complex proteins to modulate the chromatin archi-
tecture and regulate gene expression remains to be explored.
Epigenetic regulation by ASXL1 in myeloid malignancies
ASXL1 interacts with BAP1 form a PR‑DUB complex
BRCA1 associated protein 1  (BAP1) is a ubiquitin car-
boxyl-terminal hydrolase, and a member of the ubiquitin
C-terminal hydrolase (UCH) family [100]. BAP1 is located
in the nucleus and was originally discovered as a protein
interacting with the RING finger domain of BRCA1 [100].
BAP1 has three critical domains, a UCH N-terminal cata-
lytic domain with enzymatic activity, a central linker region
which modulates binding to host cell factor-1, and a C-termi-
nal ULD domain that potentially coordinates protein–protein
interactions. Loss-of-function BAP1 mutations have recently
been identified in a diverse array of solid tumor types [101,
102]. Harbour and colleagues reported inactivating muta-
tions in BAP1 occur in 47% of uveal melanomas [103].
They found a case with germline BAP1 mutation implying
gene susceptibility, and that BAP1 mutation was strongly
associated with metastasis. These clinical data indicate a
tumor suppressive role of BAP1 in cancer. Surprisingly,
unlike ASXL1 mutations, BAP1 is rarely mutated in myeloid
malignancies.
PRC1 and PRC2 mediate mono-ubiquitination on lysine
119 of histone H2A (H2AK119ub) and methylation on
lysine 27 of histone H3 (H3K27me1/me2/me3), respec-
tively [67]. BAP1 is activated by ASXL1 to deubiquitinate
mono-ubiquitinated H2A at K119 during polycomb protein-
mediated gene repression [60, 104]. ASXL1 and BAP1 form
a Polycomb-repressive deubiquitinase (PR-DUB) complex
that removes monoubiquitin from H2AK119 [57]. ASXL1
lacks a catalytic domain and generally exerts its functions
by assembling chromatin modification complexes and tran-
scription factors. As a result, ASXL1 plays an important
role in epigenetic regulation by activating or repressing the
transcription of genes involved in proliferation and differen-
tiation through its cooperative effect with other chromatin
modifiers to regulate histone modification [44, 57, 77]. As a
major component of PR-DUB complex, ASXL1 plays piv-
otal roles in controlling the levels of H2AK119ub and sub-
sequent gene regulation [57, 105]. ASXL1 binds to BAP1
via the DEUBAD domain, to assemble a PR-DUB complex,
which removes ubiquitin from H2AK119Ub [57–60]. Recent
biochemical characterization of BAP1 complexes suggests
that BAP1 forms two mutually exclusive complexes with
either ASXL1 or ASXL2 [58, 59], and both complexes can
stimulate H2AK119 deubiquitination in vitro. [60]
Dey and colleagues reported that conditional deletion of
Bap1 results in anemia, splenomegaly, leukocytosis, and
progenitor expansion, with decreased HoxA gene expres-
sion [106, 107]. Interestingly, deletion of Asxl1 or Asxl2
increases HoxA gene expression due to chromatin altera-
tions. Overexpression of truncated forms of ASXL1, but not
full-length ASXL1, in combination with overexpression of
BAP1 resulted in clear depletion of global H2AK119ub as
well as a striking reduction of H3K27me3 [104]. Further
studies showed that BAP1 is required for the mutant ASXL1
in the leukemogenesis [108], and deleting one allele of Bap1
delay the truncated ASXL1-driven myeloid malignancies
in vivo [109]. These data suggest that BAP1 has multifac-
eted functions in malignant hematopoiesis. It is unclear how
much redundancy in function occurs between ASXL1 and
ASXL2 in their cooperative effect with BAP1.
ASXL1 truncations are shown to confer an enhanced
activity on the ASXL1–BAP1 DUB complex [104], high-
lighting the significance of the ASXL1-BAP1 complex in
normal biological processes and cancer progression. We
hypothesize that the A­ SXL1aa1−587 truncation mutant pro-
motes myeloid malignancies by enhancing BAP1 DUB
activity. To test this hypothesis, we examined whether
deleting one Bap1 allele can delay or even eradicate
­ASXL1aa1−587-driven myeloid malignancies in vivo. We
generated Mx1Cre;Bap1f/+;Asxl1Y588XTg mice. PI:pC was
injected into 7  month-old Asxl1 Y588XTg (disease estab-
lished) mice to induce a single Bap1 allele inactivation in
the hematopoietic compartment. We analyzed the hemat-
opoietic phenotypes at 3 months after pI:pC injection. Bap1
hemizygous deletion in Asxl1Y588XTg mice is sufficient to
prevent the A ­ SXL1aa1−587-driven HSC/HPC dysregulation
and myeloid malignancy. These data indicate that ASXL1
truncation mutations confer gain-of-function in the patho-
genesis of myeloid malignancies by increasing BAP1 DUB
activity as reducing BAP1 activity ameliorates the abnormal
hematopoietic phenotypes in Asxl1Y588XTg mice.
ASXL1 interacts with RNAPII family members (Pol II)
ASXL1 was reported to bind to PRC2 complex and regu-
late tri-methylation of histone H3 lysine 27 (H3K27me3)
in myeloid cells [45, 46]. To determine the putative impact
of ASXL1-mediated H3K27me3 on the cell fates of bone
marrow stromal cells (BMSCs), we performed H3K27me3
and RNAPII ChIP-seq using WT and Asxl1−/− BMSCs
[110]. Surprisingly, we didn’t observe a strong associa-
tion between ASXL1 binding and H3K27me3 enrichment
on the genome of BMSCs. There is a minimal correlation
between H3K27me3 enrichment and RNAPII occupancy.
These results suggest ASXL1 regulates gene expression in
an H3K27me3-independent manner in BMSCs. To exam-
ine whether ASXL1 and RNAPII co-localize at the same
genomic regions, we performed integrative analyses to assess
the genome-wide distribution in BMSCs. The results showed
a significantly positive correlation between ASXL1 binding
and RNAPII occupancy where ASXL1 promoter binding
was highly overlapped with the RNAPII loading. Analyses
of the coverage between ASXL1 and RNAPII bound genes
showed a high degree (88.51%) of the overlay, suggesting
a role for ASXL1 in gene regulation. Biochemistry studies
13
 F.-C. Yang, J. Agosto‑Peña

verified that ASXL1 interacts with RNAPII family mem-
bers (POLR2A, POLR2B, and POLR2C) in the nuclei of
BMSCs. We further found that loss of Asxl1 alters the tar-
get gene expression by deregulating RNAPII transcriptional
activity in the nuclei of BMSCs. These studies demonstrate
multifaceted functions of ASXL1 in gene regulation by
assembling epigenetic regulators and transcription factors
at specific gene loci. These ASXL1 interacting proteins are
summarized in Fig. 1.
ASXL1 interacts with other nuclear proteins
Recent studies demonstrate a function of ASXL1 in the
modulation of H3K4 methylation, an active histone mark on
gene regulation [77]. Although phylogenetic analyses sug-
gest that the PHD domain of ASXL1 may bind to H3K4me3
[111], the exact function of the PHD domain of ASXL1
remains unclear. The role of ASXL1 in the connection of
promoters and enhancers remains to be explored.
Cooperative leukemogenic effect of ASXL1
alteration and co‑occurring somatic
alterations
ASXL1 mutation co‑occurs with RAS pathway related
gene mutations
Co-mutations of key genes are often in patients with mye-
loid malignancies. ASXL1 is known to co-occur with other
gene mutations. The matched chronic and acute stages have
shown that additional alterations associate with disease pro-
gression. Studies with NGS provide evidence that multiple
mutations can be identified in single cells of MDS patients.
These results imply a linear evolution of the disease and the
existence of a dominant clone. Integrated genomic approach
identified potential cooperating events in AML [112, 113],
as Asxl1 mutations frequently co-occur with mutations
leading to hyperactivation of Ras in myeloid malignancies
such as RAS, PTPN11, and NF1 mutations [4, 7, 114, 115].
Inactivating mutations of NF1 (neurofibromatosis type I)
are common genetic events in JMML and AML [116]. The
neurofibromin 1 gene (NF1) encodes neurofibromin, a RAS
GTPase activating protein, that when mutated, results in
hyperactive RAS signaling that is pro-leukemogenic [117].
NF1 alterations in signaling molecules can be grouped in

ASXL1 structure
238 390
DEUBAD
1 86 241 369 523 618 1099 1123 1506 1537
ASXL1-WT
ASXN ASXH NLS ASXM1 ASXM2 PHD
(1541aa)

238 390

BAP1
401 587
Not specified regions known:
1 248 Cohesin
(SMC1A,
OGT POLR2A
SMC3,
Rad21) POLR2B
N terminal
POLR2C
EZH2

1 86 241 369 523 618 646

ASXL1-MT
(range from ASXN ASXH ASXM1
~635-646aa)

1 200

BRD4

Fig. 1  Summary of wild type and mutant ASXL1 protein interactions

13
Epigenetic regulation by ASXL1 in myeloid malignancies
two major categories, a first one that is found in MPNs and
affects oncogenic tyrosine kinases (ABL1, JAK2, FGFR1,
PDGFRs) and the downstream JAK-STAT and/or PI3-kinase
pathways, and a second one that is mutated in CMML and
affect the RAS-MAP kinase pathway (RAS, PTPN11, NF1).
Expression of NRasG12D in combination with mASXL1
knockdown accelerated myeloproliferation and impaired sur-
vival compared with mice transplanted with empty vector
expressing cells in vivo, demonstrating that ASXL1 loss,
in combination with co-occurring oncogenic NRasG12D,
can lead to hematopoietic transformation and increased self-
renewal. [44]
In a cohort of 138 patients with myeloid malignancies
harboring ASXL1 mutations, we examined the gene muta-
tional profiles of these patients based on targeted sequenc-
ing of 67 frequently mutated genes in myeloid malignan-
cies and identified 35 patients with gene mutations involving
the RAS/MAPK signaling pathway, including NF1, NRAS,
KRAS, PTPN11 or CBL in these ASXL1-mutated patients
[118]. Malignancies in NF1 result from a combination of
ubiquitous NF1 heterozygosity and somatic loss of the
residual NF1 allele (i.e. loss of heterozygosity) [119, 120].
Interestingly, we did not observe loss of heterozygosity
in both ASXL1 and RAS pathway genes in these patients.
Of note, the incidence of AML was significantly higher in
patients with co-mutations of ASXL1 and RAS pathway gene
mutations than the cases without RAS pathway mutations.
According to the revised International Prognostic Scoring
System (IPSS-R) [121], the ASXL1 mutated MDS patients
with RAS pathway gene mutations had a higher frequency
of very high-risk MDS compared to patients without a RAS
pathway gene mutation. These data demonstrate that con-
comitant mutations of ASXL1 and RAS pathway genes are
likely associated with myeloid malignancies with worse
prognosis.
To determine the functional significance of co-mutations
of ASXL1 and NF1 in the disease progression of myeloid
malignancies, we intercrossed Asxl1 heterozygous (Asxl1±)
mice with Nf1 ± mice and generated Asxl1 ±;Nf1 ± mice
[118]. The survival rate of Asxl1±;Nf1± mice was signifi-
cantly lower than those of the other 3 genotypes of mice.
Examination of peripheral blood parameters showed that
a subset of Asxl1±;Nf1± mice developed profound anemia
and thrombocytopenia as compared to age-matched WT
mice. In contrast, the survival of Asxl1± mice was 83% up
to 600 days of age and the deceased Asxl1± mice died of
myeloid malignancies, such as MDS or MDS/MPN, which is
consistent with our previous report. A subset of Asxl1±;Nf1±
mice developed myeloid leukemia. Morphologic analysis of
cytospins prepared from the BM cells revealed an accumu-
lation of blastic cells in Asxl1±;Nf1± mice [118]. Necropsy
of the deceased/moribund Asxl1±;Nf1± mice demonstrated
hepatosplenomegaly and pale foot pads, indicating cachexia.
The histological analyses of BM, spleen, and liver sections
of Asxl1±;Nf1± mice showed disrupted architecture with
significant myeloid cell infiltration. The mouse models of
Asxl1 and Nf1 provide scientific evidence for the cooperative
effect of key driver mutations in disease progression. RNA-
seq analyses on Asxl1±;Nf1± HSC/HPCs revealed aberrant
transcriptional activation of multiple pathways critical for
leukemogenesis, such as MYC, NRAS, and BRD4 [47],
indicating a gain-of-function of the alterations of Asxl1 and
Nf1 in epigenetic regulation. Significantly, pharmacologi-
cal inhibition of both BET bromodomain and the MAPK
pathway prevents leukemia initiation and inhibits disease
progression [118]. This study provides a novel therapeutic
strategy for the treatment of myeloid malignancy patients
with ASXL1 and RAS pathway gene mutations.
ASXL1 alteration cooperates with JAK2V617F
to accelerate myelofibrosis
Myeloproliferative neoplasms (MPNs) are clonal hemat-
opoietic stem cell disorders. MPNs are characterized by
aberrant hematopoietic proliferation of one or more hemat-
opoietic cell lineages with increased risks of myelofibrosis
(MF) progression and leukemic transformation. [122, 123]
Somatic mutation of JAK2V617F is considered to be the
most notable landmark in the diagnosis of classic Philadel-
phia chromosome-negative MPNs and is present in > 95% of
polycythemia vera patients and in ~ 50% of essential throm-
bocythemia and primary myelofibrosis [124]. JAK2V617F
activates pro-oncogenic signaling via diverse STAT-depend-
ent and STAT-independent pathways [125]. JAK2 mutations
are associated with age-related clonal hematopoiesis [29].
Sidon et al. reported that JAK2V617F mutation was detected
in 10% of blood samples from healthy volunteers [126],
underscoring the necessity of additional genetic alterations
for progression to an MPN.
ASXL1 mutations co-occur with JAK2 mutations in patients
with myeloid malignancies [127, 128]. We reported that con-
comitent mutations of ASXL1 and JAK2 results in decreased
levels of hemoglobin (Hb), increased white blood cell (WBC)
count, increased platelet (PLT) count, palpable splenomeg-
aly, and clonal abnormal karyotypes when compared with
polycythemia vera (PV) patients with JAK2V617F mutation
only in a cohort of 95 PV patients with JAK2V617F muta-
tion [129]. In this cohort, we observed a higher proportion
of ASXL1 mutations in post-PV myelofibrosis (PPMF) (26%)
than in PV patients (4%). PV patients with co-mutations of
Asxl1 and JAK2V617F had a poor MF-free survival. Our study
demonstrated that JAK2V617F positive PV patients harbor-
ing mutated ASXL1 exhibited poor myelofibrosis-free survival,
and that mutated ASXL1 was present in 25% of post-PV mye-
lofibrosis cases compared with only 4% of PV cases without
13

myelofibrosis, evidence of a worse prognosis in patients har-
boring ASXL1 mutations in addition to JAK2V617F.
To further study the impact of Asxl1 alteration on dis-
ease progression in JAK2V617F-mediated MPNs and
JAK2V617F-mutant hematopoietic stem and progenitor cell
(HSC/HPC) function, we assessed hematopoietic pheno-
types in Asxl1±;JAK2V617F mice in vivo. [129] We found
that JAK2V617F;Asxl1± mice had significantly shorter mean
survival rates (~ 60%) than JAK2V617F or Asxl1± mice. We
found that mice with heterozygous loss of Asxl1 (Asxl1±) and
JAK2V617F expression accelerated the development of bone
marrow fibrosis and some of JAK2V617F;Asxl1± mice pro-
gressed/transformed to secondary acute myeloid leukemia
(sAML) [124]. These results indicate that heterozygous dele-
tion of Asxl1 promoted MF in JAK2V617F-driven MPN in mice,
and Asxl1 alteration cooperates with JAK2V617F mutation to
accelerate myeloid leukemic transformation.
Co‑mutation of ASXL1 and SRSF2 in myeloid
malignanices
Concomitant mutations of ASXL1 and SRSF2 are common in
MDS and CMML and are frequently found in patients with
s-AML [16, 130–134]. AML with ASXL1 and SRSF2 muta-
tions has been associated with a dismal prognosis, similar to
AML with high-risk cytogenetic features [133, 134]. John-
son and colleagues reported that ASXL1/SRSF2 co-mutated
AML was significantly more likely to express 2 or more
monocytic markers, more likely to express aberrant CD2 and
CD56, and significantly less likely to express the myeloblast
markers CD34 and CD117. These data suggest that ASXL1/
SRSF2 co-mutation results in a biased monocytic differentia-
tion [135]. ASXL1/SRSF2 co-mutated AML frequently arises
secondarily in patients with antecedent myeloid malignancy,
and even when these mutations apparently occur “de novo,”
patients may have unrecognized peripheral monocytosis prior
to leukemic onset. Their findings point to a possibility that
this subset of AML may often arise as a secondary AML
from an occult CMML-like MDS or MDS/MPN even in the
absence of a known prior myeloid neoplasm. Richardson and
colleagues reported that ASXL1mutSRSF2mut AML, in contrast
to ASXL1mutSRSF2wt AML, shares a mutational profile and
immunophenotype with CMML suggesting that this genomic
profile may identify patients with secondary AML (s-AML)
from preexisting CMML [132]. Regardless of the clinical sig-
nificance of the comutation of ASXL1 and SRSF2, the underly-
ing mechanisms remains to be investigated.
13
F.-C. Yang, J. Agosto‑Peña
Mutual exclusive gene mutations
between ASXL1 and other genes
Despite the high frequencies of several gene mutations
seen in MDS, MPN, AML and other blood malignancies,
some of these common mutations are mutually exclu-
sive. In the case of ASXL1, it has been shown to be mutu-
ally exclusive with some common and other less common
mutations, such as DNMT3A, NPM1, WT1 and ASXL2.
[12, 21, 136–139]
As mentioned before, ASXL1 is part of the ASXL fam-
ily of proteins, along with ASXL2 and ASXL3. Interest-
ingly, ASXL1 and ASXL2 mutations are mutually exclu-
sive [21]. Given the high homology on several of their
domains and the fact that ASXL1 and ASXL2 mutations
are mutually exclusive with one another, it is likely that
these two genes' mutations share a common mechanism
of transformation in AML patients. These results raise
the possibility that ASXL1 and ASXL2 mutations are both
significant and distinct in AML patients. More studies
about the differences between ASXL1 and ASXL2 are
critically needed to understand these genetically observed
differences, and can probably unravel pathogenic routes
between ASXL1 mutations and ASXL2 mutations. Daou
et al. reported that BAP1 forms two mutually exclusive
complexes with the transcriptional regulators ASXL1 and
ASXL2, which are necessary for maintaining proper pro-
tein levels of BAP1 [59]. They also found that the interac-
tion between ASXL1/2 and BAP1 requires ASXM, which
is necessary for promoting BAP1-mediated deubiquitina-
tion of its physiological substrate H2AK119ub. The phys-
ical interaction between ASXL1/2 and BAP1 and DUB
activity are required for proper coordination of cell cycle
progression. In contrast, Park and colleagues found that
while both ASXL1 and ASXL2 interact with peroxisome
proliferator-activated receptor γ, they play opposite roles
in adipogenesis via reciprocal regulation of peroxisome
proliferator-activated receptor γ. [140]
Carbuccia and colleagues reported that ASXL1 and NPM1
mutations are mutually exclusive on a study of 63 AML
patients [138]. The mutual exclusive mutations of ASXL1
and NPM1 have been also repored by Pratcorona [12, 136],
which implies that this is a common phenomenom seen in
different cohorts of ASXL1 mutated patients. ASXL1 and
NPM1 may function through a similar pathway in regulat-
ing hematopoietic cell functions. The authors reported that
9p INK4A-ARF-INK4B loci are regulated by both the poly-
comb complexes and the histone chaperone NPM1 [138].
These loci are key for the regulation of hematopoietic func-
tions, such as senescence and/or self-renewal.
In a cohort of CN-AML patients, Metzeler et  al.
reported that ASXL1  mutations were almost mutually
Epigenetic regulation by ASXL1 in myeloid malignancies
exclusive with  NPM1 mutations and  FLT3-ITD [141].
Chou and colleagues also reported an inverse association
of ASXL1 mutation with WT1 mutation and FLT3-ITD
[12]. Supprisingly, ASXL2, another member of ASXL fam-
ily, and FLT3-ITD mutations had a significant co-occur-
rence, completely opposite from the mutually exclusive
phenotype seen with ASXL1 and FLT3-ITD mutations
[136]. However, the mechanism underlying the mutually
exclusive mutations between ASXL1 and other genes in
leukemia remains to be further studied.
Challenges in therapy for ASXL1 mutated
myeloid malignancies
ASXL1 mutation was an independent risk factor associated
with worse event-free survival in chronic phase CML [142].
Although successful therapeutic strategies are developed
for several types of myeloid leukemia, including imatinib
in CML [143, 144], all trans retinoic acid (ATRA) in
almost all the cases of acute promyelocytic leukemia (APL)
[145, 146], JAK2 inhibitors in myeloproliferative diseases
[147–149], effective treatment are desperately needed for
most of the cases of myeloid malignancies, especially mye-
loid malignancies with ASXL1 mutations. ASXL1 muta-
tion frequency was significantly higher in TKI-resistant
patients. A study by Kim and colleagues revealed that
mutations in epigenetic modification pathway genes such
as DNMT3A and ASXL1 could be driver mutations in TKI
resistance-related progression of CML outside ABL1 KD
mutations [150]. A study of comparative genomic land-
scape of AML by Tyner and colleagues with a large cohort
of patients samples demonstrated that mutations in several
genes, most notably ASXL1 or TP53, were associated with a
broad pattern of drug resistance. [151]
Immunotherapy is one of the promising novel thera-
peutics targeting cancers [152]. Chimeric antigen recep-
tor (CAR) T cells using engineered T cells extracted from
patients are able to target a specific antigen on the surface
of malignant cells [153]. Using humanized mouse model
system, Kenderian and colleagues developed CART cells
to target CD33 (CART33) using the anti-CD33 single
chain variable fragment used in gemtuzumab ozogamicin
(clone My96) and tested the activity and toxicity of these
cells [154]. They found that CART33 exhibited significant
effector functions in vitro and resulted in eradication of
leukemia and prolonged survival in AML xenografts. How-
ever, CART33 also resulted in human lineage cytopenias
and reduction of myeloid progenitors in xenograft models
of hematopoietic toxicity, suggesting that CD33-specific
CART cells would have unacceptable toxicity. The authors
then designed a transiently expressed mRNA anti-CD33
CAR and found that transient expression of anti-CD33 CAR
by RNA modification has the potential to be used in patients
to avoid long-term myelosuppression.
Developing CAR T cells for AML has been hindered
by a paucity of targets expressing on the blastic cells, but
not on hematopoietic progenitor cells. Evolutionarily con-
served glucose-regulated-protein 78 (GRP78) is a key regu-
lator on the unfolded protein response [155], and residing
in the endoplasmic reticulum makes it a promising target
for AML-redirected CAR T cell therapy, since GRP78 is
translocated to the cell surface upon cell stress, a common
phenotype of malignant cells [156]. Hebbar et al. showed
the feasibility of targeting cell surface GRP78 in AML with
CAR T cells [157]. Hebbar and colleagues designed a panel
of GRP78-specific CARs (GRP78-CARs) using a peptide
that specifically binds to GRP78, and demonstrated that
GRP78 is overexpressed in a variety of AML specimens.
The GRP78-CAR T cell cultures showed minimal fratri-
cide and antigen-dependent T cell differentiation, and the
GRP78-CAR T cell have potent anti-AML activity without
hematopoietic progenitor cell (HPC) toxicity [157]. Further
investigation for developing CAR-T cells in myeloid leuke-
mia patients with ASXL1 mutations is required.
Open questions
While the advances in genomic and epigenetic research have
uncovered a central role for aberrant epigenetic regulation
in the pathogenesis of myeloid malignancies, the molecular
mechanisms underlying the alterations of epigenetic modi-
fiers in myeloid malignancies remain largely unknown. Sev-
eral studies suggest that macrophage driven inflammatory
responses are responsible for the accelerated atherosclerosis
seen in patients with clonal hematopoiesis, ASXL1 muta-
tions induce a clonal advantage of hematopoietic cells and
subsequent CH development has not been elucidated. In
addition, how ASXL1 mutations induce a clonal advantage
of hematopoietic cells and subsequent clonal hematopoie-
sis development has not been elucidated. Despite the asso-
ciation of particular gene mutations observed early in the
course of disease and response to therapy and survival, it
should be underscored that the complex dynamics of clonal
hematopoiesis are highly variable and not necessarily deter-
minative. Close monitoring of clonal hematopoiesis by both
deep sequencing and SNP-array will need to be combined
with clinical evaluation for prognosis and to guide manage-
ment of patients with aplastic anemia. It remains unknown
whether the ASXL1 or ASXL2 PHD domain binds to non-
histone proteins. Although observations in mice demonstrate
dominance of DNMT3A, ASXL1 and other mutations, the
exact mechanism of clonal selection of these mutations in
aplastic anemia is unclear. Finally, given the poor progno-
sis of myeloid malignancy patients with ASXL1 mutations,
13

effort on developing effective treatment becomes more
important.
Acknowledgements  We apologize to the researchers whose work was
not cited due to space limitations. We would also like to acknowledge
the NCI Training Grant (T32 CA148724) given by the Mays Cancer
Center for their financial and professional support of the PhD student
Joel Agosto-Peña.
Funding  NIH/NCI, HL149318, Feng-Chun Yang, R01 HL158081,
Feng-Chun Yang,CA172408, Feng-Chun Yang, T32 CA148724, Joel
Agosto-Peña.
Declarations  org/​10.​1371/​journ​al.​pone.​00430​90 (2012).
Conflict of interest  The authors declare that they have no conflict of
interest.
References
1. Hofmann WK, Koeffler HP. Myelodysplastic syndrome. Annu
Rev Med. 2005;56:1–16. https://​doi.​org/​10.​1146/​annur​ev.​med.​
56.​082103.​104704.
2. Nimer SD. Myelodysplastic syndromes. Blood. 2008;111:4841–
51. https://​doi.​org/​10.​1182/​blood-​2007-​08-​078139.
3. Bejar R, Levine R, Ebert BL. Unraveling the molecular patho-
physiology of myelodysplastic syndromes. J Clin Oncol.
2011;29:504–15. https://​doi.​org/​10.​1200/​JCO.​2010.​31.​1175.
4. Bejar R, et al. Clinical effect of point mutations in myelodysplas-
tic syndromes. N Engl J Med. 2011;364:2496–506. https://​doi.​
org/​10.​1056/​NEJMo​a1013​343.
5. Thol F, et al. Prognostic significance of ASXL1 mutations in
patients with myelodysplastic syndromes. J Clin Oncol: Off J Am
Soc Clin Oncol. 2011;29:2499–506. https://d​ oi.o​ rg/1​ 0.1​ 200/J​ CO.​
2010.​33.​4938.
6. Carbuccia N, et al. Mutations of ASXL1 gene in myeloprolifera-
tive neoplasms. Leukemia. 2009;23:2183–6. https://​doi.​org/​10.​
1038/​leu.​2009.​141.
7. Brecqueville M, et  al. Mutation analysis of ASXL1, CBL,
DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12,
and TET2 in myeloproliferative neoplasms. Genes Chromosomes
Cancer. 2012;51:743–55. https://​doi.​org/​10.​1002/​gcc.​21960.
8. Gelsi-Boyer V, et al. Mutations of polycomb-associated gene
ASXL1 in myelodysplastic syndromes and chronic myelomono-
cytic leukaemia. Br J Haematol. 2009;145:788–800. https://​doi.​
org/​10.​1111/j.​1365-​2141.​2009.​07697.x.
9. Gelsi-Boyer V, et al. ASXL1 mutation is associated with poor
prognosis and acute transformation in chronic myelomonocytic
leukaemia. Br J Haematol. 2010;151:365–75. https://​doi.​org/​10.​
1111/j.​1365-​2141.​2010.​08381.x.
10. Stieglitz E, et al. The genomic landscape of juvenile myelomono-
cytic leukemia. Nat Genet. 2015;47:1326–33. https://​doi.​org/​10.​
1038/​ng.​3400.
11. Caye A, et al. Juvenile myelomonocytic leukemia displays muta-
tions in components of the RAS pathway and the PRC2 network.
Nat Genet. 2015;47:1334–40. https://​doi.​org/​10.​1038/​ng.​3420.
12. Chou WC, et al. Distinct clinical and biological features of de
novo acute myeloid leukemia with additional sex comb-like 1
(ASXL1) mutations. Blood. 2010;116:4086–94. https://​doi.​org/​
10.​1182/​blood-​2010-​05-​283291.
13. Patel JP, et al. Prognostic relevance of integrated genetic profiling
in acute myeloid leukemia. N Engl J Med. 2012;366:1079–89.
https://​doi.​org/​10.​1056/​NEJMo​a1112​304.
13
F.-C. Yang, J. Agosto‑Peña
14. Milne TA, Sinclair DA, Brock HW. The Additional sex combs
gene of drosophila is required for activation and repression of
homeotic loci, and interacts specifically with polycomb and
super sex combs. Mol Gen Genet MGG. 1999;261:753–61.
15. Fisher CL, Berger J, Randazzo F, Brock HW. A human homolog
of additional sex combs, ADDITIONAL SEX COMBS-LIKE
1, maps to chromosome 20q11. Gene. 2003;306:115–26.
16. Gelsi-Boyer V, et al. Mutations in ASXL1 are associated with
poor prognosis across the spectrum of malignant myeloid dis-
eases. J Hematol Oncol. 2012;5:12. https://​doi.​org/​10.​1186/​
1756-​8722-5-​12.
17. Traina, F. et al. Single nucleotide polymorphism array lesions,
TET2, DNMT3A, ASXL1 and CBL mutations are present in
systemic mastocytosis. PLoS One 7, e43090, doi:https://​doi.​
18. Damaj, G. et al. ASXL1 but Not TET2 Mutations Adversely
Impact Overall Survival of Patients Suffering Systemic Mas-
tocytosis with Associated Clonal Hematologic Non-Mast-Cell
Diseases. PLoS One 9, e85362, doi:https://​doi.​org/​10.​1371/​
journ​al.​pone.​00853​62 (2014).
19. Yoshizato T, et  al. Somatic Mutations and Clonal Hemat-
opoiesis in Aplastic Anemia. N Engl J Med. 2015;373:35–47.
https://​doi.​org/​10.​1056/​NEJMo​a1414​799.
20. Prebet T, et al. Concomitant germ-line RUNX1 and acquired
ASXL1 mutations in a T-cell acute lymphoblastic leukemia.
Eur J Haematol. 2013;91:277–9. https://​doi.​org/​10.​1111/​ejh.​
12147.
21. Micol JB, et al. Frequent ASXL2 mutations in acute myeloid
leukemia patients with t(8;21)/RUNX1-RUNX1T1 chromosomal
translocations. Blood. 2014;124:1445–9. https://d​ oi.o​ rg/1​ 0.1​ 182/​
blood-​2014-​04-​571018.
22. Duployez N, et al. Comprehensive mutational profiling of core
binding factor acute myeloid leukemia. Blood. 2016;127:2451–9.
https://​doi.​org/​10.​1182/​blood-​2015-​12-​688705.
23. Inoue, D. et al. Truncation mutants of ASXL1 observed in
myeloid malignancies are expressed at detectable protein levels.
Experimental hematology 44, 172–176 e171, doi:https://​doi.​org/​
10.​1016/j.​exphem.​2015.​11.​011 (2016).
24. Inoue D, et  al. Myelodysplastic syndromes are induced by
histone methylation-altering ASXL1 mutations. J Clin Invest.
2013;123:4627–40. https://​doi.​org/​10.​1172/​JCI70​739.
25. Alvarez Argote, J. & Dasanu, C. A. ASXL1 mutations in myeloid
neoplasms: pathogenetic considerations, impact on clinical out-
comes and survival. Current Medical Research and Opinion 34,
757–763, doi:https://​doi.​org/​10.​1080/​03007​995.​2016.​12768​96
(2018).
26. Alberti MO, et al. Discriminating a common somatic ASXL1
mutation (c.1934dup; p.G646Wfs*12) From artifact in myeloid
malignancies using NGS. Leukemia. 2018;32:1874–8. https://​
doi.​org/​10.​1038/​s41375-​018-​0193-y.
27. Genovese G, et  al. Clonal hematopoiesis and blood-cancer
risk inferred from blood DNA sequence. N Engl J Med.
2014;371:2477–87. https://​doi.​org/​10.​1056/​NEJMo​a1409​405.
28. Xie M, et al. Age-related mutations associated with clonal hemat-
opoietic expansion and malignancies. Nat Med. 2014;20:1472–8.
https://​doi.​org/​10.​1038/​nm.​3733.
29. Jaiswal S, et al. Age-related clonal hematopoiesis associated with
adverse outcomes. N Engl J Med. 2014;371:2488–98. https://d​ oi.​
org/​10.​1056/​NEJMo​a1408​617.
30. Young NS. Current concepts in the pathophysiology and treat-
ment of aplastic anemia. Hematol Am Soc Hematol Educ Pro-
gram. 2013;76–81:2013. https://​doi.​org/​10.​1182/​ashed​ucati​on-​
2013.1.​76.
31. Stanley N, Olson TS, Babushok DV. Recent advances in under-
standing clonal haematopoiesis in aplastic anaemia. Br J Haema-
tol. 2017;177:509–25. https://​doi.​org/​10.​1111/​bjh.​14510.
Epigenetic regulation by ASXL1 in myeloid malignancies
32. Babushok DV, Olson TS, Bessler M. Somatic mutations
and clonal hematopoiesis in aplastic anemia. N Engl J Med.
2015;373:1673. https://​doi.​org/​10.​1056/​NEJMc​15097​03.
33. Babushok DV, et al. Emergence of clonal hematopoiesis in the
majority of patients with acquired aplastic anemia. Cancer Genet.
2015;208:115–28. https://​doi.​org/​10.​1016/j.​cance​rgen.​2015.​01.​
007.
34. Negoro E, et al. Origins of myelodysplastic syndromes after
aplastic anemia. Blood. 2017;130:1953–7. https://​doi.​org/​10.​
1182/​blood-​2017-​02-​767731.
35. Hoischen A, et al. De novo nonsense mutations in ASXL1 cause
Bohring-Opitz syndrome. Nat Genet. 2011;43:729–31. https://​
doi.​org/​10.​1038/​ng.​868.
36. Magini P, et al. Two novel patients with Bohring-Opitz syn-
drome caused by de novo ASXL1 mutations. Am J Med Genet
A. 2012;158A:917–21. https://​doi.​org/​10.​1002/​ajmg.a.​35265.
37. Russell B, et al. Clinical management of patients with ASXL1
mutations and Bohring-Opitz syndrome, emphasizing the
need for Wilms tumor surveillance. Am J Med Genet A.
2015;167A:2122–31. https://​doi.​org/​10.​1002/​ajmg.a.​37131.
38. Hastings R, et al. Bohring-Opitz (Oberklaid-Danks) syndrome:
clinical study, review of the literature, and discussion of possible
pathogenesis. Eur J Hum Genet. 2011;19:513–9. https://​doi.​org/​
10.​1038/​ejhg.​2010.​234.
39. Oberklaid F, Danks DM. The opitz trigonocephaly syndrome. a
case report. Am J Dis Child. 1975;129:1348–9.
40. Bohring A, Oudesluijs GG, Grange DK, Zampino G, Thierry
P. New cases of Bohring-Opitz syndrome, update, and critical
review of the literature. Am J Med Genet A. 2006;140:1257–63.
https://​doi.​org/​10.​1002/​ajmg.a.​31265.
41. Katoh M. Functional and cancer genomics of ASXL family mem-
bers. Br J Cancer. 2013;109:299–306. https://​doi.​org/​10.​1038/​
bjc.​2013.​281.
42. Baskind HA, et al. Functional conservation of Asxl2, a murine
homolog for the drosophila enhancer of trithorax and polycomb
group gene Asx. PLoS One. 2009;4: e4750. https://​doi.​org/​10.​
1371/​journ​al.​pone.​00047​50.
43. Fisher CL, et al. Additional sex combs-like 1 belongs to the
enhancer of trithorax and polycomb group and genetically inter-
acts with Cbx2 in mice. Dev Biol. 2010;337:9–15. https://​doi.​
org/​10.​1016/j.​ydbio.​2009.​10.​004.
44. Abdel-Wahab O, et al. ASXL1 mutations promote myeloid trans-
formation through loss of PRC2-Mediated gene repression. Can-
cer Cell. 2012;22:180–93. https://​doi.​org/​10.​1016/j.​ccr.​2012.​06.​
032.
45. Abdel-Wahab O, et al. Deletion of Asxl1 results in myelodys-
plasia and severe developmental defects in vivo. J Exp Med.
2013;210:2641–59. https://​doi.​org/​10.​1084/​jem.​20131​141.
46. Wang J, et al. Loss of Asxl1 leads to myelodysplastic syndrome-
like disease in mice. Blood. 2014;123:541–53. https://​doi.​org/​
10.​1182/​blood-​2013-​05-​500272.
47. Yang H, et al. Gain of function of ASXL1 truncating protein in
the pathogenesis of myeloid malignancies. Blood. 2018;131:328–
41. https://​doi.​org/​10.​1182/​blood-​2017-​06-​789669.
48. Fisher CL, et al. Loss-of-function Additional sex combs like 1
mutations disrupt hematopoiesis but do not cause severe myelo-
dysplasia or leukemia. Blood. 2010;115:38–46. https://​doi.​org/​
10.​1182/​blood-​2009-​07-​230698.
49. Hsu YC, et al. The distinct biological implications of Asxl1
mutation and its roles in leukemogenesis revealed by a knock-in
mouse model. J Hematol Oncol. 2017;10:139. https://d​ oi.o​ rg/1​ 0.​
1186/​s13045-​017-​0508-x.
50. Nagase R, et al. Expression of mutant Asxl1 perturbs hemat-
opoiesis and promotes susceptibility to leukemic transformation.
J Exp Med. 2018;215:1729–47. https://​doi.​org/​10.​1084/​jem.​
20171​151.
51. Katoh M, Katoh M. Identification and characterization of
ASXL2 gene in silico. Int J Oncol. 2003;23:845–50.
52. Katoh M, Katoh M. Identification and characterization of
ASXL3 gene in silico. Int J Oncol. 2004;24:1617–22.
53. Schuettengruber B, Cavalli G. Recruitment of polycomb group
complexes and their role in the dynamic regulation of cell fate
choice. Development. 2009;136:3531–42. https://​doi.​org/​10.​
1242/​dev.​033902.
54. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cav-
alli G. Genome regulation by polycomb and trithorax proteins.
Cell. 2007;128:735–45. https://​doi.​org/​10.​1016/j.​cell.​2007.​02.​
009.
55. Schuettengruber B, Martinez AM, Iovino N, Cavalli G. Trithorax
group proteins: switching genes on and keeping them active. Nat
Rev Mol Cell Biol. 2011;12:799–814. https://​doi.​org/​10.​1038/​
nrm32​30.
56. Sanchez-Pulido L, Kong L, Ponting CP. A common ancestry for
BAP1 and Uch37 regulators. Bioinformatics. 2012;28:1953–6.
https://​doi.​org/​10.​1093/​bioin​forma​tics/​bts319.
57. Scheuermann JC, et  al. Histone H2A deubiquitinase activ-
ity of the polycomb repressive complex PR-DUB. Nature.
2010;465:243–7. https://​doi.​org/​10.​1038/​natur​e08966.
58. Peng H, et al. Familial and somatic BAP1 mutations inacti-
vate ASXL1/2-mediated allosteric regulation of BAP1 deu-
biquitinase by targeting multiple independent domains. Can
Res. 2018;78:1200–13. https://​doi.​org/​10.​1158/​0008-​5472.​
CAN-​17-​2876.
59. Daou S, et al. The BAP1/ASXL2 histone H2A deubiquitinase
complex regulates cell proliferation and is disrupted in cancer.
J Biol Chem. 2015;290:28643–63. https://​doi.​org/​10.​1074/​jbc.​
M115.​661553.
60. Sahtoe DD, van Dijk WJ, Ekkebus R, Ovaa H, Sixma TK. BAP1/
ASXL1 recruitment and activation for H2A deubiquitination. Nat
Commun. 2016;7:10292. https://d​ oi.o​ rg/1​ 0.1​ 038/n​ comms​ 10292.
61. Di Carlo V, Mocavini I, Di Croce L. Polycomb complexes in nor-
mal and malignant hematopoiesis. J Cell Biol. 2019;218:55–69.
https://​doi.​org/​10.​1083/​jcb.​20180​8028.
62. Hu D, Shilatifard A. Epigenetics of hematopoiesis and hemato-
logical malignancies. Genes Dev. 2016;30:2021–41. https://​doi.​
org/​10.​1101/​gad.​284109.​116.
63. Ernst T, et al. Inactivating mutations of the histone methyltrans-
ferase gene EZH2 in myeloid disorders. Nat Genet. 2010;42:722–
6. https://​doi.​org/​10.​1038/​ng.​621.
64. Cao R, Zhang Y. SUZ12 is required for both the histone methyl-
transferase activity and the silencing function of the EED-EZH2
complex. Mol Cell. 2004;15:57–67. https://​doi.​org/​10.​1016/j.​
molcel.​2004.​06.​020.
65. Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K.
Suz12 is essential for mouse development and for EZH2 histone
methyltransferase activity. EMBO J. 2004;23:4061–71. https://​
doi.​org/​10.​1038/​sj.​emboj.​76004​02.
66. Montgomery ND, et al. The murine polycomb group protein Eed
is required for global histone H3 lysine-27 methylation. Curr
Biol: CB. 2005;15:942–7. https://d​ oi.o​ rg/1​ 0.1​ 016/j.c​ ub.2​ 005.0​ 4.​
051.
67. Margueron R, Reinberg D. The polycomb complex PRC2 and its
mark in life. Nature. 2011;469:343–9. https://​doi.​org/​10.​1038/​
natur​e09784.
68. Bachmann IM, et al. EZH2 expression is associated with high
proliferation rate and aggressive tumor subgroups in cutane-
ous melanoma and cancers of the endometrium, prostate, and
breast. J Clin Oncol. 2006;24:268–73. https://​doi.​org/​10.​1200/​
JCO.​2005.​01.​5180.
69. Nikoloski G, et al. Somatic mutations of the histone methyl-
transferase gene EZH2 in myelodysplastic syndromes. Nat Genet.
2010;42:665–7. https://​doi.​org/​10.​1038/​ng.​620.
13

70. Bejar R, et al. Validation of a prognostic model and the impact
of mutations in patients with lower-risk myelodysplastic syn-
dromes. J Clin Oncol. 2012;30:3376–82. https://​doi.​org/​10.​
1200/​JCO.​2011.​40.​7379.
71. Simon C, et al. A key role for EZH2 and associated genes in
mouse and human adult T-cell acute leukemia. Genes Dev.
2012;26:651–6. https://​doi.​org/​10.​1101/​gad.​186411.​111.
72. Souroullas GP, et al. An oncogenic Ezh2 mutation induces
tumors through global redistribution of histone 3 lysine 27
trimethylation. Nat Med. 2016;22:632–40. https://​doi.​org/​10.​
1038/​nm.​4092.
73. Mochizuki-Kashio M, et al. Ezh2 loss in hematopoietic stem
cells predisposes mice to develop heterogeneous malignan-
cies in an Ezh1-dependent manner. Blood. 2015;126:1172–83.
https://​doi.​org/​10.​1182/​blood-​2015-​03-​634428.
74. Sashida G, et al. The loss of Ezh2 drives the pathogenesis of
myelofibrosis and sensitizes tumor-initiating cells to bromo-
domain inhibition. J Exp Med. 2016;213:1459–77. https://​doi.​
org/​10.​1084/​jem.​20151​121.
75. Shimizu T, et al. Loss of Ezh2 synergizes with JAK2-V617F in
initiating myeloproliferative neoplasms and promoting myelofi-
brosis. J Exp Med. 2016;213:1479–96. https://​doi.​org/​10.​1084/​
jem.​20151​136.
76. Yang Y, Akada H, Nath D, Hutchison RE, Mohi G. Loss
of Ezh2 cooperates with Jak2V617F in the development of
myelofibrosis in a mouse model of myeloproliferative neo-
plasm. Blood. 2016;127:3410–23. https://​d oi.​o rg/​1 0.​1 182/​
blood-​2015-​11-​679431.
77. Inoue D, et al. A novel ASXL1-OGT axis plays roles in H3K4
methylation and tumor suppression in myeloid malignan-
cies. Leukemia. 2018;32:1327–37. https://​d oi.​o rg/​1 0.​1 038/​
s41375-​018-​0083-3.
78. LaJeunesse D, Shearn A. E(z): a polycomb group gene or a
trithorax group gene? Development. 1996;122:2189–97.
79. Simon JA, Tamkun JW. Programming off and on states in
chromatin: mechanisms of Polycomb and trithorax group com-
plexes. Curr Opin Genet Dev. 2002;12:210–8.
80. Sauvageau M, Sauvageau G. Polycomb group genes: keeping
stem cell activity in balance. PLoS Biol. 2008;6: e113. https://​
doi.​org/​10.​1371/​journ​al.​pbio.​00601​13.
81. Vernimmen D, et  al. Polycomb eviction as a new distant
enhancer function. Genes Dev. 2011;25:1583–8. https://​doi.​
org/​10.​1101/​gad.​16985​411.
82. Kon A, et al. Recurrent mutations in multiple components
of the cohesin complex in myeloid neoplasms. Nat Genet.
2013;45:1232–7. https://​doi.​org/​10.​1038/​ng.​2731.
83. Seitan VC, et al. Cohesin-based chromatin interactions enable
regulated gene expression within preexisting architectural com-
partments. Genome Res. 2013;23:2066–77. https://​doi.​org/​10.​
1101/​gr.​161620.​113.
84. Seitan VC, Merkenschlager M. Cohesin and chromatin organi-
sation. Curr Opin Genet Dev. 2012;22:93–100. https://​doi.​org/​
10.​1016/j.​gde.​2011.​11.​003.
85. Onn I, Heidinger-Pauli JM, Guacci V, Unal E, Koshland DE.
Sister chromatid cohesion: a simple concept with a complex
reality. Annu Rev Cell Dev Biol. 2008;24:105–29. https://​doi.​
org/​10.​1146/​annur​ev.​cellb​io.​24.​110707.​175350.
86. Losada A. The regulation of sister chromatid cohesion. Bio-
chim Biophys Acta. 2008;1786:41–8. https://​doi.​org/​10.​1016/j.​
bbcan.​2008.​04.​003.
87. Strom L, et al. Postreplicative formation of cohesion is required
for repair and induced by a single DNA break. Science.
2007;317:242–5. https://​doi.​org/​10.​1126/​scien​ce.​11406​49.
88. Watrin E, Peters JM. The cohesin complex is required for the
DNA damage-induced G2/M checkpoint in mammalian cells.
13
F.-C. Yang, J. Agosto‑Peña
EMBO J. 2009;28:2625–35. https://​d oi.​o rg/​1 0.​1 038/​e mboj.​
2009.​202.
89. Horsfield JA, et  al. Cohesin-dependent regulation of Runx
genes. Development. 2007;134:2639–49. https://​doi.​org/​10.​
1242/​dev.​002485.
90. Parelho V, et al. Cohesins functionally associate with CTCF on
mammalian chromosome arms. Cell. 2008;132:422–33. https://​
doi.​org/​10.​1016/j.​cell.​2008.​01.​011.
91. Wendt KS, et al. Cohesin mediates transcriptional insulation
by CCCTC-binding factor. Nature. 2008;451:796–801. https://​
doi.​org/​10.​1038/​natur​e06634.
92. Nasmyth K, Haering CH. Cohesin: its roles and mechanisms.
Annu Rev Genet. 2009;43:525–58. https://​d oi.​o rg/​1 0.​1 146/​
annur​ev-​genet-​102108-​134233.
93. Li Z, et  al. ASXL1 interacts with the cohesin complex to
maintain chromatid separation and gene expression for normal
hematopoiesis. Sci Adv. 2017;3: e1601602. https://​doi.​org/​10.​
1126/​sciadv.​16016​02.
94. Zuin J, et al. Cohesin and CTCF differentially affect chromatin
architecture and gene expression in human cells. Proc Natl
Acad Sci U S A. 2014;111:996–1001. https://​doi.​org/​10.​1073/​
pnas.​13177​88111.
95. Li Y, et al. The structural basis for cohesin-CTCF-anchored
loops. Nature. 2020;578:472–6. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 038/​
s41586-​019-​1910-z.
96. Davidson IF, et al. DNA loop extrusion by human cohesin.
Science. 2019;366:1338–45. https://​doi.​org/​10.​1126/​scien​ce.​
aaz34​18.
97. Cuartero S, et al. Control of inducible gene expression links
cohesin to hematopoietic progenitor self-renewal and differ-
entiation. Nat Immunol. 2018;19:932–41. https://​doi.​org/​10.​
1038/​s41590-​018-​0184-1.
98. Cuartero S, Innes AJ, Merkenschlager M. Towards a better
understanding of cohesin mutations in AML. Front Oncol.
2019;9:867. https://​doi.​org/​10.​3389/​fonc.​2019.​00867.
99. Hnisz D, Day DS, Young RA. Insulated neighborhoods: struc-
tural and functional units of mammalian gene control. Cell.
2016;167:1188–200. https://​d oi.​o rg/​1 0.​1 016/j.​c ell.​2 016.​1 0.​
024.
100. Jensen DE, et al. BAP1: a novel ubiquitin hydrolase which
binds to the BRCA1 RING finger and enhances BRCA1-medi-
ated cell growth suppression. Oncogene. 1998;16:1097–112.
101. Bott M, et al. The nuclear deubiquitinase BAP1 is commonly
inactivated by somatic mutations and 3p21.1 losses in malig-
nant pleural mesothelioma. Nat Genet. 2011;43:668–72.
https://​doi.​org/​10.​1038/​ng.​855.
102. Testa JR, et al. Germline BAP1 mutations predispose to malig-
nant mesothelioma. Nat Genet. 2011;43:1022–5. https://​doi.​
org/​10.​1038/​ng.​912.
103. Harbour JW, et al. Frequent mutation of BAP1 in metastasizing
uveal melanomas. Science. 2010;330:1410–3. https://​doi.​org/​
10.​1126/​scien​ce.​11944​72.
104. Balasubramani A, et al. Cancer-associated ASXL1 mutations
may act as gain-of-function mutations of the ASXL1-BAP1
complex. Nat Commun. 2015;6:7307. https://​doi.​org/​10.​1038/​
ncomm​s8307.
105. Daou S, et  al. Monoubiquitination of ASXLs controls the
deubiquitinase activity of the tumor suppressor BAP1.
Nat Commun. 2018;9:4385. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 038/​
s41467-​018-​06854-2.
106. Dey A, et al. Loss of the tumor suppressor BAP1 causes myeloid
transformation. Science. 2012;337:1541–6. https://​doi.​org/​10.​
1126/​scien​ce.​12217​11.
107. LaFave LM, et al. Loss of BAP1 function leads to EZH2-depend-
ent transformation. Nat Med. 2015;21:1344–9. https://d​ oi.o​ rg/1​ 0.​
1038/​nm.​3947.
Epigenetic regulation by ASXL1 in myeloid malignancies
108. Asada S, et al. Mutant ASXL1 cooperates with BAP1 to pro-
mote myeloid leukaemogenesis. Nat Commun. 2018;9:2733.
https://​doi.​org/​10.​1038/​s41467-​018-​05085-9.
109. Guo Y, et  al. Reduced BAP1 activity prevents ASXL1
truncation-driven myeloid malignancy in  vivo. Leukemia.
2018;32:1834–7. https://​doi.​org/​10.​1038/​s41375-​018-​0126-9.
110. Zhang P, et al. Loss of ASXL1 in the bone marrow niche dys-
regulates hematopoietic stem and progenitor cell fates. Cell
Discov. 2018;4:4. https://​doi.​org/​10.​1038/​s41421-​017-​0004-z.
111. Katoh M. Functional proteomics of the epigenetic regulators
ASXL1, ASXL2 and ASXL3: a convergence of proteomics and
epigenetics for translational medicine. Expert Rev Proteom-
ics. 2015;12:317–28. https://​doi.​org/​10.​1586/​14789​450.​2015.​
10334​09.
112. Hatlen MA, et al. Integrative genetic analysis of mouse and
human AML identifies cooperating disease alleles. J Exp Med.
2016;213:25–34. https://​doi.​org/​10.​1084/​jem.​20150​524.
113. Cancer Genome Atlas Research Network, et al. Genomic and
epigenomic landscapes of adult de novo acute myeloid leuke-
mia. New Engl J Med. 2013;368:2059–74. https://​doi.​org/​10.​
1056/​NEJMo​a1301​689.
114. Rocquain J, et al. Combined mutations of ASXL1, CBL, FLT3,
IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2
and WT1 genes in myelodysplastic syndromes and acute mye-
loid leukemias. BMC Cancer. 2010;10:401. https://​doi.​org/​10.​
1186/​1471-​2407-​10-​401.
115. Fernandez-Mercado M, et al. Mutation patterns of 16 genes in
primary and secondary acute myeloid leukemia (AML) with
normal cytogenetics. PLoS One. 2012;7:e42334. https://​doi.​
org/​10.​1371/​journ​al.​pone.​00423​34.
116. Ward AF, Braun BS, Shannon KM. Targeting oncogenic Ras
signaling in hematologic malignancies. Blood. 2012;120:3397–
406. https://​doi.​org/​10.​1182/​blood-​2012-​05-​378596.
117. Bollag G, et al. Loss of NF1 results in activation of the Ras
signaling pathway and leads to aberrant growth in haematopoi-
etic cells. Nat Genet. 1996;12:144–8. https://​doi.​org/​10.​1038/​
ng0296-​144.
118. Zhang P, et al. Chromatin regulator Asxl1 loss and Nf1 hap-
loinsufficiency cooperate to accelerate myeloid malignancy. J
Clin Invest. 2018;128:5383–98. https://​doi.​org/​10.​1172/​JCI12​
1366.
119. Gottfried ON, Viskochil DH, Couldwell WT. Neurofibromatosis
type 1 and tumorigenesis: molecular mechanisms and therapeutic
implications. Neurosurg Focus. 2010;28:E8. https://​doi.​org/​10.​
3171/​2009.​11.​FOCUS​09221.
120. Staser K, Yang FC, Clapp DW. Plexiform neurofibroma genesis:
questions of Nf1 gene dose and hyperactive mast cells. Curr Opin
Hematol. 2010;17:287–93. https://​doi.​org/​10.​1097/​MOH.​0b013​
e3283​39511b.
121. Greenberg PL, et al. Revised international prognostic scoring
system for myelodysplastic syndromes. Blood. 2012;120:2454–
65. https://​doi.​org/​10.​1182/​blood-​2012-​03-​420489.
122. Campbell PJ, et al. Mutation of JAK2 in the myeloproliferative
disorders: timing, clonality studies, cytogenetic associations,
and role in leukemic transformation. Blood. 2006;108:3548–55.
https://​doi.​org/​10.​1182/​blood-​2005-​12-​013748.
123. Campbell PJ, Green AR. The myeloproliferative disorders. N
Engl J Med. 2006;355:2452–66. https://​doi.​org/​10.​1056/​NEJMr​
a0637​28.
124. Guo Y, et al. ASXL1 alteration cooperates with JAK2V617F to
accelerate myelofibrosis. Leukemia. 2019;33:1287–91. https://​
doi.​org/​10.​1038/​s41375-​018-​0347-y.
125. Chen E, Mullally A. How does JAK2V617F contribute to the
pathogenesis of myeloproliferative neoplasms? Hematol Am Soc
Hematol Educ Program. 2014;268–276:2014. https://​doi.​org/​10.​
1182/​ashed​ucati​on-​2014.1.​268.
126. Sidon P, El Housni H, Dessars B, Heimann P. The JAK2V617F
mutation is detectable at very low level in peripheral blood of
healthy donors. Leukemia. 2006;20:1622. https://​doi.​org/​10.​
1038/​sj.​leu.​24042​92.
127. Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of muta-
tions in epigenetic regulators in myeloid malignancies. Nat Rev
Cancer. 2012;12:599–612. https://​doi.​org/​10.​1038/​nrc33​43.
128. Brecqueville M, et al. Array comparative genomic hybridization
and sequencing of 23 genes in 80 patients with myelofibrosis at
chronic or acute phase. Haematologica. 2014;99:37–45. https://​
doi.​org/​10.​3324/​haema​tol.​2013.​091454.
129. Guo Y, et al. ASXL1 alteration cooperates with JAK2V617F
to accelerate myelofibrosis. Leukemia. 2019. https://​doi.​org/​10.​
1038/​s41375-​018-​0347-y.
130. Zhang SJ, et  al. Genetic analysis of patients with leukemic
transformation of myeloproliferative neoplasms shows recur-
rent SRSF2 mutations that are associated with adverse out-
come. Blood. 2012;119:4480–5. https://​ d oi. ​ o rg/ ​ 1 0. ​ 1 182/​
blood-​2011-​11-​390252.
131. Yoshizato T, et al. Genetic abnormalities in myelodysplasia and
secondary acute myeloid leukemia: impact on outcome of stem
cell transplantation. Blood. 2017;129:2347–58. https://​doi.​org/​
10.​1182/​blood-​2016-​12-​754796.
132. Richardson DR, et al. Genomic characteristics and prognostic
significance of co-mutated ASXL1/SRSF2 acute myeloid leu-
kemia. Am J Hematol. 2021;96:462–70. https://​doi.​org/​10.​1002/​
ajh.​26110.
133. Papaemmanuil E, Dohner H, Campbell PJ. Genomic classifica-
tion in acute myeloid leukemia. N Engl J Med. 2016;375:900–1.
https://​doi.​org/​10.​1056/​NEJMc​16087​39.
134. Papaemmanuil E, et al. Genomic classification and prognosis
in acute myeloid leukemia. N Engl J Med. 2016;374:2209–21.
https://​doi.​org/​10.​1056/​NEJMo​a1516​192.
135. Johnson SM, et al. Acute myeloid leukemia with co-mutated
ASXL1 and SRSF2 exhibits monocytic differentiation and has
a mutational profile overlapping with chronic myelomonocytic
leukemia. Hemasphere. 2019;3:e292. https://​doi.​org/​10.​1097/​
HS9.​00000​00000​000292.
136. Pratcorona M, et al. Acquired mutations in ASXL1 in acute mye-
loid leukemia: prevalence and prognostic value. Haematologica.
2012;97:388–92. https://d​ oi.o​ rg/1​ 0.3​ 324/h​ aemat​ ol.2​ 011.0​ 51532.
137. Haferlach T, et al. Landscape of genetic lesions in 944 patients
with myelodysplastic syndromes. Leukemia. 2014;28:241–7.
https://​doi.​org/​10.​1038/​leu.​2013.​336.
138. Carbuccia N, et  al. Mutual exclusion of ASXL1 and NPM1
mutations in a series of acute myeloid leukemias. Leukemia.
2010;24:469–73. https://​doi.​org/​10.​1038/​leu.​2009.​218.
139. Schnittger S, et al. ASXL1 exon 12 mutations are frequent in
AML with intermediate risk karyotype and are independently
associated with an adverse outcome. Leukemia. 2013;27:82–91.
https://​doi.​org/​10.​1038/​leu.​2012.​262.
140. Park UH, Yoon SK, Park T, Kim EJ, Um SJ. Additional sex
comb-like (ASXL) proteins 1 and 2 play opposite roles in adi-
pogenesis via reciprocal regulation of peroxisome proliferator-
activated receptor {gamma}. J Biol Chem. 2011;286:1354–63.
https://​doi.​org/​10.​1074/​jbc.​M110.​177816.
141. Metzeler KH, et  al. ASXL1 mutations identify a high-
risk subgroup of older patients with primary cytogeneti-
cally normal AML within the ELN Favorable genetic cat-
egory. Blood. 2011;118:6920–9. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 182/​
blood-​2011-​08-​368225.
142. Bidikian A, et al. Prognostic impact of ASXL1 mutations in
chronic phase chronic myeloid leukemia. Blood Cancer J.
2022;12:144. https://​doi.​org/​10.​1038/​s41408-​022-​00742-1.
143. Druker BJ, et al. Efficacy and safety of a specific inhibitor of the
BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl
13

J Med. 2001;344:1031–7. https://​doi.​org/​10.​1056/​NEJM2​00104​
05344​1401.
144. Druker BJ, et al. Activity of a specific inhibitor of the BCR-ABL
tyrosine kinase in the blast crisis of chronic myeloid leukemia
and acute lymphoblastic leukemia with the Philadelphia chromo-
some. N Engl J Med. 2001;344:1038–42. https://d​ oi.o​ rg/1​ 0.1​ 056/​
NEJM2​00104​05344​1402.
145. Huang ME, et al. Use of all-trans retinoic acid in the treatment
of acute promyelocytic leukemia. Blood. 1988;72:567–72.
146. Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly
fatal to highly curable. Blood. 2008;111:2505–15. https://​doi.​
org/​10.​1182/​blood-​2007-​07-​102798.
147. Harrison C, et  al. JAK inhibition with ruxolitinib ver-
sus best available therapy for myelofibrosis. N Engl J Med.
2012;366:787–98. https://​doi.​org/​10.​1056/​NEJMo​a1110​556.
148. Verstovsek S, et al. A double-blind, placebo-controlled trial of
ruxolitinib for myelofibrosis. N Engl J Med. 2012;366:799–807.
https://​doi.​org/​10.​1056/​NEJMo​a1110​557.
149. Pardanani A, Tefferi A. Targeting myeloproliferative neoplasms
with JAK inhibitors. Curr Opin Hematol. 2011;18:105–10.
https://​doi.​org/​10.​1097/​MOH.​0b013​e3283​439964.
150. Kim T, et al. Exome sequencing reveals DNMT3A and ASXL1
variants associate with progression of chronic myeloid leukemia
after tyrosine kinase inhibitor therapy. Leuk Res. 2017;59:142–8.
https://​doi.​org/​10.​1016/j.​leukr​es.​2017.​06.​009.
151. Tyner JW, et al. Functional genomic landscape of acute myeloid
leukaemia. Nature. 2018;562:526–31. https://​doi.​org/​10.​1038/​
s41586-​018-​0623-z.
152. Kraehenbuehl L, Weng CH, Eghbali S, Wolchok JD, Merghoub
T. Enhancing immunotherapy in cancer by targeting emerging
immunomodulatory pathways. Nat Rev Clin Oncol. 2022;19:37–
50. https://​doi.​org/​10.​1038/​s41571-​021-​00552-7.
153. Yu S, Yi M, Qin S, Wu K. Next generation chimeric antigen
receptor T cells: safety strategies to overcome toxicity. Mol Can-
cer. 2019;18:125. https://​doi.​org/​10.​1186/​s12943-​019-​1057-4.
13
View publication stats
F.-C. Yang, J. Agosto‑Peña
154. Kenderian SS, et al. CD33-specific chimeric antigen receptor
T cells exhibit potent preclinical activity against human acute
myeloid leukemia. Leukemia. 2015;29:1637–47. https://​doi.​org/​
10.​1038/​leu.​2015.​52.
155. Ting J, Lee AS. Human gene encoding the 78,000-dalton glu-
cose-regulated protein and its pseudogene: structure, conserva-
tion, and regulation. DNA. 1988;7:275–86. https://​doi.​org/​10.​
1089/​dna.​1988.7.​275.
156. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D.
Dynamic interaction of BiP and ER stress transducers in the
unfolded-protein response. Nat Cell Biol. 2000;2:326–32. https://​
doi.​org/​10.​1038/​35014​014.
157. Hebbar N, et al. CAR T cells redirected to cell surface GRP78
display robust anti-acute myeloid leukemia activity and do
not target hematopoietic progenitor cells. Nat Commun.
2022;13:587. https://​doi.​org/​10.​1038/​s41467-​022-​28243-6.
158. Fujino T, Goyama S, Sugiura Y, et al. Mutant ASXL1 induces
age-related expansion of phenotypic hematopoietic stem cells
through activation of Akt/mTOR pathway. Nat Commun.
2021;12:1826. https://​doi.​org/​10.​1038/​s41467-​021-​22053-y.
159. Uni M, Masamoto Y, Sato T, et al. Modeling ASXL1 muta-
tion revealed impaired hematopoiesis caused by derepression
of p16Ink4a through aberrant PRC1-mediated histone modifi-
cation. Leukemia. 2019;33:191–204. https://​doi.​org/​10.​1038/​
s41375-​018-​0198-6.
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds
exclusive rights to this article under a publishing agreement with the
author(s) or other rightsholder(s); author self-archiving of the accepted
manuscript version of this article is solely governed by the terms of
such publishing agreement and applicable law.