948? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 122? ? ? Number 3? ? ? March 2012
Increased dosage of the chromosome 21
ortholog Dyrk1a promotes megakaryoblastic
leukemia in a murine model of Down syndrome
Sébastien Malinge,1 Meghan Bliss-Moreau,1 Gina Kirsammer,1 Lauren Diebold,1
Timothy Chlon,1 Sandeep Gurbuxani,2 and John D. Crispino1
1Division of Hematology/Oncology, Northwestern University, Chicago, Illinois, USA. 2Department of Pathology, University of Chicago, Chicago, Illinois, USA.
Trisomy 21 is the most common cytogenetic abnormality
observed at birth (about 1 out of 700 individuals) and one of
the most recurrent aneuploidies seen in leukemia. As an acquired
clonal chromosomal change, its incidence varies between 4.1%
and 14.8% in hematological disorders and malignant lympho-
mas (1). Supporting the link between trisomy 21 and abnormal
hematopoiesis, epidemiological studies have shown that indi-
viduals with Down syndrome (DS) have an increased frequency
of leukemia but a lower incidence of solid tumors (2). Whereas
recent studies implicated a subset of trisomic genes, including
Erg, Ets2, Adamts1, and Dscr1, in tumor growth inhibition, in part
through an altered angiogenesis (3–6), the role of trisomy 21, the
initiating event in DS leukemogenesis, and the functional impli-
cation of specific genes at dosage imbalances that predispose to
and/or participate in leukemogenesis remain unclear.
Children with DS are at an elevated risk of both acute mega-
karyoblastic leukemia (AMKL) and acute lymphoblastic leuke-
mia (ALL) (7). Moreover, epidemiological studies showed that
approximately 4%–5% of children with DS are born with tran-
sient myeloproliferative disorder (TMD), a clonal preleukemia
characterized by an accumulation of immature megakaryoblasts
in the fetal liver and peripheral blood (8, 9). Although TMD
spontaneously disappears in most cases, TMD clones reemerge
as AMKL in 20% of cases within 4 to 5 years (10). Extensive
research has focused on identifying genetic abnormalities corre-
lated with the transformation process. In addition to trisomy 21,
acquired mutations of the transcription factor GATA1 have been
observed in nearly all TMD and DS-AMKL cases (11–13). These
GATA1 mutations, localized in exon 2, prematurely terminate
translation of the full-length protein but allow for expression of
a shorter isoform named GATA1s (11). Underscoring the require-
ment for trisomy 21 in DS-AMKL, humans without DS but with
germline GATA1 mutations, analogous to those seen in TMD and
DS-AMKL, have no predisposition to leukemia (14), and GATA1
mutations are not found in pediatric leukemia in the absence of
trisomy 21. Other less frequent genetic abnormalities that lead
to activation of myeloproliferative leukemia (MPL), JAK2, JAK3,
and FLT3 have been identified in TMD/DS-AMKL samples (for
review see ref. 7). Supporting a model of oncogenic cooperation,
expression of these single genetic abnormalities, such as MPL
W515L, JAK3 A572V, or JAK2 V617F, alters proliferation and dif-
ferentiation properties of megakaryocytes but fails to reproduce
a DS-AMKL–like phenotype in vivo (15–20).
Mouse models of DS and studies of human tissues show that tri-
somy 21 is sufficient to perturb hematopoiesis and enhance mega-
karyocyte development. For example, analysis of human trisomic
fetal livers revealed a cell-autonomous enhanced proliferation of
the megakaryocytic and erythroid compartments (21, 22). Further-
more, the Tc1, Ts65Dn, and Ts1Cje murine models for DS all dis-
play alterations of the erythro/megakaryocytic compartment, with
Ts65Dn mice developing a progressive myeloproliferative disease
(23–25), further demonstrating the preleukemic role of trisomy 21.
Due to the relatively high number of trisomic genes in these par-
tially trisomic animal models, identification of specific leukemia-
predisposing genes has been challenging. While several groups have
implicated trisomy of Erg with the myeloproliferative phenotype in
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2012;122(3):948–962. doi:10.1172/JCI60455.
Related Commentary, page 807
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
Ts65Dn mice, its specific association with human DS-AMKL com-
pared with that with non–DS-AMKL remains unclear (26–29). Of
note, none of the DS mouse models develop a TMD or AMKL-like
phenotype, even when mated to mice harboring Gata1 mutations
analogous to those observed in human specimens (24, 25). These
observations suggest that trisomy 21 and GATA1s expression are
not sufficient to induce TMD or leukemia.
Here, to understand the functional implication of a partial tri-
somy 21, we used the Ts1Rhr murine model to recapitulate the
progressive acquisition of genetic abnormalities seen in human
DS specimens. We show that Ts1Rhr cooperates to some extent
with Gata1s expression in fetal and adult hematopoiesis and that
adding a third genetic event, expression of an activated allele of
MPL that has been found in humans with DS-AMKL, induces
megakaryoblastic leukemia in vivo. In addition, we demonstrate
that dosage imbalance of the human chromosome 21 (Hsa21)
gene DYRK1A, which encodes dual-specificity tyrosine-(Y)-
phosphorylation–regulated kinase 1A, itself implicated in sev-
eral DS developmental abnormalities (30) and in the decreased
solid tumor incidence in adults with DS (3), contributes to the
Ts1Rhr mice develop a progressive myeloproliferative disorder associated with thrombocytosis. (A) Monthly platelet (PLT) counts of euploid and
Ts1Rhr mice. Mean ± SD. (B) H&E staining of bone marrow and spleen sections from old Ts1Rhr mice (>12 months) and wild-type mice (origi-
nal magnification, ×400). The arrowheads point to megakaryocytes. (C) Histograms showing the percentages of myeloid progenitors of 12- to
14-month-old euploid and Ts1Rhr mice. CMP, Lin–c-kit+Sca–FcγRII/III–CD34+; GMP, Lin–c-kit+Sca–FcγRII/III+CD34+; MEP, Lin–c-kit+Sca–FcγRII/
III–CD34–. Percentages of live cells are indicated. Mean ± SD. (D) Twelve- to fourteen-month-old Ts1Rhr bone marrow and spleen (SP) give rise
to significantly more CFU-Mk colonies. Mean ± SD. (E) Histograms representing percentages of LSK populations and the percentage of CD34hi
in the LSK population. Mean ± SD. (F) Functional HSC frequency in the Ts1Rhr bone marrow (1 out of 66,182 cells) compared with that in wild-
type bone marrow (1 out of 137,284 cells), assessed by competitive transplants 8 weeks after transplantation. Mean ± SD.
950? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
increased development of DS-AMKL in children. Since trisomy
21 is one of the most recurrent aneuploidies seen in hematologi-
cal disorders, we believe that our findings will have implications
beyond DS-AMKL, for malignancies such as hyperdiploid ALL
and AML with acquired trisomy or tetrasomy of Hsa21.
33 trisomic genes are sufficient to develop a progressive thrombocytosis.
We began by studying hematopoiesis in the Ts1Rhr trisomic
mouse model of DS, which is trisomic for 33 orthologous genes
in the human DS critical region (DSCR) and spans 65%–70% of a
Genetic interaction between trisomy for 33 orthologs of Hsa21 and the Gata1 mutation in vivo. (A) CFU-Mk colony numbers from E13.5 fetal liver
cells as well as (B) representative pictures (original magnification, ×100) of the CFU-Mk colonies (n = 3–4 per group) and a histogram comparing
the large colonies/total colonies ratio. Mean ± SD. (C) Representative reticulin stains of bone marrow from 6-month-old mice (original magnifi-
cation, ×400). (D and E) Representative spleen section images of (D) H&E (original magnification, ×100; ×400 [insets]) and (E) von Willebrand
factor immunostaining (original magnification, ×400) of 6-month-old mice. The arrows point to megakaryocytes. (F) Histogram plots showing the
proportion of CD41+ splenocytes at 6 months, as determined by flow cytometry. Mean percentages ± SD (n = 2–4 per group). (G) CFU-Mk colony
number from bone marrow and spleen cells from the Gata1s and Gata1s/Ts1Rhr genetic backgrounds. Mean ± SD (n = 4–5 per group).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
minimal region recently associated with DS-TMD/AMKL (31, 32).
Monthly analysis of complete blood counts revealed that Ts1Rhr
mice have reduced red blood cell counts (Supplemental Figure
1A; supplemental material available online with this article;
doi:10.1172/JCI60455DS1) and develop a progressive thrombocy-
tosis compared to their euploid littermates (Figure 1A). Moreover,
these animals harbor an increased number of megakaryocytes, as
demonstrated by histopathology and flow cytometry (Figure 1B,
Supplemental Figure 1B, and data not shown). Ts1Rhr mice have
an altered proportion of myeloid progenitors, characterized by a
shift from megakaryocyte-erythroid progenitors (MEPs) toward
granulocyte-monocyte progenitors (GMPs) (Figure 1C and
Supplemental Figure 1C) and an increased proportion of CFU-
GM colonies (Supplemental Figure 1D). Remarkably, the CFU-
GM phenotype is reminiscent of that seen in both the Ts65Dn
murine model of DS (23) and in human trisomic fetal liver cells
(22). Moreover, bone marrow and spleen Ts1Rhr cells displayed an
increased ability to form CFU-megakaryocyte (CFU-Mk) colonies
in vitro, but not erythroid BFU-Es, compared with that in their
wild-type littermates (Figure 1D and Supplemental Figure 1D).
Eight- to twelve-week-old trisomic mice also had an increased pro-
portion of Lin–Sca+Kit+ (LSK) cells (Figure 1E) but no significant
variations in the LSK CD34hi subpopulations (containing short-
term HSCs and multipotent progenitors) (Figure 1E). Through
competitive transplant experiments in lethally irradiated mice,
Ts1Rhr adult bone marrow cells appeared to be more functional
than euploid ones (Figure 1F). In contrast to Ts1Cje mice and
human samples, which display fetal hematopoietic abnormalities
(21, 22, 24), no striking hematopoietic defects were seen in E13.5
Ts1Rhr embryos, apart from the significant increase of pheno-
typic fetal HSCs (Lin–Thy1.1loKit+Sca+Mac1+CD4–) (Supplemen-
tal Figure 1, E and F, and ref. 33).
Gata1s cooperates with Ts1Rhr in vivo. Having established that
Ts1Rhr mice fail to develop leukemia, we next mated them with
Gata1s knockin mice and assessed the oncogenic potential of these
cooperating genetic events in vivo (15). We did not observe sig-
nificant variations in proportions of the 4 genotypes at birth or
significant enhancement of the fetal megakaryopoiesis, apart from
the size of CFU-Mk colonies (Figure 2, A and B, Supplemental Fig-
ure 2, and data not shown). In line with previous reports, these
observations confirm that, unlike human fetuses, murine trisomic
fetal livers cells expressing Gata1s mutant protein do not develop a
TMD-like phenotype (25). However, we observed that adult Gata1s/
Ts1Rhr mice were mildly anemic and developed a transient throm-
bocytosis (Table 1 and Supplemental Figure 4A). Six-month-old
double-transgenic mice displayed marrow fibrosis, with increased
megakaryocytes present in clusters, splenomegaly, extensive extra-
medullary hematopoiesis, and an increased number of monocytes
and megakaryocytes as well as CFU-GM and CFU-Mk colonies in
the bone marrow and/or the spleen (Figure 2, C–G, Supplemental
Figure 3, and Supplemental Figure 4, B–F). Compound Gata1s/
Ts1Rhr mice show that Gata1 mutations synergize with partial
trisomy to enhance the fetal megakaryocytic phenotype associ-
ated with Gata1s expression and to perturb adult hematopoiesis,
emphasizing that dosage imbalance of these 33 specific genes is
specifically correlated with abnormal megakaryopoiesis. Neverthe-
less, these 2 events are not sufficient to lead to leukemia in vivo.
Trisomy is functionally implicated in DS-AMKL establishment. We next
asked what the functional impact of trisomy 21 in a more com-
plex disorder is. Given that human DS-AMKL has at least 3 known
genetic abnormalities, we attempted to reproduce the leukemia
in mice by adding a third oncogenic event to the Gata1s/Ts1Rhr
background. Since MPL and JAK2/3 mutations have been iden-
tified in human AMKL specimens (18, 34, 35), we overexpressed
these constitutively active mutant proteins in bone marrow cells
from wild-type, Gata1s knockin, Ts1Rhr, or Gata1s/Ts1Rhr mice
by retroviral transduction. To assess the specific impact of a tri-
somic cellular context on leukemia establishment, we used 6- to
8-month-old bone marrow donor cells, since no apparent pheno-
type was observed among the 4 different backgrounds. We discov-
ered that 3 genetic events — trisomy for 33 orthologs of Hsa21
genes, Gata1 mutation, and expression of MPLW515L — are suffi-
cient to cause a rapid and fatal leukemia in recipient mice (median
survival, 27 days) (Figure 3A). The disease in the triple mutant mice
was characterized by the presence of peripheral thrombocytosis
and a profound bone marrow fibrosis (Figure 3B and Supplemen-
tal Figure 5B). Whereas there was no apparent change in spleen size
(Supplemental Figure 5C), Gata1s/Ts1Rhr/W515L recipient mice
have an effacement of the splenic architecture, due to infiltration
by densely fibrotic tumor nodules comprised of megakaryoblasts
and immature megakaryocytes (Figure 3, C and D, and Supplemen-
tal Figure 5D). Flow cytometric analyses of splenic cells confirmed
the presence of the megakaryoblastic phenotype seen 4 weeks after
transplantation, compared with a variable neutrophilic disease in
the single or double mutant mice (Figure 3E and data not shown).
As determined by Southern blot analyses, the megakaryoblastic
phenotype we observed in the spleen of moribund recipient mice
is an oligoclonal disorder (Supplemental Figure 6). Interestingly,
spleen sections from moribund mice revealed that Gata1s/Ts1Rhr/
W515L mice exhibit less infiltration by mature megakaryocytes
than mice transplanted with wild-type, Ts1Rhr, or Gata1s bone
marrow cells overexpressing MPL W515L (Supplemental Figure
5E). Liver sections from the mice with all 3 genetic abnormalities
also demonstrated a substantial megakaryocytic infiltration (Sup-
plemental Figure 5F). Moreover, we observed that MPL W515L
overexpression results in an increased hematocrit in Ts1Rhr bone
marrow cells, associated with an increased Ter119-positive popula-
tion, and induces anemia and thrombocytosis when coupled with
Gata1s (Supplemental Figure 5A, Figure 3B, and data not shown).
Due to the failure associated with the rapid and profound marrow
and spleen fibrosis, we failed to transplant this DS megakaryo-
blastic leukemia (DS-MkL) in secondary recipients. We separately
overexpressed JAK3 A572V, another mutation associated with DS-
AMKL, in the 4 different backgrounds and found that it cooper-
ates with Gata1s and Ts1Rhr to lead to a fatal hematopoietic dis-
order in vivo (data not shown). However, as seen in previous bone
marrow transplantation studies (36), expression of JAK3 A572V
caused a hematolymphoid disorder characterized by a prolifera-
tion of CD8+ T cells and megakaryocytes (Supplemental Figure
7). Taken together, these data demonstrate that 3 genetic events
are sufficient to lead to a DS-MkL. To date, we believe that this
is the first murine model of megakaryoblastic leukemia involving
trisomy 21, narrowing down the list of Hsa21 leukemia predispos-
ing/promoting genes to 33 candidates and providing us with an
in vivo platform to identify novel dysregulated targets/pathways
associated with abnormal megakaryopoiesis.
Functional screening of the trisomic genes implicated in DS-AMKL.
To gain insights into the specific Hsa21 genes that promote DS-
AMKL, we designed an shRNA-based screening assay to assess the
effects of reducing expression of individual genes on cell cycle,
952? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
survival, CD41 expression, and enforced differentiation of human
megakaryoblastic leukemia cells (Figure 4A). Although this strat-
egy does not address the role of trisomy 21 outside the mega-
karyocyte lineage, the fact that Ts1Rhr mice and human fetuses
with trisomy 21 show prominent expansion of megakaryocytes
relative to that of other lineages (21) suggests that careful analy-
sis of the role of Hsa21 genes in megakaryocytes is warranted. In
addition to the 33 human orthologs of the Ts1Rhr trisomic genes,
Three oncogenic events, including a partial trisomy 21, cooperate to promote DS-MkL in vivo. (A) Survival curves of mice transplanted with dif-
ferent combinations of oncogenic events (n = 6–13 per group). (B) Platelet counts of recipient mice 4 weeks after transplantation. Mean ± SD
(n = 4–12 per group). (C) H&E-stained spleen sections of MPL W515L–overexpressing transplanted mice at 4 weeks after transplant (original
magnification, ×400). (D) von Willebrand factor immunostaining of MPL W515L–overexpressing recipient mice 4 weeks after transplant, showing
the complete megakaryocytic infiltration of the spleen only in the triple mutant mice (original magnification, ×400). (E) Representative flow cytom-
etry plots reveal that triple mutants display marked megakaryocytic expansion in the spleen, while double or single mutants show neutrophilia.
Percentages of live cells are indicated.
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ERG, DYRK1A, CHAF1B, and HLCS are leading candidate DS leukemia-promoting oncogenes. (A) Schematic representation of the strategy
used to assess the functional implication of trisomic genes in human DS-AMKL cell lines. (B) RT-PCR of the DSCR and nearby genes selected
for the functional screening in various cell lines, including DS-AMKL lines CMK and CMY (left panel). Red type indicates Ts1Rhr mice; green
type indicates Ts1Cje mice; blue type indicates Ts65Dn mice; and black type indicates Tc1 mice. D, DMSO treated; T, TPA treated. Knockdown
efficiency of the selected genes in the CMY cell lines (right panel). We hypothesize that a knockdown efficiency of at least 33% (0.66 threshold)
artificially recapitulates the disomy of euploid cells. (C) Plots of normalized values of CD42 expression and DNA content of shRNA-infected CMY
cells after treatment for 3 days with TPA. Changes outside of 2 SDs from the mean (red box) were considered significant. (D) Representative flow
cytometry plots, showing effect of the DYRK1A, CHAF1B, and HLCS knock down during TPA-induced megakaryocytic differentiation of CMK
cells. Percentages of live cells are indicated. Knockdown efficiency (KD eff) is shown. exp, expression; puro, puromycin selection.
954? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
we selected other potential candidate genes based on human seg-
mental trisomy studies and gene set enrichment analysis (GSEA)
from available DS-AMKL gene expression profiles (26, 32, 37).
We found that 32 out of the 50 selected genes were expressed in
human DS-AMKL cell lines CMY and CMK (Figure 4B and data
not shown). To analyze and select candidate genes, we normalized
the raw value observed for each shRNA on the scramble control,
calculated the average and SDs from all normalized data, and
excluded every variation contained in ±2 SD for statistical and
significant purposes. Under this stringent selection, we did not
find significant variations in survival, cell cycle, or endogenous
expression of CD41 by partial knock down of those 32 expressed
genes (Figure 4B and data not shown). However, we found that
knock down of 4 genes that are included in the DSCR — ERG,
DYRK1A, CHAF1B, and HLCS — led to significant differences (>2
or <2 SDs from the mean) in TPA-induced differentiation and
polyploidization of both CMY and CMK human DS-AMKL cell
lines (Figure 4, C and D, and Supplemental Figure 8). Whereas
DYRK1A and CHAF1B knock down showed a significant effect,
with a modest knockdown efficiency (38% and 41%, respectively,
in CMK), the functional implication of HLCS through dosage
imbalance remains unclear (71% knockdown efficiency).
Since DS-AMKL encompasses only one subtype of megakaryo-
cytic leukemia, we wondered to what extent the trisomic genes
contributed to other forms of AMKL. Since the specific region has
been specifically linked to DS leukemogenesis in murine (Ts1Rhr)
and human specimens (32), we looked for a gene expression signa-
ture of the DSCR genes in DS-AMKL, assuming that those genes
are not altered through other genetic abnormalities in non–DS-
AMKL specimens. GSEA of genes contained in the human DSCR
revealed that this entire region is moderately enriched in DS-
AMKL compared with that for non–DS-AMKL (Figure 5A). How-
ever, DYRK1A and CHAF1B are among the top-ranked genes that
are specifically enriched in the DS-AMKL subgroup, whereas HLCS
and ERG are more widely expressed in all types of human AMKL
(Figure 5B). Of note, HMGN1 and MORC3 are also enriched in DS-
AMKL from 2 independent data sets, but shRNAs targeting either
of those genes had no effect on CMY cell differentiation or poly-
ploidization (Figure 4C). Careful analysis of DYRK1A and CHAF1B
expression levels in different leukemic samples revealed that both
are significantly overexpressed in DS specimens (including DS-
AMKL and TMD) compared with those in non–DS-AMKL and/
or pediatric AML (Figure 5C). HLCS is moderately overexpressed
in DS-AMKL. Interestingly, ERG appeared to be more enriched
in non–DS-AMKL than in DS specimens (Figure 5C). Finally, we
observed an increased expression of DYRK1A, CHAF1B, and HLCS
in megakaryocytes derived from human trisomic fetal livers (Sup-
plemental Figure 9A).
DYRK1A is a megakaryoblastic tumor-promoting gene that cooperates
with Gata1s. Since ERG is a known oncogene and has been extensive-
ly studied in human and animal abnormal megakaryopoiesis (29,
38), we focused our studies here on DYRK1A (a serine/threonine
kinase), CHAF1B (a chromatin assembling factor), and HLCS (an
enzyme that catalyzes biotin binding to carboxylases and histones),
whose functions in hematopoiesis have not been yet reported. We
first assessed expression of Dyrk1a, Chaf1b, and Hlcs in our murine
model of DS-AMKL. Although it appears that they were all mod-
erately overexpressed in megakaryocytes derived from Ts1Rhr and
Gata1s/Ts1Rhr bone marrow cells, only Dyrk1a was significantly
enriched in moribund Gata1s/Ts1Rhr/MPL W515L recipient mice
(Supplemental Figure 9, B and C, and Figure 6A). In ex vivo assays
using murine bone marrow cells, Dyrk1a overexpression induced
robust expansion of low ploidy CD41-positive megakaryocytes
(Figure 6, B and C), whereas CHAF1B or HLCS overexpression pro-
duced a less potent induction of megakaryopoiesis. Of note, the
megakaryoblastic effect of Dyrk1a was enhanced in Gata1s mutant
progenitors, arguing for functional cooperation between these pro-
teins in bone marrow (Figure 6, C and D). Although Dyrk1a over-
expression increased CD41-positive megakaryocytes in wild-type
fetal liver cultures, we did not observe an enhancement in Gata1s
FL cells, likely due to the profound enhanced megakaryopoiesis in
mice of this genotype (data not shown). To test whether DYRK1A
kinase activity is required for the megakaryoblastic phenotype, we
overexpressed a catalytically inactive mutant, Dyrk1a-K179R (39),
in wild-type or Gata1s mutant bone marrow cells. Dyrk1a-K179R
failed to support a substantial expansion of CD41+/CD42+ cells in
either the wild-type or Gata1s mutant background (Figure 6D).
To determine whether the elevated megakaryoblastic prolifera-
tion driven by GATA1s and trisomy requires elevated expression
of Dyrk1a, we first attempted to mate Ts1Rhr mice with Dyrk1a
knockout mice (40) but could not obtain Dyrk1a disomic off-
spring to analyze hematopoiesis and reproduce our multistep
pathogenesis, probably due to the breeding deficiencies associ-
ated with Dyrk1a+/– mice. Next, we cultured Ts1Rhr and Gata1s/
Ts1Rhr bone marrow cells with either a Dyrk1a-targeting shRNA
or harmine, a small-molecule inhibitor of DYRK1A kinase activ-
ity (41). Knockdown of Dyrk1a reduced the proportions of mega-
karyocytes expanded from both genotypes (Figure 6E). In parallel,
we established murine cell lines by overexpression of MPL W515L
in Gata1s and Gata1s/Ts1Rhr progenitor cells, which partly repro-
duced the surface markers expression phenotype observed in
our triple mutant mice (Supplemental Figure 10C). Growth of
the trisomic Gata1s/Ts1Rhr/MPL W515L megakaryoblastic cell
lines was sensitive to harmine inhibition, while euploid Gata1s/
MPL W515L cells were not (Figure 6F). Furthermore, prolifera-
tion of human DS-AMKL cell lines was more sensitive to harmine
treatment than non-DS human cells (Figure 7A). In addition, we
verified that 5 μM harmine treatment recapitulated the pheno-
type observed with DYRK1A knockdown during TPA-induced
megakaryocytic differentiation (Figure 4, C and D, and Figure
7B). Taken together, harmine inhibition and Dyrk1a knockdown
experiments both confirm that DYRK1A is required for excessive
expansion of trisomic megakaryoblasts.
DYRK1A dosage imbalance alters the calcineurin/nuclear factor of
activated T cells pathway in DS-AMKL. DYRK1A regulates mul-
tiple cellular processes through the phosphorylation of several
substrates, including nuclear factor of activated T cells (NFAT)
(30, 42). To identify targets of DYRK1A in megakaryoblasts, we
performed global expression analysis of harmine-treated and
DYRK1A shRNA–infected human DS-AMKL cell lines during
TPA-induced differentiation. We identified 325 genes whose
expression was commonly dysregulated (Figure 7C). Ingenuity
pathway analysis revealed that the reduced activity of DYRK1A
was associated with expression changes in 2 known DYRK1A tar-
get pathways, NFAT and TP53 (Table 2). Since NFAT factors have
been implicated in megakaryopoiesis (43, 44) and because dos-
age imbalance of Dyrk1a has been functionally correlated with
common disorders of DS through NFAT pathway alteration (40,
45, 46), the calcineurin/NFAT signaling pathway is an enticing
candidate pathway for development of DS-AMKL.
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DYRK1A and CHAF1B are overexpressed in DS-TMD and DS-AMKL. (A) GSEA of the 33 trisomic human orthologs contained in Ts1Rhr mice
derived from an available gene expression profile data set (62), comparing their relative expression in non–DS-AMKL compared with that in
DS-AMKL and their relative enrichment. (B) Ranked list of chromosome 21 genes of the DSCR enriched in DS-AMKL compared with those in
non–DS-AMKL from both data set 1 (shown in A) and data set 2 (26). (C) DYRK1A (probe set 209033_s_at), CHAF1B (probe set 204775_s_at),
HLCS (probe set 209399_s_at), and ERG (probe set 211626_s_at) relative expression in pediatric AML (n = 9), non–DS-AMKL (n = 43), DS-
AMKL (n = 20), and TMD (n = 8) (GC_RMA normalized probe values extracted from ref. 26). Gene expression in each sample (individual colored
symbols), medians (horizontal bars), and P values (t test) are shown.
956? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
Phosphorylation of NFAT by DYRK1A leads to nuclear export
of activated NFAT factors and the subsequent inhibition of their
transcriptional activity. To begin dissecting the DYRK1A-regu-
lated NFAT activity in megakaryocytes, we first correlated the
increased expression levels of DYRK1A to NFATC2 and NFATC4
in megakaryocytes derived from Ts1Rhr and Gata1s/Ts1Rhr
mice as compared with those from euploid littermates (Figure 7,
D and E). To our surprise, it appears that trisomic cells had an
increased expression of NFATC2 and NFATC4. However, we cor-
related increased phosphorylation of NFATC2 transcription fac-
tors to the DYRK1A overexpression in Ts1Rhr cells, consistent
with reduced NFAT signaling in DS. The activity of DYRK1A on
NFAT transcription factors is opposed by the phosphatase calci-
neurin, which promotes the dephosphorylation/activation of the
NFAT factors and their subsequent nuclear translocation. Thus,
we predicted that treatment of cells with cyclosporine A, an inhibi-
tor of calcineurin, would give the opposite phenotype as that of
harmine treatment. To ensure the functional correlation between
Dyrk1a overexpression and NFAT signaling inhibition, we cul-
tured Gata1s/MPL W515L and Gata1s/Ts1Rhr/MPL W515L cells
with 1 μM cyclosporine A and found that euploid cells were sig-
nificantly more sensitive to growth inhibition than trisomic cells
(Figure 7F). Finally, we investigated the effect of cyclosporine A on
derivation of megakaryocytes from Ts1Rhr mice and show that
Dyrk1a knockdown trisomic cells were more sensitive than the
control infected bone marrow cells (Figure 7G). These results are
consistent with the model that DYRK1A modulates megakaryo-
blastic expansion through the inhibition of the calcineurin/NFAT
pathway in DS-AMKL.
Here we show using a mouse model of 3 oncogenic events that
trisomy of 33 gene orthologs of Hsa21, a GATA1 mutation, and
a MPL mutation are sufficient to induce DS-MkL in vivo (Figure
8). Moreover, we identified DYRK1A as a potent megakaryoblas-
tic tumor-promoting gene in the DSCR. DYRK1A has a role in
embryonic stem cell fate regulation in DS (47), and dosage imbal-
ance of the murine Dyrk1a gene is functionally correlated with
heart and neurological disorders, similar to those seen in patients
(40, 45, 46). Recently, Dyrk1a, together with another chromosome
21 ortholog, Dscr1, were shown to markedly diminish angiogen-
esis, resulting in a suppression of tumor growth (3), as observed
in patients with DS. Although the DSCR1 gene does not appear to
be overexpressed in DS-AMKL, and its knockdown did not reveal
significant variations in our screening, it is remarkable to note
that the same genes at dosage imbalance can be tumor suppres-
sors or tumor promoters in a context-dependent manner. Based
on our studies, we conclude that the differential effects of altera-
tions in pathways downstream of these genes likely account for
the discrepancy between solid tumor inhibition and leukemia
predisposition in DS (3–6).
We and others have previously shown that the Hsa21 ETS
protein ERG is a megakaryocytic oncogene that cooperates with
GATA1 mutations to enhance megakaryopoiesis (29, 38, 48). Inter-
estingly, our study shows that the ERG oncogene is not enriched
in DS-associated leukemia compared with that in other pediatric
AML specimens (Figure 6B). More surprisingly, ERG appears to be
overexpressed in non–DS-AMKL. Of note, previous studies have
shown that ERG is not specifically overexpressed in trisomic fetal
livers compared with that in euploid ones (21). The same observa-
tion has been reported for another known oncogene of the chro-
mosome 21, RUNX1 (21, 26), although, unlike Erg, Runx1 trisomy
does not seem to be implicated in the myeloproliferative disorder
developed by the Ts65Dn mice (23, 27). Those observations might
reflect a higher ERG overexpression in non–DS-AMKL driven by
other genetic abnormalities, whereas a low, increased ERG expres-
sion in DS leukemia could be sufficient to cooperate with other
trisomic genes, as demonstrated in our study, such as DYRK1A,
CHAF1B and/or HLCS. Since ERG overexpression has been associ-
ated with various types of cancer (49) and recently correlated with
promotion and maintenance of T acute lymphoblastic leukemia
(50, 51), additional experiments will be required to ensure that
ERG trisomy is directly implicated in DS-AMKL as opposed to
oncogenesis in general.
Our screening strategy identified 4 Hsa21 genes as candidates
to promote excessive megakaryopoiesis. Although we believe that
these genes represent strong candidates for the megakaryocytic
phenotype, trisomy of these genes or others may also be respon-
sible for promoting B cell acute lymphocytic leukemia and for
altering hematopoietic stem and progenitor cell expansion and/or
survival in such a way as to promote tumorigenesis. Future stud-
ies to uncover Hsa21 genes that contribute to other aspects of DS
leukemogenesis are needed.
We identified Dyrk1a as a prominent megakaryoblastic tumor-
promoting gene of the human DSCR. Although we were not able
to genetically engineer the Ts1Rhr/Dyrk1a+/+/– background by
using Dyrk1a knockout mice (40), several lines of evidence support
a direct and specific role of DYRK1A dosage imbalance in leuke-
mic predisposition in DS. First, overexpression of Dyrk1a cooper-
ates with GATA1s expression to markedly increase proliferation
of megakaryoblasts/immature megakaryocytes. Second, Dyrk1a
knockdown decreases the megakaryocytic expansion seen in tri-
somic mice. Third, inhibition of DYRK1A activity with a small-
molecule kinase inhibitor affects megakaryoblastic proliferation
from trisomic progenitors to a much greater extent than that from
euploid cells. Finally, DYRK1A expression is markedly and spe-
cifically upregulated in both human and murine DS-AMKL cells.
This latest observation correlates with a recent study showing that
DYRK1A is upregulated in trisomic fetal livers and is predicted to
be a new potential biomarker for screening DS fetuses. Interesting-
ly, it has been shown that the transcription factor E2F1 increases
DYRK1A expression by enhancing promoter activity (52). Added to
the recent observation that GATA1s protein failed to repress E2F
target genes (53), this might lead to a further increased Dyrk1a
expression and potentially explain the cooperation we observed in
the Gata1s background and account for the DYRK1A enrichment
observed in TMD and DS-AMKL compared with that in non–DS-
AMKL and pediatric AML.
The DYRK kinase family is part of the CMGC group, which
also includes mitogen-activated protein kinases (MAPKs), cyclin-
dependant kinases (CDKs), glycogen synthase kinases, and
CDK-like kinases (42). Unlike, MAPKs regulated by upstream
protein kinases, catalytic activation of DYRK proteins occurs
by autophosphorylation, which appears to occur by an intra-
molecular reaction immediately after translation (54). Since
DYRK kinases always appear to be in a catalytically active state,
their function is thought be controlled by changes in expression
levels. Thus DYRK1A dosage imbalance is predicted to directly
alter the regulation of its targets. Here we show that increased
DYRK1A dosage in trisomic megakaryocytes is linked to an inhi-
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
Dyrk1a is a prominent megakaryocytic tumor-promoting gene. (A) Fold change gene expression values of Dyrk1a, Chaf1b, and Hlcs, as
assessed by real-time PCR, in CD41-positive cells isolated from spleens of Gata1s/Ts1Rhr/MPL W515L recipient mice, compared with those
in Gata1s/MPL W515L CD41-selected spleen cells 4 weeks posttransplantation. Mean ± SD. (B) Representative flow cytometry plots depicting
the proportion of CD41+ cells derived from cultures of bone marrow progenitors infected with Dyrk1a, CHAF1B, or HLCS encoding viruses or
control vector. Percentages of live cells are indicated. (C) Overexpression of Dyrk1a leads to reduced polyploidization of megakaryocytes. Mean
± SD (n = 3–5 per group). *P < 0.004, **P < 0.0008 compared with control infected. (D) Fold change increase in percentage of CD41+ and CD42+
cells after expression of wild-type or kinase-inactive alleles of Dyrk1a in wild-type or Gata1s bone marrow progenitors. Mean percentages ± SD
(n = 2–4 per group). (E) Representative flow cytometry plots of Dyrk1a shRNA and control infected progenitors cells cultured under megakaryo-
cytic conditions. Bone marrow cells were derived from Ts1Rhr and Gata1s/Ts1Rhr mice. Percentages of live cells are indicated. (F) Treatment
of double (Gata1s/MPL W515L) and triple (Gata1s/Ts1Rhr/MPL W515L) mutant cells with harmine reveals that trisomic cells are more sensitive
to DYRK1A inhibition in vitro. Mean ± SD (n = 3–4 per group).
958? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
bition of NFAT factors activation. Interestingly, whereas the
calcineurin/NFAT pathway has been shown to favor lymphoid
leukemia progression (55), its inhibition has been associated
with a megakaryocytic accumulation, through the overexpres-
sion of the immunophilin FKBP51 in idiopathic myelofibrosis
(56). Since the calcineurin/NFAT pathway has been shown to be
active in megakaryocytes (43, 44, 57), those results emphasize
its functional implication in this lineage. Interestingly, our data
also show that murine trisomic cells have an increased expres-
sion of NFATC2 and NFATC4 transcription factors, leading that
DYRK1A dosage imbalance is correlated with NFAT pathway dysregulation in human and murine primary cells. (A) Ratio of live cells treated with
serial dilution of the Harmine inhibitor at 3 days (n = 3 per group) normalized on untreated cells. Mean ± SD. (B) Representative FACS plots,
showing the effect of 5 μM harmine on a 3-day TPA-induced megakaryocytic differentiation of CMY. (C) Venn diagram showing the number of
common genes dysregulated between treated or infected CMK and CMY cells during TPA-induced megakaryocytic differentiation. (D and E)
Representative Western blots of DYRK1A, NFATC2, phospho-NFATC2 (P-NFATC2), and NFATC4 expression (D) in CD41-enriched spleen
cells from euploid mice compared with that in Ts1Rhr mice (n = 2) and (E) in Gata1s mice (n = 2) compared with that in Gata1s/Ts1Rhr mice. (F)
Gata1s/Ts1Rhr/MPL W515L triple mutant cells are less sensitive to the NFAT/calcineurin inhibitor cyclosporine A (CsA) than the non-trisomic
Gata1s/MPL W515L cells in liquid culture. Mean ± SD (n = 5 per group). (G) Flow cytometry analysis of CD41 and CD42 populations derived
from Ts1Rhr bone marrow progenitors infected with control or DYRK1A shRNA and treated for 3 days with cyclosporine A or vehicle. Percent-
ages of live cells are indicated.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
the hypothesis that calcineurin/NFAT signaling may be altered
through multiple processes in our murine model. Of note,
NFATC4 also appears to be overexpressed in human DS-AMKL
compared with that in non–DS-AMKL (data not shown). We
also observed a modest enrichment of the calcineurin pathway
in non–DS-AMKL compared with that in DS-AMKL but failed
to find a NFAT signature using these gene expression analyses,
probably due to the fact that most of the known NFAT target
genes have been identified in cardiac and T cells. Additional
experiments will be required to establish the functional implica-
tion of this pathway in human DS-AMKL and understand how
NFAT factors expression is regulated during normal and patho-
Although our murine model reproduced most of the human DS-
AMKL pathogenesis, we only observed slight variations in the fetal
liver, which does not account for the TMD phenotype commonly
seen in humans with DS. While this discrepancy may reflect spe-
cies-specific differences, there are several other possible explana-
tions. For example, the difference may be due to the absence of
trisomic genes, miRNAs (such as miR125b-2) (58), or regulatory
elements that act during fetal hematopoiesis. Interestingly, Carmi-
chael et al. described clonogenicity and HSC function impairment
in the Ts1Cje murine model that we did not observe in the Ts1Rhr
background (24). While this discrepancy could be related to experi-
mental design, it may also emphasize the possible contribution of
trisomic genes outside of the DSCR to this hematopoietic phe-
notype. Only a few trisomic genes in the Ts1Cje mice, but not in
the Ts1Rhr mice, have a known function in fetal hematopoiesis.
Among them, however, Runx1 appears to be a relevant candidate,
because its dosage imbalance has been associated with abnormal
HSC function during fetal hematopoiesis and, more recently, with
the increased c-KIT and GATA2 expression in human DS fetal liv-
ers (59, 60). Other than Runx1, genes to consider include Dscr1,
Sod1, and Son. Alternatively, the failure to fully recapitulate the
human fetal phenotype may be due to a requirement for addi-
tional genetic abnormalities that do not spontaneously appear in
mice. In line with these hypotheses, neither DYRK1A nor CHAF1B
overexpression appears to specifically cooperate with fetal GATA1s
expression, unlike our observations in adult progenitors, arguing
for a time- and/or context-dependent cooperation in mice.
Through a retroviral integration mutagenesis strategy, Klus-
mann et al. showed that aberrant expansion of fetal, but not adult,
megakaryocytic progenitors is dependent on IGF (53). This study
emphasized the fetal origin of the target cell for transformation in
DS-AMKL but did not address the requirement of trisomy 21, the
initiating event of DS-leukemogenesis. We believe that our study
is the first to address the functional requirement of trisomy 21 in
the transformation process of Gata1s-
and/or MPL W515L–expressing pro-
genitors. Additional experiments will
be required to address the fetal origin of
the human disorder and to determine
the IGF and calcineurin/NFAT signal-
ing effects in trisomic Gata1s-expressing
fetal liver progenitors. The absence of a
fetal disease in our DS-AMKL model is
not unprecedented, as mouse models of
MLL rearrangements, which have their
origins in the fetus, consistently develop
adult not fetal leukemia.
Our study also specifically links trisomy for the 33 genes in the
Ts1Rhr mice to the previously reported red blood cell phenotype
observed in Ts65Dn, Ts1Cje, and Tc1 partially trisomic murine
models (23–25). Interestingly, we observed a functional coopera-
tion of Ts1Rhr with MPL W515L, leading to a leukocytosis and an
increased hematocrit and increased Ter119+ cells in recipient mice
(S. Malinge and J.D. Crispino, unpublished observations). These
observations are in agreement with the enhanced expression of
erythroid markers observed in DS-AMKL compared with that in
non–DS-AMKL (26, 37).
Harmine, an inhibitor of DYRK1A kinase activity, altered the
polyploidization of megakaryocytes established from murine
and human primary cells in vitro (L. Diebold and S. Malinge,
unpublished observations). Interestingly, the human AMKL cell
line CHRF, established from a patient with an acquired trisomy
21, was sensitive to harmine in the same range as that of both
of our DS-AMKL cell lines (Figure 7A). We also observed that
pediatric AMKL samples with acquired trisomy 21 and GATA1
mutations were associated with DYRK1A overexpression (data
not shown). These observations suggest that our study has
broader significance, since trisomy 21 is one of the most com-
mon cytogenetic abnormalities seen in hematological malignan-
cies. Moreover, the in vivo model of multistep DS leukemogen-
esis that we established will be a powerful tool to develop new
small-molecule inhibitors of DYRK1A activity and assess new
potential therapy for DS-AMKL.
Ts1Rhr cooperates with Gata1s expression to develop a transient thrombocytosis
1,130 (± 61)
1,045 (± 166)
1,059 (± 231)
1,122 (± 153)
1,104 (± 151)
1,727 (± 150)A
1,295 (± 334)
1,427 (± 350)
1,070 (± 193)
1,230 (± 161)
1,179 (± 230)
1,222 (± 177)
1,224 (± 216)
1,798 (± 406)B
1,876 (± 602)B
1,522 (± 643)
Platelet counts of mice from the 4 genetic backgrounds: wild-type, Ts1Rhr, Gata1s knockin, and Gata1s/
Ts1Rhr. Mean platelet counts ± SD are shown. AP < 0.01; BP = 0.02.
Canonical pathways associated with DYRK1A dysregulation
during megakaryocytic differentiation
Aryl hydrocarbon receptor signaling
Hereditary breast cancer signaling
Communication between innate and immune cells
Reelin signaling in neurons
NRF2-mediated oxidative stress response
Role of NFAT in cardiac hypertrophy
Crosstalk between DCs and NK cells
Hepatic fibrosis/hepatic stellate cell activation
Chronic myeloid leukemia
Cell cycle, G1/S checkpoint regulation
List of the canonical pathways that are significantly enriched
(AP < 0.01) for dysregulated genes, as determined by Ingenuity
Pathways Analysis software.
960? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
Mouse experiments. The partially trisomic Ts1Rhr murine model [also known
as the Dp(16Cbr1-ORF9)1Rhr model] and Gata1s knockin mice (Gata1s
mice; also known as Gata1∆ex2 mice), provided by R.H. Reeves (Johns Hopkins
University School of Medicine, Baltimore, Maryland, USA) and S.H. Orkin
(Dana-Farber Cancer Institute, Harvard, Massachusetts, USA), respectively,
have been previously described (15, 31) and genotyped using Ts1Rhr-F 5′-
CCGTCAGGACATTGTTGGA-3′ with Ts1Rhr-R 5′-CCGTAACCTCTGCC-
GTTCA-3′ and 5G1-7762 5′-GGGACAAAGGATGATGGAAGAAGAC-3′
with 3G1-8739 5′-GGTCTTCCTACAATAGTCTCTTGC-3′ for knockin
Gata1s and 5G1-7762 with Gata1-ex3R 5′-TGCATCCAAACCCTCTGGC-
3′ for the Gata1 wild-type locus. Peripheral blood (CBC) and flow cytom-
etry analyses were performed at the specific time points indicated in the
manuscript using Hemavet HV950FS (Drew Scientific) and BD LSRII,
respectively. Murine cell populations were analyzed using Mac1-PE, Gr1-
APC, Kit-PerCP-Cy5.5, CD41-PE, CD45.1-PerCP-Cy5.5, and CD45.2-APC
antibodies (all from BD Biosciences); CD61/41-APC and CD42-PE (both
form Emfret); and CD34-FITC and Sca1-PeCy7 antibodies (both from eBio-
science) and analyzed using FlowJo 8.8.6 software. In vitro CFU assays were
performed using M3234 and MegaCult (Stem Cell Technology), accord-
ing to the manufacturer’s instructions. All experiments using the Gata1s/
Ts1Rhr double-transgenic mice were done using males from at least the
third generation from different founders and by comparing littermates to
minimize background variations. For competitive transplants, 8-week-old
lethally irradiated mice were transplanted in replicate experiments via retro-
orbital injection with 15 × 103, 50 × 103, or 200 × 103 wild-type or Ts1Rhr
Ly5.2 bone marrow donor cells in combination with 200 × 103 wild-type
competitor cells (Ly5.1) (n = 8 mice per group). Engraftment was assessed at
8 weeks posttransplantation by flow cytometry for Ly5.1 and Ly5.2 mark-
ers on peripheral blood leukocytes. Stem cell frequencies were determined
using L-Calc software (Stem Cell Technologies Inc.). All animals were main-
tained in microisolator housing within a barrier facility.
Plasmids. Murine Dyrk1a and human CHAF1B and HLCS cDNAs were
amplified from the Y10 murine megakaryocytic cell line and human DS-
AMKL cells, respectively, and then subcloned into the MSCV-IRES-EGFP
(Migr) plasmid for overexpression studies. The K179R mutation was
done with the QuikChange Site-Directed Mutagenesis Kit (Stratagene,
no. 200518) according to the manufacturer instructions and subcloned
to the Migr plasmid. The murine Dyrk1a shRNA was subcloned from the
pSM2c vector (Open Biosystem) into the GFP-containing Banshee for
retroviral production (61).
Murine megakaryocyte cultures, overexpression, and knockdown studies. Whole
bone marrow cells were infected twice by retroviruses (Migr-Dyrk1a, Migr-
Dyrk1a K179R, Migr-CHAF1B, and Migr-HLCS) for overexpression stud-
ies or by Banshee constructs containing shRNA Dyrk1a (Open Biosystem,
from pSM2c no. V2MM_25405) for knockdown experiments, and cells
were then resuspended in RPMI containing 10% FBS, penicillin-strepto-
mycin, l-glutamine, 1 μM cortisol (Sigma-Aldrich, no. H6909), 10 ng/ml
mouse IL-3 (mIL-3), 5 ng/ml mIL-6, and mouse SCF (mSCF) supernatant.
On day 3, infected cells were cultured in RPMI1640, penicillin-strepto-
mycin, l-glutamine, 10% FBS, 10 ng/ml mouse thrombopoietin (mTPO),
and mSCF supernatants for an additional 3 days to promote megakaryo-
cytic differentiation. After culture, cells were stained for flow cytometry
experiments or separated by a BSA gradient (3%, 1.5%) for RNA extrac-
tion. Splenic megakaryocytes were enriched using CD41-PE antibody (BD
Biosciences, no. 558040) and PE Selection Kit (StemCell Technology, no.
18551) for RNA or protein extraction.
Bone marrow transplantation. Bone marrow transplant experiments of JAK3
wild-type, JAK3 A572V, MPL wild-type, and MPL W515L overexpression
were performed as described previously (18, 19). JAK3 and MPL constructs
were provided by D.G. Gilliland (Brigham and Women’s Hospital, Boston,
Massachusetts, USA) and R. Levine (Memorial Sloan-Kettering Cancer
Center, New York, New York, USA), respectively. Briefly, whole bone mar-
row cells were harvested from 6- to 8-month-old wild-type, Ts1Rhr, Gata1s,
Schematic representation of the multistep pathogenesis model of murine DS-MkL and phenotypes associated with the presence of the par-
tial trisomy. Trisomic genes associated with each step in murine (blue) and human samples (green) are indicated below. Note that DYRK1A
(underlined) is the only trisomic gene overexpressed at each step in both species. Triangles represent the third hit (MPL W515L in this study).
KI, knockin; T21, trisomy 21, X, GATA1 mutation.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 3 March 2012
or Gata1s/Ts1Rhr (Ly5.2) donor mice at day 0, lysed for red blood cells for
5 minutes with ammonium chloride solution (StemCell Technology, no.
07850), washed with PBS, and then resuspended overnight in RPMI1640
containing 10% FBS, cortisol (Sigma-Aldrich, no. H6909), 10 ng/ml mIL-3,
5 ng/ml mIL-6, and mSCF supernatant. On day 1, cells were spin infected
at 1,400 g, 33°C, for 99 minutes and resuspended overnight in fresh media.
On day 2, 0.5 to 1 × 106 cells were spinoculated, resuspended for 5 hours in
fresh media, and then injected through the retro-orbital vein into lethally
irradiated Ly5.1 recipient mice (2 × 5.5 Gy). Proviral integration and clon-
ality were assessed by Southern blot using 10 μg genomic DNA extracted
from splenocytes (Puregene) from moribund mice, digested by BamH1,
subjected to electrophoresis, and hybridized with an EGFP probe accord-
ing to the standard protocols.
shRNA based screening. Trisomic genes of interest were first screened for
their expression in the human CMK and CMY DS-AMKL cells lines. Primer
sequences used for the screening are shown in Supplemental Table 1. The
expressed genes were then knocked down using pGIPZ shRNA purchased
from Open Biosystem (Thermo Scientific) (Supplemental Table 2). Lenti-
viral particles were produced according the manufacturer’s instructions
using psPAX2 (gag-pol) and pMD2.G (encoding VSV-G) helper vectors, and
CMY cells were submitted to 2 rounds of spinoculation. On day 2, GFP-
positive cells were incubated with 0.5 μg/ml Hoechst33342 (Invitrogen, no.
H3570) for 90 minutes and/or stained with AnnexinV-PE (BD Biosciences,
no. 556421) and CD41-APC (BD Biosciences, no. 559777). Infected cells
were selected with 1 μg/ml puromycin for 4 days and expanded for addi-
tional 6 days prior to RNA extraction or were cultured in the same media,
RPMI1640, 10% FBS, penicillin-streptomycin, and l-glutamine with 100
nM TPA, to chemically induce megakaryocytic differentiation. Cells were
then stained for differentiation markers CD41 and CD42 (BD Biosciences,
no. 551061) and DAPI to measure DNA content.
Immunohistochemistry. Tissues were fixed for 24 hours in 10% formalin
(Sigma-Aldrich, no. HT501320) and then placed in 70% ethanol prior par-
affin embedding. Tissue sections were stained with anti-human von Wil-
lebrand factor, H&E, or reticulin. Briefly, tissue sections were deparaffinized
and incubated for 20 minutes in target retrieval buffer (10 mM Tris, 1 mM
EDTA, pH 9.0). Endogenous peroxidase activity was blocked with 3% hydro-
gen peroxide for 10 minutes. Tissue sections were blocked with normal
serum from the VECTASTAIN ABC Elite Kit (Vector Laboratories, no. PK-
6101) for 20 minutes before incubation overnight at 4°C with anti-human
Von Willebrand Factor (1:1,200; Dako, no. A0082). Secondary biotinylated
antibody and VECTASTAIN Elite ABC Reagent (Vector Laboratories) were
applied as per manufacture directions. Positive cells were detected using
diaminobenzidine (Sigma-Aldrich, no. D5905). All tissue sections were
counterstained with Harris hematoxylin (Sigma-Aldrich, no. HHS16).
Cell lines, reagents, and Western blotting. Human cell lines (CMK, CMY, CMS,
CHRF, and K562) were cultured in RPMI1640, 10% FBS, penicillin-strep-
tomycin, and l-glutamine. The murine cell lines Gata1s/MPLW515L and
Gata1s/Ts1Rhr/MPLW515L were established by MPL W515L infection of
Lin-negative bone marrow cells, selected for 4 days with 1 μg/ml puromy-
cin, and cultured in complete RPMI, 1 μM cortisol, 10 ng/ml mIL-3, 5 ng/
ml mIL-6, 5 ng/ml mTPO, and mSCF supernatant. Harmine hydrochlo-
ride inhibitor (MP Biomedicals, no. 210554) and cyclosporine A (Sigma-
Aldrich, no. C3662) were incubated with human or primary murine cells
for 3 days prior further analysis. Protein pellets were solubilized in 10 mM
TRIS/HCl (pH 7.5), 1% Triton X-100, 20% glycerol, 5 mM EDTA, 50 mM
NaCl, 50 mM NAF, 1 mM Na3OV4, and Complete lysis buffer; separated
on a Bis-Tris polyacrylamide gel (Invitrogen); and transferred to a PVDF
membrane (Millipore). Antibodies were anti-PhosphoSer326-NFATc2 (no.
sc-32994), anti-NFATc2 (no. sc-7296), anti-NFATc4 (no. sc-13036), and
anti-Hsc70 (no. sc-7298) (all from Santa Cruz Biotechnology) and anti-
DYRK1A (Abnova, no. H00001859-M01).
Statistics. Data are shown as mean ± SEM or SD as denoted. P values
were calculated using the Student’s t test (2 tailed). P ≤ 0.05 was consid-
ered to be significant.
Study approval. All animal experiments were approved by the Northwest-
ern University Animal Care and Use Committee.
The authors thank Roger Reeves for the Ts1Rhr mice, Stuart
Orkin and Zhe Li for the Gata1s knockin mice, and Mariona
Arbones (Barcelona, Spain) for the Dyrk1a+/– mice. We thank
Jeffrey Taub and Yubin Ge for the human AMKL gene expres-
sion profile data set. We thank Roger Reeves, Monika Stankie-
wicz, Lou Doré, Kim Rice, Stella Chou, and Sandra Ryeom for
their advice and helpful discussions. We also thank the Rob-
ert H. Lurie Flow Cytometry Core Facility for assistance with
flow cytometry. This work was supported by an R01 from the
National Cancer Institute (CA101774). S. Malinge is a recipi-
ent of a postdoctoral fellowship from the Leukemia and Lym-
Received for publication August 12, 2011, and accepted in revised
form December 7, 2011.
Address correspondence to: John D. Crispino, Northwestern Univer-
sity, Division of Hematology/Oncology, 303 East Superior Street,
Lurie 5-113, Chicago, Illinois 60611, USA. Phone: 312.503.1504;
Fax: 312.503.0189; E-mail: email@example.com.
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