CD117/KIT is a receptor tyrosine kinase that
regulates HSC proliferation, maintenance, and
survival after binding to its ligand, stem cell
factor ( 43 ). To analyze KIT expression in normal
hematopoiesis, FL-, CB-, and BM-derived LT-
HSCs were immunophenotypically profiled for
CD117 expression. N-FL and T21-FL LT-HSCs
contained distinct CD117-low and CD117-high
populations (Fig. 6A). By contrast, LT-HSCs
from N-CB and N-BM showed a single popu-
lation of cells with a continuum of low to
high CD117 expression. After transplantation
at limiting cell dose into NSG mice, only the
CD117-high populations and not the CD117-
low populations from N-FL and T21-FL LT-
HSCs were able to generate grafts at 20 weeks
(Fig.6,BandC).Bycontrast,bothCD117-low
and CD117-high N-CB LT-HSCs were able to
generate long-term engraftment, albeit with
different lineage outputs (Fig. 6D). These results
suggest that KIT signaling plays an essential
role in LT-HSC function during fetal develop-
ment. On the basis of the engraftment results
of disomic CB (Fig. 6, B and C), KIT signaling
may be less dependent, at least transiently, for
LT-HSC function after birth.
To investigate whether pharmacological in-
hibition of KIT can target and eliminate pre-
leukemic and leukemic blasts, mice engrafted
with 1300 T21-FL control, GATA1s, or GATA1s/
STAG2ko LT-HSCs were treated with first-,
second-, and third-generation KIT inhibitors
(50 mg/kg imatinib, 20 mg/kg dasatinib, or
7.5 mg/kg ripretinib) starting 10 weeks after
transplantation with twice-daily dosing for
2 weeks (Fig. 6E) ( 44 – 46 ). KIT inhibition did
not have a significant effect on the overall
level of CD45+engraftment for any group,
except for dasatinib-treated mice bearing T21-
FL GATA1s preleukemic grafts (P< 0.01) (Fig.
6F). KIT inhibition had no effect on the blast
population of leukemic grafts generated by
T21-FL GATA1s/STAG2ko cells (Fig. 6G). GATA1s
preleukemic grafts from mice treated with any
of the KIT inhibitors contained significantly
lower proportions of blasts as compared with
that of vehicle-treated mice (P< 0.001), with
reduction of CD117+CD45+blast populations
to levels seen in controls (Fig. 6, H and I, and
fig. S14A). KIT inhibitor–treated mice revealed
an increase in granular CD33+, CD11b+, and
CD13+myeloid cells, suggesting differentia-
tion of blast cells toward more mature myeloid
cells (fig. S14, B and C). Because some residual
CD117+blasts remained detectable in mice with
preleukemic GATA1s grafts (Fig. 6H), cells har-
vested from primary mice were serially trans-
planted at defined doses into secondary NSG
recipients to determine whether preleukemia-
initiating cells were affected. Cells from vehicle-
treated mice showed a 32-fold higher ability to
generate secondary grafts at 8 weeks compared
with cells from KIT inhibitor–treated mice
(fig. S14D). This lies in contrast to the secondary
grafts generated from KIT inhibitor–treated
GATA1s/STAG2ko–induced leukemia, which
did not show any difference in their ability to
generate secondary grafts (fig. S14E). To fur-
ther validate the sensitivity of KIT inhibition
in GATA1s-induced preleukemia, two primary
TAM samples were phenotypically char-
acterized (fig. S14, F and G, and table S10),
subsequently transplanted into mice, and treated
with KIT inhibitors starting at 6 weeks after
transplantation for 2 weeks (fig. S15A). KIT
inhibition resulted in reduced CD45+engraft-
ment with significantly reduced CD117+CD45+
blast populations (P< 0.001) and increased
granular CD33+myeloid cells (Fig. 6J and fig.
S15, B to H). However, we cannot rule out that
the effects of pharmacological KIT inhibition
could also be mediated, at least partly, through
other receptor tyrosine kinases. Altogether, our
results demonstrate as a proof of principle that
KIT inhibition targets preleukemic expansion
(fig. S15I), supporting further clinical evaluation
of the concept that preleukemic intervention
could inhibit progression to leukemia.Discussion
Our study provides insight into the cellular
and molecular mechanism of Down syndrome
leukemogenesis, from atypical hematopoiesis
associated with T21 to preleukemia initiation
and ultimately to leukemic progression. We
confirmed that the T21-FL hematopoietic sys-
tem exhibits an altered phenotypic HSPC hi-
erarchy as previously described ( 25 , 47 , 48 ).
Although earlier reports have proposed that
T21 enhances self-renewal in vitro ( 48 ), our
functional studies revealed the opposite; in-
dividual T21 HSPC subpopulations exhibited
reduced proliferation in vitro and generated
smaller grafts in xenotransplanted mice with
myeloid and megakaryocytic bias and reduced
serial transplant ability. These are likely cell-
autonomous effects and may be the basis for
the higher incidence of hematopoietic abnor-
malities such as isolated cytopenias, myelo-
dysplasia, and BM failure seen in adults with
Down syndrome ( 49 ). Despite the reduced pro-
liferative capacity of T21 LT-HSCs, our data
demonstrate that preleukemia can be initiated
in this cellular compartment, contrary to pre-
vious hypotheses that megakaryocytic-erythroid
progenitor cells are the cell of origin for pre-
leukemia, a prediction derived from their ex-
pansion in the HSPC hierarchy of T21-FL ( 50 ).
The reduced proliferative capacity of T21 LT-
HSCs is offset by the acquisition ofGATA1
mutations, providing a possible explanation
for the observed selection ofGATA1muta-
tions in the context of T21. However, the in-
creased function provided by GATA1 mutation
comes at a cost, including the development
of preleukemia and a hindrance in erythrocyte
maturation—a result consistent with the ane-
mia seen in GATA1-deficient mice and humans( 51 – 54 ). However, because we based our study
on well-characterized functional HSPC sub-
populations, we cannot exclude the possibility
that undefined populations may also be able
to initiate preleukemia ( 55 ). In contrast to
the LT-HSC origin for preleukemia, leukemic
progression can occur in multiple types of
downstream progenitors in addition to LT-
HSCs. The overall pool of progenitors is vastly
expanded owing to GATA1s-priming, provid-
ing a large reservoir for acquisition of sec-
ondary mutations in genes such asSTAG2and
thereby increasing the probability of leukemic
progression. Enhanced self-renewal mediated
by STAG2 deficiency could explain why it is
subsequently selected for during leukemic evo-
lution. Selection could also arise from STAG2
deficiency resulting in a temporary increase in
mature erythroid output, as seen in our in vitro
single-cell differentiation assays. Erythroid cells
make up the vast majority of the numerical
daily output of the blood system, raising the
possibility that there may be strong evolution-
ary pressure for the erythropoiesis-defective
GATA1s-mutated clones to reacquire erythroid
potential through additionalSTAG2mutation.
Further, this leukemic transformation can only
occur during fetal and early postnatal develop-
ment but not in adult BM stem cells. Thus, our
study reveals how critical it is to understand
the identity and the developmental stage of the
cell type that acquires genetic drivers during
leukemogenesis. Moreover, genetic drivers of
leukemia are typically distinct between pedi-
atric and adult acute myeloid leukemia ( 56 ), so
our findings uncover that the basis for the dif-
ferential leukemic potential could possibly be
the developmental status of the cell of origin.
Our findings establish that initiation of
GATA1s-induced preleukemia is dependent
on T21, which exerts its effects at least in
part through up-regulation of Chr21 miRNAs—
specifically,miR-99a,miR-125b-2, andmiR-155—
exclusively within the LT-HSC compartment.
This result refines previous suggestions that
deregulated expression of many Chr21 genes
contributes to Down syndrome leukemogenesis
and extends them to noncoding RNAs ( 57 ).
These three miRNAs are highly expressed in
leukemia-initiating populations of adult acute
myeloid leukemia ( 58 ), and miR-125b-2 has
been shown to be a potential oncomiR ( 59 ).
Children with Down syndrome are also at high
risk for developing B cell ALL ( 60 ), and it will
be important to determine whether this mech-
anism of predisposition also affects ALL de-
velopment. Although preleukemic initiation is
dependent on T21, we made the unexpected
finding that progression to leukemia is inde-
pendent of T21 and can be induced by defi-
ciency of STAG2 in combination with GATA1s.
We found that preleukemic and leukemic pop-
ulations were similar with respect to expres-
sion of lineage markers on blasts, enrichmentWagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 10 of 13
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