of genetic drivers ( 61 )lendssupporttothe
concept of convergence. Last, although our
results demonstrate that Down syndrome
leukemogenesis can be modeled by a sequence
of cell-intrinsic mechanisms, we cannot exclude
an additional role for the T21 microenviron-
ment in leukemic evolution in individuals with
Down syndrome.
Through analysis of individual preleukemic
and leukemic cell fractions isolated on the basis
of primitive stem cell markers and propagated
in serial transplants, we identified CD117/KIT
as a marker of disease-driving cells. Further,
pharmacological KIT inhibition targeted pre-
leukemic stem cells, and this proof of concept
could open an approach for therapy at the pre-
leukemic stage. This would limit the pool of
GATA1s-primed progenitors that can acquire
additional mutations and ultimately inhibit
progression to ML-DS. Such a scenario could
foreseeably work in combination with refined
clinical scores capable of identifying patients
with TAM who have risk of progression toward
leukemia subtypes that have poor outcome
( 62 ). However, this concept requires further
in-depth preclinical and clinical assessment.
Our findings not only provide insight into
Down syndrome leukemogenesis but also have
implications for pediatric leukemia in general.
Sequencing data from newborns indicated that
the first genetic alterations for many subtypes
of childhood leukemia occur during fetal de-
velopment ( 63 – 65 ). Our results potentially
suggest that the cellular origin of preleukemic
mutations in other pediatric leukemia may also
be LT-HSCs, which is supported by that it can
take years after birth until leukemia is diag-
nosed ( 66 ). Early targeting of preleukemia
during the newborn phase could become a
clinical paradigm for pediatric leukemia. Last,
numerical and structural alterations of Chr21
are extremely common in hematological malig-
nancies in both children and adults ( 67 ). Gains
of Chr21 are seen in up to 35% of cases in sev-
eral types of acute leukemia ( 68 ). Thus, it will
be important to identify mechanisms includ-
ing but not limited to dysregulation of Chr21
miRNAs that are shared among those cases.
Materials and methods summary
All mouse experiments were approved by the
University Health Network (UHN) Animal
Care Committee, and we confirm that all ex-
periments conform to the relevant regulatory
and ethical standards. All xenotransplanta-
tions were performed in 8- to 12-week-old
femaleNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG)
mice (JAX) that were sublethally irradiated
with 225cGy, 24 hours before transplanta-
tion, or in 8- to 12-week-old femaleNOD.
Cg-PrkdcscidIl2rgtm1WjlKitem1Mvw/SzJ(NSGW41)
mice that were not irradiated. Human fetal
liver samples were obtained from elective preg-
nancy terminations at Mount Sinai Hospital
with informed consent in accordance to guide-
lines approved by the Mount Sinai Hospital
Research Ethics Board (18-0093-E) and the
UHN Research Ethics Board (02-0763). Fetal
liver samples of normal disomic karyotype and
T21 were collected at 16 to 19 weeks gestation
from either sex. Fetal liver samples were pro-
cessed within 1 to 3 hours, and CD34+cells were
isolated by use of the human CD34 MicroBead
kit (Miltenyi Biotec) according to the manu-
facturer’s protocol (table S11). For all in vitro
and in vivo experiments, the full stem and pro-
genitor hierarchy was used to sort LT-HSCs,
ST-HSCs, CMPs, and MEPs ( 26 ). CRISPR/Cas9
RNP electroporations were carried out by
using chemically synthesized gRNAs (IDT),
recombinant Cas9 nuclease (IDT), and the
4D-Nucleofector (Lonza) as previously de-
scribed ( 22 ). Single-cell in vitro assays were
performed in erythro-myeloid-megakaryocytic
promoting medium. Xenotransplantations
of CRISPR/Cas9–edited LT-HSCs were per-
formed at cell doses of 300 to 400 per mouse,
unless otherwise specified, to obtain near-
clonal xenotransplantations.REFERENCESANDNOTES- H. Hasle, I. H. Clemmensen, M. Mikkelsen, Risks of leukaemia
and solid tumours in individuals with Down’s syndrome.Lancet
355 , 165–169 (2000). doi:10.1016/S0140-6736(99)05264-2;
pmid: 10675114 - M.F.Greaves,A.T.Maia,J.L.Wiemels,A.M.Ford,
Leukemia in twins: Lessons in natural history.Blood 102 ,
2321 – 2333 (2003). doi:10.1182/blood-2002-12-3817;
pmid: 12791663 - J. K. Hitzler, J. Cheung, Y. Li, S. W. Scherer, A. Zipursky, GATA1
mutations in transient leukemia and acute megakaryoblastic
leukemia of Down syndrome.Blood 101 , 4301–4304 (2003).
doi:10.1182/blood-2003-01-0013; pmid: 12586620 - I. Robertset al., GATA1-mutant clones are frequent and
often unsuspected in babies with Down syndrome:
Identification of a population at risk of leukemia.Blood 122 ,
3908 – 3917 (2013). doi:10.1182/blood-2013-07-515148;
pmid: 24021668 - J. Wechsleret al., Acquired mutations in GATA1 in the
megakaryoblastic leukemia of Down syndrome.Nat. Genet. 32 ,
148 – 152 (2002). doi:10.1038/ng955; pmid: 12172547 - S. Hoelleret al., Morphologic and GATA1 sequencing analysis
of hematopoiesis in fetuses with trisomy 21.Hum. Pathol.
45 , 1003–1009 (2014). doi:10.1016/j.humpath.2013.12.014;
pmid: 24746204 - J. W. Taubet al., Prenatal origin of GATA1 mutations may be
an initiating step in the development of megakaryocytic
leukemia in Down syndrome.Blood 104 , 1588–1589 (2004).
doi:10.1182/blood-2004-04-1563; pmid: 15317736 - M. Uffmannet al., Therapy reduction in patients with Down
syndrome and myeloid leukemia: The international ML-DS
2006 trial.Blood 129 , 3314–3321 (2017). doi:10.1182/
blood-2017-01-765057; pmid: 28400376 - J. W. Taubet al., Improved outcomes for myeloid leukemia of
Down syndrome: A report from the Children’s Oncology
Group AAML0431 trial.Blood 129 , 3304–3313 (2017).
doi:10.1182/blood-2017-01-764324; pmid: 28389462 - A. S. Gamiset al., Natural history of transient
myeloproliferative disorder clinically diagnosed in Down
syndrome neonates: A report from the Children’s Oncology
Group Study A2971.Blood 118 , 6752–6759, quiz 6996 (2011).
doi:10.1182/blood-2011-04-350017; pmid: 21849481 - M. Labuhnet al., Mechanisms of progression of myeloid
preleukemia to transformed myeloid leukemia in children with
Down syndrome.Cancer Cell 36 , 340 (2019). doi:10.1016/
j.ccell.2019.08.014; pmid: 31526763 - K. Yoshidaet al., The landscape of somatic mutations in Down
syndrome-related myeloid disorders.Nat. Genet. 45 ,
1293 – 1299 (2013). doi:10.1038/ng.2759; pmid: 24056718
13. D. Honget al., Initiating and cancer-propagating cells in
TEL-AML1-associated childhood leukemia.Science 319 , 336– 339
(2008). doi:10.1126/science.1150648; pmid: 18202291
14. J.-H. Klusmannet al., Treatment and prognostic impact of
transient leukemia in neonates with Down syndrome.Blood 111 ,
2991 – 2998 (2008). doi:10.1182/blood-2007-10-118810;
pmid: 18182574
15. A. D. Sorrellet al., Favorable survival maintained in children
who have myeloid leukemia associated with Down syndrome
using reduced-dose chemotherapy on Children’s Oncology
Group trial A2971: A report from the Children’s Oncology
Group.Cancer 118 , 4806–4814 (2012). doi:10.1002/
cncr.27484; pmid: 22392565
16. T. Tagaet al., Clinical characteristics and outcome of
refractory/relapsed myeloid leukemia in children with Down
syndrome.Blood 120 , 1810–1815 (2012). doi:10.1182/
blood-2012-03-414755; pmid: 22776818
17. J. K. Hitzleret al., Outcome of transplantation for acute
myelogenous leukemia in children with Down syndrome.Biol.
Blood Marrow Transplant. 19 , 893–897 (2013). doi:10.1016/
j.bbmt.2013.02.017; pmid: 23467128
18. J. T. Caldwell, Y. Ge, J. W. Taub, Prognosis and management of
acute myeloid leukemia in patients with Down syndrome.
Expert Rev. Hematol. 7 , 831–840 (2014). doi:10.1586/
17474086.2014.959923; pmid: 25231553
19. K. A. Alfordet al., Perturbed hematopoiesis in the Tc1 mouse
model of Down syndrome.Blood 115 , 2928–2937 (2010).
doi:10.1182/blood-2009-06-227629; pmid: 20154221
20. G. Kirsammeret al., Highly penetrant myeloproliferative
disease in the Ts65Dn mouse model of Down syndrome.Blood
111 , 767–775 (2008). doi:10.1182/blood-2007-04-085670;
pmid: 17901249
21. S. Malingeet al., Increased dosage of the chromosome 21
ortholog Dyrk1a promotes megakaryoblastic leukemia in a
murine model of Down syndrome.J. Clin. Invest. 122 , 948– 962
(2012). doi:10.1172/JCI60455; pmid: 22354171
22. E. Wagenblastet al., Functional profiling of single CRISPR/
Cas9-edited human long-term hematopoietic stem cells.
Nat. Commun. 10 , 4730–11 (2019). doi:10.1038/
s41467-019-12726-0; pmid: 31628330
23. F. Nottaet al., Evolution of human BCR-ABL1 lymphoblastic
leukaemia-initiating cells.Nature 469 , 362–367 (2011).
doi:10.1038/nature09733; pmid: 21248843
24. K. A. Alfordet al., Analysis of GATA1 mutations in Down
syndrome transient myeloproliferative disorder and myeloid
leukemia.Blood 118 , 2222–2238 (2011). doi:10.1182/
blood-2011-03-342774; pmid: 21715302
25. A. Royet al., Perturbation of fetal liver hematopoietic stem and
progenitor cell development by trisomy 21.Proc. Natl. Acad.
Sci. U.S.A. 109 , 17579–17584 (2012). doi:10.1073/
pnas.1211405109; pmid: 23045701
26. F. Nottaet al., Distinct routes of lineage development reshape
the human blood hierarchy across ontogeny.Science 351 ,
aab2116 (2016). doi:10.1126/science.aab2116; pmid: 26541609
27. S. Rahmiget al., Improved human erythropoiesis and platelet
formation in humanized NSGW41 mice.Stem Cell Reports
7 , 591–601 (2016). doi:10.1016/j.stemcr.2016.08.005;
pmid: 27618723
28. C. Langebrake, U. Creutzig, D. Reinhardt, Immunophenotype of
Down syndrome acute myeloid leukemia and transient
myeloproliferative disease differs significantly from other
diseases with morphologically identical or similar blasts.Klin.
Padiatr. 217 , 126–134 (2005). doi:10.1055/s-2005-836510;
pmid: 15858703
29. L. Wanget al., Acute megakaryoblastic leukemia associated
with trisomy 21 demonstrates a distinct immunophenotype.
Cytometry B Clin. Cytom. 88 , 244–252 (2015). doi:10.1002/
cytob.21198; pmid: 25361478
30. WHO,WHO Classification of Tumours of Haematopoietic and
Lymphoid Tissues(World Health Organization, 2008).
31. N. Bhatnagar, L. Nizery, O. Tunstall, P. Vyas, I. Roberts,
Transient abnormal myelopoiesis and AML in Down syndrome:
An update.Curr. Hematol. Malig. Rep. 11 , 333–341 (2016).
doi:10.1007/s11899-016-0338-x; pmid: 27510823
32. O. Tunstallet al., Guidelines for the investigation and management
of transient leukaemia of Down syndrome.Br. J. Haematol. 182 ,
200 – 211 (2018). doi:10.1111/bjh.15390; pmid: 29916557
33. H. Döhneret al., Diagnosis and management of AML in adults:
2017 ELN recommendations from an international expert
panel.Blood 129 , 424–447 (2017). doi:10.1182/
blood-2016-08-733196; pmid: 27895058
34. A. D. Vinyet al., Cohesin members Stag1 and Stag2 display
distinct roles in chromatin accessibility and topological control
Wagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 12 of 13
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