in secondary recipients (fig. S9, I and J). Taken
together, our results, on the basis of the assess-
ment of these defined HSPC subpopulations,
suggest that the GATA1s preleukemia-initiating
event likely occurs in LT-HSCs and not down-
stream progenitors. However, subsequent
STAG2mutations are not limited to LT-HSCs
but can be acquired further downstream in the
expanded pool of GATA1s-primed progenitor
cells, highlighting that the preleukemic andleukemic events could occur in distinct cells
of origin.
To verify whether leukemic transformation
can be induced in a stepwise transplantation
setting and to confirm whether progenitor-likeWagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 7 of 13
T21-FL
ST-HSC
GATA1s/STAG2koT21-FL
CMP
GATA1s/STAG2koT21-FL
MEP
GATA1s/STAG2koFSC-ASSC-ACD34-BV421CD117-PECD45+ Blast:Out of CD45+:Sort progenitors
from N-FL and T21-FLElectroporation of RNPs
against GATA1/STAG2Xenotransplantation
(12 weeks)Leukemic analysis:
flow cytometry + genotypingCas9+- Blast
AB CD E FGCohesinEpigeneticT21-FL
CMP+MEP
GATA1s +Average
% CD45
Engraftment
STAG2 19.8
RAD21 15.0
NIPBL 6.4
SMC1A 19.4
SMC3 2.1
CTCF -
KANSL1 2.7
EZH2 -
Control -(out of 5)Engrafted miceHSort CMP+MEP
from T21-FLElectroporation of RNPs
against GATA1 + candidateXenotransplantation
(12 weeks)Leukemic analysis:
flow cytometry + genotypingCas9+- Blast
Pool and screenT21-FL
CMP+MEP
GATA1s + KANSL1T21-FL
CMP+MEP
GATA1s + SMC3T21-FL
CMP+MEP
GATA1s + SMC1AT21-FL
CMP+MEP
GATA1s + NIPBLT21-FL
CMP+MEP
GATA1s + RAD21T21-FL
CMP+MEP
GATA1s + STAG2FSC-ASSC-ACD34-BV421CD117-PECD45+ Blast:Out of CD45+:ControlGATA1sSTAG2koGATA1s/STAG2koControlGATA1sSTAG2koGATA1s/STAG2koControlGATA1sSTAG2koGATA1s/STAG2ko0.010.1110100% CD45+ EngraftmentN-FL: ST-HSC CMP MEPControlGATA1sSTAG2koGATA1s/STAG2koControlGATA1sSTAG2koGATA1s/STAG2koControlGATA1sSTAG2koGATA1s/STAG2ko0.010.1110100% CD45+ EngraftmentT21-FL: ST-HSC CMP MEPSSC-AST-HSC
CMPMEP
ST-HSC
CMPMEP020406080100% Human cellsGATA1s/STAG2ko GATA1s/STAG2koN-FL T21-FLUnknownBlast
Myeloid cells
(not blast-like)Erythroid
precursorsLymphocytesFig. 4. Combined GATA1s and STAG2ko drive leukemic progression in
progenitors.(A) Experimental overview of sorting N-FL and T21-FL derived
progenitor cells for CRISPR/Cas9 editing and transplanting into NSGW41
mice. (B) Engraftment of N-FL ST-HSC, CMP, and MEP grafts in NSGW41 mice
(all mice are shown regardless of CD45+engraftment,n=4or5miceper
condition). (C) Engraftment as described in (B) for T21-FL (n= 5 mice per
condition). (D) Quantification of cell morphology of human cells prepared by
using cytospin in N-FL and T21-FL GATA1s/STAG2ko grafts in NSGW41 mice
from(B)and(C)(n= 400 cells per condition). (E) Flow cytometry plots
depicting the blast population out of CD45+cells in primary xenografts of
T21-FL GATA1s/STAG2ko progenitors in NSGW41 mice, as described in (C).
The CD34/CD117 profiles out of the CD45+blast populations are depicted
below. (F) Experimental overview of sorting T21-FL CMPs and MEPs to conduct
a loss-of-function screen to identify genes that endow leukemic progression
in combination with GATA1s. (G) Result of screen described in (F) showing
the number of mice with leukemic phenotypes based on average CD45+
engraftment in BM and blast appearance (>1% CD45+in BM,n= 5 mice per
condition). (H) Flow cytometry plots of blast populations out of CD45+cells in
grafts of T21-FL CMPs and MEPs edited with GATA1s and candidate gene
gRNAs as described in (F).RESEARCH | RESEARCH ARTICLE