Science - USA (2021-07-09)

of GATA1-binding sites at their promoters,
and down-regulated pathways compared with
T21 controls. Nevertheless, the addition of
STAG2ko to GATA1s-bearing cells led to en-
hanced self-renewal, as evident in the increased

frequency of leukemia-initiating cells enriched

in CD117+cells. Similar leukemic transforma-

tion was observed upon induced deficiency

of other cohesin genes in combination with

GATA1s,andthus,itispossiblethattheeffects

of these mutations converge on an increase in

self-renewal and stemness programs in gen-

eral. LSC17 stemness signature being strongly

associated with survival across a wide spectrum

of acute myeloid leukemia patients irrespective

Wagenblastet al.,Science 373 , eabf6202 (2021) 9 July 2021 11 of 13

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

0

20

40

60

80

100

% Human cells

Unknown

Blast

Myeloid cells

(not blast-like)

Erythroid

precursors

Lymphocytes

Control GATA1s

GATA1s/

STAG2ko

N-FL CD1

17 high

T21-F

L CD117 high

N-CB CD1

17 high

N-CB CD

117 low

0

10

20

30

40

50

60

% CD45+ Engraftment

LT-HSCs

*

Out of uncultured LT-HSCs:

CD117 high - 23.0%

CD34-APC-Cy7

CD117-PE

CD117 low - 77.0%

CD117 high - 14.0%

CD117 low - 86.0%

CD117 high - 74.7%

CD117 low - 19.3%

CD117 high - 61.3%

CD117 low - 30.5%

T21-FL N-BM

N-FL N-CB

Sort LT-HSCs

from T21-FL

Electroporation of RNPs

against GATA1/STAG2

Xenotransplantation

(10 weeks)

KIT inhibition

Cas9

daily for 2 weeks - Blast

Leukemic analysis:

flow cytometry

N-FL CD1

17 high

T21-FL CD1

17 high

N-CB CD1

17 high

N-CB CD1

17 low

0

20

40

60

80

100

B-Lymphoid

Myeloid

Erythroid

T-cells

Megakaryocytes

% Lineage distribution

LT-HSCs

E

AB CD

F

H I

G

J

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

0

10

20

30

40

50

% CD45+ Engraftment

**

Control GATA1s

GATA1s/

STAG2ko

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

0

20

40

60

80

% CD117+CD45+ (out of blast gate)

*** *** ***

Control GATA1s

GATA1s/

STAG2ko

*

T21-FL

GATA1s

Vehicle

T21-FL

GATA1s

Imatinib

T21-FL

GATA1s

Dasatinib

57.1% 16.4% 21.6% 8.11% 19.1% 15.1%

24.7% 1.79% 66.2% 4.02% 59.7% 6.14%

FSC-A

SSC-A

CD34-BV421

CD117-PE

CD45+ Blast:

T21-FL

GATA1s

Ripretinib

15.2% 7.99%

72.3% 4.58%

Out of CD45+:

13.7%

Blast

4.04%

Blast

7.58%

Blast

3.86%

Blast

VehicleImatinib

DasatinibRipretinib

VehicleImatinib

DasatinibRipretinib

0

20

40

60

80

% CD117+CD45+ (out of blast gate)

*** **** ***

17003 17041

*** *** ***

Sorted 1° transplantation Stem cell

population engrafted/injected frequency

20 0/5

CD117 high 80 3/5

N-FL 320 5/5

LT-HSC 20 0/5

CD117 low 80 0/4

320 1/5

20 0/5

CD117 low 80 2/5

T21-FL 320 5/5

LT-HSC 20 0/5

CD117 low 80 0/5

320 0/4

N-CB CD117 high 320 5/5 <1/320

LT-HSCCD117 low 320 4/4 <1/320

1/131

0

Source Dose

1/99

1/1,936

Fig. 6. KIT inhibition targets preleukemic-initiating cells and inhibits leukemic
progression.(A) Immunophenotypic profile of CD117 and CD34 expression of
isolated LT-HSCs from N-FL and T21-FL, normal disomic CB (N-CB), and normal
disomic BM (N-BM). (B) CD117-low and CD117-high LT-HSCs were transplanted at
defined doses into NSG mice for 20 weeks. Resulting stem cell frequencies are
depicted (>0.1% CD45+cells in BM was defined as engraftment,n=4or5miceper
condition, total 67 mice). (C) Engraftment of N-FL CD117-high, T21-FL CD117-high,
N-CB CD117-high, and N-CB CD117-low LT-HSCs transplanted into NSG mice
(n= 4 to 8 mice per condition). (D) Lineage marker distribution based on cell surface
markers of engrafted NSG mice from (C). (E) Experimental overview of control-,
GATA1s-, and GATA1s/STAG2ko–edited T21-FL LT-HSCs transplanted into NSG
mice, which were subsequently treated twice daily with small-molecule inhibitors

against KIT for 2 weeks. (F) Engraftment of T21-FL LT-HSCs transplanted into

NSG mice treated with vehicle, imatinib, dasatinib, or ripretinib (n= 4 or 5 mice

per condition). (G) Quantification of cell morphology of human cells prepared

by using cytospin in transplanted NSG mice described in (F) (n= 400 cells per

condition). (H) Flow cytometry plots showing blast populations out of CD45+

cells in primary xenografts described in (F). (I) Quantification of CD117+CD45+

blasts in transplanted NSG mice described in (F). (J) Quantification of

CD117+CD45+blasts in NSGW41 mice transplanted with primary sample TAM

17003 and NSG mice transplanted with primary sample TAM 17041 and

treated with vehicle, imatinib, dasatinib, or ripretinib (n= 5 mice per condition).

Unpaired Student’sttest: *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001;

error bars indicate standard deviation.

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