Science - 31 January 2020

thatNpm1cmutant LT-GMPs may acquire fur-
ther mutations over time, which has been shown
to occur in this and otherNpm1cknock-in
mouse models ( 7 , 12 ). Furthermore, mouse
Npm1c/Dnmt3amutant leukemia cells showed
highly up-regulatedHoxa/bexpression, which

resembled expression patterns observed in

humanNPM1cAML and otherHOX-associated

AMLs such asMLL-AF9 AML (Fig. 2E and fig.

S3, C and D). These results confirm that pre-

leukemicNpm1cmutant LT-GMPs eventually

give rise to leukemia.

We have previously shown that inhibition of

the interaction between the histone methyl-

transferase MLL1 and adaptor protein Menin

reverses leukemogenic gene expression in the

NPM1cAML cell line OCI-AML3 ( 13 ). Menin-MLL

interaction inhibitors were originally developed

Uckelmannet al.,Science 367 , 586–590 (2020) 31 January 2020 2of5

LT-HSC MPP CMP GMP

0.0

0.5

1.0

1.5

2.0

2.5

Rel.

Hoxa9

Expression (

GAPDH

)

WT

Dnmt3a

Npm1c

ns *

**

***

**

*

Npm1c/Dnmt3a

***

nsns

ns

ns

ns

GMPs

LSKs

CRE virus

Npm1cx-cA/+

Dnmt3aR878H/+

0

10

20

30

40

50

% Blood Engraftment

WT Dnmt3a Npm1c Npm1c/

Dnmt3a

4 8 12 16 4 8 12 16 4 8 12 16 4 8 12 16weeks

GMP LSK

WT Npm1c WT Npm1c

Hoxa10

Hoxa5

Fb n 1

Slc18a1

Hoxa3

Hoxaas3

Hoxa9

Ctsf

Hoxb5

Hoxa6

Hoxa2

Ra g 2

Hoxa7

Cd27

Icos

Mira

Hoxb3

Il12rb1

Hnf4a

Kif5a

Ptprf

Gpr133

Igfbp7

Hoxb2

Nkx2-3

0

2

4

6

8

Rel.

Hoxa9

Expression (

GAPDH

)

*

*

**

**

ns

ns

LSK GMP

WT

Dnmt3a

Npm1c

Npm1c/Dnmt3a

ABC

MxCreNpm1cx-cA/+

Gene

expression expressionGene

GMPs

Dnmt3a HSCs

R878H/+

pIpC

D

E

Fig. 1.Npm1cinduces self-renewal properties in myeloid progenitor
cells.(A)Hoxa9gene expression inNpm1c,Dnmt3a,andNpm1c/Dnmt3a
mutant LT-HSCs, multipotential progenitors (MPPs), common myeloid
progenitors (CMPs), and GMPs 16 weeks after pIpC injection (n≥3 mice per
group; error bars indicate mean ± SD). Rel., relative; GAPDH, glyceraldehyde
3-phosphate dehydrogenase. (B) Heatmap showing the top 25
up-regulated genes inNpm1cversus WT LSK cells and GMPs, 4 weeks
after pIpC treatment (n= 3 mice per group). (C) Relative expression ofHoxa9

3 days after in vitroCretransduction (n≥4 mice per group; error bars indicate

mean ± SEM). The dotted lines indicateHoxa9expression level from freshly

isolated LSK cells and GMPs. (D) Peripheral blood percentage CD45.2 engraftment

of WT andNpm1c,Dnmt3a,andNpm1c/Dnmt3amutant GMPs sorted 4 weeks

after pIpC induction, transplanted into lethally irradiated recipients. Error bars

indicate mean ± SEM. (E) Summary table of GMP-transplanted mouse numbers

and percentages engrafted≥1% for >12 weeks.MIG-CRE, MSCV-CRE-IRES-GFP

retrovirus. ns, not significant; *P< 0.05; **P< 0.01; ***P< 0.001.

0 100 200

0

50

100

Days elapsed

Percent survival

LSKNpm1c/Dnmt3a

GMPNpm1c/Dnmt3a

GMPNpm1c

GMPWT

ns

A

B

C

WT LSK-derived GMP-derived GMP-derived

Dnmt3a/Npm1c Npm1c

GMP

WT

GMP

Npm1c

GMP

Npm1c/Dnmt3a

LSK

Npm1c/Dnmt3a

0.0

0.2

0.4

0.6

0.8

S pleen weight [g ]

*

* ns ns

D E

Fig. 2. Myeloid progenitors are leukemia-initiating cells inNpm1cAML.(A) Experimental overview of

secondary transplantation of long-term engrafted mutant GMPs. (BtoD) Kaplan-Meyer survival analysis (B),

representative spleen images (C), and spleen weights (D) of secondary transplants ofMIG-CRENpm1c/Dnmt3a

LT-GMPs or LSK-derived cells andMxCreNpm1cLT-GMPs (n≥4 mice per group). One-way analysis of variance

(ANOVA) was performed. Error bars indicate mean ± SD. *P< 0.05; ns, not significant. (E) Top 10 up-regulated

genes inMIG-CRENpm1c/Dnmt3amutant leukemic GMPs compared with WT GMPs (n=3micepergroup).

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