Science - USA (2021-11-05)

table S2). The mutant marrow cells expressed
a stimulated inflammatory response relative
to controls, with increased expression of genes
such asfosabandchac1(fig. S7, A to C). The
source of inflammation appeared to be mutant
macrophages and neutrophils, as niche cells
were devoid of inflammatory cytokine ex-
pression (Fig. 3C and fig. S7, A and B). Elevated
inflammatory cytokine levels have been de-
scribed in clonal hematopoiesis, but their ef-
fect on clonal expansion is not known ( 13 , 14 ).
Acute inflammation enhances HSPC cycling
but can lead to exhaustion if it persists ( 15 ),
and chronic infection can potentiateDnmt3a
mutant hematopoiesis ( 16 ). We hypothesized
that mutant HSPCs moderate their response
to chronic inflammation maintained by their
mutant mature progeny, rendering them re-
sistant to replicative exhaustion. We identified
elevated gene expression of suppressors of in-
flammation, such asnr4a1andsocs3a,inmu-
tant HSPCs and progenitors (Fig. 3C and fig.
S7, A and B). We compared the gene expres-
sion of sorted marrow cells of dominant clones
from mutant zebrafish to that of total marrow
cells from control zebrafish and found simi-
larly dysregulated inflammatory gene signatures
(Fig. 3C and fig. S7D), suggesting that mutant
dominant clones express an anti-inflammatory
gene signature in response to inflammation.

Runx1deficiency has been associated with in-

flammation ( 17 ). A scRNA-seq comparison of

germline homozygousrunx1mutants with oli-

goclonal hematopoiesis to heterozygous mutants

with polyclonal hematopoiesis revealed a sim-

ilar pro-inflammatory/anti-inflammatory reg-

ulatory axis (fig. S8).

We reasoned that genetic abrogation of the

anti-inflammatory response in mutant clones

would reduce their fitness. We used TWISTR

to generate a cohort with gRNAs targeting

asxl1andnr4a1, a gene that encodes a nu-

clear receptor antagonizing immune activa-

tion and overexpressed in dominant mutant

clones ( 18 ) (Fig. 4A). We observed normal

hematopoiesis in these zebrafish (fig. S9, A

and B). We sorted 306 clusters from 20 zebra-

fish injected withasxl1-targeting gRNA and

58 zebrafish injected with gRNAs targeting

bothasxl1andnr4a1, and assessed indels (Fig.

4B). Of these, 186 harbored deleteriousasxl1

indels: 48 fromasxl1-injected and 138 from

asxl1/nr4a1-injected zebrafish. Comparing clus-

ter sizes, we observed that clusters with biallelic

deleterious indels ofnr4a1were significantly

smaller relative to those with nonr4a1indels,

in-frame/no indels, or heterozygous deleteri-

ous indels (Fig. 4C and fig. S9C).Asxl1-mutant

clones with biallelicnr4a1indels diminished

in peripheral blood over time, whereasasxl1-

mutant clones with intactnr4a1expanded

(fig. S9D). There was no difference between

wild-typeasxl1clusters acrossnr4a1genotypes,

which suggests thatnr4a1alone is not suffi-

cient to modulate clonal fitness (fig. S9E).

Within individual zebrafish, smallerasxl1-

mutant clusters were more likely than larger

asxl1-mutant clusters to harbor homozygous

deleteriousnr4a1indels (fig. S9F). These re-

sults suggest that a blunted anti-inflammatory

response inasxl1-mutant clones due tonr4a1

loss diminished their clonal fitness.

Our results provide evidence that progeny of

mutant clones generate an inflammatory envi-

ronment to which they are resistant (Fig. 4D).

Certain mutations in clonal hematopoiesis have

an association with specific cytokines, including

interleukin-6 withTET2mutations and tumor

necrosis factor–awithASXL1mutations ( 13 , 14 ).

It is plausible that there is mutation-dependent

cytokine expression in mutant mature cells, and

an up-regulation of mutation-associated anti-

inflammatory pathways in mutant progenitor

cells.Thus,clonalfitnessisachievedbyapro-

survival network engaged by mutations, which

offers an explanation for how gene mutations

with diverse functions confer similar clonal fit-

ness. Our study developed a genetic and live

clonal tracking technology to dissect the pro-

gression to clonal dominance, identifying new

SCIENCEscience.org 5 NOVEMBER 2021•VOL 374 ISSUE 6568 771

A

BCD

nr4a1

WT

-gRNA+gRNA

nr4a1

HET

0

20

40

60

80

Cluster size (%)

nr4a1

HOMO

*

*

*

nr4a1

WT

Cluster size (%)

0

10

20

30

40

50

60

70

frameshift

in-frame

Dominant clone

2bp D

9bp I

2bp D

asxl1 nr4a1

alleles wt

Small clone

alleles

5bp D

wt

2bp D

2bp D

asxl1 nr4a1

Grow to

adulthood

creER Clonal competition

drl T2

ubi

Color labeling

at 24 hpf

Sort clones

by FACS

DNA-seq

asxl1 gRNA

+/- nr4a1 gRNA

+ cas9 mRNA

Somatic mosaic mutants

3-5 months

asxl1

nr4a1

HSPCs

inflammatory

cytokines

mature progeny

asxl1 mutation

nr4a1

response

differentiation

Clonal fitness

Clusters with fs asxl1 indels nr4a1 mutation

Fig. 4. Biallelicnr4a1loss abrogates fitness ofasxl1-mutant clones.
(A) Testing the effect ofnr4a1with TWISTR. (B) Example DNA-seq analysis
of sorted clusters from a single zebrafish. D, deletion; I, insertion; bp, base pair.
(C) Size of sortedasxl1-mutant clusters in zebrafish injected with onlyasxl1
gRNA (nr4a1WT,n= 48) or withasxl1andnr4a1gRNAs that resulted in no

deleteriousnr4a1indels (nr4a1WT,n= 34), heterozygous deleterious indels

(nr4a1HET,n= 58), or homozygous deleterious indels (nr4a1HOMO,n= 46).

*P= 0.029asxl1-onlynr4a1WTversusnr4a1HOMO,P= 0.014nr4a1WTversusnr4a1HOMO,

P= 0.013nr4a1HETversusnr4a1HOMO(unpairedttest). (D) Model of inflammation-

driven clonal fitness with acquired mutations.

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