TO THE EDITOR:
Loss-o - unc ion lesions impac B-cell de elopmen and fi ness bu
a e insu ficien o d i e CLL in mouse models
Elisa en Hacken,
1,2,
*Shanye Yin,
1,2,
*Robe Redd,
3
Ma ía He n´
andez S´
anchez,
4,5
Kendell Clemen ,
6
Gab iela B uns ing Ho mann,
1
Fa a F. Regis,
1
Elizabe h Wi en,
1
Shuqiang Li,
7,8
Donna Neube g,
3
Luca Pinello,
6
Kenne h J. Li ak,
7,8
and Ca he ine J. Wu
1,2,8,9,
*
1
Depa men o Medical Oncology, Dana-Fa be Cance Ins i u e, Bos on, MA;
2
Ha a d Medical School, Bos on, MA;
3
Depa men o Da a Science, Dana-Fa be Cance
Ins i u e, Bos on, MA;
4
The Ins i u e o Cance Molecula and Cellula Biology–Cance Resea ch Cen e , Ins i u o de In es igaci ´
on Biom ´
edica de Salamanca, Uni e si y o
Salamanca, Salamanca, Spain;
5
Depa amen o Biochemis y and Molecula Biology, Pha macy School, Uni e sidad Complu ense de Mad id, Mad id, Spain;
6
Cen e o
Cance Resea ch, and Cen e o Compu a ional and In eg a i e Biology, Molecula Pa hology Uni , Massachusse s Gene al Hospi al, Bos on, MA;
7
T ansla ional
Immunogenomics Lab, Dana-Fa be Cance Ins i u e, Bos on, MA;
8
B oad Ins i u e o Massachuse s Ins i u e o Technology and Ha a d, Camb idge, MA; and
9
Depa men
o Medicine, B igham and Women’s Hospi al, Bos on, MA
Mouse models a e indispensable ools o s udy he impac o gene ic al e a ions in cance . Only a ew
s udies ha e hus a unde aken unc ional cha ac e iza ions o ecu en genomic-disco e ed gene
mu a ions o ch onic lymphocy ic leukemia (CLL)
1
despi e ecen ad ances in gene ic enginee ing ha
ha e inc easingly acili a ed an imp o ed abili y o assess he in i o e ec s o candida e disease d i e s.
In his con ex , condi ional knockin and knockou s a egies ha e been applied o he s udy o indi idual
lesions, including del(13q), modeled in he MDR mice,
2
he B-cell de elopmen al ac o Ikz 3,
3
o in he
splicing ac o S 3b1 in combina ion wi h A m dele ion,
4
all o which ecapi ula e indolen CLL de el-
opmen , consis en wi h he pheno ype o human disease.
We hypo hesize ha he mos common loss-o - unc ion (LOF) pu a i e d i e s ypical o CLL
(A m,T p53, Chd2, Bi c3, Mga, o Samhd1)
1,5
(supplemen al Figu e 1A-B) may a ec p eleukemic
signaling, hus dys egula ing B-cell–in insic p osu i al pa hways. To add ess his ques ion, we
de eloped a e sa ile pla o m o he in oduc ion o indi idual LOF lesions in i o in mu ine B cells. We
gene a ed a ansplan model ha e ficien ly deli e s single guide RNAs (sgRNAs) o hema opoie ic
s em and p ogeni o cells o s ains exp essing B-cell– es ic ed Cas9 (ie, Cd19-Cas9; Figu e 1A [le ]),
hus gene a ing gene edi s only in de eloping Cas9-exp essing B cells and allowing he in oduc ion o
inse ions-dele ions (indels) a he a ge locus, leading o LOF o he a ge gene. Fi s , we in e c ossed
mice ca ying B-cell– es ic ed C e exp ession (Cd19-c e) wi h hose ca ying condi ional Cas9–g een
fluo escen p o ein (Cas9-GFP)
6
o gene a e a s ain exp essing B-cell– es ic ed Cas9 (Cd19-Cas9)
(Figu e 1A [le ]). Nex , we op imized he in i o enginee ing o ea ly s em and p ogeni o cells (ie, Lin
-
Sca-1
+
c-ki
+
[LSK]) om Cd19-Cas9 mice using len i i us exp essing sgRNAs (mChe y
+
) a ge ing
A m,T p53, Chd2, Bi c3, Mga, o Samhd1. We chose LSKs because o hei high ansducibili y and
long- e m epopula ing po en ial and designed sgRNAs a ge ing ea ly exonic egions in each o he
6 genes o allow ea ly dis up ion o he p o ein sequence (supplemen al Table 1). Las ly, we ans-
plan ed he single sgRNA-exp essing LSKs in o suble hally i adia ed CD45.1 ecipien mice. We
confi med he consis en p esence o ~45% o 85% gene-edi ed sequences (>70% ca ying ame-
shi mu a ions, he majo i y esul ing om 1 o 2 base pai indels; supplemen al Figu e 2) in edi ed
B cells (GFP
+
mChe y
+
, isola ed om he pe iphe al blood by cell so ing), bu no in T cells o
monocy es, a 4 mon hs a e ansplan by polyme ase chain eac ion-based a ge ed deep sequencing
(analyzed by CRISPResso2 so wa e
7
;Figu e 1A [ igh ]; supplemen al Figu e 1C-D). A mino i y o
lesions (<30%) led o in- ame al e a ions (ie, ±3 nucleo ides in leng h). Rep oducible on- a ge edi ing
Submi ed 11 Oc obe 2022; accep ed 23 No embe 2022; p epublished online on
Blood Ad ances Fi s Edi ion 7 Decembe 2022; inal e sion published online 11
Augus 2023. h ps://doi.o g/10.1182/bloodad ances.2022009135.
*E.H., S.Y., and C.J.W. con ibu ed equally o his s udy.
Mouse RNA-seq da a epo ed in his a icle ha e been deposi ed in he Gene
Exp ession Omnibus da abase (accession numbe GSE197061).
Da a a e a ailable on eques om he co esponding au ho , Ca he ine J. Wu (cwu@
pa ne s.o g).
The ull- ex e sion o his a icle con ains a da a supplemen .
© 2023 by The Ame ican Socie y o Hema ology. Licensed unde C ea i e Commons
A ibu ion-NonComme cial-NoDe i a i es 4.0 In e na ional (CC BY-NC-ND 4.0),
pe mi ing only noncomme cial, nonde i a i e use wi h a ibu ion. All o he igh s
ese ed.
RESEARCH LETTER
4514 22 AUGUST 2023 •VOLUME 7, NUMBER 16
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was de ec ed ac oss he 6 coho s (wi h no de ec able gene edi s
in 24 pu a i e o - a ge s), pe single-cell DNA-sequencing analysis
(DNA-seq) o 1787 single B cells (supplemen al Figu e 1E). All
LOF mu a ions we e ound o con e a su i al ad an age o B cells
compa ed wi h he non- a ge ing sc amble con ol when exposed
o he mi ogenic s imuli lipopolysaccha ide (LPS) and in e leukin-4
(IL-4) o 72 hou s in i o (n = 5 pe g oup; P< .05; Figu e 1B).
Likewise, ci cula ing edi ed B cells om animals ollowing ans-
plan we e ele a ed ac oss he mu a ed models compa ed wi h he
non- a ge ing con ol line (N -1;P< .05; RM-ANOVA; Figu e 1C).
Despi e hese ea ly changes, mos ly ma ked a 4 mon hs a e
ansplan , a ime poin a which B cells a e conside ed o achie e
op imal hos econs i u ion, none o he single-edi ed animals
(n = 10 pe g oup) de eloped clonal B220
+
CD5
+
Igκ
+
ci cula ing
B-cell expansions o e 24-mon hs o obse a ion. Mga- and Chd2-
deple ed animals showed ajec o ies ha we e pa icula ly sho -
li ed (supplemen al Figu e 1F; P<.0002), in line wi h limi ed
in i o pe sis ence obse ed in p e ious cell line s udies.
8
These
obse a ions collec i ely sugges he dual possibili y o limi ed cell-
in insic dys egula ion o p osu i al pa hways in oduced by hese
lesions and/o insu ficien cell-ex insic con ibu ion o leukemia
pheno ypes in hese o he wise heal hy animals.
We asked whe he any o he modeled lesions (also equen d i e s
o o he agg essi e B-cell malignancies, including di use la ge B-cell
lymphoma)
9
would a ec B-cell de elopmen al ajec o ies
(supplemen al Figu e 3A; supplemen al Table 2). Al hough ansi ional
B-cell popula ion abundance was unchanged ac oss geno ype-
defined g oups (supplemen al Figu e 3B-C), Mga-deple ed mice
showed inc eased ma ginal zone (P= .003) (wi h conco dan ly
dec eased ollicula B cells) and ge minal cen e (P= .009) splenic
subpopula ions (Figu e 2A-B; supplemen al Figu e 3D). We also
iden ified an abundance o pe i oneal B1a and a conco dan
dec ease in B1b cells (P<.05;Figu e 2C; supplemen al Figu e 3E),
sugges ing a ole o Mga in egula ing ma u e B-cell de elopmen and
lineage-specifica ion. In pa icula , he inc eased spon aneous
ge minal cen e o ma ion may ela e o he lymphomogenic ac i i y o
his lesion, whe eas inc eased CD5
+
B1a pe i oneal B cells may
ins ead ela e o CLL p edisposi ion, as B1a cells a e conside ed o
be he cell o o igin o mu ine CLL.
10
Gi en he known ole o Mga as nega i e egula o o MYC a ge s
in o he cellula con ex s,
11
we u he asked whe he he obse ed
B-cell de elopmen al pheno ypes could esul om abe an MYC
a ge ac i a ion in ma u e B cells. To his end, we analyzed he
BN -1
Ga ed on li e
1
5
4
3
0
–3
2345
GFP (log)
mChe y (log)
10.9%
T p53 indel
1
5
4
3
0
–3
2345
GFP (log)
mChe y (log)
88.7%
Bi c3indel
A mindel
Samhd1indel
T p53indel
Chd2indel
Mgaindel
N -1
0123
P (all s. N -1)
< .05
n = 5/g oup
Fold inc ease a day 3 o e day 0
(edi ed s. unedi ed)
C
%GFP+mChe y+B cells
30
20
10
0
248121824
Mga P = .01
N -1
248121824
Chd2 P = .05
24 8 12 18 24
T p53 P = .04
30
20
10
0
24 8 12 18 24
Samhd1 P = .03
24 8 12 18 24
A m P = .002
24 8 12 18 24
Bi c3 P = .05
Mon hs pos - ansplan
Cd19c e/c e
Cas9/Cas9
Cd19-Cas9
(8-12 week)
X
CD45.2 LSKs CD45.2 LSKs
CD45.1
N -1
Mga
Chd2
A m
Bi c3
Samhd1
T p53
LSK isola ion
sgRNA
(single)
T ansplan
T ansduc ion
LSK enginee ing Es ablishmen o B-cell es ic ed single-edi ed coho s
A
Bi c3indel
A mindel
Samhd1indel
T p53indel
Chd2indel
Mgaindel
N -1
unedi ed
indels
0 %
89.1%
80.6%
90.8%
84.7%
45.7%
63.9%
F ameshi
In- ame
87.779.189.676.2
72
86.6
Figu e 1. Indi idual CLL LOF lesions al e ma u e B-cell su i al bu a e insu ficien o d i e leukemia in mouse models. (A) T ansplan schema and gene a ion o
singly-edi ed mouse lines. LSKs we e len i i ally ans ec ed in i o wi h sgRNAs a ge ing he single LOFs (o a non- a ge ing sc amble con ol, ie, N -1) in addi ion o he mChe y
ma ke be o e he eng a men in o suble hally i adia ed CD45.1 ecipien mice. PCR-based a ge ed deep sequencing o DNA om pe iphe al blood edi ed B cells (ie,
GFP
+
mChe y
+
) was used o confi m p esence o ~45% o 85% gene edi s (>70% ameshi mu a ions) ac oss he 6 genes. (B) In i o su i al o no mal B cells isola ed om
he spleen o 5 animals pe g oup (6 LOF-exp essing s ains and 1 N -1) and cul u ed in i o in p esence o LPS+IL4 o 3 days. Fold inc ease o e day 0 is displayed alongside
ep esen a i e flow cy ome ic plo s om 1 N -1 and 1 T p53-deple ed sample. P- alue lowe o equal o 0.05, ANOVA wi h Dunne ’s co ec ion o mul iple compa isons.
(C) Pe cen (%) GFP
+
mChe y
+
B cells as assessed on longi udinal bleeds o e he cou se o 24 mon hs, in 10 animals pe g oup. *P- alue lowe o equal o 0.05; lowe o equal
o 0.05; RM-ANOVA, epea ed measu es analysis o a iance.
22 AUGUST 2023 •VOLUME 7, NUMBER 16 RESEARCH LETTER 4515
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ansc ip omes o gene-edi ed ma u e B cells (GFP
+
mChe y
+
)
and compa ed hem wi h hose o unedi ed (GFP
+
mChe y
-
)B
cells isola ed om he same animals (2 animals pe g oup;
Figu e 2D). Analyses we e pe o med 4 mon hs a e ansplan
because his was obse ed o be he peak o edi ed B-cell abun-
dance ac oss coho s (Figu e 1C). Via En ich analysis, using
he Molecula Signa u es Da abase (MSigDB), we iden ified
en ichmen in PI3K/AKT/mTOR and No ch signaling and dimin-
ished in e e on gamma, p53, and apop o ic gene exp ession in
Mga-deple ed cells compa ed wi h hose in he con ol unedi ed
ac ion ( alse disco e y a e <0.10; Figu e 2E; supplemen al
Tables 3-4). To iden i y di ec MYC a ge s a ec ed by loss
o Mga, we pe o med an in eg a ed analysis o Mga-deple ed
RNA-seq and exis ing ch oma in immunop ecipi a ion–seq (ChIP-
seq) da a o MYC om mouse B cells exposed o LPS s imula ion
o 8 hou s
12
using CISTROME-GO,
13
which allows so ing o
di ec a ge s o specific ansc ip ion ac o s based on he in e-
g a i e anking o ansc ip ion ac o –binding (ChIP-seq) com-
bined wi h change in gene exp ession (RNA-seq). Ou esul s
showed ha Mga deple ion ac i a ed ansc ip ion o se e al MYC
a ge genes wi h ele ance o B-cell de elopmen al biology
(supplemen al Table 5), including he B-cell ecep o signaling
kinase lymphocy e cell-specific p o ein- y osine kinase (Lck), he
cell cycle and Wn egula o s S ag3 and P p s and he ibosomal
p o ein Rps29 in esponse o LPS (Figu e 2F; supplemen al
Figu e 4). No ably, di ec MYC a ge s we e mo e likely o be
N -1
–3
5
4
3
0
–3
03 4 5
CD21 (log)
CD23 (log)
9.1%
Mgaindel
–3
5
4
3
0
–3
03 4 5
CD21 (log)
CD23 (log)
18.6%
Bi c3indel
A mindel
Samhd1indel
T p53indel
Chd2indel
Mgaindel
N -1
0 5 10 15 20 25
% MZ B cells
P = .003
A
N -1
–3
5
4
3
0
–3
03 4 5
CD38 (log)
CD95 (log)
<1%
Mgaindel
–3
5
4
3
0
–3
03 4 5
CD38 (log)
CD95 (log)
3.4%
Bi c3indel
A mindel
Samhd1indel
T p53indel
Chd2indel
Mgaindel
N -1
012345
% GC B cells
P = .009
B
C
Bi c3
indel
A m
indel
Samhd1
indel
T p53
indel
Chd2
indel
Mga
indel
N -1
0 20406080
% B1a B cells
P = .0005
N -1
–3
5
4
3
0
–3
03 4 5
CD5 (log)
B220 (log)
34.3%
Mgaindel
–3
5
4
3
0
–3
03 4 5
CD5 (log)
B220 (log)
62.3%
D
–3
5B cells
4
3
0
–3
03 4 5
B220 (log)
CD45.2 (log)
–3
5Unedi ed Edi ed
4
3
0
–3
0
RNA-seq RNA-seq
Pai ed DGE/En ich
CISTROME-GO
345
GFP (log)
mChe y (log)
E
In e e on Gamma Response
Wn -be a Ca enin Signaling
In e e on Alpha Response
IL-2/STAT5 Signaling
Apop osis
p53 Pa hway
KRAS Signaling Up
PI3K/AKT/mTOR Signaling
No ch Signaling
Hypoxia
Mi o ic Spindle
42024
UPDOWN
–Log(P)
F
MYC
ChIP
Baseline
LPS
Mgaw _1
Mgaw _2
Mgaindel_1
Mgaindel_2
Lck
G
MYC a ge s
Log2FC
2
1
–1
0
n = 69
P = .001
n = 24
Figu e 2. Mga mu a ion al e s B-cell de elopmen al pa hways. (A) Pe cen (%) abundance o MZ B cells, (B) GC B cells in spleen and (C) B1a cells in pe i oneum p epa a ions
om 5 animals pe g oup, including non- a ge ing con ols. **P- alue, lowe o equal o 0.01, ***P- alue, lowe o equal o 0.001, ANOVA wi h Dunne 's co ec ion o mul iple
compa isons. (D) Flow-plo o edi ed s unedi ed ac ions analyzed ia RNA-seq and CISTROME-GO. (E) Pa hway en ichmen analysis o Mga-deple ed s unedi ed cells, pe En ich .
(F) IGV sc eensho o MYC ChIP-seq da a o mouse B cells wi h o wi hou LPS ea men and RNA-seq da a o Lck gene exp ession in edi ed (Mga
indel
) and unedi ed (Mga
WT
)
ac ions o 2 independen mice.(G) Change in exp ession (Mga
indel
s Mga
WT
) o genes ha we e di ec Myc a ge s upon LPS s imuli, as assessed by CISTROME-GO analysis.
***P- alue, lowe o equal o 0.001; χ
2
es . GC, ge minal cen e ; IGV, in eg a i e genomics iewe ; MZ, ma ginal zone.
4516 RESEARCH LETTER 22 AUGUST 2023 •VOLUME 7, NUMBER 16
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up egula ed han Mga-deple ed cells, consis en wi h MYC ac i-
a ion (P= .001; χ
2
es ; Figu e 2G). Toge he , hese esul s
suppo he no ion ha Mga plays a unique ole in egula ing MYC
a ge s in ol ed in B-cell p oli e a ion and di e en ia ion, esul ing
in a di ec impac on cell a e de e mina ion o ma u e naï e B cells.
In conclusion, we demons a e ha ou models ep esen a aluable
and e sa ile pla o m o he s udy o indi idual gene ic lesions in a
p eleukemic B-cell con ex . We show ha in oduc ion o indi idual
CLL LOF mu a ions in ma u ing B cells can al e p osu i al and B-cell
de elopmen al pa hways and inc ease cellula fi ness in i o and
in i o. We u he obse e ha indi idually hey a e insu ficien o d i e
leukemia and mo i a e s udies o e alua ing he e ec s o combina-
o ial asso men o lesions in B-cell leukemogenesis.
Acknowledgmen s: The au ho s hank membe s o he Wu lab
o he aluable discussions. The au ho s a e also g a e ul o
excellen echnical assis ance om he Dana-Fa be Cance Ins i-
u e Animal Resea ch Facili y and he Dana-Fabe Cance Ins i u e
Flow Cy ome y Co e. This s udy was suppo ed by g an s om he
Na ional Ins i u es o Heal h/Na ional Cance Ins i u e (P01
CA206978 and R01CA216273). E. .H. is a schola o he Ame -
ican Socie y o Hema ology. S.Y. is suppo ed by a esea ch
ellowship om he Lau i S auss Leukemia Founda ion and
Na ional Cance Ins i u e (R21 CA267527-01). M.H.S. has been
suppo ed by a Sa a Bo ell pos doc o al con ac (CD19/00222)
om he Ins i u o de Salud Ca los III (ISCIII), co ounded by
Fondo Social Eu opeo “El Fondo Social Eu opeo in ie e en u
u u o.”K.C. is suppo ed by Na ional Human Genome Resea ch
Ins i u e Ca ee De elopmen Awa d K99HG011658. S.L. is
suppo ed by he Na ional Cance Ins i u e Resea ch Specialis
Awa d (R50CA251956). L.P. is pa ially suppo ed by Na ional
Human Genome Resea ch Ins i u e Genomic Inno a o Awa d
R35HG010717.
Con ibu ion: E. .H., S.Y., and C.J.W. designed he s udy and w o e
he manusc ip ; E. .H. gene a ed mouse models, pe o med mos
expe imen s, and analyzed da a; S.Y. pe o med RNA-seq and ChIP-
seq analyses; S.L. pe o med he single-cell DNA-seq expe imen s;
R.R., M.H.S, K.C., L.P., K.J.L., and D.N. assis ed wi h s udy design and
da a analysis; G.B.H., F.F.R., and E.W. p o ided echnical suppo ;
and C.J.W. p o ided o e all supe ision o he s udy.
Conflic -o -in e es disclosu e: C.J.W. is an equi y holde o
BioNTech Inc. and ecei es esea ch unding om Pha macyclics.
L.P. has financial in e es s in Edily ics Inc. K.C. is an employee,
sha eholde , and o fice o Edily ics Inc. The in e es s o L.P. and
K.C. we e e iewed and a e managed by Massachuse s Gene al
Hospi al and Pa ne s Heal hCa e in acco dance wi h hei conflic -
o -in e es policies. The emaining au ho s decla e no compe ing
financial in e es s.
ORCID p ofiles: E. .H., 0000-0003-1573-8799; S.Y., 0000-
0001-9116-5238; R.R., 0000-0002-1329-5288; M.H.S., 0000-
0001-9968-2782; K.C., 0000-0003-3808-0811; G.B.H., 0000-
0001-7758-898X; K.J.L., 0000-0001-9105-5856; C.J.W., 0000-
0002-3348-5054.
Co espondence: Ca he ine J. Wu, Depa men o Medical
Oncology, Dana-Fa be Cance Ins i u e, 450 B ookline A e,
Dana Building, Room DA-520, Bos on, MA 02115; email: cwu@
pa ne s.o g.
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