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Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia

Tanaka, Atsushi 京都大学 DOI:10.14989/doctor.k24522

2023.03.23

概要

Acute myeloid leukemia (AML) with inv(3)(q21q26) or
t(3;3)(q21q26) is recognized by the World Health Organization
as a unique subtype of AML (henceforth referred to as “inv(3)/
t(3;3) AML”),1 which has a dismal median overall survival (OS) of
,1 year after diagnosis.2-5 This same genomic alteration is also
occasionally encountered in patients with myelodysplastic syndromes (MDS), where it is similarly associated with short survival.4,6 Given the poor outcome of inv(3)/t(3;3) MDS/AML, there ...

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参考文献

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Supplementary Table 1. Primer sequences and PCR conditions for mice genotyping, RT-PCR, and minigene

assays.

PCR protocol

Genotyping

Target

inv (3) transgene

Sf3b1 floxed

Mx1-Cre

Primer sequence

5'-GCAGCTGCTCTGAGACAAGTCTG-3'

5'-GCACTGTGGTCTAATTGCCTGATC-3'

5'-CTAGGCCACAGAATTGAAAGATCT-3'

5'-GTAGGTGGAAATTCTAGCATCATCC-3'

5'-TCATGGGCTGTGCTATCTTG-3'

5'-GCACTGATGGTCCGAACTTT-3'

5'-CAAGTGACAGCAATGCTGTTTCAC-3'

5'-GGCTGGCCCTGTATTCCTGAT-3'

5'-CAGGTATCTCTGACCAGAGTCATC-3'

5'-TCTTCTGACCCTTCCCTACTGAGC-3'

RT-PCR for confirming Sf3b1K700E expression

mouse Sf3b1

RT-PCR for detecting EVI1+18

human EVI1 exon 12 to 13 from

human cell lines

human EVI1 exon 12 to 13 from

mice with human inv(3) Tg

human EVI1 intron 12 inner

5'-CAACAAACCAATTTAGACAGACACC-3'

5'-GTCATCCAGAATCGCACCTG-3'

5'-GCCATTTAAGTGTCACTTATGTGATAGG-3'

5'-CTTCTTTGTCATCCAGAATCGCA-3'

5'-GCTAGTGAGAGAAGCACCTTC-3'

5'-CATAATCCAAATAAGGCCACTG-3'

5'-GTACTTCATGATTTCAGTGGTTC-3'

5'-CCTGATTTTGGCGTCAAAATGG-3'

RT-PCR for minigene assay

minigene-derived human EVI1

endogenous human EVI1

5'-TGGTCAACAAACCAATTTAGACAGAC-3'

5'-ATCAGCGAGCTCTAGCATTTAGG-3'

5'-TGGTCAACAAACCAATTTAGACAGAC-3'

5'-CTGGTCACCAAAGCCTTTTCATC-3'

EVI1 minigene cloning

EVI1 minigene, WT

MT1

MT2

MT3

MT4

MT5

MT6

MT7

MT8

MT9

MT10

MT11

MT12

MT13

MT14

MT15

MT16

Sanger sequence

polylinker of pMYs, Fw

polylinker of pcDNA3, Rv

qPCR

mBcl11a

mCd34

mHes1

mMeis1

initial

denature

annealing

extension

cycles

final

extension

2xGoTaq GreenMaster Mix

(M7123, Promega)

94℃, 2 min

94℃, 30 sec

60℃, 30 sec

72℃, 1 min

72℃, 7 min

2xGoTaq GreenMaster Mix

(M7123, Promega)

95℃, 3 min

95℃, 30 sec

59℃. 30 sec

72℃. 1 min

72℃, 5 min

2xGoTaq GreenMaster Mix

(M7123, Promega)

95℃, 2 min

95℃, 30 sec

68℃, 90 sec

72℃. 1 min

72℃, 3 min

95℃, 2 min

95℃, 30 sec

55℃, 30 sec

72℃, 1 min

72℃, 7 min

95℃, 2 min

95℃, 30 sec

55℃, 30 sec

72℃, 1 min

72℃, 7 min

95℃, 2 min

95℃, 30 sec

63℃, 30 sec

72℃, 1 min

72℃, 7 min

PCR protocol

5'-GCTGTGTGCAAAAGCAAGAAG-3'

5'-TCCTCTGTGTTGGCGGATAC-3'

RT-PCR for lariat sequencing

human EVI1 intron 12 outer

polymerase

5'-AAAAAAGGATCCATGCAAATACTGTGACAGATC-3'

5'-AAAAAACTCGAGCTCTCCTCCACATTCCTGG-3'

5'-GATAAACAATAATCTTTGTCATAAACGGCTTTCTG

CTCCACAC-3'

5'-GTGTGGAGCAGAAAGCCGTTTATGACAAAGATTA

TTGTTTATC-3'

5'-GCTAGATAAACAATAATCTTTGTCCTAAAAGGCTT

TCTGCTCCAC-3'

5'-GTGGAGCAGAAAGCCTTTTAGGACAAAGATTATTG

TTTATCTAGC-3'

5'-GTTGCTAGATAAACAATACTCTTTGTCATAAAAGG

CTTTC-3'

5'-GAAAGCCTTTTATGACAAAGAGTATTGTTTATCTA

GCAAC-3'

5'-GTTGCTAGATAAACAATAATCTTTTTCTTTTTAGG

CTTTCTGCTCCACAC-3'

5'-GTGTGGAGCAGAAAGCCTAAAAAGAAAAAGATTAT

TGTTTATCTAGCAAC-3'

5'-TTGTTTATCTAGCAACTTATTTG-3'

5'-TAATCTTTGTCATTTTAGGCTTTCTGC-3'

5'-CAAAGATTATTGTTTATCTAGC-3'

5'-TCATAAAAGGCTTTCAAAACCACACAGGTA-3'

5'-CAAAGATTATTGTTTATCTAGC-3'

5'-TCATAAAAGGCTTTCTGAAAAACACAGGTACAGC-3'

5'-CTTTGTCATAAAAGGCTTACTGCTCCACACAGGT

ACAG-3'

5'-CTGTACCTGTGTGGAGCAGTAAGCCTTTTATGAC

AAAG-3'

5'-CTTTGTCATAAAAGGCTTTATGCTCCACACAGGT

ACAG-3'

5'-CTGTACCTGTGTGGAGCATAAAGCCTTTTATGAC

AAAG-3'

5'-CTTTGTCATAAAAGGCTTAATGCTCCACACAGGT

ACAG-3'

5'-CTGTACCTGTGTGGAGCATTAAGCCTTTTATGAC

AAAG-3'

5'-CTTTGTCATAAAAGGCTAAATGCTCCACACAGGT

ACAG-3'

5'-CTGTACCTGTGTGGAGCATTTAGCCTTTTATGAC

AAAG-3'

5'-CTTTGTCATAAAAGGCAAACTGCTCCACACAGGT

ACAG-3'

5'-CTGTACCTGTGTGGAGCAGTTTGCCTTTTATGAC

AAAG-3'

5'-AATCTTTGTCATAAAAGGAAATCTGCTCCACACAG

GTAC-3'

5'-GTACCTGTGTGGAGCAGATTTCCTTTTATGACAA

AGATT-3'

5'-TTGCTAGATAAACAATcATCTTTGTCATAAAAGGC

TTTC-3'

5'-CTTATTTGGAAACTCACTCCTTATGAATTTG-3'

5'-TCTTTGTCATgAAAGGCTTTCTGC-3'

5'-TTATTGTTTATCTAGCAACTTATTTGG-3'

5'-AAGTTGCTAGgTAAACAATAATCTTTGTC-3'

5'-ATTTGGAAACTCACTCCTTATGAATTTG-3'

2xGoTaq GreenMaster Mix

(M7123, Promega)

PCR protocol

2xGoTaq GreenMaster Mix

(M7123, Promega)

2xGoTaq GreenMaster Mix

(M7123, Promega)

PCR protocol

DreamTaq Green master mix(K1081,

Thermo Fisher Scientiffic)

95℃, 2 min

95℃, 30 sec

55℃, 30 sec

72℃, 30 sec 35

72℃, 5 min

DreamTaq Green master mix(K1081,

Thermo Fisher Scientiffic)

95℃, 2 min

95℃, 30 sec

55℃, 30 sec

72℃, 30 sec 35

72℃, 5 min

PCR protocol

2xGoTaq GreenMaster Mix

(M7123, Promega)

2xGoTaq GreenMaster Mix

(M7123, Promega)

PCR protocol

Phusion High-Fidelity DNA Polymerase

(M0530S, New England Biolabs)

95℃, 2 min

95℃, 30 sec

58℃, 30 sec

72℃, 1 min

72℃, 7 min

95℃, 2 min

95℃, 30 sec

58℃, 30 sec

72℃, 1 min

72℃, 7 min

98℃, 1 min

98℃, 10 sec

60℃, 30 sec

72℃, 90 sec 35

72℃, 10 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

98℃, 10 sec

55℃, 15 sec

72℃, 5 min

72℃, 2 min

98℃, 10 sec

55℃, 15 sec

72℃, 5 min

72℃, 2 min

98℃, 10 sec

55℃, 15 sec

72℃, 5 min

72℃, 2 min

Q5 Hot Start High-Fidelity 2X Master Mix

98℃, 30 sec

(E0552, New England Biolabs)

Q5 Hot Start High-Fidelity 2X Master Mix

98℃, 30 sec

(E0552, New England Biolabs)

Q5 Hot Start High-Fidelity 2X Master Mix

98℃, 30 sec

(E0552, New England Biolabs)

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Pfu Ultra DNA polymerase

(200523, Agilent)

95℃, 30 sec

55℃, 1 min

72℃, 9 min

72℃, 15 min

Q5 Hot Start High-Fidelity 2X Master Mix

98℃, 30 sec

(E0552, New England Biolabs)

98℃, 10 sec

55℃, 15 sec

72℃, 5 min

72℃, 2 min

98℃, 10 sec

55℃, 15 sec

72℃, 5 min

72℃, 2 min

98℃, 10 sec

55℃, 15 sec

72℃, 5 min

72℃, 2 min

Q5 Hot Start High-Fidelity 2X Master Mix

98℃, 30 sec

(E0552, New England Biolabs)

Q5 Hot Start High-Fidelity 2X Master Mix

98℃, 30 sec

(E0552, New England Biolabs)

5'-CCCTTGAACCTCCTCGTTCGACC-3'

5'-TAGAAGGCACAGTCGAGG-3'

5'-CACGTCCGCACTTGAACTTG-3'

5'-GGTAGCTCTCTGCCTGATGAG-3'

5'-CCAGCCAGTGTCAACACGA-3'

5'-CATGATAGACCAGTCCAACCGA-3'

5'-AGATGAATTGTGGGAGAGCCG-3'

5'-TGGTAGGAACTGATGGGGATATT-3'

5'-AATGCCGGGAGCTATCTTTCT-3'

5'-ATTGGCTGTCCATCAGGGTTA-3'

Supplementary Table 2. Patient characteristics of EVI1 rearranged myeloid neoplasms with or without SF3B1

mutations.

Age at diagnosis (mean, SD)

Male sex (n, %)

Disease (n, %)

AML

MDS

Other (blast-phase CML, CMML)

Therapy-related MN (n, %)

Number of lines of therapy (mean, SD)

Best response to first line therapy

CR/mCR/CRi/PR/MLFS

Progressive disease/primary induction failure

not evaluable

Total cohort

(n=46)

SF3B1-mut

(n=18)

SF3B1-WT

(n=28)

P-value*

64.3 (13.4)

29 (63.0%)

62.8 (12.6)

11 (61.1%)

65.3 (14.0)

18 (64.3%)

0.55

1.00

25 (54.4%)

16 (34.8%)

5 (10.9%)

7 (15.2%)

2.2 (1.8)

10 (55.6%)

7 (38.9%)

1 (5.6%)

2.6 (1.6)

15 (53.6%)

9 (32.1%)

4 (14.3%)

6 (21.4%)

2.0 (1.9)

0.76

0.22

0.24

9 (19.6%)

7 (15.2%)

24 (52.3%)

6 (13.0%)

2 (11.1%)

3 (16.7%)

12 (66.7%)

1 (5.6%)

7 (25.0%)

4 (14.3%)

12 (42.9%)

5 (17.9%)

0.35

Allogeneic hematopoietic cell transplant (n, %)

13 (28.9%)

7 (38.9%)

6 (22.2%)

Concurrent cytogenetic abnormalities (n, %)

6 (13.0%)

6 (21.4%)

Del(5q)

22 (47.8%)

9 (50.0%)

13 (46.4%)

Monosomy 7

4 (8.7%)

1 (5.6%)

3 (10.7%)

17p abnormality or monosomy 17

12 (26.1%)

4 (22.2%)

8 (28.6%)

Complex karyotype

20 (43.5%)

5 (27.8%)

15 (53.6%)

Monosomal karyotype

3 (6.5%)

1 (5.6%)

2 (7.1%)

t(9;22)

* Student’s t-test for continuous variables and Pearson chi-square or Fisher’s exact test for categorical variables

0.32

0.07

1.00

0.74

0.13

1.00

Supplementary Table 3. EVI1-rearranged leukemia cell lines.

cell line

SF3B1

EVI1 rearrangement

age

sex

diagnosis

HNT-34

K700E

t(3;3)(q21;q26)

CMMoL overt AML

MUTZ-3

K666N

inv(3)(q21q26)

AML

YCU-AML1

K700E

t(3;3)(q21;q26.2)

AML-MRC (MDS/AML)

OCI-AML-20

wild-type

inv(3)(q21q26.2)

AML

MOLM-1

wild-type

inv(3)(q21q26)

CML-BC

Kasumi-3

wild-type

t(3:7) (q27;q22)

AML (M0)

Kasumi-4

wild-type

inv(3)(q21q26)

CML-BC

UCSD-AML1

wild-type

t(3;3)(q21;q26)

AML

CMMoL, Chronic myelomonocytic leukemia; AML-MRC, acute myeloid leukemia with myelodysplasia-related changes (AML-MRC);

CML-BC, chronic myeloid leukemia-blast crisis.

Supplementary Table 4. Mutational analysis of EVI1-rearranged leukemia cell lines.

Cell line

HNT-34

MUTZ-3

OCI-AML-20

Gene

SF3B1

PTPN11

RAD21

BCOR

SF3B1

RECQL4

ARID1A

KRAS

CREBBP

CTCF

WT1

NRAS

MAX

ATRX

DICER1

MED12

KMT2D

CDK12

Gene panel

N/A

Protein Change

K700E

T468M

A72V

T28Ifs*11

S1446Vfs*15

K666N

X766_splice

Y1101Cfs*3

G10dup

R589Kfs*15

X613_splice

L1?

Q61K

R36W

X199_splice

M1?

S63N

G1628Vfs*94

F1376Sfs*5

Annotation

OncoKB: Likely Oncogenic, level_4, resistance NA;CIViC: Predictive: 1;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: yes

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: yes

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Likely Oncogenic, level_4, resistance NA;CIViC: Predictive: 1;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Oncogenic, level NA, resistance NA;CIViC: Predictive: 20, Predisposing: 2;MyCancerGenome: present;CancerHotspot: yes;3DHotspot: yes

OncoKB: Predicted Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

Chromosome

Start Pos

198266834

112926270

112888199

117878886

39921485

198267359

145738768

27097708

25398287

3830787

67670591

32456890

115256530

65560491

76940496

95599795

70339311

49438607

37687220

End Pos

198266834

112926270

112888199

117878887

39921486

198267359

145738768

27097709

25398288

3830790

67670591

32456891

115256530

65560491

76940496

95599795

70339311

49438607

37687220

Ref

TTCC

OCI-AML-20

SMAD4

N/A

X263_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

48584615

48584709

OCI-AML-20

SUZ12

ARID2

KMT2D

N/A

X625_splice

Q1835*

X5508_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

30325677

46298856

49415653

30325677

46298856

49415653

OCI-AML-20

BRCA2

N/AR2617_I2628delinsSIWVANHYRWIV

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

32936705

32936736

OCI-AML-20

NF1

PTPRS

N/A

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

29588873

5219476

29588873

5219476

GTTGGTGCTGGCAGCCGGAACACAGCAA

GTGGGGTGGGTATCCAAGGGGCTGGAG

52719259

GTGGAACTAGCACATCAGGTCTCACTTCCC

TTTGCCTCCCTCTCCCTGCCCACAGAGT

X1574_splice

X1256_splice

OCI-AML-20

PPP2R1A

N/A

X308_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

52719147

OCI-AML-20

FOXP1

STAG2

N/A

X629_splice

X14_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

71015043 71015043

123156519 123156519

OCI-AML-20

RBM10

N/A

D20*

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

47028754

OCI-AML-20

KLF4

FOXP1

PMS2

MAP2K4

PIK3R3

N/A

X422_splice

X4_splice

X117_splice

X172_splice

X339_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

110248206 110248206

71247548 71247548

6043323

12011109 12011109

46511760 46511760

47028807

Var

CTC

Allele Freq

0.509043928

0.509186352

0.492900609

0.350584307

0.526315789

0.462167689

0.744186047

0.465397924

0.451219512

0.334801762

0.551378446

0.061452514

0.457905544

0.028225806

0.056701031

0.078431373

0.067510549

0.057894737

0.078549849

GTATGTACATACTTTAAAAAATCTTTTAAA

TAGTTGAGAAAAAAGTAGGCAGCCTTTAT

AAAAGCAAATTAACCCATGTGGGCCTTAAT

TTTTAG

0.376556017

0.062656642

0.082051282

0.053097345

AATTTGGGTTTATAATCACTATAGATGGAT CATCTGGGTTGCTAACCATTACAGGTGGAT

AGG

GACCGCTCGCAGGATGATGGTGGGGAGA TGATCGTTCACAGGATGATAGTGGAGAGA

ACCGCAGCCGAGACCACGACTACCGG

ACCACAGCTGGGACCATGATTATAGA

OCI-AML-20

SMAD4

N/A

X142_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

48575230

GGTAAGTAGACTTTGCTTTCATCCTAAGAA

ACATAAAGGGAAAAGGATCTCAATAGTGT

TTCAATTTTTGTAAGTTAAATTATAAATTT

GGAAGATAGCCCGCGACTTTAAATAAGGT

TAAAGAATCAGACATTTTTAATATACGTAT

TTAAATTTGAATTTAGGAACAAACTTATAT

TTTGACTGCTAAAACCGAGTATTGTAGTTG

48575663 ATATTTTGCCCCTTTAGAACATTGTTTTTGA

GAAATAAGATGGAAAGCATTATAAATTTG

AATACTTAGTTATGTATTGTTAGATAGCGT

TTATGCTACTTCTGAATTGAAATGGTTCAT

GAACTTTTGTAATCTTTGGTTTAAATTTAC

ATTCTCTGTTTTTAAATGGAAAATACTTTCA

TTGTAATGATTAATGTTTCATTTGTTTTCCC

CTTTAAACAATTAA

OCI-AML-20

NF2

N/A

X81_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

30035081

30035082

OCI-AML-20 HIST1H1B

N/A

A164Gfs*42

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

27834817

OCI-AML-20

KMT2D

N/A

S849Lfs*30

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

49444921

OCI-AML-20

KMT2C

N/A

X2480_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

0.055555556

0.070953437

0.06302521

0.078393881

0.077568134

0.070967742

0.078947368

0.129310345

0.117370892

0.071895425

0.052238806

0.057636888

0.066308244

0.065502183

27834818

CCGCGGGCTTCTTCGCCTTCTTCGGAGTCTT

CTTCGGCCGCCTTTTTGGGTGTGGCAGCAC

CAGTCGCCTTCTTAGGCTTCTTGGCTGCTC

0.217391304

49444922

TTCCTCAGGCCGGGGGGACAGGCATGGCT

CCTCAGACTGGGGGGACAGGCGGCAGCTC

CTTAGGTGCAGAGCTCTGGCTTGGTTCCTC

AGGAGGCTCTGCAGGCTGAGGAGGTCCA

TTTAG

0.064159292

151876921 151876921

0.057142857

CGG

0.06557377

0.997409326

0.787234043

0.400479616

0.489583333

0.495639535

0.482758621

0.259958071

0.55165692

0.518382353

0.496389892

0.492753623

0.504823151

TGGAGGTGAGGTGTGACCTGACCCTGGG

CTCCCTCCCCTGTGTCCCTTCCAAGTCCCCAT

CACCTCAGGCAGCTGGAGTTCAATTTCTCA

CGTGTTAACCCCGTGAGCCTTCTTCCCCATT

TTCCTATCCTGGCTGCTTGTGCCTGTGATG

47045461

GCCTGACCCTCAGGACCTGAGCCTCATTAG

CCAGATGGCATGCCCTGTGGGACCCCACTC

CCCATGCCTGTGTTGCCTGAGAAAAGAGA

CCAGCTCCCCAACATTCACATACACATACAA

ACTTTC

OCI-AML-20

RBM10

N/A

X731_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

47045185

MOLM-1

Kasumi-3

Kasumi-4

UCSD-AML1

TP53

RECQL4

HLA-B

TP53

GRIN2A

HLA-B

PTPN11

KIT

PTPN11

TERT

MRE11

N/A

R196*

X766_splice

E69G

X261_splice

I162F

X723_splice

E69G

Y62D

D816V

D61V

Promoter

X36_splice

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Predicted Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Predicted Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Oncogenic, level NA, resistance NA;CIViC: Prognostic: 2, Predictive: 12;MyCancerGenome: present;CancerHotspot: yes;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: yes;3DHotspot: yes

OncoKB: Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

OncoKB: Likely Oncogenic, level NA, resistance NA;CIViC: NA;MyCancerGenome: not present;CancerHotspot: no;3DHotspot: no

7578263

145738768 145738768

31324602 31324602

7577498

7578446

9892321

31324602 31324602

112888168 112888168

55599321 55599321

112888166 112888166

1295228

94226952 94226952

Supplementary Table 5. Fraction of EVI1+18 variant in SF3B1-mutated and wildtype cases.

SF3B1

SF3B1 mutated

SF3B1 wild-type

p.G740E

p.R625C

p.H662Q

p.K700E

p.K666N

p.K666T

p.K666N

wild-type

SF3B1 -mutation

VAF

35.90%

44.00%

49.12%

47.00%

51.27%

54.31%

49.00%

45.82%

22.92%

44.00%

50.00%

43.75%

55.67%

42.01%

43.60%

48.77%

(-)

EVI1

Canonical

785

381

217

282

180

258

158

1759

172

197

107

178

107

253

140

185

263

264

542

567

1282

132

781

147

298

384

162

132

146

139

114

117

130

168

467

277

127

EVI1

Novel

483

186

305

Fraction of

EVI1 novel (%)

40.30%

38.09%

37.74%

32.80%

25.17%

22.35%

19.43%

17.05%

16.23%

15.51%

14.78%

14.43%

8.45%

6.82%

5.29%

1.83%

1.37%

1.20%

1.11%

0.93%

0.78%

0.71%

0.54%

0.38%

0.37%

0.35%

0.16%

0.00%

Supplementary Figure 1. The genetic characteristics of EVI1-rearranged (EVI1-r) myeloid

neoplasms. (A) Schematic image of inv(3) resulting in EVI1 expression by GATA2 distal

hematopoietic enhancer (G2DHE). The bacterial artificial chromosome (BAC) we utilized in the

murine model is also shown. (B) Diagram of location of 18 SF3B1 mutations identified in EVI1-r

myeloid neoplasms of MSKCC cohort (n=46). HD, HEAT-repeat domain. (C) Correlations

between driver mutations in entire MDS/AML cohorts (left) and EVI1-r myeloid neoplasms

(right). Significantly co-occurring and mutually exclusive mutations are shown in red and blue

circles, respectively. Odds ratio and associated -log10(Q-value) are indicated by the color

gradient and size of circles, respectively. Q-values were calculated by Benjamini-Hochberg (BH)

adjustment from p-values obtained from Fisher's exact test. (D) Overall survival (OS) from the

time of diagnosis and from the time of inv(3) detection in inv(3) AML with (red) or without (blue)

SF3B1 mutations.

Supplementary Figure 2. The impact of combined inv(3) and Sf3b1K700E mutations on

hematopoiesis and leukemogenesis. (A) Mean number of colonies derived from bone marrow

mononuclear cells from Mx1-Cre inv(3) Sf3b1K700E/WT mice and controls. Pre-B colonies are

shown on left and BFU-E are on right. Mean + SD. (B) Peripheral blood counts overtime

following transplantation. (C) As in Figure 2E, but for LT-HSC, ST-HSC, GMP, and MEP. (D)

BM cytospins of MDS-derived AML mice indicating immature blasts and dysplastic cells (black

and red arrows, respectively) within the Mx1-Cre inv(3) Sf3b1K700E/WT group. Scale Bars, 20 μm;

x1,000 magnification. (E) The cause of death within the Mx1-Cre inv(3) Sf3b1K700E/WT group.

WBC, white blood cell count; Hb, hemoglobin; MCV, mean corpuscular volume; PLT, platelet

count; LT-HSC, long-term hematopoietic stem cells, CD150+CD48-LSK; ST-HSC, short-term

hematopoietic stem cells, CD150-CD48-LSK; MEP; megakaryocyte-erythrocyte progenitor,

CD34−FcγR−Lin-Kit+ScaI-; GMP, granulocyte-monocyte progenitor, CD34+FcγR+Lin-Kit+ScaI-. P

values were calculated by two-sided t-test, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Supplementary Figure 3. Bone marrow cytomorphology of inv(3) Sf3b1 double mutant

recipient mice and controls. (A) Hematoxylin and eosin stain of bone marrow sections of 24week-old recipient CD45.1 mice transplanted with bone marrow cells of mice with each of the

indicated genotypes. The samples were collected 12 weeks after pIpC injection. The BM of the

Mx1-Cre inv(3) Sf3b1K700E/WT animals exhibited hypercellularity and a monomorphic cell

population. Scale Bars, 100 μm; ×100 magnification. (B) BM cytospins of mice from (A)

indicating blasts (red arrows) within the Mx1-Cre inv(3) Sf3b1K700E/WT group. Scale Bars, 20 μm;

×600 magnification.

Supplementary Figure 4. The inv(3) allele rescues the self-renewal defect of mutant

SF3B1. (A) Schema of competitive transplantation of CD45.2 Mx1-Cre inv(3) Sf3b1K700E/WT mice

and single mutant controls. (B) % of CD45.2+ peripheral blood cells in primary (1o) and

secondary competitive transplantation. Mean + standard deviation shown. (C) % of donorderived (CD45.2+) B220+, CD11b+Gr1+, and LSK cells in bone marrow (BM) and/or spleen

following 5 months of transplantation. (D) As in (C), but for stem and progenitor fractions. MPP,

multipotent progenitors, CD150-CD48+LSK; CMP, common myeloid progenitor, CD34+FCgR+LinKit+ScaI-. P values were calculated by two-sided t-test, *P<0.05, **P<0.01, ***P<0.001, and

****P<0.0001.

Supplementary Figure 5. Combined impact of mutations in SF3B1 and EVI1

rearrangement on gene expression and splicing. (A) Significantly dysregulated Gene

Ontology (GO) pathways in the transcripts of Mx1-Cre inv(3), Mx1-Cre Sf3b1K700E/WT, and Mx1Cre inv(3) Sf3b1K700E/WT, compared to those of Mx1-Cre control. Three samples were

independently collected in each group. log10(P-values) are color-coded. (B) As in Figure 3F, but

for the murine model. Each circle shows the number of aberrant splicing events of the indicated

model.

Supplementary Figure 6. The roles of novel EVI1+18 variant. (A) As in Figure 4C, but for

K562 cells expressing SF3B1 wild-type (WT), K666N, K700E, and G740E. (B) As in Figure 4D,

sanger sequencing of cDNA showing that the same +18 nucleotides were inserted between

exon 12 and 13 in the transcript of Mx1-Cre Sf3b1K700E/WT; inv(3) mice. (C) The predicted

structure of the 2nd ZF domain in the EVI1+18 variant. A superposition of AlphaFold2 models of

three tandem ZF domains in the C-terminus (residues 909 - 1229) of human MECOM (UniProt

accession Q03112) wild-type (grey) and mutant (orange). The 6 amino acid residues (FLLHTG)

inserted in the mutant were highlighted by magenta. (D) The relative mRNA expression

evaluated by qRT-PCR. (E) Representative FACS plot at day0 and day14 in the competition

assay between HSPCs transduced with EVI1 (GFP+) and EVI1+18 (tdTomato+). (F) The

chimerism evaluated by flow cytometry (GFP+ vs tdTomato+) in the replating assay in M3434.

(G) GSEA plots for the comparison of the transcripts derived from K562 cells expressing

EVI1+18 (left) and EVI1 wild-type (right). P values were calculated by two-sided t-test, **P<0.01,

***P<0.001, and ****P<0.0001.

Supplementary Figure 7. S ...

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Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia

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Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia

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関連論文

Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia

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Prognostic stratification for IDH-wild-type lower-grade astrocytoma by Sanger sequencing and copy-number alteration analysis with MLPA

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