Conclusions
Introduction
ResultsSF3B1 mutations result in lineage- and disease-specific aberrant junctions with usage of alternative 3’ splice site
SF3B1K700E, but not SF3B1G742D, impairs erythroid differentiation and growth in absence of cytokines in the TF-1 cells
Expression of SF3B1MUT upregulates mutant-associated splice isoforms in TF-1 model
qPCR to confirm the expression of aberrantly splice genes in TF-1 SF3B1MUT
Normal erythroid differentiation
Gene-set enrichment analysis in SF3B1MUT MDS patient samples indicates several dysregulated pathways
E7107 is a pan SF3B1 modulator and induces apoptosis in Panc05.04
Modulation of SF3B1 in vivo leads to improved overall survival in SF3B1K700E Nalm-6 xenografts
Possible consequences of SF3B1MUT expression in cancer cells
Translation of aberrant protein
Degradation of aberrantly spliced transcripts NMD
- RNA splicing involves removal of intronic sequences from pre-mRNA and the ligation of exons to generate mature mRNA- RNA splicing increases transcript and protein diversity in cells and is controlled by the spliceosome- Recurrent heterozygous mutations in the spliceosome components including SF3B1 have been reported in several malignancies - Hotspot mutations in SF3B1 have been identified in myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), uveal melanoma, breast and pancreatic cancers- SF3B1 is part of the U2 snRNP complex which binds to the pre-mRNA branch point and is involved in early recognition and stabilization of the spliceosome at the 3’ splice site (ss)- Here, we demonstrate that SF3B1 hotspot mutations (SF3B1MUT) result in neomorphic activity with the production of aberrant splicing associated with impaired cell differentiation and propose an approach for the treatment of SF3B1MUT cancers
- Hotspot mutations in SF3B1 induce neomorphic change of splicing function resulting predominantly in use of alternate 3’ splice sites- Common and lineage specific aberrant splicing events are detected in MDS, CLL and solid tumors- Genes involved in cell differentation and epigenetic pathways are dysregulated in MDS SF3B1MUT patient samples- Expression of SF3B1K700E, but not SF3B1G742D (hotspot mutation found in CLL, but not MDS) induces block in differentiation and cytokine independent growth in TF-1 cell line- SF3B1 modulation in a xenograft model of SF3B1K700E cancer leads to improved overall survival- A lead investigational drug candidate showing selective lethality in SF3B1MUT cells and improved physiochemical properties is progressing through IND-enabling studies
GU
5’ splice site
pre-mRNA
mature mRNA
3’ splice site
Branchsite
AG Exon 2Exonic splicing enhancerExon 1 Exonic
splicing enhancer YRYYRYUACUAAC
Polypyrimidine
tract
AUGAUG
U1snRNP
U2 snRNP
SF3B1
GU
AUACUA C
p14U2AF2
ZRSR2
AGYRYYRY Exon 2Exon 1 ESE ESE
U2AF1SRSF2
AUGAUG
U1snRNP
U2 snRNP
SF3B1GU
AUACUA C
p14U2AF2
ZRSR2
AGYRYYRY Exon 2Exon 1 ESE ESE
U2AF1SRSF2
U2 snRNP
U5 snRNP U6
snRNP
AUGAUG
SF3B1
AUACUA C
p14Exon 1 ESE
AUGAUG
SF3B1
AUACUA C
AG
Exon 2
YRYYRY
ESE
SRSF2
Exon 2Exon 1 ESE ESE
MDS (25%), CLL (15%), CMML (5%), AML (5%), Breast (2%), Pancreatic (5%) Uveal melanoma (19%)
SF3B1 (several hotspots)
CMML (47%), MDS (15%), PMF (17%), AML/sAML (19%)
SRSF2 (P95 or P95-R102 indel)U2AF1 (S34 or Q157)MDS (6%), AML (5%), CMML (8%), lung (3%), uterine and pancreatic cancer (~1%)
alt 3’ss
alt 5’ss
exon inclusion
exon skipping
intron retention
Aberrant splicing associated with SF3B1MUT is observed in Nalm-6 SF3B1K700E isogenic line and Panc05.04
Nalm-6 isogenic lines(pre-B cells)
Panc05.04: SF3B1Q699H,K700E
HotspotWT
SF3B1 status
Other
Nalm-6Panc05.04Colo829BL BxPC3Capan-2CFPAC-1Nalm-6AsPC1Colo829
breast cancermelanomaCLL
Tumor specificity(patients)
< −2 −1 0 1 > 2
Aberrant splice junction expression
Patient Z-score
non-specific
CLL-specific
melanoma-specific
breast-specific
MIAPaCa-2PK-59NCI-H1792Panc04.03NCI-H1650NCI-H1975
NCI-H358NCI-H1838
Panc10.05HPAF-II
Cell lines(unless labeled)
SF3B1K700K
SF3B1K700E
AAV Recombination
WT
WT
K700K
WT
K700E
WT
PD assay (Nalm-6 SF3B1K700E 6hr) Caspase 3/7 assay
100
80
60
40
20
0
Nalm-6 SF3B1K700E
Day post treatment20 40 60 80 100 120 140 160 180 200 220 240 260
Per
cent
ani
mal
on
stud
y (%
)Nalm-6 SF3B1K700K
20 40 60 80 100 120 140 160 180 200 220 240 260Day post treatment
Per
cent
ani
mal
on
stud
y (%
) 100
80
60
40
20
0
[E7107] (nM)
0
25
50
75
100
125
1,0001001010.10.01
[E7107] (nM)
Per
cent
bin
ding
Per
cent
bin
ding
0
25
50
75
100
125
1,0001001010.10.01
IC50= 11 nM
SF3B1WT
SF3B1K700E
IC50= 13 nMIP SF3b complex and perform
compound-competition binding assay
Express FLAG-SF3B1WT or FLAG-SF3B1K700E in 293F cells
Competition binding assay for SF3b complex
Cancer-associated Mutations in SF3B1 Exhibit Neomorphic Splicing Activity and Block Erythroid Differentiation S. Buonamici1, S. Perino1, K. H. Lim1, J. Feala1, E. A. Obeng2, M. Aicher1, D. Aird1, S. Bailey1, A. Berkenblit1, B. Chan1, E. Corcoran1, L. Corson1, R. Darman1, P. Fekkes1, R. R. Furman3, G. Keaney1, P. Kumar4, K. Kunii1,
L. Lee1, C. Mackenzie4, E. Park1, X. Puyang1, A. Selvaraj1, M. Thomas1, J. Wang1, M. Warmuth1, L. Yu1, P. Zhu1, Y. Mizui1, B. L. Ebert2, P. G. Smith1 1H3 Biomedicine, Cambridge, MA, USA, 2Brigham and Women's Hospital, MA, USA, 3Weill Cornell Medical College, NY, USA, 4Eisai Inc, MA, USA
Percent of splicing events in the different diseases
Example of alternatively splice gene: Alt 3’ss
AG
Premature STOP codon
AG
mat
ure
SLC
25A
19re
lativ
e ex
pres
sion
pre-mR
NA
EIF4A
1relative expression
Canonical splicing
Aberrant splicing
[E7107] (nM)1,000100101
1.5
1.0
0.5
0.0
30
20
10
00.10.01
rela
tive
gene
exp
ress
ion 1.5
1.0
0.5
0.0
[E7107] (nM)1,0001001010.10.01
Panc10.05 - SF3B1WT
0.01 0.1 1 10 100 10000
1000
2000
3000
4000
5000
[E7107] (nM)
Cas
pase
3/7
activ
ity
Panc05.04 - SF3B1Q699H,K700E
0.01 0.1 1 10 100 10000
1000
2000
3000
4000
5000
[E7107] (nM)
Cas
pase
3/7
activ
ity
24hr
48hr
mature COASYmature ZDHHC16
VehicleE7107 1.5 mg/Kg IV, QDx5E7107 2.5 mg/Kg IV, QDx5E7107 5 mg/Kg IV, QDx5
E622D
Y623C
R625C
R625L
R625H
N626Y
H662D
H662Q
K666E
K666M
K666N
K666Q
K666R
K666T
K700E
I704N
I704F
G740E
K741N
G742D
D781G
0369121520406080100
SF3B1mutations
(%)
CLLMDS
U2AF2 binding HEAT domainSF3B14
binding
SF3B1 protein 4 5 6 7 8
HotspotWT
SF3B1 status
Other
breast cancermelanomaCLL
Tumor type
Splice junction expression
Patient z-score< −2 −1 0 1 > 2
SF3B1MUT-H
MDS
Differential splicingAverage gene expression
Tumor type specificity
Breast cancer
MDS
Melanoma
Common
Hematologic
CLL
Breast cancer CLL Melanoma MDS KnownAlt0
20
40
60
80
100
Perc
ento
falte
rnat
ive
splic
ing
even
t
anti - HA
anti - Tubulin
Western blot of TF-1 cells infected with SF3B1MUT or controls
No infe
ction
pLVX-Z
sGree
n vec
tor
HA-mxS
F3B1W
T
HA-mxS
F3B1K
700E
HA-mxS
F3B1K
700R
HA-mxS
F3B1G
742D
RNAseq profiling analysis is currently ongoing to define aberrant splice events or deregulated genes that drive SF3B1K700E vs SF3B1G742D phenotype
PercentCD71andGlyA
DoublePositiveCells
No EPO EPO (2U/mL)0
10
20
30
40Vector
SF3B1WT
SF3B1K700R
SF3B1G742D
SF3B1K700E
Day
PercentGrowth
0 5 10 15 20 25-200
0
200
400
600
800
Erythroid Differentiation Status (Day 8) Growth in absence of growth factors
GU
5’ ss
ZDHHC16canonical 3’ sscryptic 3’ ss
(15-21nt upstreamof canonical AG)
BPS
CTAAACTACCCACCAGTCTTCGCCCCTCTTTTCTTAG Exon 10Exon 9
PPT
Weak andshort PPT
CD34+
CD38-CD34+
CD38+
GEMMHSC BFU-EEPO
Erythroblast Erythrocytes
CD34+
CD71+
GlyA-
CD71+
GlyA+CD71-
GlyA+
Expression of SF3B1MUT, but not SF3B1K700R, upregulates mutant-associated splice isoforms
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0
2
4
0
2
4
0
2
4
PA
N - gene
WT isoform
MU
T isoform
Vecto
r con
trol
SF3B1W
T
SF3B1E
622D
SF3B1R
625C
SF3B1R
625H
SF3B1R
625L
SF3B1H
662Q
SF3B1K
666E
SF3B1K
666N
SF3B1K
666R
SF3B1K
666T
SF3B1K
700E
SF3B1K
700R
SF3B1G
742D
Log 2 f
old
chan
ge (v
s no
tran
sfec
tion)
α-HA
α-GAPDH
α-SF3B1
α-GAPDH
K66
6E
K66
6N
K66
6R
K66
6T
H66
2Q
K70
0E
K70
0R
G74
2D
WT
Vect
or c
ontro
l
R62
5C
R62
5H
E62
2D
HA-mxSF3B1 cDNA
No
trans
fect
ion
R62
5L
293FT cells transfected with SF3B1 cDNA
Total RNA extracted for Nanostring
48hr
Genes up-regulated in erythroid progenitor cellsDysregulation of genes involved in differentiation
Fold
chan
gere
lativ
eto
SF3B
1K70
0E
No infe
ction
pLVX-ZsG
reen
HA-mxS
F3B1W
T
HA-mxS
F3B1G
742D
HA-mxS
F3B1K
700R
HA-mxS
F3B1K
700E
0.0
0.2
0.4
0.6
0.8
1.0
1.2 aberrant COASYaberrant ZDHHC16
SPAbead
FLAG-SF3B1FLAG-SF3B1
α-FLAG
Cold competitor E7107
3H-labeled pladienolide
analog
