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