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RAF signalling in cancer:Biology and therapeutic opportunities
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• In 2002, Mike Stratton, Andy Futreal and their colleagues reported the first high-throughput re-sequencing study aimed at identifying unknown somatic cancers in human cancer (Davies et al, 2002)• The coding regions for all of the components of the RAS/RAF/ MEK/ERK signalling pathway from 545 cell lines, 340 cancer samples were sequenced• Mutations were found in RAS in the expected frequency (15%)
• Unexpectedly, mutations were also found in B-RAF in 7% of human cancers
- B-RAF was subsequently found to be mutated in
50-70% of melanoma samples~30% of thyroid cancers~30% of low-grade ovarian cancers~15% of colorectal cancers
The Cancer Genome Project
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The RAS/RAF signalling pathway
MEK
ERK
Ras
Growth Factor
proliferation, differentiation, death, senescence
Receptor
B-RAF
Mutated in 15-20% of cancers
Mutated in 7% of cancers
Mutated/amplified in cancers
Over-expressed in cancers
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RAF PROTEINS
• Serine/ threonine specific protein kinases
• Their only widely accepted substrate is MEK
• 3 paralogues in humans• A-RAF - single splice variant
• B-RAF - multiple (>10) splice variants
• C-RAF - single splice variant
Regulatory Catalytic
CR1 CR2 CR3/ Kinase
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CR1 CR3/ KinaseCR2
B-RAF is mutated in ~7% of human cancers
K A R E E A G V E L ISV V CE VLRR IDEB-RAF: VGQRIGSGSFGTV…………DFGLATVKSRWS
Glycine rich-loop
Activation segment
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B-RAF Kinase Domain
Glycine-richloop
activationsegment
Wan et al 2004, Cell
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B-RAF Kinase Domain
Glycine-richloop
activationsegment
Wan et al 2004, Cell
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B-RAF activation by mutation
activationactivationsegmentsegmentinactive
constitutivelyactive
GLR
GLR
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A-RAF/C-RAF mutations in cancer
K A R E E A G V E L ISV V CE VLRR IDEB-RAF: VGQRIGSGSFGTV…………DFGLATVKSRWS A-RAF: -------------…………--------T---C-RAF: -------------…………------------
Glycine rich-loop Activation segment
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A-RAF/C-RAF mutations in cancer
• 546 cancer cell lines screened- 45 mutations in B-RAF, none in A-RAF, 4 in C-RAF
• No V452EA-RAF or V492EC-RAF mutations (equivalent of V600EB-RAF)
K A R E E A G V E L ISV V CE VLRR IDEB-RAF: VGQRIGSGSFGTV…………DFGLATVKSRWS A-RAF: -------------…………--------T---C-RAF: -------------…………------------
Glycine rich-loop Activation segment
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WT
WT
+ R
AS
0
50
100
V492
E
C-R
AF
Kin
ase
activ
ity (
fold
WT
) Rel. WTC-RAF
C-RAF 1
V492EC-RAF 48
Rel. WTC-RAF
C-RAF 1
V492EC-RAF 48
B-RAF 60
V600EB-RAF 28,800
~600 fold
Kinase activity Relative kinase activity
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B-RAF has elevated kinase activity due to the N-region
CR1 CR2 CR3CR3
RBD CRD
C-RAF: QRDSSYYWEIE
B-RAF: RRDSSDDWEIP
N-region: Negative-charge regulatory region Marais et al, 1997 JBCMarais et al, 1997 JBC
P
P P
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The N-region determines RAF responses to mutation
0
500
1000
Kin
ase
act
ivity
(fo
ld W
T)
WT
V492
E
DD
/V49
2E
C-RAF Kinase activity
0
250
500
B-R
AF
kin
ase
act
ivity
(fo
ld W
T)
WT
V600
E
B-RAF kinase activity
DD
AAAA
/V6
00E
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Structure of B-RAF
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GLR
GLR
B-RAF and C-RAF mutations
inactive
const.active
B-RAF
N-regionN-region
activationactivationsegmentsegment
GLR
GLR
P
GLR
C-RAF
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B-RAF in cancer
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Human melanoma lines: siRNA
A-RAF
B-RAF
C-RAF
ppERK
Total ERK
Con
trol
Scr
.
A-R
AF
B-R
AF
C-R
AF
ERK activity
20,000
10,000
0[3H
]-th
ymid
ine
inco
rpo
ratio
n (c
pm)
Con
trol
Scr
.
B-R
AF
siRNA:
Proliferation
WM-266.4 cells:Melanoma cells with V600EB-RAF mutation
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WTB-RAF
myc-tag
B-RAF
ppERK
ppMEK
parental
vector
V600EB-RAF
ERKMEK
Expression of B-RAF in melan-a cells
ERK signalling Growth in nude mice
B-RAF expression in mouse melanocytes
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Oncogenic B-RAF stimulates proliferation and survival in cancer
V600EB-RAFV600EB-RAF
MEKMEK
ERKERK
survivalsurvival proliferationproliferation
V600EB-RAF
• 500 fold activated• stimulates constitutive signalling• stimulates proliferation• stimulates survival• is an excellent therapeutic target
Karasarides et al (2004)Wellbrock et al (2004a)Wan et al (2004)Garnett and Marais (2004)Wellbrock et al (2004b)
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Sorafenib (Nexavar), a multi-kinase inhibitor
• ONYX Pharmaceuticals/Bayer Corporation
• Orally available multi-kinase inhibitor (C-RAF, B-
RAF, VEGF receptor, etc)
• Inhibits V600EB-RAF: IC50 ~40nM
• However, sorafenib is ineffective against melanoma-10 patients treated at the Royal Marsden Hospital
-5 with V600EB-RAF- 4 progressive disease, 1 stable
disease
-5 with WTB-RAF- 4 progressive disease, 1 stable disease
• December 05, sorafenib was licensed for use in renal
cell carcinoma (VEGFR)
CF3
NH
NH
NH
OO
O
N
Cl
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B-RAF inhibitors
• High throughput screen- 24,000 compounds focused
against kinases
• Several hit compounds, many of which were pyrazines
• Hit was low µmolar inhibitor in vitro (IC50= 3.5µM),
and best compound has an IC50 of 800nM
H3C
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Different modes of binding
Pyrazines binds tothe active conformation
Sorafenib binds tothe inactive conformation
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A mouse model of melanoma
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V600EB-RAF inducible mouse
14 15 1615 16 17 18 NeoR
loxP loxP loxPB-RAF minigene Txnterminator
Mutantallele
V600E
cre recombinase
14 15 16
loxP
Mutantallele
V600E
17 18
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• Tyrosinase-Cre • melanocyte specific promoter• comes on at ~E9.5• B-RAF is on chromozome 7, the Tyr::Cre on the X-
chromosome
• However in over 200 live births, we did not found the
double Tyr::Cre, B-RAF targeted mutants
• Tyrosinase promoter is leaky and is active in the brain
V600EB-RAF inducible mouse
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Cell proliferation
0
100
0 5 10 15 20 25days
Cel
l n
um
ber
s (x
104
/ml)
V600EB-RAF
WTB-RAF
B-RAFC-RAF
pMEK
MEK1
WTB
-RA
F
V6
00
EB
-RA
F
MEK activity
0 10 20 30 40 50
0
100
200
300
Tumour growth
Days from inoculation
Tum
our
vol
ume
(mm
3)
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C-kit
Pax-3
Sox-10
A-MITF
GAPDH
0 6 24Time (hrs):
PD184352
cont
rol
tyrosinase
Trp-2
Cells are neuronal, but not melanocytes
M-MITF
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Inactivating B-RAF mutations in cancer
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Unexpected inactivating mutations in B-RAF in cancer
0
ACTIVITY
B-R
AF
G46
6V
1
V60
0E
480
BRaf V600E 0.2ng
Inactive in vitro …but active in vivo
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Impaired activity mutants
C-RAF activation
vector
BRAF
G466V
Fol
d ac
tivity
(c
ompa
red
to W
TB
RA
F)
10
20
30
0
WM266.4(V600D-
Activated)
H1666(G466V-Impaired)
ppERK1/2
ERK2
B-RAF
C-RAF
SC
Rx2
C-R
AF
B-R
AF
SC
Rx2
C-R
AF
B-R
AF
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B-RAF signalling in cells
B-RAF*B-RAF*
MEKMEK
ERKERK
activatedmutants
B-RAF†B-RAF†
MEKMEK
ERKERK
impairedmutants
C-RAFC-RAF
B-RAFB-RAF
MEKMEK
ERKERK
NormalB-RAF
C-RAFC-RAF
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Summary
• B-RAF is a mutated in 7% of human cancers (70% melanoma)
• The mutations destabilize the inactive conformation
• C-RAF and A-RAF are not mutated because their regulation is
fundamentally different
• Mutant B-RAF stimulates proliferation and survival and is a
validated target
• B-RAF drug discovery programme- different binding modes
• Mouse model of melanoma
• B-RAF signalling through C-RAF is a new paradigm in pathway
regulation
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Signal TransductionTeamAnnette AffolterTanya AhmadVicky EmussVanessa Gray-SchopferRobert HaywardSonja HeidornRuth KirkSareena RanaSilvy da Rocha-Diaz Slike SchepelmannSimone WalkerSteven Whittaker Claudia Wellbrock
Gene and OncogeneTargeting TeamLawrence Davies Harmen DjikstraFrank FriedlosCatherine GaulonDouglas HedleyJan MartinDan Niculescu-DuvazIon Niculescu-DuvazLesley OgilvieEsteban Roman Ian ScanlonCaroline SpringerStructural BiologyTeamPaul WanMark RoeVal GoodDavid Barford
Royal Marsden HospitalTim EisenMartin Gore
The Sanger InstituteRichard WoosterAndy FutrealMike Stratton
Leicester UniversityKatherine MercerSusan GibletCatrin Pritchard
Institut Curie, ParisVeronique DelmasLionel Larue
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