RAF signalling in cancer:
Biology and therapeutic opportunities
The Cancer Genome Project
• 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 RAS/RAF signalling pathway
Growth Factor
Receptor
Ras
Over-expressed in cancers
Mutated/amplified in cancers
Mutated in 15-20% of cancers
B-RAF Mutated in 7% of cancers
MEK
ERK
proliferation, differentiation, death, senescence
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
CR1
Regulatory
CR2
CR3/ Kinase
Catalytic
B-RAF is mutated in ~7% of human cancers
Activation segment
Glycine rich-loop
CR1
B-RAF:
CR2
CR3/ Kinase
K
A
R
E E A
G V E
L ISV V CE
VLRR IDE
VGQRIGSGSFGTV…………DFGLATVKSRWS
B-RAF Kinase Domain
Glycine-rich
loop
activation
segment
Wan et al 2004, Cell
B-RAF Kinase Domain
Glycine-rich
loop
activation
segment
Wan et al 2004, Cell
B-RAF activation by mutation
inactive
constitutively
active
activation
segment
A-RAF/C-RAF mutations in cancer
Glycine rich-loop
B-RAF:
A-RAF:
C-RAF:
Activation segment
K
A
R
E E A
G V E
L ISV V CE
VLRR IDE
VGQRIGSGSFGTV…………DFGLATVKSRWS
-------------…………--------T---------------…………------------
A-RAF/C-RAF mutations in cancer
Glycine rich-loop
B-RAF:
A-RAF:
C-RAF:
Activation segment
K
A
R
E E A
G V E
L ISV V CE
VLRR IDE
VGQRIGSGSFGTV…………DFGLATVKSRWS
-------------…………--------T---------------…………------------
• 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)
C-RAF Kinase activity (fold WT)
Kinase activity
Relative kinase activity
Rel. WTC-RAF
100
C-RAF
1
V492EC-RAF
48
B-RAF
60
V600EB-RAF
28,800
50
0
~600 fold
B-RAF has elevated kinase activity due to the N-region
RBD
CRD
CR1
CR2
CR3
P P
C-RAF: QRDSSYYWEIE
P
B-RAF: RRDSSDDWEIP
N-region:
Negative-charge
regulatory region
Marais et al, 1997 JBC
The N-region determines RAF responses to mutation
1000
500
0
B-RAF kinase activity
B-RAF kinase activity (fold WT)
Kinase activity (fold WT)
C-RAF Kinase activity
500
250
0
Structure of B-RAF
B-RAF and C-RAF mutations
B-RAF
C-RAF
N-region
inactive
activation
segment
P
const.
active
B-RAF in cancer
Human melanoma lines: siRNA
WM-266.4 cells:
Melanoma cells with V600EB-RAF mutation
Proliferation
ppERK
Total ERK
10,000
0
siRNA:
B-RAF
C-RAF
Scr.
B-RAF
20,000
Control
A-RAF
[3H]-thymidine incorporation (cpm)
Control
Scr.
A-RAF
B-RAF
C-RAF
ERK activity
Expression of B-RAF in melan-a cells
B-RAF expression in mouse melanocytes
myc-tag
B-RAF
ppMEK
ppERK
MEK
ERK
vector
parental
ERK signalling
WTB-RAF
Growth in nude mice
V600EB-RAF
Oncogenic B-RAF stimulates proliferation and survival in cancer
V600EB-RAF
MEK
V600EB-RAF
• 500 fold activated
• stimulates constitutive
signalling
• stimulates proliferation
• stimulates survival
• is an excellent therapeutic
target
ERK
survival
proliferation
Karasarides et al (2004)
Wellbrock et al (2004a)
Wan et al (2004)
Garnett and Marais (2004)
Wellbrock et al (2004b)
Sorafenib (Nexavar), a multi-kinase inhibitor
CF3
O
Cl
O
O
N
H
N
H
N
N
H
• 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)
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
Different modes of binding
Sorafenib binds to
the inactive conformation
Pyrazines binds to
the active conformation
A mouse model of melanoma
V600EB-RAF
inducible mouse
V600E
14
loxP
15 16 17 18
B-RAF minigene
NeoR
loxP
loxP
Txn
terminator
cre recombinase
loxP
14
V600E
15
Mutant
allele
16
17
18
15
Mutant
allele
16
V600EB-RAF
inducible mouse
• Tyrosinase-Cre
• melanocyte specific promoter
• comes on at ~E9.5
• B-RAF is on chromozome 7, the Tyr::Cre on the Xchromosome
• 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
100
B-RAF
C-RAF
V600EB-RAF
WTB-RAF
00
5
10
15
days
20
pMEK
25
Tumour volume (mm3)
MEK1
Tumour growth
300
200
100
0
0
10
20
30
40
Days from inoculation
50
V600EB-RAF
MEK activity
WTB-RAF
Cell numbers (x 104/ml)
Cell proliferation
PD184352
Time (hrs):
Pax-3
Sox-10
C-kit
A-MITF
tyrosinase
Trp-2
M-MITF
GAPDH
0
6
24
control
Cells are neuronal, but not melanocytes
Inactivating B-RAF mutations in cancer
Unexpected inactivating mutations in B-RAF in cancer
Inactive in vitro
…but active in vivo
ACTIVITY
480
BRaf V600E 0.2ng
V600E
G466V
0
B-RAF
1
B-RAF
SCRx2
C-RAF
H1666
(G466VImpaired)
B-RAF
Fold activity
(compared to WTBRAF)
30
WM266.4
(V600DActivated)
C-RAF
C-RAF activation
SCRx2
Impaired activity mutants
20
B-RAF
10
C-RAF
ppERK1/2
0
ERK2
B-RAF signalling in cells
activated
mutants
impaired
mutants
Normal
B-RAF
B-RAF*
B-RAF†
B-RAF
C-RAF
C-RAF
MEK
MEK
MEK
ERK
ERK
ERK
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
Signal Transduction
Team
Annette Affolter
Tanya Ahmad
Vicky Emuss
Vanessa Gray-Schopfer
Robert Hayward
Sonja Heidorn
Ruth Kirk
Sareena Rana
Silvy da Rocha-Diaz
Slike Schepelmann
Simone Walker
Steven Whittaker
Claudia Wellbrock
Gene and Oncogene
Targeting Team
Lawrence Davies
Harmen Djikstra
Frank Friedlos
Catherine Gaulon
Douglas Hedley
Jan Martin
Dan Niculescu-Duvaz
Ion Niculescu-Duvaz
Lesley Ogilvie
Esteban Roman
Ian Scanlon
Caroline Springer
Structural Biology
Team
Paul Wan
Mark Roe
Val Good
David Barford
Royal Marsden Hospital
Tim Eisen
Martin Gore
The Sanger Institute
Richard Wooster
Andy Futreal
Mike Stratton
Leicester University
Katherine Mercer
Susan Giblet
Catrin Pritchard
Institut Curie, Paris
Veronique Delmas
Lionel Larue