Ras signaling

1
RAF signalling in cancer Biology and therapeutic
opportunities
2
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

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The RAS/RAF signalling pathway
Growth Factor
Over-expressed in cancers
Receptor
Mutated/amplified in cancers
Ras
Mutated in 15-20 of cancers
B-RAF

• Mutated in 7 of cancers

MEK
ERK
proliferation, differentiation, death, senescence
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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 (gt10) splice variants
• C-RAF - single splice variant

CR1
CR2
CR3/ Kinase
Regulatory
Catalytic
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B-RAF is mutated in 7 of human cancers
Activation segment
Glycine rich-loop
CR1
CR3/ Kinase
CR2
K A
R E E A G V E
L ISV V CE VLRR IDE B-RAF
VGQRIGSGSFGTVDFGLATVKSRWS
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B-RAF Kinase Domain
Wan et al 2004, Cell
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B-RAF Kinase Domain
Wan et al 2004, Cell
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B-RAF activation by mutation
activation segment
inactive
constitutively active
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A-RAF/C-RAF mutations in cancer
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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)

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Relative kinase activity
Kinase activity
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
100
C-RAF Kinase activity (fold WT)
50
600 fold
0
WT
V492E
WT RAS
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B-RAF has elevated kinase activity due to the
N-region
RBD
CRD
CR3
C-RAF QRDSSYYWEIE B-RAF RRDSSDDWEIP
N-region Negative-charge regulatory region
Marais et al, 1997 JBC
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The N-region determines RAF responses to mutation
C-RAF Kinase activity
B-RAF kinase activity
500
1000
B-RAF kinase activity (fold WT)
Kinase activity (fold WT)
250
500
0
0
WT
DD
WT
AAAA/ V600E
V600E
V492E
DD/V492E
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Structure of B-RAF
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B-RAF and C-RAF mutations
B-RAF
C-RAF
N-region
inactive
activation segment
GLR
const. active
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B-RAF in cancer
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Human melanoma lines siRNA
WM-266.4 cells Melanoma cells with V600EB-RAF
mutation
ERK activity
Control
B-RAF
A-RAF
C-RAF
Scr.
A-RAF
B-RAF
C-RAF
ppERK
Total ERK
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Expression of B-RAF in melan-a cells
B-RAF expression in mouse melanocytes
ERK signalling
Growth in nude mice
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Oncogenic B-RAF stimulates proliferation and
survival in cancer

• 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)

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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
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A mouse model of melanoma
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V600EB-RAF inducible mouse
V600E
14
15
16
NeoR
loxP
loxP
loxP
B-RAF minigene
Txn terminator
Mutant allele
cre recombinase
V600E
loxP
14
15
16
17
18
Mutant allele
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V600EB-RAF inducible mouse

• Tyrosinase-Cre
• melanocyte specific promoter
• comes on at E9.5
• B-RAF is on chromozome 7, the TyrCre on the
  X-chromosome
• However in over 200 live births, we did not
  found the double TyrCre, B-RAF targeted mutants
• Tyrosinase promoter is leaky and is active in
  the brain

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Cells are neuronal, but not melanocytes
control
PD184352
Time (hrs)
0
6
24
Pax-3
Sox-10
A-MITF
tyrosinase
Trp-2
GAPDH
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Inactivating B-RAF mutations in cancer
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Unexpected inactivating mutations in B-RAF in
cancer
Inactive in vitro
but active in vivo
480
ACTIVITY
1
0
G466V
B-RAF
V600E
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Impaired activity mutants
C-RAF activation
30
20
Fold activity (compared to WTBRAF)
10
0
G466V
BRAF
vector
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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
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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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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
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
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
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