RAF signalling in cancer:Biology and therapeutic opportunities

• 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

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

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

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

B-RAF Kinase Domain

Glycine-richloop

activationsegment

Wan et al 2004, Cell

B-RAF Kinase Domain

Glycine-richloop

activationsegment

Wan et al 2004, Cell

B-RAF activation by mutation

activationactivationsegmentsegmentinactive

constitutivelyactive

GLR

GLR

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

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

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

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

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

Structure of B-RAF

GLR

GLR

B-RAF and C-RAF mutations

inactive

const.active

B-RAF

N-regionN-region

activationactivationsegmentsegment

GLR

GLR

P

GLR

C-RAF

B-RAF in cancer

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

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

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)

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

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

Pyrazines binds tothe active conformation

Sorafenib binds tothe inactive conformation

A mouse model of melanoma

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

• 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

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)

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

Inactivating B-RAF mutations in cancer

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

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

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

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 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