Dana Koludrovic, PhD

Beatson Institute for Cancer Research

Owen Sansom group

Presentation overview

Introduction

Results:

Study of melanoma progression and survival

Role of PREX proteins

Combinatory treatment of PI3K and MAPK

Role of mTORC2/Rictor in NRAS mediated melanoma

Study of melanoma development

Role of DUSP6

Role of FAK

Melanocytes colonize the epidermis and

the hair follicle

Neural

crest

Precursor melanoblasts

Migrating melanoblasts

Migrating melanocytes

Melanoma

Miller et al. 2006.

Molecular

Lesions

BRAFV600E

(50-70%)

NRASQ61K;R

(15-20%)

PTEN loss

CDKN2A lossCD1 increase E-cadherin loss

N-cadherin expression

Introduction

Overview of therapeutic approaches in

melanoma

Introduction

Overview of therapeutic approaches in

melanoma

Introduction

A) A metastatic melanoma patient prior to therapy.

B) Same patient after 15 weeks of therapy with the BRAFV600E-inhibitor

C) Same patient 23 weeks after therapy

Overview of therapeutic approaches in

melanoma

Introduction

Overview of therapeutic approaches in

melanoma

Introduction

Main problem

acquired resistance

Overview of therapeutic approaches in

melanoma

Introduction

Study of melanoma progression and

survival

Interconnections of signalling networks

RAC signalling pathway

PREX2

Human sequencing indicating the

importance of Rac signalling

Human sequencing indicating the

importance of Rac signalling

• Truncating mutations in PREX2 are hyperactivating

PREX2 mutations in human melanoma

PTEN, Rac1, PREX2 , PREX1 mutations are mutually exclusive

Human sequencing data

BRAF mutant mouse model of melanoma

PTEN

BRAF-/+

Tyrosinase

Melanocyte

specific Cre line

TyrCreER::BRAFV600E::PTEN-/+

Pten-floxed

Tyr-CreERBRAF-/+ ::PTEN+/+

BRAFV600E floxed

line

Tamoxifen

PREX2 loss extends survival in BRAF

mutant melanoma

Results: Role of PREX proteins

*

PREX2 combined with MEK inhibition

extends survival

Results: Role of PREX proteins

Weeks

Tum

our

volu

me (

mm

3)

Tumour volume of iMEK inhibitor treatment cohort

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

BRAFV600E::PTEN-/+ - Vehicle

BRAFV600E::PTEN-/+ - iMEK

BRAFV600E::PTEN-/+::Prex2-/- - iMEK

PREX2 combined with MEK inhibition

extends survival

Results: Role of PREX proteins

Screen of inhibitors of main signalling

nodes in melanoma cell lines

P-AKT(S473)

P-ERK

P-RSKp90K

P-AKT(S473)

P-ERK

P-RSKp90K

CHL-1 -- BRAF, NRAS WT

A375 -- BRAFV600E, PTEN+/+

P-AKT(S473)

P-ERK

P-RSKp90K

WM793 -- BRAFV600E, PTEN-/-

P-AKT(S473)

P-ERK

P-RSKp90K

SK Mel 119 – NRASQ61K , PTEN+/+

SK Mel 147– NRASQ61K , PTEN-/-

P-AKT(S473)

P-ERK

P-RSKp90K

Results: Combinatory treatment of PI3K and MAPK

Screen of inhibitors of main signalling

nodes in melanoma cell lines

Results: Combinatory treatment of PI3K and MAPK

-7-6-5-4-3-2-101234

mTORC1i mTORC1/2i AKTi MEKi PI3Ki

P-A

KT

(Lo

g2

)

PTEN+/+ vs PTEN-/- (BRAF(V600E))

A375 SKMel 28 WM793 #1

WM793 #2 WM793 #3 WM266.4

-8-7-6-5-4-3-2-1012

mTORC1i mTORC1/2i AKTi MEKi PI3Ki

P-E

RK

(Lo

g2

)

PTEN+/+ vs PTEN-/- (BRAF(V600E))

A375 SKMel 28 WM793 #1

WM793 #2 WM793 #3 WM266.4

##PTEN+/+ PTEN+/+

PTEN+/+ vs PTEN-/- (BRAFV600E)

-8-7-6-5-4-3-2-1012345

mTORC1i mTORC1/2i AKTi MEKi PI3Ki

P-A

KT

(Lo

g2

)

PTEN+/+ vs PTEN-/- (NRAS(Q61K))

SKMel 119 SKMel 119 Mel224 SKMel 147 #1 SKMel147 #2

-5

-4

-3

-2

-1

0

1

2

3

mTORC1i mTORC1/2i AKTi MEKi PI3Ki

P-E

RK

(Lo

g2

)

PTEN+/+ vs PTEN-/- (NRAS(Q61K))

SKMel 119 SKMel 119 Mel224 SKMel 147 #1 SKMel147 #2

#

#

#

#

PTEN+/+ PTEN+/+

PTEN+/+ vs PTEN-/- (NRASQ61K)

➢ PI3Kb inhibitor is highly effective in all mutation backgrounds

Combination treatment using iMEK and iPI3Kb

in melanoma cell lines

Results: Combinatory treatment of PI3K and MAPK

WM266.4 -- BRAFV600E, PTEN-/-

A2058 -- BRAFV600E, PTEN-/-

A375 -- BRAFV600E, PTEN+/+

SKMel -- BRAFV600E, PTEN+/+

Results: Combinatory treatment of PI3K and MAPK

0

0.2

0.4

0.6

0.8

1

1.2

1.4D

MSO

iMEK

iPI3

Kb

CO

MB

O

DM

SO

iMEK

iPI3

Kb

CO

MB

O

DM

SO

iMEK

iPI3

Kb

CO

MB

O

DM

SO

iMEK

iPI3

Kb

CO

MB

O

WM266.4 A2058 A375 SKMel28

BRAFV600E, PTEN-/- BRAFV600E, PTEN+/+

Combination treatment using iMEK and iPI3Kb

in melanoma cell linesA

bsorp

tion a

t 590 n

m

P-AKT(S473)

P-ERK

TOT-ERK

TOT-AKT

P-S6

TOT-S6

PREX1

Β - Actin

P-AKT(S473)

P-ERK

TOT-ERK

TOT-AKT

P-S6

TOT-S6

PREX1

Β - Actin

Results: Combinatory treatment of PI3K and MAPK

A2058 -- BRAFV600E, PTEN-/- WM266.4 -- BRAFV600E, PTEN-/-

Treatment of BRAFV600E, PTEN-/-

cell lines

P-AKT(S473)

P-ERK

TOT-ERK

TOT-AKT

P-S6

TOT-S6

PREX1

Β - Actin

PTEN

P-AKT(S473)

P-ERK

TOT-ERK

TOT-AKT

P-S6

TOT-S6

PREX1

Β - Actin

PTEN

Results: Combinatory treatment of PI3K and MAPK

A375 -- BRAFV600E, PTEN+/+ SKMel 28 -- BRAFV600E, PTEN+/+

Treatment of BRAFV600E, PTEN+/+

cell lines

Xenograft experiments using WM266.4

human cell line

0

100

200

300

400

500

600

700

800

900

1000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Tu

mo

ur

vo

lum

e (

mm

3)

VEHICLE

iMEK

iPI3Kb

COMBO

Weeks

Results: Combinatory treatment of PI3K and MAPK

Xenograft experiments – tumour volume

Results: Combinatory treatment of PI3K and MAPK

Results: Combinatory treatment of PI3K and MAPK

iMEK and iPI3Kb combination treatment in

genetic mouse melanoma model

Results: Combinatory treatment of PI3K and MAPK

0 1 0 0 2 0 0 3 0 0 4 0 0

0

5 0

1 0 0

T y r C r e E R : : B R a fV 6 0 0 E / +

: : P t e nf l / +

-

T i m e P I ( D a y s )

Ov

er

all

s

ur

viv

al

(%

)

V e h i c l e

A Z D 6 2 4 4 ( 2 5 m g k g- 1

)

A Z D 8 1 8 6 ( 5 0 m g k g- 1

)

A Z D 6 2 4 4 / A Z D 8 1 8 6

Vehicle

iMEK

iPI3Kb

Combination

iMEK and iPI3Kb combination treatment in

genetic mouse melanoma model

RNA sequencing

• Tumours from GEM model were treated short term (72h) and sampled for RNA

sequencing

• Among significantly altered transcriptional programmes:

• Cell cycle control • E2F and Myc targets, G2M checkpoint, mitotic spindle

• DNA repair

• Control of protein translation

RPPA analysis - hierarchical clustering of

individual treatments and combination

A375 -- BRAFV600E, PTEN+/+ WM266.4 -- BRAFV600E, PTEN-/-

Low HighMatt Neilson

Details of RPPA analysis of the

BRAFV600E,PTEN-/- cell line

Low High

Matt Neilson

Results: Combinatory treatment of PI3K and MAPK

Histological analysis of tumours treated with

iMEK and iPI3Kb combination treatment

Ve

hic

le

Sensitive

Resistant

Resistant

Com

bin

ation

P-S6 ribosomal proteinH&E

Future directions 1

Combinatory treatment with PI3Kb and MEK

Application of the treatment within existing clinical setting, in combination with

Vemurafenib and Dabrafenib

Generating treatment – resistant cell lines; exploring further options for resistant

tumors (imTOR, Rapamycin)

Uveal (eye) melanoma; non – responsive to treatment (conventional or targeted),

testing whether the combination treatment would be beneficial

Results: Role of mTORC2/Rictor

mTORC2 subunit Rictor deletion reduced

number of melanoblasts

Tyr::Rictor+/+ Tyr::Rictor-/-

p=0.004

William Faller

NRAS mutant mouse model of melanoma

Tyr-NRASQ61K/+ INK4A-null

TYR

NRASQ61K

knock-in line

Tyr-NRASQ61K/+ ::INK4A-/-

NRASQ61K

INK4A full body

knock – out

Causes lymphoma,

sarcoma

Results: Role of mTORC2/Rictor

Rictor deletion rescues the NRAS mutant

phenotype

Tyr::NRasQ61K/°; INK4a-/- Tyr::NRasQ1K/°; INK4a-/-; Rictor-/-

Tyr::NRasQ61K/°::INK4a-/-

Tyr::NRasQ61K/°::INK4a-/-::Rictor-/-

William Faller

Results: Role of mTORC2/Rictor

Rictor deletion has no effect in

BRAFV600E::PTEN+/- mediated melanoma

P=0.188 P=0.234

Time from induction to melanoma appearance Time from melanoma appearance to death

William Faller

Results: Role of mTORC2/Rictor

Rictor deletion in human melanoma cell lines

Rictor-/-

7%

Rictor+/+

93%

NRASQ61K::PTEN+/+

Rictor-/-

40%

Rictor+/+

60%

BRAFV600E::PTEN-/-

Rictor-/-

3%

Rictor+/+

97%

NRASQ61K::PTEN-/-

WM793

Triple deletion of Rictor, PREX1 and PREX2

Results: Role of mTORC2/Rictor

TyrCre::Rictor-/-::PREX1-/-::PREX2-/+ TyrCre::Rictor-/-::PREX1-/-::PREX2-/-

Future directions 2

Role of Rictor in melanoma

Investigation of mTORC2 regulation in NRAS and BRAF melanoma

Further elucidation of mTORC2 and PREX signaling cross-regulation

Study of melanoma development

Cell of origin of melanoma

Cell of origin of melanoma

DUSP6

model

FAK

model

Role of DUSP proteins in MAPK signaling

Extensive greying in Tyr-NRASQ61K::DUSP6

Tyr::DUSP6+/+

Tyr::N-RasQ61K

DUSP6+/+

Tyr::N-RasQ61K

DUSP6+/-

Tyr::N-RasQ61K

DUSP6-/-

Tyr::N-RasQ61K

DUSP6-/-

Results: Role of DUSP6

DUSP6 has no effect on Tyr-NRASWT

Tyr::N-RasWT

::DUSP6+/+

Tyr::N-RasWT ::DUSP6-/- Tyr::N-RasWT

::DUSP6+/+

Tyr::N-RasWT ::DUSP6-/-

Results: Role of DUSP6

Melanocytes absent form

Tyr::NRASQ61K::DUSP6-/- hair follicles

Tyr::N-RasQ61K

DUSP6+/+

Tyr::N-RasQ61K

DUSP6-/-

Results: Role of DUSP6

Dermal melanocyte proliferation in

Tyr-NRASQ61K not regulated by DUSP6

Tyr::N-RasQ61K ::DUSP6-/-

Tyr::N-RasQ61K ::DUSP6+/-

Tyr::N-RasQ61K ::DUSP6+/+

Results: Role of DUSP6

Before treatment Treated for 1 month Treated for 2 months

Tyr::N-RasQ61K::DUSP6+/+

Tyr::N-RasQ61K ::DUSP6-/-

MEK inhibition rescues the

Tyr-NRASQ61K::DUSP6-/- phenotype

Results: Role of DUSP6

Rescue of the Tyr-NRASQ61K::DUSP6-/-

phenotype is transient

Treated for 1 month Treated for 2 months

Off treatment for 2 months

Results: Role of DUSP6

BRAFV600E/+::PTENFL/+::DUSP6FL/FLBRAFV600E/+::PTENFL/+::DUSP6FL/+BRAFV600E/+::PTENFL/+::DUSP6+/+

DUSP6 deletion in BRAFV600E::PTEN-/+

melanoma model

Results: Role of DUSP6

Role of focal adhesion kinase – FAK in

melanoma

Tyr::N-RasQ61K

Tyr::Cre FAKfl/fl

Tyr::N-RasQ61K

Tyr::Cre FAKfl/+

*

Tyr::N-RasQ61K

Tyr::Cre FAKfl/+

Tyr::N-RasQ61K

Tyr::Cre FAKfl/fl

Results: Role of FAK

FAK deletion reduces dermal melanocyte

proliferation in NRAS mutant mice

Colin Lindsey

FAK deletion extends survival in the

NRAS mutant melanoma

Tyr::N-RasQ61K::FAKfl/fl (Cre-N) 6/14 41%

Tyr::N-RasQ61K::TyrCre::FAKfl/fl 6/42 12%

Results: Role of FAK

William Faller

Tyr::N-RasQ61K

Tyr::Cre FAK+/+

Tyr::N-RasQ61K

Tyr::Cre FAKfl/fl

Results: Role of FAK

Effect of FAK deletion on early dermal

mislocalisation of melanocytes – day 5

Tyr::N-RasQ61K

Tyr::Cre FAK+/+

Tyr::N-RasQ61K

Tyr::Cre FAKfl/fl

Results: Role of FAK

7 days old

Effect of FAK deletion on early dermal

mislocalisation of melanocytes – day 7

Tyr::N-RasQ61K

Tyr::Cre FAK+/+

Tyr::N-RasQ61K

Tyr::Cre FAKfl/fl

Results: Role of FAK

Effect of FAK deletion on early dermal

mislocalisation of melanocytes – day 14

Future directions

Role of DUSP proteins in melanoma

Setup TyrCreER-BRAFV600E::PTEN-/+ ::DUSP6 melanoma colony – role of DUSP6

in BRAF mutant melanoma

Setup Tyr-NRASQ61K::Cdkn2a-/- ::DUSP6 melanoma colony – role of DUSP6 in early

NRAS melanoma

Setup TyrCreER-NRASQ61R::Cdkn2a-/- ::DUSP6 melanoma colony – role of DUSP6

in adult NRAS melanoma

Role of DUSP5 in melanoma development, alone and together with DUSP6

Role of FAK in melanoma

Expanding the investigation of the effect of FAK on immune infiltration

Setup TyrCreER-BRAFV600E::PTEN-/+ ::FAK melanoma colony – role of FAK in

BRAF mutant melanoma

Acknowledgements

Owen Sansom

Andrew Campbell

Mona Foth

William Faller

All R18 & R14

Collaborators:

William Gallagher, Walter Kolch, Lan Nguyen; Sungyoung Shin Conway Institute, Dublin; University of Sydney

Lionel Larue Institut Curie, Orsay, France

Simon BarryAstra Zeneca

Cell sorting and BAIR platforms:Tom Glibey

Tim Harvey

Margaret O’Prey

Histology:Colin Nixon and co.

Common services:Andrew Keith

Billy Clark

Bioinformatics:

Ann Hedley

Matt Neilson

BSU, BRU staff

Thank you for your attention!!!