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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!!!