ucl cancer institute
Survival signalling in myeloma
Asim Khwaja
UCL Cancer Institute & Department of Haematology
Rationale for developing targeted therapies - we are likely
to be close to the limits of what can be achieved with
conventional cytotoxic drugs
Today’s talk
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Background
Potential targets for therapeutic intervention
PI3-Kinase and mTOR
PIM kinases
The molecular pathology of myeloma
Kuehl & Bergsagel, JCI 2012
Myeloma microenvironment
Kuehl & Bergsagel, JCI 2012
Potential signalling pathway targets in
myeloma
Pathway
Microenvironment
Molecular
NF-kappa B
TNF, BAFF, APRIL
Dels/amp/mut of TRAF3,
CYLD, BIRC2/3, NIK,
NFKB1/2 etc
RAS
IGF1, IL6, FGF, chemokines
Activating mutations
NRAS, KRAS, BRAF, FGFR3
PI3-kinase
IGF1, IL6, chemokines,
integrins etc
PTEN del, PIK3CA mut, RAS
mutation, (DEPTOR
overexpression)
JAK kinase
IL-6
WNT
Multiple WNTs
NOTCH
Jagged, Delta-like
NF-kappaB signalling
Alternative
Canonical
TNF
BAFF, APRIL
TRADD TRAF2
RIP1
TRAF3 TRAF2
TAK1
cIAP1/2 NIK
IKKg
IKKa
IKKa
IKKb
Proteasome
IkB
p100
RelB
p50 p65
degradation
p50 p65
processing
p52 RelB
p52 RelB
Primary MM cells display constitutive NF-κB activity that is largely resistant to high
concentrations of bortezomib.
Markovina S et al. Mol Cancer Res 2008;6:1356-1364
©2008 by American Association for Cancer Research
Possible mechanism whereby bortezomib triggers canonical NF-κB activation.
Hideshima T et al. Blood 2009;114:1046-1052
©2009 by American Society of Hematology
PI3K, AKT and mTOR signalling
GRB10
PI3K
IRS1
PIP2
RAS
INFLAMMATION
IKKB
AMP:ATP
ERK/RSK
PTEN
mTORC2
Other targets
eg RAC
PDK1
AMPK
SGK
TSC1/2
Amino
acids
PIP3
RAF/MEK
S473 T308
AKT
Rag C/D
Rag A/B
RHEB
GSK3
PRAS40
MDM2
FOXOs
mTORC1
S6K1
4EBP1
p27
S6
eIF4E
Cell growth
ULK1
ATG13
Autophagy
BIM
BAD
p53
Cyclin D1
MYC
Proliferation and Survival
Akt Signaling
Growth
Factors
R
T
K
G
P
C
R
p70S6K
PTEN
PDK-1
TSC2
ERK
Pathway
Blocks
Apoptosis
GLUT4
Vesicle
Caspase
Cascade
P
PDE3B
HSP90
P
Caspase9
CDC37
Glucose
Uptake
P
P
P
IKKs
Ser87
P
P
Akt
WNK1
Chk1
Protein
Synthesis
Cell
Survival
P
PFK2
BAD
14-3-3
Elevation of Glucose
Import
PFK1
DNA-PK
PP2A
Raf1
XIAP
SYK
PDK-1
Akt
P
PRAS40 14-3-3
P
p47Phox
p21(CIP1)
P
p27(KIP1)
14-3-3
14-3-3
P
YAP
P
MDM2
Death
Genes
GSK3
JIP1
P
ASK1
14-3-3
P
AR
14-3-3
P
Ataxin
CREB
P
NF-kB
Pathway
Insulin
Stimulated
Mitogenesis
Apoptosis
Inhibition
Respiratory
Brust
p73 Mediated
Apoptosis
Neuroprotection
Aggregation and
Neurodegeneration
Cell
Survival
FKHR
Glycogen
Synthase
P
CREB
P
Nucleus
PI3K
PIP3
TSC1
Survival
Genes
ILK
CTMP
Regulation of
Cyclic Nucleotide
FKHR
FAK
PI3K
GAB2
PIP3
eIF4E
PIP3
PI3K
mTOR
4EBP1
PIP2
Cytokine
Receptor
IRS1
PI3K
Ga
GTP
Synaptic
Signaling
Translation
Cytokines
Integrins
Ub
MDM2
p53
Ub
CcnD
Cell
Cycle
Glycogen
Synthesis
JNK
Pathway
p53 Degradation
P
eNOS
NO
Production
Cardiovascular
Homeostasis
C 2007-2009
SABiosciences.com
Targeting PI3K, Akt and mTOR
PI3Ki PIK90, GDC0941, ZSTK474
AKTi Akti1/2, AZD5363, MK2206
mTORi PP242, KU006, WYE-354
PI3K+mTORi (dual inhibitors)
PI103, BEZ235, XL765
Increased PI3K activity is associated with distinct
myeloma IgH translocation categories
Phospho-Flow Akt
***
t(4;14)
t(14;16)
t(11;14) undetectable
U266
KMS27
KMS21BM
KMS28BM
t(11;14)
LP1
JIM-1
KMS34
H929
OPM2
t(4;14)
KMS11
RPMI8226
PIP3 LEVELS
t(14;16)
MM1S
pAKT
45
40
35
30
25
20
15
10
5
0
***
KMS12BM
t(11;14)
JJN3
t(14;16)
pAKT S473
(median cell fluorescence)
t(4;14)
Increased PI3K activity correlates with high level of cell
death induced by dual PI3K/mTOR inhibition
***
***
t(14;16)
t(4;14)
t(11;14)
100
PI103
60
40
PI3K
U266
KMS27
KMS21BM
KMS12PE
KMS12BM
LP1
JIM-1
KMS34
KMS28BM
KMS18
H929
OPM2
KMS11
AKT
Patient samples
PI103-induced cell death
% control
RPMI8226
0
MM1S
20
JJN3
% live cells
80
100
r=0.813,
p=0.0001
75
50
25
0
0
10
20
pAKT (MCF)
30
40
mTOR
Optimal cell killing requires inhibition of both PI3K and mTOR
PI103
PIK90
Rapa/Evero
Bez235
PI3K
PP242 etc
mTOR
AKT
Akti1/2
% live cells
100
80
60
40
20
0
PP242
KU
WYE
EVERO
PI103
PIK90
Potent feedback activation of PI3K by
inhibitors of mTOR in myeloma cells
PI3K
*
*
pAktT308
pS6
tubulin
8 24
CON
8 24 8 24 8 24
PI
PP1 PP2
8 24
PP5
PIP3 (pmol/million cells)
pAktS473
***
RAPA
AKT
mTORC1
PIP3 LEVELS
PP242
mTORC2
There is significant PI3K-independent mTOR activity in
myeloma cells
IKKB
CON
PI103
PIK90
BEZ
RAP
ERK/RSK
PI3K
P90+RAP
pAkt S473
AMPK
pS6
mTORC1
AKT
p4EBP1
tubulin
4
8 4 8 4 8 4 8 4 8 4
8
Cell growth
Proliferation and Survival
hours
PI103
PIK90
Rapa
Bez235
PI3K
AKT
mTOR
The role of DEPTOR in modulating PI3K and mTOR
signalling in myeloma
t(11;14)
t(4;14)
t(14;16)
IKKB
Deptor
ERK/RSK
PI3K
AMPK
GAPDH
mTORC1
AKT
DEPTOR
Cell growth
Proliferation and Survival
HMCL
DEPTOR
pS6
p4EBP1
tubulin
PI3K/mTOR in myeloma
• There is a significant PI3K-independent
component leading to mTOR activation
• Dual inhibition of PI3K and mTOR kinase activities
induces maximal cytotoxic effects
• PI3K activity correlates with response to dual
inhibitors
• Cytogenetics may predict pathway activation and
hence response
• Evidence for synergy with glucocorticoids
• The role of DEPTOR is unclear
Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma
Steinbrunn et al British Journal of Haematology
Volume 159, Issue 4, pages 430-440, 17 SEP 2012 DOI: 10.1111/bjh.12039
http://onlinelibrary.wiley.com/doi/10.1111/bjh.12039/full#bjh12039-fig-0002
Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma
Steinbrunn et al British Journal of Haematology
Some conclusions
• Targeting single cell signalling pathways unlikely to be effective
• Activity of various combinations
– PI3K+MEK
– PI3K+mTOR
– PI3K/AKT+PIM
Identification of biomarkers
– mutation analysis
– cytogenetics as surrogate
– expression of signalling proteins
• Integration with existing therapies
ucl cancer institute
Acknowledgements
• Chloe Stengel
• Koremu Meja
• Ching Cheung
• Clare Shepherd
• Lolly Banerjee
• Kwee Yong