Transcript multiple myeloma: D-type cyclin and PI3k pathways — Prof

MULTIPLE MYELOMA:
D-type cyclin and PI3k pathways
UKMF Spring meeting 2015
Kwee Yong, UCL Cancer Institute
Not one, but many myelomas
Dysregulation of D-type cyclin in multiple myeloma
Early oncogenic events dysregulate a cyclin D
gene
Control of the Mammalian Cell cycle
G0
M
p15
p16
p18
p19
G1
Cyclin B-CDK1
P
pRb
G2
S
E2F
Cyclin D1-CDK4/6
Cyclin D2
Cyclin D3
P
The cyclin D-CDK4/6 complex
retinoblastoma
P
P phosphorylates
E2F
p21
pRb
Cyclin E-CDK2
protein, relieving its repressor
action, and allowing
the
p27
transcription of genes required for DNA replication, mitosis etcp57
Cyclin A-CDK2
p21
P27
p57
Expression of D-type Cyclins and their Dependent
Kinases in primary myeloma cells
Normal
BM
Multiple Myeloma
BM
Multiple Myeloma
Extra-Medullary
Cyclin D1
Cyclin D2
CDK 4
CDK 6
Phospho-Rb (K4/6)
Total pRb
p27
PCNA
Actin
• Normal PCs did not express cell cycle regulators
• MM Samples from patients with early stage, or stable disease did not express
cell cycle regulators
• Increased expression of cell cycle regulators in patients with progressive
disease (progressed on induction, florid relapse)
What is the significance of cyclin D expression in
myeloma?


Contribution of cell cycle dysregulation to disease
pathogenesis
Does cyclin D1-expressing myeloma behave
differently from D2-expressing disease?
What is the significance of cyclin D expression in
myeloma?


Contribution of cell cycle dysregulation to disease
pathogenesis
Does cyclin D1-expressing myeloma behave
differently from D2 disease?
Proliferative rate is prognostic
Thomas et al, Eur J Hematol, 2010
Cell cycle analysis of myeloma cells
Myeloma cell line
NCI-H929
Primary CD138+ Cells
G2M –
1.1%
17%
S – 1.4%
Ki67
G0/G1 – 79%
DNA Content
<G1 – 18.5%
Quinn et al, Blood 2011
Proliferative fraction of freshly isolated bone
marrow CD138+ myeloma cells
Smouldering MM = 4
De Novo MM = 30
Plateau = 8
Relapse = 88
Extramedullary disease = 9
Total = 139
Quinn et al, Blood 2011
How is cell cycling regulated in myeloma
cells?



External mitogens or growth factors
Alteration (mutations, copy number changes,
epigenetic marks) of genes encoding cell cycle
regulatory proteins
Alteration of pathways regulating cell cycle proteins
(post-translational)
How is cell cycling regulated in myeloma
cells?



External mitogens or growth factors
Dysregulation (mutations, copy number changes,
epigenetic marks) of genes encoding cell cycle
regulatory proteins
Dysregulation of pathways regulating cell cycle
proteins (post-translational)
What is the significance of cyclin D expression in
myeloma?


Contribution of cell cycle dysregulation to disease
pathogenesis
Does cyclin D1-expressing myeloma behave
differently from D2 disease?
Cyclin D2 myeloma cells respond to cytokines
Quinn et al, Blood 2011
n = 12
n= 14
Is Cyclin D1 Functional in t(11;14) MM Cells?
Immuno-precipitation experiments indicate that cyclin D1 is
found in complexes with CDK4/6
Smith D, unpublished data
Expression of cell cycle proteins in vivo:
comparing cyclin D1 with D2 tumours
CDK4
Cyclin D1
Cyclin D2
CDK6
Phospho-pRb
Is Cyclin D1 Functional in t(11;14) MM Cells?
siRNA:
(6g)
Cyclin D1
Phospho-pRb (K4/6)
% Cells in each Cell
Cycle Phase
Total pRB
100%
90%
G2/M
80%
70%
S
60%
G1
50%
40%
<2n
30%
20%
10%
0%
-
Glassford et al, Brit J Haem 2009
: siRNA
(6g)
Why are cyclin D1 tumours less proliferative?
M
p15
p16
p18
p19
G1
Cyclin B-CDC2
P
pRb
G2
E2F
S
P
P
P
Cyclin D1/CDK2 complexes are inactive
E2F
pRb
Cyclin A-CDK2
Cyclin D1-CDK4/6
Cyclin D2
Cyclin D3
Cyclin E-CDK2
p21
P27
p57
p21
p27
p57
Non-cell cycle functions of cyclin D1?


DNA repair
Survival
Jirawatnotai et al, Nature 2011
Beltran et al, PNAS 2011
How is cell cycling regulated in myeloma
cells?



External mitogens or growth factors
Dysregulation (mutations, copy number changes,
epigenetic marks) of genes encoding cell cycle
regulatory proteins
Dysregulation of pathways regulating cell cycle
proteins (post-translational)
Chromosome 1q amplification in myeloma


Frequency increases with disease progression
Associated with shorter survival
Shah et al, ASBMT 2015
Avet-Loiseau, JCO 2012
Prognostic impact of chr 1q gain may be
related to cell cycle effects of CKS1b

Gain of 1q21 associated with increased expression of CKS1b



Part of E3 ubiquitin ligase targeting p27
Associates with CDK2 to enhance kinase activity
Net result is increase in cell cycling
Freshly isolated CD138+ cells
from patients with chr1q gain
have higher levels of basal
proliferation as measured by
Ki67/PI
Quinn J, unpublished data
Myeloma cells with Cks1b amp have lower levels
of p27
p18
p27
actin
JJN3
MM1S
8
9
10
11
12
13
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. /0123$4 $' /56$$$$4 7$$$$$$$$$$$$$$4 7$$$$$$$$$$$$4 ) $$$$$$$$$$$$4 ) $$$$$$$$$$$$4 ) $$$$$$$$$$$$4 7$$$$$$$$$$$4 ) $$$$$$$$$$$4 ) $
. 89) : $$; < 5$$$$$$$!
$!
$!
!
$
Primary CD138+ cells
Quinn J, unpublished data
14
Therapeutic implications
Cell cycle Inhibitors

CDK inhibitors
Small molecule inhibitor of CDK4 and CDK6
 In vitro and in vivo pre-clinical data
 Phase 1 study with Bortezomib & Dexamethasone

Smith D, unpublished data
Cell cycle Inhibitors


Dinaciclib: CDK inhibitor (CDK1, 2, 5, 9, also cyclin D/CDK4)
Phase 1 study in RRMM completed

11% PR, 19% MR or better

PFS 3.5 mo, OS 18.8 mo
How is cell cycling regulated in myeloma
cells?



External mitogens or growth factors
Alteration (mutations, copy number changes,
epigenetic marks) of genes encoding cell cycle
regulatory proteins
Alteration of pathways regulating cell cycle proteins
(post-translational)
What does PI3k signalling have to do with
D-type cyclins and cell cycle control?
PI3k and mTOR signalling
IL-6, IGF-1, BAFF, APRIL, Integrin binding
GRB10
PI3K
IRS1
PTEN
ERK/RSK
mTORC2
PDK1
AMPK
SGK
TSC1/2
Amino
acids
PIP3
RAF/MEK
IKKB
AMP:ATP
PIP2
RAS
INFLAMMATION
S473 T308
AKT
Rag C/D
Rag A/B
RHEB
GSK3
PRAS40
MDM2
FOXOs
mTORC1
MYC
S6K1
4EBP1
p27
S6
eIF4E
Cell growth
ULK1
ATG13
Autophagy
BIM
BAD
p53
p21 Cyclin D1
Proliferation and Survival
PI3k and mTOR signalling
PI3Ki PIK90, GDC0941, ZSTK474
PI3K+mTORi (dual inhibitors)
GRB10
PI103, BEZ235, XL765
PI3K
IRS1
PIP3
RAF/MEK
IKKB
PTEN
ERK/RSK
AKTi Akti1/2,
mTORC2
AZD5363, MK2206
PDK1
AMP:ATP
Amino
acids
PIP2
RAS
INFLAMMATION
AMPK
mTORi
TSC1/2
PP242,
KU006, WYE-354
SGK
S473 T308
AKT
Rag C/D
Rag A/B
RHEB
GSK3
PRAS40
MDM2
FOXOs
Rapamycin
mTORC1
MYC
S6K1
4EBP1
p27
S6
eIF4E
Cell growth
ULK1
ATG13
Autophagy
BIM
BAD
p53
p21 Cyclin D1
Proliferation and Survival
Activation of PI3K pathway in myeloma is not
universal, may depend on D-type cyclin / IgH Tx
t(14;16)
t(4;14)
t(11;14)
pAkt
S473
pS6
S235/236
p4EBP1
T37/46
KMS27
KMS21
KMS12BM
JIM1
U266
H929
LP1
OPM2
KMS11
R8226
MM1S
JJN3
GAPDH
***
***
t(4;14)
t(14;16)
t(11;14)
U266
KMS27
KMS21BM
KMS12BM
KMS28BM
LP1
JIM-1
KMS34
H929
OPM2
KMS11
RPMI8226
Zollinger et al, Blood 2008
Stengel et al, Leukemia 2012
MM1S
45
40
35
30
25
20
15
10
5
0
JJN3
pAKT S473
(median cell fluorescence)
Phospho-Akt by flow
Effect of dual PI3K/mTOR blockade on
growth of myeloma cells
MM1.s
Cyclin D2
100
% Viable Cells in IGF-I + PI-103
90
80
70
60
50
NCI-H929
40
30
Cyclin D2
20
10
0
KMS12BM
Cyclin D1
t(11;14)
t(14;16)
t(4;14)
PI-103 (mol)
Dual PI3K/mTOR blockade decreases cell cycling
in cyclin D2 cells
G1
<2n
S G2/M
MM1-S
Cyclin D2
Control
IGF-I
IGF-I + PI103
KMS-12BM
Cyclin D1
PI3K signalling regulates cell cyclin D2 but not
cyclin D1
KMS12-BM
MM1-S
control FCS IGF-I IL-6
PI103: - + - + - + - +
control
FCS
PI103: - + - + -
IGF-I IL-6
+ - +
Cyclin D2
Cyclin D1
phospho-pRb
phospho-pRb
Total pRb
Total pRb
CDK6
CDK6
CDK4
CDK4
p27
p27
Actin
Actin
PI3k/mTOR blockade reduces proliferation in primary
MM cells expressing cyclin D2
P<0.01
H-thymidine uptake (fold change)
5
4
3
2
1
P<0.02
4
3
2
1
3
3
H-thymidine uptake (fold change)
Cyclin D2
Cyclin D1
t(11;14)
5
0
0
RPMI
IGF-I
IGF-I:
PI-103:
RPMI
IGF-I & PI-103
-
+
-
+
+
-
+
-
IGF-I
IGF-I & PI-103
+
+
Cyclin D2
phospho-Rb (K4/6)
Actin
Patient # 9
Patient # 8
t(14;16)
t(4;14)
Patient #
Patient #
5
Patient #
6
Patient #
7
8
Patient #
Patient # 10
9
Patient # 11
Patient # 12
Effect of PI3K/mTOR blockade on survival
of MM cells
HMCL
CD138+ BM cells
% live cells
100
75
50
25
0
t(14;16)
t(4;14)
t(11;14)
cytogenetic category
0h
4h
6%
84%
PI
24h
7%
70%
11%
ANN V
8h
10%
67%
23%
25%
30%
23%
44%
PI3k and mTOR signalling
PI3Ki PIK90, GDC0941, ZSTK474
PI3K+mTORi (dual inhibitors)
GRB10
PI103, BEZ235, XL765
PI3K
IRS1
PIP3
RAF/MEK
IKKB
PTEN
ERK/RSK
AKTi Akti1/2,
mTORC2
AZD5363, MK2206
PDK1
AMP:ATP
Amino
acids
PIP2
RAS
INFLAMMATION
AMPK
mTORi
TSC1/2
PP242,
KU006, WYE-354
SGK
S473 T308
AKT
Rag C/D
Rag A/B
RHEB
GSK3
PRAS40
MDM2
FOXOs
Rapamycin
mTORC1
MYC
S6K1
4EBP1
p27
S6
eIF4E
Cell growth
ULK1
ATG13
Autophagy
BIM
BAD
p53
p21 Cyclin D1
Proliferation and Survival
PI3k and mTOR signalling
PI3Ki PIK90,Buparlisib
GDC0941, ZSTK474
PI3K+mTORi (dual inhibitors)
GRB10
PI103, BEZ235, XL765
PI3K
IRS1
PIP3
RAF/MEK
IKKB
PTEN
ERK/RSK
AKTi Akti1/2,
mTORC2
AZD5363, MK2206
PDK1
AMP:ATP
Amino
acids
PIP2
RAS
INFLAMMATION
AMPK
mTORi
TSC1/2
PP242,
KU006, WYE-354
SGK
S473 T308
AKT
Rag C/D
Rag A/B
RHEB
GSK3
PRAS40
MDM2
FOXOs
Rapamycin
mTORC1
MYC
S6K1
4EBP1
p27
S6
eIF4E
Cell growth
ULK1
ATG13
Autophagy
BIM
BAD
p53
p21 Cyclin D1
Proliferation and Survival
Phase 1B Study of Buparlisib with Bortezomib in
Defined Genetic Subgroups of Patients with
Relapsed or Refractory Multiple Myeloma
Primary OBJECTIVES


To determine the MTD (and/or RP2D, recommended phase 2 dose) of buparlisib with
bortezomib (BKM-Bz) in relapsed/refractory multiple myeloma (MM) patients (dose
escalation part)
To evaluate the safety of the combination of BKM-BZ in patients with relapsed/refractory
MM (dose expansion part)
Summary and Conclusions

Cyclin D1 and D2 tumours differ
 In
cell cycle response to mitogens
 Dependence on PI3k pathway for proliferation and
survival


Exploit such differences by tailoring therapies to
particular genetic sub-types
Understand how newly acquired genetic lesions
impact on the cellular wiring imposed by type of D
cyclin