Combined Farnesyl Transferase Inhibition and
Proteasome Inhibition synergistically induce
apoptosis via Downregulation of p-AKT
Sagar Lonial, MD
Assistant Professor, Winship Cancer Institute
Director of Translational Research, B-cell Malignancy Program
Emory University School of Medicine
The Problem:
· Cancer Cells are Complex
· Cancer Cells are Smart
· Cancer Cells are Resilient
The Proteasome:
Proteolytic Enzyme With Important Impact on
Multiple Regulatory Pathways
Cyclins A,B, D & E
CDK inhibitors (p21, p27)
Protein
Ubiquitin
Protein-
Ubiquitin
p53
Topoisomerase I
Proteasome
IB/NF-B Pathway
Peptides
Ubiquitin
Adams et al. Inv New Drugs. 2000
Responses to Bortezomib in
Relapsed/Refractory MM
Summit trial
35% CR+PR+MR
Crest Trial
1.3mg/m2 50% CR+PR+MR
1.0mg/m2 33% CR+PR+MR
APEX Trial
38% CR+PR
45% CR+PR in second line
A Good Start, but there is room for improvement
Proposed Mechanism for FTI's
Tipifarnib Induces Apoptosis in
Human MM cells
S Le Gouill et al, Leukemia 2002
Clinical use of the farnesyl transferase
inhibitor R115777 (Zarnestra)
Alsina et al, Blood, May 2004
Myeloma and Stromal Cell Interactions
Myeloma Cell
Bone Marrow
Stromal Cell
Other Myeloma Cells
R115777
IL-6
IB + ubiquitin
proteosome
IL-6
IB
RAF
RAS
r
NFB
PS-341
PS-341
MEK
JAK/STAT
Free NFB
proteosome
IB
SHP2
MAPK
(Blocks Dex
IB
induced apoptosis)
IL-6
NFB
ubiquitin
Free A.A.
NFB
Thalidomide/IMiDs
Enhanced
Arsenic Trioxide
Gene
Expression
IL-6
IL-6 production
IL-6 secretion
Enhanced Gene Expression
Adhesion molecule expression
MEK/MAPK
-VLA-4
ICA
-MUC-1
M
-LFA-1
-1
Angiogenesis mediators
VC
-VEGF
AM
-bFGF
-1
From Lonial et al, Cancer Biology and Therapy, 2003
Hypothesis
Combinations of 2 Targeted Agents that
are non-overlapping in mechanism can
result in Increased MM cell death at lower
doses than either agent given alone.
MM.1S Survival with Increasing Doses of Bortezomib
0.5
0.4
Baseline Survival
0.3
(MTT)
0.2
Survival
0.1
Cell
0
0 1 2 3 4 5 10 15 20 25 30 35 40 45 50
Bortezomib (nm)
Effect of Bortezomib in combination with Tipifarnib
1.2
1
R115777
R115777+ PS-341
MTT)
by 0.8
(OD 0.6
0.4
survival
0.2
Cell
0 Untreated 250nm 500nm 750nm 1uM 2uM 3uM 4uM 5uM 8nmPS-
341
Tipifarnib alone or with Bortezomib
Control
Induction of apoptosis in MM.1R
cells with the combination
Bortezomib
Control
8nM
40%
5%
12%
21%
R115777
5µm
Combination
22%
60%
24%
28%
Time dependent apoptosis in MM.1S cells upon PS-341 + R115777 combination
8nM PS-341
Untreated- 24hrs
8nM PS-341
5µM R115777
+ 5µM R115777
1%
1%
2%
25%
PI
3%
3%
4%
11%
AnnexinV
72hrs
12
10
(%)
8
cells
24hrs
6
+
48hrs
inx 4
72hrs
enn 2
A 0 Untreated 8nmPS-341 5uMR115777 8nmPS-341+
R115777
Reduced p-AKT levels Are associated with Apoptosis
only when Combination Therapy is Used
1
2
3
4
5
6
7
8
9
10
11
AKT Phos473
AKT2
Total AKT
GSK-alpha
GSK-beta
actin
-
+
-
-
-
-
-
+
+
+
+
8nm PS-341
-
-
+
-
-
-
-
-
-
-
-
20nm PS-341
-
-
-
0.050 0.250 2.5 5.0 0.050 0.250 2.5 5.0 R115777 (µm)
FTI Alone
Combination
Primary Human MM Cells are sensitive to the
Combination
CD38
CD38
CD38
CD138
CD45
CD138
AnnexinV
AnnexinV
Untreated
8nm PS-341
5µm R115777
8nm PS-341+ R115777
CD138
AnnexinV
Activation of the Caspase Pathways is Brisk
and more pronounced in the Combination
15hrs
30hrs
1
2
3
4
5
6
1
2
3
4
5
6
Uncleaved
CFs
Caspase 8
CF
Uncleaved
Caspase 9
Intermediate forms
CF
Uncleaved
Caspase 3
Intermediate forms
CFs
Uncleaved
PARP
CF
-actin
-
+
-
-
+
-
-
+
-
-
+
-
PS-341 (8nM)
-
-
+
-
-
+
-
-
+
-
-
+
PS-341 (20nM)
-
-
-
+
+
+
-
-
-
+
+
+
SCH66336 (5µM)
MM.1S Cells Transfected with AKT are More Resistant
to Combination Therapy
GFP+ (R1)
GFP- (R2)
Untreated
Gated GFP- (R2) and GFP+ (R1) cells
4%
29.2
Untreated
Scatter
deSi
10%
35%
8nm
PS-
GFP-FITC
341
Akt (act) Akt (WT) Empty Vector
c-myc Akt
7%
31%
5µM
R115777
Phos-Akt473
8nM PS-341
10%
64%
8nm
Phos-Akt473
PS-
341+
Total Akt
5µM
Untreated
R11577
AnnexinV-APC
7
Combination therapy can overcome
the survival benefit for IL-6 and IGF-1
IL-6
RPMI8226
IGF-1
9000
Control
8000
7000
6000
5000
4000
Incorporation
3000
2000
1000
0
Thymidine
Bort (8nm)
-
+
-
+
Lona (5um)
-
-
+
+
Sequence of Administration is Critical
8nm PS-341+ 5µM
20nM PS-341+ 5µM SCH66336
90 SCH66336
80
) 70
(% 60
50
death 40
Cell 30
20
10
0
CC
P
S S
P
CC
P
S S
P
Concurrent
Sequence
Sequence
-
+
-
-
+
-
+
-
+
-
PS-341(8nM)
-
-
+
-
-
+
-
+
-
+
PS-341(20nM
-
-
-
+
+
+
+
+
-
+
SCH66336(5µM)
AKTphos473
Phos-GSK &
-actin
PS
S
P
Conclusions
· Combinations of FTI and PI are effective and
synergistic in MM cells
· Rapid Caspase dependant and independent
apoptosis is induced
· P-AKT downregulation is seen when the
combination is effective
· Sequence of Administration can effect efficacy of
the combination
· Clinical Trial design must take into account
preclinical models
Emory
DFCI
Ebeneezer David PhD
Dr Kenneth Anderson
S-Y Sun PhD
Dr Dharminder Chauhan
Fadlo Khuri MD
Dr Constantine Mitsiades
Amelia Langston MD
L.T. Heffner MD
Edmund K Waller MD, PhD