A Small Chemical Molecule Selectively Inhibiting the
Antiapoptotic and Cell Division-Controlling
Protein Survivin shows Exceptional
Antitumor Activity
Shousong Cao1,2,3,#, Xiang Ling1,#, Qiuying Cheng1, James T. Keefe1,
Youcef M. Rustum3,4 and Fengzhi Li1,4,*
Departments of Pharmacology & Therapeutics1, Medicine2 and Cancer Biology3,
Drug Discovery and Experimental Therapeutics (DDET) Program4,
Roswell Park Cancer Institute, Buffalo, New York 14263, USA
# Equal contributions to this work
Apoptosis
The the process of programmed cell death that may occur
in multicellular organisms. Biochemical events lead to
characteristic cell changes.
• Blebbing
• Loss of Cell Membrane Asymmetry and Attachment
• Cell Shrinkage
• Nuclear Fragmentation
• Chromatin Condensation
• Chromosomal DNA Fragmentation
• Extrinsic Pathway
• Initiated by extrinsic ligands binding to death receptors on
the surface of the cell.
– An example of this is the binding of tumour necrosis factor-alpha
to TNF-alpha receptor.
• The activation of the initiator caspases then initiates a
downstream cascade of events that results in the induction of
effector caspases that function in apoptosis.
• Intrinsic Pathway
• Initiated by intracellular or environmental stimuli.
• Membrane permeability of the mitochondria increases and
•
•
particular proteins are released into the cytoplasm that
facilitates the activation of initiator caspases.
Cytocochrome c is released from the mitochrondria and
binds to Apaf-1 in the cytosol resulting in activation of initiator
caspase-9.
Activation of the initiator caspases then initiates a downstream
cascade of events that results in the induction of effector
caspases that function in apoptosis.
Inhibitors of Apoptosis Proteins
• Each contain a Baculovirus IAP Repeat (~70
amino acid motif) in one to three copies.
–
–
–
–
–
–
–
–
–
X-linked IAP
cIAP1
cIAP2
Neuronal Apoptosis Inhibitor Protein
Melanoma IAP
IAP-like Protein 2
Livin
Apollon
Survivin
Survivin
• Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5).
– 142-Amino Acid, 16.5-kDa
– BIRC5 gene; 17q25
• Expressed highly in most human tumors and fetal tissue, but absent
in terminally differentiated cells.
• Inhibits caspase activation leading to negative regulation of
apoptosis or programmed cell death.
• Expression is also highly regulated by the cell cycle and is only
expressed in the G2-M phase.
• Localizes to the mitotic spindle by interaction with tubulin during
mitosis and may play a contributing role in regulating mitosis.
Mita A C et al. Clin Cancer Res 2008;14:5000-5005
©2008 by American Association for Cancer Research
Mita A C et al. Clin Cancer Res 2008;14:5000-5005
©2008 by American Association for Cancer Research
Objectives
• Identify molecules which inhibit
expression of survivin intracellularly.
– High-Throughput Screening using survivin
gene regulatory sequence driving a
luciferase reporter gene.
• Determine the effects of these
molecules on cancer cells.
– In vitro studies on cancer cell lines.
– In vivo human tumor animal models.
Compound Identification
• Cancer cell-based high throughput screening
(HTS) assay systems that used the survivin
gene regulatory sequence driving a luciferase
reporter gene were generated (US Patent
7569221, 2009).
– The HTS systems were used for discovery of
candidate compounds that could alter the
expression of survivin.
– The libraries of chemical compounds are from
multiple resources including those in the public
domain collected by the National Cancer Institute
Developmental Therapeutics Program.
• More than 4000 structurally diverse small
chemical compounds were initially screened
using genetically modified cancer cell models.
• Initial screening resulted in ~250 hit
compound candidates which showed
inhibition of luciferase activity in treated
model cells.
• Two consecutive round screening of the 250
hit candidates using a series of different
concentrations (from 0.001 nM to 1000 nM)
for individual hit compounds resulted in 20
top-hit compounds, which showed inhibition
of luciferase activity.
• Five compounds including FL118 were
selected for initial in vivo human tumor
animal model screening.
• FL118 was found to be the top compound
among the five leads, which displays
exceptional antitumor activity and
favorable toxicity profiles in the animal
models of human colon and head-&-neck
tumor xenografts.
Results
The compound FL118 possesses superior
antitumor activity to clinically used
cancer therapeutic drugs
• Using human head & neck (FaDu) and colon
(HCT-8) cancer animal models, the antitumor
activity of FL118 was compared with those of
FDA-approved, clinically used
chemotherapeutic drugs.
–
–
–
–
–
–
Irinotecan and topotecan (Topoisomerase I Poison)
Cisplatin and Oxaliplatin (DNA Alkylating Agents)
Docetaxel (Microtubule Polymerization Promoter)
Gemcitabine and 5-FU (DNA Synthesis Inhibitor)
Doxorubicin (Topoisomerase II Inhibitor)
Cyclophosphamide (Alkylating Agent)
Mean tumor volume (%)
Mean tumor volume (%)
Fig 1
a.
Control (vehicle)
FL-118 1.5 mg/kg
Irinotecan 200 mg/kg
Irinotecan
Topotecan 12.5 mg/kg
Cisplatin 10 mg/kg
Oxaliplatin 15 mg/kg
Docetaxel 60 mg/kg
Gemcitabine 200 mg/kg
5-FU 300 mg/kg
b.
Doxorubicin 12.5 mg/kg
Cytoxan 100 mg/kg
Irinotecan
FL118 effectively eradicates human tumor
without recurrence in xenografts
• The antitumor activity of FL118 with
irinotecan at their MTD in a human primary
head & neck tumor xenograft 17073 bearing
in SCID mice with the clinically relevant
schedule (weekly x 4) of irinotecan.
– Ironotecan-treated group: 2/5 mice showed
temporary tumor regression after treatment.
– FL118-treated group: 5/5 mice showed complete
response and no recurrent tumor was detected.
– Irinotecan induced an accumulated body weight
loss, while FL118 induced a reversible body weight
loss with rapid recovery after treatment.
FL118 effectively and permanently
eradicates large and advanced
human tumors
• Mice bearing maximal human xenograft
tumors (~2 gm) showed striking
responses to FL118 treatment in both
FaDu head & neck tumors and SW620
colon tumors.
– Of note, the death of the two mice was
likely a result of the Tumor Lysis Syndrome
due to treatment-induced massive tumor
necrosis.
Fig 2
Head & neck primary tumor
Control
c.
a.
Irinotecan
FL-118
b.
Control
FL-118
Irinotecan
Individual human tumor size (mg) on xenograft mice
Mean body weight change (%)
Mean tumor volume (%)
Control
Irinotecan 100mg/kg/wk x 4
d.
FL118 1.5mg/kg/wk x 4
e.
FL118 is chemically stable with a long
shelf life
• To test the chemical stability of FL118 in
solution, a ready-to-use FL118 injection
solution was prepared and stored in a +4C
refrigerator for more than 6 months.
• The old drug solution of FL118 was as
effective as the newly formulated FL118
solution against FaDu and SW620 tumors.
Individual mouse tumor size (mg)
a.
FaDu
SW620
Individual mouse tumor size (mg)
Fig 3
b.
FaDu
SW620
Inhibition of survivin by FL118 is
associated with the modulation of other
members in the IAP and Bcl-2 families
• Downregulation of survivin by FL118 was
associated with the modulation of other
members in the IAP and Bcl-2 families.
– XIAP, cIAP2 and Mcl-1
– Bcl-2 and Bcl-XL
• In contrast, treatment increases the
expression of pro-apoptotic proteins.
– Bax and Bim.
Fig 5
FL118 (10nM): 0
a. A2008
8 16 24 72h
Survivin -
Actin 24 hours
FL118 (nM): 0 10 100
Survivin -
b. PC-3
c. HCT-8
48 hours
FL118 (nM): 0 10 100
Survivin -
Mcl-1XIAP Bcl-2 Bcl-XL Bax -
Actin cIAP2 -
Bim -
Mcl-1-
Actin -
Actin -
*
FL118 differentially modulates the
phosphorylation of Akt and MAPK (Erk1/2)
signaling molecules and induces the
production of apoptotic hallmarks
• Several members in the IAP and Bcl-2 families
are regulated through Akt and/or MAPK
pathways.
• FL118 inhibits both constitutive and taxol-
induced Akt phosphorylation, but shows no
inhibitory effects on the phosphorylation of
Erk1/2.
• FL118 induced caspase activation and PARP
cleavage.
Fig 6
a.
FL118 (10nM): 0
p-Akt(473) -
1
6
12 24h
d.
FL118 (10nM): 0
Survivin -
Total Akt -
b.
FL118 (10nM): 0
p-Akt(473) -
0
Taxol 30nM
1 6 12 24h
Cleaved
caspase 3
Actin -
Total Akt -
c.
FL118 (10nM): 0
p-Erk1/2
Total Erk1/2
Actin -
0
Taxol 30nM
1
6 12
24h
Full PARP cleaved Actin -
1 12 24 48h
FL118 inhibits survivin promoter activity in
a highly selective manner
• FL118 selectively inhibits human survivin
promoter-driven luciferase activity at as low as
0.1 - 1 nM which is dependent on cancer cell
types.
• In contrast to its high inhibitory effects on
survivin promoter activity, FL118 showed no
inhibitory effects on luciferase activity driven by
gene promoters from cyclin-dependent kinase
inhibitor p21 (p21), dehydrofolate reductase
(DHFR), human thrombin receptor (HTR), and
thymidine kinase (TK) in different cancer cell
types.
Fig 4
a. PC-3
FL118
3500
3000
2500
2000
1500
1000
2000
1500
1000
500
0
e. LNCaP-LN3
d. EKVX
T
P
K-
8000
7000
6000
5000
4000
DMSO
FL118
10nM
3000
2000
1000
0
p2
1
D -P
H
FR
-P
H
TR
-P
TK
-P
Luciferase activity in
arbitrary units
c. PC-3
P
P
P
1RRp2 DHF
HT
FL118
2500
Luciferase activity in
arbitrary units
µM 0 nM 0 nM 1 nM 1 nM
1
0.
10
DMSO
FL118 10nM
DMSO
3000
0.
00
0
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
3500
1
0. nM
01
n
0. M
1
nM
1
nM
10
n
10 M
0
nM
500
1
Luciferase activity
in arbitrary units
Luciferase activity in
arbitrary units
DMSO
4000
Luciferase activity
in arbitrary units
b. A2008
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
DMSO
FL118
10nM
-P R-P R-P K-P
1
T
p2 HF HT
D
Discussion
• Several recent studies have demonstrated that
abrogation of one of the four genes (survivin,
Mcl-1, XIAP and cIAP2) inhibits tumor growth in
various in vitro and in vivo cancer models.
• Selective inhibition of the survivin gene by
FL118 in association with the modulation of
Mcl-1, XIAP and cIAP2 in cancer cells may
effectively inhibit tumor growth.
• What is the relationship of survivin inhibition by
FL118 with the associated downregulation of
Mcl-1, XIAP and cIAP2 as well as the inhibition
of Akt signaling by FL118?
• Akt signaling is linked to upregulation of survivin in
cancer.
– Suppression of the Akt/survivin pathway induces apoptosis
and increases antitumor activity. PI3K/Akt signaling also
upregulates the expression of Mcl-1, XIAP and c-IAP2 .
• FL118 inhibits HIF-1 production in hypoxic
conditions.
– HIF-1 has been shown to transcriptionally upregulate
survivin.
– Hypoxia/HIF-1 was also shown to regulate the expression
of Mcl-1, XIAP and IAP2.
• Survivin may be a transcription factor or co-factor to
inhibit p21 gene transcription in a p53-dependent
manner.
– If survivin acts as a transcription factor or a co-factor,
downregulation of survivin expression by FL118 could be
associated with the modulation of other survivin-targeted
gene expression.
Summary
We have discovered and characterized a
novel chemical molecule showing
exceptional antitumor efficacy with
favorable toxicity and chemical stability
profiles. While this molecule selectively
inhibits promoter activity and
expression of survivin, downregulation
of survivin by this molecule is
associated with the modulation of other
cancer-associated signals.
A Small Chemical Molecule Selectively Inhibiting the Antiapoptotic and Cell DivisionControlling Protein Survivin shows Exceptional Antitumor Activity
Shousong Cao1,2,3,#, Xiang Ling1,#, Qiuying Cheng1, James T. Keefe1,
Youcef M. Rustum3,4 and Fengzhi Li1,4,*
Departments of Pharmacology & Therapeutics1, Medicine2 and Cancer Biology3, Drug Discovery and Experimental
Therapeutics (DDET) Program4, Roswell Park Cancer Institute, Buffalo, New York 14263, USA
# Equal contributions to this work
Acknowledgements
• This work was sponsored in part by NIH R01 Grants (CA109481, CA133241) and
the Meso Foundation (Alexandria, VA) grant to FL, and by shared resources
supported by NCI Cancer Center Core Support Grant to Roswell Park Cancer
Institute (CA016056).
•
We thank the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics Program (DTP), Division of Cancer Treatment and Diagnosis,
National Cancer Institute (NCI) for providing chemical libraries and relevant hit
analogs in the public domain they collected and/or synthesized as a major
compound sources during the process of our drug screening and
characterization.
•
We would also like to thank Dr. Paul A. Spengler (University of Rochester) for
his critical reading of the manuscript, Ms. Lileena Johnson (Rotation graduate
student) for the involvement in repeating some of drug screening experiments.
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