A Small Chemical Molecule Selectively Inhibiting A Small Chemical Molecule Selectively Inhibiting the Antiapoptotic and Cell Division-Controlling the Antiapoptotic and Cell Division-Controlling

Protein Survivin shows Exceptional Protein Survivin shows Exceptional Antitumor ActivityAntitumor Activity

Shousong CaoShousong Cao1,2,3,#1,2,3,#, Xiang Ling, Xiang Ling1,#1,#, Qiuying Cheng, Qiuying Cheng11, James T. Keefe, James T. Keefe11, , Youcef M. RustumYoucef M. Rustum3,43,4 and Fengzhi Li and Fengzhi Li1,4,*1,4,*

Departments of Pharmacology & TherapeuticsDepartments of Pharmacology & Therapeutics11, Medicine, Medicine22 and Cancer Biology and Cancer Biology33, Drug , Drug Discovery and Experimental Therapeutics (DDET) ProgramDiscovery and Experimental Therapeutics (DDET) Program44, ,

Roswell Park Cancer Institute, Buffalo, New York 14263, USARoswell Park Cancer Institute, Buffalo, New York 14263, USA

# Equal contributions to this work # Equal contributions to this work

ApoptosisApoptosis

The the process of programmed cell death that may The the process of programmed cell death that may occur in multicellular organisms. Biochemical occur in multicellular organisms. Biochemical events lead to characteristic cell changes.events lead to characteristic cell changes.

•Blebbing Blebbing

•Loss of Cell Membrane Asymmetry and Loss of Cell Membrane Asymmetry and Attachment Attachment

•Cell Shrinkage Cell Shrinkage

•Nuclear Fragmentation Nuclear Fragmentation

•Chromatin Condensation Chromatin Condensation

•Chromosomal DNA FragmentationChromosomal 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 Inhibitors of Apoptosis ProteinsProteins• Each contain a Baculovirus IAP Repeat Each contain a Baculovirus IAP Repeat

(~70 amino acid motif) in one to three (~70 amino acid motif) in one to three copies. copies. – X-linked IAPX-linked IAP– cIAP1cIAP1– cIAP2cIAP2– Neuronal Apoptosis Inhibitor ProteinNeuronal Apoptosis Inhibitor Protein– Melanoma IAPMelanoma IAP– IAP-like Protein 2IAP-like Protein 2– LivinLivin– ApollonApollon– SurvivinSurvivin

SurvivinSurvivin

• Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5).Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5).– 142-Amino Acid, 16.5-kDa142-Amino Acid, 16.5-kDa– BIRC5BIRC5 gene; 17q25 gene; 17q25

• Expressed highly in most human tumors and fetal tissue, but Expressed highly in most human tumors and fetal tissue, but absent in terminally differentiated cells.absent in terminally differentiated cells.

• Inhibits caspase activationInhibits caspase activation leading to negative regulation of leading to negative regulation of apoptosis or programmed cell death. apoptosis or programmed cell death.

• Expression is also highly regulated by the cell cycle and is only Expression is also highly regulated by the cell cycle and is only expressed in the G2-M phase. expressed in the G2-M phase.

• Localizes to the Localizes to the mitotic spindlemitotic spindle by interaction with tubulin by interaction with tubulin during mitosis and may play a contributing role in regulating during mitosis and may play a contributing role in regulating mitosis. mitosis.

04/19/2304/19/23 Mita A C et al. Clin Cancer Res 2008;14:5000-5005

©2008 by American Association for Cancer Research

04/19/2304/19/23

Mita A C et al. Clin Cancer Res 2008;14:5000-5005

©2008 by American Association for Cancer Research

ObjectivesObjectives• Identify molecules which inhibit Identify molecules which inhibit

expression of survivin intracellularly.expression of survivin intracellularly.– High-Throughput Screening using High-Throughput Screening using

survivin gene regulatory sequence survivin gene regulatory sequence driving a luciferase reporter gene.driving a luciferase reporter gene.

• Determine the effects of these Determine the effects of these molecules on cancer cells.molecules on cancer cells.– In vitroIn vitro studies on cancer cell lines. studies on cancer cell lines.– In vivoIn vivo human tumor animal models. human tumor animal models.

• Cancer cell-based high throughput Cancer cell-based high throughput screening (HTS) assay systems that used screening (HTS) assay systems that used the survivin gene regulatory sequence the survivin gene regulatory sequence driving a luciferase reporter gene were driving a luciferase reporter gene were generated (US Patent 7569221, 2009). generated (US Patent 7569221, 2009). – The HTS systems were used for discovery of The HTS systems were used for discovery of

candidate compounds that could alter the candidate compounds that could alter the expression of survivin. expression of survivin.

– The libraries of chemical compounds are from The libraries of chemical compounds are from multiple resources including those in the multiple resources including those in the public domain collected by the National public domain collected by the National Cancer Institute Developmental Therapeutics Cancer Institute Developmental Therapeutics Program. Program.

Compound IdentificationCompound Identification

• More than 4000 structurally diverse small More than 4000 structurally diverse small chemical compounds were initially chemical compounds were initially screened using genetically modified screened using genetically modified cancer cell models.cancer cell models.

• Initial screening resulted in ~250 hit Initial screening resulted in ~250 hit compound candidates which showed compound candidates which showed inhibition of luciferase activity in treated inhibition of luciferase activity in treated model cells.model cells.

• Two consecutive round screening of the Two consecutive round screening of the 250 hit candidates using a series of 250 hit candidates using a series of different concentrations (from 0.001 nM to different concentrations (from 0.001 nM to 1000 nM) for individual hit compounds 1000 nM) for individual hit compounds resulted in 20 top-hit compounds, which resulted in 20 top-hit compounds, which showed inhibition of luciferase activity.showed inhibition of luciferase activity.

• Five compounds including FL118 were Five compounds including FL118 were selected for initial selected for initial in vivoin vivo human human tumor animal model screening. tumor animal model screening.

• FL118 was found to be the top FL118 was found to be the top compound among the five leads, compound among the five leads, which displays exceptional antitumor which displays exceptional antitumor activity and favorable toxicity profiles activity and favorable toxicity profiles in the animal models of human colon in the animal models of human colon and head-&-neck tumor xenografts. and head-&-neck tumor xenografts.

The compound FL118 possesses superior The compound FL118 possesses superior antitumor activity to clinically used antitumor activity to clinically used cancer therapeutic drugscancer therapeutic drugs

• Using human head & neck (FaDu) and colon Using human head & neck (FaDu) and colon (HCT-8) cancer animal models, the (HCT-8) cancer animal models, the antitumor activity of FL118 was compared antitumor activity of FL118 was compared with those of FDA-approved, clinically used with those of FDA-approved, clinically used chemotherapeutic drugs. chemotherapeutic drugs. – Irinotecan and topotecan Irinotecan and topotecan (Topoisomerase I Poison)(Topoisomerase I Poison)– Cisplatin and Oxaliplatin (DNA Alkylating Agents)Cisplatin and Oxaliplatin (DNA Alkylating Agents)– Docetaxel (Microtubule Polymerization Promoter) Docetaxel (Microtubule Polymerization Promoter) – Gemcitabine and 5-FU (DNA Synthesis Inhibitor) Gemcitabine and 5-FU (DNA Synthesis Inhibitor) – Doxorubicin (Topoisomerase II Inhibitor)Doxorubicin (Topoisomerase II Inhibitor)– Cyclophosphamide (Alkylating Agent) Cyclophosphamide (Alkylating Agent)

ResultsResults

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FL-118 1.5 mg/kg

Irinotecan 200 mg/kg

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

Doxorubicin 12.5 mg/kg

Cytoxan 100 mg/kg

Irinotecan

Irinotecan

FL118 effectively eradicates human tumor FL118 effectively eradicates human tumor without recurrence in xenografts without recurrence in xenografts

• The antitumor activity of FL118 with The antitumor activity of FL118 with irinotecan at their MTD in a human primary irinotecan at their MTD in a human primary head & neck tumor xenograft 17073 bearing head & neck tumor xenograft 17073 bearing in SCID mice with the clinically relevant in SCID mice with the clinically relevant schedule (weekly x 4) of irinotecan. schedule (weekly x 4) of irinotecan. – Ironotecan-treated group: 2/5 mice showed Ironotecan-treated group: 2/5 mice showed

temporary tumor regression after treatment. temporary tumor regression after treatment. – FL118-treated group: 5/5 mice showed complete FL118-treated group: 5/5 mice showed complete

response and no recurrent tumor was detected.response and no recurrent tumor was detected.– Irinotecan induced an accumulated body weight Irinotecan induced an accumulated body weight

loss, while FL118 induced a reversible body weight loss, while FL118 induced a reversible body weight loss with rapid recovery after treatment.loss with rapid recovery after treatment.

FL118 effectively and FL118 effectively and permanently eradicates large permanently eradicates large and advanced human tumorsand advanced human tumors

• Mice bearing maximal human Mice bearing maximal human xenograft tumors (~2 gm) showed xenograft tumors (~2 gm) showed striking responses to FL118 striking responses to FL118 treatment in both FaDu head & neck treatment in both FaDu head & neck tumors and SW620 colon tumors. tumors and SW620 colon tumors. – Of note, the death of the two mice was Of note, the death of the two mice was

likely a result of the Tumor Lysis likely a result of the Tumor Lysis Syndrome due to treatment-induced Syndrome due to treatment-induced massive tumor necrosis.massive tumor necrosis.

Fig 2

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Head & neck primary tumor

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Irinotecan 100mg/kg/wk x 4

FL118 1.5mg/kg/wk x 4

FL118 is chemically stable with a long FL118 is chemically stable with a long shelf lifeshelf life

• To test the chemical stability of FL118 in To test the chemical stability of FL118 in solution, a ready-to-use FL118 injection solution, a ready-to-use FL118 injection solution was prepared and stored in a solution was prepared and stored in a +4+4C refrigerator for more than 6 months. C refrigerator for more than 6 months.

• The old drug solution of FL118 was as The old drug solution of FL118 was as effective as the newly formulated FL118 effective as the newly formulated FL118 solution against FaDu and SW620 tumors.solution against FaDu and SW620 tumors.

Fig 3 In

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FaDu FaDu

SW620 SW620

Inhibition of survivin by FL118 is Inhibition of survivin by FL118 is associated with the modulation of associated with the modulation of other members in the IAP and Bcl-2 other members in the IAP and Bcl-2 familiesfamilies

• Downregulation of survivin by FL118 was Downregulation of survivin by FL118 was associated with the modulation of other associated with the modulation of other members in the IAP and Bcl-2 families.members in the IAP and Bcl-2 families.– XIAP, cIAP2 and Mcl-1 XIAP, cIAP2 and Mcl-1 – Bcl-2 and Bcl-XLBcl-2 and Bcl-XL

• In contrast, treatment increases the In contrast, treatment increases the expression of pro-apoptotic proteins.expression of pro-apoptotic proteins.– Bax and Bim.Bax and Bim.

Survivin -

Bax -

Bim -

Bcl-XL -

Mcl-1-

FL118 (nM): 0 10 100

Actin -

FL118 (10nM): 0 8 16 24 72h

a. A2008

b. PC-3

Bcl-2 -

Survivin -

Actin -

Fig 5

Mcl-1-

FL118 (nM): 0 10 100

c. HCT-8

Survivin -

Actin -

*

XIAP -

48 hours24 hours

cIAP2 -

Actin -

FL118 differentially modulates the FL118 differentially modulates the phosphorylation of Akt and MAPK phosphorylation of Akt and MAPK (Erk1/2) signaling molecules and induces (Erk1/2) signaling molecules and induces the production of apoptotic hallmarksthe production of apoptotic hallmarks

• Several members in the IAP and Bcl-2 families Several members in the IAP and Bcl-2 families are regulated through Akt and/or MAPK are regulated through Akt and/or MAPK pathways. pathways.

• FL118 inhibits both constitutive and taxol-FL118 inhibits both constitutive and taxol-induced Akt phosphorylation, but shows no induced Akt phosphorylation, but shows no inhibitory effects on the phosphorylation of inhibitory effects on the phosphorylation of Erk1/2.Erk1/2.

• FL118 induced caspase activation and PARP FL118 induced caspase activation and PARP cleavage.cleavage.

a.

b.

p-Akt(473) -

Total Akt -

FL118 (10nM): 0 1 6 12 24h

Fig 6

c.

Total Akt -

FL118 (10nM): 0 0 1 6 12 24h Taxol 30nM

FL118 (10nM): 0 0 1 6 12 24h Taxol 30nM

p-Akt(473) -

p-Erk1/2

Total Erk1/2

FL118 (10nM): 0 1 12 24 48h

Cleavedcaspase 3

Actin -

Actin -

Actin -

Survivin -

Full PARP - cleaved -

d.

FL118 inhibits survivin promoter FL118 inhibits survivin promoter activity in a highly selective manneractivity in a highly selective manner

• FL118 selectively inhibits human survivin FL118 selectively inhibits human survivin promoter-driven luciferase activity at as promoter-driven luciferase activity at as low as 0.1 - 1 nM which is dependent on low as 0.1 - 1 nM which is dependent on cancer cell types. cancer cell types.

• In contrast to its high inhibitory effects on In contrast to its high inhibitory effects on survivin promoter activity, FL118 showed survivin promoter activity, FL118 showed no inhibitory effects on luciferase activity no inhibitory effects on luciferase activity driven by gene promoters from cyclin-driven by gene promoters from cyclin-dependent kinase inhibitor p21 (p21), dependent kinase inhibitor p21 (p21), dehydrofolate reductase (DHFR), human dehydrofolate reductase (DHFR), human thrombin receptor (HTR), and thymidine thrombin receptor (HTR), and thymidine kinase (TK) in different cancer cell types.kinase (TK) in different cancer cell types.

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c. PC-3 d. EKVX e. LNCaP-LN3

• Several recent studies have demonstrated Several recent studies have demonstrated that abrogation of one of the four genes that abrogation of one of the four genes (survivin, Mcl-1, XIAP and cIAP2) inhibits (survivin, Mcl-1, XIAP and cIAP2) inhibits tumor growth in various tumor growth in various in vitroin vitro and and in vivoin vivo cancer models. cancer models.

• Selective inhibition of the survivin gene by Selective inhibition of the survivin gene by FL118 in association with the modulation of FL118 in association with the modulation of Mcl-1, XIAP and cIAP2 in cancer cells may Mcl-1, XIAP and cIAP2 in cancer cells may effectively inhibit tumor growth. effectively inhibit tumor growth.

• What is the relationship of survivin inhibition What is the relationship of survivin inhibition by FL118 with the associated downregulation by FL118 with the associated downregulation of Mcl-1, XIAP and cIAP2 as well as the of Mcl-1, XIAP and cIAP2 as well as the inhibition of Akt signaling by FL118? inhibition of Akt signaling by FL118?

DiscussionDiscussion

• Akt signaling is linked to upregulation of survivin Akt signaling is linked to upregulation of survivin in cancer. in cancer. – Suppression of the Akt/survivin pathway induces Suppression of the Akt/survivin pathway induces

apoptosis and increases antitumor activity. PI3K/Akt apoptosis and increases antitumor activity. PI3K/Akt signaling also upregulates the expression of Mcl-1, XIAP signaling also upregulates the expression of Mcl-1, XIAP and c-IAP2 . and c-IAP2 .

• FL118 inhibits HIF-1FL118 inhibits HIF-1 production in hypoxic production in hypoxic conditions.conditions.– HIF-1HIF-1 has been shown to transcriptionally upregulate has been shown to transcriptionally upregulate

survivin.survivin.– Hypoxia/HIF-1Hypoxia/HIF-1 was also shown to regulate the was also shown to regulate the

expression of Mcl-1, XIAP and IAP2. expression of Mcl-1, XIAP and IAP2.

• Survivin may be a transcription factor or co-factor Survivin may be a transcription factor or co-factor to inhibit p21 gene transcription in a p53-to inhibit p21 gene transcription in a p53-dependent manner. dependent manner. – If survivin acts as a transcription factor or a co-factor, If survivin acts as a transcription factor or a co-factor,

downregulation of survivin expression by FL118 could be downregulation of survivin expression by FL118 could be associated with the modulation of other survivin-targeted associated with the modulation of other survivin-targeted gene expression. gene expression.

We have discovered and We have discovered and characterized a novel chemical characterized a novel chemical molecule showing exceptional molecule showing exceptional antitumor efficacy with favorable antitumor efficacy with favorable toxicity and chemical stability toxicity and chemical stability profiles. While this molecule profiles. While this molecule selectively inhibits promoter activity selectively inhibits promoter activity and expression of survivin, and expression of survivin, downregulation of survivin by this downregulation of survivin by this molecule is associated with the molecule is associated with the modulation of other cancer-modulation of other cancer-associated signals.associated signals.

SummarySummary

A Small Chemical Molecule Selectively Inhibiting the Antiapoptotic and Cell Division-A Small Chemical Molecule Selectively Inhibiting the Antiapoptotic and Cell Division-Controlling Protein Survivin shows Exceptional Antitumor ActivityControlling Protein Survivin shows Exceptional Antitumor Activity

Shousong CaoShousong Cao1,2,3,#1,2,3,#, Xiang Ling, Xiang Ling1,#1,#, Qiuying Cheng, Qiuying Cheng11, James T. Keefe, James T. Keefe11, , Youcef M. RustumYoucef M. Rustum3,43,4 and Fengzhi Li and Fengzhi Li1,4,*1,4,*

Departments of Pharmacology & TherapeuticsDepartments of Pharmacology & Therapeutics11, Medicine, Medicine22 and Cancer Biology and Cancer Biology33, Drug Discovery and Experimental , Drug Discovery and Experimental Therapeutics (DDET) ProgramTherapeutics (DDET) Program44, Roswell Park Cancer Institute, Buffalo, New York 14263, USA, Roswell Park Cancer Institute, Buffalo, New York 14263, USA

# Equal contributions to this work# Equal contributions to this work

AcknowledgementsAcknowledgements• This work was sponsored in part by NIH R01 Grants (CA109481, This work was sponsored in part by NIH R01 Grants (CA109481,

CA133241) and the Meso Foundation (Alexandria, VA) grant to FL, and by CA133241) and the Meso Foundation (Alexandria, VA) grant to FL, and by shared resources supported by NCI Cancer Center Core Support Grant to shared resources supported by NCI Cancer Center Core Support Grant to Roswell Park Cancer Institute (CA016056). Roswell Park Cancer Institute (CA016056).

• We thank the Drug Synthesis and Chemistry Branch, Developmental We thank the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program (DTP), Division of Cancer Treatment and Therapeutics Program (DTP), Division of Cancer Treatment and Diagnosis, National Cancer Institute (NCI) for providing chemical libraries Diagnosis, National Cancer Institute (NCI) for providing chemical libraries and relevant hit analogs in the public domain they collected and/or and relevant hit analogs in the public domain they collected and/or synthesized as a major compound sources during the process of our drug synthesized as a major compound sources during the process of our drug screening and characterization. screening and characterization.

• We would also like to thank Dr. Paul A. Spengler (University of We would also like to thank Dr. Paul A. Spengler (University of Rochester) for his critical reading of the manuscript, Ms. Lileena Johnson Rochester) for his critical reading of the manuscript, Ms. Lileena Johnson (Rotation graduate student) for the involvement in repeating some of (Rotation graduate student) for the involvement in repeating some of drug screening experiments. drug screening experiments.

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