6th international symposium on translational research in oncology

October 11-14, 2007Dublin, Ireland

6th International Symposium on Translational Research in Oncology

Jointly sponsored by Postgraduate Institute for Medicine and Clinical Care Options, LLC

This program is supported by educational grants from

Dennis J. Slamon, MD, PhDChief, Division of Hematology/OncologyDavid Geffen School of Medicine at UCLALos Angeles, California

John Crown, MD, MPHHead, Medical Oncology ResearchSt Vincent’s HospitalElm ParkDublin, Ireland


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clinicaloptions.com/oncology


Program Overview

Now in its sixth year, this annual symposium has a firmly established reputation as a premier meeting at which the world’s leading researchers gather to present and discuss new directions in oncology research with a focus on translating the most recent laboratory developments into improved clinical outcomes for cancer patients. Under the direction of John Crown, MD, MPH, and Dennis J. Slamon, MD, PhD, the program includes didactic presentations and interactive discussions. Faculty are carefully selected from among the researchers at the forefront of the translational work in the topic, whether from academia, government, or industry. The program encourages networking and interaction between the attendees and the renowned faculty members.



DisclaimerThe materials published on the Clinical Care Options Web site reflect the views of the authors, not those of Clinical Care Options, LLC, the CME providers, or the companies providing educational grants. The materials may discuss uses and dosages for therapeutic products that have not been approved by the United States Food and Drug Administration. A qualified healthcare professional should be consulted before using any therapeutic product discussed. Readers should verify all information and data before treating patients or using any therapies described in these materials.

Users are encouraged to include these slides in their own presentations, but we ask that content and attribution not be changed. Users are asked to honor this intent.

These slides may not be published or posted online or used for any other commercial purpose without written permission from Clinical Care Options.

We are grateful to Gerry Melino, MD, PhD, the Chair of the Session, who aided in the preparation of this slideset.

We are also grateful to Donald W. Nicholson, PhD; Richard A. Knight, MD, PhD; Seamus J. Martin, PhD; Henning Walczak, PhD; and Gerry Melino, MD, PhD, who gave us permission to use a select group of their slides from the meeting to make this presentation possible.

About These Slides



Faculty and Staff Disclosures

The faculty and CCO staff reported the following financial relationships or relationships to products or devices they or their spouse/life partner have with commercial interests related to the content of this CME activity.Gerry Melino, MD, PhD, has no significant financial relationships to disclose.

Edward King, MA; Deborah Janssen, MS; Andy Bowser; Jim Mortimer; Robert S. Mocharnuk, MD, and Gordon Kelley have no significant relationships to disclose.

The following PIM clinical content reviewers, Linda Graham, RN; Jan Hixon, RN; and Trace Hutchison, PharmD, hereby state that they or their spouse/life partner do not have any financial relationships or relationships to products or devices with any commercial interests related to the content of this CME activity of any amount during the past 12 months.



Target AudienceThis activity is intended for biomedical investigators in the area of cancer research from academia and industry as well as medical, surgical, and radiation oncologists.

GoalThe goal of this activity is to provide an update on major topics in translational research to biomedical researchers and clinicians involved in the field.

Learning ObjectivesAt the conclusion of this activity, participants should be able to:

Describe how caspase activation plays a role in apoptosis Discuss how the ITCH inhibitors play a role in p63 and p73 oncogenic pathways Identify how proteolytic degradation of p53 family members by caspases plays a role in

cancer Cite the role of B-Raf in apoptosis Discuss the role of TRAIL receptor agonists as anticancer therapeutic agents



Physician Continuing Medical EducationRelease Date

Release date: January 18, 2008; valid for credit through January 17, 2009.

Accreditation This activity has been planned and implemented in accordance with the

Essential Areas and Policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of Postgraduate Institute for Medicine and Clinical Care Options, LLC. Postgraduate Institute for Medicine is accredited by the ACCME to provide continuing medical education for physicians.

Credit Designation Postgraduate Institute for Medicine designates this educational activity for a

maximum of 1.5 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.



Instructions for Credit

There are no fees for participating and receiving credit for this activity. Participation in this self-study activity should be completed in approximately 1 hour. To successfully complete this activity and receive credit, participants must follow these steps during the period from January 18, 2008, through January 17, 2009:

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Disclosure of Unlabeled Use

Statement

This educational activity may contain discussion of published and/or investigational uses of agents that are not indicated by the FDA. Postgraduate Institute of Medicine (PIM) and Clinical Care Options, LLC, do not recommend the use of any agent outside of the labeled indications.

Session III: Apoptosis and Programmed Cell Death

Gerry Melino, MD, PhDProfessorMedical Research CouncilLeicester, United Kingdom

Apoptosis Inhibitors in Cancer Therapy



Caspases

Death SignalsDeath Signals

Death Regulators Bcl-2 family IAPs and anti-IAPs Usurpins Phosphorylation

Apoptotic “Victims”Apoptotic “Victims”

Neurodegeneration

Cancer

Core Components of the Apoptotic Pathway



Extrinsic and Intrinsic Cell Death Pathways



p17 p12pro

p32

Substrate cleavage

Dormant

Active

p20

p17

Caspase Maturation and Activation



proCaspase-3 Levels in Human Colon Cancer

Colonic Adenocarcinoma vs Adjacent Normal Mucosa

N TN T N TN T N TN T

11 22 33 (Patient)(Patient)

p32p32

0

1

2

3

4

Normal Tumor

(Arb

itra

ry U

nit

s/m

g P

rote

in)

pro

Cas

pas

e-3

Co

nte

nt

(averaged data, n = 20)

6x

(Normal, Tumor)(Normal, Tumor)



prop32p32

p17p17

p17 p12

DD2828

IPTG (min):IPTG (min):

Wt

Wt

DD17

9-18

1

179-

181 AA

11 33 44 66

30306060120120 30306060120120

22 55

DDD(179-181) ONAAA OFF

Identification of an Intrinsic Regulatory “Safety Catch” Tripeptide

CC163163 DD175175



[Granzyme B][Caspase-9][proCaspase-3]

proCaspase-3

0

20

40

60

80

100

0.001 0.01 0.1 1 10 100 1000 104

Pe

rce

nta

ge

Cle

av

ed

Protease (nM)

0

2

4

6

8

10

12

0.001 0.01 0.1 1

Pe

rce

nta

ge

Cle

av

ed

proCaspase-3 (nM)

pro p17 p12

DDD(179-181) Safety catch ON

AAA(179-181) Safety catch OFF

CC163163AA

The Safety Catch Modulates Vulnerability to Activator Proteases

Caspase-9Granzyme B



Summary: Caspase “Safety Catch”

Caspase-3 dormancy maintained by “safety catch” DDD Caspase-3 dormancy maintained by “safety catch” DDD regulatory tripeptideregulatory tripeptide

– Regulates Regulates ciscis autoactivation autoactivation

– Regulates Regulates transtrans activation by “initiator” caspases (GrznB, C9) activation by “initiator” caspases (GrznB, C9)

Caspase-3 autoactivation triggered by acidificationCaspase-3 autoactivation triggered by acidification

– Destabilizes “safety catch” isoelectronic interactionsDestabilizes “safety catch” isoelectronic interactions

– Relevant to in vivo activation mechanismRelevant to in vivo activation mechanism

Therapeutics that antagonize “safety catch” DDD could Therapeutics that antagonize “safety catch” DDD could preferentially sensitize or trigger apoptotic deathpreferentially sensitize or trigger apoptotic death



A

Mitochondrion

ATP C

APAF-1C9

C3/C7C3/C7

D

ApoptosisApoptosis

IAPs

SMAC/DiabloHtrA2/Omi AVPI---

Nt

IAP Antagonism During Apoptosis



BIR3 RING Zn fBIR2BIR1XIAP/hILP/hMIHA

SESD242 AVSSDRN AVPIAQK

AVPSSPPATPFQEG

VEVD720 AAVTPEE

Smac/Diablo hOmi/HtrA2 hCaspase-9 p12

Amyloid-ß Precursor Protein

APP-AA (31mer)

XIAP-AV (12mer)

A

Caspase CleavageCaspase Cleavage

Caspase Proteolysis Generates P1’-Ala Neo Termini



M MM

SMAC/Diablo APP XIAP

Functional Antagonism of IAPs in Cell-Free Extracts

0

20

40

60

80

100

120

140

160

180

DE

VD

ase

Ac

tiv

ity

(% o

f N

on

inh

ibit

ed A

ctiv

ity)

0 12.5 50 200

Smac-20

0

20

40

60

80

100

120

0 50 200

APP-AA

APP-MGD

EV

Das

e A

cti

vit

y (

% o

f N

on

inh

ibit

ed A

ctiv

ity)

0

10

20

30

40

50

60

70

80

0 50 200 800

XIAP-AV

XIAP-MV

DE

VD

ase

Ac

tiv

ity

(%

of

No

nin

hib

ited

Act

ivit

y)



Summary: IAP AntagonismSummary: IAP Antagonism

IAP proteins are direct inhibitors of caspasesIAP proteins are direct inhibitors of caspases

– Ensure dormancy in healthy cellsEnsure dormancy in healthy cells

– Naturally antagonized by Smac and some caspase Naturally antagonized by Smac and some caspase substrates after caspase proteolysissubstrates after caspase proteolysis

IAP antagonism can be mediated by peptides and small IAP antagonism can be mediated by peptides and small moleculesmolecules

Therapeutics that antagonize IAPs could preferentially Therapeutics that antagonize IAPs could preferentially sensitize or trigger apoptotic deathsensitize or trigger apoptotic death

ITCH Inhibitors



The p53 Family

The p53 family includes 3 genes, encoding for p53, p63, p73 proteins

The oldest protein is p63, whereas p53 is the most recent

– P53 is involved in DNA damage repair

Each protein exists as different isoforms generated by distinct promoters (TA isoforms and N isoforms) or by alternative splicing (, , , etc, isoforms)

– TA isoforms induce cell death (anticarcinogenic, acting as tumor suppressor)

– N isoforms inhibit cell death (procarcinogenic, acting as oncogenes)

p73 and p63, like p53, are involved in DNA damage repair, inhibiting cell cycle progression, and inducing apoptosis

– p73 is rarely mutated in cancer (0.5% mutation rate of p73 versus > 50% mutation rate of p53)



The p53 Family (cont’d)

p73 kills cells by regulating the transcription of genes crucial to the cell cycle (eg, p21) and to apoptosis (eg, bax, PUMA, CD95), thus affecting the sensitivity of cancer cells to chemotherapy

After 30 years of research on p53, clinically exploitable targets are mostly limited to its protein degradation regulation (inhibitors of the ubiquitin E3 ligase MDM2), and ARF (DNA methyltransferase inhibitors) pathways,



DNA damage Replicative Stress

Cell Cyle ArrestApoptosis

Sensors

Effectors

Signals

Effects

Alterations

ATM ATR

p53

chk1

chk2

p21gadd45 ….

mdm2

c-abl

bax CD9514-3-3Noxa ….

MRE11rad50

BRCA1NSB1

DNA-PK PARP

cdc25

G1 S G2

Ku70/Ku86

14-3-3cdc25

Cdc2 ….

DNA Repair

HR NHEJ

NER MMR BER

p73p63

Mueller et al. Cell Death Differ. 2005;12:1564.Flinterman et al. J Biol Chem. 2005;280:5945.Vigano et al. EMBO J. 2006;25:5105.Rossi et al. PNAS. 2006;103:12753

Gong et al. Nature. 1999;399:806.Melino et al. J Biol Chem. 2004;279:8076.Bernassola et al. J Exp Med. 2004;199:1545.Gressner et al. EMBO J. 2005;24:2458.

Sayan et al. J Biol Chem. 2006;281:13566.



Regulators of p73 promoter

p73 interacting proteins

p73 trascriptional targets

Signals

p73

Effects

Trascriptionalinducers

Transcriptionaltargets

Interacting Proteins

Degradation pathways

Regulatory Proteins

Posttranslational modifications

Oncogenes: E2F1, c-Myc, RasViral proteins: E1A, TaxDrugs: retinoids

Oncogenes: c-Myc, c-Abl, Hipk2, WT1 Viral proteins: E1A, E4orf6, Tax Cell cycle genes: p300, MM1, HMBG1E3 ligases: MDM2, MDMX, Sumo1, PIAS1 Family members: p53, p63, p73

Cell cycle genes: p21WAF1, gadd45, cyclin, kip2 Apoptosis: Bax, CD95, PUMA, perp, Noxa, p53AIP1 Signaling: 14-3-3, Pig13, RTP, ADTC, p53R2, IGFBP2, jun-B, IKKa family members: DeltaN-p73Differentiation: AGP3, VEGF (neg), loricrin, involucrin, NCAM

Evolution

p73 Promoter, Proteins, and Translational Targets



p53

Senescence Apoptosis Cell cycle arrest

Oncogenes

DNA damage

Ub E3 ligase inhibitorsnutlin

ActivatorsPRIMA-1

DNA methyltransferase inhibitors

ARF

mdm2



Protein degradation of both p73 and p63 is regulated by the ubiquitin E3 ligase ITCH, a member of the HECT-containing E3s

In addition to p73 and p63, ITCH controls the degradation of c-jun, Notch, jun-B, and Flip, all involved in oncogenesis and apoptosis

– The prediction is therefore that ITCH regulation affects carcinogenesis and/or chemosensitivity

Modulation of ITCH protein levels regulates chemosensitivity in vitro, suggesting that an inhibitor of ITCH could increase chemosensitivity

How Are p63/p73 Protein Levels Controlled?



ITCH

Ubiquitin E3 ligase (NEDD4 family)

18H Mice natural Knockout (immune defects)

Interacts with atrophin

Degrades the transcription factor:Jun-B, c-Jun, Notch, Flip

HECTcWW WWC2

Ca2+ dependent lipids interaction

Protein-protein interacting domain

Catalytic domain transferring ubiquitin to substrate

CN

Phage display

TA DNA binding domain OD SAM PR PRCN

TAp73

ITCH E3 Ligase Regulates p73 Stability



504

ITCH Interacts With p63 Y504

Myc-ITCH

-ITCH



-ITCH

ITCH Interacts With p73 Y487



Is ITCH E3 Activity Regulated?

Summary

The function of ubiquitin E3 ligase ITCH is regulated by a physical interaction with a novel protein called N4BP1

N4BP1 competes with ITCH substrates (p63, p73, c-Jun) by binding on the same region of ITCH, called WW2

N4BP1 physiologically regulates ITCH and its substrates (p73, p63, c-Jun), thereby affecting cell death



N4BP1 Specifically Inhibits ITCH-Catalyzed Protein Substrates

b

d

p73p73

c-jun p53c

a



1 8 24 1 8 24 hrs

+/+ -/- N4BP1 MEFs

IB: -c-Junc-Jun

C UV C UV C UV C UV C UV C UV

IB: -p21

IB: -actin

A model for N4BP1 inhibition of ITCH substrate ubiquitylation

WWHECT

E2Ub

UbUb

UbUb

c-Jun

Ub

N4BP1

N4BP1

N4BP1

WWHECT

E2Ub

N4BP1

c-Jun

+

UV

c-Junc-Jun

c-Jun

c-Jun

Cell death

ITCH ITCH

UV-Induced Protein Stabilization of c-Jun Is Impaired in N4BP1-Deficient Cells



Tumor cell

p73Ub

p73ITCHITCH

ITCH

ITCH

Tumor cell

p73Ub

p73ITCHITCH

ITCH

DNA damage

Apoptosis

ITCH

N4BP1

p73 p73

p73

puma, noxa,CD95

p63ITCH Ub, degradation

N4BP1

p73

A Model for ITCH-Mediated Regulation of p73 Function



Small molecular inhibitors of ubiquitin E3 ligase ITCH have been identified

Potential ITCH inhibitors are currently under evaluation for their anticancer activity

Can We Inhibit ITCH E3 Activity?



p63/p73 are involved in DNA repair/cancer

p63/p73 regulate chemosensitivity in cancer

p63/p73 are ubiquitinated and degraded by ITCH

ITCH is regulated by N4BP1

Low MW ITICH inhibitors are under development

Perspective

ITCH is a candidate therapeutic target (to regulate p63/p73)

Summary

Caspase Substrates and p53



p53

Is induced by DNA damaging agents

Determines cell cycle arrest (G1/S and G2/M )

Induces apoptosis

Is frequently mutated (50%) or inactivated (20%) in all human cancers

I II III IV V

100 200 300 393

TA DBD OD



NTA DBD ODPR

C

Transcription dependent

GenomicStructure

ProteinStructure

Transcription Independent

DeathRegulation

The p53 proteins



A - 3 6 7 8

50

37

25

Caspase C - 20 50 100 200 400 nMCaspase 3

1

234

BZ-VAD-fmk +- -Etoposide +- +

50

37

25

D Caspase 3 - + - + - + - +

50

37

25

DO1 1801 FL393 C19

E - + + + +Caspase 3p53 W

T

WT

D21

AD

186A

D21

A,

D18

6A

at D21 and D186

Panel A: p53 is processed to smaller fragments after etoposide treatment and this is abrogated by z-VAD. Panel B: Caspases 3, 6, 7, and 8 cleave p53 in vitro and, panel C, as little as 50nM caspase 3 is required. Panels D/E: by epitope mapping (panel D), the cleavage sites were mapped to D21 and D186 (panel E)

p53 Is Cleaved by Caspases



p53 Is Cleaved During DNA Damage (*)

Z-VAD-fmk ++ - + - - - + - +- - - +Doxorubicin -- + + - - - - + +- - - -Cisplatin +- - - - - + + - -- - + -Etoposide -+ - - - + - - - -- + - +

P53 (C19)

HCT116 U2OS

Caspase 3

PARP

53 6 71 2 4 8 9 11 12 13 1410

p32

p19p17

p116

p85

P53 (DO1)

37

25

50

37

% apoptosis 6 37 65 14 32 1012 4 44 51 9 6 57

lane

(*) etoposidecisplatin, doxorubicin

A B



A B

Z-VAD-fmk -- +- - -Etoposide 84 8 hours- 2 6

SH-SY5Y

Caspase 3

PARPp116

p85

p32

p19p17

P53 (C19)

P53 (DO1)

37

25

50

37

TRAIL +- +HCT116

Z-VAD-fmk +- -

Caspase 3

PARP

P53 (C19)

P53 (DO1)

p53 Is Cleaved During Apoptosis (by TRAIL)



Fragments Localize to MitochondriaA B

FLp53

p53(1-186)p53(187-393)p53(22-186)p53(22-393)

21 186 393

TA PRD DBD OD CTD

1

HA

FLAGV5-His

V5-HisV5-His

V5-His

50

37

25

75

1 2 3 4 5

C

Actin

PCNA

COXII

p53

50

37

37

25

50

37

25

Fraction m m c/nc/n

p53 + + + +Cisplatin - + - +

D

Anti-p53 DAPI MergeMito-RFP

p53(1-393)

p53(1-185)

p53(187-393)

p53(22-185)

p53(22-393)

Fragments 1-186 and 22-186 localize to mitochondria by confocal (panel C) and biochemical fractionation (panel D)



B

Actin

PCNA

COXII

p53

50

37

37

25

50

37

25

Fraction m m c/nc/n

p53 + + + +Cisplatin - + - +

D

C

(2) Biochemicalfractionation

Fragments Localize to Mitochondria (cont’d)A

p53 Fragments Induce Apoptosis

. . . TranscriptionIndependent

Both 1-186 and 22-186 induce apoptosis (panel A)which is transcription independent (panel B)



. . . Contributing to Apoptosis

2 naturally occurring p53 mutants are also cleaved in a similar manner to the wt protein after cisplatin (panel B) and induce apoptosis (panel C), although their noncleavable mutants do not (circled)

Mutant p53 Is Cleaved by Caspases



DNA Damage Replicative Stress

Cell Cyle ArrestApoptosis

Sensors

Effectors

Signals

Effects

Alterations

ATM ATR

p53

chk1

chk2

p21gadd45 ….

mdm2

c-abl

bax CD9514-3-3Noxa ….

MRE11rad50

BRCA1NSB1

DNA-PK PARP

cdc25

G1 S G2

Ku70/Ku86

14-3-3cdc25

Cdc2 ….

DNA Repair

HR NHEJ

NER MMR BER

p73 and p63, members of the p53 family, cooperate with p53 in DNA damage response

What About the Other p53 Family Members (p73 and p63)?

p73/p63



TRAIL-, etoposide-induced cleavage(reverted by caspase inhibitors)

p73 is also cleaved by caspases after TRAIL and etoposide treatment

p73 Is Cleaved During Apoptosis



All N-ter isoforms cleaved All C-ter isoforms cleaved

Caspase 3 is most effective

Caspase 8 also effective

p73 Is Cleaved by Caspases in Vitro



p63 is cleaved by caspases in cells after UV-B (panel A) and in vitro (panel B). Both TA and DN isoforms are cleaved (panel C) and epitope mapping shows that D458 is the cleavage site (panels D/E)

D458 is the cleavage site

UV-induced cleavage In vitro cleavage by caspases Both N-ter isoforms cleaved

p63 Is Also a Caspase Target



Localization

Transcriptional activity

The N-terminal fragments of TA and DNp63 (TA-F and DN-F) are nuclear (panel A) and the TA fragment has enhanced transcriptional activity on 4 promoters compared to intact TAp63a

TAp63 Cleavage Increases Transcriptional Activity



. . . by UV, staurosporine, cisplatin . . . mediated by caspases(not calpain inhibitors)

Endogenous p63 Is Cleaved During Apoptosis



Summary

p53 is cleaved by caspases during apoptosis, contributing to cell death

Transcriptionally inactive natural mutants of p53 can be cleaved by caspases to produce transcriptionally inactive fragments

– Can still induce apoptosis by depolarization of mitochondria

Other members of the p53 family, p63 and p73, are also susceptible to caspase cleavage

Caspase cleavage of p63a isoforms relieves the inhibitory effects of the C-terminal transactivation inhibitory domain, resulting in

– Enhanced transcriptional activity by the TA isoform

– Abrogating the transactivational inhibitory effects of the DNp63 isoform

Granzyme B in Cancer Therapeutics



(1) Extrinsic

(2) Intrinsic

(3) Granzyme B

BH3-only proteins

CTL/NK cells

Caspase Activation Cascades

Death receptors



Bad

BimBid

PumaNoxaBmf

Hrk

Bik

p53Caspase-8

Death receptors

Granzyme B

Growth factordeprivation

Ag receptorProteasome inhibition

BH3-Only Proteins: Pathway-Specific Sensors of Stress and Damage



Malignant melanoma notoriously refractory to Malignant melanoma notoriously refractory to chemotherapy chemotherapy

~ 60% to 80% of melanomas display mutations in B-Raf~ 60% to 80% of melanomas display mutations in B-Raf

B-Raf mutations also found in colorectal, thyroid, and B-Raf mutations also found in colorectal, thyroid, and ovarian cancer ovarian cancer

Vast majority of mutations are single point mutations at Vast majority of mutations are single point mutations at V600>EV600>E

B-Raf and Melanoma



B-Raf V600E Can Block Apoptosis

Act DHeLa

Cellular target(s)?



HeLa cells

Endogenous

HEK293T cells

Endogenous

B-Raf V600E Promotes Bim and Bad Phosphorylation



Summary

Oncogenic B-Raf suppresses apoptosis

B-Raf inactivates the BH3-only proteins Bim and Bid via ERK kinase phosphorylation

B-Raf blocks Bim- and Bid-induced cell death and is required for survival



The death receptors

TRAIL-R3 TRAIL-R4 TRAIL-R1 TRAIL-R2 CD95 TRAMP TNF-R1 DR6 TNF-R2 CD40 CD30 NGF-R CD27 RANK GITR HVEM OX40 4-1BB TACI OPG SFV-T2 DcR1 DcR2 DR4 DR5 APO-1 DR3 TRID TRUNDD TRICK2 Fas APO-3 LIT KILLER Wsl

APO-2 LARD

The TRAIL Receptors Belong to the TNF Receptor Superfamily



TRAIL-R1 (DR4)/TRAIL-R2 (DR5)

TRAIL-R3(DcR1)

TRAIL-R4(DcR2)

Apoptosis-inhibitoryreceptors?

TRAIL(Apo2L)

Death domainDeath effector domainTruncated death domain

Walczak H, et al. EMBO J. 1997;16:5386-5397.Degli-Esposti MA, et al. J Exp Med. 1997;186:1165-1170.Degli-Esposti MA, et al. Immunity. 1997;7:813-820.

The TRAIL–TRAIL-R System



Apoptosis

Agonistic mAbs

TRAIL

TRAIL-R1/TRAIL-R2

TRAIL Receptor Agonists Currently in Clinical Trials



TRAIL/Apo2L: AMG 951(Genentech/Amgen)

Target validation

Target ID

1995Wiley et al. Immunity.

1999Walczak et al. Nature Med.

Pre-clinical

Phase II Phase IIIPhase ILead

validation

2001 2003 2005

TRAIL

TRAIL-R2

1997Walczak et al. EMBO J.

Target validation

Target IDPre-

clinicalPhase II Phase IIIPhase I

Lead validation

1999Walczak et al. Nature Med.

2002 2003 2005

2007

2007

TRAIL Receptor Agonists Currently in Clinical Trials (cont’d)

Anti-TRAIL-R2: AMG 655(Amgen)



TRAIL/Apo2L – Genentech/Amgen

1 agonistic antibody to TRAIL-R1 (DR4)– Human Genome Sciences

5 agonistic antibodies to TRAIL-R2 (DR5)– Human Genome Sciences

– Genentech

– Amgen

– Sankyo

– Novartis

TRAIL Receptor Agonists Currently in Clinical Trials (cont’d)



( )

Death domainDeath effector domainTruncated death domain

Ganten TM, et al. Cell Death Differ. 2004;11(suppl 1):S86-S96. Ganten TM, et al. Hepatology. 2005;43:588-597. Koschny R, et al. Hepatology. 2007;45:649-658.

TRAIL-R3

TRAIL-R4

Apoptosis-inhibitoryreceptors?TRAILTRAIL-R2/1

FLIP

Caspase-8

?

FADD

NF-B

Proteasome Inhibition Enhances TRAIL DISC Formation



Chemotherapy radiotherapyTRAIL receptor agonists

Double Hit on Tumor Cells



Only limited toxicity, even in combination with chemotherapy

The different TRAIL receptor agonists are unique

– Pharmacokinetics

– Target TRAIL-R1 and/or TRAIL-R2

Produce significant numbers of stable disease; many patients show stable disease

Response to monotherapy is rare

Long-lasting treatment possible

Conclusions From Clinical Studies With TRAIL Receptor Agonists



TRAIL/Apo2L + rituximab in CD20-positive NHL

TRAIL/Apo2L + chemotherapy with and without bevacizumab in NSCLC

Randomized phase 2 in multiple myeloma mapatumumab (anti–TRAIL-R1) with or without bortezomib

Ongoing Interesting Combination Studies (No Data Available Yet)



5 Agonistic Antibodies to TRAIL-R2 (DR5)1. ETR2 Human Genome Sciences

2 phase I studies Phase I study + chemo

2. Apomab GenentechPhase I study

3. AMG 655 AmgenPhase I study

4. LBY135 NovartisPhase I/II trialalone and with capecitabine

5. CS-1008 SankyoPhase I study



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phd chief

phd seamus

phd richard

phd henning walczak

translational work

annual symposium

clinical care options