1Sponsored by:

Participating Experts:Saul Rosenberg, Ph.D.AbbottAbbott Park, IL

Webinar SeriesWebinar SeriesScienceScience

22 September, 200922 September, 2009Brought to you by the Science/AAAS Business Office

John Abrams, Ph.D.University of Texas Southwestern Medical CenterDallas, TX

Apoptotic SignalingApoptotic Signalingin Normal and Cancer Cell Biologyin Normal and Cancer Cell Biology

Joseph T. Opferman, Ph.D.St. Jude Children's Research HospitalMemphis, TN

Apoptosis Pathways and Cancer

Saul RosenbergCancer ResearchAbbott

September 22, 2009

Apoptosis is an Essential Cellular Process

• Apoptosis (programmed cell death) is the body's normal method of disposing of damaged, unwanted, or unneeded cells

–Embryonic development–Normal cellular turnover–Expansion / contraction of cell populations–Elimination of damaged cells

• Highly regulated - characterized by a series of specific molecular events and morphological changes

• Dysregulation is implicated in a number of diseases

Cory, et al. Oncogene. 2003;22(53):8590.Skommer, et al. Leuk Res. 2007;31(3):277.Ashkenazi, Herbst. J Clin Invest. 2008;118(6):1979.Healthy cell Apoptotic cell

DermisDividing cells

Apoptoticcells shed

Epidermis

Apoptosis and Disease

proliferation > apoptosisCancer

Autoimmune disease

proliferation < apoptosisNeurodegeneration

proliferation = apoptosisSkin homeostasis

Migration

Evasion of Apoptosis is a Hallmark of Cancer

Initiation Progression Resistance

Hanahan, D., Weinberg, R.A. (2000) The Hallmarks of Cancer Cell, 100, 57-70.

Six essential alterations in cell physiology that collectively dictate malignant growth.

Cancer cells survive stresses that kill normal cells

Two Apoptosis Pathways

cytochrome C

Caspase-9‘Initiator’

Caspase-3‘Executioner’

Death receptor pathway Death receptor

FasL, TNFTRAIL

Caspase-8‘Initiator’

FADD/MORT1

Procaspase 8

Stresspathway

DNA Damage, oncogenes,

Intracellular damage

Bid Cleavage

ATPAPAF1

Procaspase 9

Apoptosome

DeathSignals

Bcl-2Family

MOMP

Pro-apoptoticFactors

Apoptosis

IAPs

Mitochondrion

Opposing Subsets of Bcl-2 Family Proteins

Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1

Bax

Bak

Bax

Bak

BH3-onlyBH3-onlyBH3-only CN BH3 CCNN BH3

Bcl-2Bcl-2Bcl-2

-likeCN TMBH1 BH2BH3BH4

Bid, Bim, Puma, Bad, Bik, Noxa, Hrk, Bmf

Anti-apoptotic

NN CCBH3 TMBH2BH1

Pro-apoptotic

Bcl-2 Family Proteins Regulate Apoptosis

Bcl-2

Bcl-2

Bax/Bak activation:

Conformation change

activatedBAX / BAK

MOMP

Cyt C

Caspaseactivation

Oligomerization

Bax

Bak

BH3-only

BaxBak

BaxBak

BaxBak

BaxBak

BH3-only

Apoptosis

Anti-apoptotic Bcl-2 proteins prevent apoptosis by

sequestering their pro- apoptotic counterparts

Letai, Cancer Cell 2, 183 (2002); Willis, Genes Dev. 19, 1294 (2005)

Bcl-2 Family Protein Interactions

CN BH3

Amphipathic -helix BH-3 domain = ligand

1 2 3 4 5 6 7Hydrophobic surface groove = binding siteAntiapoptotic Bcl- xL

CN TMBH1 BH2BH3BH4

Nature 381, 335 (1996)Science 275, 983 (1997)Prot. Sci. 9, 2528 (2000)

BH3- Only Bad

Hydrophobic groove

SA ~ 620 Å2

NPSA ~ 440 Å2L78L78

I85I85

Targeting the Bcl-xL Hydrophobic Groove

• Protein- protein interaction–Large surface area–Primarily hydrophobic interactions–Significant conformational changes

between bound and unbound state–Natural ligands bind with high affinity

• High throughput screen–Displacement of BH3 peptide–~ 10 µM sensitivity–No viable hits

• Alternate strategy–Structure-guided design

NMR Fragment-based Screening SAR by NMRScreen FragmentsScreen Fragments

Science 274, 1531 (1996)Science 278, 497 (1997)Nat. Rev. Drug Disc. 6, 211, (2007)

Detect BinderDetect Binder

Link FragmentsLink FragmentsKKdd (AB) = K(AB) = Kdd (A) * K(A) * Kdd (B) * X(L)(B) * X(L)

Identify 1st site binder

Identify 2nd site binder

1H (ppm)

15N

(ppm

)

9.4 8.7 8.0 7.3 6.6

125.

012

0.0

115.

011

0.0

105.

0

G51

V55

1H (ppm)

15N

(ppm

)

9.4 8.7 8.0 7.3 6.6

125.

012

0.0

115.

011

0.0

105.

0

1H (ppm)

15N

(ppm

)

9.4 8.7 8.0 7.3 6.6

125.

012

0.0

115.

011

0.0

105.

0

G51

V55

Small Molecule Lead Generation

SAR SAR by by

NMRNMR

Fragment-based screen

Structure GuidedStructure GuidedParallel SynthesisParallel Synthesis

J. Med. Chem. 49, 656 (2006)

1997

1998

1999

““Rational” Rational” linker designlinker design

1.4 M

300 M / 4000 M

0.1 M

Discovery of ABT-737 & ABT-263 Concept to Clinic = 10 years

J. Med. Chem. 49, 1165, (2006)

Nature 435, 677 (2005) J. Med. Chem. 50, 641,

(2007)0.1 M 0.001 M

StructureStructure--based based drug designdrug design

< 0.0001 M

1999 2002

2003

ABT-737

i.v. onlyN

N

OHN

SO O

HN

Cl

S

NHO

F3CS

OO

< 0.0001 M

ABT-263

oral

2005J. Med.

Chem. 51, 6902 (2008)FIH

2006 MedicinalMedicinalChemistryChemistry

High Binding Affinity to Bcl-2 Family Proteins

• High affinity for Bcl-xL, Bcl-2 and Bcl-w• Lower affinity for Mcl-1 and bfl-1/A1

O

N

N

SHN

OO

HN S

N

H

Cl

F3C SO

O

O

ABT-263(Oral)

O

N

N

SHN

OO

HN S

N

H

Cl

N+O–O

ABT-737(i.v.)

Oltersdorf, et al. Nature 435, 677 (2005)Tse, et al. Cancer Res. 68, 3421 (2008)

Ki (nM), Serum Free FPA TR-FRET Bcl-XL

Bcl-2 Bcl-w Mcl-1 A1 Bcl-XL hBad (25-mer) 0.5 15 33 4800 >10000 0.12

ABT-737 < 0.5 < 1 < 1 >1000 >1000 0.08 ABT-263 < 0.5 < 1 < 1 550 330 0.05

ABT-263:Bcl-2

ABT-263 is a Targeted Cytotoxic AgentSingle-agent activity against a subset of tumor typesCells dependent on Bcl-2 and/or Bcl-xL for survival

• Hematologic Malignancies– Chronic Lymphocytic Leukemia (CLL)– Acute Lymphocytic Leukemia (ALL)– Non-Hodgkin’s Lymphoma (NHL)

• Small Cell Lung Cancer (SCLC)

Broadly potentiates activity of chemotherapeutics• Hematologic Malignancies• Solid Tumors

– SCLC, NSCLC, CRC, Ovarian, Pancreatic, Gastric– Independent of single-agent activity

ABT-263 Induces Rapid Apoptosis and Regression of ALL Xenograft Tumors

RS4;11 flank xenograft

Rapid caspase activation after one dose

Robust and durable tumor regression

0

10

20

30

40

50

60

70

80

4 8 16 24 48

Hours post dose

Are

a %

cas

pase

pos

itive

cel

ls ABT-263, 100 mg/kg

Vehicle

0

500

1000

1500

2000

2500

0 10 20 30 40 50 60Days post tumor staging

Ave

. tum

or v

ol. (

mm

3 ± s

em)

ABT-263, 100 mkd, po, qd x21

Vehicle

Vehicle 100 mg/kg

Caspase-3 activationsingle dose ABT-263

, p.o.

Tse, et al. Cancer Res. 68, 3421 (2008)

ABT-263 Enhances Efficacy of Chemotherapy in Lymphoid Malignancies

DoHH2

• ABT-263 + rituximab = 70% CR

0500

100015002000

2500300035004000

10 15 20 25 30 35 40 45 50

Days post inoculation

ABT-263, 100 mg/kg, po, qd, d15-31rituximab, 10 mg/kg, iv, qd, d15ABT-263 + rituximabcombination vehicle

Mea

n Tu

mor

Vol

. (m

m3

±se

m)

0500

100015002000

2500300035004000

10 15 20 25 30 35 40 45 50

Days post inoculation

ABT-263, 100 mg/kg, po, qd, d15-31rituximab, 10 mg/kg, iv, qd, d15ABT-263 + rituximabcombination vehicle

ABT-263, 100 mg/kg, po, qd, d15-31rituximab, 10 mg/kg, iv, qd, d15ABT-263 + rituximabcombination vehicle

Mea

n Tu

mor

Vol

. (m

m3

±se

m)

ABT-263, 100 mg/kg, po, qd, d13-34

ABT-263 + R-CHOPCombination Vehicle

R-CHOP qd, d14

0

500

1000

1500

2000

3000

10 15 20 30 35 45 50 55 60Days post inoculation

25 40

2500

ABT-263, 100 mg/kg, po, qd, d13-34

ABT-263 + R-CHOPCombination Vehicle

R-CHOP qd, d14ABT-263, 100 mg/kg, po, qd, d13-34

ABT-263 + R-CHOPCombination Vehicle

R-CHOP qd, d14

0

500

1000

1500

2000

3000

10 15 20 30 35 45 50 55 60Days post inoculation

25 40

2500

• ABT-263 + R-CHOP = 100% CR

ABT-263 + rituximab in DLBCL (DoHH2) ABT-263 + R-CHOP in mantle cell lymphoma (GRANTA-519)

Tse, et al. Cancer Res. 68, 3421 (2008)

(CHOP: cyclophosphamide + doxorubicin + vincristine +

prednisone)

ABT-263 Regresses Established SCLC Tumors

0

300

600

900

1200

1500

1800

0 10 20 30 40Days post tumor staging

Ave

. tum

or v

ol. (

mm

3 ± s

em)

0

300

600

900

1200

1500

1800

0 20 40 60 80 100 120

Days post tumor staging

0

500

1000

1500

2000

0 10 20 30 40 50 60Days post tumor staging

H1963 H889 H146

• ABT-263 given at 100 mg/kg/day, po, qd x21• Significant tumor growth inhibition in 9 of 11 models• Complete tumor regression (no palpable tumor) in the three lines above• Regression maintained for > 2 months in several cases

Tse, et al. Cancer Res. 68, 3421 (2008)Shoemaker, et al. Clin. Cancer Res., 14, 3268- 3277 (2008)

ABT-263 in Multiple Phase 1/2 Clinical Trials

• Lymphoma– Follicular lymphoma t(14:18) translocation

drives Bcl-2 expression– Diffuse Large B-Cell Lymphoma (DLBCL):

Bcl-2 amplification t(14;18)

• Chronic Lymphocytic Leukemia (CLL)– > 80% overexpression of Bcl-2– Bcl-2 expression correlates with poor

prognosis

• Small Cell Lung Cancer (SCLC)– 55% to 90% overexpression of Bcl-2– Initially chemoresponsive, but rapid relapse

Lymphoma: Aggressive and indolent disease

CLL: Entry to hematologic malignancies

SCLC patients with relapsed disease

15 cm

Pre-Treatment 4 Cycles ABT-263

ABT-263 Regresses Lymphoma in Humans• Phase 1 study in subjects with refractory or relapsed lymphoid malignancies• 62 year old Female SLL/CLL S/P Fludarabine/Rituximab• CT scan results end of cycle 4• 99% total tumor reduction after Cycle 8

W. Wilson et al., ASCO (2008)

ABT-263 Regresses Lymphoma in Humans• 48-Year-old male NK-T cell lymphoma• Multiple cutaneous lesions• 75% total tumor reduction after Cycle 2

W. Wilson et al., ASCO (2008)Pre-Treatment 2 Cycles ABT-263

ABT-263 Single Agent Activity in CLL/SLL ongoing phase 1 clinical trial

• 43 patients treated to date• 35 evaluable patients

– 6 confirmed partial responses– 5 unconfirmed PR– 8 with > 50% reduction in lymphocyte

count > 2 month– 10 stable disease– 6 progressive disease

• Overall response rate = 31%– Excluding pts treated < 40mg: 38%

• 78% of patients have radiographic tumor regression

• Median progression free survival > 8.7 mo

tumorregression

tumorgrowth

• Historical Benchmarks– Campath - RR = 35%; mPFS = 4 - 7 mo– Rituxan - RR = 5-25%; mPFS ~4 mo

W. Wilson et al., ASCO (2009)

Summary

• Death is an essential part of life– Apoptosis is important in many biological processes

• Cancer cells must evade programmed cell death to survive• Bcl- 2 family proteins regulate a life/death rheostat• Protein- protein interactions can be suitable drug targets

– Structural understanding of the PPI is key: protein hot spots– Fragment-based lead generation accesses multiple hot spots

• Targeting the central apoptotic machinery is an exciting new approach to cancer therapy

Medicinal ChemistryDave Augeri

David BetebennerMilan Bruncko

Hong DingAaron KunzerChris Lynch

William McClellanThorsten Oost

Cheol-Min ParkXiaohong Song

Wang ShenSheela Thomas

Xilu WangMike Wendt

HTOS Chem.Daryl Sauer

Jurgen DingesShelley Landis

BiologyChris TseJun ChenZhui Chen

Sha JinMary Joseph

Shi-Chung Ng Paul NimmerMorey SmithSteve TahirXuifen Yang

Yu XiaoHaichao Zhang

Bob Warner

WEHIDavid Huang

Mark van DelftKylie Mason

Benjamin Kile

AcknowledgementsSteve ElmoreSteve Fesik

Structural BiologyAndrew Petros

Steve MuchmoreElizabeth FryPhil Hajduk

Clarissa JakobRob Meadows

Dave NettesheimChang Park

Kerren SwingerRussell Judge

In Vivo ModelsAlex Shoemaker

Scott AcklerJessica Adickes

David Frost Ruth Huang

Michael MittenAnatol Oleksijew

Idun PharmaceuticalTilman Oltersdorf

Robert Armstrong Ali Al-AssaadBarbara Belli

Thomas DeckwerthDarlene Martineau Kevin Tomaselli

Academic CollaboratorsTony Letai-Dana Farber

Gerry Cohen-Univ. of Leicester, UK

Abbott ColleaguesPharmacokinetics

Integrative pharmacology Preclinical safetyPharmaceutics

Process chemistryCountless others……

Acknowledgements

• Phase 1 M06-873 Clinical Collaborators– Andrew Roberts (Royal Melbourne), Jennifer R Brown (DFCI), Tom Kipps (UCSD),

John Seymour (Peter MacCallum), William Wierda (MDACC)

• Phase 1 M06-814 Clinical Collaborators– Wyndham Wilson and Louis Staudt (NCI), Anil Tulpule (USC), John Leonard

(Cornell), Owen O’Connor (Columbia), Myron Czuczman (Roswell Park), Ann LaCasce (DFCI), John Gerecitano (MSKCC), Alexandra Levine and John Kirschbaum (City of Hope)

• Abbott/Genentech Oncology Clinical Development Collaborators– Gary Gordon, Rod Humerickhouse, Sari Enschede, Andrew Krivoshik, Saul

Rosenberg, Jorge DiMartino, Iris Chan, Yi-Lin Chiu, Hao Xiong, Renee Greco, William Monte, Raymond Knight, Diane D’Amico

–

ABTABT--263 patients and their families263 patients and their families

ABT- 263 is being co- developed by Abbott and Genentech

26Sponsored by:

Participating Experts:Saul Rosenberg, Ph.D.AbbottAbbott Park, IL

Webinar SeriesWebinar SeriesScienceScience

22 September, 200922 September, 2009Brought to you by the Science/AAAS Business Office

John Abrams, Ph.D.University of Texas Southwestern Medical CenterDallas, TX

Apoptotic SignalingApoptotic Signalingin Normal and Cancer Cell Biologyin Normal and Cancer Cell Biology

Joseph T. Opferman, Ph.D.St. Jude Children's Research HospitalMemphis, TN

John Abrams

Department of Cell Biology University of Texas Southwestern

Medical Center

IAPs - gatekeepers of caspase action

A smac-mimetic exerts cross-species IAP antagonist activity: a functional probe of apoptotic networks

Smac mimetic: Li et al. 2004, Science

A smac-mimetic exerts cross-species IAP antagonist activity: a functional probe of apoptotic networks

Smac mimetic: Li et al. 2004, Science

A smac-mimetic exerts cross-species IAP antagonist activity: a functional probe of apoptotic networks

Smac mimetic: Li et al. 2004, Science

Genome scale silencing captures apoptogenic effectors

‘Hits’ reverse killing by smac mimetic

Why screen Drosophila cells? • reduced network complexity• no transfection of dsRNA needed• opportunities for in vivo validation• caspase inhibited cells survive

Genome Scale Silencing Captures Apoptotic Effectors

primary screen in rank formatprimary screen in rank format

Tango7 is required for apoptosis

Tango7 is required for apoptosis

Placing Tango7 in the apoptosis network

(reaper)IAP

antagonistsDark

Dronc

Effector Caspases

DIAPs

Cell viability

Caspase activity

SmacSmacmimeticmimetic

UVUV

CHXCHX

perhaps Tango proteins set apoptotic propensity

0

20

40

60

80

100

120

140

160

180

Normal ductalepithelium

Pa11PT Pa37PT Pa38PT Pa39PT Pa43PT

The human Tango7 counterpart is under-expressed in all five pancreatic tumors sampled by genome wide SAGE

SAGE data from Jones et al (2008, Science) compares normal pancreas to all five patient tumors sampled. Cell lines and xenografts excluded. Numbers are relevant reads per million tags.

SuKit ChewPo ChenNichole LinkKat GalindoKristi Pogue

Wan-Jin LuAlejandro D’BrotMelissa OnealSophie Tu

ReagentsXiaodong WangPatrick HarranNIG stock center, JapanBruce HayKristin White

NIGMS, NIAAA, ACS, NSF

45Sponsored by:

Participating Experts:Saul Rosenberg, Ph.D.AbbottAbbott Park, IL

Webinar SeriesWebinar SeriesScienceScience

22 September, 200922 September, 2009Brought to you by the Science/AAAS Business Office

John Abrams, Ph.D.University of Texas Southwestern Medical CenterDallas, TX

Apoptotic SignalingApoptotic Signalingin Normal and Cancer Cell Biologyin Normal and Cancer Cell Biology

Joseph T. Opferman, Ph.D.St. Jude Children's Research HospitalMemphis, TN

Anti-Apoptotic MCL-1 in Hematopoietic Development

and HomeostasisJoseph Opferman

Department of BiochemistrySt. Jude Children’s Research Hospital

NeurodegenerationImmunodeficiency

Infertility

CancerAutoimmunity

Diseases of Disordered Cell DeathDeath/Apoptosis

Regulation of the Intrinsic Apoptosis Pathway

BCL-2 Family Members In Hematopoiesis

Regulation of MCL-1 Function

• MCL-1 is required for promoting cell survival at multiple stages of hematopoiesis

• These stages require growth factor signaling to promote the survival of blood cell progenitors

• How does growth factor signaling regulate MCL-1 function?

MCL-1 Transcription is Induced by Growth Factor Signaling

MCL-1 is Regulated by Protein Degradation

His6 - Ubi:

MCL-1 is Induced by Growth Factor Signaling

MCL-1 is Eliminated in the Absence of Growth Factor Signaling

MCL-1 in Cancer Cell Survival

• High levels of MCL-1 expression are found in numerous human cancers.

• MCL-1 has not been linked to chromosomal translocation events that would enforce its expression.

• The elevated levels of MCL-1 expression in cancer cells may be due to the constitutively active signaling pathways found in these cells.

MCL-1 is Induced by Constitutive Oncogenic Signaling

MCL-1 in Cancer Cell Survival

• Understanding normal MCL- regulation by cellular signaling networks will provide critical insight into developing strategies may be critical to understanding how to down- modulate MCL-1 function in malignant cells.

Potential Therapeutic Windows to Abolish MCL-1 Function

St. Jude Children’s Research Hospital

Opferman LaboratoryDaniel Stewart, Ph.D.Rhonda Perciavalle

Brian Koss

Desiree SteimerBing Xia

Kristen Bisanz

Green LaboratoryDouglas Green, Ph.D.Ulrich Maurer, Ph.D.

Supported by:The Pew Biomedical Scholars Program

NIHAmerican Lebanese Syrian Associated

Charities (ALSAC)

60

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Sponsored by:

Brought to you by the Science/AAAS Business Office

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22 September, 200922 September, 2009

Apoptotic SignalingApoptotic Signalingin Normal and Cancer Cell Biologyin Normal and Cancer Cell Biology