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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
Webinar SeriesWebinar SeriesScienceScience
22 September, 200922 September, 2009
Apoptotic SignalingApoptotic Signalingin Normal and Cancer Cell Biologyin Normal and Cancer Cell Biology