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Page 1: Regulation of Apoptosis by BCL-2 Family Members (Prof. Thomas Kaufmann)

Regulation of Apoptosis by Bcl-2 Family Members

Thomas KaufmannInstitute of Pharmacology,

University of Bern , [email protected]

Vall d’Hebron Research InstituteNov 15 2012

and XIAP

Page 2: Regulation of Apoptosis by BCL-2 Family Members (Prof. Thomas Kaufmann)

Necrosis Apoptosisvs.

2

passive, „accident“,always pathological

active, energy-dependent,physiological + pathophys.

often death of individualcells

whole (parts of) tissue/organ affected

induces inflammationno inflammation,induces tolerance

lysis of cellscells stay intact,

cleared by phagocytosis

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PROGRAMMED CELL DEATH

NECROTIC CELL DEATH“accidents”:- lack of energy- physical damage- chemical damage

Apoptosis

Autophagic Cell Death

Cornification(“keratinization”)

Pyroptosis(casp-1)

Pyronecrosis

Necroptosis(RIPK1/3)

Anoikis

3

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BCL-2, follicular lymphoma

Tumour Cells Overexpress Anti-Apoptotic Genes

4

Follicular Lymphoma (spleen): t(14;18) IgH(14q32) BCL2(18q21)

mantle zone

germinal centre

BCL-2, control

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Effector Caspases(-3, -6 + -7)

Bax/Bak

cytochrome.c Caspase-9/Apaf-1

Bcl-2-like

Apoptosome

BH3-only proteins

cytokine deprivation, DNA- damage, hypoxia, viral infections, ER stress,anoikis, …

XIAP

(Bim, Puma, Bad,...)

(Bcl-2, Mcl-1,Bcl-xL, ...)

XIAP

tBid

Bidcaspase-8

procaspase-8 (-10)

DISC

cFLIPcIAP1/2

Death Receptor (Extrinsic Pathway)

Intrinsic Pathway

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The Bcl-2 Protein Family Regulates the Integrity of the Mitochondrial Outer Membrane

Bax-like

adaptor (APAF-1)

Bcl-2-like

BH3-only

Caspase-9

Bcl-2 Family

Cyt.cSmac/Diablo

XIAP

Caspase-3, -7 (, -6)

+cIAP1,2

MOMPMOMP: mitochondrial outer membrane permeabilisation

Vaux & Silke Nat Rev MCB 2005

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Only XIAP can directly block caspases-3 and -9

Riedl and Shi, Nat Rev MCB 2004

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Selective Interactions Between BH3-Only and Bcl-2-Like Proteins

Bim, Bid, Puma ABT-737

Obatoclax (Nguyen et al PNAS 2007)

(Oltersdorf et al Nature 2005)

Chen et al Mol Cell 2005(modified)

A1

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The Bcl-2 Family – Still Many Open Questions

9

BH3-only

Bcl-2-like

Bax/Bak

Strasser, Nat Rev Imm 2005

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BOK: A BAX/BAK-Like Protein?

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BOK: BCL-2 related ovarian killer (Hsu et al. PNAS 1997)

Ke F. et al., CDD 2012

BOK is widely expressed

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Beroukhim et al., Nature, 2010

BOK is Deleted in Human Cancers with high Frequency

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BOK IS NOT A FUNCTIONAL BAX/BAK HOMOLOGUE

Echeverry et al., in revision

• BOK induces intrinsic apoptosis upstream of BAX/BAK

• BOK localises predominantly to non-mitochondrial sites:– Golgi, ER/nuclear outer membrane– nuclear compartment

• Bok-/- cells present with aberrant ER stress response (part. BFA)

+4-OHT (h):

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Death Receptor-Induced Killing and Crosstalk to Intrinsic (mitochondrial) Apoptotic Pathway

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APOPTOSIS IS ONLY ONE OF SEVERAL POSSIBLE OUTCOMES IN DR SIGNALING

NFkB

Necroptosis

Proliferation

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FADD/C8 (?)

RIP3 RIP1

FLIP

Apoptosis

FADD/TRADD

procaspase-8

TNFa

cIAP1/2

cell survival

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TNF-R1: Not Meant to Kill

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NFkB

Necroptosis

cytokines

RIP3 RIP1

Apoptosis

FADD/TRADD

procaspase-8

cIAP1/2

anti-apoptotic genes=> cell survival

TNF-R1

TNFa

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• Bacterial LPS -> TNFa (Macrophages, Neutrophils, NK T)

• Response via soluble, circulating TNFa TNF-R1 (Pfeffer et al 1993, Rothe et al 1993, Grivennikov et al 2005)

• Sensitisation by D-(+)-galactosamine (GalN)

LPS plus Galactosamine Injection Model of TNF-R1-Mediated Fulminant Hepatitis

Maeda S et al. Immunity 2003

Kaufmann et al. (2009)

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tBidCaspase-8

Cyt.cApaf1/ Caspase-9

Effector Caspases

X?Bax/Bak

Bid

Bcl-2-like

TYPE II

Caspase-8

Effector Caspases

TYPE I

Bid

Hepatocytes are Type II Cells

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The BH3-Only Protein BIM Is Rapidly Phosphorylated in LPS/GalN-Induced Hepatitis

*

Kaufmann et al. Immunity (2009)

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Both BID and BIM are Involved in LPS/GalN-Induced Hepatitis

Kaufmann et al. Immunity (2009)

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BIM is Activated by JNK Mediated Phosphorylation

A

*

B+/- D-JNKI1 (30 mg/kg, i.p.)

Kaufmann et al. Immunity (2009)

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JNK

Bim

Both BID and BIM can Mediate a Crosstalk from Death Receptors to Mitochondria

Corazza et al. JCI (2006)JNK mediated BIM-activationdownstream of TRAIL

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Fas/CD95/Apo-1

• FasL mainly expressed on activated T cells and natural killer cells. • Critical role in the control of the immune system• Fas or FasL-mutant mice develop lymphadenopathy and SLE (systemic

lupus erythematosus)-like disease and are predisposed to lymphoma development

• Many ALPS patients have heterozygous inherited mutations in the Fas gene. (Fisher et al. Cell 1995, Rieux-Laucat et al. Science 1995)

• Only the membrane bound form of FasL is inducing cell death (O’Reilly et al. Nature 2009)

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tBid

Fas

FasL

Caspase-8

Cyt.cApaf1/ Caspase-9

Effector Caspases

X?Bax/Bak

Bid

Bcl-2-like

TYPE II

Caspase-8

Effector Caspases

Fas

FasLTYPE I

Bid

Type I or Type II Fas-Induced Apoptotic Pathway

?

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X

Many Cancer Cells Display a Mandatory Crosstalk

X

?

MOMPX

Bcl-2

XMitochondrium

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Aim: Uncoupling of Death Receptor Pathway

Bcl-2

XMOMP

drugX

25

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Combination Therapy

Bcl-2

MOMP

ABT-737ABT-737

ABT-737drug X

26

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- Viral hepatitis- Alcoholic liver disease

- Autoimmune hepatitis- Graft-vs-host disease (GvHD)

- Endotoxin-induced liver failure

- Ischemia/reperfusion-induced liver damage

Importance of Apoptosis in Liver Pathology

-> -> Hepatocellular carcinoma(HCC)

Abnormal apoptosis in hepatocytes is cause or contributing factor in:

death receptors (hepatocytes)

death ligands (activated leukocytes)

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Murine Hepatocytes are Type II-Like Cells

Jo2 anti-Fas – ALT (200 min)

wt bid-/- lpr wt bid-/- lpr

PBS Jo2

Jo2aFas

wt

bid-/-

scale bar: 50 mm

+FasL (0.25 mg/kg)

FasL (crosslinked)

Kaufmann et al. Cell 2007Jost et al. Nature 2009 (modified)

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Lack of BID Blocks Fas-Induced Apoptosis in Type II but not in Type I Cells

Jost PJ et al., 2009 (modified)

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XIAP is Stabilised in Livers of FasL-Treated WT Mice

Jost PJ et al. (2009), modified

+ FasL (0.25 mg/kg)

p<0.05

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Jost PJ et al., 2009

Absence of XIAP Re-Sensitises Bid-Deficient Mice To FasL-Induced Hepatitis

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Absence of XIAP Converts Hepatocytes Into Type I Cells

DEVD-AMC Assay

Jost PJ et al., 2009

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Effects of IAP Antagonistic Drug (BV6) Phenocopies Genetic Loss of XIAP

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Kaufmann, Strasser & Jost, CDD 2011

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mast cell

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Neutrophils: - most frequent leukocyte in human blood- major role in innate immunity- end-differentiated, short-lived- apoptotic clearance of activated

neutrophils essential

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0 24 48 72 960

20

40

60

80

100

untreatedGM-CSF 1ng/mlG-CSF 10ng/mlQ-VD oph 20uM

C57BL/6 WTS

urvi

val (

%)

Time (h)

Primary Neutrophils Die By Classical Apoptosis When Cultured In Vitro

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0 24 48 72 960

20

40

60

80

100

wt untreatedbid-/- untreatedwt TNFabid-/- TNFa

0 24 48 72 960

20

40

60

80

100

wt TNFa

bid-/- TNFa

wt TNFa + Q-VD-oph

bid-/- TNFa + Q-VD-oph

TNFα 50 ng/ml

Q-VD oph 20 μM

Neutrophils die by Apoptosis in Response to High Doses of TNFa

Time (h)

Su

rviv

al %

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FasL Induced Cell Death is Delayed In Bid-/- Neutrophils

Geering et al. Blood 2011

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n>3

FasL Trigger Both Apoptosis and Necroptosis in Neutrophils

0 24 48 72 960

20

40

60

80

100

FasL

FasL + Q-VD-oph

0 24 48 72 960

20

40

60

80

100

FasL + Q-VD-oph

FasL + Q-VD-oph + Nec

0 24 48 72 960

20

40

60

80

100

FasL

FasL + Q-VD-oph

0 24 48 72 960

20

40

60

80

100

FasL + Q-VD-oph

FasL + Q-VD-oph + Nec

Su

rviv

al %

Su

rviv

al %

Su

rviv

al %

Su

rviv

al %

wt

bid-/-

bid-/-

wt

h

FasL 100 ng/ml, Q-VD oph 20 μM, Necrostatin-1 20uM

h

hh

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Loss of BID Aggravates Dextran Sodium Sulfate-Induced Colitis

d0 d1 d2 d3 d4 d5 d6 d7 d8

DSS in drinking water water

an

ima

l we

igh

t (%

)

0 1 2 3 4 5 6 7 885

90

95

100

105

wtbid-/-

* ** ******

***

*n.s.

n.s.

days

=> BID is anti-inflammatory

DSS colitis: -> dependent of neutrophils + macrophages

-> independent of adaptive immune system

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Proinflammatory role for BID?

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Ex Vivo Generation of Neutrophils Using Conditional Hoxb8

d0d5

43

Protocol based on Wang et al. Nature Methods 2006

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Roles of XIAP in Neutrophils

0 24 48 720

20

40

60

80

100

SCF-condHoxb neutrophils

wt TNF-a

XIAP-/- TNF-a

hours

Viab

ility

[%]

TN

Fa

(pg

/ml)

SCF-condHoxb8 neutrophils

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Non-apoptotic roles of XIAP

NOD2

MDP

NFkB

cytokines

XIAP

Damgaard et al. Mol Cell 2012

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IAPs Limit Inflammasome Activation In Macrophages

Vince J. et al. Immunity (2012)

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SUMMARY

• Several poorly characterised BCL-2 family members• Besides BID, BIM can mediate a crosstalk from DR to

mitochondria• XIAP is a crucial discriminator between type I and

type II Fas-induced apoptosis• FasL triggers mix of apoptosis and necroptosis in

neutrophils• Non-apoptotic roles of IAPs and DRs become

increasingly evident (important)

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ACKNOWLEDGMENTS• Philipp Jost (Munich DE)

• Mads Gyrd-Hansen (Kopenhagen, DK)

• Christoph Borner (Freiburg i.Brsg., DE)

• Georg Häcker (Freiburg i. Brsg., DE)

• Thomas Brunner (Konstanz, DE)

• Frank Essmann (Tübingen, DE)

• Julia Fernandez-Rodriguez (Gothenburg, SE)

• Kurt Ballmer (Villigen, CH)

University of Bern (CH)• Nohemy Echeverry• Ursina Gurzeler• Tatiana Rabachini• Daniel Bachmann• Simone Wicki• Nicole Tochtermann• Laetitia Roh

• Hans-Uwe Simon• Mario Tschan• Shida Yousefi• Clemens Dahinden

WEHI, Melbourne (AU)• Andreas Strasser• Francine Ke• Paul Ekert• John Silke • David Huang• Ueli Nachbur

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LUBAC: linear ubiquitin chain assembly complex

Page 50: Regulation of Apoptosis by BCL-2 Family Members (Prof. Thomas Kaufmann)

50Andrea L 2009


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