Regulation of Apoptosis by Bcl-2 Family Members

Thomas KaufmannInstitute of Pharmacology,

University of Bern , Switzerlandthomas.kaufmann@pki.unibe.ch

Vall d’Hebron Research InstituteNov 15 2012

and XIAP

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

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

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

5

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

6

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

7

Only XIAP can directly block caspases-3 and -9

Riedl and Shi, Nat Rev MCB 2004

8

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

The Bcl-2 Family – Still Many Open Questions

9

BH3-only

Bcl-2-like

Bax/Bak

Strasser, Nat Rev Imm 2005

BOK: A BAX/BAK-Like Protein?

10

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

Ke F. et al., CDD 2012

BOK is widely expressed

Beroukhim et al., Nature, 2010

BOK is Deleted in Human Cancers with high Frequency

11

12

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):

13

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

APOPTOSIS IS ONLY ONE OF SEVERAL POSSIBLE OUTCOMES IN DR SIGNALING

NFkB

Necroptosis

Proliferation

14

FADD/C8 (?)

RIP3 RIP1

FLIP

Apoptosis

FADD/TRADD

procaspase-8

TNFa

cIAP1/2

cell survival

TNF-R1: Not Meant to Kill

15

NFkB

Necroptosis

cytokines

RIP3 RIP1

Apoptosis

FADD/TRADD

procaspase-8

cIAP1/2

anti-apoptotic genes=> cell survival

TNF-R1

TNFa

16

• 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)

17

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

18

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

*

Kaufmann et al. Immunity (2009)

19

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

Kaufmann et al. Immunity (2009)

20

BIM is Activated by JNK Mediated Phosphorylation

A

*

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

Kaufmann et al. Immunity (2009)

21

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

22

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)

23

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

?

X

Many Cancer Cells Display a Mandatory Crosstalk

X

?

MOMPX

Bcl-2

XMitochondrium

24

Aim: Uncoupling of Death Receptor Pathway

Bcl-2

XMOMP

drugX

25

Combination Therapy

Bcl-2

MOMP

ABT-737ABT-737

ABT-737drug X

26

27

- 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)

28

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)

29

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

Jost PJ et al., 2009 (modified)

30

XIAP is Stabilised in Livers of FasL-Treated WT Mice

Jost PJ et al. (2009), modified

+ FasL (0.25 mg/kg)

p<0.05

31

Jost PJ et al., 2009

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

32

Absence of XIAP Converts Hepatocytes Into Type I Cells

DEVD-AMC Assay

Jost PJ et al., 2009

33

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

Kaufmann, Strasser & Jost, CDD 2011

34

35

mast cell

36

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

neutrophils essential

37

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

38

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 %

39

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

Geering et al. Blood 2011

40

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

41

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

42

Proinflammatory role for BID?

Ex Vivo Generation of Neutrophils Using Conditional Hoxb8

d0d5

43

Protocol based on Wang et al. Nature Methods 2006

44

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

45

Non-apoptotic roles of XIAP

NOD2

MDP

NFkB

cytokines

XIAP

Damgaard et al. Mol Cell 2012

46

IAPs Limit Inflammasome Activation In Macrophages

Vince J. et al. Immunity (2012)

47

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)

48

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

49

LUBAC: linear ubiquitin chain assembly complex

50Andrea L 2009