The document summarizes recent research findings on apoptosis regulation by Bcl-2 family members and XIAP. Key points include: 1) BID and BIM can mediate crosstalk from death receptors to mitochondria to induce apoptosis. 2) XIAP is a crucial factor in determining whether Fas induces type I or type II apoptosis. 3) FasL can trigger both apoptosis and necroptosis in neutrophils. 4) Non-apoptotic roles of IAPs and death receptors are becoming increasingly evident and important.

1. Valld’Hebron Research Institute
Nov 15 2012
Regulation of Apoptosis by Bcl-2 Family Members
and XIAP
Thomas Kaufmann
Institute of Pharmacology,
University of Bern , Switzerland
thomas.kaufmann@pki.unibe.ch

2. Necrosis vs. Apoptosis
passive, „accident“, active, energy-dependent,
alwayspathological physiological + pathophys.
cellsstayintact,
lysis of cells
clearedbyphagocytosis
whole (parts of) tissue/ oftendeath of individual
organaffected cells
no inflammation,
inducesinflammation
inducestolerance
2

3. PROGRAMMED CELL DEATH
Apoptosis
Necroptosis
(RIPK1/3)
Autophagic Cell Death NECROTIC
Pyroptosis CELL DEATH
Anoikis “accidents”:
(casp-1) - lack of energy
- physical damage
Cornification Pyronecrosis - chemical damage
(“keratinization”)
3

4. TumourCellsOverexpressAnti-Apoptotic Genes
Follicular Lymphoma (spleen): t(14;18) IgH(14q32) BCL2(18q21)
BCL-2, follicular
BCL-2,control lymphoma
germinal
centre
mantlezone
4

5. Death Receptor (Extrinsic Pathway)
Intrinsic Pathway
cytokine deprivation, DNA- damage,
hypoxia, viral infections, ER stress,
DISC anoikis, …
procaspase-8 (-10)
BH3-only proteins
(Bim, Puma, Bad,...)
cFLIP caspase-8 Bid
cIAP1/2
(Bcl-2, Mcl-1,
tBid Bcl-2-like Bcl-xL, ...)
Bax/Bak
Effector Caspases cytochrome.c
XIAP
(-3, -6 + -7) Caspase-9/Apaf-1
Apoptosome
XIAP
5

6. The Bcl-2 Protein Family Regulates the Integrity of the
Mitochondrial Outer Membrane
BH3-only
Bcl-2-like Bcl-2 Family
Bax-like
MOMP: mitochondrial outer
membrane permeabilisation MOMP
Smac/Diabl
cIAP1,2 Cyt.c + adaptor (APAF-1)
o
XIAP
Vaux &Silke Nat Rev MCB 2005
Caspase-3, -7 (, -6) Caspase-9
6

7. Only XIAP can directly block caspases-3 and -9
Riedl and Shi, Nat Rev MCB 2004
7

8. Selective Interactions Between BH3-Only and Bcl-2-Like Proteins
Bim, Bid, Puma ABT-737 (Oltersdorf et al
Nature 2005)
Obatoclax (Nguyen et al
PNAS 2007)
A1
Chen et al Mol Cell 2005
(modified)
8

9. The Bcl-2 Family – Still Many Open Questions
BH3-only
Bcl-2-like
Bax/Bak
Strasser, Nat Rev Imm 2005
9

10. BOK: A BAX/BAK-Like Protein?
BOK: BCL-2 related ovarian killer (Hsu et al. PNAS 1997)
BOK is widely expressed
Ke F. et al., CDD 2012
10

11. BOK is Deleted in Human Cancers with high Frequency
Beroukhim et al., Nature, 2010
11

12. BOK IS NOT A FUNCTIONAL BAX/BAK HOMOLOGUE
• 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):
Echeverry et al., in revision
12

13. Death Receptor-Induced Killing and Crosstalk to
Intrinsic (mitochondrial) Apoptotic Pathway
13

14. APOPTOSIS IS ONLY ONE OF SEVERAL POSSIBLE OUTCOMES IN DR SIGNALING
FADD/C8 (?)
Proliferation
cIAP1/2
Apoptosis
Necroptosis
NFkB
TNFa
cell survival
14

15. TNF-R1: Not Meant to Kill
TNFa
TNF-R1
RIP1
Apoptosis cIAP1/2
Necroptosis
NFkB
anti-apoptotic genes
cytokines
=> cell survival
15

16. LPS plus GalactosamineInjectionModelof TNF-R1-MediatedFulminant Hepatitis
• 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)
Maeda S et al. Immunity 2003
Kaufmann et al. (2009)
16

17. Hepatocytesare Type IICells
TYPE I TYPE II
Bid
Caspase-8
tBid
Bid Caspase-8 Bcl-2-like
? X
Bax/Bak
Effector
Caspases
Effector Cyt.c
Caspases Apaf1/ Caspase-9
17

18. The BH3-Only Protein BIM IsRapidlyPhosphorylated in LPS/GalN-Induced Hepatitis
*
Kaufmann et al. Immunity (2009) 18

19. Both BID and BIM areInvolved in LPS/GalN-InducedHepatitis
Kaufmann et al. Immunity (2009)
19

20. BIM isActivatedby JNK MediatedPhosphorylation
A B
+/- D-JNKI1 (30 mg/kg, i.p.)
*
Kaufmann et al. Immunity (2009) 20

21. Both BID and BIM canMediate a Crosstalk fromDeathReceptors to Mitochondria
JNK
Corazza et al. JCI (2006)
Bim JNK mediated BIM-activation
downstream of TRAIL
21

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
Fasgene. (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)
22

23. Type I or Type II Fas-Induced Apoptotic Pathway
FasL FasL
TYPE I TYPE II
Fas Fas
? Bid
Caspase-8
tBid
Bid Caspase-8 Bcl-2-like
? X
Bax/Bak
Effector
Caspases
Effector Cyt.c
Caspases Apaf1/ Caspase-9
23

24. ManyCancerCells Display a Mandatory Crosstalk
Bcl-2
? X
Mitochondrium
X
MOMP
X
X
24

25. Aim: Uncoupling of DeathReceptorPathway
Bcl-2
drugX
X
MOMP
25

26. CombinationTherapy
ABT-737 Bcl-2
ABT-737
drug X
ABT-737
MOMP
26

27. Importance of Apoptosis in Liver
Pathology
Abnormal apoptosis in hepatocytes is cause or contributing factor in:
- Viral hepatitis -> -> Hepatocellular carcinoma
- Alcoholic liver disease (HCC)
- Autoimmune hepatitis
- Graft-vs-host disease (GvHD)
- Endotoxin-induced liver failure
- Ischemia/reperfusion-induced liver damage
death ligands (activated leukocytes)
death receptors (hepatocytes)
27

28. MurineHepatocytesare Type II-LikeCells
Jo2 anti-Fas – ALT (200 min) Jo2aFas
wt
wt bid-/- lpr wt bid-/- lpr
PBS Jo2
FasL (crosslinked)
bid-/-
+FasL (0.25 mg/kg)
scale bar: 50 mm
Kaufmann et al. Cell 2007
Jost et al. Nature 2009 (modified) 28

29. Lack of BID Blocks Fas-InducedApoptosis in Type II but not in Type I Cells
Jost PJ et al., 2009 (modified)
29

30. XIAP isStabilised in Livers of FasL-Treated WT Mice
+ FasL (0.25 mg/kg)
p<0.05
Jost PJ et al. (2009), modified
30

31. Absence of XIAP Re-SensitisesBid-DeficientMice To FasL-Induced Hepatitis
Jost PJ et al., 2009
31

32. Absence of XIAP ConvertsHepatocytesInto Type I Cells
DEVD-AMC Assay
Jost PJ et al., 2009
32

33. Effects of IAP Antagonistic Drug (BV6) Phenocopies Genetic Loss of XIAP
33

34. Kaufmann, Strasser&Jost, CDD 2011
34

35. mast cell
35

36. Neutrophils: - most frequent leukocyte in human blood
- major role in innate immunity
- end-differentiated, short-lived
- apoptotic clearance of activated neutrophils essential
36

37. Primary Neutrophils Die By Classical Apoptosis When Cultured In Vitro
C57BL/6 WT
100
80
Survival (%)
60 untreated
GM-CSF 1ng/ml
G-CSF 10ng/ml
40
Q-VD oph 20uM
20
0
0 24 48 72 96 Time (h)
37

38. Neutrophils die by Apoptosis in Response to High Doses of TNFa
100
80 wt untreated
bid-/- untreated
Survival %
60
wt TNFa
40 bid-/- TNFa
20
0
0 24 48 72 96 Time (h)
100
80
60 wt TNFa
TNFα 50 ng/ml bid-/- TNFa
Q-VD oph 20 μM 40
wt TNFa + Q-VD-oph
20 bid-/- TNFa + Q-VD-oph
0
0 24 48 72 96 38

39. FasLInduced Cell Death is Delayed In Bid-/-Neutrophils
Geeringet al.Blood 2011
39

40. FasL Trigger Both Apoptosis and Necroptosis in Neutrophils
100 100
wt bid-/-
80 80
Survival %
Survival %
FasL FasL
60 60
FasL + Q-VD-oph FasL + Q-VD-oph
40 40
20 20
0 0
0 24 48 72 96 h 0 24 48 72 96 h
100
wt 100 bid-/-
80 FasL + Q-VD-oph
80 FasL + Q-VD-oph
Survival %
60 FasL + Q-VD-oph + Nec Survival %
60 FasL + Q-VD-oph + Nec
40
40
20
20
0
0
0 24 48 72 96 h n>3
0 24 48 72 96 h
FasL 100 ng/ml, Q-VD oph 20 μM, Necrostatin-1 20uM 40

41. Loss of BID Aggravates Dextran Sodium Sulfate-Induced Colitis
DSS colitis: -> dependent of neutrophils + macrophages
-> independent of adaptive immune system
DSS in drinking water water
d0 d1 d2 d3 d4 d5 d6 d7 d8
105
animal weight (%)
100 * ** ***
***
95 *** wt
n.s. bid-/-
90 n.s. *
85
0 1 2 3 4 5 6 7 8
days
=> BID is anti-inflammatory
41

42. Proinflammatory role for BID?
42

43. Ex Vivo Generation of Neutrophils Using Conditional Hoxb8
Protocol based on Wang et al. Nature Methods 2006
d5 d0
43

44. Roles of XIAP in Neutrophils
SCF-condHoxb neutrophils SCF-condHoxb8 neutrophils
100
80 wt TNF-a
TNFa (pg/ml)
XIAP-/- TNF-a
Viability [%]
60
40
20
0
0 24 48 72
hours
44

45. Non-apoptotic roles of XIAP
MDP
NOD2 XIAP
NFkB
cytokines Damgaard et al. Mol Cell 2012
45

46. IAPs Limit Inflammasome Activation In Macrophages
Vince J. et al. Immunity (2012)
46

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)
47

48. ACKNOWLEDGMENTS
University of Bern (CH) WEHI, Melbourne (AU) • Philipp Jost(Munich DE)
• NohemyEcheverry •Andreas Strasser • MadsGyrd-Hansen (Kopenhagen, DK)
• UrsinaGurzeler •Francine Ke • ChristophBorner (Freiburg i.Brsg., DE)
• Tatiana Rabachini •Paul Ekert
• Daniel Bachmann • Georg Häcker(Freiburg i. Brsg., DE)
•John Silke
• Simone Wicki • Thomas Brunner (Konstanz, DE)
•David Huang
• Nicole Tochtermann • Frank Essmann(Tübingen, DE)
•UeliNachbur
• LaetitiaRoh
• Julia Fernandez-Rodriguez
(Gothenburg, SE)
• Hans-Uwe Simon
• Mario Tschan • Kurt Ballmer (Villigen, CH)
• ShidaYousefi
• Clemens Dahinden
48

49. LUBAC: linear ubiquitin chain assembly complex
49

50. Andrea L 2009
50