Induced Apoptosis

APOPTOSIS INDUCTION AND IMMUNOTHERAPY-HOW TO IMPROVE RESULTS OF IMMUNOTHERAPY

C/ Professor Peter Hersey University of Newcastle NSW Australia IMMUNOTHERAPY DEPENDS ON INDUCTION OF APOPTOSIS!

• IF WE UNDERSTAND THE RESISTANCE MECHANISMS AGAINST APOPTOSIS WE CAN TARGET THESE AND IMPROVE THE RESULTS OF IMMUNOTHERAPY CELL KILLING MECHANISMS USED BY LYMPHOCYTES DEPEND ON INDUCTION OF APOPTOSIS 1. Granzyme – Perforin Mediated Killing CD8 CTL (CD4 CTL) NK Cells and ADCC

2.Death Ligand Mediated Killing TRAIL, FasL, TNF CD4 T Cells Monocytes, Dendritic Cells CURRENTCURRENT CONCEPTSCONCEPTS ININ APOPTOSISAPOPTOSIS TRAIL, Granz B P53.-NOXA, BID CYTOSKELETON PUMA,BAD,BID BIM,BMF

Bcl-2,Bcl-xL.Mcl-1 ER Stress, Bik,PUMA,Noxa Bax,Bak Mitochondria Smac,Omi Cyto c, Casp 9 IAPs Classical Pathway

Effector Caspases

3,7 MITOCHONDRIAL PATHWAYS TO APOPTOSIS ARE REGULATED BY BCL-2 FAMILY PROTEINS • Pro-apoptotic BH3 only damage sensor proteins (Bid, Bik,Bim, Bmf, Noxa,Puma,Bad,Hrk) • Pro-apoptotic multidomain proteins: BAX, BAK • Anti-apoptotic proteins: BCL-2, BCL-Xl, MCL-1, Bcl-W,A1

WE ALREADY HAVE AGENTS THAT TARGET THE ANTI APOPTOTIC PROTEINS! Targetting Anti Apoptotic Proteins • Genasense against Bcl-2.

• Inhibition of production of the IAP protein Survivin YM155 (Astellas Pharm

• BH3 mimics that bind Bcl-2 proteins(Abbott ABT-737) Targetting Anti-Apoptotic Proteins • AT-101 (Gossypol) Oral inhibitor of Bcl-2 Bcl- XL,Mcl-1 . Ascenta Therapeutics

• TW37- Small mw mimic of Bim that inhibits Bcl-2, Bcl-XL,Mcl-1. Univ Michigan

• Obatoclax (GX015-070) Small mw BH3 mimic . Inhibits Bcl-2,Bcl-XL,Mcl-1. (Gemin X) TRAIL INDUCED KILLING REQUIRES DEATH RECEPTORS! TRAIL Induces Apoptosis in the Majority of Melanoma Cell Lines

100 90 80 70 60 50 40 30 % Apoptotic Cells % Apoptotic 20 10 0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930

Melanoma Cell Lines TRAIL-R1 & R2 Expression Correlates with Degree of Apoptosis

A 120 120

T 100 100

b 80 80

d e 60 60 c

d 40 40

s 20 20

o 0 0 t

p -20 o -20

A -10 10 30 50 70 90

-10 10 30 50 70 90

% TRAIL-R1 TRAIL-R2

Zhang et al. Cancer research.59:2747 1999 Zhuang et al. Human Pathology 37,1286-94 .2006

CAN TRAIL RECEPTORS BE UPREGULATED? AGENTS THAT UPREGULATE TRAIL DEATH RECEPTORS • Tunicamycin- Glycosylation inhibitor. Induces ER Stress • Cox 2 Inhibitors. Upregulates Gadd 153(CHOP) • Dipyrimadole-nucleoside transport inhibitor Upregulates Gadd153 • Curcumin-Upregulates Gadd153 • Perifosine (AKT inhibitor) in some cells TunicamycinTunicamycin selectivelyselectively upregulatesupregulates TRAILTRAIL--R2R2

Golgi TR-2 Overlay + DAPI

IgR3 Untreated

IgR3 Tunicamycin

Golgi TR-2 Overlay + DAPI

Mel RM Untreated

Mel RM Tunicamycin Tunicamycin sensitizes melanoma cells to TRAIL- induced apoptosis

100 Tunicamycin 90 TRAIL 80 Tunicamycin + TRAIL 70 60 50 40 30 20 % Apoptotic cells 10 0

3 V 8 0 s 2 1 0 200 -C - -FH te 0 IgR l l-1 y c W2 Mel -Mel Me o Mel-RM MM Me4405 k O HUVECs S lan L Sk-Me F Me Jiang et al Canc Res 67:5880 2007 STATUS OF CURRENT TRIALS WITH TRAIL (ASCO 2006,2007)

Genentech/Amgen. 58 patients with various cancers. 7 with melanoma. 1 hr infusion, 5 days each 3 weeks. Well tolerated Abst3013

HGS. ETR2,Lexatumumab.Up to 10mg/kg each 2 weeks.31 patients Abst3012 HGS. ETR1,Mapatumumab. 1CR,2PR in follicular Lymphoma. Now in phase 2 studies in NHL Amgen. AMG655 Ab.Dose finding in16 patients IS MANIPULATION OF THE BCL-2 FAMILY ENOUGH?

NO-THE MEK/ERK & Akt SIGNAL PATHWAYS SHUT DOWN APOPTOSIS THE RAS/RAF/MEK/ ERK1/2 INHIBITORY PATHWAY IN MELANOMA • May be activated by mutations in BRAF

• Cytokines such as TRAIL, chemotherapy such as the TAXOLS

• Adhesion molecule interactions

• ER Stress

• Activation of ERK related to the PKC epsilon phenotype in Melanoma cells (Mhaidat et.al. Mol. Can Res 5:1073 2007) Zhuang et al J Clin Path 58:1163 2005 U0126 Sensitises Melanoma to TRAIL-Induced Apoptosis U0126 100 TRAIL U0126+TRAIL

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S M THE ERK1/2 PATHWAY BLOCKS APOPTOSIS AT MULTIPLE SITES

• Inhibits Bim EL by phosphorylation Ser 69 • Phosphorylates Bad • Induces Mcl-1 (Wang et al 2007) • Induces GRP78-(GRP78 binds Bik, casp4) • Induces IL-8 and upregulation of ICAM • Increases HIF-1A expression ADDITIONAL WAYS THE RAS /MEK PATHWAY INHIBITS CTL ACTIVITY

• Downregulation of Mart-1, gp100 and Tyrosinase in Melanoma (Kono et al 2006)

• MEK inhibitors decreased production of IL- 10,VEGF,IL-6 from melanoma cells (Sumimoto et al 2006)

• Knockdown of BRAF reduced IL-8 and ICAM- 1 expression and reduced Melanoma cell extravasation (Liang et al 2007). WE ALREADY HAVE MANY AGENTS WHICH TARGET THE ANTI APOPTOTIC PATHWAYS RAF Signal Pathway Inhibitors • Sorafenib/Nexavar RAF/VEGFR2,3 Onyx/Bayer

• KOS-953(17-AAG).Tanespimycin. Geldanomycin derivative. Hsp90 inhibitor. Raf,Akt,and others (Kosan/ Roche)

• Chir-265. Mutant BRAF/VEGFR2 (Novartis)

• PLX4032. Mutant BRAF ,Plexxicon/Roche MEK & RTK Signal Pathway Inhibitors • AZD6244, Specific MEK inhibitor (Astra Zeneca)

• PD0325901,MEK inhibitor (Pfizer)

• RTKinases, Imatinib, Sutent,Erlotinib

• Farnesyl transferase Inhibitors R11577 THE MOST IMPORTANT CAUSE OF FAILURE OF IMMUNOTHERAPY ARE ADAPTIVE PROCESSES IN CANCER CELLS AGAINST ER STRESS

GRP78 CHAPERONE PROTEIN EXPRESSION IS AN INDICATOR OF ER STRESS GRP78+ thick melanoma GRP78+ thin melanoma

GRP78+ subcutaneous met GRP78(-) compound naevus Cultured melanomacellsarerelativelyresistant to 67:9750 2007 Res Jiang et alCanc % apoptotic cells 100

Me 20 40 60 80 0 l- R MM M ER stress-inducedapoptosis 2 00 IgR3 M e l-CV

Me TG TM 4 Sk-M 4 0 5 e l-2 Mel-FH8 Sk- M el- 1 Me101 M 0 e la n 0 ocy 7 F ib te roblastss ER STRESS INDUCES ANTI APOPTOTIC EFFECTS • Upregulation of BCL-XL,Mcl-1. Down of Bcl-2

• Activation of Akt., (MEK/ERK in normal cells)

• Upregulation of GRP78

• Downregulation of p53 (via?HDM2,viaGSK3b)

• Glycolysis and acidification of the microenvironment Down regulation of Bcl-2 during progression of melanoma

Mcl-1 mean IRS Bcl-xl mean IRS Bcl-2 mean IRS e *** 30 p<0.001 *** ** ** ** *** 25 * * 20 * 15

Mean immunoreactive scor Mean immunoreactive 0 n=10Compound n=10 N Dysplastic n=17 N Thin M n=24 Thick M n=21 Subcutaneous n=17 LN Met M Met M

Melanocytic Lesions Zhuang et al Mod Path 20:416 2007 KNOCKDOWN OF MCL-1 RATHER THAN BCL-2 IS MORE EFFECTIVE IN SENSITISING MELANOMA CELLS TO ER STRESS

Mel-RM MM200 Mel-RM MM200 Con siRNA - + - - + - Con siRNA - + - - + - Bcl-2 siRNA - - + - - + Mcl-1siRNA - - + - - + -Mcl-1 -Bcl-2 -GAPDH -GAPDH

70 70 60 60 50 50 40 40 30 30

20 apoptotic cells % 20 % apoptotic cells % 10 10 0 0 Con siRNA + - + - + - + - Con siRNA + - + - + - + - Mcl-1 siRNA - + - + - + - + Bcl-2 siRNA - + - + - + - + TG + + - - + + - - TG + + - - + + - - TM - - + + - - + + TM - - + + - - + + Mel-RM MM200 Mel-RM MM200 Inhibition of MEK, Akt, or PKC sensitizes melanoma cells to ER stress-induced apoptosis

90 80 70 60 50 40 30 20

% Apoptotic cells 10 0 Tunicamycin + - + - + + + U0126 - + + - - - - LY294002 - - - + + - + Bis - - - - - + + SHOULD WE USE ADDITIONAL AGENTS TO TARGET ER STRESS INDUCED RESISTANCE TO APOPTOSIS? • Agents that target HDM2 & increase p53 Eg Nutlin 3a

• Inhibitors of GSK3beta that targets S 315,376 on p53 eg DW1/2

• Inhibitors of GRP78,IRE1a eg Irestatin

• VEGF R inhibitors,eg AZD 2171,

• Proton pump inhibitors? Eg Omeprazole CONCLUSION - THE TUMOR HAS EVOLVED INHERENT RESISTANCE MECHANISMS THAT NEED TO BE INHIBITED BEFORE IMMUNOTHERAPY WILL BE EFFECTIVE

DOES THE WAY A CELL DIES INFLUENCE THE IMMUNE RESPONSE? • Necrosis said to release inflammatory mediators which stimulate NF-Kb,ERK and STAT 1,3 ,release of TNF,IL-1,IL-6, IL- 8, IL-23 cytokines which can stimulate tumor growth and or induce Th17 helper T cells.

• HMGB1(High Mobility group B1) and Heat shock proteins may be key stimulators of DC maturation and immune responses ?

• Apoptosis said to be a more silent death which delivers antigens to APC without the marked release of Inflammatory mediators and no generation of CD4 Tcells.

• Resulting “Help-less” CD8 Tcells said to be tolerant and to prevent auto immune disease CONCLUSION LETS HEAR WHAT THE PANEL SAYS!!

How is UPR initiated?

dimerization/oligomerization of IRE1, PERK, and ATF6 upon released from Bip initiates UPR signaling

IRE, inositol-requiring enzyme

PERK, double-stranded RNA-activated protein kinase-like ER kinase

ATF, activating transcription factor b-ZIP, basic leucine zipper

Bip, also called GRP78; GRP, glucose-regulated protein ER stress up-regulates the expression of pro-apoptotic proteins

Mel-RM MM200 TG TM TG TM 0 16 24 36 48 16 24 36 48 0 16 24 36 48 16 24 36 48 -PUMA

-NOXA

-GAPDH

16 PUMA TG 16 PUMA TG PUMA TM PUMA TM 14 Noxa TG 14 Noxa TG 12 Noxa TM 12 Noxa TM 10 10 8 8 mRNA 6 mRNA 6 4 4 2 2 Relative Abundance of Abundance Relative Relative Abundance of Abundance Relative 0 0 Hours 0 1 3 6 162436 Hours 0 1 3 6 16 24 36 Mel-RM MM200 Akt –A KEY PLAYER IN ER STRESS • Down regulates p53 by facilitating HDM2 entry into Nucleus

• Phosphorylates FOXO and decreases its entry into nucleus &BIM transcription

• Activates NF-kB

• Inhibits GSK3beta?? Akt related Signal Pathway inhibitors • PI3K inhibitors-PI 103 (Workman ICR UK) • Akt3 inhibitors- Perifosine , CMEP (Zhang Virginia) • mTOR Inhibitor, RAD 001,CCI- 779,Everolimus(Novartis) • GSK3beta inhibitors SB216763?DW1/2 • Nutlin-3a HDM2 inhibitor Regulation of the expression of anti-apoptotic proteins by the ER stress inducers, TG and TM

TM TG

Mel-RM MM200 Mel-RM MM200 Hours 0 16 24 36 0 16 24 36 0 16 24 36 0 16 24 36 -GRP78

-GAPDH

Mel-RM MM200 TG TM TG TM 0 16 24 36 48 16 24 36 48 0 16 24 36 48 16 24 36 48

-Mcl-1

-Bcl-2

-Bcl- xL -GAPDH STRATEGY TO OVERCOME ER STRESS INDUCED RESISTANCE • Use signal pathway inhibitors particularly those targetting ERK1/2 and Akt pathways • Combine with Anti apoptotic strategies.In particular agents like TW37 ,Obatoclax or ?AT101 which can bind Mcl-1 • Target metabolic pathway?Anti LDH-A,, DCA (dichloroacetate),PTK/XK, Anti VEGFR eg AZD 2171 Ult53 ? N tli 3DW1/2 SUMMARY

• The immune system and ER stress are key drivers of progression and are responsible for the resistance of melanoma to treatment.

• Key roles for signal pathways ERK1/2,Akt and possibly GRP78 . Down regulation of p53 likely to be a consequence of ER stress

• Heterogeneity in the disease implies single agents unlikely to have much impact.

• We have a wealth of new agents waiting to be applied but we need better ways of selecting treatments for patient subgroups Figure 3">

Ellerman, J. E. et al. Clin Cancer Res 2007;13:2836-2848

Glucose Hypoxia mutant depletion proteins

Alterations in disturbances of calcium Redox and/or flux glycosylation ER STRESS

Death Intrinsic Resistance apoptotic to receptors pathways Progression chemotherapy of melanoma

Apoptosis What about the serine protease inhibitor 9 against Granzyme B?

• Induced by Estrogens • High levels in some melanoma and breast carcinoma cells The TRAIL-Induced Apoptotic Pathway in Melanoma Cells

TRAIL TRAIL-R1 or/and -R2

Bax/Bak FADD

Pro-caspase-8 Bcl-2

Activated Bid tBid caspase-8 Smac/DIABLO

Caspase-3 activation XIAP

apoptosis Voskoboinik et al. Nature Reviews Immunology 6, 940–952 (December 2006) | doi:10.1038/nri1983 TUNICAMYCIN SELECTIVELY UPREGULATES TRAIL-R2

Intact cells Permeabilized cells

TRAIL-R2TRAIL-R1 Fas TNF-R1 TRAIL-R2

Mel-RM

Mee1007 1007

Mel-VD

Jiang et al Cancer Research In press, 2007

Intact or permeabilized melanoma cells without (dotted lines) or with (solid lines) treatment with tunicamycin for 16 hours. were stained with mAB against TRAIL-R2, Fas, or TNF-R1 and analyzed by flow cytometry The filled hisgrams are isotype controls. Figure 2">

Ellerman, J. E. et al. Clin Cancer Res 2007;13:2836-2848

• XIAP, IAP1,2, ML-IAP

• XIAP BINDS TO ACTIVATED CASPASE 3,6,7 & INHIBITS ACTIVATION OF CASPASE 9.

• IAP1&2 MAY HAVE AN INDIRECT EFFECT UPSTREAM OF CASPASE 8