sot hemangiosarcoma 2006 dacc

8/8/2019 SOT Hemangiosarcoma 2006 DACC

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Investigative Approaches to Understandingthe Mode of Action and Human Relevance ofPPAR Agonist-Induced Hemangiosarcomas

DACC Spring Meeting21-April-2006

Richard D. Storer, Ph.D.

Dept. of Laboratory Sciences and Investigative Toxicology

Merck Research Laboratories

West Point, PA

Slide 1


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PCAF

PPARs: Ligand-Regulated Transcription Factors

Endogenous ligands:arachidonic acid metabolites, sat. & unsaturated FA Ligand-induced conformational change co-activator recruitment Heterodimerize with RXR and bind PPRE DR-1 enhancer sites Transcriptional activation Alterations in gene expression major biological actions

CBPSRC

AGGTCA-X-AGGTCA

PPAR RXR

RNAPol.

TBP

TFII-B

histone acetylation

transcription


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PPAR Agonist Therapeutics for Type II Diabetes

PPAR

Thiazolidinediones

(TZDs)

Synthetic Ligands

insulin sensitization glucose lowering lipid lowering

Adipocytes


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PPAR

Fibrates,

hypolipidaemics

Synthetic Ligands

PPAR insulin sensitization glucose lowering lipid lowering

fatty acid oxidation TG, LDL lowering HDL raising

peroxisome proliferation

Thiazolidinediones

(TZDs)

Dual Agonists

Partial Agonists

Liver Adipocytes


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PPAR

Synthetic Ligands

Fibrates,

hypolipidaemics

PPAR insulin sensitization glucose lowering lipid lowering

fatty acid oxidation TG, LDL lowering HDL raising

peroxisome proliferation

Thiazolidinediones

(TZDs)

lipid metabolism TG lowering lipoprotein profiles

metabolic rate

Dual Agonists

Partial Agonists

GW501516

GW0742

Liver Adipocytes Skeletal Muscle

PPAR


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PPARagonist

gene expressionin adipocytes

FA uptake lipolysis FFAs

Insulin sensitizingfactorse.g. adiponectin, visfatin

Expression ofinsulin resistancefactorse.g. resistin, TNF, IL6

insulin actionin muscle / liver

Insulin-sensitivesmall adipocytes

PPAR Efficacy:Insulin Sensitization and Adipogenesis

visceraladiposity

subcutaneousadiposity


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Investigative Approaches to Understanding theMode of Action for, and Human Relevance of, PPAR

Agonist-Induced Hemangiosarcomas

Slide 7

Overview

Background: PPAR agonists for type II diabetes

Hemangiosarcoma in mice spontaneous tumor incidence in CD-1, B6C3F1

nuclear receptor agonist (PPAR, retinoids) induced tumors

S


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PPAR Agonists and Sarcomas in Rodents

Normal vs PPAR-Mediated Adipogenesis & Angiogenesis

Normal Stimuli(High caloric intake)

Pericytes

Normal Pathology

Normal Adipogenesis

Adipogenesis

PPAR Mesenchymalstem cells

Adipocyte

Capillary

endothelium

Monocyte/macrophage

Pre-adipocyte

PPAR A i d S i R d


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PPAR Agonist(Therapy)




Pericytes

Liposarcoma/FibrosarcomaNormal Pathology

Normal Adipogenesis

Adipogenesis


Adipocyte

Capillary

endothelium

Monocyte/macrophage

Rat

Pre-adipocyte

3/6 PPAR5/6 PPAR duals

PPAR A i t d S i R d t


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PPAR Agonist(Therapy)




Pericytes

Hemangiosarcomas

(subcutaneous, liver,

bone marrow, spleen)

Normal Pathology

Normal Adipogenesis

Adipogenesis


Adipocyte

Capillary

endothelium

Monocyte/macrophage

Rat

Mouse

Pre-adipocyte

3/5 PPAR3/6 PPAR duals

Liposarcoma/Fibrosarcoma


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Spontaneous Hemangiosarcoma Incidence in Mice*Most Common Sites

CD-1 Outbred Mice* Males Females

Liver 48 09% 25 0-6%

Spleen 35 08% 18 05%

Whole Body/Multiple Organ 29 012% 25 012%

Uterus - - 15 04%

Bone Marrow 3 0-3% 5 0-3%

*Giknis, R and Clifford, C. (2005) Charles River Laboratories. Total # of tumors in 52 () or 54() studies and % range in studies

** D. Malarkey, NIH/NIEHS: Total # of tumors and range in 28 studies, all routes.

0-8%18-4Skin

0-4%70-4%13Bone Marrow

0-8%180-6%26Spleen

0-4%120-6%35Liver

FemalesMalesB6C3F1 Mice**


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TroglitazoneTroglitazone

Oral Carcinogenicity Study in B6C3F1 MiceOral Carcinogenicity Study in B6C3F1 Mice**

Distribution of Hemangiosarcomas

Females (60/group) Males (60/group)

Treatment Groups(mkd)

VC EC 50 400 800 VC EC 50 400 800

Skin 0 1 0 10 12 1 2 1 4 8

Liver 1 3 0 1 2 2 1 0 4 7

Spleen 4 2 1 5 4 3 1 1 4 6Bone marrow 1 1 0 4 2 0 0 0 2 2

Adipose tissue 0 0 0 2 0 2 1 1 1 1

Heart 0 1 2 0 0 0 0 0 1 2

Other tissues 1 1 4 6 1 1 0 1 1 3

*Reference: Duddy, S.K., et al., Toxicol. Appl. Pharmacol. 156: 106-112, 1999

42% multicentric, 58% single tumors spontaneous incidence

10% in males and females (combined controls) 2-4x higher than historical (literature values)

H i I d i i Mi b R i id


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Hemangiosarcoma Induction in Mice by Retinoids

Slide 13

Retinyl acetate (principal supplement form of Vitamin A) hemangiomas in C3H mice (pRAR; not RAR, RXR

PPAR activation reported

anti-angiogenic effects described, potent apoptosis inducer

RAR/RXR independent mechanisms? (ROS, ceramide)

PPAR A i t I d d H i


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PPAR Agonists Induced HemangiosarcomasCurrent Weight of Evidence & Mode of Action

Species-specific effect (mice & hamsters not rats) genetic control of susceptibility in mice

non-genotoxic mode of action no consistent evidence of genotoxicity in in vitroor in vivotests

tumorigenicity seen with structurally diverse molecules

no mutations in ras or p53 in tro-induced tumors in B6C3F1 mice*

Vascular tumor promotion is by an indirect mechanism PPAR-dependent process

dysregulation of adipogenesisassociated angiogenic responses

Off-target PPAR-independent effects may play a role PPAR-mediated effects on cell survival

mitochondrial dysfunction

ROS and stress responses

anti-inflammatory effects

*Duddy, S.K., et al. (1999) Toxicol Appl. Pharmacol 156:106-112 & Toxicol Appl. Pharmacol 160:133-140.


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Receptor-Independent Effects of PPAR TZDs*

*Feinstein, D.L. et al., Biochem Pharmacol. 70(2):177-88, 2005

I i i A h U d di h


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Slide 16

Overview



nuclear receptor agonist (PPAR, retinoids) induced tumors

PPAR agonist-induced adipogenesis and angiogenesis


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Adipocytes, Adipokines and Angiogenesis

Rajala, M.W. & Scherer, P.E., Endocrinology 144(9): 3765-3773, 2003

Regulation of Vascular Morphogenesis


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Regulation of Vascular Morphogenesis,Maintenance and Remodeling by RTKs and Their Ligands*

*Hanahan, D. (1997) Science 277(5322):48-50.

PPAR Agonists and Hemangiosarcomas in Mice


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PPAR Agonists and Hemangiosarcomas in MiceHypotheses for Investigational Studies

Excessive PPAR agonism results in dose-dependent non-neoplasticpathologies in adipose tissue in multiple species

increased amount of fat in WAT depots & bone marrow

cytoplasmic atrophy

fibrosis (R,M)

capillary dilation, edema, plasma volume expansion

In mice, dysregulation of key factors (eg. VEGF, FGFs, angiopoietins)and cell-mediated processes regulating endothelial cell proliferationand capillary endothelial maturation leads to progressive vascularabnormalities

vascular congestion chronic activation of hypoxia response pathways

vascular hyperplasia

Vascular tumors

PPAR A i t I d


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PPAR Agonists InducePlasma Volume Expansion & Capillary Enlargement in

Rosiglitazone Treated fa/fa Rats

Dose-dependent increase in plasma volume and extracellularfluid volume after 7 days

Progressive increase in capillary enlargement Similar effects with several PPARg agonists

Plasma Volume Expansion Capillary Enlargement

Vehicle

4 days

2 days

7 days

7 days

Capillary Enlargement in Adipose Tissue of C57Bl/6 Mice


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vehicle rosi

Transmission Electron Microscopy

Capillary Enlargement in Adipose Tissue of C57Bl/6 Mice

Adipose Treated With Rosiglitazone for 14 Days

a normal capillary is just slightly larger than a single RBC

presence of multiple RBC's demonstrate enlarged capillary

PPAR agonist treatment increases diameter of capillary lumen in inguinaladipose tissue

no effect seen in skeletal muscle control

Capillary Enlargement in Adipose Tissue of CD-1 Mice


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Capillary Enlargement in Adipose Tissue of CD 1 Mice

Adipose Treated With Various PPAR Agonists For 14 Days

PPAR selective compounds all caused enlarged capillaries Neither PPAR nor PPAR selective compounds had any effect

100mpk

rosiglitazonevehicle

compound B(PPAR agonist) compound C(PPAR agonist)

Capillary Enlargement in Adipose Tissue of CD-1 Mice


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Adipose Tissue Depots:

Dosage epididymal inguinal perirenal Drug exposure(mpk) (# enlarged / # examined) (tAUC,mM*h)

vehicle --- 0/5 1/5 0/5 ---

Rosiglitazone () 10 1/5 0/5 0/5 71100 2/5 5/5 1/5 562

Compound B () 30 3/5 5/5 1/5 98300 3/5 4/5 0/5 177

Compound C () 30 2/5 2/5 0/5 186300 2/5 4/5 0/5 617

Compound D () 10 0/5 0/5 1/5 8.7100 0/5 0/5 1/5 104

Fenofibrate () 300 0/5 0/5 0/5 1709

PPARg selective compounds all caused enlarged capillaries

Neither PPAR or PPAR selective compounds had any effect

Number Of CD-1 Mice With Enlarged Capillaries

Capillary Enlargement in Adipose Tissue of CD 1 Mice

Adipose Treated With Various PPAR Agonists For 14 Days

Capillary Enlargement in White Adipose Tissue in PPAR


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Capillary Enlargement in White Adipose Tissue in PPARAgonist-Treated CD-1 Mice

Effect is restricted to capillaries within adipose tissue likely mediated by specific factors released by PPAR-activated

adipocytes

Hypothesis: edema results from the transient hyper-permeability concomitant

with vascular remodeling and incorporation of new endothelial cells

into capillary matrix in susceptible species (eg. mice), chronic PPAR agonism may

further dysregulate the angiogenic response

sub-optimal (deficient) or supra-optimal (exaggerated) capillary

endothelial cell proliferation need to compare PPAR agonists that are hemangiosarcomagens with

those that are not

Investigative Approaches to Understanding the


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Slide 25

Overview



nuclear receptor agonist (PPAR, retinoids) inducedtumors

PPAR agonist-induced adipogenesis and angiogenesis In vitro model systems for investigation of PPAR

agonist effects


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In Vitro Cell Models

Develop and characterize models for cell proliferation, apoptosis, differentiation endpoints

stromal cells: 3T3L1; mouse embryo fibroblasts (MEFs)

microvascular endothelial cells in vitro (HMVECS) mouse embryonic stem cells

differentiated towards adipocyte or endothelial lineages

Examine effects of PPAR agonists on growth factor/cytokineproduction mRNA & protein expression

angiogenesis assays for angiogenic factors in conditioned medium

PPAR subtype-specific agonist effects and interactions do PPAR or PPAR agonism play a role?

Off-target effects/PPAR independent effects


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Control

(undiff)

+1M

Rosi

Control

(Diff)

Adipocyte Differentiation in 3T3 L-1 Cells


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Assessment of Adipocytic Markers

Time (Days)

0 2 4 6 8 10

FoldIncrease

0

500

1000

1500

2000

2500

3000w/o Rosiw/ Rosi

0 2 4 6 8 10

FoldIncrease

0

5000

10000

15000

20000

w/o Rosiw/ Rosi

Time (Days)

Fabp (Ap2) Adiponectin

Differential Regulation of Adipokine Gene


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Time (Days)

0 2 4 6 8

FoldInc

rease

0

2

4

6

8

10w/o Rosiw/ Rosi

Time (Days)

0 2 4 6 8

FoldInc

rease

0.0

0.5

1.0

1.5

2.0w/o Rosiw/ Rosi

VEGF-A HGF

g p

Expression During Adipocyte Differentiation

R i lit R l t bFGF I d d tl f


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0 2 4 6 8

FoldIncrease

0.0

0.5

1.0

1.5

2.0

w/o Rosiw/ Rosi

Time (Days)

Rosiglitazone Regulates bFGF Independently ofAdipocyte Differentiation

No effect of Rosi on aFGF mRNA


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Conclusions: PPAR Effects in In Vitro Models

Mouse 3T3L1 Pre-Adipocytes marked effects of rosi on adipocyte differentiation in 3T3 L-1 cells.

Oil Red O accumulation & adiopcyte marker expression

marked effects of rosiglitazone on angiogenic growth factormRNA expression during differentiation

VEGFA

HGF bFGF mRNA levels independent of differentiation.

only a PPAR agonist (Rosi) affected mature adipocytes bFGF and slight in VEGF

Human microvascular cells (HMVECS) no evidence thus far that PPAR agonists are :

directly mitogenic to ECs.

promote EC cell survival (PPAR agonist) conditions optimized to detect increased tube formation by VEGF

Investigative Approaches to Understanding the


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g pp gMode of Action for, and Human Relevance of, PPAR


Slide 32

Overview


Hemangiosarcoma in mice spontaneous tumor incidence in CD-1, B6C3F1 nuclear receptor agonist (PPAR, retinoids) induced tumors

PPAR agonist-induced adipogenesis and angiogenesis In vitro model systems for investigation of PPAR agonist effects Lessons from tumor angiogenesis: role of vascular and

hematopoietic stem cells

Mobilization of Circulating Endothelial Progenitors and

H t i ti St C ll d P it


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Hematopoietic Stem Cells and Progenitorsin Tumor Angiogenesis

Rafii, S, Nature Reviews/Cancer 2: 826-835, 2002.

Reproduced with permission from Macmillan Magazines LTD

Stimulation of Adipogenesis in Subcutaneous Adipose


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Tissue and Bone Marrow by PPAR AgonistsBone Marrow Circulation Subcutaneous adipose

Osteobla

sticZone

VascularZone

Mature adipocytes

Pre-adipocytes

Mesenchymal cells

Capillary endothelial cells

CE Progenitor cellsMonocyte/macrophage

Pericyte

HSCs Angioblasts

Hemangioblast?

PPAR agonist

Mesenchymal

Stem Cell

Angiogenic Growth Factors Upregulated in Subcutaneous


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Adipose Tissue and Bone Marrow by PPAR AgonistsBone Marrow Circulation Subcutaneous adipose

Osteobla

sticZone

VascularZone

Mature adipocytes

Pre-adipocytes

Mesenchymal cells



Pericyte

HSCs Angioblasts

Hemangioblast?

PPAR agonist

Mesenchymal

Stem Cell

VEGFA

Activation and Recruitment of HSCs and Angioblasts from


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Bone Marrow by Colony Stimulating Factors

Bone Marrow Circulation Subcutaneous adipose

Osteobla

sticZone

VascularZone

Mature adipocytes

Pre-adipocytes

Mesenchymal cells



Pericyte

HSCs Angioblasts

Hemangioblast?

PPAR agonist

Mesenchymal

Stem Cell

VEGFA

GM-CSF

Stimulation of Angiogenesis and Capillary Sprouting by


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Tie2 -Expressing Monocytes (TEMS)


Osteobla

sticZone

VascularZone

Mature adipocytes

Pre-adipocytes

Mesenchymal cells



Pericyte

HSCs Angioblasts

Hemangioblast?

PPAR agonist

Mesenchymal

Stem Cell

VEGFA

GM-CSF

Stimulation of Capillary Spouting and Endothelial CellP lif i b Ti 2 E i M (TEMS)


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Proliferation by Tie2 -Expressing Monocytes (TEMS)


Osteobla

sticZone

VascularZone

Mature adipocytes

Pre-adipocytes

Mesenchymal cells



Pericyte

HSCs Angioblasts

Hemangioblast?

PPAR agonist

Mesenchymal

Stem Cell

VEGFA

GM-CSF

Investigative Approaches to Understanding theM d f A ti f d H R l f PPAR


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Mode of Action for, and Human Relevance of, PPARAgonist-Induced Hemangiosarcomas

Slide 39

Overview


Hemangiosarcoma in mice spontaneous tumor incidence in CD-1, B6C3F1 nuclear receptor agonist (PPAR, retinoids) induced tumors

PPAR agonist-induced adipogenesis and angiogenesis

In vitro model systems for investigation of PPAR agonist effects Lessons from tumor angiogenesis: role of vascular and hematopoietic

stem cells

Genetic heterogeneity of angiogenic responses in mice

Mouse Strain Differences in AngiogenicR i & Ci l ti E d th li l P it


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Responsiveness & Circulating Endothelial Progenitors

Strain Vessel Area

180 ng VEGF

129 SvI/mJ 2.2 0.4**DBA2/J 1.4 0.3**

Balbc/J 1.0 0.2**

FVB/NJ 0.9 0.1*

CBA/J 0.8 0.1

C57BL6/J 0.8 0.2

C3H/HeJ 0.8 0.1

CD-1 0.8 0.2

Genetic Heterogeneity of The Vasculogenic Phenotype Parallels Angiogenesis:Implications For Cellular Surrogate Marker Analysis Of Antiangiogenesis

Shaked, Y, et al., Cancer Cell 7:101-111, 2005

Corneal Micropocket Assay

Strain-Dependent Angiogenesis Induced by bFGF*


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*Chan, C. et al., Investigative Ophthamology &Visual Science 45(2): 441-447, 2004

Genetic Loci Controlling VEGF and bFGFAngiogenic Responses in Mice*


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Angiogenic Responses in Mice

Quantitative trait loci identified which influence the angiogenicresponse to bFGF and VEGF

Correlation of QTLs for bFGF angiogenesis with other QTL-correlated effects Body size

Wound healing

Lung tumor susceptibility Increased tumor burden in polyoma middle T transgenic mice

Mammary tumor growth

* Rogers, M., et al. (2004) FASEB J. 18:1050-1059.

Investigative Approaches to Understanding theMode of Action for and Human Relevance of PPAR


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Mode of Action for, and Human Relevance of, PPARAgonist-Induced Hemangiosarcomas

Slide 43

Summary

Species specificity of sarcoma response to PPAR agonists in commonrodent test species

liposarcomas in rats

hemangiosarcomas in mice

Commonality in mode of action based on stimulation of adipogenesis

Dysregulation of adipogenesis-associated angiogenic responsein miceconfers predisposition to vascular proliferative lesions short-term in vivo models of PPAR-induced vascular changes in adipose

tissue, blood, and bone marrow likely to inform for mode of action

in vitro models useful for investigating PPAR agonist sub-type specificeffects in adipocyte and endothelial cell lineages

new insights into role of progenitor cells (monocytes, CEPs, MSCs) in tumorangiogenesis suggests role in adipose angiogenesis

Investigation of genetic basis for heterogeneity of angiogenic responsesin different mouse strains may yield clues to species-specific effects

Contributors


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Safety Assessment

Tim JohnsonDiane Umbenhauer

Chunhua QinBridget YkorukShu ShiDanette Pascarella

Alan KaczorKim BleicherFrank Sistare

Target Validation

John WoodsEmanuel Zycband

Ching Chang

Metabolic Disorders-Diabetes

Joel Berger

Medicinal Chemistry

Peter Meinke

sot hemangiosarcoma 2006 dacc

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