……at an average cost of $1B per drug

Current Cancer Drug Development

adapted from: Sharpless and DePinho; Nature Reviews Drug Discovery ‘06

Work Horse Mouse in Cancer Drug Development

(xenograft: cells from one species transplanted into another)

in cancer drug testing: subcutaneous injection of established human cancer cell lines into immunocompromized mice

adapted from: Sharpless and DePinho; Nature Reviews Drug Discovery ‘06

TUMOR

TUMOR

“We’ve cured plenty of cancers in mice, but only very few in

humans….The mouse is not a good model”

many reasonable people

Cancer is a Dynamic and Evolutionary Process

adapted from Reilly and Van Dyke, Cell 2008adapted from Hanahan and Weinberg, Cell 2000

Mutant Mice in Integrated Disease Analyses:Two Decades of Development

cell biology & cell imaging

genomics, proteomics

cell-based testshistopathology, physiology

animal imaging

0% 50% 100%

simple and complex genetics

p107p130

INK4s cdk4,6cyclin Ds pRb E2Fs

G1

S

inactivated inactivatedactivatedor or

most human cancers

Rb Pathway Commonly Aberrant in Human Cancers

pRb, p107, p130Inactivation

SV40 T Ag

T121

Rb107130

p53

A Tool for Cell-Specific Inactivation of pRb Function

p300/CBP

Rb107130

N C

normal cell

pRb

proliferation + cell death

evolutionary selection for impaired cell

death

aberrant signals to proliferate

progression

evolution/ selection

loss of many biological controls

p53

Ptenor

common amongmost cell types

cell type/tissue specific

Cancer models

Choroid plexusMammary/breast

ProstateOvarian

Astrocytoma

Cancer Evolution as Deciphered in GEM

poorly differentiated

high mitotic index

diffuse invasion

angiogenesis

pseudopalisading necrosis

High-grade Astrocytoma

Grade III: anaplastic astrocytomaGrade IV: glioblastomamost common brain tumorspoor prognosis no effective treatments

RB orCDK4 orINK4a

EGFR or PDGFRPten

(K-Ras )

Astrocytoma Model Engineering

stop

Lac Z T121GFAP

stopG12D

Pten+/fl

mouse genotype

NORMAL

4 5 6

K-Ras+/lslG12D

TgGZT121

Qian Zhang

* *** **

* * * * * *** **

**

**

****** **

X GFAP-CreERTAM

(K. McCarthy UNC)+ 4-OHTam

T121GFAP

G12DKRas

Pten+/-

*adultastrocyte genotype

Cancer-associated

TgGdZT121

2 3 6

K-Ras+/G12D

Rbf

Pten

Ras

Diffuse grade II-III astrocytoma

AA

GBM

Rbf

Rbf, Ras

Rbf, Ras Pten

Inducible Astrocytoma Model Assessment

2 mo 4 mo 1 yr

Normal

Normal

K-Ras

Pten

Diffuse grade II-III astrocytoma acceleratedRbf Pten

Ras Pten

GBMRbf, Ras Pten+/-

time post 4OHT:

no BT sk. tu.

+sk. tu.

angiogenesisinvasion

angiogenesisinvasionnecrosis

Diffuse grade II-III astrocytoma

AA

GBM

Rbf

Rbf, Ras

Rbf, Ras Pten

angiogenesis

Inducible Astrocytoma Models

2 mo 4 mo 1 yr4OHT

angiogenesisnecrosisinvasion

GBMRbf, Ras Pten+/-

angiogenesisnecrosisinvasion

N N

V

800m

V

N

200m

N

200m

T121;K-RasG12D T121;K-RasG12D;Pten+/- T121;K-RasG12D;Pten-/-

GEM Astrocytoma: Human Disease Properties

50m

Qian Zhang, Chao YinR. Miller; D. Louis

Malignant Vessels in GEM-GBM

Bullitt, Lin, Van Dyke UNC

inducible cell-type-targeted events

* *** *** * * * * *** ****

******

** **

widespread induction

Scheme for Integrated Disease Analysis

**

focal induction

GEM in Clinical Translation

Cancer genomeCancer transcriptomeCancer proteome

Serum proteome

frequent aberrations

Cancer initiation and progression

genometranscriptomeproteomebiomarker discoverytarget discoverytarget validationpreclinical trials

Engineer programmed aberrationscancer patient

GEM model

In Vivo Pathway Analyses:

What are the Critical Cause-Effect Relationships in the

Context of Natural Microenvironment?

Diffuse grade II-III astrocytoma

AA

GBM

Rbf

Rbf, Ras

Rbf, Ras Pten

angiogenesis

Inducible Astrocytoma Models

2 mo 4 mo 1 yr4OHT

angiogenesisnecrosisinvasion

GBMRbf, Ras Pten+/-

angiogenesisnecrosisinvasion

Ras activates multiple effector signaling pathways

Ras

Protein KinaseCascades

Raf

MEK

ERK

P

P

P

PI3K PLC

PIP2 DAG + IP3

Ca2+PKC

PIP2 PIP3

AKT

Phospholipid and SecondMessenger Metabolism

Tiam1

GTPaseCascades

RacGDP

RacGTP

RalGEF

RalGTP

RalGDP

MLK-3

MKK4

JNK

P

P

P

PAK RalBP1

courtesy of Channing Der, UNC

Pathways to Astrocytoma

RasT121

pRb

RTKs

ERKP

E2Fs

proliferation

PI3KPIP3

AKTJNK

Pten

P

PIP2

PPKC

cell survival

metabolism,growth

motility,invasion

RasT121

pRb

RTKs

ERKP

E2Fs

proliferation

PI3KPIP3

AKTJNK

Pten

P

PIP2

PPKC

cell survival

metabolism,growth

motility,invasion

Pathways to Astrocytoma

In Vitro Pathway Analyses:

What are the likely MechanisticCause-Effect Relationships of Pathways Perturbed In Vivo?

What are the Critical Therapeutic Targets?

inducible cell-type-targeted events

* *** *** * * * * *** ****

******

** **

widespread induction

Scheme for Integrated Disease Analysis

**

focal induction

Inducible primary cultures

Induced primary cultures

primary tumor

cultures

orthotopic syngeneic transplants

inducible cell-type-targeted events

* *** *** * * * * *** ****

******

** **

widespread induction

Scheme for Integrated Disease Analysis

**

focal induction

Inducible primary cultures

Induced primary cultures

primary tumor

cultures

Orthotopic Syngeneic Transplant Model for “Rapid” Pathway/Microenvironment Assessment

Ryan Bash, Natalie Karpinich, Ryan Miller

no 1º Ab T121

4X

20X

inducible cell-type-targeted events

* *** *** * * * * *** ****

******

** ****

widespread induction

focal induction

Inducible primary cultures

Induced primary cultures

primary tumor

cultures

orthotopic syngeneic transplants

Scheme for Integrated Disease Analysis

poorly differentiated

high mitotic index

diffuse invasion

angiogenesis

pseudopalisading necrosis

High-grade Astrocytoma

RB orCDK4 orINK4a

EGFR or PDGFRPten

(K-Ras ) ?

Ras

EGFR

EGFR ligands

EGFR Signal Activation via Ras Activation

Targeting EGFR in Cancer

adapted from Ciardiello & Tortora, 2002; courtesy of David Threadgill (UNC)

and/or Etc.

Diffuse grade II-III astrocytoma

AA

GBM

Rbf

Rbf, Ras

Rbf, Ras Pten

angiogenesis

Inducible Astrocytoma Models

2 mo 4 mo 1 yr4OHT

angiogenesisnecrosisinvasion

GBMRbf, Ras Pten+/-

angiogenesisnecrosisinvasion

* *** **

* * * * * *** **

**

**

****** **

T121;K-RasG12D;EGFR-/-

Q. Zhang, D. Threadgill

EGFR+/+ EGFR+/- EGFR-/-

CD310

5

10

15

20

25

30

TR (n=11) TRE+/- (n=5) TRE-/- (n=5)

EGFR Inactivation INCREASES Severity

EGFR

PDGFR

Cortex Tu

185340 TRE+/-

Cortex Tu

185571 TRE+/-

b-actin

TUM

OR

VO

LUM

E

Pathways to Astrocytoma

RasT121

pRb

RTKs

ERKP

E2Fs

proliferation

PI3KPIP3

AKTJNK

Pten

P

PIP2

PPKC

cell survival

metabolism,growth

motility,invasion

Prog

ress

ion-

Free

Rat

e

Surv

ival

Rat

e

Chemo Alone

Chemo + Erlotinib

Chemo Alone

Chemo + Erlotinib

Months Months

Disease Models at the Frontiers of Basic and Clinical Discovery

cell biology & imaging

biochemistrygenomics

histopathology, physiology

animal imaging

0% 50%100%

complex genetics

Target discoveryDiagnosisEarly detectionMonitor treatment

Therapeutic development

Diagnosis

DiagnosisMonitor treatment

Risk assessmentTherapy response

Pharmacogenomics

Target validationTherapeutic testing HT cell-based screens

genomics,proteomics

Why Have Spontaneous Cancer Models not been Incorporated into Drug Discovery Preclinical

Assessment?

DuPont

FDA

old dogs and new tricks

requires major expertise in cancer mechanisms, pathways,GEMM, genetics, drug development and clinical care

expensive compared to xenografts

academic-private technology transfer

requires uncommon research culture

NCI-CAPRCenter for Advanced Preclinical

Studies….to facilitate the improvement of

preclinical assessment and clinical trial design for effective cancer diagnosis and treatment

drug developers

clinicians/physician scientists

basic researchscientists

drug developers

clinicians/physician scientists

basic researchscientists

NCI-CAPR

Current Interactome Projected Interactomea new paradigm for translational science

NCI-CAPRCenter for Advanced Preclinical Studies

•Predict possible outcomes/patient stratification to inform clinical trial design •Therapeutic target discovery and validation

•Biomarker/molecular signature identification via comparative (human, canine, murine) analyses

•Cancer model and “tool” mouse development for UNMET needs. •Annotated tissue/fluids/nucleic acids banks

•Consultation.•Integrated preclinical/clinical LIMS development

•Develop effective preclinical testing strategies in murine cancer models (GEM, humanized orthotopic xenografts)

•Comparative assessment of predictive power among murine cancer models •Develop molecular/cellular imaging strategies for therapeutic/diagnostic

assessment • Develop technologies to overcome barriers to scale up and throughput while limiting sacrifice in predictive power.

NCI-CAPRPreclinical

Trials R&D MacromolecularStudies

project managersproject teams

HistopathologyMolecular pathology

Sm Animal

Imaging

Mouse engineering

(SAIC and MCGP)

MolecularTechnologies

Pathophysiology

Cell Imaging

DCTD/DTPPhase 0

Tox/PK/PD

Molecular Targets(HTP cell based screens)

Chemical biology

Advanced computing

COTC(canine trials)

MMHCCNano

AllianceExtramural

Investigators PharmaFound-ations

IntramuralInvestigators

Tissue/fluids/nucleic acids Bank