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……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
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 ) ?
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
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