Basisvorlesung (BVO) Modelling cancer using transgenic ... · Modelling Cancer Using Transgenic...

Center of Physiology and Pharmacology

Modelling Cancer Using Transgenic Mice and Beyond

Basisvorlesung (BVO)


Transgenic Modelsin Cancer Research

Hanahan and Weinberg. Cell 2011

Hanahan and Weinberg. Cell 2000

Cancer Cells can be Considered as a Darwinian System


1mutation

Cancer is a Complex and Heterogeneous Disease

1000 mutations

>500.000 mutations

100 mutations

Vogelstein et al., Science 2013

Alexandrov et al., Nature 2013

Stewart et al., Nature 2012


140 genetic “drivers”

2-8 “drivers” per tumor


Different Animals Models to Study Cancer

D. D. melanogaster rerio M. musculus R. norvegicus

Lower complexity Higher complexity

Close to humans (85% homology)

Test therapeutic drugs

Modeling human diseases

Fast and cheap

Far away from humans

Time consuming

Genetic tools available

Genetic tools available




Forward genetics

Reverse genetics

Chemical mutagenesis (ENU)

Insertional mutagenesis (transposons, retrovirus, lentivirus, etc.)

Loss of function Gain of function/overexpression

“Humanization of mice” Chromosomal translocations, Inversions, etc. Point mutations

Mario R. Capecchi

Sir Martin J. Evans

Oliver Smithies

"for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells“

Nobel Prize 2007

Embryonic stem cells

Modeling Cancer: A Transgenic Mouse Genetic Tool Box



Modeling Cancer: Forward GeneticsFrom Phenotype to Genotype

N-ethyl-N-nitrosoureaENU

RetrovirusLentivirusEtc.

Appleby & Ramsdell Nature Reviews Immunology 2003



Forward Genetics: Searching for the Needle in the Haystack

Problems: Time consumingExpensiveENU induce multiple mutations, confusing resultsValidation need it!

Forward Genetics:Very useful in simpler organisms!



“Collaborative Cross” Project: Clever Forward Genetics

360 inbreed mice, in the future 1000



Trai

t, e.

g. c

ance

r sus

cept

ibili

ty

“Collaborative Cross”: All Inbreed Mice are Equal, but Some More than Others

Inbreed mice



Reverse Genetics in Mice

From Genotype to Phenotype

e.g. Transgenics

Genomic modification:

Random integration Target integration



Modifying the Mouse Genome by Random Integration

Pronuclear injection


Albumin c-Myc pA

Random integration: a Simple Approach to Study Tumorigenesis


Pronuclear Injection

Founders

Expressing transgenic line

Tumor model (e.g. drug screening)

Basic tumor biology, e.g. inactivate a tumor gene candidate


Random integration: a Simple but Inefficient Approach to Study Tumorigenesis


Albumin c-MYC pA Pronuclear Injection

X

No expression Expression No expression

XExpression is random

Miss(ectopic)expression

Un-physiological levels

Several Tg lines need to be generated


Chromatin effects Chromatin effects

Expression vector

Random integration: a Simple but Inefficient Approach Due to Chromatin effects



Improved Random Integration Using Bacterial Artificial Chromosomes (BACs)


200 kbAlbumin BAC

22 kb Albumin locus

cMYC pA

*ATG

Large vectorsNot affected by surrounding chromatinFaithfull expression


Inducible systems in cancer: Rever


Reversible Inducible System: The “TET system (Random/Targeted Integration)


Tet OFF

Tet ONmCMV

mCMV

mCMV


The “TET system: a Two Mice System



The “TET” System to Study Cancer and Oncogene Addiction


cMyc

Oncogene addition

cMYC Tumor regression?

Shachaf et al., Nature 2004

PLAP tTA pA

TetO cMyc pA

dox


Modifying the Mouse Genome by Targeted Integration

1. Loss of function (knock-out)

2. Tissue specific knock-out

3. Gain of function (knock-in)



Targeted Integration Needs HomologsRecombination and Embryonic Stem Cells


Targeting construct

Endogenous locus

Targeted locus


Targeted Integration Needs HomologousRecombination and Embryonic Stem Cells



Loss of Function, e.g. p53 Knock-out

1 2 3 4

1 4

1 4

p53 endogenous locus

p53 “targeting construct”

p53 Knock-out

OsteosarcomaAdapted from Donehower et al., Nature 1992


Li–Fraumeni syndrome


Tissue Specific knock-outs: The Cre/loxP System



The Cre/loxP System: A Two Mice System



Conditional Mutagenesis: e.g. Prostate Specific Pten Knock-out

1 2 3 4

“Floxed” Pten mouse

Probasin Cre

1 2 3 4

1 4loxP loxP

Prostate expression of Cre recombinase

Mouse 1

Mouse 2

intercrossed

“Floxed” Pten

Null Pten

Cre recombinase in prostate

Prostate specific deletion of Pten

Normal prostate Prostate Pten deletionAdapted from Wang et al., Cancer cell, 2003



“Silenced” KrasG12D mutation

loxP loxP

Mouse 1

G12D

Exon 1

* 2 G12D* 2

G12D* 2

Cre recombinase

+Adenovirus-mediated delivery ofthe Cre recombinase in the Lung

+ Cre recombinasein the lung

Gain of Function Point Mutations: KrasG12D Induction of Lung tumors

Jackson et al., Genes and Dev. 2001



In vivo Killing Cells and Cancer



In vivo Killing Cells: Diphtheria Toxin System




How Can We Exploit Transgenic Mice in Cancer Research?

Tumor samples

Analysis of somatic aberrations

Engineeredtransgenic mice

Gene function, Drug testing



How Can We Exploit Transgenic Mice in Cancer Research?Example: Role of Stat3 in Kras mutated lung cancer


Lung Cancer Facts


Kaiser J., Science 2009

Stat3?

Estimated Cancer Deaths in the US in 2015

Lung & bronchus 28%Prostate 9%Colon & rectum 8%Pancreas 7%Liver & 5%intrahepatic bile ductLeukemia 5%Esophagus 4%Urinary bladder 4%Non‐Hodgkin 4%lymphomaKidney & renal pelvis 3%Other 23 %

26% Lung and bronchus15% Breast9% Colon & rectum7% Pancreas

3% Liver & intrahepatic bile duct

5% Ovary

4% Leukemia4% Uterine corpus3% Non‐Hodgkin lymphoma

2% Brain & other nervous system22% Other

Men 312,150

Women 277,280

©2015, American Cancer Society, Inc.

The Cancer Genome Atlas 2014


Why to Study STAT3 in Lung Cancer?


Looyenega et al., 2012Kluge et al., 2009Weerashinghe et al., 2007Gao et al.,2007Haura et al, 2005

Role of STAT3 in lung tumorigenesis in vivo? A transgenic mouse approach

STAT3 is considered to be an oncogene in lung cancer (in vitro studies)

STAT3 inhibitors against solid tumors are being developed



Lung induced KrasG12D mutation

G12D* 2 G12D* 2

18

KrasG12D

Lung induced KrasG12D mutation

Lung Stat3 knock-out (Dr. Poli)

KrasG12D

Stat3 KO21

Kras Kras-S3KO

Lung Tumors More or Less Lung Tumors ?

Mouse Model to Study the Role of Stat3 in K-RasG12D Induced Lung Cancer in vivo



STAT3 is a Tumor Suppressor in K-RasG12D Induced Lung Cancer

Kras

Kras-S3KO

Kra

sK

ras-

S3K

O



STAT3 Could be a Tumor Suppressor in Mutated K-RAS Human Lung Tumors


Validated Stat3 as tumor suppressor in Kras mutated lung tumors

Kras mutated lung human tumors have reduce levels of Stat3

Kras mutations could predict resistance to Stat3 blockers in lung tumors

Conclusion

Kras mutated Kras wt

Stat3 inhibitors

No Response Response

Combination of transgenic mice and analysis of human tumors : Powerful approach to study cancer!



Hanahan and Weinberg Cell 2000

4-8 Key mutations


Normal cell

Metastasis

Of Mice, Men and Tumors

2 mutations: Medium

3 mutations: Challenging

1 mutation: Simple

Metastasis?

X

Generating transgenic mice with five or more mutations in one step!

X

Breeding



One-Step Generation of Mice with Five Mutations: CRISPR/Cas9-Mediated Gene Editing

CRISPR/Cas9-mediated gene editing

Induction of targeted DNA double strand breaks

“Adaptive immune system” of Streptococcus pyogenes

Sánchez-Rivera and Jacks. Nat. Rev. Cancer. 2015

Wang et al. Cell. 2013

edc

ba

edcba Metastasis ?

Multiple Gene targeting in mouse ES Cells

edcba

Tumor drivers



The CRISPR/Cas9 System: A Revolution in Biology

Multiple knock-outs (loss of function)

Multiple knockings (point mutations)

Chromosomal inversions/deletions/duplications

Gene repression

Gene activation

In vitro and in vivo ! Gene correction/gene therapy

Whole genome screenings

Num

ber o

f pub

licat

ions

0100200300400500600700800900


The CRISPR/Cas9 System: A “flox” Mouse in One Month



The CRISPR/Cas9 System:In vivo Induced Chromosomal Rearranges


Blasco et al., Cell Rep. 2014


The CRISPR/Cas9 System:In vivo Induced loss of Tumor Suppressors


Xue et al., Nature 2014



Yoshimi et al., Nature Communications 2016

The CRISPR/Cas9 System:Mouse humanization: knock-in of a human 200 kb BAC



The CRISPR/Cas9 System: A Revolution in Biology

E. Charpentier

F. Zhang

J. Doudna

G. Church

“I’ve seen two huge developments since I’ve been in science: CRISPR andPCR,” says John Schimenti, a geneticist at Cornell University in Ithaca.

The CRISPR/Cas System: Precise Molecular Scissors to Fight Cancer?

Taken from “Insect Genetic Technologies Research”



Costa et al. 2007

EGFR

GTC AAG ATC ACA GAT TTT GGG CTG GCC AAA CTG V K I T D F G L A K L 858

GTC AAG ATC ACA GAT TTT GGG CGG GCC AAA CTG V K I T D F G R* A K L 858

Cas9 PAM NGG

Cas9

Normal cell

Cancer cell

The CRISPR/Cas System: Precise Molecular Scissors to Fight Cancer in 15-20 Years?


Thank you very much for your attention!

Basisvorlesung (BVO) Modelling cancer using transgenic ... · Modelling Cancer Using Transgenic...

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