resistance to classical and targeted anti-cancer drugs

Resistance to classical and targeted anti-cancer drugs: insights from a mouse model for hereditary

breast cancer

Sven Rottenberg, Netherlands Cancer Institute, [email protected]

Immune cells Stroma Angiogenesis WT cells

Blood

lymph

OncogenesTumor suppressor genes

Metastasis

Primary tumor

Normal cells

Factors controlling tumorigenesis

Reproducing sporadic humancancer in mice

controlled switching of multiple mutations

controlled switching of a single mutation

Recombinase-mediated gene switching

TS protein

TS protein1 2 3

1

3

1 3

2

2

+

+ Cre

loxP loxP

4 x pApromoter oncogene pAIVS

+ Cre

loxP loxP

promoter oncogene pAIVS

oncoprotein

Conditional mouse tumor models at theNetherlands Cancer Institute

Cre viruses

Adeno-CreRetro-CreRetro-CreERT2

Lenti-Cre

Cre transgenics

Actin-CreIRBP-CreK14-CreR26-CreERT2

WAP-CreP0-CrePOMC-Cre

ConditionalTSG knockouts

Atr F

Brca1 F

Brca2 F

Ecad F

Ink4a/Arf F

Nf2 F

p53 F

Pten F

Rb F

Conditionaltransgenics

LSL-KrasV12G

+

Conditionalmouse tumormodels

Skin cancer

Breast cancer

Prostate cancer

NSCLC

SCLC

Mesothelioma

Glioblastoma

Medulloblastoma

Pituitary cancer

=

Characteristics KCre, Brca1F/F, p53F/F mouse model

• mean latency of mammary tumor development of 213 days

• stochastic tumor development

• superficial tumors, accessible for measurement and sampling

• carcinoma of undifferentiated (‘basal’) cells with similar features as BRCA1-associated and basal-like breast cancer in humans

• expression of basal markers (Keratin 5, 6, 14, p63)

• genomic instability

(J. Jonkers group)

Tn

orthotopic transplantation of tumor fragments (1mm)

into wt animalstumor size

~200mm3, therapy start (MTD)

doxorubicin

docetaxel

topotecan

T*ncon

T*ndox-res

T*ndoce-res

T*ntopo-res

generation of dox/doce/topo-resistant tumors, repeated

sensitivity to cisplatin

cisplatin

T*ncismean latency

of 4 weeks

Brca1/p53-deficient tumors can be transplanted orthotopically

0

200

400

600

800

1000

1200

0 2 4 6 8 10 12

days

tum

or s

ize

in %

Growth of mammary tumors in K14-Cre, Brca1flox/flox, p53flox/flox mice

X-rayscisplatin

MMCdoxorubicin

UVPolycyclic aromatic

hydrocarbons

Replication errors

Alkylatingagents

X-raysRadicalsAlkylating

agents

HRNHEJ

NER MMR AGT BER

DSBBulky adduct

CPD

MismatchInsertionDeletion Alkylguanine

UracilAbasic site

SSB

Intervention with doxorubicin

Intervention with the MTD of doxorubicin

days

tum

or s

ize

in %

Mechanisms of drug resistance

(mainly based on in vitro studies):

C C*

X ATP ADP

metabolism

DNA damagerepair

C

C C*

drug targets

efflux

influx inhibition of apoptosis/

senescence

XC

compartmentalization

Pgp/MDR1BCRPMRP1

number of upregulated genes: 45Δ = 0.865false discovery rate: 1.95%

Significance analysis of microarrays (SAM) of 13 doxorubicin-resistant tumors in comparison to

untreated tumors

Rottenberg et al., PNAS 2007

doxorubicin-resistant tumors

RT-MLPA analysis of 13 doxorubicin-resistant mouse mammary tumors

Mdr1a

1 2 3 4 5 6 7 8 9 10 11 12 130

10

2575

125

ratio

resi

stan

t/sen

sitiv

e

Mdr1b

1 2 3 4 5 6 7 8 9 10 11 12 130

10

2575

125

Mrp1

1 2 3 4 5 6 7 8 9 10 11 12 130

10

20


Mdr1a

duodenum

ileum

caec

umco

lonliv

erad

renal

kidney

splee

n T1

T1 dox r

es T2

T2 dox r

es T3

T3 dox r

es T4

T4 dox r

es T5

T5 dox r

es T6

T6 dox r

es T7

T7 dox r

es T8

T8 dox r

es T9

T9 dox r

esT10

T10 dox r

es 1

T11 dox r

es 2

T12 dox r

es 3

T13 dox r

es 4

0

2

4

610

20

30

40

50

ratio

Mdr

1a /

actin

β

Mdr1b

duodenum

ileum

caec

umco

lonliv

erad

renal

kidney

splee

n T1

T1 dox r

es T2

T2 dox r

es T3

T3 dox r

es T4

T4 dox r

es T5

T5 dox r

es T6

T6 dox r

es T7

T7 dox r

es T8

T8 dox r

es T9

T9 dox r

esT10

T10 dox r

es 1

T10 dox r

es 2

T10 dox r

es 3

T10 dox r

es 4

0

2

4

610

20

30

40

50

ratio

Mdr

1a /

actin

β

A moderate increase in Mdr1a/1b expression associates with in vivo resistance

doxorubicin-resistant tumor

untreated tumor

normal tissue

0100200300400500600700800900

0 10 20 30 40

days

tum

or s

ize

in %

untreated control doxorubicintariquidar doxorubicin+tariquidar

T*23 dox res 2

0100200300400500600700800900

0 10 20 30 40days

tum

or s

ize

in %


T*23 dox res 3

0100200300400500600700800900

0 10 20 30 40

days

tum

or s

ize

in %


T7 dox res

Drug resistance can be reversed by the 3rd

generation P-glycoprotein inhibitor tariquidar

Mdr1a: 17-foldMdr1b: 46-fold



X-rayscisplatin

MMCdoxorubicin


hydrocarbons

Replication errors

Alkylatingagents

X-raysAlkylating

agents topotecan

HRNHEJ

NER MMR AGT BER

DSBBulky adduct

CPD


UracilAbasic site

SSB

Intervention with topotecan

days

tum

or s

ize

in %

0100200300400500600700800900

0 50 100 1500

100200300400500600700800900

0 50 100 1500

100200300400500600700800900

0 50 100 150

= untreated matched control tumors

= topotecan MTD treated tumors

= 1 treatment course of 4mg/kg topotecan IP on 5 consecutive days

Growth responses of Brca1-/-;p53-/- tumors to the MTD of topotecan

= ratio of absolute M-values resistant/sensitive (p53 reference pool)

= RT-MLPA values: ratio resistant/sensitive (internal reference Actinβ)

Comparison of MEEBO versus RT-MLPA data

ratio

resi

stan

t/sen

sitiv

e0

1

2

3

4

5

6

7

8

9

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17

tumors

SAM analysis for 17 Brca1-/-;p53-/- tumors: topotecan-resistant versus matched untreated controls

δ-value = 0.72

sign. genes = 237

false sign. genes = 20.6

Bcrp1

Increased transcript levels of the ABC drug efflux transporter Bcrp1 in topotecan-resistant tumors

Confirmation of increased Bcrp1 levels by q RT-PCR

ABCG2/BCRP1 content in topotecan-resistant tumors

topotecan-resistant

α-BCRP1 (BXP-53)

control

tumor 1 tumor 2 tumor 3

Ablation of Bcrp1 in the mouse tumor model

xKCre;Brca1F/F;p53F/F

Bcrp1-/-

KCre;Brca1F/F;p53F/F;Bcrp1-/-

orthotopictransplantation of tumor

fragment into wt animals

MTD of topotecan

KCre;Brca1F/F;p53F/Forthotopic

transplantation of tumor fragment into wt

animals

MTD of topotecan

difference ?

Bcrp1 proficient

Bcrp1 deficient

0

20

40

60

80

100

120

0 50 100 150 200 250

days

surv

ival

%Bcrp1-/-,Brca1-/-,p53-/- Brca1-/-,p53-/-untreated untreated

n=9 n=9n=11

n=11

p<0.003

Increased survival in response to topotecan in animals bearing Bcrp1-/- tumors

X-raysCisplatin

MMC


hydrocarbons

Replication errors

Alkylatingagents

HRNHEJ

NER MMR AGT BER

DSBBulky adduct

CPD


UracilAbasic site

SSB

(PARP inhibition)(cisplatin, carboplatin)

X-raysAlkylating

agents topotecan

HR-deficient cell targeting by inhibition of poly(ADP-ribose) polymerase1 (PARP1)

Intervention with the PARP1 inhibitor AZD2281 (KuDOS) in Brca1/p53-deficient tumors

AZD2281 28d

0

2

4

6

8

10

0 20 40 60 80 100days

RTV

T1 T2 T3 T4 T5 T6 T7

AZD2281 100d

0

2

4

6

8

10

0 100 200 300 400days

RTV

T1 T2 T3 T4 T5 T6 T7

untreated control

0

2

4

6

8

10

0 5 10days

RTV

T1 T2 T3 T4 T5T6 T7 T8 T9

0

20

40

60

80

100

120

0 0.5 2 6 24hours

051015202530354045

average % Parp1 activityaverage conc AZD2281 (nM)


0

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400

days

over

all s

urvi

val %

untreated control AZD2281 100d

Intervention with AZD2281+cisplatincisplatin 6mg/kg i.v., AZD2281 50mg/kg i.p.

n=9 n=7


n=9

n=7

n=9

n=7

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400

days

over

all s

urvi

val %

untreated control AZD2281 100d cisplatin

n=9 n=9 n=7


n=9

n=7

n=90

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400days

over

all s

urvi

val %

untreated control AZD2281 100d

cisplatin cisplatin+AZD2281 100d

n=9 n=7n=9

n=7

Response of Brca/p53-deficient tumors to docetaxel

Can we predict the outcome beforehand?

Why are predictive signatures elusive, whereasprognostic signatures flourish?

Chemotherapy response can be prevented by small changesin only one of many proteins:- the drug target (HER2, Herceptin; cABL, Gleevec; Topo II,

doxorubicin)- drug disposition

- failing drug activation- increased drug inactivation- decreased cellular drug accumulation

- reduced damage response (reactivation BRCA1/2).

These alterations may not be easily picked up by geneexpression profiling (sensitivity; posttranscriptionalmodifications) or proteomics (sensitivity), given the highdegree of genetic variation in the patients and their tumors.

Why use the mouse tumors to generatepredictive tests?

1. No patient genetic heterogeneity (inbred mice);little tumor heterogeneity (they all start with the same geneticallyengineered mutations).

2. All forms of secondary resistance can be deduced bycomparing tumor samples before and after resistance arises. Provides inventory of possible mechanisms andtools to detect them (directly or indirectly).

3. Differences in the initial response to drug can be correlatedwith differences in gene expression levels, protein levels,DNA alterations (deep sequencing).

4. Insertional mutagenesis may be used to tag genes that conferacquired drug resistance

The mouse as surrogate patient

Treatment

Follow up

Diagnostics

RadiologyMolecular imaging

PathologyMolecular diagnostics

RadiotherapyChemotherapy

Targeted therapyCombination therapy

Response monitoringDetection of residual disease

Detection of tumor recurrenciesDetection of therapy resistance

Acknowledgements

Piet BorstMarina Pajic

Ariena KersbergenJanneke Jaspers

Serge Zander

Jos JonkersXiaoling Liu

Eline van der Burg Jan SchellensSjoerd Rodenhuis

Jonkers groupMedical oncology

Borst group

Animal facility

Anders O. NygrenJan Schouten

MRC-Holland

many

KuDOS/AstraZenecaNiall Martin

Mark O’ConnorAaron Cranston

Alan Lau

Diagnostic oncologyOlaf van Tellingen

Susan BatesTito Fojo

NIH

Microarray facilityRon KerkhovenMike Heimerikx

Arno Velds

resistance to classical and targeted anti-cancer drugs

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