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Presentation Abstract

Abstract Number:

22

PresentationTitle:

Cyclin E amplification, a novel mechanism of resistance to trastuzumab in HER2amplified breast cancer

Presentation Time:

Sunday, Apr 18, 2010, 2:25 PM - 2:40 PM

Location: Room 103, Washington Convention Center

AuthorBlock:

Maurizio Scaltriti1, Pieter Eichhorn1, Magui Gili1, Marta Guzman1, Claudia Aura1, José Jimenez1, Ludmila Prudkin1, Simon R. Green2, Javier Cortés1, Sarat Chandarlapaty3, Sabine Mai4, Clifford A. Hudis3, Neal Rosen3, José Baselga1. 1Vall d'Hebron Univ. Hospital, Barcelona, Spain;2Cyclacel, Dundee, United Kingdom; 3Memorial Sloan-Kettering Cancer Center, New York, NY; 4Manitoba Institute of Cell Biology, Winnipeg, MB, Canada

Abstract Body:

HER2 amplification/overexpression occurs in 20% of breast cancers and is associated with poor prognosis. Therapeutic agents against HER2, including monoclonal antibodies and tyrosine kinase inhibitors, have shown to improve the survival of these patients. However, primary and acquired resistance to these agents is a major barrier to the effective treatment of this disease.Aiming to identify the molecular pathways responsible for resistance to anti-HER2 therapy we have established HER2 positive breast cancer cell lines refractory to the anti-proliferative effects of the therapeutic antibody trastuzumab. We have studied their acquired genetic aberrations byperforming gene expression microarray and genome single nucleotide polymorphisms analyses. In trastuzumab resistant cells we identified an increase in copy number variation in locus 19q12. This chromosome region comprises seven genes, among them cyclin E1 (CCNE1). We further confirmed Cyclin E over-expression in trastuzumab resistant cells by western blot.To test the hypothesis that increased cyclin E expression could play a role in the acquisition of trastuzumab resistance we have demonstrated that ablation of cyclin E expression by siRNArestores trastuzumab sensitivity in HER2 positive resistant cells. Furthermore, we have determined that trastuzumab resistant cells are highly sensitive to the specific CDK inhibitor seliciclib and its more potent derivative Cmpd. 5 (Cyclacel) both in vitro and in vivo. In addition, we have analyzed a cohort of HER2 positive breast cancer patients treated with trastuzumab-based therapy and identified a proportion of patients refractory to trastuzumab that harbor amplification of the cyclin E gene. Our results suggest that cyclin E amplification decreases trastuzumab sensitivity and may provide the rationale to test the efficacy of CDK2 inhibitors in breast cancer patients with cyclin E amplification that escaped trastuzumab therapy.

American Association for Cancer Research615 Chestnut St. 17th Floor

Philadelphia, PA 19106

Page 1 of 1Abstract Print View

Cyclin E amplification, a novel mechanism of resistance to trastuzumab in HER2 amplified breast cancer

Maurizio Scaltriti, PhDExperimental Therapeutics ProgramVall d’Hebron Institute of Oncology

Barcelona, Spain

Disclosures:

Nothing to disclose

-Trastuzumab

Other HERs HER2

Ligands

Cell Cycle, SurvivalProliferation

RAS/RAF/MEK/ERK PI3K/AKT/mTORp27

Mechanisms of trastuzumab activity

Immune Response(ADCC)

Mechanisms of resistance to trastuzumab

•Hyperactivation of the PI3K PathwayNagata et al, 2004 Cancer Cell Berns et al, 2007 Cancer Cell Eichhorn et al, 2008 Cancer Res

•Presence of truncated forms of HER2 (p95HER2) Scaltriti et al, 2007 JNCI

•Loss of HER2 expression Mittendorf et al, 2009 Clin Cancer Res

Confirmed mechanisms in the clinic

BT474

Trastuzumab (nM)5 100 50 100

Rel

ativ

e P

rolif

erat

ion

BT474R do not respond to the antiproliferative effects of trastuzumab in vitro

Selection of trastuzumab resistant BT474 (BT474R) cells

Trastuzumab

18 months

BT474


Rel

ativ

e P

rolif

erat

ion



BT474R


Rel

ativ

e P

rolif

erat

ion

Trastuzumab

18 months

(IC50>1uM)

BT474


Rel

ativ

e P

rolif

erat

ion



BT474R cells do not exhibit previously known mechanisms of trastuzumab resistance

Characterization of BT474R cells

BT474 BT474R

G1SG2

Tras (nM)0 01 10 100 1 10 100

100

90

80

70

Per

cent

age

of c

ells

0

50

60

Tras (100nM)+- ++ - -BT474 BT474R BT474R2

pHER2

p27

pIGFR

MAPK

Akt

actin

HER2

pAkt

pMAPK

IGFR

Cyclin D

BT474 BT474R

G1SG2

Tras (nM)0 01 10 100 1 10 100

100

90

80

70

Per

cent

age

of c

ells

0

50

60

Tras (100nM)+- ++ - -BT474 BT474R BT474R2

pHER2

p27

pIGFR

MAPK

Akt

actin

HER2

pAkt

pMAPK

IGFR

Cyclin D

BT474R cells do not exhibit previously known mechanisms of trastuzumab resistance


BT474R cells present cyclin E amplification and overexpression

CNV2CNV1 CNV1 (Chr.19)UQCRFS1POP4PLEKHF1C19orf12CCNE1C19orf2ZNF536

CNV2 (Chr.14)

GALCGPR65

Tras (100nM)

+- ++ - -BT474 BT474R BT474R2

Cyclin E

actin

BT474 BT474R

IHC: Cyclin E

MKN7

pRB(S780)

RB

p27


Cyclin E expression modulates trastuzumab sensitivity

Cyclin E/CDK2 addiction of BT474R cells

Cyclin E

tubulin

siCCNE1- - + +

BT4

74

BT4

74

BT4

74R

BT4

74R

siCCNE1sicontrol + Tras

sicontrol siCCNE1+ Tras

Rel

ativ

e A

BS

570

nm

20

40

80

100

60

0

BT474R

BT474

Tras 100nMcontrol

20

40

80

100

60

0

Rel

ativ

e A

BS

570

nm

BT474 BT474CE1 BT474CE2

IP:Cyclin EWB:Cyclin E

BT4

74

BT4

74 C

E1

BT4

74 C

E2Overexpression

Knockdown

Cmpd5 Ratio to CDK2

CDK2 0.005

CDK5 0.021 4

CDK9 0.026 5

CDK3 0.029 6

LKB1 0.063 13

CDK7 0.193 39

CDK4 0.232 46

CLK2 0.252 50

CLK1 0.549 110

CDK1 0.578 116

Trascontrol

Abs

orba

nce

at 5

70nM

BT-474R

BT-474

Cdk2i T+C

CDK2 inhibition decreases proliferation of BT474R cells

CDK2 inhibitor: Cmpd 5 (Cyclacel) is trisubstituted purine derivative of seliciclib. IC50 �300nM

BT-474 CEBT-474

Trascontrol

Abs

orba

nce

at 5

70nM

Cdk2i T+C


CDK2 inhibition leads to G1 arrest and increases cell death of BT474R cells

Tras 10 nM- -+ - ++ -+Cdk2i 300 nM- - + ++ +- -

G1

SG2

100

90

80

70

Per

cent

age

of c

ells

0

50

60

25

BT474 BT474R

-++

- -+

+-

BT474RBT474

Cdk2i(300nM)Tras(100nM)

Sub G1

Annexin V

BT474RBT474

Cdk2i(300nM)+- -- -+ +

+Tras(100nM)

05

30

2025

1015

% C

ells

% C

ells

25

0

15

10

5

20


Cdk2i(300nM)+-++

- -

BT474 BT474R

++- - -

- -+ ++

Tras(100nM)

cleaved Parp

HER2

pRB(S807)

p27

pRB(S780)

Rb

pHER2

CDK2 inhibition decreases pRb and promotes cleavage of Parp


CDK2 inhibition reduces tumor growth of BT474R-derived xenografts


Cyclin E positive: FISH ratio � 1.5 and IHC nuclear staining � 30 H-Score

Cyclin E amplification/overexpression in HER2+ patients

Patient 1: CCNE1 WT

Patient 2: CCNE1 Amp

DAPICCNE1 probe

Cyclin E amplification/overexpression in HER2+ patientsand resistance to trastuzumab

*Breslow test

Cohort of patients with cyclin E + (n=16)

No ciclin E (n=8)High ciclin E (n=13)

Median PFS: 4 months vs 13 months

4 m 13 m

Months

PFS

pro

babi

lity

• Metastatic HER2 + breast cancer• Cyclin E amplification/overexpression• Treated with 1st-line trastuzumab-containing therapy• Compared to similar cohort of patients cyclin E – (n=8)

Blue: no cyclin E amplif/nuclear stainingGreen: cyclin E amplif/nuclear staining

P value .046*

�

��

��

��

��

��

��

��

��

��

��

�� !"12.5

68.8

87.5

31.3

Pat

ient

s (%

)

P value .030

Clinical benefit rate

Clinical benefit rate: CR+PR+SD>6 months.

* nuclear staining � 30 H score.

Cyclin E amplification/overexpression in HER2+ patientsand resistance to trastuzumab

No cyclin E

High cyclin E

Conclusions

• BT474 cells with acquired resistance to trastuzumab presentamplification and overexpression of cyclin E

• Cyclin E overexpression modulates sensitivity to trastuzumab

• BT474R cells are addicted to cyclin E-dependent CDK2 activity

• Amplification/overexpression (nuclear staining) of cyclin E correlates with lower therapeutic response to trastuzumab-based therapy

• CDK2 inhibition could be a valid strategy to target cyclin Eamplified/overexpressing HER2 positive breast cancer

Acknowledgments

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