Mutated p53 correlates with extreme sensitivity to Yondelis in low passaged cell lines explanted from chemonaive

sarcoma patients

11th Annual CTOS Meeting November 19–21, 2005

V. Moneo, B.G. Serrelde, M. Taron, J. Fominaya, J.F. Martinez-Leal, C. Blanco-Aparicio, L. Romero, M. Sánchez-Beato,J.C Cigudosa,

J.C Tercero, M.A. Piris, J. Jimeno, A. Carnero

CNIO Dpt. Of Molecular Biology, Madrid, Spain

PharmaMar R&D, Colmenar Viejo, Madrid, Spain

Proposed mechanisms of action ofET-743

Interference DNA repair pathways

Interaction with transcription factors and DNA binding proteins

Binding to the minor groove of DNA

ET-743 (Yondelis)

Ecteinascidia turbinata

Objective Remissions and Tumor Control in patients with Progressive STS Treated with Yondelis

PFS-6 CR+PR

Yondelis Long Lasting Objective Response in a patient with Anthracycline Resistant High Grade Liposarcoma

Courtesy of Dr G Demetri

Generation and molecular characterization of the low passage sarcoma cell lines

Tumorsample

Disaggregate Serialpassage Immortalisation

Cytogeneticcharacterisation

Molecularcharacterisation

Pharmacologicalcharacterisation

CELL LINESFOR PRECLINICAL

STUDIES

XENOGRAFT MODELFOR PRECLINICAL

VALIDATION

Tumorsample

Disaggregate Serialpassage Immortalisation

Cytogeneticcharacterisation

Molecularcharacterisation

Pharmacologicalcharacterisation

CELL LINESFOR PRECLINICAL

STUDIES

XENOGRAFT MODELFOR PRECLINICAL

VALIDATION

- In vitro Cytotoxicity assays- MTT assay 96h- Clonogenic assay

- Q-RT-PCR: mRNA expression- Western: protein expression- full gene p-53 sequencing

96 h 96 h

1 CNI O CE RABDOMYOSARCOMA >100 R >300 R

2 CNI O BN FI BROHI STI OCI TOMA >100 R >300 R

3 CNI O BC MPNST >100 R >300 R

4 CNI O BB MPNST >100 R 232 R

5 CNI O BM HI BERNOMA 10 R >300 R

6 CNI O BK LI POSARCOMA 9 R >300 R

7 CNI O AY LEI OMYOSARCOMA 9 R 44 S

8 CNI O AZ FI BROUS TUMOR 5 R/ S 14 S

9 CNI O BJ OSTEOSARCOMA 2 R >300 R

10 A673 EWI NG’s SARCOMA 1 Cut off 50 Cut off

11 CNI O AW LI POSARCOMA 0,7 S 45 S

12 CNI O AX LI POSARCOMA 0,7 S 44 S

13 SW872 LI POSARCOMA 0,5 S >300 R

14 CNI O AA LEI OMYOSARCOMA 0,4 S 21,5 S

15 CNI O BP OSTEOSARCOMA 0,3 S >300 R

16 CNI O BF OSTEOSARCOMA 0,3 S 15 S

17 CNI O BG MI XOI D FI BROSARCOMA 0,3 S 22 S

18 SAOS-2 OSTEOSARCOMA 0,11 S >300 R

19 CNI O BI GI ST 0,1 S 50 S

20 1455 LI POSARCOMA 0,1 S >300 R

ET-743

response

Doxo nM Doxo

responseNº

CELL LI NE

(code)TUMOUR ORI GI N

ET-743 nM

Sarcoma primary cell line panelET-743 vs Doxorubicin IC50

Molecular characterization sarcoma cell lines

Apaf-1

p73

PTEN

p85

-actin

E-cad

APC

-catMSH-2

MLH-1

-actin

p 21cip1

p 27kip1

p16INK4a

p14ARF

p15INK4b

RS

0306

CA

1010

SW

872

SR

2103

/A

SR

2103

/B

SR

2205

SR

2410

SR

2910

A67

3

LS

0904

SR

0312

SR

0406

RS

0306

CA

1010

SW

872

SR

2103

/1A

SR

2103

/1B

SR

2205

SR

2410

SR

2910

A67

3

LS

0904

SR

0312

SR

0406

RS SSS S S S SR RR RS SSS S S S SR RR

mRNA expression

Cyc D1

cdk4

Tubulina

p27

MDM2

MUT p53

RS

0306

CA

1010

SW

872

SR

2103

/A

SR

2103

/B

SR

2205

SR

2410

SR

2910

A67

3

LS

0904

SR

0312

SR

0406

RS SSS S S S SR RR

S

A. Carnero, AARC-04

Protein expression

Molecular characterization sarcoma cell lines

Sarcoma primary cell line panelp53 mutational status

96 h 96 h

1 CNI O CE RABDOMYOSARCOMA >100 R >300 R WT

2 CNI O BN FI BROHI STI OCI TOMA >100 R >300 R WT MDM2 Amplif

3 CNI O BC MPNST >100 R >300 R WT MDM2 Amplif

4 CNI O BB MPNST >100 R 232 R WT

5 CNI O BM HI BERNOMA 10 R >300 R WT MDM2 Amplif

6 CNI O BK LI POSARCOMA 9 R >300 R WT MDM2 Amplif

7 CNI O AY LEI OMYOSARCOMA 9 R 44 S WT

8 CNI O AZ FI BROUS TUMOR 5 R/ S 14 S WT

9 CNI O BJ OSTEOSARCOMA 2 R >300 R WT MDM2 Amplif

10 A673 EWI NG’s SARCOMA 1 Cut off 50 Cut off WT MDM2 Amplif

11 CNI O AW LI POSARCOMA 0,7 S 45 S 273H

12 CNI O AX LI POSARCOMA 0,7 S 44 S 273H

13 SW872 LI POSARCOMA 0,5 S >300 R 251N

14 CNI O AA LEI OMYOSARCOMA 0,4 S 21,5 S 273H

15 CNI O BP OSTEOSARCOMA 0,3 S >300 R R72P, R175H

16 CNI O BF OSTEOSARCOMA 0,3 S 15 S 273H

17 CNI O BG MI XOI D FI BROSARCOMA 0,3 S 22 S DEL/ 273H *

18 SAOS-2 OSTEOSARCOMA 0,11 S >300 R DEL

19 CNI O BI GI ST 0,1 S 50 S 273H

20 1455 LI POSARCOMA 0,1 S >300 R R72P

ET-743

response

Doxo nM Doxo

responseP53 mut/ MDM2 overexNº

CELL LI NE

(code)TUMOUR ORI GI N

ET-743 nM

Mut. p53Mut. p53

-2 -1 0 1 2 3 4 5 630

40

50

60

70

80

90

100

110

120P53+/+

ET-743 (log pM)

P53-/-

4.92

16.63

Expt 1

3.22

11.30

Expt 2

5.05P53 null

15.89P53 wt

Expt 3HCT116

IC50 (nM)

Impact of p53 knock-out on cytotoxicity of Yondelis

Conclusions

• This primary sarcoma panel has established a set of cell lines with differential extreme sensitivity to Yondelis (IC50 < 1nM).Such concentration range is reacheable in plasma well below the recommended dose.

• Yondelis lacks complete cross-resistance with doxorubicin.

• The extreme sensitivity to Yondelis is correlated with mp53 genotype(p<0.01).

• This data might be of clinical relevance since it can provide a tool to characterize the response/resistance to Yondelis in patients.

• Correlative studies in tumor samples from sarcoma patients treated with Yondelis are ongoing.