11th Annual CTOS Meeting November 19–21, 2005
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Transcript of 11th Annual CTOS Meeting November 19–21, 2005
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.