1. Connective Tissue Oncology Society, October 18, 2014
Sensitization of leiomyosarcoma cell lines to chemotherapy by inhibition of BCL family members
Marieke A. de Graaff1, Marije A. de Rooij1, Fréderic Chibon2, Adrian Marino-Enriquez3, Jonathan A. Fletcher3, Anne-Marie Cleton-Jansen1, Judith Bovée1
1Pathology, Leiden University Medical Center, Leiden, ZH, Netherlands; 2Molecular Pathology, Institut Bergonie, Bordeaux, France; 3Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States
Connective Tissue Oncology Society, October 18, 2014

2. Disclosures
No disclosures.
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3. Leiomyosarcoma
Aggressive soft tissue sarcoma, derived from smooth muscle cells
Complex genetic background
Sub classification:
Soft tissue
Uterine
Intimasarcoma
Radiotherapy and chemotherapy resistance
Desmin
Age 5th - 6th decade, 16% of all sarcomas
Surgery most effective
Need for new treatment strategies
Abraham et al, J Bone Joint Surg, 2012
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4. Expression of BCL family members
High protein expression of BCL family members (Bcl-w, Bcl-xL and Bcl-2) in soft tissue leiomyosarcomas
Bcl-2 Leiomyosarcoma
Bcl-2 Uterine leiomyoma
Bcl-xL Leiomyosarcoma
Bcl-xL Uterine leiomyoma
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5. ABT-737 is a BH3 mimetic inhibitor
Top: in the basal state, Bax and Bak proteins are monomeric and BH3-only proteins are bound by Bcl-xL. Cytochrome c is retained in the mitochondria. Bottom: in cancer cells, ABT-737 promotes pore formation and apoptotic cell death by binding to Bcl-xL, causing it to dissociate from BH3-only proteins. The unsequestered BH3-only proteins induce oligomerization of Bax and Bak, leading to large pore formation. Cytochrome c enters the cytosol through the pore and triggers caspase activation, leading to cell death.
Can ABT-737 sensitize leiomyosarcoma cell lines for doxorubicin treatment?
Cotter, Nature Reviews, 2009; Swanson, Nature Neuroscience, 2012
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6. Panel of 4 leiomyosarcoma cell lines
LMS04
LMS05
IP566
JA192
Western blots
Expression of Bcl-xl, Bcl-2
LMS04
LMS05
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7. Effect of ABT-737 versus doxorubicin
Minimal effect of ABT-737 alone on cell viability after 72 hr treatment
Doxorubicin affects cell viability of leiomyosarcoma cells
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8. Combination of ABT-737 and doxorubicin
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9. Combinational index of both drugs
Table 1: Combinational index (CI) according to the Chou & Talalay method for synergistic relationships per concentration analysed.  
Dox
0.05 µM
JA192
LMS-04
0.1µM
IP566
0.2 µM
0.5µM
LMS-05
0.7µM
0.9 µM
1 µM
ABT-737
1.35
0.80
0.89
0.56
0.67
0.27
0.44
5µM
1.21 
0.98
1.05
0.63
0.31
0.54
0.09
0.12
10µM
0.78
1.17
1.38
0.88
0.16
0.45
0.03
0.04
Combinational index
Blue: >0.80
Green:
Light red:
Red: <0.20
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10. Discussion Unique panel of leiomyosarcoma cell lines
High expression of anti-apoptotic proteins in leiomyosarcomas
BH3 mimetic drug increases leiomyosarcoma sensitivity to doxorubicin in vitro
No/minimal effect of treatment with ABT-737 alone
Synergy of both drugs observed in all 4 investigated cell lines
Future directions
Evaluate down regulation of BCL family proteins after treatment with Western blot
Investigate apoptosis
Next research goals
-investigate apoptosis
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11. Acknowledgements Marije de Rooij Inge Briaire-de Bruijn
Pauline Wijers-Koster
René Zwartbol
Yvonne de Jong
Johnny Suijker
Anne-Marie Cleton-Jansen
Judith Bovée
Fréderic Chibon
Adrian Marino-Enriquez
Jonathan Fletcher
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12. 11/18/2018

13. Characteristics of the 4 cell lines
Table 3: The characteristics of the 4 cell lines used.
Cell lines
Passage number
Diagnosis
Patient
TP53
PTEN
LMS-04
P45
LMS III$
F/54
Homozygous deletion
No expression
LMS-05
P34
LMS II ST
M/76
Wild type: exons 4-9
Expression
IP566
P21 -
Deletion: exon 2 and 3
JA192
P22
Wild type: Exon 1-11
LMS: Leiomyosarcoma, $ Retroperitoneal metastasis from uterine LMS. ST: soft tissue.
-: not known
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15. Material and methods 4 leiomyosarcoma cell lines Dose response curves
LMS-04, LMS-05, JA192 and IP566
Dose response curves
Cell viability assay (Alamar blue)
24h, 48h and 72h
µM ABT-737 or doxorubicin
Treatment with both ABT-737 and doxorubicin
ABT-737 (24h)  doxorubicin (48h)
ABT-737 and doxorubicin (72 h)
doxorubicin (24h)  ABT-737 (48h)
Western blot
Bcl-2, Bcl-XL and Mcl-1
Cleaved PARP
PTEN
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16. Percentage difference between cells treated with only doxorubicin and both doxorubicin and ABT-737
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17. Mcl-1 protein expression after doxorubicin treatment
Reduction Mcl-1 prostate cancer cells
 Sensitivity depends on the ability of doxorubicin to decrease Mcl-1 expression
LMS-05 the highest reduction in Mcl-1
4 Parrondo R. et al. PeerJ. 2013
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18. Conclusions ABT-737 does sensitize leiomyosarcoma for doxorubicin
Almost no effect single agent
All 4 cell lines show synergy combinational treatment
IP566 and JA192 slightly lower Bcl-2 and Bcl-XL expression
Other cell lines no clear differences
Likely sensitivity depends on the ability of doxorubicin to decrease the expression of Mcl-1
Most sensitive cells in this study
PTEN expression
wild type TP53 gene
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19. Clinical phase I studies of ABT-737
Thrombocytopenia
grade III and IV in 2 out of 5 patients.
marrow compensation platelet counts20
Diarrhea (40%), vomiting (36%), nausea (34%) fatigue (34%) 20,24
24 Leena Gandhi D. et al. J. clin. Oncol. 2011
20Rudin C.M. et al.Clin. Cancer Res. 2012
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