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

Disclosures No disclosures. 11/18/2018

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 11/18/2018

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 11/18/2018

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 11/18/2018

Panel of 4 leiomyosarcoma cell lines LMS04 LMS05 IP566 JA192 Western blots Expression of Bcl-xl, Bcl-2 LMS04 LMS05 11/18/2018

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 11/18/2018

Combination of ABT-737 and doxorubicin 11/18/2018

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: 0.50-0.80 Light red: 0.20-0.50 Red: <0.20 11/18/2018

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 11/18/2018

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 11/18/2018

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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 11/18/2018

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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 0.01-10µM 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 11/18/2018

Percentage difference between cells treated with only doxorubicin and both doxorubicin and ABT-737 11/18/2018

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 11/18/2018

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 11/18/2018

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 11/18/2018