12 th Annual CTOS Meeting 2006 SESSION 11 Molecular biology for the patients: Define new targets? Predict Outcome? Moderators: Ole Steen Nielsen & Irene Andrulis 22 Posters – all worth seeing! Selected 4 posters: –# 526 Ueda et al, WT1 in STS –# 557 Hannay et al, VEGF in STS –# 578 Keschman et al, PI3K/AKT –# 579 Shor et al, SRC kinase

12 th Annual CTOS Meeting 2006 INTRODUCTION: WT1 gene, first identified from Wilms’ tumor patients, encodes a zinc finger transcription factor, regulating the genes related to cell differentiation, proliferation and apoptosis, such as PDGF- α, IGF-II, c-myc and bcl-2. WT1 was originally categorized as a tumor-suppressor gene, but recently has been emphasized of its oncogenic properties. MATERIALS & METHODS: Tissue samples; 52 primary soft-tissue sarcoma (STS) frozen samples & 13 normal soft-tissue samples (control) To analyze WT1 mRNA expression level using quantitative real-time RT-PCR & univariate/multivariate surival analysis according to various clinicopathological factors including WT1 mRNA expression level. AIM of the STUDY: To evaluate the prognostic implication of WT1 mRNA expression in patients with STS. Tissue samples 52 primary soft-tissue sarcoma frozen samples # 526: Ueda et al: Prognostic significance of WT1 expression in STS

12 th Annual CTOS Meeting 2006 RESULTS: The levels of WT1 mRNA expression in various STSs were significantly higher than those in normal soft-tissue samples. No significant correlation was observed between WT1 expression level and various clinico- pathological factors. The disease-specific survival of patients with high WT1 levels was found to be significantly worse than those with low WT1 levels (p=0.0182). Multivariate analysis further indicated that WT1 expression level is a significant prognostic factor (RR 2.6; p=0.0488), independent of other prognostic factors including age, histological grade, and distant metastasis at presentation. CONCLUSIONS: WT1 mRNA is frequently overexpressed in various types of STS, and its expression level is a significant prognostic indicator for STS patients. These results strongly suggest that WT1 can be a candidate for a potent molecular marker to predict patient prognosis, and a molecular target for tumor-specific therapy against STS.

12 th Annual CTOS Meeting 2006 Introduction, materials & methods, and aim of study To better elucidate the role of VEGF165 in soft tissue sarcoma (STS) growth, metastasis and chemoresistance we… –Stably transfected leiomyosarcoma (SKLMS-1) and rhabdomyosarcoma (RD) human STS cell lines with VEGF 165 to generate a panel of SK-SE, and RD-SE daughter clones. –Assessed tumor growth, invasion, metastasis, angiogenesis, and chemoresistance phenotypes of parental and daughter lines in vivo in SCID mice, and HUVEC stimulation in vitro. –Assessed the effect of VEGFR2 inhibition with DC101 anti-VEGFR2 mAb on STS growth, angiogenesis, metastasis, and response to doxorubicin in parental and daughter lines in vivo, and HUVEC stimulation in vitro. # 557 Hannay et al: Targeting soft tissue sarcoma-associated endothelial cell chemoresistance via VEGFR inhibition: A strategy to prevent tumor growth and metastasis

12 th Annual CTOS Meeting 2006 Results, discussion & conclusions We found that: –VEGF165 transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. –Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 mAb enhanced doxorubicin chemoresponse; inhibiting tumor growth and decreased pulmonary metastases without overt toxicity. –Combined therapy reduced microvessel counts while increasing vessel maturation index. –VEGF overexpression did not impact on the sarcoma cells per-se, however, CM from VEGF tranfectants caused increased endothelial cell proliferation, migration, and chemoresistance. –DC101 induced EC sensitivity to doxorubicin and suppressed the activity of MMPs secreted by EC. We conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor associated endothelial cells. STS growth and metastasis can be interrupted by combined low dose doxorubicin and anti- VEGFR2, a strategy that attacks STS associated EC.

12 th Annual CTOS Meeting 2006 Introduction and Aim of Study The PI3K-Akt signaling pathway regulates cell survival, proliferation, angiogenesis, translation, and apoptosis, and is known to be altered in sarcomas LY acts on the ATP-binding site of the PI3K enzyme, which inhibits the PI3K-Akt binding complex LY has been used successfully to induce apoptosis in cancer cells, and this has previously been ascribed to its PI3K-Akt blocking activity The aim of this study was to investigate the effects of LY on cell cycle proliferation, cell growth, and apoptosis in leiomyosarcoma, osteosarcoma, Ewing’s sarcoma, and high-grade undifferentiated sarcoma, and to determine the pathway by which this inhibitor induces apoptosis in these cells # 578: Keschman et al: LY induces apoptosis in sarcoma independent of the PI3K-AKT pathway

12 th Annual CTOS Meeting 2006 Results and Conclusions LY and wortmannin both inhibit Akt phosphorylation, however LY induces apoptosis in sarcoma cells much more effectively, in both a time and dose-dependent manner After 24 hour treatment with LY294002, cells were arrested in G 0 -G 1 phase, according to flow cytometry analysis At higher doses and after 48 hours, cells treated with LY were killed by apoptosis, as evidenced by PARP cleavage, TUNEL staining, and DNA fragmentation ELISA assay These results indicate that LY induces apoptosis in leiomyosarcoma, osteosarcoma, Ewing’s sarcoma, and high-grade undifferentiated sarcoma by some mechanism independent of the PI3K-Akt pathway Figure 1. LY induces apoptosis more effectively than wortmannin, as evidenced by DNA fragmentation of cell nuclei in SKLMS-1 leiomyosarcoma cells.

12 th Annual CTOS Meeting 2006 Introduction and Aim of Study Sarcomas frequently possess abnormalities in growth factor receptor signaling pathways, including PDGF-R, c-KIT, c-MET and IGF1-R. A common point of signal convergence downstream of many of these pathways is Src kinase, the first oncoprotein identified as a solid tumor virus, Rous sarcoma virus, which induces sarcomas in chickens. Src is regulated by growth factors, cytokines, cell adhesion and antigen receptor activation, and is involved in controlling a myriad of fundamental cellular processes including cell proliferation, migration, invasion and survival. Several studies have demonstrated the activity of dasatinib, a Src kinase inhibitor, in epithelial tumor cell lines and early phase clinical trials have established the safety and efficacy of dasatinib for treatment of imatinib-resistant chronic myelogenous leukemia. However, the responses and mechanisms of action of dasatinib in mesenchymally-derived tumor cell lines have not been described previously. The aim of this study was to evaluate the activity of dasatinib in sarcoma cell lines and determine the role for Src kinase in the survival of sarcoma cell lines. # 579 Shor et al: Action of the SRC kinase inhibitor Dasatinib on human sarcoma cell lines

12 th Annual CTOS Meeting 2006 Results and Conclusions Src kinase is activated in sarcoma tissue and cell lines. Dasatinib inhibits Src activation and downstream signaling, cell motility and invasion in sarcoma cell lines and induces apoptosis in bone sarcoma cell lines. Furthermore, siRNA to c-Src induces apoptosis in a subset of bone sarcoma cell lines. Taken together, our results suggest that dasatinib will provide clinical benefit to soft tissue and other sarcomas by preventing metastasis, which may be further augmented in bone sarcomas by induction of apoptosis. Table 1. Summary of response to dasatinib in human sarcoma cell lines.