Abstract 5837: Phosphoproteomic landscape of sarcoma: novel subgroups and actionable targets
Authors: Fleuren Emmy D. , Vlenterie Myrella , Graaf Winette van der , Hillebrandt-Roeffen Melissa , Cebeci Sabri , Ven Amy van de , Flucke Uta , Wu Jianmin , Versleijen-Jonkers Yvonne , Daly Roger 
Published: July 2017

Abstract
Sarcomas comprise a heterogeneous group of malignant tumors of the connective tissue, and disproportionally affect the young. Despite intensive, multimodal treatments, survival is still limited and accompanied by many early and late side effects. In contrast to advances in carcinomas, specific targetable driver mutations have not or rarely been found in sarcoma and targeted therapies have met with disappointing clinical results, stressing the need for an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. We utilized a mass-spectrometry (MS)-based phosphoproteomics screening approach to profile the largest and most heterogeneous set of sarcoma cell lines (n=20) to date, including both adult (gastrointestinal stromal tumor (GIST) and angiosarcoma (AS)) and multiple pediatric/adolescent and young adult (AYA; synovial sarcoma (SS), Ewing sarcoma (ES) and rhabdomyosarcoma (RMS)) sarcomas, as well as soft-tissue sarcomas (STS) and bone sarcomas. We specifically included cell lines and SS and AS subtypes that were never phosphoprofiled before, to reveal completely novel molecular insights into sarcoma pathology. Validations were performed in vitro, in vivo, and on patient tumor material. Our unique tyrosine phosphorylation data revealed a novel subclassification of sarcoma, and showed that particularly the pediatric/AYA sarcoma models did not cluster within their histological subgroup. Particular tyrosine kinases exhibited enhanced phosphorylation in particular subtypes, and we identified driver kinases with outlier levels of activation. ALK was identified as a completely novel and unexpected driver in the Aska-SS SS cell line, which was explained by an underlying ALK translocation. Functional ALK dependency was confirmed with in vitro proliferation assays, showing high sensitivity for the ALK inhibitor TAE684 (IC50 26±3 nM) and the ALK/MET inhibitor crizotinib (IC50 46±5 nM). Patient specimens confirmed a clinical role for ALK in SS, with ALK immunopositivity in 14% (6/43) of patients and one 16y old patient harboring an ALK translocation, present in both her primary tumor as in 4 metastases. Other SS cell lines and sarcoma subtypes exhibited multiple activated tyrosine kinases, providing a rationale for combined targeting approaches. Indeed, high MET and PDGFRα phosphorylation in Yamato-SS SS cells predicted sensitivity to crizotinib and pazopanib (PDGFR) in vitro and in vivo. The clinical significance was again confirmed in a cohort of SS patients, in which MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with co-expression in 56%. Altogether, this is the first study to provide a deep insight into activated networks and molecular clustering patterns in sarcoma. Our integrated approach systematically identified and validated ALK and MET as novel and actionable drivers in SS.
Citation Format: Emmy D. Fleuren, Myrella Vlenterie, Winette van der Graaf, Melissa Hillebrandt-Roeffen, Sabri Cebeci, Amy van de Ven, Uta Flucke, Jianmin Wu, Yvonne Versleijen-Jonkers, Roger Daly. Phosphoproteomic landscape of sarcoma: novel subgroups and actionable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5837. doi:10.1158/1538-7445.AM2017-5837