Abstract 2780: Tyro3 and Axl receptors tyrosine kinase as potential therapeutic targets in leiomyosarcoma
Authors: Dantas-Barbosa Carmela , Loarer François Le , Mendiola Marta , Treilleux Isabelle , Chibon Frederic , Sayadi Hyba E. , Coindre Jean Michel , Alberti Laurent , Blay Jean-Yves 
Published: May 2015

Abstract
Leiomyosarcoma (LMS) represents 15% of adult sarcomas. Once metastatic, curative options are limited and innovative strategies are needed. TYRO3 and AXL proto-oncogenes belong to TAM family (Tyro3/Axl/Mer) of tyrosine kinase receptors, with oncogenic properties that are often overexpressed or activated in various malignancies. Using kinexus phosphoprotein microarrays (KAM-1.1) and western blot analyses, we evidenced that Tyro3 and Axl are overexpressed in LMS along with Gas6, their ligand. Interestingly LMS samples harbouring high levels of Gas6 and its receptors also presented AKT phosphorylation on S473 residue keeping with previous studies that highlighted AKT- mTOR activation in leiomyosarcomas. Based on these premises, we asked whether Tyro3-Axl axis may drive LMS proliferation and could represent potential targets in these tumors. The overexpression of Tyro3 was found to be associated with a specific miRNA signature, notably with an overexpression of miR22 in LMS tumors. Furthermore Tyro3 and Axl were found overexpressed in a panel of 6 LMS cell lines (SK-LMS-1 from ATCC and IB112, IB118, IB133, IB134 and IB136 from Institut Bergonié), compared with normal human smooth muscle. Tyro3 or Axl knockdown via shRNA retroviral vector reduced cell proliferation by up to 50 and 64% respectively and decreased soft agar colony-forming ability. Crizotinib and foretinib are two tyrosine kinase inhibitors (TKI) targeting Met and Axl. Treatment of LMS cell lines with both TKIs resulted in a significant increase in cell death, with IC50 values ranging from 3.6 to 14.6µM and 0.7 to 1.8µM, respectively. Annexin V and PI staining revealed that cell death was mainly due to apoptosis. Flow cytometric cell cycle analysis demonstrates that both TKIs altered cell cycle phase distribution. A sub-G1 phase accumulation was observed for one of the cell lines; meanwhile the other cell lines presented a G2 arrest and 4n chromosomes accumulation accompanied by an increase in cell and nucleus sizes. Only foretinib induced nuclear fragmentation consistent with late apoptosis. Pharmacological inhibition also reduced migration and colony formation in soft agar. Drug combinations with an AKT inhibitor led to significant increase in growth inhibition in all tested cell lines. Crizotinib and foretinib reduced activation of Tyro3 or Axl-mediated downstream signaling including MAPK/ERK, PI3K/AKT along with an inhibition of Axl phosphorylation in a dose dependent manner. By immunoprecipitation we observed also a reduction in Tyro3 phosphorylation, following exposure to both agents, showing that Tyro3 is a new target for those drugs. IHC analysis of Tyro3, Axl and Gas6 expression in a cohort of LMS patient is ongoing. In vivo analysis wil be performed. In conclusion these results strongly suggest that Tyro3 and Axl are relevant therapeutic targets for LMS treatment and will be investigated in a proof of concept clinical trial.
Citation Format: Carmela Dantas-Barbosa, François Le Loarer, Marta Mendiola, Isabelle Treilleux, Frederic Chibon, Hyba El Sayadi, Jean Michel Coindre, Laurent Alberti, Jean-Yves Blay. Tyro3 and Axl receptors tyrosine kinase as potential therapeutic targets in leiomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2014-2780