SERUM DEPRIVATION INDUCES EWING SARCOMA CELLS TO ADOPT A MORE INVASIVE PHENOTYPE AND TO UP REGULATE RHO-MKL TRANSCRIPTIONAL SIGNALING Merlin Airik, Melanie Krook, Christopher Scannell, Andrew Haak, Richard R. Neubig, Elizabeth R. Lawlor Departments of Pediatrics, Pathology and Pharmacology University of Michigan, Ann Arbor, Michigan, USA

Merlin Airik, PhD Post-doctoral fellow Melanie Krook Graduate student Chris Scannell Graduate student Andrew Haak Graduate student Richard R. Neubig, MD, PhD Dept of Pharmacology Sunbeam Foundation, Russell G. Adderley Endowment UM SARC Sarcoma SPORE (NIH), Liddy Shriver Sarcoma Initiative

Ewing Sarcoma: Metastasis Can be an early or late event Many patients with localized disease relapse at metastatic sites months and even years following an initial clinical remission Phenotypic plasticity of cancer cells is well-characterized in epithelial malignancies: cells transition back and forth between metastatic and non-metastatic phenotypes Epithelial to mesenchymal transition (EMT) is implicated in the initiation of metastasis of epithelial malignancies Does phenotypic plasticity exist in Ewing sarcoma? What molecular pathways drive metastasis of Ewing sarcoma cells?

Serum deprivation induces cell cycle arrest & morphologic change in Ewing sarcoma cells 10%FBS 0%FBS A673 CHLA9 10%0% 10%0% A. C. B. D. A673 CHLA9 + 10% x 2.5hr

A.B. Serum deprived cells exhibit enhanced motility and migration 10% 0% 10%0% C.D. xCelligence CIM-plate migration assay24hr end-point migration assay

Rho-Transcriptional Signaling Rho-GTPases contribute to cell movement and cancer metastasis – Change so actin cytoskeleton directly in cytoplasm – Activation of downstream transcriptional signaling via translocation of MKL/MRTF to nucleus Rho-MKL transcriptional axis is required for breast cancer and melanoma metastasis (Medjkane, Nature Cell Biol 2009) – MYL9 and MYH9 implicated as metastasis effectors Nature Reviews Molecular Cell Biology 11, (May 2010) MKL

Serum starved Ewing sarcoma cells show dramatic changes in arrangement of the actin cytoskeleton A. B. 10% FBS0% FBS DAPI Phalloidin

Nuclear localization of MKL & upregulation of MYL9 in serum starved Ewing sarcoma cells MYL9 10%0%+10% x24h Normalized ratio MYL9MYH9 10% 0% Normalized ratio A. C. B.

Inhibition of the Rho-MKL transcriptional axis restores morphology & actin cytoskeleton MKL CCG-1423 CCG-1423: a small molecule inhibitor of RhoA transcriptional signaling Evelyn,…Neubig. Molecular Cancer Therapy 2007 A.B. 10% FBS 0% FBS + 800nM CCG-1423 DAPI Phalloidin

0h 24h 12h A673 SFM A673 SFM + 800nM CCG-1423 Pharmacologic inhibition of the Rho-MKL transcriptional axis blocks Ewing sarcoma cell motility

Summary Phenotypic plasticity exists in Ewing sarcoma cells in vitro Serum starvation induces a rapid and reversible switch to a more migratory phenotype The migratory phenotype is associated with increased nuclear localization of transcriptional co-activator MKL and increased expression of the metastasis-associated MKL target gene MYL9 Small molecule inhibitors of Rho-MKL transcriptional axis block Ewing cell migration Metastatic Relapse Activation of Rho-MKL signaling Opportunity for metastasis prevention? Growth factor deprivation Diminished blood supply? Rapid tumor growth? Altered microenvironment? Post-chemotherapy?