In patients with solid cancers, the development of metastases is frequently associated with therapy
resistance and, in consequence, bad prognosis. Therefore, there is a burning need for new therapies
specifically preventing metastatic spread. This applies also to Ewing sarcoma, a malignant bone
cancer affecting predominantly adolescents. Metastasis is based upon marked cellular plasticity,
which allows tumor cells to reshape their cytoskeleton thus enabling invasion into neighboring tissue,
entrance into the circulation, homing to distant organs and proliferation at the metastatic site. Ewing
sarcoma growth is driven by an oncogenic fusion protein, which is expressed as consequence of
rearrangement between the EWS and FLI1 genes. The resulting fusion protein binds DNA and alters
the regulation of a large number of genes by various distinct mechanisms. Previous results suggested
that fluctuations in EWS-FLI1 expression may promote Ewing sarcoma plasticity. In addition, in
primary Ewing sarcomas, a small number of tumor cells was described, which experimentally is
associated with low EWS-FLI1 levels and increased metastatic potential. We seek to test this
hypothesis and uncover factors driving EWS-FLI1 fluctuations. We will identify EWS-FLI1 modulatory
signaling pathways and their upstream environmental stimuli, and study their influence on tumor
plasticity and metastasis. On the other hand, we will interrogate the influence of distinct EWS-FLI1
concentrations on downstream gene regulatory mechanisms, and study ensuing consequences for
tumor cell growth and metastasis. To that end, we will apply a new Ewing sarcoma model expressing
EWS-FLI1 fused to a fluorescent protein domain and tagged with a so-called degron, which allows
rapid, tunable partial or complete pharmacological EWS-FLI1 degradation. This way, we will be able
to decrease EWS-FLI1 levels gradually and determine limiting concentrations for proliferation,
invasion and migration of tumor cells, as well as characterize distinct gene sets with different EWS-
FLI1 affinity. Automated monitoring of declining fluorescence of the fusion protein will allow us to
identify in high throughput format molecular factors, gene activities and novel drugs modulating
EWS-FLI1 protein concentrations and consequently tumor cell invasion and migration. Thus, this
project will for the first time investigate causes and consequences of oncogene fluctuations in a
cancer using Ewing sarcoma as a paradigm, and define new therapeutic avenues to prevent
metastasis of solid tumors.