Characterization of the function of a novel lncRNA in Ewing’s sarcoma using genomics and proteomics

Ewing’s sarcoma is a devastating disease that affects children and young adults. Current therapies for this disease are highly toxic and lead to significant side effects.  Thus, new approaches to therapy are urgently needed.  The most common driving oncogenic event in Ewing’s sarcoma is a translocation known as EWS-FLI-1 in which the RNA binding protein EWS is translocated with the ets transcription factor FLI-1. Recent data from our laboratory demonstrates that one consequence of EWS-FLI-1 expression is the upregulation of several long non-coding RNAs (lncRNAs). As lncRNAs are increasingly recognized as regulators of many cellular processes, this observation may provide new avenues for identifying novel targets for treatment of Ewing’s sarcoma. Functional studies demonstrate that one lncRNA,  lnc277, plays an important role in mediating EWS-FLI-1 driven oncogenesis.  Knock-down of lnc277 is deleterious to Ewing’s sarcoma cells and decreases growth of xenografts in vivo. In this proposal, we will elucidate the molecular mechanisms through which lnc277 regulates Ewing’s sarcoma pathogenesis.  This will be done using three approaches.  First, we will identify proteins that bind to lnc277.  Using protein arrays, we have already identified several proteins that bind lnc277 in vitro.  We will compliment these studies using mass spectrometry to validate true lnc277 interacting partners in vivo.  Promising protein interactions will be further analyzed using biochemical and functional methods.  Second, we will use RNAseq to identify genome-wide changes that occur as a consequence of lnc277 knock-down. The effects of lnc277 knock-down will be compared to the effects of EWS-FLI-1 knockdown to elucidate the effects of lnc277 on the genetic network established by expression of the translocation.  These studies will help elucidate the complex mechanism through which EWS-FLI-1 leads to oncogenesis.  Perhaps more importantly, they may identify novel roles for lncRNAs in oncogenesis and they may have important implications for other sarcomas in which ets family translocations occur.  Identification of lnc277 binding proteins may also point to novel potential therapeutic opportunities for treatment of Ewing’s sarcoma.