(P 229) CHARACTERIZING THE MICROSATELLITE-BINDING FUNCTION OF EWS-FLI AND ETV6 IN EWING SARCOMA

Objective: Ewing sarcoma is an aggressive bone cancer most frequently diagnosed in adolescents and young adults. Molecularly, Ewing sarcoma is characterized by a chimeric E26 transformation specific (ETS) family transcription factor, EWS-FLI, which deregulates gene expression to drive tumorigenesis. Previous studies by our lab and others have found that, unlike canonical ETS factors, EWS-FLI binds DNA at repetitive GGAA-microsatellites that are scattered throughout the human genome. EWS-FLI acts as a “pioneer factor”, which alters the heterochromatin state of GGAA microsatellites, transforming them into de novo promoters and enhancers for downstream oncogenes. In this study, we sought to determine the structural features of EWS-FLI that are necessary and sufficient for microsatellite binding in Ewing sarcoma.

Methods: We performed CUT& Tag and RNA sequencing to compare the DNA binding and expression profiles of 3 canonical ETS factors, ETV6, ELF2, and ELK4, to that of EWS-FLI in A673 cells. A knockdown/rescue approach was used to assess the effects of mutations in the ETV6 pointed domain on its ability to bind microsatellites. Additionally, we generated a series of ETS domain fusion genes to determine if forced polymerization of ETS factors is sufficient for microsatellite binding.

Results: We found that ETV6, which forms polymers on DNA via its self-associating PNT domain, co-localizes with EWS-FLI at both microsatellites and high-affinity ETS motifs (5′-GGAA/T-3′) in Ewing sarcoma cells. ETV6 binding to microsatellites is dependent upon EWS-FLI, which is consistent with EWS-FLI’s pioneering function. In contrast, ELF2 and ELK4, which are monomeric, only bind high-affinity ETS sites. We found that mutation of the ETV6 PNT domain abrogates DNA binding, but only has a modest effect on gene expression in Ewing sarcoma cells. We are currently testing a series of ETV6-ETS and EWS-ETS fusion proteins for their ability to bind microsatellites in vivo.

Conclusion: Our finding that ETV6 localizes to microsatellites while ELK4 and ELF2 do not suggests that a shared structural feature of ETV6 and EWS-FLI enables microsatellite binding. Both the PNT domain of ETV6 and the EWS domain of EWS-FLI have been shown to polymerize or aggregate on DNA, suggesting that the polymerization of an ETS factor may be sufficient to bind microsatellites. This hypothesis is currently being tested via a series of engineered ETV6-ETS and EWS-ETS fusion proteins. Our findings will reveal the structural characteristics of EWS-FLI that enable aberrant microsatellite binding and subsequent pioneering activity in Ewing sarcoma. This knowledge is critical for finding new and effective ways to target EWS-FLI.