Investigating mechanistic roles of a novel onco-fusion-driven transcript isoform, named DLG2η3, in Ewing sarcoma

Ewing sarcoma (EwS) is an aggressive type of bone cancer that disproportionately affects children and young adults. EwS is characterized by a chromosomal translocation that produces a de novo oncofusion between members of the FET-ETS gene families. The two most commonly identified gene fusions in EwS are EWSR1::FLI1 (EWS::FLI1) and EWS::ERG, in which their respective proteins bind to GGAA repeat sequences to induce or repress enhancers acting as an aberrant transcription factor and rewiring transcriptional programs essential for tumor development. Understanding the function of dysregulated genes is crucial for developing targeted treatments in EwS. I recently identified that a novel truncated isoform of DLG2 is highly expressed in EwS with no prior characterization in cancer. I identified that EwS is among the highest DLG2 expressing cancers, and more specifically, that EwS expresses a truncated isoform of DLG2, identified as DLG2η3, likely through a de novo enhancer bound by EWS::FLI1. Based on H3K27Ac data, I observed that the DLG2η3 gene locus is the third strongest super enhancer only when EWS::FLI1 is expressed, supporting a direct role of EWS::FLI1 in inducing DLG2η3 gene expression. Additionally, I showed that DLG2η3 mRNA and protein can be altered depending on the availability of free intracellular iron. I then showed that DLG2η3 mRNA contains two likely iron responsive elements, suggesting an iron-dependent translational control mechanism. I wanted to identify if DLG2η3 contributed to the oncogenic phenotype of EwS. Here, I showed that DLG2η3-depleted EwS cells are less capable of forming 3D growth in both in vitro and in vivo model systems. To determine the oncogenic role of DLG2η3, I assessed the relationship between DLG2η3, iron, and survival in EwS cells. Not only did DLG2η3-depleted EwS cells show an increase in mitochondrial superoxides, but there was also an apparent increase in lipid peroxide levels and cell death under ambient and high iron conditions in comparison to control cells. Addition of ferrostatin-1 entirely rescued the observed phenotype, suggesting that DLG2η3 depleted cells are entering ferroptosis at higher rates. Collectively, this data profiles the pro-tumorigenic role of DLG2η3 and its potential downstream involvement in ferroptosis protection in Ewing sarcoma.