Mentor Name: Shasha Chong

Ewing sarcoma is an aggressive pediatric bone and soft tissue cancer driven by the fusion transcription factor (TF) EWS::FLI1 composed of the intrinsically disordered, low-complexity domain (LCD) of EWSR1 and the DNA-binding domain of FLI1. EWS::FLI1 has been shown to form local, high-concentration hubs at GGAA microsatellites via multivalent LCD-LCD interactions that concentrate transcriptional machinery at target genes, thereby driving oncogenesis. Also, recent studies have implicated O-GlcNAcylation, a dynamic glycosylation post-translational modification (PTM) that targets serine and threonine residues, in the regulation of TF hubs; further, an in vitro study of the EWSR1 LCD has shown that its synthetic O-GlcNAcylation can decrease EWSR1 LCD-LCD interaction strength, resulting in altered physical properties such as decreased phase separation propensity and viscosity. Our lab is actively investigating how O-GlcNAcylation affects the physical properties of endogenous EWS::FLI1 in patient-derived cell lines.

We hypothesize that increasing O-GlcNAcylation will reduce EWS::FLI1's ability to drive transcription of its target genes by weakening the multivalent interactions required to maintain hubs and recruit transcriptional machinery. To test this hyposthesis, we will link changes in EWS::FLI1 O-GlcNAcylation and physical properties to changes in EWS::FLI1-driven transcription. We will chemically manipulate O-GlcNAcylation levels in the cell using established inhibitors for the sole pair of enzymes responsible for cycling O-GlcNAc on and off: OSMI, which reduces O-GlcNAcylation by inhibiting O-GlcNAc transferase, and TMG, which increases O-GlcNAcylation by inhibiting O-GlcNAcase. We will measure changes in the transcriptional function of EWS::FLI1 using two complementary approaches. We will use a luciferase reporter assay in which a reporter gene driven by a GGAA microsatellite will quantify EWS::FLI1-driven transcription in an overexpression system. We will also use RT-qPCR to measure changes in transcription of known endogenous target genes of EWS::FLI1. Combining these results with our single-molecule imaging experiments that quantify the dynamic interactions of endogenous EWS::FLI1, we will obtain a comprehensive picture of how O-GlcNAcylation alters the physical properties of EWS::FLI and changes its transcription functions.

Overall, this project will establish a mechanistic link between O-GlcNAcylation and oncogenic gene regulation in Ewing sarcoma. By understanding how O-GlcNAcylation affects EWS::FLI1 function, this work may help guide future therapeutic strategies to target transcriptional dysregulation in pediatric cancers.The proposed project leverages a clinically implemented, clinical-grade targeted sequencing assay (LBSeq4Kids) applied to AH-derived DNA to characterize RB1 first and second hits in retinoblastoma patients. The study will survey RB1 mutation detection and characterize the spectrum of pathogenic alterations observed in AH samples, including single-nucleotide variants, insertions and deletions, and copy number alterations. By examining these genomic features across a clinical cohort, this work aims to define how aqueous humor liquid biopsy captures RB1 two-hit inactivation and variant diversity. Overall, this project provides a foundational framework for understanding RB1 genomic alterations detectable through aqueous humor liquid biopsy and supports ongoing efforts to integrate clinically derived genomic data into the molecular characterization and clinical management of retinoblastoma.