Modeling, Characterizing, and Targeting Aggressive, STAG2-mutated Ewing Sarcoma

Loss of function mutations of STAG2 occur in 10-15% of Ewing sarcoma (ES) patients. This is clinically significant, as loss of STAG2 is associated with poor clinical outcomes. STAG2 encodes a protein that is central to the cohesin complex, a highly conserved chromosome-associated multi-subunit protein complex which mediates chromosome segregation during mitosis. STAG2 is also implicated in DNA repair and gene expression but exactly how its loss affects ES tumorigenesis and biology is unclear. Recent studies have shown that tumors containing STAG2 loss of function mutations are highly susceptible to inhibition of its paralog STAG1, representing a synthetic lethal interaction. In ES lines, loss of STAG1 abrogated sister chromatid cohesion in STAG2 mutated, but not in STAG2 wild-type ES cell lines, leading to defective cell division and apoptosis. However, targeting STAG1 is technically challenging and no STAG1-specific drugs exist. Here, we propose studies to better characterize and target the aggressive, STAG2-mutated subset of ES, as outlined in two specific aims: AIM 1. Clarify the role of STAG2 loss in the biology of Ewing sarcoma through CRISPR-Cas9 genomic editing. We will use CRISPR-Cas9 genomic editing to model STAG2 loss in ES lines; use the resulting isogenic pairs to evaluate growth rate and cellular behavior, perform copy number analysis, gene expression analysis, promoter methylation analysis, and EWSR1-FLI1 location analysis by chromatin immunoprecipitation. The generation of isogenic ES cell lines differing only in STAG2 status using CRISPR-Cas9 will provide a robust system to study the biology of STAG2 as well as an important resource for future CRISPR/Cas9 and/or drug screens to discover novel STAG2-associated vulnerabilities. We will use the CRISPR-Cas9 targeting for STAG2 knockout in the ES cell lines CHP100, ES-6, CHLA-32, and RD-ES. AIM 2. Therapeutic targeting of STAG1 as a synthetic lethal vulnerability in STAG2-mutated Ewing sarcoma using next generation antisense oligonucleotides (ASO). We will design and evaluate next generation ASOs for the direct targeting of STAG1 mRNA. The synthetic lethality of STAG1 and STAG2 provides a potential therapeutic window to target ES lacking STAG2. Advances in ASO-based technology have led to a new generation of ASOs with greatly improved pharmacokinetic and systemic pharmacodynamic properties. As several ASO-based therapeutics have recently obtained FDA approval, this class of agents is now accepted to have a path to the clinic. To target STAG1 with ASOs, we will undertake a tiling approach for the targeting of STAG1 mRNA, through the synthesis of overlapping gapmer ASOs. We have successfully used such a Gapmer ASO strategy to target other genes in vitro and in xenograft models. Successful completion of the work proposed in this Aim 2 could open a novel therapeutic avenue for the treatment of STAG2-mutated ES, the most aggressive molecular subset of this sarcoma.