RNA-Based Therapeutics Targeting the Oncogenic Splicing Factor TRA2 beta Rewire Glioblastoma Cell Transcriptomes and Inhibit Their Proliferation

INTRODUCTION: Glioblastomas are an aggressive brain tumor that, despite current treatment paradigms, carry a poor prognosis. Novel therapies aimed at specific glioblastoma alterations are needed. Alternative RNA splicing is a critical step in gene expression that allows a single gene to encode multiple RNA and subsequently protein isoforms. Alternative splicing becomes dysregulated in human tumors, including glioblastoma, through alterations in upstream regulatory splicing factor proteins such as TRA2β, an oncogene that enhances glioma cell proliferation. Despite these known alterations, targeting alternative splicing in glioblastomas has not been leveraged therapeutically. METHODS: U87MG glioblastoma cells were treated with splice-switching antisense oligonucleotides (ASOs) targeting the TRA2β transcript (ASO-1570) or non-targeting control (ASO-CTL). Cancer-relevant phenotypes, including proliferation and apoptosis, were measured using high-content imaging. Transcriptomic splicing signatures were defined using high-depth RNA-sequencing with computational tools that quantify percent splicing event inclusion (PSI) and validated using RT-PCR and western blotting. RESULTS: ASO-1570 treatment increased inclusion of a poison-exon in TRA2β leading to decreased coding transcripts and subsequent reductions in TRA2β protein expression compared to ASO-CTL. ASO-1570 significantly decreased cell proliferation and increased apoptosis. Mechanistically, ASO-1570 treated cells exhibited significant differences in alternatively spliced events, impacting genes involved in cancer-relevant pathways such as p53 and mTOR signaling. For example, ASO-1570 led to increased exon skipping in MDM2 leading to decreased MDM2 protein expression and increased p53 wild-type expression. ASO-1570 treatment also impacted growth of U87MG 3D-neurospheres, suggesting efficacy in advanced models that better capture in vivo conditions. CONCLUSIONS: Glioblastoma cells are sensitive to ASO-mediated TRA2β knockdown, which mechanistically induced transcriptome wide changes that impact cancer-related pathways. Further testing of this ASO in vivo may open novel avenues for its use as a targeted glioblastoma therapeutic.