Decoy and antisense RNA oligonucleotides targeting the EGFR exon 3 splice site suppress proliferation of A172 glioblastoma cells

Three or more documented clinical failures match this mechanism, or a Phase 3 efficacy failure is on record.

Abstract excerpt

Short RNA oligonucleotides directed at the 3' splice site region of EGFR exon 3 reduced the proliferation of A172 glioblastoma cells, while the same oligonucleotides had no detectable effect on A549 lung adenocarcinoma cells. Glioblastoma carries EGFR amplification in roughly a third of IDH-wildtype cases, and the A172 line harbours a tandem duplication in the EGFR tyrosine kinase domain. Because exon definition in human genes depends on RNA-binding proteins that engage exonic sequence near splice sites, we used the SpliceAid database to identify candidate RNA-binding proteins at EGFR exons 2 and 3. Y-box binding protein 1 (YB-1, encoded by YBX1), reported to drive glioblastoma growth through its RNA-binding function, scored highly immediately adjacent to the exon 3 3' splice site. We designed four 2'-modified RNA oligonucleotides carrying a nuclear localisation signal: two antisense oligonucleotides against the exon 3 region (asRNA1, spanning the splice site; asRNA2, downstream of it), a decoy complementary to the asRNA2 target (dRNA2), and a decoy carrying the YB-1 consensus binding sequence (dYB-1). In A172 cells, all four reduced EGFR protein in a single Western blot experiment, and asRNA1, asRNA2 and dRNA2 reduced proliferation relative to scrambled RNA at days 1 to 3; dYB-1 produced a comparable reduction at day 3. None of these effects was seen in A549 cells. YB-1 protein level and subcellular distribution were unchanged by the oligonucleotides, arguing against an indirect route through altered YB-1 abundance. These observations are preliminary. Every experiment reported here was performed once: the proliferation data come from single plates with three wells per group, analysed by Dunnett's test against the scrambled-RNA control, and the Western blot, RT-PCR and immunocytochemistry panels are single runs presented as representative observations. Direct binding of YB-1 to the targeted EGFR sequence has not been demonstrated. We post this work as a preprint so that the design strategy and the initial observations are available while these questions remain open.