Abstract A036: Synthetic RNA-guided combinatorial targeting of glioblastoma adaptive resistance: Dual suppression of PI3K-AKT and mesenchymal transition escape pathways

Abstract Glioblastoma remains one of the most treatment-refractory solid tumors due to rapid adaptive resistance across multiple convergent molecular pathways. In particular, persistent activation of the PI3K-AKT axis combined with mesenchymal transition (EMT) reprogramming contributes to rapid failure of standard therapeutic regimens, including temozolomide, radiation, and immunotherapeutics. To address this clinical gap, we developed a synthetic RNA-guided dual-targeting system designed to simultaneously suppress both canonical PI3K-AKT activation and mesenchymal escape signatures, using strand-engineered molecules optimized for combinatorial silencing. Using patient-derived glioblastoma stem cell lines and isogenic resistance models, we performed transcriptomic profiling to identify co-expressed resistance-driving nodes. Computational integration of CRISPR perturbation datasets, RNA interference screens, and synthetic lethality maps enabled selection of targetable PI3K-AKT intermediates and EMT regulators including SOX2, TWIST1, and ZEB1. Synthetic RNA molecules were designed to co-suppress both vertical (PI3K-AKT) and horizontal (EMT plasticity) resistance axes, with in vitro validation demonstrating >70% knockdown efficiency and significant synergistic reduction in proliferation (p<0.001) across diverse resistance phenotypes. Mechanistic assays revealed reversal of mesenchymal signatures with restored epithelial markers (E-cadherin upregulation; vimentin downregulation) alongside AKT dephosphorylation. Combination treatment with low-dose temozolomide further amplified cell death, suggesting sensitization to standard chemotherapy. In vivo murine xenograft models exhibited >60% reduction in tumor volume compared to controls (p<0.01), without evidence of off-target cytotoxicity or systemic toxicity. This study demonstrates the feasibility of multi-axis synthetic RNA-guided combinatorial targeting to suppress glioblastoma therapeutic resistance. These findings support future clinical translation of modular RNA-based therapeutics capable of overcoming the heterogeneity and plasticity that characterize high-grade gliomas. Citation Format: Shivi Kumar. Synthetic RNA-guided combinatorial targeting of glioblastoma adaptive resistance: Dual suppression of PI3K-AKT and mesenchymal transition escape pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A036.