G-quadruplex stabilizer CX-5461 effectively combines with ionizing radiation to selectively target ATRX-deficient malignant glioma [RNA-seq]
Mutational inactivation of a-thalassaemia/mental retardation X-linked (ATRX) represents a defining molecular feature in large subsets of adult and pediatric malignant glioma. ATRX deficiency gives rise to abnormal G-quadruplex (G4) DNA secondary structures at GC-rich regions of the genome, altering chromatin accessibility and enhancing DNA damage. Building on earlier work, we sought to assess the extent to which pharmacological G4 stabilization selectively enhances DNA damage and cell death in preclinical models of ATRX-deficient glioma. Deploying the G4 stabilizer CX-5461 in patient-derived glioma stem cells (GSCs) in vitro and in GSC murine flank and intracranial xenografts in vivo, we evaluated efficacy as both a single agent and in combination with ionizing radiation (IR), a central element of current treatment standards. CX-5461 promoted dose-sensitive lethality in ATRX-deficient GSCs relative to ATRX-intact controls. Mechanistic studies revealed that CX-5461 disrupted histone variant H3.3 deposition, enhanced replication stress and DNA damage pathways, activated p53-independent apoptosis, and induced G2/M arrest selectively in ATRX-deficient GSCs. These data were corroborated in vivo, where we notably demonstrated that combinational treatment leads to profound tumor growth delay and prolonged survival exclusively in ATRX-deficient flank tumors. In its totality, our work substantively demonstrates efficacy and defines mechanisms of action for a novel therapeutic strategy targeting ATRX-deficient malignant glioma, laying the groundwork for clinical translation. Overall design: Comparative gene expression profiling analyses of RNA-seq data for patient-derived glioma stem cells (GSCs) treated for 48 hours with either Vehicle (DMSO) or G4 stabilizer CX-5461 (1 µM). GSC lines were treated in triplicates for each condition (VEH or CX) and are as follows: TS 543-sh590 (ATRX-/-), TS 543 (ATRX wt), TS 603 (ATRX wt), GS 5-22 (ATRX -/-), and GS 8-18 (ATRX -/-).