EXTH-62. COMBINATIONAL TREATMENT OF G-QUADRUPLEX STABILIZER CX-5461 AND IONIZING RADIATION POTENTIATES SELECTIVE LETHALITY IN PRECLINICAL ATRX-DEFICIENT GLIOMA MODELS

Abstract

Abstract Mutations in α-thalassaemia/mental retardation X-linked (ATRX) are a critical molecular marker for fatal high-grade glioma (HGG). Standard of care has remained stagnant in the last 30 years, with no therapies approved specifically for ATRX-deficient glioma. ATRX mutations give rise to abnormal DNA secondary G-quadruplex (G4) structures at GC-rich regions of the genome, altering genome-wide accessibility of chromatin and enhancing DNA damage. We aim to use G4 stabilizers, a class of novel small molecule compounds that selectively bind to and stabilize G4s, to enhance DNA damage and induce cell death in ATRX-deficient glioma. Using patient-derived glioma stem cells (GSCs) and the G4 stabilizing compound CX-5461 (Senhwa Biosciences), we evaluated the efficacy of G4 stabilization as a single agent and in combination with ionizing radiation (IR). As IR is standard of care and DNA-damaging, it may hold strong potential to synergize with CX-5461. Here, we report that ATRX-deficient GSCs demonstrate dose-sensitive lethality to CX-5461, compared to controls. Mechanistic studies reveal that CX-5461 activates p53-independent apoptosis, exhibits G2/M arrest in ATRX-deficient GSCs, and upregulates G4s (BG4), replication stress (ATR, RPA32) and DNA damage (ATM, γH2AX) pathways specifically in ATRX-deficient GSCs. These functional findings are corroborated by ATRX-deficient and -intact GSC flank xenograft mouse models, which were separated into the following treatment groups: 1) vehicle, 2) CX-5461 alone, 3) IR alone, 4) CX-5461 and IR (n = 20/group). Excitingly, we demonstrate that combinational treatment leads to profound tumor growth delay exclusively in ATRX-deficient flank tumors compared to vehicle (p = 0.0004), CX-5461 alone (p = 0.0001), or IR alone (p = 0.0002) groups. Multiplex immunohistochemistry of CX-5461 alone and the combinational treatment tumors shows enhanced induction of G4s, replication stress, and DNA damage, recapitulating in vitro findings. Taken together, our work defines mechanisms of action and efficacy for a novel therapeutic strategy for pre-clinical ATRX-deficient HGG, with strong implications for clinical translation.