2340 - Leveraging H3K27M-Driven ATR Dependency to Enhance the Efficacy of Radiotherapy for Diffuse Midline Gliomas

Purpose/Objective(s): Diffuse midline gliomas (DMGs) are aggressive pediatric brain tumors with dismal survival rates. Radiation therapy prolongs survival but never offers a cure. H3K27M mutation—the defining oncogenic driver of DMGs—increases replication stress (RS) by driving hypertranscription. This increased RS creates a dependency on ATR causing H3K27M DMG cells to exhibit hypersensitivity to ATR inhibition (ATRi). Since radiation is also known to induce RS, we hypothesized that combining radiation with ATR inhibition would synergistically amplify replication stress and DMG cell death.

Materials/Methods: To investigate whether ATR inhibition enhances radiation-induced replication stress, DNA damage, and cell death in H3K27M-mutant DMG cells, we treated H3K27M-mutated and H3 wild-type human isogenic pediatric glioma cell line pairs, murine isogenic DMG lines, and patient-derived DMG cell lines in vitro with the brain-penetrant ATR inhibitor alnodesertib, ionizing radiation, or combination thereof. We quantified RS (chromatin-bound RPA32), DNA damage (gamma-H2AX), genomic instability (micronuclei formation), and apoptosis (caspase 3/7 activation), and analyzed drug-radiation synergy with Combenefit modeling. To evaluate the efficacy of combination therapy in vivo and its translational potential, we treated mice harboring orthotopic SU-DIPG13P* tumors with ATRi, radiation, or combination, assessing overall survival by Kaplan-Meier analysis.

Results: Compared to their wild-type counterparts, H3K27M-mutant cells exhibit higher basal replication stress and ATR activation and are therefore hypersensitive to ATR inhibition. Since radiation is the standard of care for DMG patients, we combined radiation treatment with ATR inhibition and observed synergistic DMG cell killing, with a more pronounced effect in H3K27M mutated cells. We further document that the synergistic cell killing was a consequence of enhanced RS, DNA damage, genomic instability, and apoptosis in the combination treatment relative to either monotherapy alone. Of note, combination treatment led to a greater increase in RS, DNA damage, and apoptosis in H3K27M cells compared to wild-type cells. These in vitro findings were recapitulated in vivo with mice bearing DMG intracranial xenografts, showing significant survival benefit with the combination treatment of ATRi and radiation compared to either monotherapy.

Conclusion: H3K27M-driven DMGs exhibit high levels of replication stress, creating a therapeutically exploitable reliance on ATR. We demonstrate that ATR inhibition sensitizes H3K27M-mutant DMG cells to radiation-induced DNA damage, driving synergistic tumor cell death both in vitro and in vivo. These findings establish ATR inhibition as a promising radiosensitizing strategy, supporting the clinical translation of ATR inhibitor-radiotherapy combinations in children with H3K27M-driven DMGs.