ID #939 Targeting DNA Damage Response Pathways to Enhance Radiotherapy in Diffuse Midline Glioma, H3 K27-Altered

Background

Diffuse midline glioma (DMG), H3 K27-altered, is a WHO grade IV pediatric brain tumor with a median survival of approximately 8-11 months. Radiotherapy remains the only treatment providing transient clinical benefit.

Hypothesis

TP53 mutations occur in approximately 40–77% of DMG cases[1], indicating exploitable defects in cell cycle control following irradiation. Based on this rationale, we propose that inhibition of ataxia telangiectasia and Rad3-related kinase (ATR) in combination with radiotherapy may represent a promising novel treatment strategy for DMG.

Methods

A broad-spectrum compound screen was performed in DMG cell lines to identify agents with radiosensitizing potential. From this hypothesis-generating screen, ATR inhibitors emerged as leading candidates and were prioritized for further validation in vitro and in the chicken chorioallantoic membrane (CAM) model. Mechanistic analyses included evaluation of ATR pathway signaling by Western blot, cell cycle distribution by flow cytometry, micronuclei formation by light and electron microscopy, and radiation-induced DNA damage by fluorescence microscopy.

Results

Two clinically advanced ATR inhibitors demonstrated robust radiosensitization in vitro and in the CAM model, showing consistent treatment responses across both platforms. Mechanistically, ATR inhibition suppressed downstream signaling, abrogated radiation-induced G2 arrest, increased micronuclei formation, and led to persistent DNA damage following irradiation.

Conclusion

ATR inhibition effectively radiosensitizes DMG cells by disrupting cell cycle checkpoints and increasing radiation-induced DNA damage, with successful validation in the CAM model supporting further translational development.

Acknowledgement

This work was supported by a grant from the DFG (GRK2762/1).

1. Buczkowicz P, Bartels U, Bouffet E, Becher O, Hawkins C. Histopathological spectrum of paediatric diffuse intrinsic pontine glioma: diagnostic and therapeutic implications. Acta Neuropathol. 2014 Oct;128(4):573-81. doi: 10.1007/s00401-014-1319-6. Epub 2014 Jul 22. PMID: 25047029; PMCID: PMC4159563.