# Berzosertib enhances the sensitivity of pediatric diffuse midline glioma H3K27-altered cells to radiotherapy

## TL;DR

This study shows that berzosertib, an ATR inhibitor, can make pediatric brain cancer cells more sensitive to radiotherapy, potentially improving treatment outcomes.

## Contribution

The study identifies berzosertib as a novel radiosensitizer for H3K27-altered diffuse midline glioma cells.

## Key findings

- Berzosertib significantly reduced proliferation and clonogenic survival of DMG cells when combined with radiation.
- The drug delayed spheroid growth and suppressed tumor formation in an in ovo model.
- ATR inhibition emerged as a promising strategy to enhance radiotherapy efficacy in DMG.

## Abstract

Diffuse midline glioma H3K27M-altered (DMG) remains a fatal pediatric brain cancer driven by a global loss of histone H3K27 trimethylation. Radiotherapy comprises the most important treatment modality and significantly improves overall survival. Novel therapeutic strategies for DMG patients without or with radiotherapy are urgently needed. Here, we aimed to gain insights into potential radiation response modulators. To identify modulators of radiation response, we performed a high-throughput drug screening (HTS) in seven representative DMG cell lines using conventional chemotherapeutic drugs and phase I-IV drugs (n = 687), followed by irradiation with 0 or 2 × 4 Gray (Gy). The ataxia–telangiectasia and Rad3-related (ATR) inhibitor berzosertib emerged as a potent radiosensitizer. Its effects were validated in three DMG cell lines using short-term proliferation assays, long-term limiting dilution assays (LDA), 3D spheroid cultures, and the chorioallantoic membrane (CAM) assay in ovo. Across all three tested DMG cell line models, berzosertib enhanced the antineoplastic effects of clinically relevant radiation doses, significantly reducing proliferation and clonogenic survival, delaying spheroid growth, and suppressing tumor formation in ovo. These findings provide strong preclinical evidence that ATR inhibition increases the sensitivity of DMG cells to radiotherapy. They highlight a novel therapeutic vulnerability and support further exploration of ATR inhibitors in rational combination strategies to improve radiotherapy efficacy for this deadly disease.