Targeted Delivery of Napabucasin with Radiotherapy in Diffuse Midline Glioma

Background Introduction

Diffuse Midline Glioma (DMG) is the most aggressive primary brain tumor in children, with a median survival typically less than one year. Although radiation therapy (RT) is the current standard treatment, it only provides transient symptom relief and fails to significantly extend patient survival. Additionally, due to the location of DMG in midline structures such as the brainstem, surgical resection carries high risks, and the efficacy of systemic therapies is limited by the presence of the blood-brain barrier (BBB). Therefore, identifying a therapeutic strategy that can synergize with RT has become a critical direction in DMG research.

Napabucasin is an NAD(P)H quinone oxidoreductase 1 (NQO1)-bioactivatable reactive oxygen species (ROS) inducer, which has shown potential therapeutic effects in various cancer types. This study aims to explore the application of Napabucasin as a radiosensitizer in DMG and to overcome BBB limitations through focused ultrasound (FUS) and convection-enhanced delivery (CED) technologies, thereby enhancing the therapeutic efficacy of the drug.

Source of the Paper

This paper was co-authored by Matthew Gallitto, Xu Zhang, and other researchers from multiple institutions, including Columbia University and Virginia Tech, and was published in Neuro-Oncology in 2025. The title of the paper is “Targeted Delivery of Napabucasin with Radiotherapy Improves Outcomes in Diffuse Midline Glioma.”

Research Process and Results

1. Expression of NQO1 in DMG

The study first analyzed the expression levels of NQO1 in 76 DMG patient tissue samples and normal brain tissues using RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq). The results showed that NQO1 expression was significantly higher in DMG patient tissues compared to normal brain tissues. Additionally, through Western blot analysis, the research team confirmed high NQO1 expression in multiple DMG cell lines, suggesting that NQO1 could be a potential therapeutic target for DMG.

2. ROS-Inducing Effects of Napabucasin

The research team tested the cytotoxic effects of Napabucasin on DMG cells in vitro and found that it exhibited significant cytotoxicity in multiple DMG cell lines, with half-maximal inhibitory concentration (IC50) values ranging from 0.80 to 1.44 µM. Through Gene Ontology (GO) analysis and Viper-inferred protein activity analysis, the team discovered that ROS-related pathways were significantly upregulated after Napabucasin treatment. Further experiments quantified ROS levels using the DCFDA reagent, demonstrating that Napabucasin induced ROS production in a dose-dependent manner.

3. Radiosensitizing Effects of Napabucasin

The research team hypothesized that Napabucasin might exhibit enhanced therapeutic effects under RT conditions. Through clonogenic assays, the team found that Napabucasin significantly increased the sensitivity of DMG cells to RT. Additionally, ROS quantification experiments revealed that the combination of Napabucasin and RT led to a significant increase in ROS levels, indicating a synergistic effect between the two.

4. NQO1 Dependency Experiments

To verify whether the effects of Napabucasin depend on NQO1, the research team knocked out the NQO1 gene in DMG cells using CRISPR-Cas9 technology. The results showed that NQO1 knockout significantly reduced the cytotoxicity of Napabucasin, as well as its ROS-inducing and radiosensitizing effects. These findings confirm that the mechanism of Napabucasin is indeed dependent on NQO1.

5. In Vivo Experiments: Subcutaneous Xenograft Model

The research team established a subcutaneous xenograft model by implanting DMG cells into nude mice. The experimental results showed that Napabucasin monotherapy had no significant impact on tumor volume, but when combined with RT, it significantly improved local tumor control. This result further validates the radiosensitizing effects of Napabucasin.

6. In Vivo Experiments: Orthotopic Xenograft Model and CED Technology

To overcome the limitations of the BBB, the research team employed CED technology to directly deliver Napabucasin to the DMG tumor site. The experimental results demonstrated that CED significantly increased the drug concentration in tumor tissues and, when combined with RT, significantly prolonged the survival of mice. These findings indicate that CED technology can effectively enhance the therapeutic efficacy of Napabucasin and provide important evidence for its clinical application.

Conclusions and Significance

The study concludes that Napabucasin, as an NQO1-dependent ROS inducer, exhibits significant radiosensitizing effects in DMG. By utilizing CED technology, the research team successfully overcame BBB limitations, significantly improved the therapeutic efficacy of Napabucasin, and extended the survival of DMG mice. This research provides new insights into the treatment of DMG and lays the groundwork for future clinical studies.

Research Highlights

1. NQO1 as a Potential Therapeutic Target: This study is the first to confirm the high expression of NQO1 in DMG and to reveal its critical role in the mechanism of Napabucasin.
2. Radiosensitizing Effects of Napabucasin: Through in vitro and in vivo experiments, the research team validated the potential of Napabucasin as a radiosensitizer, offering a new option for combination therapy in DMG.
3. Application of CED Technology: This study is the first to demonstrate the feasibility of CED technology in DMG treatment and to prove that it can significantly enhance drug delivery efficiency, providing a new technical approach to overcoming the BBB.

Additional Valuable Information

The research team also explored the application of focused ultrasound (FUS) technology in drug delivery. Although FUS did not significantly increase the brain concentration of Napabucasin in this study, it has shown potential in other research and remains worthy of further exploration.

Through multidisciplinary collaboration, this study provides new ideas and methods for the treatment of DMG, holding significant scientific value and clinical application prospects.