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Background <p>Neuroblastoma, the most common extracranial solid tumor in children, is often associated with high-risk features and poor outcomes. Despite intensive multimodal therapies, survival remains unsatisfactory, underscoring the need for new treatments. Checkpoint kinase 1 (CHK1) and checkpoint kinase 2 (CHK2) are key regulators of the DNA damage response (DDR) and cell cycle checkpoints. We investigated the antitumor effect of their combined inhibition in neuroblastoma.</p> Methods <p>Neuroblastoma cell lines were treated with the CHK1 inhibitor LY2606368 and CHK2 inhibitor PV1019, either individually or in combination. Cell viability was measured using the WST-8 assay. Cell cycle distribution was analyzed by flow cytometry following propidium iodide staining. DNA damage and replication stress were assessed by immunofluorescence and western blotting for γH2AX and phospho-RPA2 (S8), respectively. Apoptosis was evaluated using sub-G1 population analysis, detection of cleaved-PARP and cleaved–caspase-3, and TUNEL staining.</p> Results <p>Co-treatment showed synergistic activity against both <i>MYCN</i>-amplified and non-amplified cell lines. Combination treatment reduced cell numbers more effectively than either agent alone, increased S-phase entry, and enhanced DNA damage, replication stress, and apoptosis marker expression.</p> Conclusion <p>Dual CHK1/CHK2 inhibition disrupts the DDR and checkpoints, leading to replication stress, DNA damage, and apoptosis, representing a promising therapeutic strategy for neuroblastoma.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>Combined CHK1/CHK2 inhibitor therapy shows a synergistic anti-tumor effect against neuroblastoma cells.</p> </ItemContent> <ItemContent> <p>Combination therapy impairs DNA damage response pathways and drives accelerated cell cycle progression in neuroblastoma cells.</p> </ItemContent> <ItemContent> <p>Combination therapy increases DNA damage, replication stress, and apoptosis marker expression.</p> </ItemContent> <ItemContent> <p>Combined CHK1/CHK2 inhibition holds therapeutic potential for the treatment of neuroblastoma.</p> </ItemContent> </UnorderedList></p>

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Combination of CHK1 and CHK2 inhibitors exerts synergistic antitumor effects against neuroblastoma cells

  • 期刊论文

  • 发表日期：

    2026年6月2日

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  • Rina Kato,
  • Hiromasa Aoki,
  • Kohki Toriuchi,
  • Kazuya Izumi,
  • Mai Nagasaka,
  • Yasumichi Inoue,
  • Hidetoshi Hayashi,
  • Satoru Takeshita,
  • Hiroki Kakita,
  • Yasumasa Yamada,
  • Mineyoshi Aoyama

摘要

Background

Neuroblastoma, the most common extracranial solid tumor in children, is often associated with high-risk features and poor outcomes. Despite intensive multimodal therapies, survival remains unsatisfactory, underscoring the need for new treatments. Checkpoint kinase 1 (CHK1) and checkpoint kinase 2 (CHK2) are key regulators of the DNA damage response (DDR) and cell cycle checkpoints. We investigated the antitumor effect of their combined inhibition in neuroblastoma.

Methods

Neuroblastoma cell lines were treated with the CHK1 inhibitor LY2606368 and CHK2 inhibitor PV1019, either individually or in combination. Cell viability was measured using the WST-8 assay. Cell cycle distribution was analyzed by flow cytometry following propidium iodide staining. DNA damage and replication stress were assessed by immunofluorescence and western blotting for γH2AX and phospho-RPA2 (S8), respectively. Apoptosis was evaluated using sub-G1 population analysis, detection of cleaved-PARP and cleaved–caspase-3, and TUNEL staining.

Results

Co-treatment showed synergistic activity against both MYCN-amplified and non-amplified cell lines. Combination treatment reduced cell numbers more effectively than either agent alone, increased S-phase entry, and enhanced DNA damage, replication stress, and apoptosis marker expression.

Conclusion

Dual CHK1/CHK2 inhibition disrupts the DDR and checkpoints, leading to replication stress, DNA damage, and apoptosis, representing a promising therapeutic strategy for neuroblastoma.

Impact

Combined CHK1/CHK2 inhibitor therapy shows a synergistic anti-tumor effect against neuroblastoma cells.

Combination therapy impairs DNA damage response pathways and drives accelerated cell cycle progression in neuroblastoma cells.

Combination therapy increases DNA damage, replication stress, and apoptosis marker expression.

Combined CHK1/CHK2 inhibition holds therapeutic potential for the treatment of neuroblastoma.