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Updated: Jun 8, 2025
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Combined inhibition of histone methyltransferases EZH2 and DOT1L is an effective therapy for neuroblastoma
Janith A Seneviratne1,2, Daenikka Ravindrarajah1,2, Daniel R Carter1,3
1Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, UNSW Sydney, Kensington, New South Wales, Australia.
Background:
The child cancer, neuroblastoma (NB), is characterised by a low incidence of mutations and strong oncogenic embryonal driver signals. Many new targeted epigenetic modifier drugs have failed in human trials as monotherapy.
Methods:
We performed a high-throughput, combination chromatin-modifier drug screen against NB cells. We screened 13 drug candidates in 78 unique combinations.
Results:
We found that the combination of two histone methyltransferase (HMT) inhibitors: GSK343, targeting EZH2, and SGC0946, targeting DOT1L, demonstrated the strongest synergy across 8 NB cell lines, with low normal fibroblast toxicity. High mRNA expression of both EZH2 and DOT1L in NB tumour samples correlated with the poorest patient survival. Combination HMT inhibitor treatment caused activation of ATF4-mediated endoplasmic reticulum (ER) stress responses. In addition, glutathione and several amino acids were depleted by HMT inhibitor combination on mass spectrometry analysis. The combination of SGC0946 and GSK343 reduced tumour growth in comparison to single agents.
Conclusion:
Our results support further investigation of HMT inhibitor combinations as a therapeutic approach in NB.
Insights
Combining histone methyltransferase (HMT) inhibitors targeting EZH2 and DOT1L shows promise for neuroblastoma (NB) treatment, demonstrating synergy and reduced tumor growth with low toxicity.
Area of Science:
- Oncology
- Epigenetics
- Drug Discovery
Background:
- Neuroblastoma (NB) is a childhood cancer with few mutations but strong oncogenic signals.
- Epigenetic therapies targeting NB have shown limited success as monotherapy.
Purpose of the Study:
- To identify effective combination therapies for neuroblastoma by screening epigenetic drugs.
- To evaluate the synergistic effects of chromatin-modifier drugs in NB cell lines.
Main Methods:
- High-throughput screening of 13 drug candidates in 78 combinations against NB cells.
- Assessed drug synergy, normal cell toxicity, gene expression, metabolic changes, and tumor growth reduction.
Main Results:
- Combination of EZH2 inhibitor GSK343 and DOT1L inhibitor SGC0946 demonstrated significant synergy in 8 NB cell lines with low toxicity.
- High EZH2 and DOT1L expression correlated with poor patient survival.
- Combination treatment induced ATF4-mediated endoplasmic reticulum stress and depleted glutathione and amino acids, reducing tumor growth.
Conclusions:
- Histone methyltransferase (HMT) inhibitor combinations warrant further investigation as a therapeutic strategy for neuroblastoma.
- Targeting EZH2 and DOT1L simultaneously may overcome limitations of monotherapy in NB treatment.
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