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MGMT function determines the differential response of ATR inhibitors with DNA-damaging agents in glioma stem cells
Vincent W S Leong1, Sabbir Khan1, Pratibha Sharma1
1Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Background:
The most prevalent cancer treatments cause cell death through DNA damage. However, DNA damage response (DDR) repair pathways, initiated by tumor cells, can withstand the effects of anticancer drugs, providing justification for combining DDR inhibitors with DNA-damaging anticancer treatments.
Methods:
Cell viability assays were performed with CellTiter-Glo assay. DNA damage was evaluated using Western blotting analysis. RNA-seq and single-cell level expression were used to identify the DDR signatures. In vivo, studies were conducted in mice to determine the effect of ATris on TMZ sensitization.
Results:
We found a subpopulation of glioma sphere-forming cells (GSCs) with substantial synergism with temozolomide (TMZ) using a panel of 3 clinical-grade ataxia-telangiectasia- and Rad3-related kinase inhibitors (ATRis), (elimusertib, berzosertib, and ceralasertib). Interestingly, most synergistic cell lines had O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, indicating that ATRi mainly benefits tumors with no MGMT repair. Further, TMZ activated the ATR-checkpoint kinase 1 (Chk1) axis in an MGMT-dependent way. TMZ caused ATR-dependent Chk1 phosphorylation and DNA double-strand breaks as shown by increased γH2AX. Increased DNA damage and decreased Chk1 phosphorylation were observed upon the addition of ATRis to TMZ in MGMT-methylated (MGMT-) GSCs. TMZ also improved sensitivity to ATRis in vivo, as shown by increased mouse survival with the TMZ and ATRi combination treatment.
Conclusions:
This research provides a rationale for selectively targeting MGMT-methylated cells using ATRis and TMZ combination. Overall, we believe that MGMT methylation status in GBM could serve as a robust biomarker for patient selection for ATRi combined with TMZ.
Insights
Combining ataxia-telangiectasia- and Rad3-related kinase inhibitors (ATRis) with temozolomide (TMZ) shows promise for treating O6-methylguanine-DNA methyltransferase (MGMT) methylated glioblastoma. This combination enhances DNA damage and improves survival in preclinical models.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- DNA damage response (DDR) pathways enable cancer cells to resist DNA-damaging chemotherapy.
- Inhibiting DDR is a rational strategy to enhance anticancer drug efficacy.
- Ataxia-telangiectasia- and Rad3-related kinase inhibitors (ATRis) target a key DDR pathway.
Purpose of the Study:
- To investigate the synergistic potential of ATRis combined with temozolomide (TMZ) in cancer treatment.
- To identify biomarkers predicting response to ATRi and TMZ combination therapy.
- To evaluate the efficacy of ATRi and TMZ in preclinical glioblastoma models.
Main Methods:
- Cell viability assays (CellTiter-Glo) and Western blotting for DNA damage assessment.
- RNA sequencing (RNA-seq) to identify DDR signatures.
- In vivo studies in mice to assess TMZ sensitization by ATRis.
Main Results:
- A subpopulation of glioma sphere-forming cells (GSCs) exhibited significant synergy with TMZ when treated with ATRis (elimusertib, berzosertib, ceralasertib).
- Synergistic effects were predominantly observed in cell lines with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, suggesting ATRi benefits tumors lacking MGMT repair.
- TMZ activated the ATR-Chk1 axis in an MGMT-dependent manner, leading to DNA double-strand breaks; ATRis enhanced TMZ-induced DNA damage and reduced Chk1 phosphorylation in MGMT-methylated GSCs.
- Combination treatment in vivo increased mouse survival, demonstrating TMZ's ability to sensitize tumors to ATRis.
Conclusions:
- The study provides a rationale for selectively targeting MGMT-methylated glioblastoma cells with a combination of ATRis and TMZ.
- MGMT methylation status in glioblastoma (GBM) can serve as a predictive biomarker for patient selection in clinical trials combining ATR inhibitors with TMZ.
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