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Updated: Mar 31, 2026
Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts
Published on: July 25, 2020
mTORC1 and DNA-PKcs as novel molecular determinants of sensitivity to Chk1 inhibition
Andrew J Massey1, Peter Stephens2, Rebecca Rawlinson1
1Vernalis Research, Granta Park, Cambridge, CB21 6GB, UK.
Background:
Chk1 inhibitors are currently under clinical evaluation as single agents and in combination with cytotoxic chemotherapy. Understanding determinants of sensitivity and novel combinations is critical for further clinical development.
Methods:
Potentiation of mTOR inhibitor cytotoxicity by the Chk1 inhibitor V158411 was determined in p53 mutant colon cancer cells. DNA damage response, expression levels of repair proteins, cell cycle effects and the contribution of alternative DSB repair pathways were further evaluated by western blotting and high content analysis.
Results:
mTOR inhibitors AZD8055, RAD-001, rapamycin and BEZ235 induced synergistic cytotoxicity with the Chk1 inhibitor V158411 in p53 mutant colon cancer cells. Reduced FANCD2, RAD51 and RPA70, core proteins in homologous recombination repair (HRR) and interstrand crosslink repair (ICLR), following inhibition of mTOR was associated with increased V158411 induced DSBs and caspase 3-independent cell death. Dual mTOR and Chk1 inhibition activated DNA-PKcs. Cells defective in DNA-PKcs exhibited increased resistance to V158411 with Chk1 expression closely correlated to DNA-PKcs expression in various types of cancer.
Conclusions:
Down regulation of proteins involved in HRR or ICLR by mTOR inhibitors is associated with increased sensitivity of human tumours to Chk1 inhibitors such as V158411. High levels of DNA-PKcs may be a potential biomarker to stratify patients to Chk1 inhibitor therapy alone or in combination with mTOR inhibitors.
Insights
Combining mTOR and Chk1 inhibitors enhances cancer cell death, particularly in p53 mutant colon cancers. This synergy is linked to DNA repair pathway alterations and DNA-PKcs expression, suggesting potential biomarkers for targeted therapies.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- Checkpoint kinase 1 (Chk1) inhibitors are in clinical trials for cancer treatment.
- Identifying sensitivity factors and novel drug combinations is crucial for Chk1 inhibitor development.
Purpose of the Study:
- To investigate the synergistic effects of mTOR inhibitors and a Chk1 inhibitor (V158411) on p53 mutant colon cancer cells.
- To elucidate the underlying mechanisms of drug sensitivity and identify potential biomarkers.
Main Methods:
- Assessed cytotoxicity of mTOR inhibitors (AZD8055, RAD-001, rapamycin, BEZ235) combined with V158411 in p53 mutant colon cancer cells.
- Evaluated DNA damage response, repair protein expression (FANCD2, RAD51, RPA70), cell cycle effects, and DNA double-strand break (DSB) repair pathways using western blotting and high-content analysis.
Main Results:
- Synergistic cytotoxicity was observed between mTOR inhibitors and V158411 in p53 mutant colon cancer cells.
- mTOR inhibition led to reduced homologous recombination repair (HRR) and interstrand crosslink repair (ICLR) proteins, increasing V158411-induced DSBs and cell death.
- Dual inhibition activated DNA-protein kinase catalytic subunit (DNA-PKcs); cells deficient in DNA-PKcs showed resistance to V158411.
Conclusions:
- Downregulation of HRR/ICLR proteins by mTOR inhibitors enhances tumor sensitivity to Chk1 inhibitors like V158411.
- High DNA-PKcs expression may serve as a biomarker for patient stratification in Chk1 inhibitor-based therapies, alone or combined with mTOR inhibitors.
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