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Published on: May 27, 2021
Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition
John Paul Shen1,2, Rohith Srivas1,3, Andrew Gross4
1Department of Medicine, University of California San Diego, La Jolla, CA, USA.
Abstract:
Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function.
Insights
RAD17 loss-of-function sensitizes cancer cells to checkpoint kinase inhibitors like CHEK1 and WEE1. This discovery identifies RAD17 as a potential biomarker for predicting patient response to these targeted cancer therapies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Checkpoint kinases (CHEK1, CHEK2) are critical in DNA damage response and cancer progression.
- Targeting these kinases shows therapeutic promise, but patient stratification is needed.
- Biomarkers are essential for identifying patients likely to benefit from checkpoint kinase inhibitors.
Purpose of the Study:
- To identify molecular biomarkers for predicting response to CHEK1 and CHEK2 inhibitors.
- To screen for synthetic lethal interactions between tumor suppressor genes and checkpoint kinase inhibitors.
- To evaluate the role of RAD17 in sensitivity to checkpoint kinase inhibition.
Main Methods:
- Screening of 112 tumor suppressor genes for synthetic lethality with CHEK1/CHEK2 inhibitors.
- Clonogenic assays using RAD17 knockdown cell lines.
- Large-scale functional shRNA screen across 100+ cancer cell lines.
- Yeast-based interaction studies.
- Analysis of DNA damage marker (γH2AX) accumulation.
Main Results:
- Identified eight synthetic lethal interactions, including with RAD17.
- RAD17 knockdown significantly increased sensitivity (3.5-fold) to checkpoint kinase inhibition.
- CHEK1/CHEK2 mutant cell lines showed unexpected sensitivity to RAD17 knockdown.
- RAD17 knockdown potentiated the synergistic effects of CHEK1/2 and WEE1 inhibition.
- RAD17 knockdown magnified DNA damage marker accumulation upon kinase inhibition.
Conclusions:
- RAD17 loss-of-function is synthetically lethal with inhibition of CHEK1/CHEK2 and WEE1.
- RAD17 functions as a potential predictive biomarker for response to checkpoint kinase inhibitors.
- CHEK1 or WEE1 inhibitors may be more effective in tumors with RAD17 loss-of-function.
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