Emily M. Schleicher, Ashna Dhoonmoon, Lindsey M. Jackson, Kristen E. Clements, Coryn L. Stump, Claudia M. Nicolae, George-Lucian Moldovan
The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis.
Proper response to DNA damage and replication stress is critical for all organisms. Replication stress occurs upon arrest of the DNA replication machinery at sites of DNA damage, or during replication of endogenous difficult to replicate DNA sequences such as microsatellite regions . The ataxia-telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases are in primary control of the cellular responses to replication stress and DNA damage . The activation of these kinases is critical to arrest the cell cycle and allow time for proper execution of DNA replication and repair prior to cell division . ATR is activated by single-stranded DNA (ssDNA) formed upon replication fork arrest. ATR activation leads to downstream phosphorylation of Chk1, resulting in stabilization of the replication fork, suppression of origin firing, and cell cycle arrest. ATM is triggered by the presence of double-stranded DNA breaks, and phosphorylates p53 and Chk2 leading to cell cycle arrest to allow for the DNA to be repaired before proceeding through the cell cycle.
Materials and methods
HeLa and 8988T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 1% Pen/Strep. MCF10A cells were grown in DMEM/F12 supplemented with 5% fetal calf serum, 1% Pen/Strep, 20ng/mL hEGF, 0.5 mg/mL Hydrocortisone, 100 ng/mL Cholera Toxin, and 10 μg/mL Insulin.
Gene knockdown was performed using Lipofectamine RNAiMAX transfection reagent. Cells were treated with siRNA for two consecutive days. The following SilencerSelect oligonucleotides (ThermoFisher) were used for gene knockdown: KNTC1 (ID: s18776); LUC7L3 (ID: s226748); SOD2 (ID: s13267); LIAS (ID: s223178); EEF1B2 (ID: s194388); RETSAT (ID: s29671); MED12 (ID: s19362); MED7 (ID: s18080), MED13 (ID: s19365), CCNC (ID: s391), CDK8 (ID: s2831), TGFBR2 (ID: s14077), RNASEH2 (ID: s20656), RBM25 (ID: s33912). AllStars Negative Control siRNA (Qiagen 1027281) was used as control.
Detailed information on the genetic make-up of tumors will help to better treat patients on a personalized basis. Identification of markers that lead to resistance to ATRi is critical to advance the use of ATRi in cancer therapy. With the emerging use of ATRi in clinical trials, there has been renewed interest in determining predictors to ATRi response. Moreover, beyond the treatment of patients, the effects of ATRi on cell biology and pathways that regulate responses to ATRi are still not well understood.
Citation: Schleicher EM, Dhoonmoon A, Jackson LM, Clements KE, Stump CL, Nicolae CM, et al. (2020) Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors. PLoS Genet 16(11): e1009176. https://doi.org/10.1371/journal.pgen.1009176
Editor: Dmitry A. Gordenin, National Institute of Environmental Health Sciences, UNITED STATES
Received: April 6, 2020; Accepted: October 2, 2020; Published: November 2, 2020.
Copyright: © 2020 Schleicher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by: NIH R01ES026184 and R01GM134681 (to GLM) and 1F31CA243301 (to EMS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.