Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors

Autoři: Emily M. Schleicher aff001;  Ashna Dhoonmoon aff001;  Lindsey M. Jackson aff001;  Kristen E. Clements aff001;  Coryn L. Stump aff001;  Claudia M. Nicolae aff001;  George-Lucian Moldovan aff001
Působiště autorů: Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America aff001
Vyšlo v časopise: Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors. PLoS Genet 16(11): e32767. doi:10.1371/journal.pgen.1009176
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009176


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. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.

Klíčová slova:

Apoptosis – Cancer treatment – CRISPR – DNA replication – Genetic screens – HeLa cells – Library screening – Small interfering RNA


1. Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nat Cell Biol. 2014;16(1):2–9. doi: 10.1038/ncb2897 24366029; PubMed Central PMCID: PMC4354890.

2. Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harbor perspectives in biology. 2013;5(9). Epub 2013/09/05. doi: 10.1101/cshperspect.a012716 24003211; PubMed Central PMCID: PMC3753707.

3. Brown EJ, Baltimore D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev. 2003;17(5):615–28. Epub 2003/03/12. doi: 10.1101/gad.1067403 12629044; PubMed Central PMCID: PMC196009.

4. Gaillard H, Garcia-Muse T, Aguilera A. Replication stress and cancer. Nat Rev Cancer. 2015;15(5):276–89. doi: 10.1038/nrc3916 25907220.

5. Toledo LI, Murga M, Fernandez-Capetillo O. Targeting ATR and Chk1 kinases for cancer treatment: a new model for new (and old) drugs. Mol Oncol. 2011;5(4):368–73. Epub 2011/08/09. doi: 10.1016/j.molonc.2011.07.002 21820372; PubMed Central PMCID: PMC3590794.

6. Fokas E, Prevo R, Hammond EM, Brunner TB, McKenna WG, Muschel RJ. Targeting ATR in DNA damage response and cancer therapeutics. Cancer Treat Rev. 2014;40(1):109–17. Epub 2013/04/16. doi: 10.1016/j.ctrv.2013.03.002 23583268.

7. Hall AB, Newsome D, Wang Y, Boucher DM, Eustace B, Gu Y, et al. Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970. Oncotarget. 2014;5(14):5674–85. Epub 2014/07/11. doi: 10.18632/oncotarget.2158 25010037; PubMed Central PMCID: PMC4170644.

8. Mohni KN, Thompson PS, Luzwick JW, Glick GG, Pendleton CS, Lehmann BD, et al. A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments. PLoS One. 2015;10(5):e0125482. Epub 2015/05/13. doi: 10.1371/journal.pone.0125482 25965342; PubMed Central PMCID: PMC4428765.

9. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA, et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011;7(7):428–30. Epub 2011/04/15. doi: 10.1038/nchembio.573 21490603.

10. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell. 2007;11(2):175–89. Epub 2007/02/13. doi: 10.1016/j.ccr.2006.11.024 17292828; PubMed Central PMCID: PMC2742175.

11. Vendetti FP, Lau A, Schamus S, Conrads TP, O'Connor MJ, Bakkenist CJ. The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo. Oncotarget. 2015;6(42):44289–305. Epub 2015/10/31. doi: 10.18632/oncotarget.6247 26517239; PubMed Central PMCID: PMC4792557.

12. Maya-Mendoza A, Petermann E, Gillespie DA, Caldecott KW, Jackson DA. Chk1 regulates the density of active replication origins during the vertebrate S phase. EMBO J. 2007;26(11):2719–31. Epub 2007/05/12. doi: 10.1038/sj.emboj.7601714 17491592; PubMed Central PMCID: PMC1888675.

13. Liu P, Barkley LR, Day T, Bi X, Slater DM, Alexandrow MG, et al. The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism. J Biol Chem. 2006;281(41):30631–44. Epub 2006/08/17. doi: 10.1074/jbc.M602982200 16912045.

14. Chen YH, Jones MJ, Yin Y, Crist SB, Colnaghi L, Sims RJ, 3rd, et al. ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress. Mol Cell. 2015;58(2):323–38. Epub 2015/04/07. doi: 10.1016/j.molcel.2015.02.031 25843623; PubMed Central PMCID: PMC4408929.

15. Couch FB, Bansbach CE, Driscoll R, Luzwick JW, Glick GG, Betous R, et al. ATR phosphorylates SMARCAL1 to prevent replication fork collapse. Genes Dev. 2013;27(14):1610–23. Epub 2013/07/23. doi: 10.1101/gad.214080.113 23873943; PubMed Central PMCID: PMC3731549.

16. Charrier JD, Durrant SJ, Golec JM, Kay DP, Knegtel RM, MacCormick S, et al. Discovery of potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase as potential anticancer agents. Journal of medicinal chemistry. 2011;54(7):2320–30. Epub 2011/03/19. doi: 10.1021/jm101488z 21413798.

17. Fokas E, Prevo R, Pollard JR, Reaper PM, Charlton PA, Cornelissen B, et al. Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation. Cell Death Dis. 2012;3:e441. Epub 2012/12/12. doi: 10.1038/cddis.2012.181 23222511; PubMed Central PMCID: PMC3542617.

18. Kim H, Xu H, George E, Hallberg D, Kumar S, Jagannathan V, et al. Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models. Nat Commun. 2020;11(1):3726. Epub 2020/07/28. doi: 10.1038/s41467-020-17127-2 32709856; PubMed Central PMCID: PMC7381609.

19. Mei L, Zhang J, He K, Zhang J. Ataxia telangiectasia and Rad3-related inhibitors and cancer therapy: where we stand. J Hematol Oncol. 2019;12(1):43. Epub 2019/04/26. doi: 10.1186/s13045-019-0733-6 31018854; PubMed Central PMCID: PMC6482552.

20. Mittra A, Coyne GHOS, Do KT, Piha-Paul SA, Kummar S, Takebe N, et al. Safety and tolerability of veliparib, an oral PARP inhibitor, and M6620 (VX-970), an ATR inhibitor, in combination with cisplatin in patients with refractory solid tumors. Journal of Clinical Oncology. 2019;37(15_suppl):3067-. doi: 10.1200/JCO.2019.37.15_suppl.3067

21. Konstantinopoulos PA, Medical Oncology DFCIBMAUSA, Wahner Hendrickson AE, Medical Oncology MCRMNUSA, Penson RT, Medical Oncology MGHBMAUSA, et al. LBA60Randomized phase II (RP2) study of ATR inhibitor M6620 in combination with gemcitabine versus gemcitabine alone in platinum-resistant high grade serous ovarian cancer (HGSOC). Annals of Oncology. 2020;30(Supplement_5). doi: 10.1093/annonc/mdz394.057

22. Miranda Suzanne C, Fang Y, Sharon K, Marina P, John P, et al. Abstract PR14: Phase I trial of first-in-class ataxia telangiectasia-mutated and Rad3-related (ATR) inhibitor VX-970 as monotherapy (mono) or in combination with carboplatin (CP) in advanced cancer patients (pts) with preliminary evidence of target modulation and antitumor activity. 2015. doi: 10.1158/1535-7163.TARG-15-PR14

23. Hustedt N, Alvarez-Quilon A, McEwan A, Yuan JY, Cho T, Koob L, et al. A consensus set of genetic vulnerabilities to ATR inhibition. Open Biol. 2019;9(9):190156. Epub 2019/09/12. doi: 10.1098/rsob.190156 31506018; PubMed Central PMCID: PMC6769295.

24. Ruiz S, Mayor-Ruiz C, Lafarga V, Murga M, Vega-Sendino M, Ortega S, et al. A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors. Mol Cell. 2016;62(2):307–13. Epub 2016/04/14. doi: 10.1016/j.molcel.2016.03.006 27067599; PubMed Central PMCID: PMC5029544.

25. Wang C, Wang G, Feng X, Shepherd P, Zhang J, Tang M, et al. Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition. Oncogene. 2019;38(14):2451–63. Epub 2018/12/12. doi: 10.1038/s41388-018-0606-4 30532030; PubMed Central PMCID: PMC6450769.

26. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184–91. doi: 10.1038/nbt.3437 26780180; PubMed Central PMCID: PMC4744125.

27. Konig R, Chiang CY, Tu BP, Yan SF, DeJesus PD, Romero A, et al. A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods. 2007;4(10):847–9. Epub 2007/09/11. doi: 10.1038/nmeth1089 17828270.

28. Smith J, Tho LM, Xu N, Gillespie DA. The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res. 2010;108:73–112. Epub 2010/11/03. doi: 10.1016/B978-0-12-380888-2.00003-0 21034966.

29. Puig O, Bragado-Nilsson E, Koski T, Seraphin B. The U1 snRNP-associated factor Luc7p affects 5' splice site selection in yeast and human. Nucleic Acids Res. 2007;35(17):5874–85. Epub 2007/08/30. doi: 10.1093/nar/gkm505 17726058; PubMed Central PMCID: PMC2034479.

30. Zhou A, Ou AC, Cho A, Benz EJ Jr., Huang SC. Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5' splice site selection. Molecular and cellular biology. 2008;28(19):5924–36. Epub 2008/07/30. doi: 10.1128/MCB.00560-08 18663000; PubMed Central PMCID: PMC2546994.

31. Gong D, Yang F, Li F, Qian D, Wu M, Shao Z, et al. Crystal structure and functional characterization of the human RBM25 PWI domain and its flanking basic region. The Biochemical journal. 2013;450(1):85–94. Epub 2012/11/30. doi: 10.1042/BJ20121382 23190262; PubMed Central PMCID: PMC3553564.

32. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74(4):597–608. Epub 1993/08/27. doi: 10.1016/0092-8674(93)90508-n 8358789.

33. Mutreja K, Krietsch J, Hess J, Ursich S, Berti M, Roessler FK, et al. ATR-Mediated Global Fork Slowing and Reversal Assist Fork Traverse and Prevent Chromosomal Breakage at DNA Interstrand Cross-Links. Cell Rep. 2018;24(10):2629–42 e5. Epub 2018/09/06. doi: 10.1016/j.celrep.2018.08.019 30184498; PubMed Central PMCID: PMC6137818.

34. Soutourina J. Transcription regulation by the Mediator complex. Nat Rev Mol Cell Biol. 2018;19(4):262–74. Epub 2017/12/07. doi: 10.1038/nrm.2017.115 29209056.

35. Allison DF, Wang GG. R-loops: formation, function, and relevance to cell stress. Cell Stress. 2019;3(2):38–46. Epub 2019/06/22. doi: 10.15698/cst2019.02.175 31225499; PubMed Central PMCID: PMC6551709.

36. Al-Hadid Q, Yang Y. R-loop: an emerging regulator of chromatin dynamics. Acta Biochim Biophys Sin (Shanghai). 2016;48(7):623–31. Epub 2016/06/03. doi: 10.1093/abbs/gmw052 27252122; PubMed Central PMCID: PMC6259673.

37. Hamperl S, Bocek MJ, Saldivar JC, Swigut T, Cimprich KA. Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses. Cell. 2017;170(4):774–86 e19. Epub 2017/08/13. doi: 10.1016/j.cell.2017.07.043 28802045; PubMed Central PMCID: PMC5570545.

38. Wahba L, Amon JD, Koshland D, Vuica-Ross M. RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Mol Cell. 2011;44(6):978–88. Epub 2011/12/27. doi: 10.1016/j.molcel.2011.10.017 22195970; PubMed Central PMCID: PMC3271842.

39. Fant CB, Taatjes DJ. Regulatory functions of the Mediator kinases CDK8 and CDK19. Transcription. 2019;10(2):76–90. Epub 2018/12/27. doi: 10.1080/21541264.2018.1556915 30585107; PubMed Central PMCID: PMC6602567.

40. Huang S, Holzel M, Knijnenburg T, Schlicker A, Roepman P, McDermott U, et al. MED12 controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling. Cell. 2012;151(5):937–50. Epub 2012/11/28. doi: 10.1016/j.cell.2012.10.035 23178117; PubMed Central PMCID: PMC3672971.

41. Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun. 2018;9(1):5416. Epub 2018/12/24. doi: 10.1038/s41467-018-07901-8 30575746; PubMed Central PMCID: PMC6303322.

42. Moiseeva TN, Yin Y, Calderon MJ, Qian C, Schamus-Haynes S, Sugitani N, et al. An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication. Proc Natl Acad Sci U S A. 2019;116(27):13374–83. Epub 2019/06/19. doi: 10.1073/pnas.1903418116 31209037; PubMed Central PMCID: PMC6613105.

43. Zhong Y, Nellimoottil T, Peace JM, Knott SR, Villwock SK, Yee JM, et al. The level of origin firing inversely affects the rate of replication fork progression. The Journal of cell biology. 2013;201(3):373–83. doi: 10.1083/jcb.201208060 23629964; PubMed Central PMCID: PMC3639389.

44. Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat Rev Mol Cell Biol. 2017;18(10):622–36. Epub 2017/08/16. doi: 10.1038/nrm.2017.67 28811666; PubMed Central PMCID: PMC5796526.

45. Schoonen PM, Talens F, Stok C, Gogola E, Heijink AM, Bouwman P, et al. Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells. Nat Commun. 2017;8:15981. doi: 10.1038/ncomms15981 28714471; PubMed Central PMCID: PMC5520019.

46. Brunen D, Willems SM, Kellner U, Midgley R, Simon I, Bernards R. TGF-beta: an emerging player in drug resistance. Cell Cycle. 2013;12(18):2960–8. Epub 2013/08/27. doi: 10.4161/cc.26034 23974105; PubMed Central PMCID: PMC3875670.

47. Pal D, Pertot A, Shirole NH, Yao Z, Anaparthy N, Garvin T, et al. TGF-beta reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells. Elife. 2017;6. Epub 2017/01/17. doi: 10.7554/eLife.21615 28092266; PubMed Central PMCID: PMC5345931.

48. Liu L, Zhou W, Cheng CT, Ren X, Somlo G, Fong MY, et al. TGFbeta induces "BRCAness" and sensitivity to PARP inhibition in breast cancer by regulating DNA-repair genes. Mol Cancer Res. 2014;12(11):1597–609. Epub 2014/08/12. doi: 10.1158/1541-7786.MCR-14-0201 25103497; PubMed Central PMCID: PMC4233161.

49. Zhang H, Kozono DE, O'Connor KW, Vidal-Cardenas S, Rousseau A, Hamilton A, et al. TGF-beta Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia. Cell stem cell. 2016;18(5):668–81. Epub 2016/04/08. doi: 10.1016/j.stem.2016.03.002 27053300; PubMed Central PMCID: PMC4860147.

50. Liu Q, Ma L, Jones T, Palomero L, Pujana MA, Martinez-Ruiz H, et al. Subjugation of TGFbeta Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End Joining. Clin Cancer Res. 2018;24(23):6001–14. Epub 2018/08/09. doi: 10.1158/1078-0432.CCR-18-1346 30087144.

51. Kirshner J, Jobling MF, Pajares MJ, Ravani SA, Glick AB, Lavin MJ, et al. Inhibition of transforming growth factor-beta1 signaling attenuates ataxia telangiectasia mutated activity in response to genotoxic stress. Cancer Res. 2006;66(22):10861–9. Epub 2006/11/09. doi: 10.1158/0008-5472.CAN-06-2565 17090522.

52. Kanamoto T, Hellman U, Heldin CH, Souchelnytskyi S. Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair. EMBO J. 2002;21(5):1219–30. Epub 2002/02/28. doi: 10.1093/emboj/21.5.1219 11867550; PubMed Central PMCID: PMC125881.

53. Horikoshi N, Pandita RK, Mujoo K, Hambarde S, Sharma D, Mattoo AR, et al. beta2-spectrin depletion impairs DNA damage repair. Oncotarget. 2016;7(23):33557–70. Epub 2016/06/02. doi: 10.18632/oncotarget.9677 27248179; PubMed Central PMCID: PMC5085102.

54. Schoonen PM, van Vugt M. Never tear us a-PARP: Dealing with DNA lesions during mitosis. Mol Cell Oncol. 2018;5(1):e1382670. Epub 2018/02/07. doi: 10.1080/23723556.2017.1382670 29404385; PubMed Central PMCID: PMC5791853.

55. Nicolae CM, Aho ER, Vlahos AH, Choe KN, De S, Karras GI, et al. The ADP-ribosyltransferase PARP10/ARTD10 interacts with proliferating cell nuclear antigen (PCNA) and is required for DNA damage tolerance. J Biol Chem. 2014;289(19):13627–37. Epub 2014/04/04. doi: 10.1074/jbc.M114.556340 24695737; PubMed Central PMCID: PMC4036367.

56. Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nature protocols. 2017;12(4):828–63. Epub 2017/03/24. doi: 10.1038/nprot.2017.016 28333914; PubMed Central PMCID: PMC5526071.

57. Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, Shanks E, et al. Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods. 2009;6(8):569–75. doi: 10.1038/nmeth.1351 19644458; PubMed Central PMCID: PMC2789971.

58. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25–9. Epub 2000/05/10. doi: 10.1038/75556 10802651; PubMed Central PMCID: PMC3037419.

59. The Gene Ontology C. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. 2019;47(D1):D330–D8. Epub 2018/11/06. doi: 10.1093/nar/gky1055 30395331; PubMed Central PMCID: PMC6323945.

60. Li Z, Li Q, Han L, Tian N, Liang Q, Li Y, et al. Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines. Oncol Rep. 2016;35(2):1013–9. Epub 2016/01/01. doi: 10.3892/or.2015.4465 26718027.

61. Sun Q, Li S, Li J, Fu Q, Wang Z, Li B, et al. Homoharringtonine regulates the alternative splicing of Bcl-x and caspase 9 through a protein phosphatase 1-dependent mechanism. BMC Complement Altern Med. 2018;18(1):164. Epub 2018/05/24. doi: 10.1186/s12906-018-2233-6 29788973; PubMed Central PMCID: PMC5964699.

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 11
Nejčtenější tento týden
Nejčtenější v tomto čísle

Zvyšte si kvalifikaci online z pohodlí domova

Třikrát z interní medicíny
nový kurz
Autoři: MUDr. Jana Kubátová

Pokročilá Parkinsonova nemoc − úskalí a možnosti léčby
Autoři: doc. MUDr. Marek Baláž, Ph.D.

Léčba diabetes mellitus 2. typu pomocí GLP- 1 RA

Depresivní porucha a zánětlivé procesy
Autoři: MUDr. Juraj Tkáč

Methotrexát a jeho formy podávání v revmatologii
Autoři: MUDr. Liliana Šedová

Všechny kurzy
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se