CRISPR editing of sftb-1/SF3B1 in Caenorhabditis elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing

English version

Autoři: Xènia Serrat ^aff001; Dmytro Kukhtar ^aff001; Eric Cornes ^aff002; Anna Esteve-Codina ^aff003; Helena Benlloch ^aff001; Germano Cecere ^aff002; Julián Cerón ^aff001
Působiště autorů: Modeling human disease in . Group, Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge–IDIBELL, Barcelona, Spain ^aff001; Mechanisms of Epigenetic Inheritance, Department of Developmental and Stem Cell Biology, Institut Pasteur, UMR3738, CNRS, Paris, France ^aff002; CNAG‐CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain ^aff003; Universitat Pompeu Fabra (UPF), Barcelona, Spain ^aff004
Vyšlo v časopise: CRISPR editing of sftb-1/SF3B1 in Caenorhabditis elegans allows the identification of synthetic interactions with cancer-related mutations and the chemical inhibition of splicing. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008464
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008464

Souhrn

SF3B1 is the most frequently mutated splicing factor in cancer. Mutations in SF3B1 likely confer clonal advantages to cancer cells but they may also confer vulnerabilities that can be therapeutically targeted. SF3B1 cancer mutations can be maintained in homozygosis in C. elegans, allowing synthetic lethal screens with a homogeneous population of animals. These mutations cause alternative splicing (AS) defects in C. elegans, as it occurs in SF3B1-mutated human cells. In a screen, we identified RNAi of U2 snRNP components that cause synthetic lethality with sftb-1/SF3B1 mutations. We also detected synthetic interactions between sftb-1 mutants and cancer-related mutations in uaf-2/U2AF1 or rsp-4/SRSF2, demonstrating that this model can identify interactions between mutations that are mutually exclusive in human tumors. Finally, we have edited an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B or herboxidiene. Thus, we have established a multicellular model for SF3B1 mutations amenable for high-throughput genetic and chemical screens.

Klíčová slova:

Alleles – Caenorhabditis elegans – CRISPR – Larvae – Mutant strains – RNA interference – Sequence motif analysis – Missense mutation

Zdroje

1. Urbanski LM, Leclair N, Anczuków O. Alternative-splicing defects in cancer: Splicing regulators and their downstream targets, guiding the way to novel cancer therapeutics. WIREs RNA. 2018;9: e1476. doi: 10.1002/wrna.1476 29693319

2. Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478: 64–69. doi: 10.1038/nature10496 21909114

3. Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D, et al. Somatic SF3B1 Mutation in Myelodysplasia with Ring Sideroblasts. N Engl J Med. 2011;365: 1384–1395. doi: 10.1056/NEJMoa1103283 21995386

4. Malcovati L, Karimi M, Papaemmanuil E, Ambaglio I, Jädersten M, Jansson M, et al. SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood. 2015;126: 233–241. doi: 10.1182/blood-2015-03-633537 25957392

5. Quesada V, Conde L, Villamor N, Ordóñez GR, Jares P, Bassaganyas L, et al. Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat Genet. 2012;44: 47–52. doi: 10.1038/ng.1032 22158541

6. te Raa G, Derks I, Navrkalova V, Skowronska A, Moerland P, van Laar J, et al. The impact of SF3B1 mutations in CLL on the DNA-damage response. Leukemia. 2015;29: 1133–1142. doi: 10.1038/leu.2014.318 25371178

7. Wang L, Lawrence MS, Wan Y, Stojanov P, Sougnez C, Stevenson K, et al. SF3B1 and Other Novel Cancer Genes in Chronic Lymphocytic Leukemia. N Engl J Med. 2011;365: 2497–2506. doi: 10.1056/NEJMoa1109016 22150006

8. Furney SJ, Pedersen M, Gentien D, Dumont AG, Rapinat A, Desjardins L, et al. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov. 2013;3: 1122–1129. doi: 10.1158/2159-8290.CD-13-0330 23861464

9. Harbour J, Roberson E, Anbunathan H, Onken M, Worley L, Bowcock M. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat Genet. 2013;45: 133–135. doi: 10.1038/ng.2523 23313955

10. Martin M, Maßhöfer L, Temming P, Rahmann S, Metz C, Bornfeld N, et al. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat Genet. 2013;45: 933–936. doi: 10.1038/ng.2674 23793026

11. Biankin A V., Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012;491: 399–405. doi: 10.1038/nature11547 23103869

12. Maguire SL, Leonidou A, Wai P, Marchiò C, Ng CK, Sapino A, et al. SF3B1 mutations constitute a novel therapeutic target in breast cancer. J Pathol. 2015;235: 571–580. doi: 10.1002/path.4483 25424858

13. Darman RB, Seiler M, Agrawal AA, Lim KH, Peng S, Aird D, et al. Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3’ Splice Site Selection through Use of a Different Branch Point. Cell Rep. 2015;13: 1033–1045. doi: 10.1016/j.celrep.2015.09.053 26565915

14. DeBoever C, Ghia EM, Shepard PJ, Rassenti L, Barrett CL, Jepsen K, et al. Transcriptome sequencing reveals potential mechanism of cryptic 3’ splice site selection in SF3B1-mutated cancers. PLoS Comput Biol. 2015;11: e1004105. doi: 10.1371/journal.pcbi.1004105 25768983

15. Liberante FG, Lappin K, Barros EM, Vohhodina J, Grebien F, Savage KI, et al. Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1-mutant myelodysplastic syndromes as a therapeutic vulnerability. Sci Rep. 2019;9: 2678. doi: 10.1038/s41598-019-39591-7 30804405

16. Alsafadi S, Houy A, Battistella A, Popova T, Wassef M, Henry E, et al. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat Commun. 2016;7:10615. doi: 10.1038/ncomms10615 26842708

17. Gupta AK, Murthy T, Paul K V, Ramirez O, Fisher JB, Rao S, et al. Degenerate minigene library analysis enables identification of altered branch point utilization by mutant splicing factor 3B1 (SF3B1). Nucleic Acids Res. 2019;47: 970–980. doi: 10.1093/nar/gky1161 30462273

18. Shiozawa Y, Malcovati L, Gallì A, Sato-Otsubo A, Kataoka K, Sato Y, et al. Aberrant splicing and defective mRNA production induced by somatic spliceosome mutations in myelodysplasia. Nat Commun. 2018;9: 3649. doi: 10.1038/s41467-018-06063-x 30194306

19. Xu JJ, Smeets MF, Tan SY, Wall M, Purton LE, Walkley CR. Modeling human RNA spliceosome mutations in the mouse: not all mice were created equal. Exp Hematol. 2019;70: 10–23. doi: 10.1016/j.exphem.2018.11.001 30408513

20. Bonnal S, Vigevani L, Valcárcel J. The spliceosome as a target of novel antitumour drugs. Nat Rev Drug Discov. 2012;11: 847–59. doi: 10.1038/nrd3823 23123942

21. Effenberger KA, Urabe VK, Jurica MS. Modulating splicing with small molecular inhibitors of the spliceosome. WIREs RNA. 2017;8: e1381. doi: 10.1002/wrna.1381 27440103

22. DeNicola AB, Tang Y. Therapeutic approaches to treat human spliceosomal diseases. Curr Opin Biotechnol. 2019;60: 72–81. doi: 10.1016/j.copbio.2019.01.003 30772756

23. Seiler M, Yoshimi A, Darman R, Chan B, Keaney G, Thomas M, et al. H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers. Nat Med. 2018;24: 497–504. doi: 10.1038/nm.4493 29457796

24. Yokoi A, Kotake Y, Takahashi K, Kadowaki T, Matsumoto Y, Minoshima Y, et al. Biological validation that SF3b is a target of the antitumor macrolide pladienolide. FEBS J. 2011;278: 4870–4880. doi: 10.1111/j.1742-4658.2011.08387.x 21981285

25. Teng T, Tsai JH, Puyang X, Seiler M, Peng S, Prajapati S, et al. Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A-SF3b complex. Nat Commun. 2017;8: 15522. doi: 10.1038/ncomms15522 28541300

26. Hansen SR, Nikolai BJ, Spreacker PJ, Carrocci TJ, Hoskins AA. Chemical Inhibition of Pre-mRNA Splicing in Living Saccharomyces cerevisiae. Cell Chem Biol. 2019;26: 443–448. doi: 10.1016/j.chembiol.2018.11.008 30639260

27. Loerch S, Leach JR, Horner SW, Maji D, Jenkins JL, Pulvino M, et al. The pre-mRNA splicing and transcription factor Tat-SF1 is a functional partner of the spliceosome SF3b1 subunit via a U2AF homology motif interface. J Biol Chem. 2018;294: 2892–2902. doi: 10.1074/jbc.RA118.006764 30567737

28. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003;421: 231–237. doi: 10.1038/nature01278 12529635

29. Cretu C, Schmitzová J, Ponce-Salvatierra A, Dybkov O, De Laurentiis EI, Sharma K, et al. Molecular Architecture of SF3b and Structural Consequences of Its Cancer-Related Mutations. Mol Cell. 2016;64: 307–319. doi: 10.1016/j.molcel.2016.08.036 27720643

30. Carrocci TJ, Zoerner DM, Paulson JC, Hoskins AA. SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast. Nucleic Acids Res. 2017;45: 4837–4852. doi: 10.1093/nar/gkw1349 28062854

31. Wang L, Brooks AN, Fan J, Wan Y, Gambe R, Li S, et al. Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia. Cancer Cell. 2016;30: 750–763. doi: 10.1016/j.ccell.2016.10.005 27818134

32. Pellagatti A, Armstrong RN, Steeples V, Sharma E, Repapi E, Singh S, et al. Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations. Blood. 2018;132: 1225–1240. doi: 10.1182/blood-2018-04-843771 29930011

33. Shen S, Park JW, Lu Z, Lin L, Henry MD, Wu YN, et al. rMATS: Robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci U S A. 2014;111: E5593–601. doi: 10.1073/pnas.1419161111 25480548

34. Kerins JA, Hanazawa M, Dorsett M, Schedl T. PRP-17 and the Pre-mRNA Splicing Pathway Are Preferentially Required for the Proliferation Versus Meiotic Development Decision and Germline Sex Determination in Caenorhabditis elegans. Dev Dyn. 2010;239: 1555–1572. doi: 10.1002/dvdy.22274 20419786

35. Mantina P, MacDonald L, Kulaga A, Zhao L, Hansen D. A mutation in teg-4, which encodes a protein homologous to the SAP130 pre-mRNA splicing factor, disrupts the balance between proliferation and differentiation in the C. elegans germ line. Mech Dev. 2009;126: 417–429. doi: 10.1016/j.mod.2009.01.006 19368799

36. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, et al. Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing. Cell. 2012;150: 1107–1120. doi: 10.1016/j.cell.2012.08.029 22980975

37. Seiler M, Peng S, Agrawal AA, Palacino J, Teng T, Zhu P, et al. Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types. Cell Rep. 2018;23: 282–296.e4. doi: 10.1016/j.celrep.2018.01.088 29617667

38. Ilagan JO, Ramakrishnan A, Hayes B, Murphy ME, Zebari AS, Bradley P, et al. U2AF1 mutations alter splice site recognition in hematological malignancies. Genome Res. 2015;25: 14–26. doi: 10.1101/gr.181016.114 25267526

39. Lee SC-W, North K, Kim E, Jang E, Obeng E, Lu SX, et al. Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations. Cancer Cell. 2018;34: 225–241.e8. doi: 10.1016/j.ccell.2018.07.003 30107174

40. Cretu C, Agrawal AA, Cook A, Will CL, Fekkes P, Smith PG, et al. Structural Basis of Splicing Modulation by Antitumor Macrolide Compounds. Mol Cell. 2018;70: 265–273.e8. doi: 10.1016/j.molcel.2018.03.011 29656923

41. Lagisetti C, Palacios G, Goronga T, Freeman B, Caufield W, Webb TR. Optimization of antitumor modulators of pre-mRNA splicing. J Med Chem. 2013;56: 10033–10044. doi: 10.1021/jm401370h 24325474

42. Finci LI, Zhang X, Huang X, Zhou Q, Tsai J, Teng T, et al. The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action. Genes Dev. 2018;32: 309–320. doi: 10.1101/gad.311043.117 29491137

43. Dickinson DJ, Goldstein B. CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering. Genetics. 2016;202: 885–901. doi: 10.1534/genetics.115.182162 26953268

44. Nance J, Frøkjær-Jensen C. The Caenorhabditis elegans Transgenic Toolbox. Genetics. 2019;212: 959–990. doi: 10.1534/genetics.119.301506 31405997

45. An M, Henion PD. The zebrafish sf3b1b460 mutant reveals differential requirements for the sf3b1 pre-mRNA processing gene during neural crest development. Int J Dev Biol. 2012;56: 223–237. doi: 10.1387/ijdb.113383ma 22562198

46. Isono K, Mizutani-Koseki Y, Komori T, Schmidt-Zachmann MS, Koseki H. Mammalian Polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1. Genes Dev. 2005;19: 536–541. doi: 10.1101/gad.1284605 15741318

47. Zhou Q, Derti A, Ruddy D, Rakiec D, Kao I, Lira M, et al. A chemical genetics approach for the functional assessment of novel cancer genes. Cancer Res. 2015;75: 1949–1958. doi: 10.1158/0008-5472.CAN-14-2930 25788694

48. Paolella BR, Gibson WJ, Urbanski LM, Alberta JA, Zack TI, Bandopadhayay P, et al. Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability. Elife. 2017;6: e23268. doi: 10.7554/eLife.23268 28177281

49. Dolatshad H, Pellagatti A, Fernandez-Mercado M, Yip B, Malcovati L, Attwood M, et al. Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells. Leukemia. 2015;29: 1092–1103. doi: 10.1038/leu.2014.331 25428262

50. Obeng EA, Chappell RJ, Seiler M, Chen MC, Campagna DR, Schmidt PJ, et al. Physiologic Expression of Sf3b1K700E Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation. Cancer Cell. 2016;30: 404–417. doi: 10.1016/j.ccell.2016.08.006 27622333

51. Mupo A, Seiler M, Sathiaseelan V, Pance A, Yang Y, Agrawal A, et al. Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts. Leukemia. 2017;31: 720–727. doi: 10.1038/leu.2016.251 27604819

52. Yin S, Gambe RG, Sun J, Martinez AZ, Cartun ZJ, Regis FFD, et al. A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion. Cancer Cell. 2019;35: 283–296.e5. doi: 10.1016/j.ccell.2018.12.013 30712845

53. Dalton WB, Helmenstine E, Walsh N, Gondek LP, Kelkar DS, Read A, et al. Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation. J Clin Invest. 2019; doi: 10.1172/JCI125022 31393856

54. Morton JJ, Blumenthal T. RNA Processing in C. elegans. Methods in Cell Biology. Second Edi. 2011. pp. 187–217. doi: 10.1016/B978-0-12-544172-8.00007–4

55. Tourasse NJ, Millet JRM, Dupuy D. Quantitative RNA-seq meta-analysis of alternative exon usage in C. elegans. Genome Res. 2017;27: 2120–2128. doi: 10.1101/gr.224626.117 29089372

56. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008;40: 1413–1415. doi: 10.1038/ng.259 18978789

57. Ragle JM, Katzman S, Akers TF, Barberan-Soler S, Zahler AM. Coordinated tissue-specific regulation of adjacent alternative 3’ splice sites in C. elegans. Genome Res. 2015;25: 982–94. doi: 10.1101/gr.186783.114 25922281

58. Kfir N, Lev-Maor G, Glaich O, Alajem A, Datta A, Sze SK, et al. SF3B1 association with chromatin determines splicing outcomes. Cell Rep. 2015;11: 618–29. doi: 10.1016/j.celrep.2015.03.048 25892229

59. Wu G, Fan L, Edmonson MN, Shaw T, Boggs K, Easton J, et al. Inhibition of SF3B1 by molecules targeting the spliceosome results in massive aberrant exon skipping. RNA. 2018;24: 1056–1066. doi: 10.1261/rna.065383.117 29844105

60. Carrocci TJ, Paulson JC, Hoskins AA. Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex. RNA. 2018;24: 1028–1040. doi: 10.1261/rna.065664.118 29752352

61. McDiarmid TA, Au V, Loewen AD, Liang J, Mizumoto K, Moerman DG, et al. CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance. Dis Model Mech. 2018;11: dmm036517. doi: 10.1242/dmm.036517 30361258

62. Vicencio J, Martínez-Fernández C, Serrat X, Cerón J. Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans. Genetics. 2019;211: 1143–1154. doi: 10.1534/genetics.119.301965 30696716

63. Porta-de-la-Riva M, Fontrodona L, Villanueva A, Cerón J. Basic Caenorhabditis elegans Methods: Synchronization and Observation. J Vis Exp. 2012;64: e4019. doi: 10.3791/4019 22710399

64. Stiernagle T. Maintenance of C. elegans. WormBook. 2006. pp. 1–11. doi: 10.1895/wormbook.1.101.1 18050451

65. Notredame C, Higgins DG, Heringa J. T-coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000;302: 205–217. doi: 10.1006/jmbi.2000.4042 10964570

66. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2-A multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25: 1189–1191. doi: 10.1093/bioinformatics/btp033 19151095

67. Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL. CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. PLoS One. 2015;10: e0124633. doi: 10.1371/journal.pone.0124633 25909470

68. Kim H, Ishidate T, Ghanta KS, Seth M, Conte D, Shirayama M, et al. A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics. 2014;197: 1069–80. doi: 10.1534/genetics.114.166389 24879462

69. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29: 15–21. doi: 10.1093/bioinformatics/bts635 23104886

70. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12: 323. doi: 10.1186/1471-2105-12-323 21816040

71. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 25516281

72. Cvitkovic I, Jurica MS. Spliceosome database: A tool for tracking components of the spliceosome. Nucleic Acids Res. 2013;41: D132–D141. doi: 10.1093/nar/gks999 23118483

73. Rual J-F, Ceron J, Koreth J, Hao T, Nicot A-S, Hirozane-Kishikawa T, et al. Toward Improving Caenorhabditis elegans Phenome Mapping With an ORFeome-Based RNAi Library. Genome Res. 2004;14: 2162–2168. doi: 10.1101/gr.2505604 15489339

74. Moore BT, Jordan JM, Baugh LR. WormSizer: High-throughput Analysis of Nematode Size and Shape. PLoS One. 2013;8: e57142. doi: 10.1371/journal.pone.0057142 23451165

75. Forbes SA, Beare D, Boutselakis H, Bamford S, Bindal N, Tate J, et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 2017;45: D777–D783. doi: 10.1093/nar/gkw1121 27899578