A kinome-wide shRNA screen uncovers vaccinia-related kinase 3 (VRK3) as an essential gene for diffuse intrinsic pontine glioma survival

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Abstract

Diffuse intrinsic pontine glioma (or DIPG) are pediatric high-grade gliomas associated with a dismal prognosis. They harbor specific substitution in histone H3 at position K27 that induces major epigenetic dysregulations. Most clinical trials failed so far to increase survival, and radiotherapy remains the most efficient treatment, despite only transient tumor control. We conducted the first lentiviral shRNA dropout screen in newly diagnosed DIPG to generate a cancer-lethal signature as a basis for the development of specific treatments with increased efficacy and reduced side effects compared to existing anticancer therapies. The analysis uncovered 41 DIPG essential genes among the 672 genes of human kinases tested, for which several distinct interfering RNAs impaired cell expansion of three different DIPG stem-cell cultures without deleterious effect on two control neural stem cells. Among them, PLK1, AURKB, CHEK1, EGFR, and GSK3A were previously identified by similar approach in adult GBM indicating common dependencies of these cancer cells and pediatric gliomas. As expected, we observed an enrichment of genes involved in proliferation and cell death processes with a significant number of candidates belonging to PTEN/PI3K/AKT and EGFR pathways already under scrutiny in clinical trials in this disease. We highlighted VRK3, a gene involved especially in cell cycle regulation, DNA repair, and neuronal differentiation, as a non-oncogenic addiction in DIPG. Its repression totally blocked DIPG cell growth in the four cellular models evaluated, and induced cell death in H3.3-K27M cells specifically but not in H3.1-K27M cells, supporting VRK3 as an interesting and promising target in DIPG.

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References

  1. 1.

    Cohen KJ, Jabado N, Grill J. Diffuse intrinsic pontine gliomas-current management and new biologic insights. Is there a glimmer of hope? Neuro Oncol. 2017;19:1025–34.

  2. 2.

    Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily M-A, et al. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med. 2015;21:555–9.

  3. 3.

    Hargrave D, Bartels U, Bouffet E. Diffuse brainstem glioma in children: critical review of clinical trials. Lancet Oncol. 2006;7:241–8.

  4. 4.

    Warren KE. Diffuse intrinsic pontine glioma: poised for progress. Front Oncol. 2012;2: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531714/

  5. 5.

    Taylor KR, Mackay A, Truffaux N, Butterfield YS, Morozova O, Philippe C, et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nat Genet. 2014;46:457–61.

  6. 6.

    Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet. 2012;44:251–3.

  7. 7.

    Misuraca KL, Hu G, Barton KL, Chung A, Becher OJ. A novel mouse model of diffuse intrinsic pontine glioma initiated in Pax3-expressing cells. Neoplasia. 2016;18:60–70.

  8. 8.

    Plessier A, Le Dret L, Varlet P, Beccaria K, Lacombe J, Mériaux S, et al. New in vivo avatars of diffuse intrinsic pontine gliomas (DIPG) from stereotactic biopsies performed at diagnosis. Oncotarget. 2017;8:52543–59.

  9. 9.

    Truffaux N, Philippe C, Paulsson J, Andreiuolo F, Guerrini-Rousseau L, Cornilleau G, et al. Preclinical evaluation of dasatinib alone and in combination with cabozantinib for the treatment of diffuse intrinsic pontine glioma. Neuro Oncol. 2015;17:953–64.

  10. 10.

    Cheng P, Phillips E, Kim S-H, Taylor D, Hielscher T, Puccio L, et al. Kinome-wide shRNA screen identifies the receptor tyrosine kinase AXL as a key regulator for mesenchymal glioblastoma stem-like cells. Stem Cell Rep. 2015;4:899–913.

  11. 11.

    Gargiulo G, Cesaroni M, Serresi M, de Vries N, Hulsman D, Bruggeman SW, et al. In vivo RNAi screen for BMI1 targets identifies TGF-β/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis. Cancer Cell. 2013;23:660–76.

  12. 12.

    Goidts V, Bageritz J, Puccio L, Nakata S, Zapatka M, Barbus S, et al. RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival. Oncogene. 2012;31:3235–43.

  13. 13.

    Kulkarni S, Goel-Bhattacharya S, Sengupta S, Cochran BH. A large-scale RNAi screen identifies SGK1 as a key survival kinase for GBM stem cells. Mol Cancer Res. 2018;16:103–14.

  14. 14.

    Sa JK, Yoon Y, Kim M, Kim Y, Cho HJ, Lee J-K, et al. In vivo RNAi screen identifies NLK as a negative regulator of mesenchymal activity in glioblastoma. Oncotarget. 2015;6:20145–59.

  15. 15.

    Tandle AT, Kramp T, Kil WJ, Halthore A, Gehlhaus K, Shankavaram U, et al. Inhibition of polo-like kinase 1 in glioblastoma multiforme induces mitotic catastrophe and enhances radiosensitisation. Eur J Cancer. 2013;49:3020–8.

  16. 16.

    Wurdak H, Zhu S, Romero A, Lorger M, Watson J, Chiang C-Y, et al. An RNAi screen identifies TRRAP as a regulator of brain tumor-initiating cell differentiation. Cell Stem Cell. 2010;6:37–47.

  17. 17.

    Yang J, Fan J, Li Y, Li F, Chen P, Fan Y, et al. Genome-wide RNAi screening identifies genes inhibiting the migration of glioblastoma cells. PLoS ONE 2013;8:e61915.

  18. 18.

    Hart T, Brown KR, Sircoulomb F, Rottapel R, Moffat J. Measuring error rates in genomic perturbation screens: gold standards for human functional genomics. Mol Syst Biol. 2014;10:733.

  19. 19.

    Hart T, Tong AHY, Chan K, Van Leeuwen J, Seetharaman A, Aregger M, et al. Evaluation and design of genome-wide CRISPR/SpCas9 knockout screens. G3 (Bethesda). 2017;7:2719–27.

  20. 20.

    Varlet P, Debily M-A, Teuff GL, Tauziede-Espariat A, Pages M, Andreiuolo F. et al. DIPG-20 pre-randomisation central review and real-time biomarkers screening in the multicentre biological medicine for dipg eradication (biomede)trial: lessons learnt from the first 120 biopsies. Neuro Oncol. 2018;20:i52–3.

  21. 21.

    Zarghooni M, Bartels U, Lee E, Buczkowicz P, Morrison A, Huang A, et al. Whole-genome profiling of pediatric diffuse intrinsic pontine gliomas highlights platelet-derived growth factor receptor α and poly (ADP-ribose) polymerase as potential therapeutic targets. J Clin Oncol. 2010;28:1337–44.

  22. 22.

    Ding Y, Hubert CG, Herman J, Corrin P, Toledo CM, Skutt-Kakaria K, et al. Cancer-specific requirement for BUB1B/BUBR1 in human brain tumor isolates and genetically transformed cells. Cancer Discov. 2013;3:198–211.

  23. 23.

    Farhan M, Wang H, Gaur U, Little PJ, Xu J, Zheng W. FOXO signaling pathways as therapeutic targets in cancer. Int J Biol Sci 2017;13:815–27.

  24. 24.

    Shriver M, Marimuthu S, Paul C, Geist J, Seale T, Konstantopoulos K, et al. Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit. Oncotarget. 2016;7:45414–28.

  25. 25.

    Grill J, Puget S, Andreiuolo F, Philippe C, MacConaill L. Critical oncogenic mutations in newly diagnosed pediatric diffuse intrinsic pontine glioma. Pediatr Blood Cancer. 2012;58:489–91.

  26. 26.

    Lee N, Kim D-K, Han SH, Ryu HG, Park SJ, Kim K-T, et al. Comparative interactomes of VRK1 and VRK3 with their distinct roles in the cell cycle of liver cancer. Mol Cells 2017;40:621–31.

  27. 27.

    Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017;45:W98–102.

  28. 28.

    Song H, Kim W, Kim S-H, Kim K-T. VRK3-mediated nuclear localization of HSP70 prevents glutamate excitotoxicity-induced apoptosis and Aβ accumulation via enhancement of ERK phosphatase VHR activity. Sci Rep. 2016;6:38452.

  29. 29.

    Chen J, Li Y, Yu T-S, McKay RM, Burns DK, Kernie SG. et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature. 2012;488:522–6.

  30. 30.

    Filbin MG, Tirosh I, Hovestadt V, Shaw ML, Escalante LE, Mathewson ND. et al. Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq. Science.2018;360:331–5.

  31. 31.

    Du Z, Song X, Yan F, Wang J, Zhao Y, Liu S. Genome-wide transcriptional analysis of BRD4-regulated genes and pathways in human glioma U251 cells. Int J Oncol. 2018. https://doi.org/10.3892/ijo.2018.4324.

  32. 32.

    Tang Y, He W, Wei Y, Qu Z, Zeng J, Qin C. Screening key genes and pathways in glioma based on gene set enrichment analysis and meta-analysis. J Mol Neurosci 2013;50:324–32.

  33. 33.

    Korur S, Huber RM, Sivasankaran B, Petrich M, Morin P, Hemmings BA, et al. GSK3beta regulates differentiation and growth arrest in glioblastoma. PLoS ONE 2009;4:e7443.

  34. 34.

    Kotliarova S, Pastorino S, Kovell LC, Kotliarov Y, Song H, Zhang W, et al. Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation. Cancer Res. 2008;68:6643–51.

  35. 35.

    Di Stefano AL, Fucci A, Frattini V, Labussiere M, Mokhtari K, Zoppoli P, et al. Detection, characterization, and inhibition of FGFR-TACC fusions in IDH wild-type glioma. Clin Cancer Res. 2015;21:3307–17.

  36. 36.

    Kang M-S, Choi T-Y, Ryu HG, Lee D, Lee S-H, Choi S-Y, et al. Autism-like behavior caused by deletion of vaccinia-related kinase 3 is improved by TrkB stimulation. J Exp Med. 2017;214:2947–66.

  37. 37.

    Fedorov O, Marsden B, Pogacic V, Rellos P, Müller S, Bullock AN, et al. A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases. Proc Natl Acad Sci USA. 2007;104:20523–8.

  38. 38.

    O’Rahilly R, Müller F, Hutchins GM, Moore GW. Computer ranking of the sequence of appearance of 73 features of the brain and related structures in staged human embryos during the sixth week of development. Am J Anat. 1987;180:69–86.

  39. 39.

    sgRNA/shRNA/ORF PCR for Illumina Sequencing Protocol, GPP Web Portal. http://portals.broadinstitute.org/gpp/public/resources/protocols

  40. 40.

    Barde I, Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc Neurosci. 2010.

  41. 41.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.

  42. 42.

    Chang JT, Nevins JR. Gather: a systems approach to interpreting genomic signatures. Bioinformatics. 2006;22:2926–33.

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Acknowledgements

DC, JG, and MAD acknowledge financial support from Canceropole Ile de France, INCa, La Ligue Contre le Cancer (projet DILESS—DM/CB/003–17) and the charity l’Etoile de Martin. The authors are grateful to the Necker hospital tumor and DNA banks and the Necker operating room nurses/assistants for their technical assistance. CSE acknowledges financial support from National Council for Scientific and Technological Development (CNPq)/ Program “Science without borders” in Brasil. We thank N. Droin and the genomic core facility of Gustave Roussy.

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Correspondence to Marie-Anne Debily.

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