Phenotypic Screening for Inhibitors of a Mutant Thrombopoietin Receptor

  • Anna Ngo
  • Ann Koay
  • Christian Pecquet
  • Carmen C. Diaconu
  • David A. Jenkins
  • Andrew K. Shiau
  • Stefan N. Constantinescu
  • Meng Ling Choong
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1787)

Abstract

An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.

Key words

Myeloproliferative neoplasms Thrombopoietin receptor Phenotypic screening Cell-based assay Cell viability ATP 

Notes

Acknowledgments

There is no financial conflict of interest among the authors. Support for A.N., A.K., and M.L.C was from an A*STAR core funding to the Experimental Therapeutics Centre. Support for C.C.D. was from UEFISCDI PCCA PN II 133/2012 and OPERATIONAL COMPETITIVITATY PROGRAM 2014-2020 POC-A1-A1.1.4-E-2015/P_37_798/149/2016. S.N.C. and A.K.S. received funding from the Ludwig Institute for Cancer Research. S.N.C. also received supports from FRS-FNRS, Salus Sanguinis Foundation, the Action de Recherche Concertée project ARC10/15-027 of the Université catholique de Louvain, the Fondation contre le Cancer, the PAI Programs BCHM61B5, and Belgian Medical Genetics Initiative.

References

  1. 1.
    Levine RL, Pardanani A, Tefferi A, Gilliland DG (2007) Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer 7:673–683CrossRefGoogle Scholar
  2. 2.
    Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M et al (2006) MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood 108:3472–3476CrossRefGoogle Scholar
  3. 3.
    Pecquet C, Staerk J, Chaligne R, Goss V, Lee KA, Zhang X et al (2010) Induction of myeloproliferative disorder and myelofibrosis by thrombopoietin receptor W515 mutants is mediated by cytosolic tyrosine 112 of the receptor. Blood 115:1037–1048CrossRefGoogle Scholar
  4. 4.
    Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD et al (2013) Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 369:2379–2390CrossRefGoogle Scholar
  5. 5.
    Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al (2013) Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 369:2391–2405CrossRefGoogle Scholar
  6. 6.
    Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C et al (2016) Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood 127:1325–1335CrossRefGoogle Scholar
  7. 7.
    Hobbs GS, Rampal RK (2015) JAK2 mutations and JAK inhibitors in the management of myeloproliferative neoplasms. Contemp Oncol 7:22–28Google Scholar
  8. 8.
    Ngo A, Koay A, Pecquet C, Diaconu CC, Ould-Amer Y, Huang Q et al (2016) A phenotypic screen for small-molecule inhibitors of constitutively active mutant Thrombopoietin receptor implicated in myeloproliferative neoplasms. Comb Chem High Throughput Screen 19:824–833CrossRefGoogle Scholar
  9. 9.
    Defour JP, Itaya M, Gryshkova V, Brett IC, Pecquet C, Sato T et al (2013) Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation. Proc Natl Acad Sci U S A 110:2540–2545CrossRefGoogle Scholar
  10. 10.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Anna Ngo
    • 1
  • Ann Koay
    • 1
  • Christian Pecquet
    • 2
  • Carmen C. Diaconu
    • 3
  • David A. Jenkins
    • 4
  • Andrew K. Shiau
    • 4
  • Stefan N. Constantinescu
    • 2
  • Meng Ling Choong
    • 1
  1. 1.Experimental Therapeutics Centre, Agency for Science Technology and Research (A*STAR)SingaporeSingapore
  2. 2.Ludwig Institute for Cancer ResearchUniversité catholique de Louvain and de Duve InstituteBrusselsBelgium
  3. 3.Stefan S. Nicolau Institute of Virology, Romanian AcademyBucharestRomania
  4. 4.Small Molecule Discovery Program, Ludwig Institute for Cancer ResearchLa JollaUSA

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