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  • Original Article
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Sensitivity and Resistance to Therapy

Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment

Leukemia volume 22, pages 1395–1401 (2008)Cite this article

Abstract

Relapse in acute myeloid leukaemia (AML) is mediated by survival of leukaemic stem cells following remission-induction chemotherapy. It would therefore be useful to identify therapeutic agents that target leukaemic stem cells. We devised a flow cytometric chemosensitivity assay allowing 48 h culture of leukaemic blasts in a defined microenvironment followed by enumeration of viable CD34+CD38−CD123+ leukaemic stem and progenitor cells (LSPC). The assay was used to investigate the LSPC response to cytosine arabinoside (Ara-C) and to the FLT3 inhibitor AG1296. There was a 3.6-fold increase in Ara-C-treated LSPC survival under defined ‘niche-like’ conditions compared to culture without microenvironmental support. Nine AML samples with internal tandem duplications of FLT3 (FLT3/ITDs) were treated with AG1296. Three samples were very sensitive (>50% kill) and 4 were moderately sensitive (10–50% kill) in bulk suspension culture without microenvironmental support. However, under defined ‘niche-like’ conditions, the survival of LSPC was enhanced rather than inhibited by AG1296 treatment. We conclude that an interaction between LSPC and a defined in vitro microenvironment models a chemoresistant niche. Our data point to a need to investigate more novel chemotherapeutic agents under these stringent conditions to identify agents that may be suitable to target minimal residual disease in AML.

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References

  1. Gilliland DG, Griffin JD . The roles of FLT3 in hematopoiesis and leukemia. Blood 2002; 100: 1532–1542.

    Article  CAS  PubMed  Google Scholar 

  2. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1, 612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties. Blood 1998; 92: 2322–2333.

    CAS  PubMed  Google Scholar 

  3. Gale RE, Hills R, Kottaridis PD, Srirangan S, Wheatley K, Burnett AK et al. No evidence that FLT3 status should be considered as an indicator for transplantation in acute myeloid leukemia (AML): an analysis of 1135 patients, excluding acute promyelocytic leukemia, from the UK MRC AML10 and 12 trials. Blood 2005; 106: 3658–3665.

    Article  CAS  PubMed  Google Scholar 

  4. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645–648.

    Article  CAS  PubMed  Google Scholar 

  5. Bonnet D, Dick JE . Human acute leukemia is organised as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–737.

    Article  CAS  PubMed  Google Scholar 

  6. Hope KJ, Jin L, Dick JE . Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 2004; 5: 738–743.

    Article  CAS  PubMed  Google Scholar 

  7. Wang JC, Dick JE . Cancer stem cells: lessons from leukemia. Trends Cell Biol 2005; 15: 494–501.

    Article  CAS  PubMed  Google Scholar 

  8. van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005; 11: 6520–6527.

    Article  CAS  PubMed  Google Scholar 

  9. Jordan CT, Upchurch D, Szilvassy SJ, Guzman ML, Howard DS, Pettigrew AL et al. The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia 2000; 14: 1777–1784.

    Article  CAS  PubMed  Google Scholar 

  10. Arai F, Hirao A, Suda T . Regulation of hematopoiesis and its interaction with stem cell niches. Int J Hematol 2005; 82: 371–376.

    Article  CAS  PubMed  Google Scholar 

  11. Taichman RS . Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem-cell niche. Blood 2005; 105: 2631–2639.

    Article  CAS  PubMed  Google Scholar 

  12. Hazlehurst LA, Landowski TH, Dalton WS . Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death. Oncogene 2003; 22: 7396–7402.

    Article  CAS  PubMed  Google Scholar 

  13. Matsunaga T, Takemoto N, Sato T, Takimoto R, Tanaka I, Fujimi A et al. Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia. Nat Med 2003; 9: 1158–1165.

    Article  CAS  PubMed  Google Scholar 

  14. Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE, McCubrey JA . JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 2004; 18: 189–218.

    Article  CAS  PubMed  Google Scholar 

  15. Ailles LE, Gerhard B, Hogge DE . Detection and characterization of primitive malignant and normal progenitors in patients with acute myelogenous leukemia using long-term coculture with supportive feeder layers and cytokines. Blood 1997; 90: 2555–2564.

    CAS  PubMed  Google Scholar 

  16. Guan Y, Hogge DE . Proliferative status of primitive hematopoietic progenitors from patients with acute myelogenous leukemia (AML). Leukemia 2000; 14: 2135–2141.

    Article  CAS  PubMed  Google Scholar 

  17. Sutherland H, Blair A, Vercauteren S, Zapf R . Detection and clinical significance of human acute myeloid leukaemia progenitors capable of long-term proliferation in vitro. Br J Haematol 2001; 114: 296–306.

    Article  CAS  PubMed  Google Scholar 

  18. Hatfield K, Ryningen A, Corbascio M, Bruserud O . Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts. Int J Cancer 2006; 119: 2313–2321.

    Article  CAS  PubMed  Google Scholar 

  19. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004; 118: 149–161.

    Article  CAS  PubMed  Google Scholar 

  20. Smith BD, Levis M, Beran M, Giles F, Kantarjian H, Berg K et al. Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. Blood 2004; 103: 3669–3676.

    Article  CAS  PubMed  Google Scholar 

  21. Stone RM, DeAngelo DJ, Klimek V, Galinsky I, Estey E, Nimer SD et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105: 54–60.

    Article  CAS  PubMed  Google Scholar 

  22. Levis M, Murphy KM, Pham R, Kim KT, Stine A, Li L et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood 2005; 106: 673–680.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Birkenkamp KU, Geugien M, Lemmink HH, Kruijer W, Vellenga E . Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts. Leukemia 2001; 15: 1923–1931.

    Article  CAS  PubMed  Google Scholar 

  24. Tse KF, Allebach J, Levis M, Smith BD, Bohmer FD, Small D . Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor. Leukemia 2002; 16: 2027–2036.

    Article  CAS  PubMed  Google Scholar 

  25. Hunter HM, Pallis M, Seedhouse CH, Grundy M, Gray C, Russell NH . The expression of P-glycoprotein in AML cells with FLT3 internal tandem duplications is associated with reduced apoptosis in response to FLT3 inhibitors. Br J Haematol 2004; 127: 26–33.

    Article  CAS  PubMed  Google Scholar 

  26. Pallis M, Syan J, Russell NH . Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34. Cytometry 1999; 37: 308–313.

    Article  CAS  PubMed  Google Scholar 

  27. Altman D . Practical statistics for medical research. Chapman and Hall: London, 1991, 162pp.

    Google Scholar 

  28. Seedhouse CH, Hunter HM, Lloyd-Lewis B, Massip AM, Pallis M, Carter GI et al. DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412. Leukemia 2006; 20: 2130–2136.

    Article  CAS  PubMed  Google Scholar 

  29. van Rhenen A, Moshaver B, Kelder A, Feller N, Nieuwint AW, Zweegman S et al. Aberrant marker expression patterns on the CD34+CD38− stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission. Leukemia 2007; 21: 1700–1707.

    Article  CAS  PubMed  Google Scholar 

  30. Van Der Pol MA, Feller N, Roseboom M, Moshaver B, Westra G, Broxterman HJ et al. Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells. Haematologica 2003; 88: 983–993.

    PubMed  Google Scholar 

  31. Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 2007; 25: 1315–1321.

    Article  CAS  PubMed  Google Scholar 

  32. Florian S, Sonneck K, Hauswirth AW, Krauth MT, Schernthaner GH, Sperr WR et al. Detection of molecular targets on the surface of CD34+/CD38− stem cells in various myeloid malignancies. Leuk Lymph 2006; 47: 207–222.

    Article  CAS  Google Scholar 

  33. Guan Y, Gerhard B, Hogge DE . Detection, isolation and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML). Blood 2002; 5: 5.

    Google Scholar 

  34. Tao M, Li B, Nayini J, Andrews CB, Huang RW, Devemy E et al. SCF, IL-1beta, IL-1ra and GM-CSF in the bone marrow and serum of normal individuals and of AML and CML patients. Cytokine 2000; 12: 699–707.

    Article  CAS  PubMed  Google Scholar 

  35. Guzman ML, Neering SJ, Upchurch D, Grimes B, Howard DS, Rizzieri DA et al. Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells. Blood 2001; 98: 2301–2307.

    Article  CAS  PubMed  Google Scholar 

  36. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA et al. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001; 98: 1752–1759.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the NCRN adult working party for the permission to use trial samples. The method of analysis of rare events in flow cytometry was suggested to us by Dr T Hoy, Department of Haematology, University of Cardiff. All research was carried out in the Division of Haematology, University of Nottingham. This study was funded by a project grant from the Leukaemia Research Fund. Ullas Mony was in receipt of an Overseas Research Scholarship.

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  1. Division of Haematology, University of Nottingham, Nottingham, UK

    U Mony, M Jawad, C Seedhouse & N Russell

  2. Department of Haematology, Nottingham University Hospitals, Nottingham, UK

    N Russell & M Pallis

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  1. U Mony
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  2. M Jawad
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  3. C Seedhouse
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Correspondence to M Pallis.

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Mony, U., Jawad, M., Seedhouse, C. et al. Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment. Leukemia 22, 1395–1401 (2008). https://doi.org/10.1038/leu.2008.125

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