Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Therapy

Combinations of the FLT3 inhibitor CEP-701 and chemotherapy synergistically kill infant and childhood MLL-rearranged ALL cells in a sequence-dependent manner

Abstract

Mixed lineage leukemia (MLL) rearrangements occur in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL). These cases have a poor prognosis with current therapy. The FLT3 kinase is overexpressed and constitutively activated in MLL-rearranged ALL cells. The FLT3 inhibitor CEP-701 selectively kills these cells, but is unlikely to be curative if used as monotherapy. To identify potentially synergistic combination strategies, we studied CEP-701 and six standard chemotherapeutic agents in three sequences of exposure (S1: chemotherapy followed by CEP-701, S2: simultaneous exposure to both; and S3: CEP-701 followed by chemotherapy) using MLL-rearranged ALL cell lines and patient bone marrow samples. MTT cytotoxicity and annexin V binding apoptosis assays were used to assess antileukemic effects. Combination indices (CI) were calculated for each combination (CI<0.9 – synergistic; CI 0.9–1.1 – additive; CI>1.1 – antagonistic). A striking pattern of sequence-dependent synergy was observed: S1 was markedly synergistic (mean CI=0.59±0.10), S2 was additive (mean CI=0.99±0.09) and S3 was antagonistic (mean CI=1.23±0.10). The sequence dependence is attributable to the effect of CEP-701 on cell cycle kinetics, and is mediated specifically by FLT3 inhibition, as these effects are not seen in control cells without activated FLT3.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Chen CS, Sorensen PH, Domer PH, Reaman GH, Korsmeyer SJ, Heerema NA et al. Molecular rearrangements on chromosome 11q23 predominate in infant acute lymphoblastic leukemia and are associated with specific biologic variables and poor outcome. Blood 1993; 81: 2386–2393.

    CAS  PubMed  Google Scholar 

  2. Pui CH, Behm FG, Downing JR, Hancock ML, Shurtleff SA, Ribeiro RC et al. 11q23/MLL rearrangement confers a poor prognosis in infants with acute lymphoblastic leukemia. J Clin Oncol 1994; 12: 909–915.

    Article  CAS  PubMed  Google Scholar 

  3. Rubnitz JE, Link MP, Shuster JJ, Carroll AJ, Hakami N, Frankel LS et al. Frequency and prognostic significance of HRX rearrangements in infant acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood 1994; 84: 570–573.

    CAS  PubMed  Google Scholar 

  4. Cimino G, Rapanotti MC, Rivolta A, Lo Coco F, D'Arcangelo E, Rondelli R et al. Prognostic relevance of ALL-1 gene rearrangement in infant acute leukemias. Leukemia 1995; 9: 391–395.

    CAS  PubMed  Google Scholar 

  5. Heerema NA, Arthur DC, Sather H, Albo V, Feusner J, Lange BJ et al. Cytogenetic features of infants less than 12 months of age at diagnosis of acute lymphoblastic leukemia: impact of the 11q23 breakpoint on outcome: a report of the Childrens Cancer Group. Blood 1994; 83: 2274–2284.

    CAS  PubMed  Google Scholar 

  6. Heerema NA, Sather HN, Ge J, Arthur DC, Hilden JM, Trigg ME et al. Cytogenetic studies of infant acute lymphoblastic leukemia: poor prognosis of infants with t(4;11) – a report of the Children's Cancer Group. Leukemia 1999; 13: 679–686.

    Article  CAS  PubMed  Google Scholar 

  7. Reaman GH, Sposto R, Sensel MG, Lange BJ, Feusner JH, Heerema NA et al. Treatment outcome and prognostic factors for infants with acute lymphoblastic leukemia treated on two consecutive trials of the Children's Cancer Group. J Clin Oncol 1999; 17: 445–455.

    Article  CAS  PubMed  Google Scholar 

  8. Pui CH, Gaynon PS, Boyett JM, Chessells JM, Baruchel A, Kamps W et al. Outcome of treatment in childhood acute lymphoblastic leukaemia with rearrangements of the 11q23 chromosomal region. Lancet 2002; 359: 1909–1915.

    Article  PubMed  Google Scholar 

  9. Pui CH, Chessells JM, Camitta B, Baruchel A, Biondi A, Boyett JM et al. Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements. Leukemia 2003; 17: 700–706.

    Article  CAS  PubMed  Google Scholar 

  10. Behm FG, Raimondi SC, Frestedt JL, Liu Q, Crist WM, Downing JR et al. Rearrangement of the MLL gene confers a poor prognosis in childhood acute lymphoblastic leukemia, regardless of presenting age. Blood 1996; 87: 2870–2877.

    CAS  PubMed  Google Scholar 

  11. Felix CA, Lange BJ . Leukemia in infants. Oncologist 1999; 4: 225–240.

    CAS  PubMed  Google Scholar 

  12. Chessells JM . Relapsed lymphoblastic leukaemia in children: a continuing challenge. Br J Haematol 1998; 102: 423–438.

    Article  CAS  PubMed  Google Scholar 

  13. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden MD et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet 2002; 30: 41–47.

    Article  CAS  PubMed  Google Scholar 

  14. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Mahfouz R et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002; 1: 133–143.

    Article  CAS  PubMed  Google Scholar 

  15. Ross ME, Zhou X, Song G, Shurtleff SA, Girtman K, Williams WK et al. Classification of pediatric acute lymphoblastic leukemia by gene expression profiling. Blood 2003; 102: 2951–2959.

    Article  CAS  PubMed  Google Scholar 

  16. Armstrong SA, Kung AL, Mabon ME, Silverman LB, Stam RW, Den Boer ML et al. Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification. Cancer Cell 2003; 3: 173–183.

    Article  CAS  PubMed  Google Scholar 

  17. Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA et al. FLT3 mutations in childhood acute lymphoblastic leukemia. Blood 2004; 103: 3544–3546.

    Article  CAS  PubMed  Google Scholar 

  18. Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R et al. FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood 2004; 103: 1085–1088.

    Article  CAS  PubMed  Google Scholar 

  19. Brown P, Small D . FLT3 inhibitors: a paradigm for the development of targeted therapeutics for paediatric cancer. Eur J Cancer 2004; 40: 707–721 (discussion 722–724).

    Article  CAS  PubMed  Google Scholar 

  20. Brown P, Levis M, Shurtleff S, Campana D, Downing J, Small D . FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression. Blood 2005; 105: 812–820.

    Article  CAS  PubMed  Google Scholar 

  21. Stam RW, den Boer ML, Schneider P, Nollau P, Horstmann M, Beverloo HB et al. Targeting FLT3 in primary MLL-gene-rearranged infant acute lymphoblastic leukemia. Blood 2005; 106: 2484–2490.

    Article  CAS  PubMed  Google Scholar 

  22. Levis M, Allebach J, Tse KF, Zheng R, Baldwin BR, Smith BD et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 2002; 99: 3885–3891.

    Article  CAS  PubMed  Google Scholar 

  23. Chou TC, Talalay P . Generalized equations for the analysis of inhibitions of Michaelis–Menten and higher-order kinetic systems with two or more mutually exclusive and nonexclusive inhibitors. Eur J Biochem 1981; 115: 207–216.

    Article  CAS  PubMed  Google Scholar 

  24. Levis M, Pham R, Smith BD, Small D . In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects. Blood 2004; 104: 1145–1150.

    Article  CAS  PubMed  Google Scholar 

  25. Tkachuk DC, Kohler S, Cleary ML . Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691–700.

    Article  CAS  PubMed  Google Scholar 

  26. Greil J, Gramatzki M, Burger R, Marschalek R, Peltner M, Trautmann U et al. The acute lymphoblastic leukaemia cell line SEM with t(4;11) chromosomal rearrangement is biphenotypic and responsive to interleukin-7. Br J Haematol 1994; 86: 275–283.

    Article  CAS  PubMed  Google Scholar 

  27. Stong RC, Korsmeyer SJ, Parkin JL, Arthur DC, Kersey JH . Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics. Blood 1985; 65: 21–31.

    CAS  PubMed  Google Scholar 

  28. Ramakers-van Woerden NL, Beverloo HB, Veerman AJ, Camitta BM, Loonen AH, van Wering ER et al. In vitro drug-resistance profile in infant acute lymphoblastic leukemia in relation to age, MLL rearrangements and immunophenotype. Leukemia 2004; 18: 521–529.

    Article  CAS  PubMed  Google Scholar 

  29. Pieters R, den Boer ML, Durian M, Janka G, Schmiegelow K, Kaspers GJ et al. Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia – implications for treatment of infants. Leukemia 1998; 12: 1344–1348.

    Article  CAS  PubMed  Google Scholar 

  30. Pieters R, Kaspers GJ, van Wering ER, Huismans DR, Loonen AH, Hahlen K et al. Cellular drug resistance profiles that might explain the prognostic value of immunophenotype and age in childhood acute lymphoblastic leukemia. Leukemia 1993; 7: 392–397.

    CAS  PubMed  Google Scholar 

  31. Kim KT, Baird K, Ahn JY, Meltzer P, Lilly M, Levis M et al. Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival. Blood 2005; 105: 1759–1767.

    Article  CAS  PubMed  Google Scholar 

  32. 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 

  33. Tse KF, Novelli E, Civin CI, Bohmer FD, Small D . Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia 2001; 15: 1001–1010.

    Article  CAS  PubMed  Google Scholar 

  34. Minami Y, Yamamoto K, Kiyoi H, Ueda R, Saito H, Naoe T . Different antiapoptotic pathways between wild-type and mutated FLT3: insights into therapeutic targets in leukemia. Blood 2003; 102: 2969–2975.

    Article  CAS  PubMed  Google Scholar 

  35. Jonsson M, Engstrom M, Jonsson JI . FLT3 ligand regulates apoptosis through AKT-dependent inactivation of transcription factor FoxO3. Biochem Biophys Res Commun 2004; 318: 899–903.

    Article  PubMed  Google Scholar 

  36. Scheijen B, Ngo HT, Kang H, Griffin JD . FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins. Oncogene 2004; 23: 3338–3349.

    Article  CAS  PubMed  Google Scholar 

  37. Srinivas G, Kusumakumary P, Joseph T, Pillai MR . In vitro drug sensitivity and apoptosis induction in newly diagnosed pediatric acute lymphoblastic leukemia: correlation with overall survival. Pediatr Hematol Oncol 2004; 21: 465–473.

    Article  CAS  PubMed  Google Scholar 

  38. Holleman A, den Boer ML, Kazemier KM, Janka-Schaub GE, Pieters R . Resistance to different classes of drugs is associated with impaired apoptosis in childhood acute lymphoblastic leukemia. Blood 2003; 102: 4541–4546.

    Article  CAS  PubMed  Google Scholar 

  39. Haarman EG, Kaspers GJ, Veerman AJ . Glucocorticoid resistance in childhood leukaemia: mechanisms and modulation. Br J Haematol 2003; 120: 919–929.

    Article  CAS  PubMed  Google Scholar 

  40. De Abreu RA, Trueworthy RC, van Kuilenburg AB, Vogels-Mentink TM, Lambooy LH, van Gennip AH . Combination therapy in childhood leukaemia: in vitro studies of thiopurines and inhibitors of purine metabolism on apoptosis. Ann Clin Biochem 2003; 40: 70–74.

    Article  CAS  PubMed  Google Scholar 

  41. Wuchter C, Ruppert V, Schrappe M, Dorken B, Ludwig WD, Karawajew L . In vitro susceptibility to dexamethasone- and doxorubicin-induced apoptotic cell death in context of maturation stage, responsiveness to interleukin 7, and early cytoreduction in vivo in childhood T-cell acute lymphoblastic leukemia. Blood 2002; 99: 4109–4115.

    Article  CAS  PubMed  Google Scholar 

  42. Liu T, Raetz E, Moos PJ, Perkins SL, Bruggers CS, Smith F et al. Diversity of the apoptotic response to chemotherapy in childhood leukemia. Leukemia 2002; 16: 223–232.

    Article  CAS  PubMed  Google Scholar 

  43. Groninger E, de Graaf SS, Meeuwsen-de Boer GJ, Sluiter WJ, Poppema S . Vincristine-induced apoptosis in vivo in peripheral blood mononuclear cells of children with acute lymphoblastic leukaemia (ALL). Br J Haematol 2000; 111: 875–878.

    CAS  PubMed  Google Scholar 

  44. Campana D, Manabe A, Evans WE . Stroma-supported immunocytometric assay (SIA): a novel method for testing the sensitivity of acute lymphoblastic leukemia cells to cytotoxic drugs. Leukemia 1993; 7: 482–488.

    CAS  PubMed  Google Scholar 

  45. Manabe A, Yi T, Kumagai M, Campana D . Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs I. Cytotoxicity of interferon alpha in acute lymphoblastic leukemia. Leukemia 1993; 7: 1990–1995.

    CAS  PubMed  Google Scholar 

  46. Kumagai M, Manabe A, Coustan-Smith E, Blakley RL, Beck WT, Santana VM et al. Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs II. Potent cytotoxicity of 2-chloro-deoxyadenosine in acute lymphoblastic leukemia. Leukemia 1994; 8: 1116–1123.

    CAS  PubMed  Google Scholar 

  47. Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, Muller C et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 2000; 96: 3907–3914.

    CAS  PubMed  Google Scholar 

  48. Zhang S, Fukuda S, Lee Y, Hangoc G, Cooper S, Spolski R et al. Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling. J Exp Med 2000; 192: 719–728.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Quentmeier H, Reinhardt J, Zaborski M, Drexler HG . FLT3 mutations in acute myeloid leukemia cell lines. Leukemia 2003; 17: 120–124.

    Article  CAS  PubMed  Google Scholar 

  50. Mizuki M, Schwable J, Steur C, Choudhary C, Agrawal S, Sargin B et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations. Blood 2003; 101: 3164–3173.

    Article  CAS  PubMed  Google Scholar 

  51. Choudhary C, Schwable J, Brandts C, Tickenbrock L, Sargin B, Kindler T et al. AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations. Blood 2005; 106: 265–273.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Bruce Ruggeri and Susan Jones-Bolin of Cephalon Inc. for providing CEP-701. This work was supported by grants from the Children's Cancer Foundation (PB), the Burroughs Wellcome Fund (DS) and the National Cancer Institute (NCI; CA90668, CA70970, CA100632, DS). PB is a Richard S Ross Clinician Scientist Award recipient. DS is the Douglas Kroll Research Foundation Translational Researcher of the Leukemia and Lymphoma Society and the recipient of the Kyle Haydock Professorship in Oncology.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to P Brown.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brown, P., Levis, M., McIntyre, E. et al. Combinations of the FLT3 inhibitor CEP-701 and chemotherapy synergistically kill infant and childhood MLL-rearranged ALL cells in a sequence-dependent manner. Leukemia 20, 1368–1376 (2006). https://doi.org/10.1038/sj.leu.2404277

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2404277

Keywords

This article is cited by

Search

Quick links