Annals of Hematology

, Volume 91, Issue 3, pp 331–344 | Cite as

3,4-Diarylmaleimides—a novel class of kinase inhibitors—effectively induce apoptosis in FLT3-ITD-dependent cells

  • Florian H. Heidel
  • Thomas S. Mack
  • Elena Razumovskaya
  • Marie-Christine Blum
  • Daniel B. Lipka
  • Anne Ballaschk
  • Jan-Peter Kramb
  • Stanislav Plutizki
  • Lars Rönnstrand
  • Gerd Dannhardt
  • Thomas Fischer
Original Article

Abstract

FLT3 kinase has become an attractive drug target in AML with up to 30% of cases harboring internal-tandem-duplication (ITD) mutations. For these, conferring a worse prognosis and decreased overall survival, several FLT3 tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials. However, when using these drugs as monotherapy, the problem of short duration of remissions and high incidence of TKI resistance has emerged. Here, we investigated two members of a novel class of tyrosine kinase inhibitors, 3,4-diarylmaleimides, for their efficacy on mutated FLT3 kinase. These compounds inhibit FLT3 kinase in an ATP-competitive manner and effectively inhibit phosphorylation of downstream targets. 3,4-Diarylmaleimides (DHF125 and 150) induce apoptosis in FLT3-ITD-dependent cells lines and patient blasts at low micromolar concentrations. They are retained in the cytoplasm of exposed cells for more than 24 h and synergize with chemotherapy and midostaurin. Both 3,4-diarylmaleimides show inhbition of FLT3-ITD-related kinase autophosphorylation at distinct tyrosine residues when compared to midostaurin. In conclusion, this novel group of compounds shows differential inhibition patterns with regard to FLT3 kinase and displays a promising profile for further clinical development. Currently, experiments evaluating toxicity in murine models and unraveling the exact binding mechanism are under way to facilitate a potential clinical application.

Keywords

AML FLT3 Tyrosine kinase inhibitor Tyrosine phosphorylation 

Notes

Acknowledgement

We thank Fian Mirea and Ann-Kathrin Borrmann for technical assistance and Dr. D. Strand (Department of Gastroenterology, University Hospital Mainz) for support with confocal microscopy. This work was supported by grants from the Johannes-Gutenberg-University (MAIFOR, Mainz Research Funding no. 9728249, to F.H.H.) and the German Cancer Aid (DKH 108218 and DKH 108401 (TP6) to T.F.).

Disclosures

F. Heidel, J.-P. Kramb, S. Plutitzki, G. Dannhardt, and T. Fischer filed a patent on the use of 3,4-diarylmaleimides in leukemia.

Supplementary material

277_2011_1311_Fig7_ESM.jpg (32 kb)
Fig. S1

Synergy of DHF compounds with either daunorubicin or cytarabine as investigated in apoptosis assays. Co-incubation of cells with DHF125 or DHF150 and daunorubicin (left panel) or cytarabine (right panel) demonstrated increased efficacy with regard to apoptosis induction. Synergy could be calculated using concentrations above the IC10 for daunorubicin and all effective doses applied for cytarabine. (JPEG 32 kb)

277_2011_1311_MOESM1_ESM.tiff (1.1 mb)
High-resolution image (TIFF 1,092 kb)
277_2011_1311_Fig8_ESM.jpg (49 kb)
Fig. S2

3,4-Diarylmaleimide compounds induce apoptosis in FLT3-ITD harboring AML blasts at micromolar concentrations while leaving normal progenitor cells largely unaffected. diarylmaleimide compounds show induction of apoptosis in primary AML blasts. Primary cells were incubated for 72 h with different inhibitor concentrations. Apoptosis was determined as the amount of cells subG1 exceeding the baseline apoptosis rate of AML cells in culture (baseline apoptosis is graphed as 0%). As indicated for every single patient sample DHF125 (a, left panel) led to increase of basal apoptosis by 10–17% while DHF150 (a, right panel) elevated the rate of apoptotic cells by 17–30%. Using bone marrow cells from healthy donors, colony formation was analyzed upon increasing doses of diarylmaleimide inhibitors—in methylcellulose supplemented with cytokines—for 10 days. Impairment of colony formation was not detectable up to a dose of 5 μM of either compound. Incubation of these cells with 10 μM of either inhibitor led to almost complete loss of colony formation, suggesting toxicity on healthy hematopoietic stem and progenitor cells. (JPEG 49 kb)

277_2011_1311_MOESM2_ESM.eps (100 kb)
High-resolution image (EPS 99 kb)

References

  1. 1.
    Godwin JE, Kopecky KJ, Head DR, Willman CL, Leith CP, Hynes HE, Balcerzak SP, Appelbaum FR (1998) A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest Oncology Group Study (9031). Blood 91(10):3607–3615PubMedGoogle Scholar
  2. 2.
    Goldstone AH, Burnett AK, Wheatley K, Smith AG, Hutchinson RM, Clark RE (2001) Attempts to improve treatment outcomes in acute myeloid leukemia (AML) in older patients: the results of the United Kingdom Medical Research Council AML11 trial. Blood 98(5):1302–1311. doi: 10.1182/blood.V98.5.1302 PubMedCrossRefGoogle Scholar
  3. 3.
    Lowenberg B, Suciu S, Archimbaud E, Haak H, Stryckmans P, de Cataldo R, Dekker AW, Berneman ZN, Thyss A, van der Lelie J, Sonneveld P, Visani G, Fillet G, Hayat M, Hagemeijer A, Solbu G, Zittoun R (1998) Mitoxantrone versus daunorubicin in induction-consolidation chemotherapy–the value of low-dose cytarabine for maintenance of remission, and an assessment of prognostic factors in acute myeloid leukemia in the elderly: final report. European Organization for the Research and Treatment of Cancer and the Dutch-Belgian Hemato-Oncology Cooperative Hovon Group. J Clin Oncol 16(3):872–881PubMedGoogle Scholar
  4. 4.
    Mayer RJ, Davis RB, Schiffer CA, Berg DT, Powell BL, Schulman P, Omura GA, Moore JO, McIntyre OR, Frei E, The Cancer and Leukemia Group B (1994) Intensive postremission chemotherapy in adults with acute myeloid leukemia. N Engl J Med 331(14):896–903. doi: 10.1056/nejm199410063311402 PubMedCrossRefGoogle Scholar
  5. 5.
    Stone RM, Berg DT, George SL, Dodge RK, Paciucci PA, Schulman P, Lee EJ, Moore JO, Powell BL, Schiffer CA, The Cancer and Leukemia Group B (1995) Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. N Engl J Med 332(25):1671–1677. doi: 10.1056/nejm199506223322503 PubMedCrossRefGoogle Scholar
  6. 6.
    Mrozek K, Heerema NA, Bloomfield CD (2004) Cytogenetics in acute leukemia. Blood Rev 18(2):115–136PubMedCrossRefGoogle Scholar
  7. 7.
    Langer C, Radmacher MD, Ruppert AS, Whitman SP, Paschka P, Mrozek K, Baldus CD, Vukosavljevic T, Liu CG, Ross ME, Powell BL, de la Chapelle A, Kolitz JE, Larson RA, Marcucci G, Bloomfield CD (2008) High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: a Cancer and Leukemia Group B (CALGB) study. Blood 111(11):5371–5379PubMedCrossRefGoogle Scholar
  8. 8.
    Marcucci G, Maharry K, Whitman SP, Vukosavljevic T, Paschka P, Langer C, Mrozek K, Baldus CD, Carroll AJ, Powell BL, Kolitz JE, Larson RA, Bloomfield CD (2007) High expression levels of the ETS-related gene, ERG, predict adverse outcome and improve molecular risk-based classification of cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B Study. J Clin Oncol 25(22):3337–3343PubMedCrossRefGoogle Scholar
  9. 9.
    Mrozek K, Dohner H, Bloomfield CD (2007) Influence of new molecular prognostic markers in patients with karyotypically normal acute myeloid leukemia: recent advances. Curr Opin Hematol 14(2):106–114PubMedCrossRefGoogle Scholar
  10. 10.
    Heuser M, Beutel G, Krauter J, Dohner K, von Neuhoff N, Schlegelberger B, Ganser A (2006) High meningioma 1 (MN1) expression as a predictor for poor outcome in acute myeloid leukemia with normal cytogenetics. Blood 108(12):3898–3905PubMedCrossRefGoogle Scholar
  11. 11.
    Dohner K, Schlenk RF, Habdank M, Scholl C, Rucker FG, Corbacioglu A, Bullinger L, Frohling S, Dohner H (2005) Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood 106(12):3740–3746PubMedCrossRefGoogle Scholar
  12. 12.
    Frohling S, Schlenk RF, Stolze I, Bihlmayr J, Benner A, Kreitmeier S, Tobis K, Dohner H, Dohner K (2004) CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations. J Clin Oncol 22(4):624–633PubMedCrossRefGoogle Scholar
  13. 13.
    Paschka P, Marcucci G, Ruppert AS, Whitman SP, Mrozek K, Maharry K, Langer C, Baldus CD, Zhao W, Powell BL, Baer MR, Carroll AJ, Caligiuri MA, Kolitz JE, Larson RA, Bloomfield CD (2008) Wilms tumor 1 gene mutations independently predict poor outcome in adults with cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B Study. J Clin Oncol 26(28):4595–4602PubMedCrossRefGoogle Scholar
  14. 14.
    Frohling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis K, Dohner H, Dohner K (2002) Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood 100(13):4372–4380PubMedCrossRefGoogle Scholar
  15. 15.
    Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U, Wermke M, Bornhauser M, Ritter M, Neubauer A, Ehninger G, Illmer T (2002) Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 99(12):4326–4335PubMedCrossRefGoogle Scholar
  16. 16.
    Breitenbuecher F, Schnittger S, Grundler R, Markova B, Carius B, Brecht A, Duyster J, Haferlach T, Huber C, Fischer T (2009) Identification of a novel type of ITD mutations located in nonjuxtamembrane domains of the FLT3 tyrosine kinase receptor. Blood 113(17):4074–4077PubMedCrossRefGoogle Scholar
  17. 17.
    Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S (1996) Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10(12):1911–1918PubMedGoogle Scholar
  18. 18.
    Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T, Sonoda Y, Abe T, Kahsima K, Matsuo Y, Naoe T (1997) Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia 11(10):1605–1609PubMedCrossRefGoogle Scholar
  19. 19.
    Gale RE, Green C, Allen C, Mead AJ, Burnett AK, Hills RK, Linch DC (2008) The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood 111(5):2776–2784PubMedCrossRefGoogle Scholar
  20. 20.
    Kayser S, Schlenk RF, Correa Londono M, Breitenbuecher F, Wittke K, Du J, Groner S, Spath D, Krauter J, Ganser A, Dohner H, Fischer T, Dohner K (2009) Insertion of FLT3 internal tandem duplication in the tyrosine kinase domain-1 is associated with resistance to chemotherapy and inferior outcome. Blood 114(12):2386–2392PubMedCrossRefGoogle Scholar
  21. 21.
    Griffith J, Black J, Faerman C, Swenson L, Wynn M, Lu F, Lippke J, Saxena K (2004) The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Mol Cell 13(2):169–178PubMedCrossRefGoogle Scholar
  22. 22.
    Kelly LM, Liu Q, Kutok JL, Williams IR, Boulton CL, Gilliland DG (2002) FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 99(1):310–318PubMedCrossRefGoogle Scholar
  23. 23.
    Cools J, Mentens N, Furet P, Fabbro D, Clark JJ, Griffin JD, Marynen P, Gilliland DG (2004) Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia. Cancer Res 64(18):6385–6389PubMedCrossRefGoogle Scholar
  24. 24.
    Heidel F, Solem FK, Breitenbuecher F, Lipka DB, Kasper S, Thiede MH, Brandts C, Serve H, Roesel J, Giles F, Feldman E, Ehninger G, Schiller GJ, Nimer S, Stone RM, Wang Y, Kindler T, Cohen PS, Huber C, Fischer T (2006) Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain. Blood 107(1):293–300PubMedCrossRefGoogle Scholar
  25. 25.
    Graf C, Heidel F, Tenzer S, Radsak MP, Solem FK, Britten CM, Huber C, Fischer T, Wolfel T (2007) A neoepitope generated by an FLT3 internal tandem duplication (FLT3-ITD) is recognized by leukemia-reactive autologous CD8+ T cells. Blood 109(7):2985–2988PubMedGoogle Scholar
  26. 26.
    Dannhardt G, Fischer T, Heidel F, Peifer C, Plutizki S, Kramb JP (2007) Use of 3-(4-indolyl)- or 3-(azaindolyl)-4-arylmaleimide derivatives in leukemia management. Patent no. WO 2009/071620 A1.Google Scholar
  27. 27.
    Peifer C, Krasowski A, Hammerle N, Kohlbacher O, Dannhardt G, Totzke F, Schachtele C, Laufer S (2006) Profile and molecular modeling of 3-(indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1 H-pyrrole-2,5-dione (1) as a highly selective VEGF-R2/3 inhibitor. J Med Chem 49(25):7549–7553PubMedCrossRefGoogle Scholar
  28. 28.
    Peifer C, Stoiber T, Unger E, Totzke F, Schachtele C, Marme D, Brenk R, Klebe G, Schollmeyer D, Dannhardt G (2006) Design, synthesis, and biological evaluation of 3,4-diarylmaleimides as angiogenesis inhibitors. J Med Chem 49(4):1271–1281PubMedCrossRefGoogle Scholar
  29. 29.
    Razumovskaya E, Masson K, Khan R, Bengtsson S, Ronnstrand L (2009) Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation. Exp Hematol 37:979–989PubMedCrossRefGoogle Scholar
  30. 30.
    Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58(3):621–681PubMedCrossRefGoogle Scholar
  31. 31.
    Lipka DB, Hoffmann LS, Heidel F, Markova B, Blum MC, Breitenbuecher F, Kasper S, Kindler T, Levine RL, Huber C, Fischer T (2008) LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells. Mol Cancer Ther 7(5):1176–1184PubMedCrossRefGoogle Scholar
  32. 32.
    Weisberg E, Boulton C, Kelly LM, Manley P, Fabbro D, Meyer T, Gilliland DG, Griffin JD (2002) Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell 1(5):433–443PubMedCrossRefGoogle Scholar
  33. 33.
    Heidel F, Lipka DB, Mirea FK, Mahboobi S, Grundler R, Kancha RK, Duyster J, Naumann M, Huber C, Bohmer FD, Fischer T (2009) Bis(1H-indol-2-yl)methanones are effective inhibitors of FLT3-ITD tyrosine kinase and partially overcome resistance to PKC412A in vitro. Br J Haematol 144(6):865–874PubMedCrossRefGoogle Scholar
  34. 34.
    Kasper S, Breitenbuecher F, Hoehn Y, Heidel F, Lipka DB, Markova B, Huber C, Kindler T, Fischer T (2008) The kinase inhibitor LS104 induces apoptosis, enhances cytotoxic effects of chemotherapeutic drugs and is targeting the receptor tyrosine kinase FLT3 in acute myeloid leukemia. Leuk Res 32(11):1698–1708PubMedCrossRefGoogle Scholar
  35. 35.
    Fischer T, Stone RM, Deangelo DJ, Galinsky I, Estey E, Lanza C, Fox E, Ehninger G, Feldman EJ, Schiller GJ, Klimek VM, Nimer SD, Gilliland DG, Dutreix C, Huntsman-Labed A, Virkus J, Giles FJ (2010) Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3. J Clin Oncol 28(28):4339–4345. doi: 10.1200/JCO.2010.28.9678 PubMedCrossRefGoogle Scholar
  36. 36.
    Stone RM, DeAngelo DJ, Klimek V, Galinsky I, Estey E, Nimer SD, Grandin W, Lebwohl D, Wang Y, Cohen P, Fox EA, Neuberg D, Clark J, Gilliland DG, Griffin JD (2005) Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 105(1):54–60. doi: 10.1182/blood-2004-03-0891 PubMedCrossRefGoogle Scholar
  37. 37.
    Levis M, Allebach J, Tse KF, Zheng R, Baldwin BR, Smith BD, Jones-Bolin S, Ruggeri B, Dionne C, Small D (2002) A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood 99(11):3885–3891PubMedCrossRefGoogle Scholar
  38. 38.
    Zarrinkar PP, Gunawardane RN, Cramer MD, Gardner MF, Brigham D, Belli B, Karaman MW, Pratz KW, Pallares G, Chao Q, Sprankle KG, Patel HK, Levis M, Armstrong RC, James J, Bhagwat SS (2009) AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood 114(14):2984–2992PubMedCrossRefGoogle Scholar
  39. 39.
    Shah NP, Kasap C, Weier C, Balbas M, Nicoll JM, Bleickardt E, Nicaise C, Sawyers CL (2008) Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 14(6):485–493PubMedCrossRefGoogle Scholar
  40. 40.
    Huber S, Oelsner M, Decker T, Zum Buschenfelde CM, Wagner M, Lutzny G, Kuhnt T, Schmidt B, Oostendorp RA, Peschel C, Ringshausen I (2011) Sorafenib induces cell death in chronic lymphocytic leukemia by translational downregulation of Mcl-1. Leukemia 25(5):838–847. doi: 10.1038/leu.2011.2 PubMedCrossRefGoogle Scholar
  41. 41.
    Rocnik JL, Okabe R, Yu JC, Lee BH, Giese N, Schenkein DP, Gilliland DG (2006) Roles of tyrosine 589 and 591 in STAT5 activation and transformation mediated by FLT3-ITD. Blood 108(4):1339–1345PubMedCrossRefGoogle Scholar
  42. 42.
    Vempati S, Reindl C, Wolf U, Kern R, Petropoulos K, Naidu VM, Buske C, Hiddemann W, Kohl TM, Spiekermann K (2008) Transformation by oncogenic mutants and ligand-dependent activation of FLT3 wild-type requires the tyrosine residues 589 and 591. Clin Cancer Res 14(14):4437–4445PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Florian H. Heidel
    • 1
  • Thomas S. Mack
    • 1
  • Elena Razumovskaya
    • 2
  • Marie-Christine Blum
    • 1
  • Daniel B. Lipka
    • 1
  • Anne Ballaschk
    • 1
  • Jan-Peter Kramb
    • 3
  • Stanislav Plutizki
    • 3
  • Lars Rönnstrand
    • 2
  • Gerd Dannhardt
    • 3
  • Thomas Fischer
    • 1
  1. 1.Department of Hematology and Oncology, Medical CenterOtto-von-Guericke UniversityMagdeburgGermany
  2. 2.Experimental Clinical Chemistry, Wallenberg Laboratory, Department of Laboratory MedicineSkåne University Hospital, Lund UniversityMalmöSweden
  3. 3.Department of PharmacyJohannes-Gutenberg-UniversityMainzGermany

Personalised recommendations