Abstract
Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G4S)2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) ⩾10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocyte–macrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three- to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kantarjian HM, Keating MJ, Talpaz M . Chronic myelogenous leukemia in blast crisis Am J Med 1987 83: 445–454
Keating MJ, Kantarjian H, Smith TL, Estey E, Walters R, Andersson B, Beran M, McCredie KB, Freireich EJ . Response to salvage therapy and survival after relapse in acute myelgenous leukemia J Clin Oncol 1989 7: 1071–1080
List AF . Role of multidrug resistance and its pharmacological modulation in acute myeloid leukemia Leukemia 1996 10: 937–942
Frankel AE, Hall PD, Burbage C, Vesely J, Willingham MC, Bhalla K, Kreitman RJ . Modulation of the apoptotic response of human myeloid leukemia cells to a diphtheria toxin granulocyte–macrophage colony-stimulating factor fusion protein Blood 1997 90: 3654–3661
Hogge DE, Willman CL, Kreitman RJ, Berger M, Hall PD, Kopecky KJ, McLain C, Tagge EP, Eaves CJ, Frankel AE . Malignant progenitors from patients with acute myelogenous leukemia are sensitive to a diphtheria toxin–granulocyte–macrophage colony-stimulating factor fusion protein Blood 1998 92: 589–595
Frankel AE, Lilly M, Kreitman RJ, Hogge D, Beran M, Freedman MH, Emanuel PD, McLain C, Hall PD, Tagge EP, Berger M, Eaves C . Diphtheria toxin fused to granulocyte–macrophage colony-stimulating factor is toxic to blasts from patients with juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia Blood 1998 92: 4279–4286
Frankel AE, Ramage J, Latimer A, Feely T, Delatte S, Hall PD, Tagge EP, Kreitman RJ, Willingham MC . High-level expression and purification of the recombinant diphtheria fusion toxin DTGM for phase I clinical trials Protein Expression Purification 1999 16: 190–201
Suda T, Suda J, Ogawa M, Ihle JN . Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hematopoietic progenitors in culture J Cell Physiol 1985 124: 182–190
Ogata H, Taniguchi S, Inaba M, Sugawara M, Ohta Y, Inaba K, Mori J, Ikehara S . Separation of hematopoietic stem cells into two populations and their characterization Blood 1992 80: 91–95
Park LS, Waldron PE, Friend D, Sassenfeld HM, Price V, Anderson D, Cosman D, Andrews RG, Bernstein ID, Urdal DL . Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness Blood 1989 74: 56–65
Miyauchi J, Kelleher CA, Wong GG, Yang YY, Clark SC, Minkin S, Minden MD, McCulloch EA . The effects of combinations of the recombinant growth factors GM-CSF, G-CSF, IL-3, and CSF-1 on leukemic blast cells in suspension culture Leukemia 1988 2: 382–387
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 alyers and cytokines Blood 1997 90: 2555–2564
Jian X, Lopez A, Holyoake T, Eaves A, Eaves C . Autocrine production and action of IL-3 and G-CSF in chronic myeloid leukemia Proc Natl Acad Sci USA 1999 96: 12804–12809
Steelman LS, Algate PA, Blalock WL, Wang XY, Prevost KD, Hoyle PE, McCubrey JA . Oncogenic effects of overexpression of the interleukin-3 receptor on hematopoietic cells Leukemia 1996 10: 528–542
Hara T, Miyajima A . Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3) EMBO J 1992 11: 1875–1884
Nicola NA, Peterson L, Hilton DJ, Metcalf D . Cellular processing of murine colony-stimulating factor (Multi-CSF, GM-CSF, G-CSF) receptors by normal hematopoietic cells and cell lines Growth Factors 1988 1: 41–49
Kreitman RJ, Pastan I . Recombinant toxins containing human granulocyte–macrophage colony-stimulating factor and either Pseudomonas exotoxin or diphtheria toxin kill gastrointestinal cancer and leukemia cells Blood 1997 90: 252–259
Collier RJ, Kandel J . Structure and activity of diphtheria toxin J Biol Chem 1971 246: 1496–1503
Hwang J, FitzGerald DJ, Adhya S, Pastan I . Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli Cell 1987 48: 129–136
Greenfield L, Johnson VG, Youle RJ . Mutations in diphtheria toxin separate binding from entry and amplify immunotoxin selectivity Science 1987 238: 536–539
Ellman GL . Tissue sulfhydryl groups Arch Biochem Biophys 1959 82: 70–75
Wallin R, Culp EN, Coleman DB, Goodman SR . A structural model of human erythrocyte band 2.1: alignment of chemical and functional domains Proc Natl Acad Sci USA 1984 81: 4095–4099
Matsudaira P . A Practical Guide to Protein and Peptide Purification for Microsequencing Academic: San Diego, CA 1993
Kim CN, Bhalla K, Kreitman RJ, Willingham MC, Hall P, Tagge EP, Jia T, Frankel AE . Diphtheria toxin fused to granulocyte–macrophage colony-stimulating factor and Ara-C exert synergistic toxicity against human AML HL-60 cells Leukemia Res 1999 23: 527–538
Chan CH, Blazar BR, Greenfield L, Kreitman RJ, Vallera DA . Reactivity of murine cytokine fusion toxin, diphtheria toxin390 murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells Blood 1996 88: 1445–1456
Liger D, vanderSpek JC, Gaillard C, Cansier C, Murphy JR, Leboulch P, Gillet D . Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins, DAB389-mIL3 and DAB389-(Gly4Ser)2-mIL-3 FEBS Lett 1997 406: 157–161
Gsur A, Zochbauer S, Gotzl M, Kyrle PA, Lechner K, Pirker R . MDR1 RNA expression as a prognostic factor in acute myeloid leukemia: an update Leuk Lymphoma 1993 12: 91–94
List AF, Spier C, Grogan T, Johnson C, Roe D, Greer J, Wolff S, Broxterman H, Scheffer G, Scheper R, Dalton W . Overexpression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia Blood 1996 87: 2464–2469
Russo D, Marie JP, Zhou DC, Faussat AM, Melli C, Damiani D, Michelutti A, Michieli M, Fanin R, Baccarani M . Evaluation of the clinical relevance of the anionic glutathione-S-transferase (GST pi) and multidrug resistance (mdr-1) gene coexpression in leukemias and lymphomas Leuk Lymphoma 1994 15: 453–468
Falnes PO, Olsnes S . Modulatiion of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway EMBO J 1998 17: 615–625
Mashal R, Shtalrid M, Talpaz M, Kantarjian H, Smith L, Beran M, Cork A, Trujillo J, Gutterman J, Deisseroth A . Rearrangement and expression of p53 in the chronic phase and blast crisis of chronic myelogenous leukemia Blood 1990 75: 180–189
McCurrach ME, Connor TM, Knudson CM, Korsmeyer SJ, Lowe SW . Bax-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis Proc Natl Acad Sci USA 1997 94: 2345–2349
Orashima M, Teoh G, Chauhan D, Ogata A, Shirahama S, Chiharu KK, Matsuzaki M, Matsushima H, Akiyama M, Yuza Y, Maekawa K, Anderson K . MDM2 protein overexpression inhibits apoptosis of TF-1 granulocyte–macrophage colony-stimulating factor-dependent acute myeloblastic leukemia cells Blood 1998 92: 959–967
Hertler A, Schlossman D, Borowitz M, Blythman H, Casellas P, Frankel AE . An anti-CD5 immunotoxin for chronic lymphocytic leukemia: enhancement of cytotoxicity with human serum albumin-monensin Int J Cancer 1989 43: 215–219
Kreitman RJ, Chaudhary VK, Waldmann T, Willingham MC, FitzGerald DJ, Pastan I . The recombinant immunotoxin anti-Tac (Fv)-Pseudomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia Proc Natl Acad Sci USA 1990 87: 8291–8295
Cannistra SA, Koenigsmann M, DiCarlo J, Groshek P, Griffin JD . Differentiation associated expression of two functionally distinct classes of granulocyte–macrophage colony-stimulating factor receptor by human myeloid cells J Biol Chem 1990 265: 12656–12563
Ghielmini M, Colli E, Bosshard G, Pennella G, Geroni C, Torri V, D'Incalci M, Cavalli F, Sessa C . Hematotoxicity on human bone marrow- and umbilical cord blood-derived progenitor cells and in vitro therapeutic index of methoxymorpholinyldoxorubicin and its metabolites Cancer Chemother Pharmacol 1998 42: 235–240
Ghielmini M, Bosshard G, Capolongo L, Geroni MC, Pesenti E, Torri V, D'Incalci M, Cavalli F, Sessa C . Estimate of the haematological toxicity of minor groove alkylators using tests on human cord blood cells Br J Cancer 1997 75: 878–883
Leglise MC, de Tailly PD, Vignot JL, Le Bot MA, Le Roux AM, Riche C . A cellular model for drug interactions on hematopoiesis Cell Biol Toxicol 1996 12: 39–53
Morikawa Y, Tohya K, Hara T, Kitamura T, Miyajima A . Expression of IL3 receptor in testis Biochem Biophys Res Commun 1996 226: 107–112
Korpelainen EI, Gamble JR, Vadas MA, Lopez AF . IL3 receptor expression, regulation and function in cells of the vasculature Immunol Cell Biol 1996 74: 1–17
Tabira T, Chui DH, Fan JP, Shirabe T, Konishi Y . Interleukin-3 and interleukin-3 receptors in the brain Ann NY Acad Sci 1998 840: 107–116
Frankel AE, Hall PD, McLain C, Safa AR, Tagge EP, Kreitman RJ . Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by targeted toxins Bioconj Chem 1998 9: 490–496
Kreitman RJ . IBC's International Conference on novel therapeutic proteins for oncology (drug discovery and development) Exp Opin Invest Drugs 1998 7: 1–8
Hendrie PC, Miyazawa K, Yang YC, Langefeld CD, Broxmeyer HE . Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of the human factor-dependent cell line, M07e Exp Hematol 1991 19: 1031–1037
Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miyagawa K, Piao YF, Miyazono K, Urabe A, Takaku F . Establishment and characterization of a unique human line that proliferates dependently on GM-CSF, IL-3 or erythropoietin J Cell Physiol 1989 140: 323–334
Dalton WT, Ahearn M, McCredie KB, Freireich EJ, Stass SA, Trujillo JM . HL-60 cell line was derived from a patient with FAB-M2 and not FAB-M3 Blood 1988 71: 242–247
Uzman BG, Foley GE, Farber S, Lazarus H . Morphologic variations in human leukemic lymphoblasts (CCRF-CEM) after long term culture and exposure to chemotherapeutic agents Cancer 1966 19: 1725–1742
Lange B, Valtieri M, Santoli D, Caracciolo D, Mavilio F, Gemperlein I, Griffin C, Emanuel B, Finan J, Nowell P, Rovera G . Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines Blood 1987 70: 192–199
Klein E, Ben-Bassat H, Neumann H, Ralph P, Zeuthen J, Polliack A, Vanky F . Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia Int J Cancer 1976 18: 421–431
Ralph P, Nakoinz I . Phagocytosis and cytolysis by a macrophage tumour and its cloned cell line Nature 1975 257: 393–394
Liber HL, Thilly WG . Mutation assay at the thymidine kinase locus in diploid human lymphoblasts Mutat Res 1982 94: 467–485
Isogai C, Murate T, Tamiya-Koizumi K, Yoshida S, Ito T, Nagai H, Kinoshita T, Kagami Y, Hotta T, Hamaguchi M, Saito H . Analysis of bax protein in sphringosine-induced apoptosis in the human leukemic cell line TF1 and its bcl-2 transfectants Exp Hematol 1998 26: 1118–1125
McCubrey JA, Steelman LS, Hoyle PE, Blalock WL, Weinstein-Oppenheimer C, Franklin RA, Cherwinski H, Bosch E, McMahon M . Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells Leukemia 1998 12: 1903–1929
Acknowledgements
This work was supported in part by Leukemia Society of America Grant No. 6114–98 (to AF), NIH Grants No. R01CA76178 (to AF), R01CA54116 (to ET), R01CA51025 (to JM) and grants from the National Cancer Institutes of Canada (NCIC) to DH with funds from the Terry Fox Run.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Frankel, A., McCubrey, J., Miller, M. et al. Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias. Leukemia 14, 576–585 (2000). https://doi.org/10.1038/sj.leu.2401743
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.leu.2401743
Keywords
This article is cited by
-
Updates in Novel Therapies for Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)
Current Hematologic Malignancy Reports (2019)
-
AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells
Journal of Hematology & Oncology (2015)
-
CD 123 is a membrane biomarker and a therapeutic target in hematologic malignancies
Biomarker Research (2014)
-
A Novel Fusion Protein Diphtheria Toxin–Stem Cell Factor (DT-SCF)—Purification and Characterization
Applied Biochemistry and Biotechnology (2010)
-
Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy
Leukemia (2008)